Clinical Applications of Mass Spectrometry in Drug Analysis

Ryan C. Schofield, Lakshmi V. Ramanathan, Kazunori Murata, Martin Fleisher, Melissa S. Pessin, Dean C. Carlow, Schofield, Ryan C., Ramanathan, Lakshmi V., Murata, Kazunori, Fleisher, Martin, Pessin, Melissa S., Carlow, Dean C.

Methotrexate (MTX) is a folic acid antagonist that is widely used as an immunosuppressant and chemotherapeutic agent. After high-dose administration of MTX serum levels must be monitored to determine when to administer leucovorin, a folic acid analog that bypasses the enzyme inhibition caused by MTX and reverses its toxicity. We describe a rapid and simple turbulent flow liquid chromatography (TFLC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of MTX, 7-hydroxymethotrexate (7-OH MTX), and 4-amino-4-deoxy-N(10)-methylpteroic acid (DAMPA) concentrations in serum. MTX is isolated from serum samples (100 μL) after protein precipitation with a methanolic solution containing internal standard (MTX-D3) followed by centrifugation. The supernatant is injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFLC-ESI-MS/MS) and quantified using a six-point calibration curve. For MTX, 7-OH MTX, and DAMPA the assays were linear from 20 to 1000 nmol/L. Dilutions of 10-, 100-, and 1000-fold were validated giving a clinically reportable range of 20 to1.0 × 10(6) nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10 % for all three analytes.