PAPER SPRAY MASS SPECTROMETRY FOR RAPID DRUG SCREENING by Rachel Jett A Thesis Submitted to the Faculty of Purdue University In Partial Fulfillment of the Requirements for the degree of Master of Science Department of Chemistry Indianapolis, Indiana August 2017 ii THE PURDUE UNIVERSITY GRADUATE SCHOOL STATEMENT OF COMMITTEE APPROVAL Dr. Nick Manicke, Chair Department of Chemistry and Chemical Biology Dr. John Goodpaster Department of Chemistry and Chemical Biology Dr. Michael McLeish Department of Chemistry and Chemical Biology Approved by: Dr. Eric C. Long Head of the Graduate Program iii To the CreatorWho places the mysteries of the world before us and the curiosities of the mind within us iv ACKNOWLEDGMENTS This project would not have been possible without the input, support, and feedback of the Manicke and Goodpaster Groups: Nick, Chengsen, Brandon, Greta, Josiah, Grace, John, Dana, Will, Jordan, Clinton Thank you. v TABLE OF CONTENTS LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ............................................................................................................ x LIST OF ABBREVIATIONS .......................................................................................... xiv ABSTRACT ................................................................................................................. xv CHAPTER 1: BACKGROUND ......................................................................................... 1 Toxicology...................................................................................................................... 1 Forensic Toxicology ....................................................................................................... 2 Human Performance Toxicology ................................................................................ 3 Doping Control ............................................................................................................ 3 Workplace Drug Testing ............................................................................................. 3 Postmortem Investigations .......................................................................................... 4 Analytical Strategy ......................................................................................................... 4 Targeted Analysis ........................................................................................................ 4 General Unknown Screening....................................................................................... 5 Practical Toxicology.................................................................................................... 6 The Need for Something New ........................................................................................ 7 CHAPTER 2: INTRODUCTION ....................................................................................... 9 Project Overview ............................................................................................................ 9 Paper Spray-Mass Spectrometry .................................................................................. 10 Selectivity ..................................................................................................................... 14 Mass Spectrometer Selection .................................................................................... 14 Fragmentation ............................................................................................................ 15 Selectivity in Paper Spray ......................................................................................... 16 Special Considerations ................................................................................................. 18 Postmortem blood...................................................................................................... 19 Matrix Effects ............................................................................................................ 20 CHAPTER 3: MATERIALS AND METHODS .............................................................. 23 Chemicals and Reagents ............................................................................................... 23 vi Mass Spectrometer and Materials ................................................................................ 23 Method.......................................................................................................................... 23 Manual Method ......................................................................................................... 24 Automated Method .................................................................................................... 26 CHAPTER 4: METHOD DEVELOPMENT.................................................................... 27 Solvent Selection .......................................................................................................... 27 Paper Shape and Sample Loading ................................................................................ 29 Placement of Sample................................................................................................ 30 Loading Capacity and Blood Dilution ..................................................................... 30 Tuning .......................................................................................................................... 36 Acquisition Parameter .................................................................................................. 38 Screening Identification Criteria .................................................................................. 39 CHAPTER 5: RESULTS .................................................................................................. 45 Results Summarized ..................................................................................................... 46 CHAPTER 6: INTERFERENCE STUDY ....................................................................... 67 Exogenous interferences............................................................................................... 68 Intra-target interferences .............................................................................................. 68 Conclusions and Future Work ................................................................................. 76 CHAPTER 7: CONCLUSIONS ....................................................................................... 78 Summary of Conclusions ............................................................................................. 78 Discussion .................................................................................................................... 79 Future work .................................................................................................................. 80 BIBLIOGRAPHY ............................................................................................................. 82 APPENDIX A: PURPOSED ANALYTICAL TARGETS WITH CUTOFF VALUES FROM AFT .............................................................................................. 89 APPENDIX B: DETAILED RESULTS FROM LOADING CAPACITY AND BLOOD DILUTION STUDY ................................................................................. 108 APPENDIX C: MASS SPEC PARAMETERS .............................................................. 113 APPENDIX D: SPIKING SOLUTION COCKTAILS .................................................. 122 APPENDIX E: DETAILED DATA BY COCKTAIL ................................................... 126 APPENDIX F: EXOGENOUS INTERFERENCES ...................................................... 185 vii LIST OF TABLES Table 1: Screening cutoff values for the target anlaytes in the project ranged from less than 5 ng/mL to over 1000 ng/mL. ...........................................................11 Table 2: MS-MS based identification criteria for various applications .........................18 Table 3: 47 different solvent systems were tested for use with PS-MS in an effort to identify a solvent that would produce a steady Taylor cone, prevent electrical discharge, and effectively extract target analytes. ............................28 Table 4: Relative AUC of fragment ions at different positions on the long paper strip. The lowest AUC was normalized to 1.00 for comparative purposes and the position on the paper strip with the largest AUC is denoted in green. ................................................................................................................31 Table 5: Four dilutions schemes used to test the effect of diluting blood on the signal to blank ratio of five analytical targets. .................................................32 Table 6: Results from thirteen analytes show that using long paper with diluted blood generally gives lower blank signals and higher analyte response than using whole blood and 3mm punches. The first seven analytes (in blue) were run in one set of samples and the last 6 (in red) were run in a second set of samples. ..................................................................................................35 Table 7: A list of targets run at concentrations other than those proposed by AFT compared to normal, toxic, and fatal levels2 Normal levels are defined as the effective dose where no or minimal side effects occur. Toxic levels are those at which side effects or negative symptoms arise, and fatal levels are the concentrations which result in either coma or death. ................................ 48 Table 8: Summary of results for all 154 proposed analytical targets ............................ 49 Table 9: Cocktail A-Manual experiments had 26 scans per target and experiments run on the automated ionization source on the laser-cut paper had 16 scans per target. Red shading indicates failure to meet detection criteria. .............. 52 viii Table 10: Cocktail B-Manual experiments had 35 scans per target and experiments run on the automated ionization source on the laser-cut paper had 16 scans per target. Red shading indicates failure to meet detection criteria. ............... 53 Table 11: Cocktail C-Manual experiments had 32 scans per target and experiments run on the automated ionization source on the laser-cut paper had 16 scans per target. Red shading indicates failure to meet detection criteria. ............... 54 Table 12: Cocktail D- Manual experiments had 20 scans per target and experiments Run on the automated ionization source had 15 scans per target. Red shading indicates failure to meet detection criteria......................................... 55 Table 13: Cocktail E- Manual experiments had 20 scans per target and experiments run on the automated ionization source had 15 scans per target. Red shading indicates failure to meet detection criteria......................................... 57 Table 14: Cocktail F-Manual experiments had 27 scans per target and experiments run on the automated ionization source had 20 scans per target. Red shading indicates failure to meet detection criteria......................................... 59 Table 15: Cocktail G-Manual experiments had 24 scans per target and experiments Run on the automated ionization source had 20 scans per target. Red shading indicates failure to meet detection criteria......................................... 60 Table 16: Cocktail H Manual experiments had 28 scans per target and experiments run on the automated ionization source had 21 scans per target. .................... 61 Table 17: Cocktail I-Manual experiments had 28 scans per target and experiments run on the automated ionization source had 21 scans per target. Red shading indicates failure to meet detection criteria......................................... 62 Table 18: Cocktail J-Manual experiments had 19 scans per target and experiments run on the automated ionization source had 18 scans per target. Red shading indicates failure to meet detection criteria......................................... 63 Table 19: Cocktail K-Manual experiments had 30 scans per target and experiments run on the automated ionization source had 18 scans per target. Red shading indicates failure to meet detection criteria......................................... 64 ix Table 20: Cocktail L-Manual experiments had 14 scans per target and experiments run on the automated ionization source had 19 scans per target. Red shading indicates failure to meet detection criteria......................................... 65 Table 21: Cocktail M- Manual experiments had 13 scans per target and experiments run on the automated ionization source had 18 scans per target. Red shading indicates failure to meet detection criteria......................................... 66 Table 22: Targets that share the same parent ion and could potentially interfere with one another. ............................................................................................. 69 Table 23: Using an ESI spray source, targets were analyzed at 1000 ng/mL for the transitions used in the analysis of other targets which share the same parent ion. S:B less than 3:1 indicated that the target would not interfere with the channels scanned for the other target, while a greater signal routed targets through additional interference testing. The following 25 targets were found to have S:B greater than 3:1 for the transitions recorded below ................................................................................................ 71 Table 24: Targets with the same parent ion were each added at 100 ng/mL to the spray solvent and sprayed over blank paper. The fragmentation ion for each target was monitored and those that differed from their established ratio by greater than the permissible deviation of ±30% were flagged, as shown in red. The remaining targets were concluded to not interfere with one another............................................................................................................. 75 x LIST OF FIGURES Figure 1: Drug classes represented in this project. The size of each block is proportional to the number of targets in that class ...........................................10 Figure 2: Schematic of paper spray .................................................................................12 Figure 3: (A) During paper spray, a piece of paper cut into a sharp tip is positioned 5 mm away from the inlet of a mass spectrometer. (B) Solvent is added and allowed to saturate the paper while the voltage remains off. (C) When high voltage is applied, a Taylor cone forms at the paper’s tip, emitting a plume of charged droplets that quickly evaporate, leaving behind gaseous ions that enter the inlet of the mass spectrometer. ...........................................12 Figure 4: Unlike typical chromatograms produced in LC-MS, the “peaks” produced in PS-MS are boxy rather than Gaussian and are called “chronograms”. This is because essentially no chromatography occurs during PS-MS. When the voltage is turned on, all extracted ions enter the mass spectrometer at the same time, leading to an almost instantaneous rise in ion intensity. When the voltage is turned off, the Taylor cone collapses and ionization stops. An example chronogram of the transition from m/z 172 -> 119 for gabapentin in blank blood and at 500 ng/mL is depicted. ...........................................................................................................13 Figure 5: (A) An in-house designed reusable cartridge was used for manual experiments. Using this set up 3µL blood spots were dried onto pentagon-shaped papers hand-cut from Whatman 31ET chromatography paper and inserted into a slot at the front of the cartridge. In this design, solvent is applied through a well directly over blood spot. (B) Commercial Velox cartridges from Prosolia were used for automated experiments. In this set up, 12µL blood spots are dried onto precut paper stored inside individual disposable cartridges. Solvent is applied into well behind blood spot and wicks through the paper and blood spot. .............24 xi Figure 6: (A) Using the manual method, 3µL of whole blood was used to saturate a 3.5mm round paper punch (B) Pentagonal paper was hand-cut with razor blades and the dried blood spot was placed on top of the paper. (C) The automated method utilized a larger, different shaped paper design than the manual method. This precut paper was housed in single-use Prosolia cartridges and 12µL of whole blood was directly applied to the paper for analysis. ............................................................................................................25 Figure 7: Paper shape and sample loading capacity were tested on two different paper designs. (A) Traditional pentagonal-shaped papers provided a base-line reference while (B) 30mm long pointed paper strips were used to test the effects of higher loading capacities .................................................29 Figure 8: Plots of signal to blank ratio using blood diluted 1:1 on 30mm long paper for (A) Zolpidem (B) Morphine (C) Clonazepam (D) Fentanyl (E) Buprenorphine indicate an optimal loading capacity of 20 µL for diluted blood .................................................................................................................33 Figure 9: Plots of signal to blank ratio using whole blood on long paper for (A) Zolpidem (B) Morphine (C) Clonazepam (D) Fentanyl (E) Buprenorphine. Although analytes do not follow the same trend, t hey generally show an optimal loading capacity of ~20 uL of whole blood.................................................................................................................34 Figure 10: An example of some of the data outputted from the tuning software. In the curves produced for Flunitrazepam above, the 4 most intense fragment ions are plotted against collision energy. Some fragments, like m/z 268 are more sensitive to changes in collision energy, as is evident in the narrow peak in the curve, while other fragments with broad peaks, like m/z 183, are less effected by changes in collision energy. ...............................................................................................................36 xii Figure 11: When selecting appropriate fragment ions to monitor, fragmentation ratio stability over a range of concentrations was considered. For example, while m/z 83 was found to be the most abundant fragment for norbuprenorphine during tuning, ratios that use fragment m/z 83 do not behave proportionally, especially near the target detection level (1ppb). In cases like this, a lesser abundant fragment is used to provide fragmentation ratios that are more consistent over a range of concentrations ..................................................................................................38 Figure 12: Running average plot of fragmentation ratios for targets with ratios > 50 show ratios generally stabilize after 15 scans on each fragment ion channel .............................................................................................................40 Figure 13: Running average plot of fragmentation ratios for targets with ratios < 50 show ratios generally stabilize after 15 scans on each fragment ion channel .............................................................................................................41 Figure 14: Deviation between solvent-established fragmentation ratios and fragmentations ratios in whole blood at cutoff concentrations for target analytes where both fragments were detected with a S:B ≥3 ..........................43 Figure 15 Number of analytical targets detected using three different methods. Out of the 133 targets analyzed using the manual method, 86% met detection criteria; for the 131 targets analyzed using the automatic method with laser-cut paper, 76% met detection criteria. Of the 68 targets analyzed using the automatic method with die-cut paper, 87% met detection criteria ............................................................................................................. 47 Figure 16. Stacked column graphs representing the reason that targets failed to meet detection criteria for each of the three methods. When the manual method was used, 53% of failed detections were due to ratio deviations outside of the permitted ±30% window. For the automatic method using laser-cut paper 53% of failures were due to ratio deviations, and for die-cut experiments, the failure due to ratio deviations reached 75%. ....................... 51 xiii Figure 17: (A) Of the 68 targets run using all three method, 16 were detected in at least one method, but not in all three. There was one instance (desmethylclomipramine ) where the target failed to be detected in any of the three methods. Venn diagram A depicts the methods where these 17 targets failed to meet detection criteria. (B) Of the 63 targets run using only the manual and laser-cut methods 23 total targets were not detected, 7 of which were detected in neither method. Venn diagram B specifies in which method these failures to meet detection criteria occurred. ................... 51 xiv LIST OF ABBREVIATIONS AFT CE CID ELISA EMIT ESI FPIA Axis Forensic Toxicology GC-MS GUS Gas Chromatography-Mass Spectrometry MTS PMR PS PS-MS QqQ RIA S:B SRM STA Multi-Target Screening Collision Energies Collision Induced Dissociation Enzyme-linked Immunosorbent Assays Enzyme-multiplied Immunoassay Technique Electro Spray Ionization Fluorescence Polarization Immunoassays General Unknown Screening HPLC-MS High Performance Liquid Chromatography Mass Spectrometry HR-MS High Resolution Mass Spectrometry IS Internal Standards KIMS Kinetic Interaction of Microparticles in Solution LC-UV Liquid chromatography with Ultra Violet Detection LLE Liquid-Liquid Extraction LLOQ Lower Limit of Quantitation LOD Limit of Detection WADA XIC Post-mortem Redistribution Paper Spray Paper Spray Mass Spectrometry Triple Quadrupole Mass Spectrometers Radioimmunoassays Signal to Blank Ratio Selected Reaction Monitoring Systematic Toxicological Analysis World Anti-Doping Agency Extracted Ion Chronograms xv ABSTRACT Author: Jett, Rachel MS Institution: Purdue University Degree Received: August 2017 Title: Paper Spray Mass Spectrometry for Rapid Drug Screening Major Professor: Nick Manicke Paper spray mass spectrometry is an alternative technique for toxicological screening that is able to quickly and adequately screen for compounds encountered in postmortem investigations with little sample handling and no sample preparation. For analysis of dried blood spots using a triple quadrupole mass spectrometer, detection criteria were defined to align with relevant regulatory guidelines while considering how fragment ion selection, method sensitivity, and fragment ion ratio tolerances are best utilized in paper spray mass spectrometry. For analysis, drugs and drug metabolites relevant to postmortem investigations were spiked into drug-free blood, and by monitoring two fragment ion channels in selected reaction monitoring mode, as well as the ratio between the two fragment ions, a method was developed capable of detecting over 120 drug and drug metabolites at concentrations relevant to postmortem drug screening. Total analysis time for the developed method is less than 8 minutes, and less than 50µL of sample and 5mL of solvent are consumed during analysis. 1 1 BACKGROUND The following project was undertaken with the goal of developing a multi-target toxicological screening process that would be applicable for use in forensic postmortem investigation cases. While the same method may be applied in different areas of forensics or even in different fields within toxicology, it was postmortem investigation that provided the context that shaped this project. As such, a brief overview of how postmortem toxicology relates to other disciplines and how analysis is typically carried out within this context will provide a basis for understanding how this project advances the field of forensic toxicology and why certain decisions were made along the way. 1.1 Toxicology The word, “toxicology” is derived from the Greek root toxicos, meaning “poisonous.” At its core, toxicology is simply the study of poisons. Hundreds of years ago, however, one of the fathers of toxicology, Paracelsus, famously stated that any physical or chemical agent can be defined as a poison, and that, “Solely the dose determines that a thing is not a poison.”4 Toxicology then, is actually a very broad field that draws from organic and analytical chemistry, genetics, biochemistry, ecology, pharmacology, pathology, physiology, and other areas of study in order to identify what physical agents and chemical substances cause toxic effects in living organisms, at what dose those effects arise, the mechanisms through which those effects take place, and how to limit or treat exposure to these poisons. The diverse background necessary to understand the complexities of toxicology also allow for its application into a wide variety of situations. From determining how many aspirin we take for headache relief to what chemicals OSHA allows us to handle at work, the impact of toxicology is woven throughout our daily lives. Although there are a wide range of applications for toxicology, a few subdisciplines represent much of the prominent work in the field. Clinical and medicinal toxicology are concerned with the development and safe use of medicines and pharmaceuticals that are used to treat, manage, or prevent disease. Environmental 2 toxicology looks at how industrial waste and byproducts can adversely affect humans and other living creatures in the environment. Food toxicology promotes food safety by monitoring and setting limitations on what pesticides and/or veterinary medicines are safe for human consumption. Forensic toxicology is used when toxicological effects may have legal ramifications, and can include anything from determining a person’s cause of death to detecting the use of illegal performance-enhancing drugs in racehorses. 1.2 Forensic Toxicology While other branches of toxicology are concerned with why a compound is toxic or the mechanisms by which it affects an organism, forensic toxicology is used mostly in situations where the presence or absence of a substance has legal implications. While forensic toxicologists must still understand the hows and whys of toxicology, their primary focus is on the detection, identification, and quantification of toxic agents in the body. There are four essential disciplines within forensic toxicology: human performance investigation, doping control, workplace drug testing, and postmortem investigations.9 1.2.1 Human Performance Toxicology Human performance toxicology is concerned with identifying the presence and concentration of drugs and chemicals which are known to affect the way a person reasons and behaves. Identifying the presence of these chemicals can aid in different types of investigations ranging from collision investigations where a driver is suspected to have been impaired, to drug facilitated crimes such sexual assault cases, and even to child welfare cases. The primary analytical targets of these investigations tend to be alcohol and drugs of abuse. 