Development and validation of a turbulent flow chromatography and tandem mass spectrometry method for the quantitation of methotrexate and its metabolites 7-hydroxy methotrexate and DAMPA in serum

Abstract

A rapid and simple turbulent flow liquid chromatography (TFC–LC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of methotrexate (MTX), 7-hydroxy methotrexate (7-OH MTX), and 4-amino-4-deoxy-N10-methylpteroic acid (DAMPA) concentrations in serum was developed. MTX was isolated from serum samples (100 μL) after protein precipitation with methanol containing formic acid and internal standard (MTX-D3) followed by centrifugation. The supernatant was injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFC–LC–MS/MS) and quantified using a six-point calibration curve. For MTX and DAMPA the assays were linear from 10 to 1000 nmol/L and for 7-OH MTX from 20 to 2000 nmol/L. Dilutions of 10, 100 and 1000-fold were validated giving a clinically reportable range of 10 nmol/L to 5 × 105 nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10% for all three analytes. MTX, DAMPA and 7-OH MTX were sufficiently stable under all relevant analytical conditions. No significant matrix effect was observed during the method validation. The TFC–LC-MS/MS MTX method was also compared with three other clinically validated MTX assays: a dihydrofolate reductase (DHFR) inhibition assay, an immunoassay based on fluorescence polarization and a previously developed LC–MS/MS assay.

A Simple and Sensitive Method for Quantitative Measurement of Methylmalonic Acid by Turbulent Flow Chromatography and Tandem Mass Spectrometry

“…An in-depth description of the system was previously published [16]. The triple quadrupole mass spectrometer was a Thermo Scientific TSQ Quantum Ultra (San Jose, CA, USA) and it implemented a heated electrospray ionization probe that was maintained at 380°C.…”

“…To evaluate matrix effects a post column infusion experiment was performed using an experimental set-up as described previously [16]. A tee was inserted between the outlet of the analytical column and a syringe was inserted into the tee to allow for the infusion of analyte into the mass spectrometer.…”

Antineoplastic drugs and their analysis: a state of the art review

“…4,9,10 Most of the reported methods are based on the use of liquid chromatography (LC) coupled with UV spectrophotometry (UV) 11,12,13,14 or mass spectrometry (MS), when there was a need for high selectivity and/or sensitivity. [15][16][17][18][19][20][21][22]23,24,25,26,27,28,29,30,31,32,33,34 Limits of detection 51 In our opinion, ELSD is not recommended for analysing pemetrexed since this molecule is UV-active and ELSD is often less sensitive and selective than the commonly used UV detectors. A methodology including a PP step followed by LC-MS analysis was also developed for the determination of raltitrexed in human plasma with a limit of quantification (LOQ) of 2 ng mL −1 .…”

On‐line approaches for the determination of residues and contaminants in complex samples

“…Methotrexate, a folic acid antagonist widely used as an immune suppressant and chemotherapeutic agent, was determined in serum using a Cyclone‐P (50 mm × 0.5 mm) as a first LC dimension and a Hypersil Gold C8 column (2.1 × 50 mm, 5 μm) with MS in tandem applied as detection technique . The flow rate applied to the loading step was 2.0 mL/min and 50 μL was the injection volume chose.…”