Simultaneous Quantification of Vincristine and Its Major M1 Metabolite from Dried Blood Spot Samples of Kenyan Pediatric Cancer Patients by UPLC-MS/MS

Abstract

Vincristine (VCR) is an integral part of chemotherapy regimens in the US and in developing countries. There is a paucity of information about its disposition and optimal therapeutic dosing. VCR is preferentially metabolized to its major M1 metabolite by the polymorphic CYP3A5 enzyme, which may be clinically significant as CYP3A5 expression varies across populations. Thus, it is important to monitor both VCR and M1 and characterize their dispositions. A previously developed HPLC-MS/MS method for VCR quantification was not sensitive enough to quantify the M1 metabolite beyond 1 hr. post VCR dose (not published). Establishing a highly sensitive assay is a pre-requisite to simultaneously quantify and monitor VCR and M1, which will enable characterization of drug exposure and dispositions of both analytes in a pediatric cancer population. The addition of formic acid during the extraction process enhanced M1 extraction from DBS samples. A sensitive, accurate, and precise UPLC-MS/MS assay method for the simultaneous quantification of VCR and M1 from human dried blood spots (DBS) was developed and validated. Chromatographic separation was performed on Inertsil ODS-3 C18 column (5 μm, 3.0 x 150 mm). A gradient elution of mobile phase A (methanol-0.2% formic acid in water, 20:80 v/v) and mobile phase B (methanol-0.2% formic acid in water, 80:20 v/v) was used with a flow rate of 0.4 mL/min and a total run time of 5 min. The analytes were ionized by electrospray ionization in the positive ion mode. The linearity range for both analytes in DBS were 0.6-100 ng/ml for VCR and 0.4-100 ng/ml for M1. The intra- and inter-day accuracies for VCR and M1 were 93.10-117.17% and 95.88-111.21%, respectively. While their intra- and inter-day precisions were 1.05 to 10.11% and 5.78 to 8.91%, respectively. The extraction recovery of VCR from DBS paper was 35.3 – 39.4% and 10.4 – 13.4% for M1, with no carryover observed for both analytes. This is the first analytical method to report the simultaneous quantification of VCR and M1 from human DBS. For the first time, concentrations of M1 from DBS patient samples were obtained beyond 1 -h post VCR dose. The developed method was successfully employed to monitor both compounds and perform pharmacokinetic analysis in a population of Kenyan pediatric cancer patients.