1.2.2 Doping Control In competitive sports, using certain performance enhancing substances is illegal and is regulated and monitored by organizations like the World Anti-Doping Agency 3 (WADA). While most of these drugs have legitimate medical application, banning their use provides a level playing field for athletes and protects their health. These substances, which include steroids, diuretics, and stimulants, are generally not illegal for the general public, but because of the rules and regulations of the professional sports associations, detecting them in athletes can have legal consequences.9 1.2.3 Workplace Drug Testing In 1988, Congress passed the Drug-Free Workplace Act that mandates that federal employees or employees of a company operating on federal money are prohibited from using recreational drugs. Many other companies and organizations, especially those where workers preform potentially dangerous tasks, have chosen to comply with these standards in order to promote workplace safety. Before being hired and during random screenings, employees will give a urine sample which is typically screened for five major classes of abused drugs.9 If a toxicologist identifies the presence of one of these drugs, there can be occupational and legal consequences for the parties involved. 1.2.4 Postmortem Investigations The toxicity of a chemical can produce adverse effects ranging anywhere from a headache or a rash to loss of fine motor skills or blindness. In the most severe cases, however, the toxicity of a substance or the dose administered is so extreme that it results in coma or death. In death investigation cases, also known as postmortem investigations, toxicologists work with medical examiners and coroners to help determine cause and manner of death. Unlike in other areas of toxicology, toxicologists investigating postmortem cases must consider a wide range of compounds instead of a small, targeted list. In these cases, toxicologists are presented with a sample that is truly unknown and is presented with the difficult job of detecting, identifying, and quantifying any and all possible toxicants present within any given sample. 4 1.3 Analytical Strategy The analytical strategies employed by a forensic toxicologist are often dictated by practicality rather than ideology. Legal goals, limitations within the laboratory, and the circumstances surrounding a case all shape how each sample is processed. Individual labs determine an analytical strategy that is fit for the purpose of each case and is an effective use of the time, money, and resources available to it. In theory, there are two basic analytical strategies: targeted analysis and general unknown screening. In practice, however, many laboratories operate on a hybrid of these methods and use a two-fold method of screening and confirmatory testing. 1.3.1 Targeted Analysis In cases where there are a finite number of compounds of interest, such as in doping control or workplace drug testing, a targeted, or directed analytical approach may be used. The result of this type of analysis is a list of compounds whose presence in a sample is either confirmed or excluded at certain concentrations. The list of targets analyzed can range from a handful of compounds to several hundred compounds. Targeted analysis is widely employed in forensic laboratories for a number of reasons. First, forensic toxicology is not an isolated science; it occurs in context. There is often non-analytical information that can serve as a jumping off point for the toxicological examination. If a known drug abuser’s body is found surrounded by paraphernalia, a lot of time and money can be saved by first testing for the abused drugs suggested by the investigation. Another reason that targeted analysis is widely used is that a very small number of compounds are typically responsible for death in the majority of postmortem cases. One laboratory reported that 70% of fatal drug poisoning cases in their lab were the result of the less than 30 individual drugs.10 Even though only a few dozen compounds are commonly encountered in typical forensic toxicology cases, the list of potential toxic substances is practically limitless. Additionally, new designer drugs are continually developing, and toxicology laboratories are responsible for detecting them, even if it is a new drug that has never been seen before. Because of this, toxicology laboratories cannot solely rely on targeted analytical 5 techniques, and must incorporate methods to detect a broad range of compounds that are either new or not commonly encountered in typical case work. 1.3.2 General Unknown Screening The alternative analytical approach to targeted analysis is untargeted analysis, also known as general unknown screening (GUS). The daunting task of untargeted analysis is to identify any and all potentially harmful substances, even when their identity is unknown and their presence is uncertain11. In order to provide a framework for untargeted analysis, toxicologists utilize what is known as systematic toxicological analysis (STA), as a guide through the analytical process. The International Association of Forensic Toxicologists’ Committee of Systematic Toxicological Analysis defines systematic toxicological analysis as, “the application of an adequate analytical strategy for the detection and identification of as many as possible potentially toxic compounds and their metabolites in biological samples.”12 The goal of STA is to use a panel of analytical techniques to detect, identify, and if necessary, quantify a very broad range of targets. A few decades ago, this approach meant throwing a sample through every test the lab was capable of because techniques were not as selective or sensitive as they are today.13 Modern advances in instrumentation and computational software are opening doors to make general unknown screening a much more viable option. In mass spectrometry, data-dependent, or information-dependent acquisition modes allow for adaptive data acquisition. Developments in high-resolution mass spectrometry are also advancing the field by opening the door for a posteriori data analysis to detect compounds that were not originally targeted. The downside of GUS is that it is often labor intensive and requires extensive sample preparation. In its truest form, STA means running a prescribed panel of tests independently. In practice, toxicologists can save time and money by allowing the results of each test to help direct further testing instead of running everything independently. 6 1.3.3 Practical Toxicology In an effort to use their resources efficiently, most forensic toxicology laboratories use a two-step process to detect toxicants in biological samples. The first step is screening, and is analogous to STA in that a variety of analytical procedures are employed to detect a broad range of targets. The best screening tests require little sample manipulation, are fast, inexpensive, sensitive, selective, and cover a broad range of targets. Historically, analysis using immunoassays, liquid chromatography coupled to ultra-violet detection, and gas chromatography mass spectrometry have been used for screening purposes, but liquid chromatography mass spectrometry has become a more common screening technique in recent years.14 Screening tests may be based on multitarget screening (MTS) or may screen for a certain classification of drugs or toxins, and are generally qualitative in nature. After qualitative identification is achieved in the screening process, the second step a toxicology laboratory will take is confirmatory analysis. This step is analogous to targeted screening because only those compounds detected in the initial screen are intentionally analyzed. While screening tests are used to detect toxins, confirmatory analysis focuses on identifying and quantifying them. According to the guidelines published by the Society of Forensic Toxicologists in conjunction with the American Academy of Forensic Science, a confirmatory test should be more specific than the screening test and based upon a different chemical principal.15 1.4 The Need for Something New Traditionally, toxicology laboratories performing postmortem analyses use immunoassays, gas chromatography either alone or coupled with mass spectrometry, or liquid chromatography with ultra-violet detection to perform their screening tests. Each of these techniques suffer from various limitations including lack of specificity, difficulty of incorporating new targets, and labor intensive sample preparation and instrument maintenance. Immunoassays are commonly used for presumptive screening in forensic toxicology because they are inexpensive, simple, fast, and automatable. Several types of 7 immunoassays are utilized in casework, including: fluorescence polarization immunoassays (FPIA), radioimmunoassays (RIA), enzyme-multiplied immunoassay technique (EMIT), kinetic interaction of microparticles in solution (KIMS), and enzyme-linked immunosorbent assays (ELISA).16 Immunoassays are good at detecting a class of compounds, but have poor specificity due to the cross reactivity of the binding sites of the antibodies, which prohibits immunoassays from being used to identify specific compounds. In order for immunoassays to provide a broad range of coverage, several analyses have to be run, and it is difficult to add new targets into these analyses. Even in multiplex systems, the coverage provided by immunoassays is far from comprehensive and they have a problematic false positive rate which can waste a laboratory’s time and resources by unnecessarily routing truly negative samples to confirmatory testing. Gas chromatography, typically coupled with mass spectrometry (GC-MS) has typically been considered the so-called “gold standard” for toxicological analysis. Gas chromatography is able to separate compounds well, and when it is coupled with mass spectrometry, the technique gains a high level of specificity. GC-MS also benefits from being able to compare laboratory generated results to externally generated libraries and databases due to the reproducible ion fragmentation that is produced when using a hard ionization technique like electron impact. While GC-MS is an excellent analytical process for confirmatory testing, its use as a screening test is less than ideal because of the labor and time involved in sample preparation. Because only volatile, thermallystable compounds can be analyzed via GC-MS, sample clean up, extraction, and derivatization is often necessary. Sample preparation in GC-MS will vary depending on the analytes of interest, making it necessary to use several different derivatizations to provide adequate coverage, making GC-MS a labor and time intensive option for postmortem toxicological screening.14 Liquid chromatography with ultra violet detection (LC-UV) has historically been used as a screening technique, but has recently been fading in popularity due to its limitations. LC-UV can require long run times for peak resolution and does not have good specificity. Instead of LC-UV, more toxicology laboratories are currently opting to run high performance liquid chromatography coupled with mass spectrometry (HPLC- 8 MS). HPLC-MS has good sensitivity and specificity, especially when high resolution or tandem mass spectrometry is used. It is also able to screen for a broad spectrum of analytes, and is not limited to volatile or thermally stable compounds like GC-MS. Because of this, HPLC-MS is rapidly gaining prominence in toxicology laboratories for confirmatory testing.14 Its use as a screening technique, however, is less ideal for several reasons. First, HPLCs are comparatively expensive, which limits their use in public laboratories where funding is tight. This problem is compounded by the fact that HPLCs require much more oversite than most instruments in order to keep them functioning properly. When using an HPLC system, a lot of time and expertise is spent troubleshooting leaks and fluctuating backpressure, monitoring retention shifts, and preventing carryover and column degradation. The oversight and money necessary to implement HPLC-MS into routine screening testing is often beyond the capabilities of small laboratories, and even when using HPLC-MS is a viable option, the time spent in sample preparation makes HPLC-MS much better suited for confirmatory analysis rather than for initial screening. The use of immunoassays, GC-MS, LC-UV, and HPLC-MS in conjunction with one another is an effective way for toxicology laboratories to detect a broad range of compounds commonly encountered in postmortem cases, however, they represent a significant investment of both time and money in a system that is notoriously strained by tight budgets and substantial backlogs. It would be advantageous then, if new techniques could be developed that provided a simpler, faster, and cheaper screening method that would allow toxicology laboratories to operate more efficiently. 9 2 INTRODUCTION Paper spray mass spectrometry (PS-MS) is an ambient ionization technique that drastically simplifies traditional sample preparation, allowing for the rapid analysis of samples. This technique was first published in 2010 as a new method for detecting drugs and other small molecules in biofluids.17 Originally, paper spray research was directed towards dried blood spot analysis,18 but it has since been utilized in areas including: pharmacokinetic studies,19 food safety,20-23 tissue analysis,24 and the profiling of algae and bacteria.25,26 The ability of PS-MS to detect drugs and pharmaceuticals has been widely investigated,27-34 routinely allowing for low to sub-ng/mL limits of quantitation.16,18,35-38 While the focus of paper spray’s application has historically been the quantitative analysis of a small panel of targets,16 it may be well suited as an alternative for toxicological drug screening procedures as well. 2.1 Project Overview The goal of this project was to develop a PS-MS based method to use as a screening procedure in postmortem toxicology. In order to narrow down the nearly infinite list of possible toxic substances that forensic toxicologists may encounter, the original scope for this project was defined based on consultations with the Axis Forensic Toxicology (AFT). AFT is a national toxicology lab that processes over 100 postmortem samples a day, and is accredited by the American Board of Forensic Toxicologists. AFT provided the project with a list of targets that represent their mid-grade, extended screening panel, which covers 99.5% of targets they encounter in postmortem investigations.39 They also provided screening cutoff values for the project that generally fall at the low-end of what a therapeutic dose of the drug would be. These detection levels have since been raised at AFT, but will serve as a guide for this project. The compiled list consisted of 154 target 10 compounds (Appendix A) that represent a variety of drugs, pharmaceuticals, and metabolites. The staked column graph in Other Figure 1 describes the relative representation of different classes of compounds within the list of analytical targets. Classes of targets represented in the “other” category include: amphetamines, cannabinoids, cocaine, fentanyl, Antidepressants gastrointestinal, methadone, neurologicals, stimulants, and urologicals. The screening cutoff levels for these targets set by AFT range from 1 to 30,000 ng/mL and are summarized in Analgesics Table 1. 2.2 Paper Spray-Mass Spectrometry Benzodiazepines Using paper spray (PS) as a new approach to drug Anticonvulsants screening may allow toxicology laboratories to operate more efficiently for several reasons. First, paper spray would allow Cardiovascular for faster data turnaround by eliminating the time spent in sample cleanup and pretreatment, and by shortening analytical run times. Being a direct analysis technique, there is no sample Antipsychotics Sedatives Opiates Barbiturates Anesthetics Narcotics Antihistamines Barbiturates Figure 1 Drug classes represented in this project. The size of each block is proportional to the number of targets in that class preparation for PS analysis, and run times are typically less than two minutes. Comparatively, the currently employed methods of GC-MS, IA’s, or LC-MS have analytical run times of 10 minutes to upwards of an hour,40-42 without considering the time spent in sample clean up and derivatization. The disposable paper substrate used in PS-MS analysis would also allow laboratories to recover the time that is currently spent troubleshooting by eliminating problems caused by carryover, clogs, and leaks. Another reason that developing a PS-MS method for toxicological screening would be advantageous is that it provides a cheaper alternative to current techniques by 11 Table 1 Screening cutoff values for the target anlaytes in the project ranged from less than 5 ng/mL to over 1000 ng/mL. Cutoff (ng/mL) Number of Targets Percentage of Total Targets ≤5 10 20 25 50 100 500 4 17 26 13 43 14 13 3% 11% 17% 8% 28% 9% 8% 1000+ 24 16% reducing cost in several different areas. First, the amount of solvent used in PS-MS analysis is typically only around 100 microliters and is completely consumed during analysis, leaving behind absolutely no solvent waste. This reduces the amount of money laboratories have to spend on reagents, as well as eliminates the costs associated with waste management and removal. Using PS-MS can also reduce the cost associated with properly storing and shipping biological samples since the dried blood spots used do not have to be carefully refrigerated like liquid whole blood or plasma samples. Another added advantage to paper spray’s use of dried blood spots for sampling is that samples stored as dried blood spots are more stable than liquid samples.43,44 The potential to lower cost, limit sample preparation, and increase sample throughput make paper spray a valuable option for toxicological screening, but there are a few things that must be considered if PS-MS is to be implemented into routine toxicology work. To understand these issues, it is important to understand the principals of paper spray-mass spectrometry. Paper spray mass spectrometry belongs to a family of ambient ionization techniques that simplify analysis by removing the chromatography that is typically carried out prior to mass spectrometry. To accomplish this using paper spray, a sample is dried onto a piece of paper that has been cut to a point at one end. Typical sample matrices used in 12 Extraction/spray solvent Paper 4.0 kV Inlet to mass spectrometer Charged droplets containing analyte Figure 2 Schematic of paper spray PS-MS analysis include whole blood, plasma, and urine. The sample is typically allowed to dry prior to analysis, although analysis of wet samples has been reported.38 Once the sample is dried, solvent is applied to the back of the paper, which is positioned a few millimeters away from the inlet of the mass spectrometer at atmospheric pressure. As the solvent wicks through the paper and permeates the sample, it extracts the soluble components from the matrix, and they travel with the solvent front to the sharp tip of the paper. Once the paper is completely saturated, a high voltage is applied, which generates ions through electrospray ionization by inducing a Taylor cone at the sharp tip of the paper. A schematic of this experimental set up illustrated in Figure 2 is depicted visually by the images in Figure 3. A B C Figure 3 (A) During paper spray, a piece of paper cut into a sharp tip is positioned 5 mm away from the inlet of a mass spectrometer. (B) Solvent is added and allowed to saturate the paper while the voltage remains off. (C) When high voltage is applied, a Taylor cone forms at the paper’s tip, emitting a plume of charged droplets that quickly evaporate, leaving behind gaseous ions that enter the inlet of the mass spectrometer. 13 The data that is generated though PS-MS analysis looks different than typical frontend chromatography techniques, although it is processed similarly. Because there is no intentional chromatography performed during PS-MS analysis, the term “chromatogram” is a misnomer in this application. Instead, the word “chronogram” is used to describe the ion signal over time. In typical chromatography based methods, ion signals appear as more or less Gaussian peaks. As shown in the chronogram in Figure 4, the “peaks” produced by PS-MS do not follow this trend. As soon as a high voltage is applied, the analytes extracted by the spray solvent are ionized and are detected by the mass spectrometer. As long as the paper remains wet enough to produce an electrospray, the signal will continue until the voltage is turned off. Much like typical chromatograms, chronograms are analyzed by quantifying the area under the curve of extracted ion chronograms (XIC) or selected reaction monitoring (SRM) channels. Voltage On Relative Abundance Voltage On Voltage Off Blank 1 Voltage Off Gabapentin at 500 ng/mL 172 -> 119 2 3 4 5 Time (min) Figure 4. Unlike typical chromatograms produced in LC-MS, the “peaks” produced in PS-MS are boxy rather than Gaussian and are called “chronograms”. This is because essentially no chromatography occurs during PS-MS. When the voltage is turned on, all extracted ions enter the mass spectrometer at the same time, leading to an almost instantaneous rise in ion intensity. When the voltage is turned off, the Taylor cone collapses and ionization stops. An example chronogram of the transition from m/z 172 -> 119 for gabapentin in blank blood and at 500 ng/mL is depicted. 6 14 2.3 Selectivity Because no chromatography is performed in paper spray, there is a burden of specificity that is placed on the mass spectrometer. During PS-MS, all compounds that are extracted by the solvent co-elute, therefore the mass spectrometer must be able to differentiate compounds with the same nominal mass in order to discriminate between a target analyte and any of similar mass/charge that could interfere with the signal produced by the analyte of interest. In PS-MS, this is accomplished by high resolution mass spectrometry (HR-MS) and/or tandem mass spectrometry. Typical mass spectrometers that have been used for PS-MS analysis include: ion trap, orbitrap, time-offlight, and triple quadrupole mass spectrometers.16 2.3.1 Mass Spectrometer Selection While a variety of types of mass spectrometers have been used to perform PS-MS experiments, they are not all suitable for use in forensic toxicology laboratories. Ion trap mass spectrometers are able to achieve adequate specificity for PS-MS by using their MSn feature. Because they are tandem in time, however, they are too slow to be used for screening procedures because of the need to quickly acquire data for a large list of targets. High resolution mass spectrometry is able to distinguish between co-eluting compounds based on exact mass measurements. Their high mass resolution reduces the possibility of interference from non-isomeric compounds, and they also have the unique feature of being able to retrospectively interrogate data for targets that were not originally specified. The detection limits on a HRMS are generally higher than on other mass spectrometers, however, and they are much more expensive to purchase and require daily calibration. Triple quadrupole (QqQ) mass spectrometers achieve specificity much like ion traps, in that they are able to discriminate targets by monitoring characteristic fragment ions. Triple quadrupoles are low resolution instruments and can only measure mass to about +/- 0.5 m/z units. However, because QqQ’s are tandem in space rather than time, they are able to quickly scan a large list of targets by using selected reaction monitoring mode. QqQ’s can also attain adequately low detection limits for toxicological 15 purposes and are more accessible to forensic laboratories due to their lower price. Because of these reasons, a triple quadrupole mass spectrometer was chosen as the mass spectrometer to be used during method development in this project. 2.3.2 Fragmentation When used in selected reaction monitoring (SRM) mode, triple quadrupole instruments are able to enhance selectivity by using two sets of quadrupoles as mass filters. The first set of quadrupoles is set to only allow ions within a certain window of mass-to-charge ratio (m/z) to pass through. This window, normally 0.7 m/z units wide, filters out all other ions that have been generated by the ionization source. The ions produced by electrospray ionization and typically selected to pass through the first quadrupole are the protonated or deprotonated molecular ions ([M + H]+ or [M - H]-) or small adduct ions, such as potassium, sodium, or ammonium. The ions with the selected m/z, known as either parent or precursor ions, enter the second set of quadrupoles where they are bombarded with argon gas. When the ions collide with the gas molecules, they fragment via Collision Induced Dissociation (CID). The ions produced by this fragmentation process are known as daughter or product ions. The product ions then enter the third set of quadrupoles which is used to as a mass filter. In SRM mode, a characteristic fragment at a specific m/z is specified, and only that fragment is allowed to pass through the third quadrupole and reach the detector. A decade ago, the detection of one unique fragment ion was generally seen as sufficient grounds for quantitative analysis.45 It is more common nowadays, however, to enhance selectivity even further by monitoring more than one fragment ion per parent ion. If full specificity is required, the third quadrupole can be operated in full scan mode, in which case all of the fragments produced in the second quadrupole would be detected. For most HPLC-MS/MS applications, adequate specificity is generally achieved by monitoring two fragments for each parent ion. The most abundant of these fragment ions is referred to as the quantifier ion, while the lesser abundant is termed the qualifier ion. Typically the two most abundant ions are monitored, however, due to interfering compounds or matrix effects, other fragments can be monitored instead. Oftentimes, 16 however, the specificity of the chosen fragment ions is not considered due to the perceived specificity that can be gained by monitoring the ratio between different fragment ions. The idea behind using fragmentation ratios is that under specified conditions, a parent ion should fragment reproducibly, and the abundance of the fragment ions produced under those conditions should be relatively consistent from run to run for a specific target analyte. Fragmentation ratios can be established by either taking the ratio of the intensities of the SRM transitions for each product ion or by taking the ratio of the area under the curves. There are several conditions under which fragmentation ratios may be obtained. A common method is to optimize instrumental conditions for each fragment ion separately and use the resulting ion intensities to calculate the fragmentation ratio. This method is advantageous when signal to blank response is poor or when interferences are present, as it provides an optimized signal for each product ion. Another method is to monitor two fragments under identical conditions. Other methods used include monitoring the same fragment ion under varying conditions and monitoring fragments from different precursor ions.45 The use of the later, though, has been discouraged recently because of indications that asymmetrical signal suppression can interfere with fragmentation ratios.46 When ionization suppression is present, competitive ionization within the matrix can hinder analytes from completely ionizing, leading to a less intense signal. 2.3.3 Selectivity in Paper Spray By using a triple quadrupole instrument and monitoring a parent ion, two fragment ions, and the fragment ion ratio, good selectivity is achievable for paper spray even without the advantage of chromatography. Using this method should allow for differentiation between structural isomers that fragment differently. For example, although the isomers methamphetamine and 4-methylamphetamine have the same exact mass, they fragment uniquely. Methamphetamine undergoes transitions from m/z 150 to m/z 119 and 91, while 4-methylamphetamine fragments from m/z 150 to ions with m/z 133, 105, 103, 77, and 79.16 While both compounds would produce a parent ion with m/z 150 that would pass through the first set of quadrupoles, the unique fragments generated 17 in the collision cell would allow one compound to be detected without interference from the other. Even in cases where no unique fragments exist, fragmentation ratios have been able to distinguish between two isomers.45 Because of this, fragmentation ratios not only add a level of confidence to compound identification, but can also help differentiate between otherwise interfering compounds. However, in the case where isomers with the same nominal mass share the same fragment ions, unique fragments should be used if possible. There are several established organizations that use fragments and fragmentation ratios as the basis for identification. The requirements these organizations have set for analytical identification using these ratios vary depending on application and have evolved over time. Some relevant identification criteria for LC-MS based assays are summarized in Table 2. One thing to consider when looking at these requirements is that all of these are based on the assumption that chromatography is being performed. With this, there is the assumption that co-eluting compounds will be somewhat limited, but this is not the case in paper spray. Some of these organizations acknowledge and sometimes discourage the use of commonly produced fragment ions, but none of them ban them from being used for identification purposes. Another variable to note is the tendency to base the acceptable deviation of the fragment ion ratio on the relative abundance of the two ions. The precedence for this decision is based on the European Union decision that was made by a panel of experts in the 90’s.49 Since that time, however, instrumentation has evolved and fragmentations ratios have been found to be analyte and instrument specific and vary widely based on concentration and matrix. 15,47,50,51 The variability of fragment ion ratios in some instances has caused some to suggest recently that it is better to simply rely on the presence or absence of fragment ions for compound identification.52 Many other authors have suggested a “fitness of purpose” approach that tailors criteria to fit the analytical specificity needs of each particular project. Using fragmentation ratios in PSMS analysis has historically been done to add a level of specificity lost by not preforming chromatography. During the method development phase of this project, setting the acceptable tolerances for these ratios will be an area of interest. 18 Table 2 MS-MS based identification criteria for various applications Ion Requirements Fragment Ion Ratio Tolerance Live animals and animal products ≥2 product ions Ratio tolerance is dependent relative abundance of ion to base peak; ranges from ±20% to ±50% Pesticides in food products and animal feed ≥2 product ions Ratio tolerance is ±30% Sports doping 1 product if unique; if not unique, ≥2 product ions Ratio tolerance is dependent on relative abundance of ion to base peak; ranges from ±10% to ±50% EWDTS6 Workplace drug testing ≥3 ions Ratio tolerance is ±20% UNODC7 Illegal drugs ≥3 ions Ratio tolerance is ±20% Forensic Toxicology ≥2 product ions Ratio is tolerance is ±25% to ±30% Organization/Code EU: 2002/657/EC1 EU: SANCO/12571/20133 WADA TD2010DCR5 SOFT/AAFS8 Applies To 2.4 Special Considerations There are several areas that require special consideration if paper spray mass spectrometry is to be applied to toxicological postmortem screening. Two of these are the characteristics of postmortem blood as compared to healthy blood and the matrix effects that can arise during PS-MS analysis. 19 2.4.1 Postmortem blood While this project used single-donor drug free blood, there are several unique aspects of postmortem blood that will affect this project as it undergoes method validation. One important, well-recognized phenomenon that continues to be the subject of current research is the process by which drug concentrations change after death, known as post-mortem redistribution (PMR). During PMR tissue-bound drugs diffuse into adjacent blood vessels, increasing the drug concentrations in blood, especially for basic and lipophilic drugs. The extent of PMR varies with each drug, with some drugs showing highly time-dependent concentration, while other drug concentrations remain more or less stable over time.53 Understanding the effects of PMR is one of the most important considerations for medical examiners in selecting blood sampling sites that are the truest representation of drug concentrations in the blood at the time of death. If any, the most likely effect that PRM will have on this project is an increase in basic drug concentration levels. While exact concentrations are not important for screening methods, increased concentrations could improve performance for drugs near the limit of detection when real postmortem blood is used. Another aspect for consideration is the difference in matrix effects between antemortem and post-mortem blood caused by the decomposition process. Saar et al. studied the differences in matrix effects and liquid-liquid extraction (LLE) efficiency for antipsychotic drugs in ante-mortem and post-mortem blood. They found considerable differences in both extraction efficiency and matrix effects between the different types of blood and suggested that post-mortem methods be validated using drug-free postmortem blood as opposed to pooled blood bank ante-mortem blood.54 They also found that while the matrix effects for decomposed post-mortem blood were similar to ante-mortem blood, they were much more variable. Similarly, Rosano et al found that both the variability of matrix effects and the ion suppression in post-mortem blood was higher than in bank blood.55 Because of these differences, Peters et al. echoed Saar’s proposition to use postmortem blood in method validation and additionally suggested that non-decomposed post-mortem blood and decomposed blood be separately evaluated.56 20 In this project, higher variability in drug recovery in post-mortem blood could lead to a “hit-or-miss” scenario that could require multiple samples to be run in certain cases where the concentrations are near the limit of identification. Higher ion suppression could also be problematic for targets that either do not ionize well or exist at low concentrations. Both of these could potentially be somewhat counteracted by preconcentration techniques, such as integrating SPE, as described by Zhang et al.57 2.4.2 Matrix Effects Matrix effects are a common phenomenon which cause instrumental response to be altered due to components within the matrix in which the analyte is contained. Both the ionization technique and the analytical separation technique can affect which matrix effects are observed. For example, electrospray ionization is more affected by matrix effects than atmospheric-pressure chemical ionization.56 In chromatography-based techniques like GC and LC, matrix effects commonly arise from co-eluting compounds and can cause either ion suppression or ion enhancement. This issue is compounded even further for non-chromatography based techniques like paper spray due to the fact that all compounds elute simultaneously. There are two principal forms of matrix effects that affect the performance of paper spray mass spectrometry: recovery and ion suppression. Recovery refers to the percentage of the analyte that is extracted from the matrix. Ion suppression occurs when ionization efficiency for an analyte is lowered due to competitive ionization between the analyte and other co-extracted matrix components. Factors that influence competitive ionization include: access to a droplet’s surface during electrospray, surface tension of the solvent, sample pH, and compound polarity.58 Both of these affect the amount of ions that reach the mass spectrometer and are therefore available for detection. As such, they have a direct effect on the limits of detection. Ion suppression and recovery depend on both the specific analyte of interest and the matrix from which the analyte is extracted. The spray solvent used will also play a role in ion suppression and recovery based on how soluble the analyte and potentially coextracted matrix components are in the solvent system. Out of the typical biofluids 21 analyzed in PS-MS (urine, plasma, and whole blood), blood has been reported to typically have the lowest recovery. This is offset, however, by the fact that ion suppression is generally lowest in blood.18,59 One study investigated the donordependency of ion suppression and recovery in blood using PS-MS and found that neither were significantly different in the 33 patient samples that were tested.60 This suggests that within whole blood samples, the variability of matrix effects will be due mostly to compound-specific qualities. Compounds that are known to ionize well in paper spray are hydrophobic molecules with basic aliphatic amine groups.18 These compounds do not suffer from ion suppression nearly as much as poor ionizers.59 Poor ionizers are hydrophilic (logP >~2) and lack basic aliphatic amine groups. Out of the 154 targets in this project, 48 do not have aliphatic amines and 97 have logP values that are greater than two. That means that 64% of the targets have the potential to ionize poorly. However, only 15% of the targets are both hydrophobic and lack a basic aliphatic amine. The challenge to drug recovery in this project will be in maintaining the simplicity of the process. Recovery may be improved by optimizing the solvent system, but using multiple solvent systems would add time to the analytical process. However, even if multiple solvents must be run for full target coverage, the time required for PS-MS would still be competitive with current screening techniques. One of the challenges for the project, then, is to find a solvent system that efficiently extracts as many targets as possible at relevant concentrations while minimizing the possibility of extracting interfering compounds from the matrix. Ion suppression is also a potential problem, especially for poor ionizers with low target cutoff concentrations. Incorporating a preconcentration technique with PS-MS has been shown to improve signal in some of these cases and is an option to help improve detection in future method development.57 22 While recovery and ion suppression are the primary matrix effects of concern in paper spray, other minor effects could play a role in this project, one of which is the formation of protomers. Protomers are ions that differ only by the site at which they are protonated. Where the proton is attached to the molecule is determined by the chemical environment, and solvent composition has been suggested as a contributing factor.61 Solvent characteristics that have been found to influence the formation of protomers include: pH, aqueous-organic ratio, and ionic strength.62 23 3 MATERIALS AND METHODS 3.1 Chemicals and Reagents Analytical grade methanol, acetic acid, and water were purchased from Fisher Scientific (Hampton, NH, USA). All targets (Appendix A) were purchased as standards from Cerilliant (Reston, VA, USA) with the exception of: acetaminophen, metaxalone, salicylic acid, etomidate, carbamazepine, valproic acid, fluvoxamine, hydroxyzine, aripiprazole, secobarbital, amlodipine, papaverine, metoclopramide, benztropine, donepezil, ropinirole, methocarbamol, bupivacaine, levetiracetam, and labetalol, which were purchased from Sigma–Aldrich (St. Louis, MO, USA). Drug-free human blood was collected in K2EDTA blood collection tubes from a single donor. Both analytes and blood were stored at -20°C. 3.2 Mass Spectrometer and Materials Experiments for this project were carried out on a triple quadrupole, TSQ Vantage mass spectrometer (Thermo Scientific, San Jose, CA, USA) operated in MS/MS mode. Manually run experiments were performed using an in-house designed cartridge (Figure 5-A) and Whatman grade 31ET chromatography paper purchased from Whatman (Piscataway, NJ, USA). A TM-200 miniature CCD camera was purchased from JAI PULNiX (San Jose, CA, USA) and used to visually monitor paper and electrospray quality during manual experiments. Automated experiments were run using a Velox 360 sample handling and ionization source and Velox sample cartridges (Figure 5-B) from Prosolia, Inc. (Indianapolis, IN, USA). 3.3 Method Two sets of methods were used during this project. Method development and initial testing were done using the manual method outlined below. After a functional method was developed using the manual method, the parameters were used to inform a method that used an automated ionization source and disposable cartridges. 24 A B Figure 5: (A) An in-house designed reusable cartridge was used for manual experiments. Using this set up 3 µL blood spots were dried onto pentagon-shaped papers hand-cut from Whatman 31ET chromatography paper and inserted into a slot at the front of the cartridge. In this design, solvent is applied through a well directly over blood spot. (B) Commercial Velox cartridges from Prosolia were used for automated experiments. In this set up, 12 µL blood spots are dried onto precut paper stored inside individual disposable cartridges. Solvent is applied into well behind blood spot and wicks through the paper and blood spot. For both the manual and automated experiments, analytical targets were combined together from their original stocks into 13 different cocktail solutions according to Appendix D. Each cocktail contained anywhere from 8 to 14 target analytes at 20x each analyte’s target detection concentration. While the analytes were grouped according to their target detection concentrations, care was taken to ensure isomers were separated into different cocktails to avoid introducing interferences. The diluent used to bring each cocktail to the appropriate concentration was 95:5:0.01 methanol:water:formic acid, and the cocktails were stored at -20° C. For both the manual and automated methods, 300 µL of drug-free blood was aliquoted into plastic microcentrifuge tubes and put through two freeze-thaw cycles to try to mimic the matrix decomposition that may occur in postmortem blood samples. 15 µL of one of the cocktail solutions was then added to the thawed blood so that the total organic content was less than or equal to 5%. This helped prevent the blood from congealing and limited protein precipitation which caused the blood to become heterogeneous and difficult to pipette. For blanks, 15 µL of 95:5:0.01 methanol:water:acetic acid was added to blank blood to keep organic content consistent between blank and spiked samples. The samples were then inverted 30 times, and 25 allowed to incubate for 45 minutes at room temperature before being aliquoted onto paper. 3.3.1 Manual Method During manually run experiments, pentagonal papers (Figure 6-B) were razor-cut from Whatman 31ET chromatography paper to fit into an in-house designed cartridge (Figure 5-A). After the analytes were incubated in the blood for 45 minutes, the samples were mixed and 3 µL of blood was used to saturate 3.5mm paper punches (Figure 6-A) and allowed to dry at room temperature for two hours. For analysis, the pentagonal papers were loaded though the side slot in the cartridge and then the dried blood spots were placed on top of the paper via the well on the top of the cartridge. The solvent system 95:5:0.01 methanol:water:formic acid was delivered in three 15µL aliquots to saturate the paper and then 4000 V was applied through a metal contact to induce electrospray. Data was collected for 60 s for each sample, and four replicate dried blood spots were run for each target cocktail. Between each sample, the voltage was turned off, and the cartridge was rinsed with methanol and air dried before loading the next sample. Replicate samples were run in succession followed by replicate blanks. A C B 12 µL 3 µL 10 mm 5 mm 3.5 mm 5 mm Figure 6 (A) Using the manual method, 3 µL of whole blood was used to saturate a 3.5 mm round paper punch (B) Pentagonal paper was hand-cut with razor blades and the dried blood spot was placed on top of the paper. (C) The automated method utilized a larger, different shaped paper design than the manual method. This precut paper was housed in single-use Prosolia cartridges and 12 µL of whole blood was directly applied to the paper for analysis. 26 4.3.2 Automated Method During experiments run using the automated ionization source, blood samples were prepared following the same procedure as the manual experiments. Instead of applying the spiked blood to a paper disk and setting the disk on top of the paper tip, the spiked blood was applied directly to the paper housed inside each disposable cartridge. Two types of paper were investigated in the automated experiments, laser-cut and die-cut. Prosolia typically cuts their paper tips with lasers, but a slight brown discoloration was observed on the edges of the paper of the laser-cut tips, so die-cut tips were provided as well in order to investigate whether the laser-cutting process affected results. Because the paper size was larger, 12 µL aliquot volumes were used in the automated experiments, and instead of a spot of blood, a band of blood was created across the width of the paper (Figure 6-C). Creating a “band” of blood instead of a spot was necessary because the automated source delivers the solvent behind the dried sample rather than on top of it. Forming a band of blood ensures that solvent must flow through the blood and will not flow around it, bypassing the sample. The larger size of the paper also led to using 120 µL of solvent rather than 45 µL, but like the manual method, the solvent was delivered in several aliquots to allow the solvent time to soak through the paper. Like the manual method, the solvent system used was 95:5:0.01 methanol:water:formic acid, and 4000 V were applied to induce electrospray. Due to the automatic ionization source, however, the spray was not able to be monitored visually for samples run in the disposable cartridges. Unlike in the manual method, no clean up was necessary between samples while using the automatic method. Sample were allowed to dry on the papers contained within each cartridge and then the cartridges were loaded into a magazine. This magazine attached into the automated ionization source so that the samples could be queued up and left to run automatically. 27 4 METHOD DEVELOPMENT Several preliminary sets of experiments were run for method development. These included a series of experiments to optimize the solvent system, paper layout, and sample loading. Experiments were also run to select appropriate SRM channels for each target analyte, establish the amount of data needed to reliably represent results, and to establish acceptable fragment ion ratio tolerances. 4.1 Solvent Selection Solvent selection is the primary means though which both selectivity and sensitivity are manipulated during paper spray experiments. One of the challenges in this project was to find one solvent mixture that could effectively extract and spray the entire panel of analytical targets. Typically, paper spray solvents are mostly organic in composition, which allows hydrophobic small molecules like drugs to be extracted while the hydrophilic matrix components remain trapped on the paper. Examples of commonly used extraction/spray solvents include 90:10 methanol:water and 90:10 acetonitrile:water.18 Oftentimes, acetic acid or formic acid is added as a solvent modifier at approximately 0.01% to help encourage the formation positively charged ions and to increase spray stability. In negative mode, ammonium hydroxide can be used to encourage the formation of deprotonated ions. In order to choose a spray solvent for this project, a subset of 16 targets was chosen to represent a range of target cutoff levels, hydrophobicities, and ionizabilities. A set of two-solvent systems made by combining either methanol, acetonitrile, ethanol, isopropanol, water, dichloromethane, or tetrahydrofuran were made in proportions of 35:65, 40:60, 50:50, 65:35, and 95:5 and included 100 ppm acetic acid as a solvent modifier to encourage positive ion formation. This yielded 47 different solvent combinations that were tested on the subset of 16 targets that were spiked at their cutoff concentrations into blood. Observations for some of these solvent systems can be found in Table 3. 28 Table 3. 47 different solvent systems were tested for use with PS-MS in an effort to identify a solvent that would produce a steady Taylor cone at 4000 V, prevent electrical discharge, and effectively extract target analytes. Solvent Observations 60:40 Ethanol:Water Didn't produce cone 75:25 Ethanol:Water Unstable cone, discharge at higher voltages 95:5 Ethanol:Water Discharge that isn't solved by lowering voltage 60:40 Isopropanol:Water 75:25 Isopropanol:Water 95:5 Isopropanol:Water Cone wasn't visualized, but spray was; wicked slowly Cone wasn't visualized, but spray was; both blank and target signals elevated Cone wasn't visualized, but spray was 60:40 Acetonitrile:Water Cone wasn't visualized 75:25 Acetonitrile:Water Cone only formed in some trials 95:5 Acetonitrile:Water Taylor cone stable until 30s 60:40 Methanol:Water Stable cone 75:25 Methanol:Water Stable cone 60:40 Methanol:Acetonitrile Cone only formed in some trials 75:25 Methanol:Acetonitrile Produced good cone, failed after 30s 95:5 Methanol:Acetonitrile 60:40 Ethanol: Acetonitrile 75:25 Ethanol: Acetonitrile Cone failure after 20-30 seconds; Tendency to discharge Cone failure after 15 seconds, but spray still visualized for up to a minute Sometimes spray was seen without a cone forming 95:5 Ethanol: Acetonitrile Sprayed for up to a minute, even without cone formation 60:40 Isopropanol:Acetonitrile Cone failure quickly and tendency to discharge 75:25 Isopropanol:Acetonitrile Cone wasn't visualized, but spray was 95:5 Isopropanol:Acetonitrile Discharges badly 40:60 Methanol:Dicholormethane Neither cone nor spray consistently 65:35 Methanol:Dicholormethane Unstable spray 95:5 Methanol:Dicholormethane Cone failure after 30s and discharge 40:60 Ethanol:Dicholormethane Neither cone nor spray consistently 65:35 Ethanol:Dicholormethane Sputtering discharge throughout 95:5 Ethanol:Dicholormethane Sprayed at times, but discharged 40:60 Isopropanol:Dicholormethane Cone wasn't visualized but spray was at times 65:35 Isopropanol:Dicholormethane Cone wasn't visualized, tendency to discharge 95:5 Isopropanol:Dicholormethane 35:65 Methanol:Tetrahydrofuran 50:50 Methanol:Tetrahydrofuran 65:35 Methanol:Tetrahydrofuran Cone wasn't visualized but spray was Cone wasn't visualized but spray was; Solvent depleted quickly Cone wasn't visualized but spray was; Solvent depleted quickly Spray dries up quickly 35:65 Ethanol:Tetrahydrofuran Cone wasn't visualized but spray was 50:50 Ethanol:Tetrahydrofuran Cone failure after 35s; tendency to discharge 35:65 Isopropanol:Tetrahydrofuran 50:50 Isopropanol:Tetrahydrofuran 65:35 Isopropanol:Tetrahydrofuran Cone wasn't visualized but spray was Cone wasn't visualized but spray was; tendency to discharge Cone wasn't visualized but spray was; tendency to discharge 29 Some of these solvent systems did not produce a stable Taylor cone and had a tendency to produce an electrical discharge, which was indicated by the presence of a glowing A point at the tip of the paper, a spike in the spray current, and at 10 mm times an audible clicking. Other solvent systems produced unreasonably high blank signals which would not allow for 5 mm adequate sensitivity. In the end, the solvent system that 5 mm produced a stable electrospray and yielded the best extraction results for the panel of drugs tested was 95:5:0.01 B methanol:water:acetic acid. This solvent was used for all subsequent experiments. 4.2 Paper Shape and Sample Loading 30 mm Historically, paper spay experiments have been performed on paper that is shaped more or less like an equilateral triangle on top of a square (Figure 7-A). However, during method development, elongating the paper was investigated because Vega et al. found that as the distance between the sample and the tip of the paper increased, both ionization suppression and recovery decreased.59 This could potentially benefit targets that are bad ionizers by reducing ionization suppression, at the expense, however, of decreasing recovery. The published experiments increased the distance between the sample and the paper tip by stacking blank “spacer” discs of paper between the sample and the paper that was cut into a point. Rather than taking this approach during method development, a strip of paper 30 mm long, as depicted in Figure 7-B, was used to increase the distance from the sample to the paper tip. Figure 7: Paper shape and sample loading capacity were tested on two different paper designs. (A) Traditional pentagonal-shaped papers provided a base-line reference while (B) 30 mm long pointed paper strips were used to test the effects of higher loading capacities 30 4.2.1 Placement of Sample The first set of experiments run using the long paper studied the effect of the position of the blood on the paper. To do this, 20 µL of whole blood was loaded at 13, 17, and 22 mm from the tip of the paper. All of the blood was loaded onto the paper at a single point, resulting in a sample front that was rounded due to the way that the blood wicked though the paper, but always resulting in bands of blood 11 mm wide. Five targets (morphine, zolpidem, clonazepam, fentanyl, and buprenorphine) were monitored during these experiments. The area under the curve for several fragment ions from these targets was recorded and normalized using the lowest area in order to easily compare which position produced the strongest signal. The results presented in Table 4 show that for most of the fragments, the central position at 17 mm produced the highest signal by a factor of approximately 1.5-2. During these experiments, it was observed that the cartridge design allowed some of the solvent to wick around the sides of the paper rather than through the sample, so a new cartridge was designed for future experiments. The new cartridge supported the length of the paper on small pegs rather than on channeled grooves on the sides, so that the solvent would be forced though the sample. 4.2.2 Loading Capacity and Blood Dilution Using the small paper punches, a set of experiments were run to find the effect of diluting blood, which could improve ion suppression and/or analyte recovery. To this end, triplicate samples were run at blood:water ratios of 1:0.5, 1:1, 1:5, and 1:10. The results of the five drugs investigated (morphine, zolpidem, clonazepam, fentanyl, and buprenorphine) shown in Table 5 indicate that a dilution factor of 1:1 could increase signal to blank. Another set of experiments were run using both diluted blood and whole blood on 30mm long paper. The longer paper allows for a larger volume of blood to be loaded onto the paper, which increases the absolute amount of drug available to be recovered during paper spray. Therefore, while using the elongated paper, the amount of sample loaded onto the paper was also investigated. The larger volumes used in these experiments meant that it was not feasible to position the samples at 17mm from the tip 31 as in previous experiments, since this would cause the sample to wick all the way to the tip of the paper and inhibit the solvent from spraying. Instead, the front edge of the blood spot was positioned so that it was always 6mm from the tip of the paper. When 5µL of whole blood was used, the band of blood produced on the strip of paper was 6mm long. The same sized blood band was produced when diluted blood was used as well. The amount of blood used was increased incrementally up to 40µL, which was found to be the maximum volume of blood that the long paper strips could accommodate and maintain a good spray. The length of the blood band formed on the paper increased as the loading volume increased, with the final volume of 40µL producing a blood band 20mm long. The results of these experiments are presented in Appendix B and summarized in Figure 8 and Figure 9. The results indicate that the Table 4 Relative AUC of fragment ions at different positions on the long paper strip. The lowest AUC was normalized to 1.00 for comparative purposes and the position on the paper strip with the largest AUC is denoted in green. Relative Area Under the Curve Position m/z of fragments Morphine at 40 ng/mL 13 mm 17 mm 21 mm 152 1.00 1.58 1.40 165 1.00 1.48 1.33 13 mm 17 mm 235 1.07 1.00 92 1.00 1.12 21 mm 1.37 1.27 13 mm 17 mm 21 mm 270 1.00 1.84 1.29 214 1.00 2.19 1.74 241 1.00 1.68 1.28 13 mm 17 mm 21 mm 188 1.00 1.07 1.23 105 1.00 1.53 1.30 79 1.00 1.79 1.15 13 mm 17 mm 396 1.00 2.08 414 1.00 1.92 21 mm 1.79 1.55 m/z of fragments Zolpidem at 10 ng/mL m/z of fragments Clonazepam 20 ng/mL m/z of fragments Fentanyl at 10 ng/mL m/z of fragments Buprenorphine at ng/mL 201 1.09 1.00 1.02 151 1.00 1.71 1.07 205 1.00 1.92 1.38 206 1.00 2.94 1.56 207 1.00 2.19 1.53 101 1.00 2.47 187 1.00 1.65 55 1.00 1.81 211 1.00 1.59 225 1.00 2.17 1.49 1.22 1.23 1.44 1.88 190 1.00 2.64 1.68 32 optimal loading capacity using the 30mm long paper is 20µL and that using a dilution factor of 1:1 can increase a target’s signal to noise ratio by lowering the blank signal. In order to test the effects of the optimized method of using 20µL of blood diluted 1:1 with water and placed in the center of a 30mm long paper, an experiment was run that tested this method against the original method (loading 3.5µL of whole blood onto a 3mm punch placed on top of a 10mm long paper). A panel of 13 targets was split into two groups and tested using both the original method and the optimized method. The targets from each group were cocktailed together and tested at each analyte’s target cutoff concentration. Two cocktails of targets were necessary in this case to avoid introducing intra-target interferences from targets who share parent ions in the same sample, i.e. morphine and 7-aminoclonazepam. The results of these experiments, shown in Table 6 indicate that long paper with diluted blood generally gives lower blank signals and higher response to target SRM channels, resulting in better signal to noise ratios. While in some cases, this improved S:B enough to make a target be considered “detected” due to raising the S:B ratio above 3:1, the improvements are not drastic enough to warrant adapting the entire experimental set up in favor of the simpler original method. Since the end goal of this project is to be able to run a fully automated process using the automated ionization source, the results of Table 5 Four dilutions schemes used to test the effect of diluting blood on the signal to blank ratio of five analytical targets. The results indicate an optimal dilution factor of 1:1 Morphine Zolpidem Clonazepam Fentanyl Buprenorphine Dilution Factor Fragment 286-> 152 286-> 165 308 -> 92 308 -> 235 316 -> 214 316 -> 241 337 -> 105 337 -> 188 468-> 396 468-> 414 1:10 1:5 1:1 1:0.5 S:B 1.7 1.5 1.8 9.4 1.6 2.4 1.3 2.7 1.2 1.1 S:B 1.7 1.5 2.2 18 1.8 2.6 1.6 4.7 1.2 1.1 S:B 3.5 2.3 5.2 39 1.5 4.0 2.5 15 2.1 2.7 S:B 3.0 2.3 3.7 6.8 2.0 4.0 2.1 13 1.5 1.7 33 the dilution and placement survey will serve to inform future work, but in this project, undiluted blood and commercially available cartridges will be tested. A. C. E. B. D. Figure 8: Plots of signal to blank ratio using blood diluted 1:1 on 30mm long paper for (A) Zolpidem (B) Morphine (C) Clonazepam (D) Fentanyl (E) Buprenorphine indicate an optimal loading capacity of 20µL for diluted blood 34 A. B. C. D. E. Figure 9: Plots of signal to blank ratio using whole blood on long paper for (A) Zolpidem (B) Morphine (C) Clonazepam (D) Fentanyl (E) Buprenorphine. Although the analytes do not follow the same trend, they generally show an optimal loading capacity of ~20µL of whole blood Fentanyl Cocaethylene Clonazepam Buprenorphine 7-Aminoclonazepam Alprazolam Topiramate Zolpidem Morphine Meprobamate Ketamine Normeperdine Gabapentin Fragment m/z 119 137 42 56 125 89 158 180 152 165 92 235 207 265 281 205 94 121 396 214 241 196 82 105 188 Long Blank Avg AUC 1.5E+04 3.9E+04 2.2E+03 4.4E+03 4.1E+03 3.6E+03 7.5E+03 8.0E+03 1.4E+04 5.8E+03 6.6E+03 1.0E+03 3.3E+03 8.4E+03 8.6E+03 8.3E+03 9.8E+03 6.8E+03 2.0E+03 6.0E+03 3.6E+03 5.3E+04 2.4E+04 7.3E+04 9.3E+03 Long Blank Error 1400 3800 160 280 330 400 280 510 1200 420 460 84 220 760 2200 2000 980 980 340 1100 700 25000 8800 11000 860 Punch Blank Avg AUC 3.2E+04 9.1E+04 2.4E+03 6.6E+03 8.0E+03 5.2E+03 5.3E+03 3.9E+04 1.5E+04 5.6E+03 7.1E+03 4.0E+03 8.2E+03 1.6E+04 6.3E+03 6.8E+03 7.2E+03 5.7E+03 2.3E+03 5.8E+03 3.2E+03 1.8E+04 1.2E+04 7.5E+04 1.4E+04 Punch Blank Error 1000 2800 290 450 2700 1400 620 18000 2400 1100 850 1540 2600 5200 1000 860 800 670 250 940 500 2600 1000 6900 450 Long Drug Avg AUC 2.0E+05 6.0E+05 2.2E+04 4.5E+04 9.7E+05 2.6E+05 1.3E+04 6.6E+04 2.4E+04 1.2E+04 9.8E+04 4.9E+05 6.7E+05 1.2E+06 5.7E+04 5.1E+04 2.0E+04 3.0E+04 2.2E+03 1.2E+04 8.7E+03 1.9E+06 5.8E+05 7.9E+04 1.9E+04 Long Drug Error 23000 69000 2300 5300 1.3E+05 34000 960 8300 3300 890 12000 79000 1.1E+05 2.1E+05 6900 6300 1000 2100 260 1000 780 2.8E+05 82000 5900 1000 Punch Drug Agv AUC 1.6E+05 4.8E+05 1.9E+04 4.2E+04 8.5E+05 2.3E+05 1.2E+04 9.1E+04 1.7E+04 8.2E+03 1.1E+05 5.8E+05 9.6E+05 1.7E+06 1.5E+04 1.3E+04 9.4E+03 1.5E+04 1.9E+03 5.9E+03 3.7E+03 1.1E+06 3.5E+05 8.7E+04 2.0E+04 Punch Drug Error 11000 37000 870 1800 28000 8200 520 2400 2300 490 7900 45000 34000 58000 1500 1700 1100 1600 273 1100 600 91000 29000 16000 830 Long S:B 13 15 10 10 240 72 1.7 8.2 1.7 2.0 15 470 200 150 6.6 6.2 2.1 4.4 1.1 2.0 2.4 35 24 1.1 2.0 Punch S:B 4.9 5.2 8.1 6.3 110 44 2.2 2.3 1.2 1.5 16 150 120 100 2.3 2.0 1.3 2.7 0.8 1.0 1.2 61 28 1.2 1.4 Table 6 Results from thirteen analytes show that using long paper with diluted blood generally gives lower blank signals and higher analyte response than using whole blood and 3mm punches. The first seven analytes (in blue) were run in one set of samples and the last 6 (in red) were run in a second set of samples. 35 36 4.3 Tuning Thermo TSQ Tune Master was used to automatically identify the prominent fragment ions produced for each analytical target and to optimize instrumental parameters for those fragments. During tuning, the voltage was set to 4000 V, the collision gas pressure was held at 1.5 mTorr, and the software was set to exclude fragments resulting from loss of water and ammonium. From there, the software optimized instrumental conditions for the parent ion, identified the 4 most intense fragment ions, and found optimal collision energies (CE) for each fragment ion (Figure 10). For each analyte, it also optimized the S-lens, which focuses the initial gas plume that enters the inlet of the mass spectrometer into a concentrated beam of ions that enter the high-vacuum region of the mass spec. Initial tuning was performed using targets cocktailed in 95:5:0.01 methanol:water:acetic acid at 1ppm and an H-ESI source. The results generated were the basis of selecting appropriate fragment ions and establishing fragmentation ratios and instrument parameters for each analyte. After several experiments were done, each analyte was re-tuned by spraying it in a neat solvent over blank paper at each analyte’s respective target cutoff concentration. This was Figure 10: An example of some of the data generated from the tuning software. In the curves produced for Flunitrazepam above, the 4 most intense fragment ions are plotted against collision energy. Some fragments, like m/z 268 are more sensitive to changes in collision energy, as is evident in the narrow peak in the curve, while other fragments with broad peaks, like m/z 183, are less effected by changes in collision energy. 37 done in part because using a 1ppm solution in some cases saturated the detector, but also because of experiments performed after the initial tuning where fragmentation ratios were found to be somewhat concentration dependent. This resulted in a few minor adjustments of the fragment ions selected and the established fragment ion ratios. In most cases, the protonated molecular ion was selected as the precursor ion, although for some targets, like topiramate, a sodiated precursor ion was used. An effort was made to encourage all targets to ionize positively due to complications that arise when paper spray is used in negative ionization mode, however, the following targets were only found at suitable intensities as deprotonated ions: all barbiturates (amobarbital, butabarbital, butalbital, pentobarbital, phentobarbital, and secobarbital), as well as furosemide, hydrochlorothiazide, warfarin, ibuprofen, salicylic acid, valproic acid, and tadalafil. Although not typically required because of chromatography, in this project the specificity of the product ions used for identification was considered. In general, the two most abundant fragments from the automated tuning were selected, as they provide greatest sensitivity near the limit of detection. However, to enhance selectivity, larger, more complex fragments were chosen over smaller fragments when possible. Commonly produced fragments were avoided for compounds with amino side chains including m/z 58, 86, and 100.47 Similarly, the isopropyl side chains present in many cardiovascular drugs produce common fragments at m/z 43, 60, 116, 98, 74, and 56, which were avoided if possible.48 In some cases, the most abundant fragment found during tuning was not used because it caused unacceptably low signal to blank ratios. This is expected in instances where the most abundant fragment is a commonly produced fragment like those outlined in Section 2.3.3. In a few instances, the most abundant fragment was not used because it caused the fragmentation ratios to behave disproportionately over a range of concentrations. While this was not observed in most cases, an example of this behavior was seen in the fragmentation of norbuprenorphine and depicted in Figure 10. Circumstances such as this have lead some to suggest that ratios should be established near the intended analytical concentration.46 A list of the final parent/fragment ions chosen, instrumental conditions, and the fragmentation ratios used for each target analyte in this project can be found in Appendix C. 38 Fragmentation Ratios for Norbuprenorphine from 0.25 ng/mL to 100 ng/mL 95 Ratio 85 75 65 55 45 35 0.10 1.00 10.00 Concentration in ng/mL 101/211 187/211 83/101 100.00 83/187 Figure 11: When selecting appropriate fragment ions to monitor, fragmentation ratio stability over a range of concentrations was considered. For example, while m/z 83 was found to be the most abundant fragment for norbuprenorphine during tuning, ratios that use fragment m/z 83 do not behave proportionally, especially near the target detection level (1 ng/mL). In cases like this, a lesser abundant fragment is used to provide fragmentation ratios that are more consistent over a range of concentrations 4.4 Acquisition Parameter In tandem-in-time mass spectrometers, there are inevitable small variations in the signal intensity of fragment ions between scans. Averaging a large number of scans for each SRM channel will produce a more consistent, representative response, but at the expense of increasing run time. The variability of individual SRM channel responses becomes a compounded concern when two SRM’s are used to calculate a fragmentation ratio. In order to reach an accurate ratio and to minimize the time required for sample analysis, it is necessary to determine the minimum number of scans needed in order to provide a consistent fragmentation ratio. To this end, data from 26 different targets that had shown moderate to severe ratio deviations were analyzed. The targets were analyzed in cocktails in solution using 95:5:0.01 methonol:water:acetic acid as a solvent. The number of scans over which 39 data was recorded ranged from 32 to 60, and each scan lasted 0.1 seconds. The data was analyzed by monitoring the running average of the ratio of the intensities of two fragment ions per target analyte. The ratio produced after all scans were collected was considered the “true” ratio, and this ratio was compared to the ratio obtained for each target after 10, 15, 20, 25, and 30 scans. The running averages are shown for the first 50 scans in Figure 12 and Figure 13. 62% of targets showed no significant difference between their “true” fragmentation ratio and the ratio acquired after 15 scans (1.5 seconds/channel). 92% of targets showed less than ±5% variation between their true ratio and the ratio obtained after 15 scans. Doubling the number of scans to 30 only increases that percentage to 96%. As such, 15 was chosen as the minimum number of 0.1 second scans needed to provide a reproducible fragmentation ratio. Defining the minimum number of scans needed per SRM channel has direct implications on the analysis time for paper spray. If there are 154 target analytes that each have two fragment ion channels and if each of these channels needs to be scanned for 1.5 seconds, total analysis time to screen the entire panel of targets is set at a minimum of 7.7 minutes. If run times are capped at two minutes to eliminate the need to replenish solvent mid-run, at least four samples will need to be run to provide full coverage for the purposed targets. 4.5 Screening Identification Criteria One of the final tasks during method development for this project was to establish screening identification criteria by which the targets would be classified as “detected” or “not detected”. These criteria need to be stringent enough that they support specificity and low false positive rates, but liberal enough that they allow for the detection of targets with possible interferes from co-eluting matrix components or similarly structured targets. Because paper spray cannot rely on chromatographic retention times, specificity when using a triple quadrupole instrument is obtained from identifying the presence of two known fragment ions for each target, as well as the fragmentation ratio between these ions. Confirmative results are obtained when both fragments are present in a sample and 40 Cumulative Ratio Average In Solution at Cutoff Concentrations for Targets with Ratios > 50 95 90 Ratio Out of 100 85 80 <- Average after 15 scans 75 70 65 60 0 10 20 30 Cumulative Average after N Scans 40 50 Baclofen Clomipramine Desalkylflurazepam Dextromethorphan Etomidate Lamotrigine mCPP MDA Meprobamate Metoprolol Norclozapine Nordoxepin Trimipramine Figure 12: Running average plot of fragmentation ratios for targets with ratios > 50 show ratios generally stabilize after 15 scans on each fragment ion channel 41 Cumulative Ratio Average In Solution at Cutoff Concentrations for Targets with Ratios < 50 45 40 Ratio Out of 100 35 30 <- Average after 15 scans 25 20 15 10 0 Benztropine Desipramine Metaxalone Norvenlafaxine TFMPP 10 20 30 40 Cumulative Average after N Scans Benzylpiperazine Desmethylclomipramine Methylclomipramine Olanzapine Ziprasidone 50 Cyclobenzaprine Gabapentin Methylphenidate Ropinirole Zolpidem Figure 13: Running average plot of fragmentation ratios for targets with ratios < 50 show ratios generally stabilize after 15 scans on each fragment ion channel 60 42 when their fragmentation ratio is within some permitted tolerance of the expected ratio for the analyte. A signal to blank threshold of 3:1 is commonly used to define the limit of detection, and will be adopted in this project. Detecting two fragment ions with a signal to blank ratio of greater than 3, will be the first criteria that must be met to establish the detection of an analyte, although a higher threshold may be needed depending on future method validation experiments. It is also necessary to establish an appropriate tolerance for fragmentation ratio variance. Establishing a fragmentation ratio tolerance that is fit for paper spray was informed by both past paper spray research and published guidelines from various organizations (Table 2). The first major guideline to be published on this topic and which many others are based off of was the European Union decision of 2002, which allowed for tolerances as high as ±50% for fragmentation ratios3. While these guidelines were used as a basis for establishing suitable tolerance levels for PS-MS, they are based on assumptions that do not apply to paper spray, namely the assumption that chromatography is being performed for quantitative analysis. Paper spray does not enjoy the selectivity that comes from chromatography, so the possibilities of interferences arising is higher, which suggests the need for a broader ion ratio tolerance to decrease the chances of false negatives. A broad tolerance is also supported by the findings of Mol et al. who investigated ion ratio deviations in 120 pesticides in 21 food matrices in five different laboratories. The authors found that fragment ion ratio deviations are dependent upon detector response, and that near the limit of detection it is typical for ion ratios to diverge as much as ±45%.49 Historically a ratio tolerance of ±20% has been used for paper spray analysis, however, qualitative screening on a large group of targets has not been done before. During quantitative analysis of eight drugs using paper spray, Espy et al. saw ratio variations as high as 19%, in the case of morphine. This variation occurred at a concentration of 38 ng/mL34 (double this project’s target LOD). Deviations at lower concentrations would be expected to be even greater because of lower detector response and would deviate even further if the ratios were derived from solvent standards and were not matrix-matched. The problem of variable ratios near LOD is illustrated by the data compiled in Figure 13. While it is possible a higher tolerance range may prove suitable 43 after method validation, a range of ±30% for fragment ion ratios was used in this project, as it allows more variability than previous paper spray experiments, yet still falls within the guidelines published by the society of forensic toxicologists and the academy of forensic scientists. S:B vs Ratio Deviation % Deviation from established ratio 60 50 40 30 20 10 0 -10 0 50 100 150 200 -20 -30 -40 -50 S:B of least abundant fragment Figure 14: Deviation between solvent-established fragmentation ratios and fragmentations ratios in whole blood at cutoff concentrations for target analytes where both fragments were detected with a S:B ≥3 44 5 RESULTS At the beginning of this project, 154 different analytical targets were proposed to investigate the feasibility of paper-spray mass spectrometry as an effective means of rapid drug screening. After method development, 14 of the originally proposed targets were shown to only ionize well in negative mode and will be studied in future experiments optimized for negative ionization. The 14 negatively ionizing targets included all of the barbiturates (amobarbital, butabarbital, butalbital, pentobarbital, phentobarbital, secobarbital), as well as: furosemide, hydrochlorothiazide, ibuprofen, salicylic acid, thiopental, tadalafil, valproic acid, and warfarin. In addition to the negatively ionizing targets, delta-9-THC and its secondary metabolite 11-nor-9-carboxyTHC, as well as buprenorphine and its metabolite norbuprenorphine were also not analyzed in these experiments because they failed to be detected in preliminary optimization experiments, even at 250 times higher than their proposed cutoff levels. Previously published paper spray studies have shown that delta-9-THC and 11-nor-9carboxy-THC require a specialized solvent system for detection.34 In addition to these targets, hydroxychloroquine and 10-monohydroxyoxcarbazepine have been excluded from this data set due to the availability of the compounds during the time frame in which the experiments were conducted. Excluding all of these compounds from the panel of analytical targets originally proposed by AFT left 134 targets that were analyzed to determine if PS-MS could be an effective alternative method for drug screening in forensic toxicology. Experiments were run on paper cut manually with razors and reusable cartridges, as well as on laser-cut and die-cut paper in disposable cartridges. Only 69 of the targets, however, were run using the die-cut paper because there were only a limited number of die-cut cartridges available. Although 134 target analytes were included in the cocktails that were analyzed, results will only be reported for 133 targets using the manual method and 131 targets using the laser-cut cartridges due to an error in transposing the correct SRM channels to the mass spec acquisition program for topiramate, papaverine, and etomidate. Out of the 134 targets included in the cocktails, 28 had to be run at concentrations 45 other than the cutoffs specified by AFT during project development. Six out of these 28 targets (acetaminophen, benzylpiperazine, naproxen, phenytoin, topiramate, and zonisamide) were run at concentrations lower than those originally proposed by AFT in order to avoid swamping the detector at the upper end of the calibration curve. The other 22 targets were found to have limits of detection higher than the proposed cutoff concentrations from AFT. These differences in the experimental LOD and proposed cutoffs from AFT are outlined in Table 7. Only four targets of the 23 targets whose limits of detection were found to be higher than AFT’s, however, were run at concentrations outside of the expected normal therapeutic concentration range as defined by data compiled by Schultz et al. (See Table 7). The four targets were: amphetamine, buspirone, tramadol, and demoxepam. 5.1 Results A summary of the results for all 154 proposed targets is found in Table 8 and more detailed results sorted by cocktail are found in Table 9 – Table 21. For full results, see Appendix E. Out of the 133 targets run using the manual method, 114 met the detection requirements when spiked into drug free whole blood at the targeted cut-off concentration; the detection criteria were having a signal to blank ratio of greater than three and deviated less than 30% in their established fragmentation ratio. Nineteen of the targets failed to meet these criteria, half of the time because of low signal to blank and half of the time due to ratio variability. When the same targets (minus papaverine and etomidate whose SRM channels were wrongly transcribed) were run using the automated source and the laser-cut paper, 99 satisfied the detection criteria. In the 32 cases where the target was not detected at the cutoff or the experimental limit of detection, 15 were attributed to low signal to blank ratios and 17 to ratio variability outside of the permitted tolerance. When comparing the two methods, there were 22 instances where targets were detected manually but not when using the automated source and the laser-cut paper, and there were 7 instances where targets were detected with the automated source but not manually (See Figure 17). 46 Of the 68 targets (etomidate excluded due to incorrect SRM channels being monitored) that were analyzed, using the die-cut paper, 87% were detected at cutoff or LOD. Of the 8 targets that did not meet detection criteria using the die-cut paper, only two were due to low signal, while the rest were excluded due to ratio variability outside of the permitted ±30% window. There were seven instances where targets were detected using die-cut paper that were not previously detected using the automated method on laser-cut paper. 99 n=133 n=131 n=68 Figure 15 Number of analytical targets detected using three different methods. Out of the 133 targets analyzed using the manual method, 86% met detection criteria; for the 131 targets analyzed using the automatic method with laser-cut paper, 76% met detection criteria. Of the 68 targets analyzed using the automatic method with die-cut paper, 87% met detection criteria 47 Table 7: A list of targets run at concentrations other than those proposed by AFT compared to normal, toxic, and fatal levels2 Normal levels are defined as the effective dose where no or minimal side effects occur. Toxic levels are those at which side effects or negative symptoms arise, and fatal levels are the concentrations which result in either coma or death. Concentration in mg/L Target LOD found during project AFT proposed cutoff level Normal Toxic Fatal 6-acetylmorphine 0.03 0.02 - - - 7-aminoclonazepam 0.05 0.010 - - - Acetaminophen 10 20 10-25 100-150 200-300 Amphetamine 0.2 0.05 0.02-0.1 0.2 0.5-1 Aripiprazole 0.1 0.05 0.15-0.5 1 - Benzylpiperazine 0.025 0.05 - - - Buspirone 0.025 0.01 0.001-0.004 .008 - Clonazepam 0.025 0.01 0.02-0.08 0.1 - Demoxepam 0.075 0.05 0.5-0.74 1 2.7 Desmethylclomipramine 0.1 0.02 - - - Fentanyl 0.01 0.001 0.003-0.3 0.003-0.2 - Fluvoxamine 0.025 0.02 0.060-0.23 0.5-0.65 2.8 Hydroxyzine 0.075 0.01 0.05-0.1 0.1 39 MDA 0.1 0.05 - 1.5 1.8-2 Mescaline 0.1 0.05 - - - Morphine 0.025 0.02 0.01-0.1 0.1 0.1-4 Naproxen 3 30 20-50 200-400 - Norsertraline 2 0.1 - - - Nortramadol 1 0.1 - - - Oxycodone 0.1 0.02 0.005-0.1 0.2 0.6 Oxymorphone 0.025 0.02 - - - Paroxetine 0.025 0.02 0.01-0.05 0.35-0.4 3.7-4 Phenytoin 2.5 5 5-15 20-25 43 Topiramate 1 2 2-10 16 - Tramadol 2 0.1 0.1-1 1 2 Ziprasidone 0.1 0.01 0.05-0.2 0.4 - Zonisamide 7.5 10 10-40 40-70 100 Zopiclone 0.025 0.01 0.01-0.05 0.15 0.6-1.8 48 Detected Die-Cut? Cocktail Run In Diazepam Diltiazem Diphenhydramine Donepezil Doxepin Doxylamine Duloxetine EDDP Ephedrine/ Pseudoephedrine Etomidate Felbamate Fentanyl Flecainide Flunitrazepam Fluoxetine Flurazepam Fluvoxamine Furosemide Gabapentin Haloperidol Hydrochlorothiazide Hydrocodone Hydromorphone Hydroxychloroquine Hydroxyzine Ibuprofen Ketamine Labetalol Lamotrigine Levetiracetam Lidocaine Lorazepam mCPP MDA MDMA MDPV Meperidine Mephedrone Meprobamate Mescaline Metaxalone Methadone Methamphetamine Methocarbamol Methylone Methylphenidate Metoclopramide Detected Laser-cut? G G J K A G G K L J C D L B L J C H C G I B B A G I G M J I G G M M K E G J D K Target Detected Manually? Cocktail Run In Not run Not run No Yes Yes Yes No Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes No No Negative ionizer Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes No No Yes Yes Yes Yes Yes Yes Not run Yes Yes Yes Yes Yes Yes Negative ionizer Negative ionizer Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No No Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Not run No No Yes Yes No No No No No No No Yes Yes - Detected Die-Cut? 10-monohydroxyoxcarbazepine 11-nor-9-Carboxy-THC 6-acetylmorphine 7-aminoclonazepam 7-aminoflunitrazepam 9-hydroxyrisperidone Acetaminophen Alfentanil Alpha-PVP Alprazolam Amitriptyline Amlodipine Amobarbital Amphetamine Aripiprazole Atenolol Baclofen Benzoylecgonine Benztropine Benzylpiperazine Brompheniramine Bupivacaine Buprenorphine Bupropion Buspirone Butabarbital Butalbital Carbamazepine Carbamazepine-10,11-epoxide Carisoprodol Chlordiazepoxide Chlorpheniramine Chlorpromazine Citalopram Clomipramine Clonazepam Clozapine Cocaethylene Cocaine Codeine Cyclobenzaprine delta9-THC Demoxepam Desalkylflurazepam Desipramine Desmethylclomipramine Dextromethorphan Detected Laser-cut? Target Detected Manually? Table 8: Summary of results for all 154 proposed analytical targets; "-" indicates not analyzed Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes L G H F M H J H Yes Yes Yes E No Yes Yes Yes Yes Yes Yes Yes No No No Yes Yes Yes Yes Yes Yes Negative ionizer Yes No Yes Yes Negative ionizer Yes No Yes No Not Run Yes Yes Yes Negative ionizer Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes D A K C J J H I C K L M E D F B A C H J D F F H F B D B I F B F J D 49 Tramadol Trazodone Triazolam Trimipramine Valproic Acid Vardenafil Venlafaxine Verapamil Warfarin Zaleplon Ziprasidone Zolpidem Zonisamide Zopiclone Cocktail Run In F F F I A F M J J D H E A B L E E M D I C I H E A C A H E E E C K K D D M E A Detected Die-Cut? Cocktail Run In Detected Die-Cut? No Yes Yes Yes Yes Yes No Yes Yes Not run Yes No No Yes Yes Yes Yes no Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes Yes Yes No No Yes Yes No No No No Yes Yes Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Negative ionizer Negative ionizer Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Negative ionizer Negative ionizer Yes Yes Yes Yes Yes Yes Negative ionizer No No Yes No No Negative ionizer - Detected Laser-cut? Yes Yes Yes Yes Yes Target Detected Manually? Metoprolol Midazolam Mirtazapine Morphine Naproxen Norbuprenorphine Norclozapine Nordiazepam Nordoxepin Norfluoxetine Norketamine Normeperidine Norpropoxyphene Norsertraline Nortramadol Nortriptyline Norvenlafaxine Olanzapine Oxazepam Oxycodone Oxymorphone Papaverine Paroxetine PCP Pentazocine Pentobarbital Phenobarbital Phenytoin Pregabalin Primidone Promethazine Propoxyphene Propranolol Quetiapine Ranitidine Risperidone Ropinirole Salicylic Acid Secobarbital Sertraline Sildenafil Tadalafil Temazepam TFMPP Thiopental Topiramate Detected Laser-cut? Target Detected Manually? Table 8 Continued Yes Yes Yes Yes No Yes Yes No Yes - A D J J Negative ionizer - Yes Yes Yes Yes Yes Yes Yes Yes Yes Negative ionizer Yes No Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes D E E I D K B I 50 Manual Laser-Cut Ratio Ratio R at i o L o w S : B Low S:B Low S:B n= 32 n= 19 Die-Cut Ratio Low S:B n= 8 Figure 16. Stacked column graphs representing the reason that targets failed to meet detection criteria for each of the three methods. When the manual method was used, 53% of failed detections were due to ratio deviations outside of the permitted ±30% window. For the automatic method using laser-cut paper 53% of failures were due to ratio deviations, and for die-cut experiments, the failure due to ratio deviations reached 75%. A. B. Manual 0 2 Laser-Cut 7 1 7 Figure 17: (A) Of the 68 targets run using all three methods, 16 were detected in at least one method, but not in all three. There was one instance (desmethylclomipramine ) where the target failed to be detected in any of the three methods. Venn diagram A depicts the methods where these 17 targets failed to meet detection criteria. (B) Of the 63 targets run using only the manual and laser-cut methods 23 total targets were not detected, 7 of which were detected in neither method. Venn diagram B specifies in which method these failures to meet detection criteria occurred. 51 7500 213 Primidone 5000 219 Naproxen 3000 231 Felbamate 10000 239 Phenytoin 2500 253 Carisoprodol 2000 261 Tramadol 2000 264 Norsertraline 2000 275 Topiramate 1000 362 26 6% 48 5% 72 3% 28 -9% 61 -5% 66 7% 69 -17% 35 17% 7 -37% - - 139 55 48 85 671 31 292 2 7 9 52 791 6 12 29 19 2 5159 57 66 - True Ratio Zonisamide -5% Ratio Deviation 171 28 Run Ratio 2000 29 25 23 20 35 20 40 15 21 7 14 37 44 43 4 4 10 39 35 29 - S:B Levetiracetam 110 65 126 154 132 77 162 91 170 185 117 178 104 182 55 97 264 58 124 159 265 207 Ratio Deviation 152 Lasercut Run Ratio 10000 Manual S:B Precursor m/z Acetaminophen Product m/z Concentration in blood (ng/mL) Table 9: Cocktail A-Manual experiments had 26 scans per target and experiments run on the automated ionization source on the laser-cut paper had 16 scans per target. Red shading indicates failure to meet detection criteria. 28 -6% 30 26 10% 24 47 3% 46 85 21% 70 30 -1% 30 64 -1% 64 76 25% 61 78 -6% 83 17 6 120% 30 7 -36% 11 - - 52 52 1000 222 Carbamazepine 1000 237 Methocarbamol 1000 242 Nortramadol 1000 250 Carbamazepine10,11-epoxide 1000 253 Lamotrigine 1000 256 88 9% 27 4% 22 -25% 21 -10% 21 -17% 39 8% 62 0% 11 35 496 24 9 113 208 888 109 94 5 21 291 179 83 181 True Ratio Metaxalone -4% Ratio Deviation 219 84 Run Ratio 2000 94 78 208 25 24 118 186 181 108 65 5 34 246 91 77 84 S:B Meprobamate 151 115 158 97 105 161 192 194 118 199 232 42 180 210 145 211 Ratio Deviation 214 Lasercut Run Ratio 1000 S:B Precursor m/z Baclofen Manual Product m/z Concentration in blood (ng/mL) Table 10: Cocktail B-Manual experiments had 35 scans per target and experiments run on the automated ionization source on the laser-cut paper had 16 scans per target. Red shading indicates failure to meet detection criteria. 89 2% 88 87 8% 81 28 10% 26 22 -25% 30 21 -11% 23 23 -7% 25 38 7% 36 64 1% 63 53 160 Gabapentin 500 172 Benzylpiperazine 25 177 Lidocaine 500 235 Bupivacaine 500 289 Ranitidine 500 315 Papaverine 500 340 Flecainide 500 415 -6% 467 0% 32 0% 21 70% 10 -16% 17 -2% 51 4% 105 4% 40 6% S:B S:B 23 6 47 12 7 34 147 41 34 59 7086 13927 2130 10319 713 2025 5159 True Ratio 500 9 22 68 28 103 116 3 16 311 410 335 284 447 225 347 342 310 335 Ratio Deviation Pregabalin 91 119 124 97 119 137 85 91 58 86 140 98 102 176 324 202 301 98 Run Ratio 136 Lasercut Ratio Deviation 200 Manual Run Ratio Amphetamine Product m/z Precursor m/z Concentration in blood (ng/mL) Table 11: Cocktail C-Manual experiments had 32 scans per target and experiments run on the automated ionization source on the laser-cut paper had 16 scans per target. Red shading indicates failure to meet detection criteria. 21 -15% 25 22 6% 21 814 2444% 32 12 -1% 12 10 -16% 12 17 -2% 17 51 4% 49 - - 101 40 5% 38 100 100 100 100 100 100 100 100 100 100 Mescaline Aripiprazole Desmethylclomipramine Etomidate Ketamine Metoclopramide Norketamine Oxycodone Sertraline Concentration in blood (ng/mL) MDA Precursor m/z 306 316 224 300 238 245 301 448 212 180 Product m/z 133 135 165 180 176 285 227 72 141 95 125 89 184 227 125 179 212 241 159 275 S:B 17 25 48 6 35 74 11 108 20 5 141 91 89 110 108 67 19 29 31 81 Run Ratio 81 65 45 37 25 105 24 29 64 93 Ratio Deviation -4% 4% 2% -4% 1% 41% 39% 1% -20% 3% S:B 23 23 55 4 45 391 30 1720 29 1544 209 1451 6443 543 159 25 61 72 351 Run Ratio 84 68 45 39 25 - 26 28 62 100 Ratio Deviation -1% 7% 2% 1% 3% - 51% 2% -23% 10% S:B 12 12 7 2 56 215 13 1232 33 157 31 249 1591 118 32 10 33 47 349 Run Ratio 82 69 46 38 26 - 23 28 30 100 Ratio Deviation -3% 10% 5% -3% 6% - 37% 1% -63% 10% 85 63 44 39 24 66 17 28 81 91 True Ratio Table 12: Cocktail D- Manual experiments had 20 scans per target and experiments run on the automated ionization source had 15 scans per target. Red shading indicates failure to meet detection criteria. Manual Diecut Lasercut 54 100 100 100 100 Trazodone Vardenafil Ziprasidone Concentration in blood (ng/mL) Sildenafil Precursor m/z 413 489 372 475 Product m/z 100 283 148 176 151 312 159 194 S:B 143 25 76 71 39 99 6 129 81 40 83 69 Run Ratio Ratio Deviation 121% -9% -1% 12% S:B 469 47 914 316 268 474 8 125 Diecut 44 42 83 66 Run Ratio Manual Ratio Deviation 19% -2% -2% 6% S:B 499 20 167 433 207 235 6 88 Lasercut 45 41 83 68 Run Ratio Table 12 Continued Ratio Deviation 22% -5% -2% 9% True Ratio 37 43 84 62 55 50 50 50 50 50 50 75 50 50 TFMPP Propranolol Norvenlafaxine Venlafaxine Pentazocine Demoxepam Norpropoxyphene Olanzapine Concentration in blood (ng/mL) Ephedrine/Pseudoephedrine Precursor m/z 313 308 287 286 278 264 260 231 166 Product m/z 115 117 118 188 116 183 107 58 121 58 218 69 180 207 128 44 198 256 S:B 26 27 22 42 44 23 13 17 20 93 72 11 6 3 20 61 11 28 Run Ratio 27 49 79 24 26 32 89 24 83 Ratio Deviation 3% 3% 27% 8% 4% 34% 1% 4% -3% S:B 44 50 20 35 558 72 6 487 16 894 774 17 18 10 71 852 2 39 33 49 67 22 26 26 91 79 83 Run Ratio Diecut Ratio Deviation 22% 2% 2% 2% 5% 9% 3% 242% -4% S:B 59 21 4 76 88 33 2 40 5 171 186 11 4 3 17 416 5 32 Lasercut 40 51 70 24 32 34 91 8 85 Run Ratio Manual Ratio Deviation 48% 6% 8% 8% 26% 40% 3% -64% -2% 27 48 65 22 25 24 88 23 86 True Ratio Table 13: Cocktail E- Manual experiments had 20 scans per target and experiments run on the automated ionization source had 15 scans per target. Red shading indicates failure to meet detection criteria. 56 50 75 50 50 Hydroxyzine Quetiapine Verapamil Concentration in blood (ng/mL) Propoxyphene Precursor m/z 455 384 375 340 Product m/z 128 266 165 201 221 253 150 165 S:B 10 31 56 28 81 66 80 34 37 64 83 21 Run Ratio Ratio Deviation 2% 0% -3% 3% S:B 22 425 1903 124 812 1080 1463 647 Diecut 37 65 84 20 Run Ratio Manual Ratio Deviation 3% 1% -2% -1% S:B 10 227 702 135 284 648 241 335 Lasercut 37 65 84 21 Run Ratio Table 13 continued Ratio Deviation 3% 1% -2% 6% True Ratio 36 64 86 20 57 50 50 50 50 50 50 50 50 50 50 50 Mephedrone MDMA Methylone Mirtazapine Metoprolol MDPV Norclozapine Midazolam Labetalol Donepezil Concentration in blood (ng/mL) Methamphetamine Precursor m/z 380 329 326 313 276 268 266 208 194 178 150 S:B 98 51 112 78 40 114 36 58 101 109 70 11 84 92 68 59 44 45 41 81 62 75 Product m/z 119 91 144 145 105 163 132 160 194 195 116 77 135 175 192 270 249 291 162 91 243 91 Run Ratio 16 95 28 74 89 75 40 53 36 75 38 Ratio Deviation -2% 10% 3% 2% 0% 10% -1% 1% 5% -2% -4% S:B 278 69 279 230 23 676 45 216 52 329 985 18 90 309 181 84 1487 11116 51 351 701 622 16 107 28 137 89 72 41 53 37 75 38 Run Ratio Diecut S:B 330 105 114 -1% 132 20 5% 392 16 1% 31 26 -1% 168 365 6% 6 31 0% 162 90 87% 45 337 3% 3530 24 23% 349 248 0% 272 Ratio Deviation -2% Lasercut 16 105 28 135 94 89 42 54 38 75 38 Run Ratio Manual Ratio Deviation 2% 21% 4% 85% 6% 31% 2% 4% 9% -1% -2% 16 87 27 73 89 68 41 52 35 76 39 True Ratio Table 14: Cocktail F-Manual experiments had 27 scans per target and experiments run on the automated ionization source had 20 scans per target. Red shading indicates failure to meet detection criteria. Error! Not a valid link. 58 50 50 50 50 50 50 50 50 30 50 50 Bupropion 7-aminoclonazepam Desalkylflurazepam Chlordiazepoxide Cocaethylene Chlorpromazine Clozapine 6-acetylmorphine Diltiazem Alfentanil Concentration in blood (ng/mL) Alpha-PVP Precursor m/z 417 415 328 327 319 318 300 289 286 240 232 S:B 54 24 47 49 19 9 6 44 26 18 62 42 19 22 77 64 2 2 76 56 27 13 Product m/z 91 77 184 131 121 94 140 226 227 255 196 82 214 58 270 192 165 211 178 150 268 197 Run Ratio 68 43 42 64 30 27 24 48 39 56 41 Ratio Deviation 10% -1% -26% 0% 18% -1% 5% -27% 9% 1% 1% S:B 423 49 155 198 12 5 13 96 59 10 799 846 97 107 1634 864 6 6 8926 2166 458 622 62 44 52 65 385 26 25 150 44 56 41 Run Ratio Diecut Ratio Deviation 1% -1% -9% 1% 1380% -3% 14% 127% 21% 2% 0% S:B 126 10 35 46 2 1 8 11 19 4 350 127 11 50 306 214 7 4 823 263 318 272 67 43 59 64 365 27 27 241 68 57 42 88% 3% 3% Ratio Deviation 7% -1% 4% 0% 1305% -1% 21% 266% Lasercut Run Ratio Manual 62 44 57 64 26 27 22 66 36 55 41 True Ratio Table 15: Cocktail G-Manual experiments had 24 scans per target and experiments run on the automated ionization source had 20 scans per target. Red shading indicates failure to meet detection criteria. 59 25 25 25 25 25 25 25 25 25 25 PCP Meperidine Diphenhydramine Doxylamine EDDP Promethazine Brompheniramine Lorazepam Flurazepam Concentration in blood (ng/mL) Normeperidine Precursor m/z 388 321 319 285 278 271 256 248 244 234 Product m/z 160 56 91 159 220 174 167 165 167 182 234 249 86 198 274 167 276 167 315 317 S:B 11 9 9 8 16 12 12 16 16 18 12 13 6 6 21 9 22 11 17 12 Run Ratio 23 49 50 43 41 88 44 57 55 23 Ratio Deviation 5% 2% 5% -1% -1% 0% 3% -1% -3% 1% S:B 39 81 28 93 253 99 200 230 126 307 1410 2742 17 16 719 248 295 268 1975 572 23 47 48 44 41 85 44 60 51 22 Run Ratio Ratio Deviation 3% -1% 2% 2% -2% -3% 2% 3% -9% -2% S:B 7 14 19 60 29 14 110 93 67 106 491 619 17 12 200 98 159 111 761 48 Lasercut 23 47 49 45 41 88 45 62 48 20 Run Ratio Diecut 4% -1% 4% 4% -3% 0% 4% 6% -15% -7% Ratio Deviation Manual 22 48 47 43 42 88 43 58 56 22 True Ratio Table 16: Cocktail H Manual experiments had 28 scans per target and experiments run on the automated ionization source had 21 scans per target. 60 25 25 25 25 25 25 25 25 25 25 Morphine Oxymorphone Zaleplon Methadone Clonazepam Fluvoxamine Paroxetine Buspirone Zopiclone Concentration in blood ( ng/mL) Chlorpheniramine Precursor m/z 389 386 330 319 316 310 306 302 286 275 S:B 43 28 4 3 8 8 16 15 36 23 5 10 10 4 9 6 36 5 5 18 Product m/z 230 167 152 165 227 198 236 264 265 105 270 214 71 200 192 70 122 95 245 112 Run Ratio 35 23 59 25 29 33 88 77 79 51 Ratio Deviation -11% 28% 13% 16% -8% 1% 0% 6% 12% -4% S:B 1192 1324 4 5 12 7 18 8 1763 306 3 4 37 13 26 11 1977 91 7 7 37 18 57 24 24 33 99 100 63 50 Run Ratio Ratio Deviation -6% 1% 9% 13% -26% 3% 13% 37% -12% -5% S:B 26 96 2 2 3 1 1 3 251 59 1 1 11 4 8 8 118 15 4 11 Lasercut 33 19 51 23 44 32 68 112 62 50 Run Ratio Diecut -15% 3% -3% 10% 39% 1% -23% 53% -12% -5% Ratio Deviation Manual 39 18 52 21 32 32 88 73 71 53 True Ratio Table 17: Cocktail I-Manual experiments had 28 scans per target and experiments run on the automated ionization source had 21 scans per target. Red shading indicates failure to meet detection criteria. 61 62 20 266 Desipramine 20 267 7-aminoflunitrazepam 20 284 Trimipramine 20 295 Norfluoxetine 20 296 Duloxetine 20 298 Benztropine 10 308 Fluoxetine 20 310 Flunitrazepam 20 314 Clomipramine 20 315 Triazolam 20 343 Amlodipine 20 409 13 5% 59 -1% 37 79% 51 0% 78 10% 19 -25% 95 10% 45 -1% 16 30% 32 -16% 49 -33% 85 11% 77 14% 8 4 114 5 10 36 249 5 9 8 25 26 4 76 14 3 195 14 219 3 2 5 10 16 336 13 17 2 True Ratio Nordoxepin -9% Ratio Deviation 234 81 Run Ratio 20 6 13 12 6 8 14 17 6 11 13 8 8 3 10 2 2 13 13 15 6 3 7 4 5 13 5 2 2 S:B Methylphenidate 154 118 84 56 107 235 72 193 135 227 193 208 134 30 44 154 167 165 44 148 268 239 227 242 308 239 238 294 Ratio Deviation 197 Lasercut Run Ratio 20 S:B Precursor m/z mCPP Manual Product m/z Concentration in blood (ng/mL) Table 18: Cocktail J-Manual experiments had 19 scans per target and experiments run on the automated ionization source had 18 scans per target. Red shading indicates failure to meet detection criteria. 90 1% 89 13 4% 13 56 -7% 60 27 34% 20 55 8% 51 100 41% 71 19 -28% 26 88 1% 87 1 -98% 45 17 34% 13 23 -40% 38 67 -8% 73 80 6% 76 79 18% 67 63 10 276 Zolpidem 10 308 Alprazolam 5 309 Fentanyl 10 337 Haloperidol 10 376 Risperidone 10 411 9-hydroxyrisperidone 10 427 77 1% 39 -1% 19 5% 87 0% 76 9% 93 0% 8 3% 22 1% 8 4 23 11 150 23 25 23 6 4 13 26 39 40 141 19 79 16 True Ratio Cyclobenzaprine 3% Ratio Deviation 272 22 Run Ratio 10 5 4 5 5 5 5 4 5 3 3 5 4 4 4 3 3 3 3 S:B Dextromethorphan 114 132 215 147 215 231 92 235 205 281 188 105 123 165 191 110 207 110 Ratio Deviation 261 Lasercut Run Ratio 10 S:B Precursor m/z Ropinirole Manual Product m/z Concentration in blood (ng/mL) Table 19: Cocktail K-Manual experiments had 30 scans per target and experiments run on the automated ionization source had 18 scans per target. Red shading indicates failure to meet detection criteria. 90 327% 21 81 7% 76 42 7% 39 20 11% 18 81 -7% 87 75 7% 70 91 -3% 93 8 4% 8 24 9% 22 64 20 278 Diazepam 50 285 Benzoylecgonine 50 290 Hydrocodone 20 300 70 -10% 62 1% 78 -3% 38 16% 40 10% 55 23 45 151 37 46 19 34 77 4 17 5 True Ratio Amitriptyline 6% Ratio Deviation 267 45 Run Ratio 100 31 12 6 12 21 14 18 22 11 4 19 10 S:B Atenolol 233 117 145 190 233 117 154 222 168 77 199 171 Ratio Deviation 264 Lasercut Run Ratio 20 S:B Precursor m/z Nortriptyline Manual Product m/z Concentration in blood (ng/mL) Table 20: Cocktail L-Manual experiments had 14 scans per target and experiments run on the automated ionization source had 19 scans per target. Red shading indicates failure to meet detection criteria. 44 4% 42 77 -1% 78 59 -4% 61 81 0% 81 51 55% 33 56 54% 37 65 20 286 Oxazepam 50 287 Codeine 20 300 Temazepam 50 301 Cocaine 50 304 Citalopram 10 325 48 -8% 77 11% 89 511% 74 23% 26 -17% 23 8% 78 66% 25 16 28 28 3 3 2 2 4 5 7 3 97 49 28 12 True Ratio Hydromorphone 6% Ratio Deviation 280 72 Run Ratio 20 44 29 12 15 6 4 2 1 10 4 2 3 46 14 16 6 S:B Doxepin 140 208 107 165 185 157 241 77 215 199 177 239 182 82 262 234 Ratio Deviation 271 Lasercut Run Ratio 50 S:B Precursor m/z Nordiazepam Manual Product m/z Concentration in blood (ng/mL) Table 21: Cocktail M- Manual experiments had 13 scans per target and experiments run on the automated ionization source had 18 scans per target. Red shading indicates failure to meet detection criteria. 72 -6% 68 51 -2% 52 105 53% 69 188 1151% 15 46 -25% 61 47 48% 32 22 2% 21 68 44% 47 66 6 INTERFERENCE STUDY In order for paper spray to be a viable approach to post-mortem toxicological screening it must be able to differentiate target analytes from endogenous substances that exist within a matrix, and it must also be able to differentiate target analytes from other exogenous compounds. For example, PCP is one of the analytical targets of the developed screening method and is categorized as a schedule II controlled substance in the US, but it shares the same molecular weight as frovatriptan, a prescription migraine medication. In this instance, the legal medication could affect the results for a controlled substance by producing a parent ion with the same mass to charge ratio. It is therefore necessary to investigate the specificity of the developed method in producing accurate results in the presence of potentially interfering compounds. Interferences to the target analytes can arise from several different situations. Both exogenous and endogenous isomers of an analyte may produce common daughter fragments with the target analyte, which would result in skewed fragmentation ratios and could create false negative results. Compounds that do not share the same mass as the target could even interfere by producing adduct ions more complex than [M+H]+ that have the same mass to charge ratio as the protonated adduct of the target analyte. Isotopic interferences could also arise when molecules with moieties containing heavily abundant isotopes, such as chlorine or bromine, are present. To a lesser extent, isotopic interferences in larger molecules could even arise due to the C-13 isotope if the interfering compound is found at a much higher concentration than the target analyte. Although this makes the list of potential interferences for the project’s 154 different analytical targets practically unquantifiable, investigating, to a reasonable extent, potentially interfering compounds will help inform data interpretation. To investigate compounds that could cause interferences with the developed drug screening method, potential interferences were sorted into two classifications: interferences that arise from exogenous substances potentially contained within blood, and intra-target interferences that arise from isomeric or isotopic parent ion overlap between the 154 analytical targets defined in the study. 67 6.1 Exogenous interferences While there are compounds that could produce isotopic interferences by generating the same parent ions as the target analytes in this project, these compounds would have to contain elements with relatively abundant isotopes (i.e. chlorine, bromine) and would have to be fairly concentrated within the matrix to become a true concern. As the scope of identifying potentially interfering compounds is already wide, at this time the search has been limited to compounds that are biologically available to the matrix and have the same molecular weight as the target analytes. To this end, two publically available databases were used to compile a list of potentially interfering compounds: the Human Metabolome Database63-65, a database of small molecule metabolites, and DrugBank, a searchable database that contains over 8232 drugs.66 Using these databases, 450 potentially interfering exogenous compounds were identified that have the same molecular weight at the target analytes of this project. These compounds are outlined in Appendix F. 6.2 Intra-target interferences Out of the 154 analytical targets in this study, 43 positively ionizing analytes were identified that that have parent ions that share the same mass to charge ratio as another analyte, and could therefore be a source of interference. These target analytes are presented in Table 22. For an analyte to be a true interference to another target, it must produce at least one fragment as another target with the same parent ion, and it must produce this fragment in an abundance such that it alters the expected fragmentation ratio of the target analyte. To this end, each of the 43 potentially interfering targets were spiked at 1ppm into 95:5:0.01 methanol:water:acetic acid and introduced into the mass spec via an ESI ionization source, where they were scanned for 60s for the daughter fragments of all the potentially interfering target analytes. Each infusion was followed by a 60 second infusion of blank solvent to avoid carryover. Topiramate was added to each solution at 100 ng/mL as an internal standard to monitor ionization. The signal-to-blank for topiramate never fell below 15:1 during all of the interference experiments, and the fragmentation ratio never deviated more than 8% from the established ratio. 68 During these preliminary experiments, 25 target analytes were identified that had a signal-to-blank greater than 3:1 when monitoring the SRM channels for a different analyte. The results for the initial experiments using the ESI spray source are presented in Table 23. These targets were investigated further, while the other 18 analytes were concluded to not be true interferences since they added no significant increase in signal at 1000 ng/mL, a concentration much higher than the cutoff levels for most of the targets. Table 22: Targets that share the same parent ion and could potentially interfere with one another. Cutoff in ng/mL MW Name 5000 218 Primidone 2000 218 Meprobamate 500 226 Amobarbital 500 226 Pentobarbital 25 233 Normeperidine 20 233 Methylphenidate 5000 252 Phenytoin Carbamazepine-10,111000 252 epoxide 1000 255 Lamotrigine 25 255 Diphenhydramine 2000 260 Carisoprodol 10 260 Ropinirole 20 265 Nordoxepin 50 265 Mirtazapine 100 291 Norsertraline 25 274 Chlorpheniramine Parent Ion 219.087 219.096 225.116 225.116 234.121 234.122 253.081 Product Product Ion 1 Ion 2 162.111 91.055 158.134 97.105 182.139 42.066 182.144 42.043 160.129 56.068 84.086 56.072 182.110 104.046 Ion Ratio 100:70 100:101 100:47 100:45 100:22 100:12 100:65 253.088 180.092 210.110 100:36 256.010 256.129 261.144 261.145 266.120 266.133 275.030 275.104 210.993 167.098 97.097 114.128 107.042 195.104 158.974 230.082 144.949 165.073 55.065 86.099 235.118 194.091 123.990 167.072 100:65 100:48 100:81 100:25 100:57 100:41 100:26 100:54 69 Table 22 Continued Cutoff Name MDPV Cyclobenzaprine EDDP Venlafaxine 7-aminoclonazepam Morphine Pentazocine Desalkylflurazepam Bupivacaine Chlordiazepoxide Metoclopramide Sertraline Zaleplon Zolpidem Benztropine Norpropoxyphene Fluoxetine Methadone Norclozapine Olanzapine Clomipramine Ranitidine delta9-THC Clonazepam Oxycodone Brompheniramine Chlorpromazine Fluvoxamine in ng/mL 50 10 25 50 10 20 50 50 500 50 100 100 25 10 10 50 20 25 50 50 20 500 10 10 20 25 50 20 Parent Product Product Ion MW Ion Ion 1 Ion 2 Ratio 275 276.127 175.086 135.041 100:91 275 276.141 215.092 216.106 100:56 277 278.146 234.128 249.166 100:51 277 278.154 58.083 121.063 100:26 285 286.057 222.134 121.109 100:85 285 286.107 152.057 165.062 100:70 285 286.162 218.155 69.068 100:29 288 289.035 140.015 226.092 100:66 288 289.165 140.126 98.094 100:19 299 300.070 227.044 241.056 100:27 299 300.125 227.057 184.026 100:51 305 306.067 158.971 275.068 100:84 305 306.106 236.088 264.123 100:85 307 308.136 235.158 92.076 100:19 307 308.149 167.074 165.069 100:68 325 308.155 100.064 44.064 100:71 309 310.106 44.069 148.105 100:6 309 310.156 265.166 105.027 100:34 312 313.096 192.062 270.081 100:70 312 313.124 256.070 198.025 100:49 314 315.120 86.087 58.072 100:37 314 315.124 176.061 102.026 100:53 314 315.167 193.130 123.032 100:65 315 316.000 270.156 214.002 100:36 315 316.115 241.094 256.128 100:82 318 319.060 274.026 167.065 100:52 318 319.084 86.090 58.075 100:46 318 319.126 71.047 200.007 100:24 70 Table 23: Using an ESI spray source, targets were analyzed at 1000 ng/mL in solution for the transitions used in the analysis of other targets which share the same parent ion. S:B less than 3:1 indicated that the target would not interfere with the channels scanned for the other target, while a greater signal routed targets through additional interference testing. The following 25 targets were found to have S:B greater than 3:1 for the transitions recorded below Target Present in Neat Solution Table 23 Continued Target whose fragments were SRM scanned for Methylphenidate Normeperdine 324 -> 56 Lamotrigine Diphenhydramine 256 -> 165 315 -> 86 Ranitidine Clomipramine 315 -> 58 310 -> 44 Methadone Fluoxetine 310 ->148 234 -> 84 Normeperidine Methylphenidate 234 ->56 286 -> 94 7-Aminoclonazepam 286 -> 121 Pentazocine 286 -> 152 Morphine 286 -> 165 234 -> 84 Normeperidine Methylphenidate 234 -> 56 AUC Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference 5.4E+03 4.6E+07 8.0E+03 9.6E+06 6.2E+04 2.5E+05 2.6E+04 2.3E+06 6.0E+01 3.8E+03 3.0E+01 1.9E+02 5.6E+03 2.5E+04 3.4E+03 2.5E+07 1.8E+03 1.1E+05 4.2E+03 2.1E+06 1.0E+03 3.2E+06 1.7E+03 1.3E+05 5.4E+03 2.5E+04 3.4E+03 2.5E+07 S:B 8586 1201 4 90 64 6 5 7478 63 497 3059 77 5 7478 71 Target Present in Neat Solution Table 23 Continued Target whose fragments were SRM scanned for 308 -> 92 Benzotropine Zolpidem 308 -> 44 286 -> 94 Morphine 7-Aminoclonazepam 286 -> 121 316 -> 270 Oxycodone Clonazepam 316 -> 214 Norpropoxyphene Benzotropine 308 -> 165 319 ->71 Bromopheniramine Fluvoxamine 319 -> 200 319 -> 274 Brompheniramine 319 -> 167 Chlorpromazine 319 -> 71 Fluvoxamine 319 -> 200 313 -> 256 Norclozapine Olanzapine 313 -> 198 286 -> 152 7-Aminoclonazepam Morphine 286 -> 165 AUC Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference 1.8E+03 9.6E+04 1.5E+03 5.7E+04 9.0E+02 1.3E+05 2.0E+03 6.7E+05 1.4E+04 1.0E+06 2.7E+03 3.3E+05 6.6E+05 3.1E+06 2.8E+05 9.9E+05 6.2E+04 2.2E+05 4.1E+05 2.1E+07 2.2E+05 5.1E+06 2.7E+05 1.9E+06 6.0E+04 3.0E+05 2.8E+02 1.1E+06 7.7E+01 1.8E+04 3.2E+04 1.7E+06 2.5E+04 1.4E+05 S:B 54 38 140 327 74 123 5 3 4 52 23 7 5 3978 229 52 6 72 Target Present in Neat Solution Chlordiazepoxide Table 23 Continued Target whose fragments were SRM scanned for Metoclopramide 300 -> 227 316 -> 241 Clonazepam Oxycodone 316 -> 212 289 -> 140 Desalkylflurazepam Bupivicaine 289 -> 98 276 -> 175 Cyclobenzapirine MDPV 276 -> 135 Cabamazepine, 10-,11-epoxide 253 -> 182 Phenytoin 253 -> 104 315 ->176 Clomipramine Ranitidine 315 -> 102 319 -> 274 Brompheniramine 319 -> 167 Fluvoxamine Chlorpromazine 319 -> 58 266 -> 107 Mirtazapine Nordoxepin 266 ->235 AUC Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference Blank Interference S:B 5.7E+05 300 1.7E+08 1.1E+03 941 1.1E+06 1.1E+03 13 1.4E+04 1.5E+03 11121 1.7E+07 1.4E+03 3 4.7E+03 3.1E+03 11 3.4E+04 3.5E+03 3 1.1E+04 3.5E+02 25237 8.9E+06 3.8E+02 119 4.5E+04 3.6E+03 8 2.9E+04 7.0E+02 36 2.5E+04 2.5E+03 21 5.2E+04 5.2E+03 7 3.5E+04 7.3E+03 5 3.8E+04 1.7E+03 1196 2.0E+06 3.0E+02 47460 1.4E+07 73 Target Present in Neat Solution Table 23 Continued Target whose fragments were SRM scanned for 266 -> 195 Nordoxepin Mirtazapine 266 -> 194 AUC Blank Interference Blank Interference 1.3E+06 1.5E+07 7.0E+05 9.8E+06 In the next round of testing, the 25 targets identified using the ESI spray source were tested using paper spray. Targets sharing the same parent ion were combined at 100ppb in the spray solvent (95:5:0.01 methanol:water:acetic acid) and sprayed over blank paper. All of the fragment ions were scanned and targets ratios were monitored to see if the presence of the interfering compound caused the target’s ratio to vary more than the permitted ±30%. The results of these experiments, found in Table 24, indicate that out of the 20 target pairs/groups, only 9 targets may produce false negatives in the presence of another target that shares a parent ion with it. These targets are: phenytoin, norsertraline, desalkylflurazepam, chlordizepoxide, metoclopramide, zaleplon, benztropine, fluoxetine, and olanzapine. The transitions belonging to methadone were unintentionally left out of this series of tests, and no conclusion is made about the affect of the presence of fluoxetine on methadone at this time. S:B 12 14 74 Table 24 Targets with the same parent ion were each added at 100 ng/mL to the spray solvent and sprayed over blank paper. The fragmentation ion for each target was monitored and those that differed from their established ratio by greater than the permissible deviation of ±30% were flagged, as shown in red. The remaining targets were concluded to not interfere with one another Table 24 continued Primidone 219 162 2.77E+06 91 Ratio Ratio Deviation (%) 1.99E+06 72 2.7 Meprobamate 219 158 1.21E+06 97 1.24E+06 102 1.2 Amobarbital 225 182 1.27E+08 42 6.12E+07 48 0.0 Pentobarbital 225 182 1.27E+08 42 6.12E+07 48 0.1 Normeperidine 234 160 4.80E+07 56 1.22E+07 25 15.3 Methylphenidate 234 84 8.50E+07 56 1.22E+07 14 19.3 Phenytoin Carbamazepine-10,11epoxide Lamotrigine 253 182 2.75E+06 104 5.44E+05 20 -69.6 253 180 1.50E+07 210 5.69E+06 38 5.6 256 211 1.48E+07 145 9.20E+06 62 -4.2 Diphenhydramine 256 167 8.80E+07 165 3.98E+07 45 -5.8 Carisoprodol 261 97 1.56E+06 55 1.31E+06 84 3.7 Ropinirole 261 114 1.31E+08 86 3.03E+07 23 -7.4 Nordoxepin 266 107 6.79E+07 235 4.25E+07 63 9.8 Mirtazapine 266 195 1.85E+08 194 7.06E+07 38 -6.8 Norsertraline 275 159 1.99E+07 124 1.41E+06 7 -72.7 Chlorpheniramine 275 230 1.40E+08 167 7.26E+07 52 -4.3 MDPV 276 175 7.22E+07 135 6.56E+07 91 -0.2 Cyclobenzaprine 276 215 1.83E+08 216 1.01E+08 55 -1.6 EDDP 278 234 2.90E+08 249 1.16E+08 40 -21.7 Venlafaxine 278 58 6.41E+07 121 1.65E+07 26 -1.3 7-aminoclonazepam 286 222 1.13E+07 121 9.02E+06 80 -5.8 Morphine 286 152 6.45E+06 165 3.92E+06 61 -13.1 Pentazocine 286 218 1.08E+10 69 2.46E+09 23 -21.7 Desalkylflurazepam 289 140 2.02E+08 226 4.84E+06 2 -96.4 Bupivacaine 289 140 2.02E+08 98 4.34E+07 21 12.8 Chlordiazepoxide 300 227 2.88E+08 241 3.14E+07 11 -59.7 Metoclopramide 300 227 6.70E+10 184 8.86E+07 0 -99.7 Sertraline 306 159 5.43E+07 275 4.55E+07 84 -0.2 Zaleplon 306 236 9.78E+06 264 5.16E+06 53 -37.9 Name Parent Product Ion Ion 1 Peak Area Product Ion 2 Peak Area 75 Table 24 continued Zolpidem 308 235 3.17E+08 92 Ratio Ratio Deviation (%) 5.71E+07 18 -5.4 Benztropine 308 167 3.92E+08 165 1.81E+08 46 -32.0 Norpropoxyphene 308 100 2.40E+07 44 1.61E+07 67 -5.1 Fluoxetine 310 44 1.83E+07 148 2.32E+06 13 111.8 Methadone 310 265 - 105 7.04E+07 - - Norclozapine 313 192 1.24E+08 270 8.86E+07 72 2.4 Olanzapine 313 256 1.99E+07 198 5.39E+06 27 -44.7 Clomipramine 315 86 1.06E+08 58 3.78E+07 36 -3.3 Ranitidine 315 176 4.24E+07 102 2.22E+07 52 -1.3 Clonazepam 316 270 1.19E+07 214 3.83E+06 32 -10.8 Oxycodone 316 241 1.53E+07 256 1.15E+07 75 -8.3 Brompheniramine 319 274 1.29E+08 167 6.25E+07 48 -7.1 Chlorpromazine 319 86 1.20E+08 58 5.35E+07 45 -2.9 Fluvoxamine 319 71 2.57E+07 200 5.51E+06 21 -10.6 Name Parent Product Ion Ion 1 6.3 Peak Area Product Ion 2 Peak Area Conclusions and Future Work Using database searches, 540 compounds were found who could potentially interfere with the target analytes of this study. They would only be true interferences though, if they exist at detectable concentrations, ionize via protonation and produce a daughter fragment with the same mass to charge ratio as one that is monitored for one of the analytical targets. Nine targets were identified who could interfere with other targets and provide false negatives by skewing the fragmentation ratios outside of the permitted ±30% deviation. Carbamazepine-10,11-epoxide was identified as a potential interference for phenytoin, and chlorphenirameine was identified as a potential interferences for norsertraline. Bupivacaine was identified as a potential interference to desalkylflurazepam, which is to be assumed to be a true interference since both analytes monitor the fragment with m/z 140. Likewise, chlordiazepoxide and metoclopramide interfere with one another because they both share the fragment with m/z 227. Sertraline was identified as a potential 76 interference for zaleplon, and methadone was identified as a potential interference to fluoxetine. Finally, norclozapine was identified as a potential interference to the detection of olanzapine. A final set of experiments needs to be run to confirm the effect of intra-target interferences. The nine analytes that were identified as potential interferences in neat solution need to be analyzed in blood along with the targets that seem to cause their fragmentation ratios to skew. The extraction efficiencies and the ionization suppression will likely be different for each target, so the targets should be run together with the interfering compound at a very high concentration to test for a “worst case scenario,” and they should also be run at the cutoff levels for a more realistic assessment of the extent to which they may interfere. 77 7 CONCLUSIONS Drug screening is a necessary tool in postmortem investigations that focuses analytical efforts, time, and resources. Currently, drug screening itself can consume huge amounts of time and money in sample clean-up and analysis, and adding newly emerging compounds can be cumbersome. Paper Spray Mass Spectrometry provides an alternative method for drug screening that drastically simplifies the screening process, allowing for rapid detection of a wide variety of drugs. While paper spray analysis has historically been applied to a small focused panel of targets, by adapting existing techniques, it has been found to be suitable to use in large-panel screening as well. 7.1 Summary of Conclusions Two methods were developed during this project: the first was a manually run method that used hand-cut paper. By detecting two daughter fragments and by monitoring the ratio between their relative abundancies, this method was able to detect 114 drugs and metabolites at toxicologically relevant concentrations in blood. Using this method, screening the entire panel of drugs can be accomplished in three sixty second runs, requiring only 10.5 µL of blood. Screening these 114 targets, this “manual” method would require approximately 9 minutes total analysis time. In order to really make paper spray a viable alternative, a second, more automated and commercially available method was developed as well. The second method used commercially available disposable cartridges on an automated ionization source. Under the same detection criteria used in the manually run experiments, this method was able to successfully detect a panel of 100 drugs and metabolites at toxicologically relevant concentrations in blood. This method would also require 3 replicate dried blood spot samples due to the time needed for the mass spectrometer to scan through all of the SRM channels. Because of the larger paper size, this method uses almost four times as much blood as the manual method, but the total volume of blood required is still less than 50 µL. The automated method has the same analytical run time 78 as the manual method, however, there is very little down time between samples since the cartridges do not have to be cleaned, so the entire screening process can be accomplished in about 4 minutes. In addition to the successively developed methods, a list of potentially interfering compounds was compiled that can inform future data interpretation. Within the analytes on the panel tested, 9 were identified that could potentially interfere with one another and lead to false negatives if both a present in a sample. 7.2 Discussion The target levels of detection used in developing these methods were based on the lower limit of quantitation for methods used at a local toxicology laboratory. While the level of detection for paper spray was found to be higher than the LLOQ presented by the toxicology laboratory in some instances, other times targets were run at lower concentrations than the reported LLOQ’s. In the instances where PS-MS was not sufficiently sensitive, it will be important to determine if the LOD is at a toxicologically relevant concentration; in the cases where it is more sensitive, paper spray could help properly route samples into confirmatory testing which could potentially be false negatives when analyzed using other methods. The concentrations, whether run at proposed cutoff levels or experimental LOD’s, used during analysis were established in method development using the manual method. The higher number of target analytes that were ultimately able to be detected in the manual method vs the automated method using the laser-cut paper suggest room for further method optimization and development for the automated method. Although only about half as many targets were analyzed on die-cut paper as laser-cut paper, the percent of targets detected on the die-cut paper (87%) is much more congruent with the manual method (86%) than the laser-cut paper is (76%). It is possible that the laser cutting approach used during manufacturing requires some refinement. If this is the case, a new LOD may need to be established using the laser-cut paper, especially in the 11 cases where a target was detected using the manual method, but not using the automated 79 method with the laser-cut paper. Revisiting blood spot placement and dilution could also prove advantageous for future experiments run on the laser-cut paper. In this project, 14 of the originally proposed analytical targets were not included because preliminary tests showed that they only ionized well in negative mode and would require further optimization in order to reach acceptable limits of detection. This work was completed in a separate project, by McKenna et. al. where an optimized solvent of 90:10:0.01 MeOH:CCl4:NH4OH was used to successfully detect all 14 targets, 4 of which were determined to have LOD’s slightly higher than the purposed screening cutoff concentrations.67 This work was done on an exact mass instrument, but would be translated to a triple quadrupole instrument. 7.3 Future work The next set of experiments that need to be run are establishing calibration curves for each of the analytes. This will establish an experimental LOD and can help inform acceptance criteria. One of the largest hurdles faced in this project was establishing a permissible ratio tolerance. Ratio tolerances are always discussed in the context of chromatography, and usually at concentrations well above the LOD. Since no intentional chromatography is performed during PS and this project deals with some analytes near or at their LOD, running calibration curves will help establish the robustness of fragment ion ratios in a context that has yet to be established. This could lead to justifying either broadening or constricting the current ±30% permissible deviation, or even a ratio tolerance that is tied to signal to blank ratio. To run a set of calibration curves, internal standards (IS) will need to be chosen. Since it is impractical to use a stable-labeled internal standard for every target, work will need to be done to find a reasonable number of analogue standards that can act as internal standards for the entire panel of targets. Ideally, these IS’s could be cocktailed together and spiked directly into a sample. In addition to running calibration curves, a final blinded study of postmortem samples should be run to determine the effectiveness of the assay on true postmortem samples. 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In Press APPENDIX A Purposed Analytical Targets with Cutoff Values from AFT Target Drug Class Target Screening Cutoff (ng/mL) Acetaminophen Analgesic 20000 Alfentanil Narcotic 50 Alpha-PVP Stimulant 50 Alprazolam Benzodiazepine 5 Amitriptyline Antidepressant 20 Amlodipine Cardiovascular 20 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) Amobarbital Barbiturate 500 Amphetamine Amphetamine 50 Aripiprazole Antipsychotic 50 Atenolol Cardiovascular 100 Baclofen Analgesic 1000 Benzoylecgonine Cocaine 50 Benztropine Neurological 10 Benzylpiperazine Miscellaneous 50 Brompheniramine Antihistamine 25 Bupivacaine Anesthetic 500 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) Buprenorphine Analgesic 1 Bupropion Antidepressant 50 Buspirone Antipsychotic 10 Butabarbital Barbiturate 500 Butalbital Barbiturate 500 Carbamazepine Anticonvulsant 1000 Carbamazepine-10,11epoxide Anticonvulsant 1000 Carisoprodol Analgesic 2000 Chlordiazepoxide Benzodiazepine 50 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) Chlorpheniramine Antihistamine 25 Chlorpromazine Antipsychotic 50 Citalopram Antidepressant 10 Clomipramine Antidepressant 20 Clonazepam Benzodiazepine 10 Clozapine Antipsychotic 50 Cocaethylene Cocaine 50 Cocaine Cocaine 50 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) Codeine Opiate 20 Cyclobenzaprine Analgesic 10 delta9-THC Cannabinoid 10 Demoxepam Benzodiazepine 50 Desalkylflurazepam Benzodiazepine 50 Desipramine Antidepressant 20 Desmethylclomipramine Antidepressant 20 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) Dextromethorphan Narcotic 10 Diazepam Benzodiazepine 50 Diltiazem Cardiovascular 50 Diphenhydramine Antihistamine 25 Donepezil Neurological 50 Doxepin Antidepressant 20 Doxylamine Antihistamine 25 Duloxetine Antidepressant 20 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) EDDP Methadone 25 Ephedrine/Pseudoephedrine Amphetamine 50 Etomidate Anesthetic 100 Felbamate Anticonvulsant 10000 Fentanyl Fentanyl 1 Flecainide Cardiovascular 500 Flunitrazepam Sedative/Hypnotic 20 Fluoxetine Antidepressant 20 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) Flurazepam Benzodiazepine 25 Fluvoxamine Antidepressant 20 Furosemide Cardiovascular 1000 Gabapentin Anticonvulsant 500 Haloperidol Antipsychotic 10 Hydrochlorothiazide Cardiovascular 100 hydrocodone Opiate 20 hydromorphone Opiate 20 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) Hydroxychloroquine Analgesic 2000 Hydroxyzine Antihistamine 10 Ibuprofen Analgesic 10000 Ketamine Anesthetic 100 Labetalol Cardiovascular 50 Lamotrigine Anticonvulsant 1000 Levetiracetam Anticonvulsant 2000 Lidocaine Anesthetic 500 Lorazepam Benzodiazepine 25 mCPP Antidepressant 20 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) MDA Amphetamine 50 MDMA Amphetamine 50 MDPV Stimulant 50 Meperidine Narcotic 25 Mephedrone Stimulant 50 Meprobamate Analgesic 2000 Mescaline Miscellaneous 50 Metaxalone Analgesic 1000 Methadone Methadone 25 Methamphetamine Amphetamine 50 Methocarbamol Analgesic 1000 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) Methylone Stimulant 50 Methylphenidate Stimulant 20 Metoclopramide Gastrointestinal 100 Metoprolol Cardiovascular 50 Midazolam Anesthetic 50 Mirtazapine Antidepressant 50 Morphine Opiate 20 Naproxen Analgesic 30000 Norbuprenorphine Analgesic 1 Norclozapine Antipsychotic 50 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) Nordiazepam Benzodiazepine 50 Nordoxepin Antidepressant 20 Norfluoxetine Antidepressant 20 Norketamine Anesthetic 100 Normeperidine Narcotic 25 Norpropoxyphene Propoxyphene 50 Norsertraline Antidepressant 100 Nortramadol Analgesic 100 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) Nortriptyline Antidepressant 20 Norvenlafaxine Antidepressant 50 Olanzapine Antipsychotic 50 Oxazepam Benzodiazepine 50 Oxycodone Opiate 20 Oxymorphone Opiate 20 Papaverine Cardiovascular 500 Paroxetine Antidepressant 20 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) PCP Phencyclidine 25 Pentazocine Narcotics 50 Pentobarbital Barbiturate 500 Phenobarbital Barbiturate 2000 Phenytoin Anticonvulsant 5000 Pregabalin Anticonvulsant 500 Primidone Anticonvulsant 5000 Promethazine Sedative/Hypnotic 25 Propoxyphene Propoxyphene 50 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) Propranolol Cardiovascular 50 Quetiapine Antipsychotic 50 Ranitidine Gastrointestinal 500 Risperidone Antipsychotic 10 Ropinirole Neurological 10 Salicylic Acid Analgesic 9000 Secobarbital Barbiturate 500 Sertraline Antidepressant 100 Sildenafil Urological 100 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) Tadalafil Urological 100 Temazepam Benzodiazepine 50 TFMPP Miscellaneous 50 Thiopental Sedative/Hypnotic 2000 Topiramate Anticonvulsant 2000 Tramadol Analgesic 100 Trazodone Antidepressant 100 Triazolam Benzodiazepine 20 Trimipramine Antidepressant 20 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) Valproic Acid Anticonvulsant 5000 Vardenafil Urological 100 Venlafaxine Antidepressant 50 Verapamil Cardiovascular 50 Warfarin Cardiovascular 500 Zaleplon Sedative/Hypnotic 25 Ziprasidone Antipsychotic 10 Zolpidem Sedative/Hypnotic 10 Zonisamide Anticonvulsant 10000 Chemical Structure Target Drug Class Target Screening Cutoff (ng/mL) Zopiclone Sedative/Hypnotic 10 10Anticonvulsant monohydroxyoxcarbazepine 1000 11-nor-9-Carboxy-THC Cannabinoid 10 6-acetylmorphine Opiate 20 7-aminoclonazepam Benzodiazepine 10 7-aminoflunitrazepam Sedative/Hypnotic 20 9-hydroxyrisperidone Antipsychotic 10 Chemical Structure 105 APPENDIX B Detailed results from loading capacity and blood dilution study for the five targets analyzed in Chapter 4. Morphine Whole Blood 40 uL 20 uL 10 uL 5 uL Fragment Diluted Blood 286 -> 152 Whole Blood Diluted Blood 286 -> 165 Whole Blood Diluted Blood 286 -> 201 Avg Blank 7.7E+03 5.6E+03 4.8E+03 4.7E+03 3.1E+05 3.7E+05 Stdev Avg Drug Stdev 4.3E+03 6.0E+04 6.5E+03 1.0E+03 7.5E+04 7.6E+03 2.5E+03 4.1E+04 3.6E+03 5.3E+02 5.3E+04 4.8E+03 7.2E+04 3.9E+05 8.3E+04 4.4E+04 4.1E+05 1.5E+04 S:B 7.8 14 8.4 11 1.3 1.1 Avg Blank Stdev Avg Drug Stdev 8.5E+03 6.7E+03 5.6E+04 3.1E+03 4.1E+03 2.6E+03 6.5E+04 2.1E+03 4.9E+03 3.3E+03 4.0E+04 1.8E+03 2.4E+03 1.3E+03 4.7E+04 1.7E+03 5.0E+05 7.3E+04 4.5E+05 1.1E+05 2.5E+05 1.7E+04 3.5E+05 5.7E+04 S:B 6.6 16 8.1 20 0.90 1.4 Avg Blank 3.4E+03 8.7E+02 2.0E+03 6.3E+02 2.0E+03 1.2E+05 Stdev 1.2E+03 4.3E+02 6.2E+02 3.4E+02 6.2E+02 4.6E+04 Avg Drug Stdev 4.6E+04 1.4E+04 5.3E+04 1.0E+04 3.2E+04 9.1E+03 3.7E+04 7.2E+03 3.2E+04 9.1E+03 2.6E+05 7.4E+04 S:B 13 61 16 9 16 2.3 Avg Blank Stdev Avg Drug Stdev 3.9E+03 3.5E+03 5.4E+04 6.5E+03 8.8E+02 1.3E+02 3.7E+04 6.2E+02 2.6E+03 2.2E+03 3.8E+04 4.4E+03 6.8E+02 2.3E+02 2.6E+04 3.2E+02 3.7E+05 7.5E+04 3.3E+05 2.8E+04 9.8E+04 3.3E+04 2.1E+05 5.9E+04 S:B 14 42 15 38 0.89 2.2 106 Zolpidem Whole Blood 40 uL 20 uL 10 uL 5 uL Fragment Diluted Blood 308 -> 92 Whole Blood Diluted Blood 308 -> 235 Avg Blank Stdev Avg Drug Stdev 2.9E+04 1.3E+04 1.7E+05 1.3E+04 1.7E+04 5.8E+03 1.3E+05 1.9E+04 1.3E+05 6.4E+04 8.9E+05 6.7E+04 7.2E+04 2.9E+04 6.7E+05 9.3E+04 S:B 6.0 8.0 6.7 9.3 Avg Blank Stdev Avg Drug 1.3E+04 9.5E+03 2.0E+05 8.0E+03 3.6E+03 1.4E+05 4.4E+04 3.5E+04 1.1E+06 3.2E+04 1.6E+04 7.3E+05 Stdev 1.3E+04 1.3E+04 8.2E+04 8.1E+04 S:B 15 17 24 23 Avg Blank 9.6E+03 2.9E+03 4.2E+04 1.2E+04 Stdev 3.8E+03 1.2E+03 1.8E+04 5.3E+03 Avg Drug Stdev 1.6E+05 5.4E+04 1.0E+05 1.4E+04 8.5E+05 2.9E+05 5.5E+05 7.3E+04 S:B 17 36 20 44 Avg Blank Stdev Avg Drug 1.6E+04 1.5E+04 1.8E+05 4.0E+03 3.7E+02 6.7E+04 7.9E+04 7.3E+04 9.6E+05 2.0E+04 4.5E+02 3.5E+05 Stdev 2.0E+04 2.7E+03 1.0E+05 1.3E+04 S:B 11 17 12 18 107 Fentanyl Whole Blood 40 uL 20 uL 10 uL 5 uL Fragment Diluted Blood 337 -> 79 Whole Blood Diluted Blood 337 -> 105 Whole Blood Diluted Blood 337 -> 188 Avg Blank Stdev 3.4E+04 1.7E+04 2.6E+04 2.8E+03 4.7E+04 2.1E+04 3.4E+04 3.0E+03 2.5E+04 1.0E+04 1.3E+04 5.1E+03 Avg Drug Stdev 7.6E+04 1.7E+04 8.1E+04 1.5E+04 1.8E+05 2.7E+04 1.5E+05 1.2E+04 1.5E+05 2.0E+04 1.0E+05 1.8E+03 S:B 2.3 3.1 3.9 4.5 6.0 7.8 Avg Blank Stdev Avg Drug Stdev 4.2E+04 2.6E+04 7.5E+04 6.1E+03 1.9E+04 7.1E+03 5.9E+04 8.6E+03 3.9E+04 2.4E+04 2.4E+05 2.3E+04 1.9E+04 7.5E+03 1.4E+05 7.0E+03 7.8E+03 5.4E+03 2.2E+05 2.5E+04 5.2E+03 2.3E+03 1.1E+05 1.6E+04 S:B 1.8 3.2 6.2 7.6 28 22 Avg Blank 1.6E+04 4.6E+03 1.6E+04 5.8E+03 6.0E+03 2.8E+03 Stdev 2.1E+03 2.7E+03 3.5E+03 2.8E+03 2.1E+03 1.3E+03 Avg Drug 6.4E+04 4.4E+04 6.4E+04 1.3E+05 1.6E+05 1.1E+05 Stdev 1.8E+04 6.7E+03 1.8E+04 1.3E+04 5.0E+04 1.1E+04 S:B 4.0 9.4 3.9 22 27 38 Avg Blank Stdev Avg Drug Stdev 1.5E+04 1.0E+04 6.7E+04 3.3E+03 3.6E+03 4.8E+02 2.9E+04 2.9E+02 1.9E+04 1.5E+04 2.4E+05 2.1E+04 6.6E+03 5.6E+01 9.4E+04 3.8E+03 1.1E+04 9.0E+03 2.1E+05 2.2E+04 5.0E+03 5.0E+02 8.7E+04 2.2E+03 S:B 4.6 8.0 12 14 19 17 5 uL 10 uL 20 uL 40 uL 4.7 6.5E+2 2.3E+2 3.1E+3 2.9E+2 4.7 1.7E+2 7.9E+1 2.1E+3 4.9E+2 12 1.8E+2 4.2E+1 9.0E+2 2.1E+2 5.0 3.9 1.0E+3 5.2E+02 3.2E+3 3.2E+2 3.1 5.5E+2 1.3E+2 2.5E+3 9.1E+2 4.6 4.6E+2 3.1E+2 2.5E+3 5.6E+2 5.4 Avg Blank Stdev Avg Drug Stdev S:B Avg Blank Stdev Avg Drug Stdev S:B Avg Blank Stdev Avg Drug Stdev S:B Avg Blank Stdev Avg Drug Stdev S:B 0.99 5.6E+3 4.3E+2 5.6E+3 4.0E+2 1.5 7.2E+03 2.0E+03 3.5E+2 5.0E+3 1.4 8.3E+3 9.6E+1 6.1E+3 1.3E+3 1.3 7.0E+3 1.1E+3 8.9E+3 7.7E+2 8.5E+2 7.1E+1 4.0E+3 3.2E+2 1.0E+3 4.2E+2 3.8E+3 8.2E+2 Fragment 0.95 5.7E+3 4.2E+2 5.3E+3 1.5E+3 1.9 8.9E+3 7.3E+2 7.3E+2 4.5E+3 1.35 1.1E+4 6.1E+2 8.0E+3 1.1E+3 1.4 6.4E+3 4.3E+2 9.1E+3 8.9E+2 Whole Diluted Blood Blood 468-> 187 Whole Diluted Blood Blood 468-> 101 13 4.3E+2 3.3E+2 5.5E+3 1.3E+3 12 5.3E+3 1.7E+3 2.2E+1 4.3E+2 6.7 6.8E+3 1.2E+3 1.0E+3 5.1E+2 6.9 9.7E+2 4.5E+2 6.7E+3 1.6E+3 30 7.3E+1 9.4E+0 2.2E+3 3.6E+2 36 4.4E+3 1.5E+3 6.2E+1 1.2E+2 11 5.0E+3 2.3E+2 4.5E+2 1.6E+2 8.9 9.1E+2 8.2E+1 8.1E+3 1.3E+3 Whole Diluted Blood Blood 468-> 396 Buprenorphine 17 3.0E+2 2.2E+2 5.3E+3 1.4E+3 22 4.6E+3 1.8E+3 5.2E+1 2.1E+2 5.5 6.0E+3 1.1E+3 1.1E+3 6.5E+2 6.2 9.8E+2 5.2E+2 6.1E+3 1.2E+3 30 6.4E+1 1.1E+1 1.9E+3 4.1E+2 84 4.0E+3 1.2E+3 1.9E+1 4.8E+1 13 4.7E+3 1.2E+2 3.6E+2 1.8E+2 6.3 9.9E+2 3.1E+1 6.2E+3 3.8E+2 Whole Diluted Blood Blood 468-> 414 108 5 uL 10 uL 20 uL 40 uL Diluted Blood 7.4E+03 4.8E+03 1.5E+04 7.0E+03 2.0 1.2E+04 9.0E+03 4.9E+03 1.6E+03 0.42 4.0E+03 1.1E+03 5.6E+03 2.6E+03 1.4 2.3E+03 1.7E+03 2.7E+03 8.6E+02 1.2 S:B Avg Blank Stdev Avg Drug Stdev S:B Avg Blank Stdev Avg Drug Stdev S:B Avg Blank Stdev Avg Drug Stdev S:B 3.0 4.0E+02 5.2E+02 2.0E+02 1.6E+03 4.3 1.0E+03 7.4E+02 4.5E+03 1.2E+03 3.4 1.4E+04 7.2E+03 4.1E+03 2.6E+03 4.6 4.8E+03 9.6E+02 2.2E+04 6.1E+03 316 -> 151 Avg Blank Stdev Avg Drug Stdev Fragment Whole Blood Diluted Blood 2.7 1.0E+03 1.0E+03 7.6E+02 2.7E+03 5.1 9.2E+02 3.8E+02 4.7E+03 1.9E+03 1.5 4.3E+03 1.4E+03 2.9E+03 2.1E+03 5.4 1.9E+03 1.3E+03 1.0E+04 3.4E+03 7.8 2.7E+02 1.5E+02 6.8E+01 1.2E+03 18 2.1E+02 1.2E+02 3.7E+03 1.2E+03 8.0 8.1E+03 3.3E+03 1.0E+03 5.5E+02 7.1 2.1E+03 2.4E+02 1.5E+04 1.6E+03 316 -> 214 Whole Blood Diluted Blood 3.6 7.0E+02 5.1E+02 4.5E+02 1.8E+03 8.1 3.6E+02 1.3E+02 2.9E+03 1.2E+03 2.0 2.7E+03 7.8E+02 1.4E+03 1.0E+03 7.3 8.9E+02 6.8E+02 6.5E+03 2.3E+03 19 2.0E+02 3.6E+01 2.0E+01 6.6E+02 27 8.7E+01 5.3E+01 2.4E+03 7.6E+02 12 5.5E+03 2.2E+03 4.7E+02 2.4E+02 14 6.6E+02 7.8E+01 9.3E+03 1.2E+03 316 -> 241 Whole Blood Clonazepam Diluted Blood 1.4 2.4E+03 8.0E+03 4.4E+03 1.1E+04 2.0 1.0E+04 1.9E+03 2.0E+04 6.5E+03 0.75 1.8E+04 4.5E+03 2.5E+04 1.5E+04 2.2 1.9E+04 1.1E+04 4.2E+04 1.4E+04 2.5 1.5E+03 2.4E+03 7.3E+02 6.0E+03 4.8 3.4E+03 1.7E+03 1.6E+04 4.4E+03 3.1 3.5E+04 1.4E+04 1.1E+04 4.3E+03 3.5 1.6E+04 9.6E+02 5.7E+04 1.2E+04 316 -> 270 Whole Blood 109 110 APPENDIX C Mass spec parameters for purposed targets after tuning on triple quadrupole Target Q1 Q3 CE S lens Ion ratio Acetaminophen 152 110 16 59 100:30 65 30 268 17 95 100:62 197 26 91 24 69 100:41 77 43 205 41 111 100:87 281 25 233 16 81 100:61 117 30 238 11 58 100:67 294 11 91 17 34 100:25 119 6 285 25 146 100:28 176 31 145 26 85 100:78 190 18 151 18 52 100:88 115 45 167 29 106 100:45 165 50 168 19 85 100:33 77 48 91 22 59 100:12 85 16 274 19 62 100:47 167 41 140 20 73 100:17 98 34 Alfentanil Alpha-PVP Alprazolam Amitriptyline Amlodipine Amphetamine Aripiprazole Atenolol Baclofen Benztropine Benzoylecgonine Benzylpiperazine Brompheniramine Bupivacaine 417 232 309 278 409 136 448 267 214 308 290 177 319 289 111 Target Q1 Q3 CE S lens Ion ratio Bupropion 240 184 12 53 100:55 131 27 122 31 127 100:18 95 50 194 19 82 100:30 192 23 180 30 58 100:36 210 15 97 16 42 100:83 55 29 227 25 75 100:22 255 20 230 17 58 100:53 167 41 214 39 78 100:26 58 35 262 19 95 100:47 234 28 227 40 75 100:73 242 26 270 24 136 100:32 214 36 270 22 88 100:64 192 43 196 19 78 100:27 82 32 182 19 94 100:21 82 31 215 25 97 100:61 199 30 215 40 87 100:39 231 17 180 22 113 100:65 207 36 Buspirone Carbamazepine Carbamazepine-10,11-epoxide Carisoprodol Chlordiazepoxide Chlorpheniramine Chlorpromazine Citalopram Clomipramine Clonazepam Clozapine Cocaethylene Cocaine Codeine Cyclobenzaprine Demoxepam 386 237 253 261 300 275 319 325 315 316 327 318 304 300 276 287 112 Target Q1 Q3 CE S lens Ion ratio Desalkylflurazepam 289 140 29 91 100:66 226 27 72 16 68 100:20 193 38 72 17 73 100:17 227 37 215 23 107 100:76 147 30 154 26 94 100:81 222 26 178 24 94 100:44 150 41 167 15 46 100:43 165 39 91 36 112 100:16 243 26 107 23 89 100:52 165 56 167 34 54 100:88 182 16 44 14 41 100:87 154 5 234 31 89 100:42 249 23 115 27 44 100:86 117 19 141 10 45 100:66 95 24 117 16 40 100:64 178 5 188 22 104 100:70 105 35 301 32 127 100:38 98 29 Desipramine Desmethylclomipramine Dextromethorphan Diazepam Diltiazem Diphenhydramine Donepezil Doxepin Doxylamine Duloxetine EDDP Ephedrine/Pseudoephedrine Etomidate Felbamate Fentanyl Flecainide 267 301 272 285 415 256 380 280 271 298 278 166 245 239 337 415 113 Target Q1 Q3 CE S lens Ion ratio Flunitrazepam 314 268 25 91 100:38 239 33 44 14 56 100:13 148 6 315 22 90 100:22 317 18 71 16 67 100:21 200 21 137 15 51 100:32 119 17 123 37 101 100:93 165 23 199 29 112 100:37 171 38 185 30 97 100:69 157 40 201 19 71 100:86 165 61 89 54 62 100:24 125 30 162 25 71 100:87 91 36 211 26 104 100:63 145 38 126 15 33 100:24 154 5 86 18 66 100:12 58 35 276 18 65 100:48 167 41 154 19 71 100:89 118 34 135 19 39 100:91 133 18 Fluoxetine Flurazepam Fluvoxamine Gabapentin Haloperidol Hydrocodone Hydromorphone Hydroxyzine Ketamine Labetalol Lamotrigine Levetiracetam Lidocaine Lorazepam mCPP MDA 310 388 319 172 376 300 286 375 238 329 256 171 235 321 197 180 114 Target Q1 Q3 CE S lens Ion ratio MDMA 194 163 12 48 100:35 105 24 175 22 87 100:89 135 27 220 21 81 100:58 174 20 145 20 54 100:76 144 31 158 5 39 100:81 97 14 180 18 44 100:81 165 24 161 11 57 100:26 105 27 265 14 77 100:32 105 29 91 19 50 100:39 119 11 118 11 47 100:23 199 5 160 17 59 100:52 132 27 84 20 69 100:13 56 41 227 18 75 100:39 184 31 116 18 83 100:68 77 52 291 26 98 100:27 249 37 195 26 74 100:41 194 42 152 60 84 100:71 165 40 MDPV Meperidine Mephedrone Meprobamate Mescaline Metaxalone Methadone Methamphetamine Methocarbamol Methylone Methylphenidate Metoclopramide Metoprolol Midazolam Mirtazapine Morphine 276 248 178 219 212 222 310 150 242 208 234 300 268 326 266 286 115 Target Q1 Q3 CE S lens Ion ratio Naproxen 231 185 14 63 100:30 170 26 192 41 90 100:73 270 24 140 28 99 100:68 208 27 107 22 76 100:60 235 15 134 5 43 100:26 30 13 125 25 54 100:44 179 16 160 16 64 100:22 56 23 44 20 60 100:48 128 43 159 18 77 100:11 124 41 232 5 48 100:25 42 53 233 14 77 100:42 117 20 58 19 69 100:24 107 34 256 22 96 100:27 198 41 241 22 88 100:15 77 51 241 28 86 100:63 212 41 227 28 84 100:73 198 43 324 30 114 100:101 202 26 Norclozapine Nordiazepam Nordoxepin Norfluoxetine Norketamine Normeperidine Norpropoxyphene Norsertraline Nortramadol Nortriptyline Norvenlafaxine Olanzapine Oxazepam Oxycodone Oxymorphone Papaverine 313 271 266 296 224 234 308 275 250 264 264 313 287 316 302 340 116 Target Q1 Q3 CE S lens Ion ratio Paroxetine 330 192 20 88 100:52 70 30 91 34 43 100:56 159 13 218 20 83 100:22 69 26 182 18 63 100:61 104 34 97 23 50 100:21 124 15 162 13 51 100:70 91 32 86 17 60 100:43 198 27 266 5 50 100:20 128 45 116 18 76 100:88 183 18 253 22 97 100:64 221 36 176 17 77 100:49 102 34 191 28 113 100:8 110 47 114 19 77 100:21 132 33 159 29 50 100:85 275 12 100 29 158 100:62 283 38 177 38 87 100:32 239 45 188 22 76 100:23 118 39 PCP Pentazocine Phenytoin Pregabalin Primidone Promethazine Propoxyphene Propranolol Quetiapine Ranitidine Risperidone Ropinirole Sertraline Sildenafil Temazepam TFMPP 244 286 253 160 219 285 340 260 384 315 411 261 306 475 301 231 117 Target Q1 Q3 CE S lens Ion ratio Topiramate 362 265 18 93 100:52 207 18 264 15 60 100:30 58 15 176 23 101 100:84 148 33 308 26 146 100:76 239 41 193 41 74 100:71 208 25 151 47 152 100:43 312 37 58 18 61 100:25 121 32 165 28 115 100:36 150 37 236 26 97 100:88 264 22 194 27 115 100:37 159 40 92 51 103 100:18 235 33 132 15 61 100:46 77 33 245 16 59 100:39 112 54 165 38 100 100:57 211 25 121 30 106 100:36 94 40 135 27 100 100:51 227 25 207 26 108 100:22 110 40 Tramadol Trazodone Triazolam Trimipramine Vardenafil Venlafaxine Verapamil Zaleplon Ziprasidone Zolpidem Zonisamide Zopiclone 6-acetylmorphine 7-aminoclonazepam 7-aminoflunitrazepam 9-hydroxyrisperidone 264 372 343 295 489 278 455 306 413 308 213 389 328 286 284 427 118 APPENDIX D Spiking solutions A-C were made by combining stock solutions into 95:5:0.01 methanol:water:acetic acid. Spiking solutions D-J were made by combining stock solutions into 95:5 methanol:water at 200x cutoff and then diluting 1:10 with 95:5:0.01 methanol:water:acetic acid. Solution K was made by combining stock solutions into 95:5 methanol:water at 2000x cutoff and then diluting 1:100 with 95:5:0.01 methanol:water:acetic acid. Solution A Solution B Acetaminophen•† Carisoprodol Felbamate Levetiracetam• Naproxen† Norsertraline† Phenytoin† Baclofen Carbamazepine• Carbamazepine-10,11-epoxide Lamotrigine Meprobamate Metaxalone Methocarbamol• Primidone Topiramate† Tramadol† Zonisamide† Nortramadol† 119 Solution C Solution D Amphetamine† Benzylpiperazine† Bupivacaine• Flecainide Gabapentin Lidocaine Papaverine• Pregabalin Aripiprazole•† Desmethylclomipramine† Etomidate• Ketamine MDA† Mescaline† Metoclopramide• Norketamine Ranitidine• Oxycodone† Sertraline Sildenafil Trazodone Vardenafil Ziprasidone† Solution E Solution F Demoxepam† Ephedrine/Pseudoephedrine Hydroxyzine•† Norpropoxyphene Norvenlafaxine Olanzapine Pentazocine Donepezil• Labetalol• MDMA MDPV Mephedrone Methamphetamine Methylone Propoxyphene Propranolol Quetiapine Metoprolol Midazolam Mirtazapine TFMPP Venlafaxine Verapamil Norclozapine 120 Solution G Solution H 6-acetylmorphine† 7-aminoclonazepam† Alfentanil Alpha-PVP Bupropion Chlordiazepoxide Chlorpromazine Clozapine Brompheniramine Diphenhydramine Doxylamine EDDP Flurazepam Lorazepam Meperidine Normeperidine Cocaethylene Desalkylflurazepam Diltiazem PCP Promethazine Solution I Solution J Buspirone† Chlorpheniramine Clonazepam† Fluvoxamine•† 7-aminoflunitrazepam Amlodipine• Benztropine• Clomipramine Methadone Morphine† Oxymorphone† Paroxetine† Zaleplon Zopiclone† Desipramine Duloxetine Flunitrazepam Fluoxetine mCPP Methylphenidate Nordoxepin Norfluoxetine Triazolam Trimipramine 121 Solution K Solution L 9-hydroxyrisperidone Alprazolam Cyclobenzaprine Dextromethorphan Fentanyl† Haloperidol Risperidone Ropinirole• Amitriptyline Atenolol Benzoylecgonine Diazepam Hydrocodone Nortriptyline Zolpidem Solution M Citalopram Cocaine Codeine Doxepine Hydromorphone Nordiazepam Oxazepam Temazepam • Original solutions made by dissolving powdered Sigma reagents † Target run at a concentration other than that specified by AFT 122 APPENDIX E Detailed data by cocktail Acetaminophen Cocktail A Spiked Average m/z 110 m/z 65 1.02E+07 2.87E+06 19% 19% 3.49E+05 1.16E+05 173% 144% 1.75E+07 4.96E+06 49% 51% 1.26E+05 8.96E+04 29% 27% n 4 Spiked RSD Manual Blank Average 4 Blank RSD Spiked Average 4 Spiked RSD Laser-cut Blank Average 4 Blank RSD Levetiracetam Cocktail A Spiked Average m/z 126 m/z 154 9.37E+05 2.40E+05 10% 9% 4.01E+04 1.21E+04 167% 143% 3.78E+06 9.94E+05 43% 47% 7.95E+04 1.17E+04 42% 37% n 4 Spiked RSD Manual Blank Average 4 Blank RSD Spiked Average 4 Spiked RSD Laser-cut Blank Average 4 Blank RSD 123 Zonisamide Cocktail A Spiked Average m/z 132 m/z 77 4.54E+06 2.19E+06 23% 23% 1.30E+05 1.11E+05 172% 110% 6.52E+06 3.08E+06 59% 59% 9.71E+03 9.83E+04 33% 22% n 4 Spiked RSD Manual Blank Average 4 Blank RSD Spiked Average 4 Spiked RSD Laser-cut Blank Average 4 Blank RSD Primidone Cocktail A Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 162 m/z 91 2.55E+06 1.83E+06 33% 32% 6.32E+04 1.20E+05 172% 86% 3.48E+06 4.09E+06 56% 44% 1.19E+04 2.27E+06 35% 19% n 4 4 4 4 Naproxen Cocktail A Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 185 m/z 170 1.12E+06 3.09E+05 15% 17% 1.66E+05 1.50E+04 58% 105% 3.48E+06 5.42E+05 56% 56% 1.19E+04 7.32E+04 35% 21% n 4 4 4 4 124 Felbamate Cocktail A Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 117 m/z 178 7.17E+05 4.37E+05 22% 23% 5.26E+04 1.17E+04 76% 173% 1.68E+06 1.07E+06 48% 48% 3.24E+04 1.35E+03 18% 35% n 4 4 4 4 Phenytoin Cocktail A Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 182 m/z 104 5.49E+05 3.60E+05 80% 80% 1.29E+04 8.19E+03 121% 95% 2.75E+05 2.10E+05 67% 69% 2.25E+04 3.61E+04 28% 34% n 4 4 4 4 Carisoprodol Cocktail A Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 97 m/z 55 5.46E+05 3.75E+05 37% 29% 1.42E+05 8.43E+04 33% 45% 1.59E+06 1.25E+06 31% 34% 8.38E+04 4.26E+04 22% 22% n 4 4 4 4 125 Tramadol Cocktail A Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 264 m/z 58 1.58E+07 4.50E+07 46% 48% 1.55E+06 1.16E+06 52% 178% 7.75E+07 1.36E+08 18% 17% 3.40E+07 2.64E+04 21% 25% n 4 4 4 4 Norsertraline Cocktail A Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 158 m/z 123 3.43E+06 2.40E+05 41% 43% 1.17E+05 6.87E+03 81% 93% 1.12E+07 7.89E+05 11% 12% 1.68E+05 1.38E+04 19% 37% n 4 4 4 4 126 Meprobamate Cocktail B Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 158 m/z 97 5.77E+05 5.09E+05 20% 20% 2.78E+03 2.05E+04 51% 30% 9.12E+05 7.95E+05 21% 21% 1.84E+03 3.28E+04 31% 18% n 4 4 4 4 Metaxalone Cocktail B Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 161 m/z 105 1.42E+06 3.83E+05 21% 21% 1.20E+04 1.58E+04 29% 32% 1.77E+06 5.04E+05 19% 19% 1.57E+04 5.78E+04 27% 25% n 4 4 4 4 Carbamazepine Cocktail B Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 194 m/z 192 1.09E+07 2.45E+06 14% 14% 6.04E+04 1.32E+04 49% 47% 2.57E+07 5.76E+06 25% 24% 2.90E+04 2.76E+04 35% 38% n 4 4 4 4 127 Methocarbamol Cocktail B Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 118 m/z 199 3.49E+05 7.21E+04 23% 26% 3.24E+03 1.12E+03 30% 29% 6.46E+05 1.33E+05 24% 24% 5.92E+03 1.41E+03 27% 26% n 4 4 4 4 Nortramadol Cocktail B Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 232 m/z 42 4.78E+05 9.98E+04 11% 20% 9.97E+04 2.96E+03 14% 23% 1.29E+06 2.98E+05 16% 16% 2.59E+05 1.44E+04 30% 32% n 4 4 4 4 Carbamazepine-10,11-epoxide Cocktail B Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 180 m/z 210 3.58E+06 1.39E+06 17% 17% 1.45E+04 1.53E+04 51% 27% 1.01E+07 3.86E+06 26% 26% 3.46E+04 2.15E+04 20% 28% n 4 4 4 4 128 Lamotrigine Cocktail B Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 210 m/z 144 2.97E+06 1.85E+06 16% 16% 3.52E+04 2.39E+04 66% 61% 4.53E+06 2.88E+06 16% 16% 2.50E+04 3.47E+04 24% 21% n 4 4 4 4 Baclofen Cocktail B Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 151 m/z 115 2.82E+06 2.37E+06 11% 10% 2.99E+04 3.06E+04 48% 34% 7.84E+05 7.02E+05 32% 31% 2.24E+04 6.51E+04 39% 20% n 4 4 4 4 129 Amphetamine Cocktail C Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 91 m/z 119 2.83E+06 6.61E+05 13% 16% 3.17E+05 2.99E+04 17% 23% 4.53E+06 9.61E+05 16% 14% 7.48E+05 2.05E+04 22% 31% n 4 4 4 4 Pregabalin Cocktail C Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average n m/z 97 m/z 124 1.26E+05 5.88E+05 9% 10% 4.48E+03 8.69E+03 24% 36% 1.11E+05 4.98E+05 27% 27% 1.70E+04 4.26E+04 4 m/z 301 m/z 98 n 6.75E+06 2.73E+06 23% 24% 2.17E+04 8.15E+03 78% 86% 3.47E+07 1.39E+07 19% 18% 1.71E+04 6.47E+03 15% 15% 4 4 4 Flecainide Cocktail C Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD 4 4 4 4 130 Gabapentin Cocktail C Spiked Average Manual m/z 119 1.80E+06 5.70E+05 9% 9% 1.55E+04 5.54E+03 36% 27% 1.78E+06 1.45E+07 30% 9% 1.21E+04 4.30E+05 44% 19% Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut m/z 137 Spiked RSD Blank Average Blank RSD n 4 4 4 4 Benzylpiperazine Cocktail C Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 91 m/z 85 4.92E+06 1.03E+06 18% 9% 3.16E+05 3.00E+05 39% 19% 1.45E+07 1.73E+06 9% 9% 4.30E+05 4.21E+04 19% 42% n 4 4 4 4 Lidocaine Cocktail C Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 86 m/z 58 2.19E+07 2.22E+06 13% 13% 5.35E+04 7.14E+03 118% 101% 6.09E+07 6.14E+06 13% 14% 8.59E+03 1.04E+05 22% 25% n 4 4 4 4 131 Bupivacaine Cocktail C Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 140 m/z 98 4.26E+07 7.09E+06 24% 24% 1.27E+05 2.50E+04 101% 87% 1.48E+08 2.47E+07 17% 17% 1.06E+04 1.16E+04 25% 33% n 4 4 4 4 Ranitidine Cocktail C Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 176 m/z 102 7.79E+06 3.96E+06 28% 28% 3.46E+04 8.87E+03 47% 97% 3.92E+07 1.99E+07 33% 35% 5.50E+04 1.93E+03 7% 55% n 4 4 4 4 Papaverine Cocktail C Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 324 m/z 202 2.55E+07 2.67E+07 34% 33% 7.35E+04 7.81E+04 99% 98% n 4 4 4 4 132 Mescaline Cocktail D Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Die-cut Spiked RSD Blank Average Blank RSD m/z 180 m/z 165 3.42E+05 2.19E+05 30% 43% 5.28E+04 4.59E+03 28% 51% 9.03E+05 2.74E+05 10% 8% 5.71E+05 3.98E+04 17% 32% 3.70E+05 2.30E+05 - - 9.96E+04 4.18E+03 32% 57% n 4 4 3 2 1 3 Norketamine Cocktail D Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Die-cut Spiked RSD Blank Average Blank RSD m/z 125 m/z 179 1.36E+06 6.12E+05 41% 40% 1.27E+04 9.18E+03 79% 56% 2.32E+06 1.07E+06 13% 15% 1.97E+04 3.37E+04 11% 5% 2.96E+06 1.33E+06 - - 5.46E+03 8.34E+03 47% 42% n 4 4 3 2 1 3 133 Ketamine Cocktail D Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Die-cut Spiked RSD Blank Average Blank RSD m/z 89 m/z 125 4.91E+05 1.99E+06 31% 30% 5.42E+03 1.41E+04 77% 104% 1.22E+06 4.72E+06 23% 21% 3.89E+04 3.00E+04 14% 15% 1.84E+06 7.34E+06 - - 8.81E+03 4.75E+03 71% 51% n 4 4 3 2 1 3 Etomidate Cocktail D m/z 141 m/z 95 2.15E+06 2.27E+06 29% 9% 1.08E+05 4.46E+05 49% 67% Spiked Average - 2.47E+06 Spiked RSD - 29% Blank Average - 7.45E+04 Blank RSD - 10% Spiked Average - 2.52E+06 Spiked RSD - - Blank Average - 8.75E+04 Blank RSD - 47% Spiked Average Manual Spiked RSD Blank Average Blank RSD Laser-cut Die-cut n 4 4 3 2 1 3 134 Metoclopramide Cocktail D Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Die-cut Spiked RSD Blank Average Blank RSD m/z 227 m/z 184 7.39E+06 2.75E+06 34% 34% 6.70E+04 3.10E+04 107% 77% 3.02E+07 1.14E+07 32% 31% 1.90E+04 4.57E+04 16% 6% 7.51E+07 2.96E+07 - - 1.16E+04 2.04E+04 60% 65% n 4 4 3 2 1 3 Desmethylclomipramine Cocktail D Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Die-cut Spiked RSD Blank Average Blank RSD m/z 72 m/z 227 3.77E+05 9.10E+04 36% 28% 3.49E+03 8.18E+03 68% 32% 1.52E+06 3.55E+05 6% 13% 1.23E+03 2.77E+04 6% 11% 1.98E+06 5.09E+05 - - 1.15E+03 1.70E+04 83% 78% n 4 4 3 2 1 3 135 Sertraline Cocktail D Spiked Average Manual m/z 275 2.66E+05 2.16E+05 33% 37% 8.49E+03 2.66E+03 44% 65% 8.95E+05 7.37E+05 6% 6% 1.89E+04 2.11E+03 4% 16% 9.17E+05 7.68E+05 - - 1.27E+04 2.19E+03 3 m/z 135 m/z 133 n 2.86E+05 2.66E+05 33% 34% 1.15E+04 1.54E+04 38% 28% 4.48E+05 4.48E+05 5% 5% 3.78E+04 3.78E+04 22% 22% 4.87E+05 4.87E+05 - - 2.08E+04 2.08E+04 46% 46% Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Die-cut Spiked RSD Blank Average n m/z 158 4 4 3 2 1 MDA Cocktail D Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Die-cut Spiked RSD Blank Average Blank RSD 4 4 3 2 1 3 136 Oxycodone Cocktail D Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Die-cut Spiked RSD Blank Average Blank RSD m/z 241 m/z 212 2.84E+05 1.84E+05 36% 33% 9.80E+03 9.51E+03 36% 39% 4.93E+05 3.42E+05 17% 19% 1.51E+04 3.31E+04 19% 13% 4.52E+05 3.06E+05 - - 7.39E+03 1.24E+04 66% 70% n 4 4 3 2 1 3 Trazodone Cocktail D Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Die-cut Spiked RSD Blank Average Blank RSD m/z 176 m/z 148 2.52E+06 2.09E+06 36% 35% 3.54E+04 2.76E+04 52% 53% 9.91E+06 8.19E+06 23% 24% 2.29E+04 4.90E+04 6% 7% 1.33E+07 1.10E+07 - - 4.19E+04 1.20E+04 35% 62% n 4 4 3 2 1 3 137 Ziprasidone Cocktail D Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Die-cut Spiked RSD Blank Average Blank RSD m/z 194 m/z 159 7.30E+05 5.93E+05 49% 23% 5.68E+03 9.76E+04 69% 68% 1.32E+06 5.98E+05 16% 16% 1.50E+04 9.83E+04 6% 0% 7.82E+05 3.43E+05 - - 6.23E+03 4.46E+04 54% 34% n 4 4 3 2 1 3 Aripiprazole Cocktail D Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Die-cut Spiked RSD Blank Average Blank RSD m/z 285 m/z 176 8.62E+05 2.46E+05 46% 43% 1.16E+04 7.07E+03 48% 27% 1.93E+06 5.46E+05 11% 11% 8.94E+03 9.71E+03 4% 20% 1.50E+06 4.28E+05 - - 3.85E+03 9.49E+03 50% 52% n 4 4 3 2 1 3 138 Sildenafil Cocktail D Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Die-cut Spiked RSD Blank Average Blank RSD m/z 100 m/z 283 2.16E+05 1.50E+05 42% 35% 1.51E+03 6.02E+03 92% 36% 2.09E+05 1.41E+05 22% 24% 4.20E+02 7.15E+03 3% 3% 1.63E+05 1.07E+05 - - 3.47E+02 2.29E+03 97% 74% n 4 4 3 2 1 3 Vardenafil Cocktail D Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 151 m/z 312 5.39E+05 2.14E+05 40% 43% 1.40E+04 2.17E+03 36% 90% 1.20E+06 4.88E+05 28% 26% 5.76E+03 2.08E+03 4% 9% 9.63E+05 4.05E+05 - - 3.59E+03 8.53E+02 66% 70% n 4 4 3 2 1 3 139 Ephedrine/Pseudoephedrine Cocktail E Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 115 m/z 117 1.85E+05 1.54E+05 4% 6% 7.04E+03 5.66E+03 112% 108% 3.55E+05 3.07E+05 36% 37% 3.87E+03 9.06E+03 1% 28% 4.39E+05 3.62E+05 13% 13% 9.95E+03 7.31E+03 41% 56% n 4 4 4 2 4 3 TFMPP Cocktail E Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 188 m/z 118 6.26E+05 1.50E+05 11% 10% 1.49E+04 6.71E+03 96% 66% 7.98E+05 2.45E+05 11% 25% 3.16E+04 4.23E+04 7% 10% 1.20E+06 2.85E+05 15% 17% 2.19E+04 1.42E+04 63% 63% n 4 4 4 2 4 3 140 Propranolol Cocktail E Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 116 m/z 183 3.13E+05 2.80E+05 8% 8% 7.13E+03 1.23E+04 125% 73% 8.76E+05 7.77E+05 21% 22% 6.21E+03 1.49E+04 15% 1% 1.04E+06 9.41E+05 12% 11% 1.86E+03 1.31E+04 67% 36% n 4 4 4 2 4 3 Norvenlafaxine Cocktail E Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 58 m/z 107 5.46E+05 1.30E+05 12% 12% 3.26E+04 1.04E+04 123% 52% 1.95E+06 6.53E+05 53% 57% 3.13E+04 1.94E+05 41% 19% 2.39E+06 6.22E+05 21% 22% 4.90E+03 9.72E+04 39% 68% n 4 4 4 2 4 3 141 Venlafaxine Cocktail E Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 58 m/z 121 9.77E+05 2.56E+05 17% 17% 1.05E+04 1.26E+04 150% 56% 3.70E+06 1.34E+06 47% 46% 1.61E+04 1.74E+05 21% 9% 5.80E+06 1.52E+06 24% 24% 6.48E+03 9.21E+04 75% 68% n 4 4 4 2 4 3 Pentazocine Cocktail E Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 218 m/z 69 1.58E+06 3.75E+05 15% 14% 2.18E+04 3.47E+04 132% 40% 6.86E+06 1.60E+06 40% 40% 2.55E+04 1.06E+05 7% 11% 9.67E+06 2.17E+06 25% 25% 1.25E+04 1.26E+05 52% 70% n 4 4 4 2 4 3 142 Demoxepam Cocktail E Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 179 m/z 207 1.91E+04 1.52E+04 21% 18% 3.17E+03 5.62E+03 76% 68% 4.53E+04 3.14E+04 56% 59% 7.20E+03 6.92E+03 16% 7% 4.43E+04 2.95E+04 41% 45% 2.40E+03 2.93E+03 62% 64% n 4 4 4 2 4 3 Norpropoxyphene Cocktail E Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 44 m/z 128 2.62E+05 1.28E+05 20% 19% 4.31E+03 6.32E+03 115% 72% 1.39E+06 7.25E+05 27% 22% 2.32E+03 2.94E+04 17% 8% 1.98E+06 9.67E+05 31% 32% 2.32E+03 1.37E+04 52% 55% n 4 4 4 2 4 3 143 Olanzapine Cocktail E Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 256 m/z 198 1.12E+05 3.07E+04 16% 14% 4.02E+03 2.78E+03 66% 52% 5.66E+05 1.74E+05 32% 41% 8.51E+03 2.20E+04 19% 3% 4.10E+05 1.35E+05 22% 29% 1.05E+04 7.75E+04 57% 83% n 4 4 4 2 4 3 Propoxyphene Cocktail E Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 266 m/z 128 3.23E+05 6.67E+04 14% 13% 1.03E+04 6.59E+03 64% 70% 1.26E+06 2.68E+05 48% 47% 3.76E+03 1.89E+04 5% 1% 1.73E+06 3.43E+05 25% 25% 4.07E+03 1.59E+04 15% 75% n 4 4 4 2 4 3 144 Hydroxyzine Cocktail E Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 201 m/z 165 2.20E+06 1.82E+06 10% 10% 7.89E+04 3.24E+04 63% 109% 8.73E+06 7.46E+06 40% 42% 4.40E+04 7.12E+03 14% 4% 1.19E+07 1.00E+07 20% 20% 9.63E+04 5.27E+03 48% 65% n 4 4 4 2 4 3 Quetiapine Cocktail E Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 253 m/z 221 2.36E+06 1.51E+06 11% 10% 3.58E+04 1.87E+04 109% 128% 9.93E+06 6.44E+06 52% 53% 9.03E+03 1.34E+04 13% 5% 1.08E+07 6.97E+06 25% 25% 9.96E+03 8.58E+03 83% 84% n 4 4 4 2 4 3 145 Verapamil Cocktail E Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 165 m/z 1.57E+06 5.76E+05 10% 10% 4.59E+04 7.21E+03 72% 138% 8.45E+06 3.16E+06 14% 14% 1.72E+04 8.86E+03 6% 28% 1.16E+07 4.30E+06 26% 26% 1.80E+04 2.94E+03 44% 54% n 4 4 4 2 4 3 146 Mephedrone Cocktail F Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 145 m/z 144 1.09E+06 8.11E+05 8% 9% 1.39E+04 7.25E+03 42% 77% 1.85E+06 1.39E+06 20% 19% 1.40E+04 1.22E+04 25% 44% 4.78E+06 3.58E+06 3% 4% 2.08E+04 1.28E+04 61% 34% n 4 4 4 2 4 3 MDMA Cocktail F Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 163 m/z 105 1.11E+06 4.03E+05 7% 7% 9.74E+03 1.01E+04 68% 42% 2.26E+06 8.64E+05 17% 19% 5.76E+03 4.23E+04 31% 30% 3.58E+06 1.32E+06 10% 11% 5.30E+03 5.83E+04 95% 80% n 4 4 4 2 4 3 147 Methylone Cocktail F Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 160 m/z 132 1.09E+06 5.76E+05 8% 9% 1.88E+04 1.59E+04 43% 18% 2.08E+06 1.13E+06 23% 26% 6.75E+04 7.23E+04 7% 13% 4.00E+06 2.10E+06 13% 13% 1.85E+04 4.70E+04 112% 80% n 4 4 4 2 4 3 Mirtazapine Cocktail F Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 195 m/z 194 1.60E+06 6.46E+05 9% 9% 1.47E+04 6.39E+03 57% 43% 3.77E+06 1.57E+06 26% 27% 2.25E+04 6.16E+04 1% 19% 6.69E+06 2.73E+06 7% 7% 2.03E+04 5.24E+04 49% 46% n 4 4 4 2 4 3 148 Metoprolol Cocktail F Spiked Average Manual Blank Average Spiked Average Spiked RSD Blank Average Blank RSD Spiked Average Die-cut m/z 77 2.78E+05 2.08E+05 8% 7% 4.00E+03 1.85E+04 77% 33% 8.10E+05 7.23E+05 10% 20% 2.22E+03 1.24E+05 34% 18% 1.14E+06 8.18E+05 11% 11% 1.15E+03 4.61E+04 110% 59% Spiked RSD Blank RSD Laser-cut m/z 116 Spiked RSD Blank Average Blank RSD n 4 4 4 2 4 3 MDPV Cocktail F Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 175 m/z 135 1.26E+06 1.12E+06 18% 18% 1.37E+04 1.33E+04 40% 47% 2.83E+06 2.66E+06 38% 39% 1.74E+04 8.56E+04 28% 10% 5.39E+06 4.82E+06 22% 23% 1.74E+04 5.36E+04 65% 40% n 4 4 4 2 4 3 149 Norclozapine Cocktail F Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 192 m/z 270 3.97E+05 2.93E+05 10% 10% 5.86E+03 4.94E+03 53% 52% 6.90E+05 5.11E+05 21% 21% 7.64E+03 1.12E+04 16% 67% 9.77E+05 7.15E+05 14% 15% 5.41E+03 8.48E+03 73% 45% n 4 4 4 2 4 3 Midazolam Cocktail F Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 291 m/z 249 1.18E+06 3.33E+05 10% 10% 2.63E+04 7.59E+03 89% 83% 2.84E+06 7.99E+05 38% 37% 8.05E+02 2.37E+03 27% 21% 5.40E+06 1.51E+06 14% 13% 4.86E+02 1.01E+03 80% 80% n 4 4 4 2 4 3 150 Labetalol Cocktail F Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 162 m/z 91 3.31E+05 3.16E+05 21% 18% 8.10E+03 3.04E+04 61% 40% 3.22E+05 3.06E+05 28% 30% 1.34E+04 4.01E+04 8% 19% 3.84E+05 3.58E+05 16% 15% 7.53E+03 3.84E+04 63% 84% n 4 4 4 2 4 3 Donepezil Cocktail F Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 91 m/z 243 1.75E+06 2.81E+05 11% 12% 2.35E+04 4.56E+03 48% 44% 6.20E+06 1.01E+06 18% 18% 2.28E+04 4.06E+03 6% 11% 1.19E+07 1.91E+06 12% 12% 1.92E+04 2.73E+03 39% 64% n 4 4 4 2 4 3 151 Methamphetamine Cocktail F Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 91 m/z 119 1.14E+06 4.28E+05 8% 9% 2.21E+04 4.37E+03 44% 64% 2.28E+06 8.72E+05 15% 18% 2.16E+04 2.64E+03 11% 8% 4.00E+06 1.53E+06 3% 3% 5.82E+04 5.50E+03 55% 98% n 4 4 4 2 4 3 152 Alpha-PVP Cocktail G Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 91 m/z 77 1.82E+06 7.53E+05 14% 14% 3.34E+04 3.16E+04 104% 61% 2.01E+04 1.08E+05 18% 26% 2.52E+06 1.07E+06 25% 23% 2.14E+04 7.54E+04 51% 52% 9.05E+06 3.72E+06 31% 30% n 4 4 4 2 4 3 Bupropion Cocktail G Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 184 m/z 131 1.37E+06 7.63E+05 11% 10% 2.89E+04 1.54E+04 96% 93% 4.96E+04 2.16E+04 35% 20% 1.75E+06 9.88E+05 15% 14% 4.66E+04 2.04E+04 56% 57% 7.20E+06 4.04E+06 33% 34% n 4 4 4 2 4 3 153 7-aminoclonazepam Cocktail G Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 121 m/z 94 1.76E+05 6.92E+04 18% 18% 9.32E+03 7.98E+03 61% 53% 3.57E+04 5.48E+04 34% 22% 7.70E+04 5.21E+04 26% 31% 1.92E+04 2.17E+04 51% 68% 2.37E+05 1.03E+05 40% 38% n 4 4 4 2 4 3 Desalkylflurazepam Cocktail G Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 140 m/z 226 3.19E+05 1.54E+05 16% 33% 5.43E+04 3.51E+03 52% 125% 4.50E+03 8.07E+03 22% 12% 3.69E+04 8.90E+04 20% 19% 1.06E+04 2.16E+03 85% 73% 1.39E+05 2.08E+05 21% 26% n 4 4 4 2 4 3 154 Chlordiazepoxide Cocktail G Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 227 m/z 255 6.81E+05 1.61E+05 11% 12% 2.61E+04 9.06E+03 74% 44% 1.28E+04 1.80E+04 24% 2% 2.42E+05 6.41E+04 22% 20% 8.80E+03 1.31E+04 53% 57% 5.17E+05 1.30E+05 29% 28% n 4 4 4 2 4 3 Cocaethylene Cocktail G Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 196 m/z 82 3.53E+06 9.50E+05 10% 10% 5.74E+04 2.28E+04 103% 79% 1.73E+04 1.27E+04 2% 16% 6.06E+06 1.62E+06 19% 20% 3.33E+04 8.27E+03 84% 57% 2.66E+07 7.00E+06 27% 27% n 4 4 4 2 4 3 155 Chlorpromazine Cocktail G Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 214 m/z 58 3.65E+04 1.37E+05 10% 11% 1.90E+03 6.27E+03 62% 48% 8.04E+03 6.49E+03 12% 27% 8.90E+04 3.25E+05 19% 18% 2.15E+03 7.47E+03 73% 56% 2.08E+05 8.02E+05 26% 26% m/z 270 m/z 192 1.32E+06 8.41E+05 10% 10% 1.72E+04 1.32E+04 118% 104% 8.55E+03 7.83E+03 1% 24% 2.61E+06 1.68E+06 25% 25% 3.76E+03 4.60E+03 57% 77% 6.15E+06 3.97E+06 36% 37% n 4 4 4 2 4 3 Clozapine Cocktail G Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD n 4 4 4 2 4 3 156 6-acetylmorphine Cocktail G Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 165 m/z 211 8.69E+04 3.66E+04 11% 14% 3.76E+04 1.64E+04 36% 79% 9.03E+03 1.07E+04 14% 7% 6.61E+04 3.93E+04 24% 21% 1.62E+04 9.08E+03 109% 70% 9.86E+04 5.12E+04 26% 25% n 4 4 4 2 4 3 Diltiazem Cocktail G Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Spiked RSD Die-cut Blank Average Blank RSD m/z 178 m/z 150 1.83E+06 7.92E+05 9% 9% 2.40E+04 1.41E+04 123% 100% 5.97E+03 8.12E+03 31% 18% 4.91E+06 2.13E+06 27% 27% 2.17E+03 3.92E+03 94% 77% 1.94E+07 8.48E+06 31% 32% n 4 4 4 2 4 3 157 Alfentanil Cocktail G Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 268 m/z 197 1.71E+05 1.17E+05 11% 10% 6.35E+03 8.68E+03 61% 39% 6.27E+02 2.05E+03 16% 14% 1.99E+05 1.33E+05 32% 30% 3.07E+03 3.79E+03 100% 74% 1.41E+06 8.76E+05 42% 39% n 4 4 4 2 4 3 158 PCP Cocktail H Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 91 m/z 159 4.76E+05 2.60E+05 9% 9% 5.58E+04 3.10E+04 61% 58% 1.37E+06 6.51E+05 33% 37% 7.27E+04 1.09E+04 73% 32% 1.34E+06 6.85E+05 30% 31% 4.80E+04 7.39E+03 39% 50% n 4 4 4 2 4 3 Meperidine Cocktail H Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 220 m/z 174 8.86E+05 5.04E+05 1% 2% 5.66E+04 4.36E+04 94% 73% 2.51E+06 1.55E+06 26% 27% 8.62E+04 1.14E+05 59% 67% 2.61E+06 1.57E+06 28% 31% 1.03E+04 1.58E+04 55% 52% n 4 4 4 2 4 3 159 Diphenhydramine Cocktail H Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 167 m/z 165 8.30E+05 3.63E+05 2% 1% 6.65E+04 2.33E+04 73% 92% 2.33E+06 1.04E+06 28% 30% 2.11E+04 1.11E+04 30% 52% 2.49E+06 1.09E+06 26% 27% 1.25E+04 4.73E+03 43% 50% n 4 4 4 2 4 3 Doxylamine Cocktail H Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 167 m/z 182 1.60E+06 1.41E+06 7% 8% 9.87E+04 7.62E+04 84% 96% 3.68E+06 3.22E+06 37% 34% 5.48E+04 3.03E+04 60% 5% 4.15E+06 3.53E+06 32% 31% 3.29E+04 1.15E+04 43% 34% n 4 4 4 2 4 3 160 EDDP Cocktail H Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 234 m/z 249 2.81E+06 1.15E+06 9% 10% 2.26E+05 9.19E+04 118% 118% 1.45E+07 5.95E+06 28% 28% 2.96E+04 9.61E+03 54% 54% 1.46E+07 6.05E+06 30% 31% 1.04E+04 2.21E+03 36% 61% n 4 4 4 2 4 3 Promethazine Cocktail H Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 86 m/z 198 3.07E+05 1.31E+05 1% 1% 5.14E+04 2.13E+04 34% 39% 8.55E+05 3.82E+05 30% 32% 5.14E+04 3.27E+04 0% 16% 7.36E+05 3.23E+05 28% 29% 4.40E+04 1.98E+04 26% 23% n 4 4 4 2 4 3 161 Brompheniramine Cocktail H Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 274 m/z 167 7.88E+05 3.90E+05 5% 5% 3.67E+04 4.20E+04 93% 38% 2.64E+06 1.29E+06 32% 34% 1.32E+04 1.31E+04 69% 77% 2.45E+06 1.17E+06 27% 28% 3.41E+03 4.74E+03 63% 63% n 4 4 4 2 4 3 Lorazepam Cocktail H Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 276 m/z 167 7.82E+05 3.80E+05 5% 4% 3.51E+04 3.46E+04 95% 47% 2.54E+06 1.20E+06 29% 30% 1.60E+04 1.08E+04 43% 66% 2.41E+06 1.14E+06 26% 26% 8.18E+03 4.27E+03 30% 65% n 4 4 4 2 4 3 162 Normeperidine Cocktail H Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 160 m/z 56 5.68E+05 1.30E+05 6% 6% 5.18E+04 1.41E+04 70% 76% 1.02E+06 2.08E+05 10% 10% 1.41E+05 1.51E+04 53% 64% 1.15E+06 2.47E+05 17% 17% 2.92E+04 3.06E+03 29% 50% n 4 4 4 2 4 3 Flurazepam Cocktail H Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 315 m/z 317 9.48E+05 2.14E+05 2% 3% 5.66E+04 1.79E+04 106% 69% 4.32E+06 9.91E+05 34% 34% 5.68E+03 2.06E+04 68% 87% 3.66E+06 8.32E+05 34% 32% 1.85E+03 1.45E+03 33% 38% n 4 4 4 2 4 3 163 Morphine Cocktail I Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut m/z 152 m/z 165 1.53E+04 1.22E+04 18% 22% 3.64E+03 3.80E+03 70% 56% 3.70E+04 2.30E+04 15% 18% 24154.03 10725.94 62% 70% 2.56E+04 1.60E+04 - - 6.67E+03 3.31E+03 50% 67% Spiked RSD Blank Average Blank RSD n 4 4 4 2 1 3 Oxymorphone Cocktail I Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 227 m/z 198 3.15E+04 2.43E+04 9% 11% 3.76E+03 2.99E+03 72% 71% 4.18E+04 4.68E+04 16% 19% 13437.55 32548.87 68% 72% 3.74E+04 3.75E+04 - - 3.06E+03 5.25E+03 58% 43% n 4 4 4 2 1 3 164 Zaleplon Cocktail I Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut m/z 236 m/z 264 5.80E+04 5.11E+04 41% 41% 3.72E+03 3.41E+03 91% 66% 5.74E+04 3.91E+04 31% 25% 40471.3 13342.55 64% 60% 6.25E+04 6.21E+04 - - 3.55E+03 7.67E+03 64% 88% Spiked RSD Blank Average Blank RSD n 4 4 4 2 1 3 Methadone Cocktail I Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 265 m/z 105 1.34E+06 4.41E+05 11% 10% 3.69E+04 1.89E+04 148% 111% 3.88E+06 1.26E+06 32% 33% 15483.3 21467.25 51% 74% 1.11E+07 3.66E+06 - - 6.30E+03 1.20E+04 64% 50% n 4 4 4 2 1 3 165 Clonazepam Cocktail I Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut m/z 270 m/z 214 6.41E+04 1.88E+04 50% 51% 1.17E+04 1.90E+03 52% 70% 5.63E+04 2.50E+04 29% 31% 71418.77 29487.67 70% 74% 5.33E+04 1.27E+04 - - 2.09E+04 3.07E+03 48% 44% Spiked RSD Blank Average Blank RSD n 4 4 4 2 1 3 Fluvoxamine Cocktail I Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 71 m/z 200 1.02E+05 2.55E+04 4% 9% 1.00E+04 6.20E+03 63% 44% 1.76E+05 4.06E+04 9% 8% 16634.52 10218.4 86% 6% 3.50E+05 8.35E+04 - - 9.53E+03 6.28E+03 42% 24% n 4 4 4 2 1 3 166 Paroxetine Cocktail I Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut m/z 192 m/z 70 7.46E+04 4.37E+04 5% 2% 8.35E+03 6.79E+03 61% 54% 1.29E+05 6.51E+04 11% 12% 16409.67 7930.324 39% 72% 1.40E+05 7.93E+04 - - 5.33E+03 7.07E+03 44% 69% Spiked RSD Blank Average Blank RSD n 4 4 4 2 1 3 Buspirone Cocktail I Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 122 m/z 95 7.62E+05 1.74E+05 12% 2% 2.09E+04 3.20E+04 131% 43% 3.14E+06 5.83E+05 24% 24% 26690.31 38870.77 68% 69% 1.13E+07 2.05E+06 - - 5.71E+03 2.26E+04 58% 48% n 4 4 4 2 1 3 167 Chlorpheniramine Cocktail I Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut m/z 230 m/z 167 9.67E+05 4.96E+05 11% 9% 2.23E+04 1.77E+04 149% 113% 2.48E+06 1.25E+06 26% 26% 93527.49 13034.49 83% 73% 1.04E+07 5.25E+06 - - 8.75E+03 3.96E+03 45% 63% Spiked RSD Blank Average Blank RSD n 4 4 4 2 1 3 Zopiclone Cocktail I Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD Spiked Average Die-cut Spiked RSD Blank Average Blank RSD m/z 245 m/z 111 1.40E+05 4.89E+04 5% 4% 2.80E+04 2.72E+03 29% 75% 1.25E+05 4.17E+04 17% 17% 34498.15 3883.349 34% 28% 9.94E+04 3.65E+04 - - 1.45E+04 5.05E+03 33% 80% n 4 4 4 2 1 3 168 Methylphenidate Cocktail J Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 84 m/z 56 9.00E+05 1.18E+05 7% 7% 7.69E+04 1.96E+04 43% 36% 2.67E+06 3.60E+05 25% 20% 2.34E+04 7.49E+04 40% 38% n 4 4 4 3 Nordoxepin Cocktail J Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 107 m/z 235 1.06E+05 6.27E+04 4% 5% 1.27E+04 4.58E+03 55% 72% 2.73E+05 1.53E+05 6% 6% 2.64E+04 4.20E+03 50% 68% n 4 4 4 3 Desipramine Cocktail J Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 72 m/z 193 1.54E+05 5.64E+04 5% 6% 8.80E+03 9.66E+03 69% 32% 4.34E+05 1.16E+05 6% 5% 1.74E+03 2.35E+04 59% 53% n 4 4 4 3 169 7-aminoflunitrazepam Cocktail J Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 135 m/z 227 1.17E+05 5.96E+04 11% 6% 1.11E+04 4.76E+03 38% 58% 2.42E+05 1.33E+05 24% 26% 2.79E+04 1.60E+04 52% 47% n 4 4 4 3 Trimipramine Cocktail J Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 193 m/z 208 5.64E+04 4.11E+04 6% 7% 6.94E+03 5.44E+03 47% 31% 1.53E+05 1.53E+05 19% 19% 6.00E+03 5.99E+03 51% 51% n 4 4 4 3 Norfluoxetine Cocktail J Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 134 m/z 30 1.47E+04 2.81E+03 10% 12% 5.42E+03 2.70E+02 28% 84% 1.41E+04 2.64E+03 7% 6% 3.61E+03 3.47E+01 44% 73% n 4 4 4 3 170 Duloxetine Cocktail J Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 44 m/z 154 1.16E+04 1.11E+04 37% 31% 5.94E+03 5.66E+03 66% 66% 1.70E+04 1.49E+04 9% 4% 1.21E+03 4.43E+03 82% 46% n 4 4 4 3 Benztropine Cocktail J Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 167 m/z 165 4.06E+05 1.82E+05 7% 8% 3.03E+04 1.44E+04 38% 41% 1.97E+06 8.94E+05 18% 19% 1.01E+04 7.58E+03 59% 64% n 4 4 4 3 Fluoxetine Cocktail J Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 44 m/z 148 4.47E+04 7.32E+03 4% 7% 3.01E+03 1.26E+03 70% 12% 1.03E+05 1.79E+04 4% 7% 4.71E+02 6.06E+03 61% 49% n 4 4 4 3 171 Flunitrazepam Cocktail J Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 268 m/z 239 8.77E+04 2.80E+04 14% 15% 2.56E+04 3.91E+03 47% 56% 1.38E+05 3.16E+04 18% 22% 5.66E+04 6.22E+03 56% 54% n 4 4 4 3 Clomipramine Cocktail J Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 227 m/z 242 3.20E+04 2.05E+04 3% 5% 8.74E+03 4.26E+03 33% 37% 1.11E+05 7.42E+04 11% 10% 1.09E+04 4.75E+03 69% 57% n 4 4 4 3 Triazolam Cocktail J Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 308 m/z 239 4.48E+04 3.81E+04 12% 12% 3.35E+03 7.35E+03 85% 42% 1.83E+05 1.47E+05 27% 24% 5.43E+02 1.14E+04 66% 60% n 4 4 4 3 172 Amlodipine Cocktail J Spiked Average Manual m/z 238 m/z 294 4.44E+04 3.40E+04 3% 1% 1.79E+04 1.58E+04 13% 15% 4.00E+04 3.16E+04 8% 12% 2.38E+03 1.41E+04 27% 15% Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD n 4 4 4 3 mCPP Cocktail J Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 154 m/z 118 1.27E+05 1.03E+05 5% 5% 1.98E+04 7.94E+03 56% 62% 1.53E+05 1.37E+05 8% 5% 1.99E+04 3.15E+04 44% 43% n 4 4 4 3 173 Cyclobenzaprine Cocktail K Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 215 m/z 231 1.32E+06 5.16E+05 121% 121% 2.8E+05 1.1E+05 53% 51% 6.30E+05 2.64E+05 31% 32% 4.19E+03 1.13E+04 65% 55% n 4 3 3 3 Zolpidem Cocktail K Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 92 m/z 235 9.54E+05 5.07E+06 103% 104% 2.1E+05 1.1E+06 49% 51% 6.59E+05 3.34E+06 42% 41% 2.69E+04 5.62E+03 51% 54% n 4 3 3 3 Alprazolam Cocktail K Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 205 m/z 281 4.81E+05 5.52E+05 78% 80% 1.7E+05 1.9E+05 13% 13% 5.68E+04 7.02E+04 64% 61% 9.88E+03 1.91E+04 51% 47% n 4 3 3 3 174 Fentanyl Cocktail K Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 188 m/z 105 2.23E+06 1.70E+06 114% 108% 4.6E+05 4.0E+05 54% 43% 1.46E+06 1.10E+06 37% 37% 1.08E+05 4.19E+04 19% 37% n 4 3 3 3 Haloperidol Cocktail K Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 122 m/z 165 1.30E+06 1.20E+06 115% 117% 3.1E+05 2.8E+05 51% 52% 6.23E+05 5.64E+05 36% 36% 1.60E+04 1.42E+04 52% 39% n 4 3 3 3 Risperidone Cocktail K Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 191 m/z 110 2.45E+06 1.96E+05 100% 96% 7.3E+05 6.2E+04 56% 48% 2.11E+06 1.75E+05 33% 35% 1.50E+04 9.35E+03 45% 62% n 4 3 3 3 175 9-hydroxyrisperidone Cocktail K Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 207 m/z 110 1.75E+06 3.88E+05 82% 84% 5.8E+05 1.4E+05 55% 54% 1.49E+06 3.58E+05 36% 38% 1.90E+04 2.20E+04 31% 48% n 4 3 3 3 Ropinirole Cocktail K Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 114 m/z 132 1.31E+06 2.90E+05 112% 111% 2.8E+05 6.5E+04 53% 49% 5.49E+05 1.72E+05 33% 34% 7.84E+03 4.82E+04 35% 49% n 4 3 3 3 Dextromethorphan Cocktail K Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 215 m/z 147 8.50E+05 6.58E+05 119% 118% 1.9E+05 1.5E+05 52% 52% 4.84E+05 3.93E+05 28% 29% 2.06E+04 3.54E+04 50% 50% n 4 3 3 3 176 Benzoylecgonine Cocktail L Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 168 m/z 77 4.30E+05 1.65E+05 42% 37% 4.05E+04 3.76E+04 62% 92% 7.02E+05 3.58E+05 84% 75% 9.14E+03 8.12E+04 85% 85% n 4 4 4 4 Amitriptyline Cocktail L Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 233 m/z 117 2.30E+05 1.42E+05 17% 17% 1.07E+04 1.01E+04 67% 77% 7.67E+05 4.51E+05 43% 41% 2.06E+04 9.81E+03 53% 59% n 4 4 4 4 Nortriptyline Cocktail L Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 233 m/z 117 1.83E+05 8.16E+04 16% 12% 5.82E+03 6.54E+03 84% 66% 3.87E+05 1.70E+05 18% 14% 7.10E+03 7.29E+03 21% 69% n 4 4 4 4 177 Atenolol Cocktail L Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 145 m/z 190 9.32E+05 6.53E+05 18% 19% 1.47E+05 5.46E+04 84% 103% 9.65E+05 7.43E+05 3% 4% 2.15E+04 4.91E+03 66% 63% n 4 4 4 4 Hydrocodone Cocktail L Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 199 m/z 171 1.87E+05 7.52E+04 18% 16% 9.96E+03 7.35E+03 67% 70% 1.57E+05 8.85E+04 10% 36% 9.32E+03 1.68E+04 89% 86% n 4 4 4 4 Diazepam Cocktail L Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 154 m/z 222 2.53E+05 1.98E+05 26% 23% 1.40E+04 8.84E+03 91% 105% 1.67E+05 1.35E+05 25% 27% 8.90E+03 4.00E+03 83% 81% n 4 4 4 4 178 Temazepam Cocktail M Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 177 m/z 239 5.10E+04 1.35E+04 14% 9% 2.44E+04 4.00E+03 70% 56% 4.33E+04 2.05E+04 27% 30% 6.43E+03 5.90E+03 93% 74% n 4 4 4 4 Doxepine Cocktail M Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 107 m/z 165 2.08E+05 9.94E+04 17% 18% 1.76E+04 6.45E+03 73% 79% 5.21E+05 2.65E+05 24% 24% 1.83E+04 9.39E+03 51% 75% n 4 4 4 4 Nordiazepam Cocktail M Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 140 m/z 208 2.05E+05 1.47E+05 25% 25% 4.64E+03 5.02E+03 29% 46% 8.33E+04 6.05E+04 33% 33% 3.40E+03 3.71E+03 99% 75% n 4 4 4 4 179 Citalopram Cocktail M Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 262 m/z 234 3.01E+04 2.35E+04 17% 11% 1.83E+03 3.90E+03 63% 58% 7.36E+04 4.99E+04 17% 16% 2.62E+03 4.18E+03 73% 49% n 4 4 4 4 Oxazepam Cocktail M Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 241 m/z 77 1.49E+05 1.33E+05 20% 31% 8.73E+04 9.94E+04 65% 83% 1.24E+05 2.33E+05 30% 23% 5.67E+04 1.23E+05 64% 46% n 4 4 4 4 Hydromorphone Cocktail M Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 185 m/z 157 4.71E+04 3.63E+04 10% 11% 7.64E+03 8.64E+03 42% 61% 7.12E+04 7.50E+04 16% 24% 1.53E+04 2.84E+04 67% 66% n 4 4 4 4 180 Codeine Cocktail M Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 215 m/z 199 4.74E+04 3.53E+04 14% 20% 4.67E+03 9.95E+03 57% 67% 9.30E+04 4.25E+04 24% 21% 2.54E+04 9.31E+03 76% 88% n 4 4 4 4 Cocaine Cocktail M Spiked Average Manual Spiked RSD Blank Average Blank RSD Spiked Average Laser-cut Spiked RSD Blank Average Blank RSD m/z 182 m/z 82 2.40E+06 5.54E+05 18% 18% 5.27E+04 3.97E+04 12% 81% 5.73E+06 1.26E+06 40% 40% 5.89E+04 2.55E+04 183% 104% n 4 4 4 4 181 APPENDIX F Exogenous interferences identified for each target analyte using the Human Metabolome Database and DrugBank Target Analyte Acetaminophen Alfentanil Alpha-PVP Alprazolam Amitriptyline; Venlafaxine, EDDP Interference Compound Formula Molecular Weight 2-Amino-3-methylbenzoate C8H9NO2 151.1 Dopamine quinone C8H9NO2 151.1 Cathine C9H13NO 151.1 Phenylpropanolamine C9H13NO 151.1 4-Hydroxyamphetamine C9H13NO 151.1 Amantadine C10H17N 151.1 Trovafloxacin C20H15F3N4O3 416.1 Ramipril C23H32N2O5 416.2 Forasartan C23H28N8 416.2 Paracetamol sulfate C8H9NO5S 231.0 Isocarboxazid C12H13N3O2 231.1 Fenfluramine C12H16F3N 231.1 Dexfenfluramine C12H16F3N 231.1 Aminophenazone C13H17N3O 231.1 Pinazepam C18H13ClN2O 308.1 Fluoxetine glucuronide C16H16N6O 308.1 Phenylbutazone C19H20N2O2 308.2 8-Hydroxycarteolol C16H24N2O4 308.2 Indecainide C20H24N2O 308.2 Oxybuprocaine C17H28N2O3 308.2 DOPA sulfate C9H11NO7S 277.0 Azathioprine C9H7N7O2S 277.0 Ethylmethylthiambutene C15H19NS2 277.1 Entecavir C12H15N5O3 277.1 Maprotiline C20H23N 277.2 N-Desmethylterbinafine C20H23N 277.2 Perhexiline C19H35N 277.3 Melatonin glucuronide C19H24N2O8 408.2 Tamsulosin C20H28N2O5S 408.2 Homocysteine C4H9NO2S 135.0 Adenine C5H5N5 135.1 Aripiprazole Hydromorphone-3-glucoside C23H29NO8 447.2 Baclofen Chloroxine C9H5Cl2NO 213.0 Amlodipine Amphetamine 182 Target Analyte Benzoylecgonine Benztropine; Zolpidem Benzylpiperazine Brompheniramine; Chlorpromazine, Fluvoxamine Buprenorphine Buprenorphine Bupropion Interference Compound Formula Molecular Weight Carmustine C5H9Cl2N3O2 213.0 Droxidopa C9H11NO5 213.1 Phenazopyridine C11H11N5 213.1 Guanadrel Sulfate C10H19N3O2 213.1 Quinethazone C10H12ClN3O3S 289.0 Chlophedianol C17H20ClNO 289.1 Clofedanol C17H20ClNO 289.1 Norcocaine C16H19NO4 289.1 Chloropyramine Hydroxylated N-acetyl desmethyl frovatriptan Hyoscyamine C16H20ClN3 289.1 C15H19N3O3 289.1 C17H23NO3 289.2 Donepezil metabolite M4 C17H23NO3 289.2 Atropine C17H23NO3 289.2 Dyclonine C18H27NO2 289.2 2-oxobrimonidine C11H10BrN5O 307.0 Nitazoxanide C12H9N3O5S 307.0 Histamine Phosphate C5H15N3O8P2 307.0 Glutathione C10H17N3O6S 307.1 Tolnaftate C19H17NOS 307.1 Alcaftadine C19H21N3O 307.2 Ibopamine C17H25NO4 307.2 Hydroxyterbinafine C21H25NO 307.2 Betaxolol C18H29NO3 307.2 Fingolimod C19H33NO2 307.3 Pemoline C9H8N2O2 176.1 4-Methylaminorex C10H12N2O 176.1 Cotinine C10H12N2O 176.1 Clotiazepam C16H15ClN2OS 318.1 fluvoxamino acid C14H17F3N2O3 318.1 5-Hydroxyemedastine C17H26N4O2 318.2 Glycopyrrolate C19H28NO3 318.2 Tridihexethyl C21H36NO 318.3 N-Desmethylrosuvastatin C21H26FN3O6S 467.2 4-Hydroxytamoxifen sulfate C26H29NO5S 467.2 Tobramycin C18H37N5O9 467.3 Tiropramide C28H41N3O3 467.3 9-Carboxymethoxymethylguanine C8H9N5O4 239.1 Salbutamol C13H21NO3 239.2 Moprolol C13H21NO3 239.2 183 Target Analyte Buspirone Carbamazepine Carbamazepine-10,11-epoxide; Phenytoin Carisoprodol; Ropinirole Chlordiazepoxide; Metoclopraminde; Codeine; Hydrocodone Interference Compound Formula Molecular Weight Isoetharine C13H21NO3 239.2 Benzphetamine C17H21N 239.2 5-hydroxylansoprazole C16H14F3N3O3S 385.1 Hydroxylansoprazole C16H14F3N3O3S 385.1 Lansoprazole sulfone C16H14F3N3O3S 385.1 Phosphatidylserine C13H24NO10P 385.1 Nilvadipine C19H19N3O6 385.1 Isosorbide Dinitrate C6H8N2O8 236.0 Zileuton C11H12N2O2S 236.1 Didanosine C10H12N4O3 236.1 Hexobarbital C12H16N2O3 236.1 O-Desmethyl-lacosamide C12H16N2O3 236.1 Procaine C13H20N2O2 236.2 Hydroxyzileuton C11H12N2O3S 252.1 Zileuton sulfoxide C11H12N2O3S 252.1 Oxcarbazepine C15H12N2O2 252.1 2-Hydroxycarbamazepine C15H12N2O2 252.1 Epoxy-hexobarbital C12H16N2O4 252.1 3'-Hydroxyhexobarbital C12H16N2O4 252.1 Cimetidine C10H16N6S 252.1 Talbutal C13H20N2O3 252.1 Ifosfamide C7H15Cl2N2O2P 260.0 Cyclophosphamide C7H15Cl2N2O2P 260.0 Fenspiride C15H20N2O2 260.2 Oxymetazoline C16H24N2O 260.2 Imipenem C12H17N3O4S 299.1 N-desalkylpropafenone C18H21NO3 299.2 N-depropylpropafenone C18H21NO3 299.2 184 Target Analyte Chlorpheniramine Citalopram Clomipramine; Ranitidine; Delta-9-THC Clonazepam; Oxycodone Clozapine Interference Compound Formula Molecular Weight 4-Ketoifosfamide C7H13Cl2N2O3P 274.0 4-Ketocyclophosphamide C7H13Cl2N2O3P 274.0 5-Hydroxythalidomide C13H10N2O5 274.1 Thalidomide arene oxide C13H10N2O5 274.1 cis,trans-5'-Hydroxythalidomide C13H10N2O5 274.1 N2-Succinoylarginine C10H18N4O5 274.1 Ropivacaine 5-Methoxy-N,Ndiisopropyltryptamine Dorzolamide C17H26N2O 274.2 C17H26N2O 274.2 C10H16N2O4S3 324.0 4-hydroxyalprazolam C17H13ClN4O 324.1 Alpha-hydroxyalprazolam C17H13ClN4O 324.1 Prazepam C19H17ClN2O 324.1 Acetohexamide C15H20N2O4S 324.1 Dolasetron C19H20N2O3 324.1 Ditazole C19H20N2O3 324.1 Valaciclovir C13H20N6O4 324.2 Quinidine C20H24N2O2 324.2 Quinine C20H24N2O2 324.2 Diampromide C21H28N2O 324.2 Clorazepate C16H11ClN2O3 314.0 Dantrolene C14H10N4O5 314.1 Sulfaphenazole C15H14N4O2S 314.1 Pergolide C19H26N2S 314.2 Bromazepam C14H10BrN3O 315.0 Efavirenz C14H9ClF3NO2 315.0 Chlorprothixene C18H18ClNS 315.1 Codeine N-oxide C18H21NO4 315.1 Rotigotine C19H25NOS 315.2 Alizapride C16H21N5O2 315.2 Mitiglinide C19H25NO3 315.2 Saxagliptin C18H25N3O2 315.2 Niclosamide 5-Fluorodeoxyuridine monophosphate Hydroxyhexamide C13H8Cl2N2O4 326.0 C9H12FN2O8P 326.0 C15H22N2O4S 326.1 Aceprometazine C19H22N2OS 326.1 Acepromazine C19H22N2OS 326.1 Ajmaline C20H26N2O2 326.2 185 Target Analyte Cocaethylene Cocaine Cocaine Cyclobenzaprine; MDPV Demoxepam; Oxazepam Desalkylflurazepam; Bupivacaine Desipramine; Atenolol Interference Compound Formula Molecular Weight Nilutamide C12H10F3N3O4 317.1 N2-Monodes-methylnizatidine C11H19N5O2S2 317.1 Arbutamine C18H23NO4 317.2 beta-oxycodol C18H23NO4 317.2 Nateglinide C19H27NO3 317.2 Tetrabenazine C19H27NO3 317.2 Butenafine C23H27N 317.2 Clofarabine C10H11ClFN5O3 303.1 Chlorambucil C14H19Cl2NO2 303.1 Flumazenil C15H14FN3O3 303.1 Pipemidic acid C14H17N5O3 303.1 Ezogabine C16H18FN3O2 303.1 Phenoxybenzamine C18H22ClNO 303.1 Fenoterol C17H21NO4 303.1 7-Hydroxyetodolac C17H21NO4 303.1 alpha-noroxycodol C17H21NO4 303.1 Scopolamine C17H21NO4 303.1 Hydromorphinol C17H21NO4 303.1 Vildagliptin C17H25N3O2 303.2 Iobenguane C8H10IN3 275.0 Lorcaserin sulfamate C11H14ClNO3S 275.0 4'-hydroxypropanolol C16H21NO3 275.2 Physostigmine C15H21N3O2 275.2 Alphameprodine C17H25NO2 275.2 norclobazam C15H11ClN2O2 286.1 nortemazepam C15H11ClN2O2 286.1 3-Hydroxynordiazepam C15H11ClN2O2 286.1 Phenytoin dihydrodiol C15H14N2O4 286.1 4-Hydroxy tolbutamide C12H18N2O4S 286.1 Abacavir 10-alpha-methoxy-9,10dihydrolysergol Tetrazepam C14H18N6O 286.2 C17H22N2O2 286.2 C16H17ClN2O 288.1 Sodium lauryl sulfate C12H25NaO4S 288.1 Carbamazepine-O-quinone C15H10N2O3 266.1 Nevirapine C15H14N4O 266.1 Practolol C14H22N2O3 266.2 Cyclizine C18H22N2 266.2 186 Target Analyte Desmethylclomipramine; Temazepam Dextromethorphan Diphenhydramine; Lamotrigine Diphenhydramine; Lamotrigine Donepezil Doxepin Duloxetine Interference Compound Formula Molecular Weight Tazobactam C10H12N4O5S 300.1 Clobazam C16H13ClN2O2 300.1 Promazine 5-sulfoxide C17H20N2OS 300.1 Chlorcyclizine 6,7-Dichloro-3-hydroxy-1,5 dihydroimidazo[2,1-b]quinazolin-2-one 4-hydroxy ketorolac C18H21ClN2 300.1 C10H7Cl2N3O2 271.0 C15H13NO4 271.1 Norhydromorphone C16H17NO3 271.1 Normorphine C16H17NO3 271.1 Bupranolol C14H22ClNO2 271.1 Desomorphine C17H21NO2 271.2 4-Hydroxyatomoxetine C17H21NO2 271.2 Gallium nitrate GaN3O9 254.9 Anagrelide C10H7Cl2N3O 255.0 Sulfathiazole C9H9N3O2S2 255.0 Pranoprofen C15H13NO3 255.1 Ketorolac C15H13NO3 255.1 Ganciclovir C9H13N5O4 255.1 Hydroxybupropion C13H18ClNO2 255.1 Atomoxetine C17H21NO 255.2 N-Demethyl orphenadrine C17H21NO 255.2 Tripelennamine C16H21N3 255.2 Trichlormethiazide C8H8Cl3N3O4S2 378.9 6-Thioguanosine monophosphate C10H14N5O7PS 379.0 6-Thioguanylic acid C10H14N5O7PS 379.0 Droperidol C22H22FN3O2 379.2 7-Hydroxyticlopidine C14H14ClNOS 279.0 Ticlopidine S-oxide C14H14ClNOS 279.0 2-Oxoticlopidine C14H14ClNOS 279.0 Cidofovir C8H14N3O6P 279.1 Oxamniquine C14H21N3O3 279.2 E-10-Hydroxynortriptyline C19H21NO 279.2 Etamiphylline C13H21N5O2 279.2 Sibutramine C17H26ClN 279.2 Cisplatin Cl2H4N2Pt 296.9 2-Chloroticlopidine C14H13Cl2NS 297.0 Albendazole sulfone C12H15N3O4S 297.1 Nelarabine C11H15N5O5 297.1 mono-isopropyl-disopyramide C18H23N3O 297.2 187 Target Analyte Ephedrine; Pseudoephedrine Etomidate Interference Compound Formula Molecular Weight Benzocaine C9H11NO2 165.1 L-Phenylalanine C9H11NO2 165.1 4-Methoxyamphetamine C10H15NO 165.1 4-Hydroxymethamphetamine C10H15NO 165.1 Hordenine C10H15NO 165.1 Apraclonidine C9H10Cl2N4 244.0 Ribavirin C8H12N4O5 244.1 Azacitidine C8H12N4O5 244.1 Biotin C10H16N2O3S 244.1 Carbidopa C10H16N2O5 244.1 Rufinamide C10H8F2N4O 238.1 Captopril-cysteine disulfide C12H20N2O5S2 336.1 Berberine C20H18NO4 336.1 Acebutolol C18H28N2O4 336.2 Acetyl-alpha-methylfentanyl C22H28N2O 336.2 Miconazole C18H14Cl4N2O 414.0 Flecainide; Diltiazem Nafcillin C21H22N2O5S 414.1 N-desalkyl delavirdine C19H22N6O3S 414.1 Fluoxetine; Methadone Lamivudine-monophosphate 2,8-bis-Trifluoromethyl-4-quinoline carboxylic acid 3-oxobrimonidine C8H12N3O6PS 309.0 C12H5F6NO2 309.0 C11H12BrN5O 309.0 Hydroxylumiracoxib C15H13ClFNO3 309.1 Glycodiazine C13H15N3O4S 309.1 Ketotifen C19H19NOS 309.1 Metixene C20H23NS 309.2 Felbamate Fentanyl Fluoxetine; Methadone Flurazepam Gabapentin Nadolol C17H27NO4 309.2 Metipranolol C17H27NO4 309.2 Diphenidol C21H27NO 309.2 Dicyclomine C19H35NO2 309.3 5'-Hydroxylornoxicam C13H10ClN3O5S2 387.0 triazolopyridinone epoxide C19H22ClN5O2 387.1 4'-hydroxytrazodone C19H22ClN5O2 387.1 Terazosin C19H25N5O4 387.2 Tamoxifen N-oxide C26H29NO2 387.2 4-Hydroxytamoxifen C26H29NO2 387.2 alpha-Hydroxytamoxifen C26H29NO2 387.2 3-Hydroxytamoxifen (Droloxifene) C26H29NO2 387.2 Metronidazole C6H9N3O3 171.1 Rasagiline C12H13N 171.1 188 Target Analyte Haloperidol Hydroxychloroquine Hydroxyzine Interference Compound Formula Molecular Weight Azidocillin C16H17N5O4S 375.1 Tiagabine C20H25NO2S2 375.1 Gatifloxacin C19H22FN3O4 375.2 Benzylmorphine C24H25NO3 375.2 Almotriptan C17H25N3O2S 335.2 Naratriptan C17H25N3O2S 335.2 Bromhexine C14H20Br2N2 374.0 Etoricoxib 1'-N'-oxide C18H15ClN2O3S 374.0 6-Hydroxymethyletoricoxib C18H15ClN2O3S 374.0 N-Desmethylzopiclone C16H15ClN6O3 374.1 omega-hydroxyfinasteride C22H34N2O3 374.3 2-Amino-5-benzoylbenzimidazole C14H11N3O 237.1 7-hydroxyolanzapine C17H20N4OS 328.1 2-hydroxymethylolanzapine C17H20N4OS 328.1 Tiapride C15H24N2O4S 328.1 Methotrimeprazine C19H24N2OS 328.2 Labetalol 7-hydroxygranisetron C18H24N4O2 328.2 Stanozolol C21H32N2O 328.3 Levetiracetam Propylthiouracil C7H10N2OS 170.1 p-Chlorobenzene sulfonyl urea C7H7ClN2O3S 234.0 4,4'-methanol-bisbenzonitrile C15H10N2O 234.1 4'-hydroxymephenytoin C12H14N2O3 234.1 S-4-Hydroxymephenytoin C12H14N2O3 234.1 Epirizole C11H14N4O2 234.1 Enoxacin C15H17FN4O3 320.1 3,7-Dimethyluric acid C7H8N4O3 196.1 1,9-Dimethyluric acid C7H8N4O3 196.1 7,9-Dimethyluric acid C7H8N4O3 196.1 1,3-Dimethyluric acid C7H8N4O3 196.1 1,7-Dimethyluric acid C7H8N4O3 196.1 Hippuric acid C9H9NO3 179.1 Acetylisoniazid C8H9N3O2 179.1 Glucosamine C6H13NO5 179.1 Ketamine Labetalol Lidocaine Lorazepam mCPP MDA Mexiletine C11H17NO 179.1 Methylephedrine C11H17NO 179.1 Methoxyphenamine C11H17NO 179.1 Rimantadine C12H21N 179.2 Memantine C12H21N 179.2 189 Target Analyte MDMA Meperidine Mephedrone Mescaline Mescaline Metaxalone Methamphetamine Formula Molecular Weight C10H11NO3 193.1 C10H11NO3 193.1 C10H11NO3 193.1 C15H21NO2 247.2 desmethylprodine C15H21NO2 247.2 Ketobemidone C15H21NO2 247.2 Phenmetrazine C11H15NO 177.1 Bethanidine C10H15N3 177.1 N-Methylnicotinium 2,4-Dihydroxy-7-methoxy-2H-1,4benzoxazin-3(4H)-one Milrinone; Milrinone Lactate C11H17N2 177.1 C9H9NO5 211.0 C12H9N3O 211.1 5-Hydroxylorcaserin C11H14ClNO 211.1 1-Hydroxylorcaserin C11H14ClNO 211.1 7-Hydroxylorcaserin 4-(4-chlorophenyl)-4hydroxypiperidine Methyldopa C11H14ClNO 211.1 C11H14ClNO 211.1 C10H13NO4 211.1 3-O-Methyl-a-methyldopa C10H13NO4 211.1 Interference Compound 3-Carbamoyl-2phenylpropionaldehyde 4-Anilino-4-oxobutanoic acid 4-Hydroxy-5-phenyltetrahydro-1,3oxazin-2-one Pethidine Zalcitabine C9H13N3O3 211.1 Varenicline C13H13N3 211.1 Pramipexole C10H17N3S 211.1 Isoprenaline C11H17NO3 211.1 Orciprenaline C11H17NO3 211.1 Isoproterenol C11H17NO3 211.1 N-Acetyl-D-glucosamine C8H15NO6 221.1 N,N,O-Tridesmethyl-tramadol C13H19NO2 221.1 Procarbazine C12H19N3O 221.2 Tapentadol C14H23NO 221.2 NAPQI C8H7NO2 149.0 Penicillamine C5H11NO2S 149.1 L-Methionine C5H11NO2S 149.1 Sevelamer C6H12ClNO 149.1 Cathinone C9H11NO 149.1 Phentermine C10H15N 149.1 190 Target Analyte Methocarbamol Methylone Methylphenidate; Normeperidine Metoprolol Metoprolol Mirtazapine; Nordoxepine Mirtazapine; Nordoxepine Naproxen; TFMPP Interference Compound Formula Molecular Weight Moxonidine C9H12ClN5O 241.1 3'-Amino-3'-deoxythimidine C10H15N3O4 241.1 Mefenamic acid C15H15NO2 241.1 Tetrahydrobiopterin C9H15N5O3 241.1 N,N-Didemethyl orphenadrine C16H19NO 241.1 N-Desmethyldiphenhydramine C16H19NO 241.1 triazolopropionic acid C9H9N3O3 207.1 Isoniazid pyruvate C9H9N3O3 207.1 Carbamazepine iminoquinone C14H9NO 207.1 N-isopropylterephthalamic acid 4-(Methylnitrosamino)-1-(3-pyridyl)1-butanone Ciclopirox C11H13NO3 207.1 C10H13N3O2 207.1 C12H17NO2 207.1 N-Desmethyl tapentadol C13H21NO 207.2 Dopamine 3-O-sulfate C8H11NO5S 233.0 Dopamine 4-sulfate C8H11NO5S 233.0 Lomustine C9H16ClN3O2 233.1 N,N-Didesmethyltramadol C15H23NO 233.2 Sulfisoxazole C11H13N3O3S 267.1 Sulfamoxole C11H13N3O3S 267.1 Zidovudine C10H13N5O4 267.1 Adenosine C10H13N5O4 267.1 Vidarabine C10H13N5O4 267.1 4-amino-MX C12H17N3O4 267.1 Apomorphine C17H17NO2 267.1 Voglibose C10H21NO7 267.1 Desacetyl-nitazoxanide C10H7N3O4S 265.0 Isoniazid alpha-ketoglutaric acid C11H11N3O5 265.1 Albendazole C12H15N3O2S 265.1 Streptozocin C8H15N3O7 265.1 Thiamine C12H17N4OS 265.1 E-10-Hydroxydesmethylnortriptyline C18H19NO 265.1 Antazoline C17H19N3 265.2 Oxprenolol C15H23NO3 265.2 4-Hydroxy-alprenolol C15H23NO3 265.2 Diazoxide C8H7ClN2O2S 230.0 Guanabenz C8H8Cl2N4 230.0 Hydralazine pyruvate hydrazone C11H10N4O2 230.1 cyclic Melatonin C13H14N2O2 230.1 Ibudilast C14H18N2O 230.1 191 Target Analyte Norbuprenorphine Norclozapine; Olanzapine Nordiazepam; Doxylamine Norfluoxetine Norfluoxetine Norketamine Norpropoxyphene; Midazolam Interference Compound Formula Molecular Weight Desacetylcefotaxime C14H15N5O6S2 413.0 Dihydroetorphine C25H35NO4 413.3 Ethopropazine C19H24N2S 312.2 Praziquantel C19H24N2O2 312.2 Granisetron C18H24N4O 312.2 Oseltamivir C16H28N2O4 312.2 Sulfamethizole C9H10N4O2S2 270.0 Leflunomide C12H9F3N2O2 270.1 A771726 C12H9F3N2O2 270.1 10,11-Dihydroxycarbamazepine C15H14N2O3 270.1 Tolbutamide C12H18N2O3S 270.1 Chloroprocaine C13H19ClN2O2 270.1 N-Desmethylpromazine C16H18N2S 270.1 Chlorothiazide C7H6ClN3O4S2 294.9 Meclofenamic acid C14H11Cl2NO2 295.0 Diclofenac C14H11Cl2NO2 295.0 N4-Acetylsulfamethoxazole C12H13N3O4S 295.1 Mebendazole C16H13N3O3 295.1 Citalopram aldehyde C18H14FNO2 295.1 Nor-ketotifen C18H17NOS 295.1 Sumatriptan C14H21N3O2S 295.1 (E)-2-hydroxydoxepin C19H21NO2 295.2 Doxepin N-oxide C19H21NO2 295.2 Tertatolol C16H25NO2S 295.2 Esmolol C16H25NO4 295.2 Cerulenin C12H17NO3 223.1 Neostigmine C12H19N2O2 223.1 2,5-Dimethoxy-4-ethylamphetamine C13H21NO2 223.2 Amdinocillin C15H23N3O3S 325.1 Ergonovine C19H23N3O2 325.2 dinor-Levomethadyl acetate C21H27NO2 325.2 Bisoprolol C18H31NO4 325.2 Dapiprazole C19H27N5 325.2 Tolterodine C22H31NO 325.2 192 Target Analyte Norsertraline Nortramadol Norvenlafaxine; Nortrityline, Tramaol Oxymorphone Oxymorphone Paroxetine Interference Compound Formula Molecular Weight Brimonidine C11H10BrN5 291.0 Orciprenaline-3-O-sulfate C11H17NO6S 291.1 Diethylthiambutene C16H21NS2 291.1 Desethylchloroquine C16H22ClN3 291.2 Cyclopentolate C17H25NO3 291.2 Levobunolol C17H25NO3 291.2 Terbinafine C21H25N 291.2 Penbutolol C18H29NO2 291.2 Alendronate C4H13NO7P2 249.0 Norepinephrine sulfate C8H11NO6S 249.0 Sulfapyridine C11H11N3O2S 249.1 Epinastine C16H15N3 249.1 Desmethylnortriptyline C18H19N 249.2 Tenocyclidine C15H23NS 249.2 Alprenolol C15H23NO2 249.2 N,O-Didesmethylvenlafaxine C15H23NO2 249.2 Amfecloral C11H12Cl3N 263.0 Epinephrine sulfate C9H13NO6S 263.0 Ticlopidine C14H14ClNS 263.1 Gemcitabine C9H11F2N3O4 263.1 Dimethylthiambutene C14H17NS2 263.1 desethylzaleplon C15H13N5 263.1 Protriptyline C19H21N 263.2 demethylmaprotiline 2-Ethyl-5-methyl-3,3-diphenyl-1pyrroline Protriptyline C19H21N 263.2 C19H21N 263.2 C19H21N 263.2 Lisdexamfetamine C15H25N3O 263.2 Carboxybupranolol C14H20ClNO4 301.1 Noroxycodone C17H19NO4 301.1 Dihydrocodeine C18H23NO3 301.2 Dobutamine C18H23NO3 301.2 Trihexyphenidyl C20H31NO 301.2 Nitisinone C14H10F3NO5 329.1 8-Hydroxyamoxapine C17H16ClN3O2 329.1 Dopamine glucuronide C14H19NO8 329.1 Cloperastine C20H24ClNO 329.2 Trilostane C20H27NO3 329.2 N-desethyloxybutynin C20H27NO3 329.2 193 Target Analyte PCP Pregabalin Primidone; Meprobamate Promethazine; Diazepam Propoxyphene; Papaverine; Topiramate Propranolol Propranolol Interference Compound Formula Molecular Weight Cytarabine C9H13N3O5 243.1 Agomelatine C15H17NO2 243.1 Frovatriptan C14H17N3O 243.1 Pargyline C11H13N 159.1 Mephenytoin C12H14N2O2 218.1 N-Acetylserotonin C12H14N2O2 218.1 N-despropyl ropinirole C13H18N2O 218.1 6-Thioinosinic acid C10H12N4O4S 284.1 Mazindol C16H13ClN2O 284.1 Phenytoin catechol C15H12N2O4 284.1 arabinofuranosylguanine C10H14N5O5 284.1 Etozoline C13H20N2O3S 284.1 Articaine C13H20N2O3S 284.1 Promazine C17H20N2S 284.1 Tropicamide C17H20N2O2 284.2 Cefacetrile C13H13N3O6S 339.1 Methylhydroxygliclazide C15H21N3O4S 339.1 7-Hydroxygliclazide C15H21N3O4S 339.1 Alogliptin C18H21N5O2 339.2 Methylergonovine C20H25N3O2 339.2 Methylergometrine C20H25N3O2 339.2 noracymethadol C22H29NO2 339.2 nor-Levomethadyl acetate C22H29NO2 339.2 Disopyramide C21H29N3O 339.2 Hexetidine 4-Bromo-2,5dimethoxyphenethylamine Mizoribine C21H45N3 339.4 C10H14BrNO2 259.0 C9H13N3O6 259.1 Lenalidomide C13H13N3O3 259.1 Clobenzorex C16H18ClN 259.1 norzolmitripan C14H17N3O2 259.1 Ramelteon C16H21NO2 259.2 Primaquine C15H21N3O 259.2 Eperisone C17H25NO 259.2 194 Target Analyte Quetiapine Risperidone Sertraline; Zaleplon Sildenafil Trazodone Triazolam Trimipramine Verapamil Ziprasidone Zopiclone Interference Compound Formula Molecular Weight clofarabind-5'-monophosphate C10H12ClFN5O6P 383.0 Ceftizoxime C13H13N5O5S2 383.0 Brinzolamide C12H21N3O5S3 383.1 Felodipine C18H19Cl2NO4 383.1 Pantoprazole C16H15F2N3O4S 383.1 Meropenem C17H25N3O5S 383.2 Prazosin C19H21N5O4 383.2 Butoconazole C19H17Cl3N2S 410.0 Ceftibuten C15H14N4O6S2 410.0 Fenoldopam C16H16ClNO3 305.1 Entacapone C14H15N3O5 305.1 Vandetanib C22H24BrFN4O2 474.1 Lornoxicam C13H10ClN3O4S2 371.0 Carboplatin C6H12N2O4Pt 371.0 Berberine chloride C20H18ClNO4 371.1 Camazepam C19H18ClN3O3 371.1 Isradipine C19H21N3O5 371.1 Tamoxifen C26H29NO 371.2 5-Fluorouridine monophosphate C9H12FN2O9P 342.0 Etizolam C17H15ClN4S 342.1 Clozapine N-oxide C18H19ClN4O 342.1 Estazolam C16H11ClN4 294.1 Sulfacytine C12H14N4O3S 294.1 Rosoxacin C17H14N2O3 294.1 Aspartame C14H18N2O5 294.1 Alosetron C17H18N4O 294.1 Proparacaine C16H26N2O3 294.2 Dimetacrine C20H26N2 294.2 Sitaxentan C18H15ClN2O6S2 454.0 Cefazolin C14H14N8O4S3 454.0 Methotrexate C20H22N8O5 454.2 Zileuton O-glucuronide C17H20N2O8S 412.1 O-Deethylated candesartan C22H16N6O3 412.1 Cinalukast C23H28N2O3S 412.2 Trandolapril-d5 Diketopiperazine C24H32N2O4 412.2 Sulfinpyrazone sulfide C23H20N2O2S 388.1 Bepotastine C21H25ClN2O3 388.2 195 Target Analyte Zopiclone 10-monohydroxyoxcarbazepine 6-acetylmorphine 7-aminoclonazepam; Hydromorphone, Morphine 7-aminoflunitrazepam 9-hydroxyrisperidone Interference Compound Formula Molecular Weight Cetirizine C21H25ClN2O3 388.2 Bepotastine C21H25ClN2O3 388.2 Nisoldipine C20H24N2O6 388.2 Trimethobenzamide C21H28N2O5 388.2 Ramiprilat C21H28N2O5 388.2 N-acetyl zonisamide C10H10N2O4S 254.0 Acetaminophen cystein C11H14N2O3S 254.1 2-Hydroxyfelbamate C11H14N2O5 254.1 Dyphylline C10H14N4O4 254.1 Nepafenac C15H14N2O2 254.1 Thiamylal C12H18N2O2S 254.1 Midodrine C12H18N2O4 254.1 Diloxanide C14H11Cl2NO4 327.0 Acetaminophen glucuronide C14H17NO8 327.1 Desethylamodiaquine C18H18ClN3O 327.1 Naloxone C19H21NO4 327.1 Dimenoxadol C20H25NO3 327.2 Butorphanol C21H29NO2 327.2 Norelgestromin C21H29NO2 327.2 Butorphanol C21H29NO2 327.2 Cladribine C10H12ClN5O3 285.1 Faropenem C12H15NO5S 285.1 Letrozole C17H11N5 285.1 Probenecid C13H19NO4S 285.1 Isothipendyl C16H19N3S 285.1 Norcodeine C17H19NO3 285.1 Norhydrocodone C17H19NO3 285.1 N-Monodesmethyl-rizatriptan C15H19N5O 285.2 Mepyramine; Pyrilamine C17H23N3O 285.2 Risedronate C7H11NO7P2 283.0 Oxtriphylline; Choline theophylline C12H21N5O3 283.2 Cadralazine C12H21N5O3 283.2 alpha-Hydroxymetoprolol C15H25NO4 283.2 Levallorphan C19H25NO 283.2 N-Dealkylated tolterodine C19H25NO 283.2 Iloperidone C24H27FN2O4 426.2 Darifenacin C28H30N2O2 426.2