Abstract
Objective To establish and validate a simple, sensitive, and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of methotrexate (MTX) and its major metabolite 7-hydroxy-methotrexate (7-OH-MTX) in human plasma. Method The chromatographic separation was achieved on a Zorbax C18 column (3.5 μm, 2.1 × 100 mm) using a gradient elution with methanol (phase B) and 0.2% formic acid aqueous solution (phase A). The flow rate was 0.3 mL/min with analytical time of 3.5 min. Mass spectrometry detection was performed in a triple-quadruple tandem mass spectrometer under positive ion mode with the following mass transitions: m/z 455.1/308.1 for MTX, 471.0/324.1 for 7-OH-MTX, and 458.2/311.1 for internal standard. The pretreatment procedure was optimized with dilution after one-step protein precipitation. Results The calibration range of methotrexate and 7-OH-MTX was 5.0-10000.0 ng/mL. The intraday and interday precision and accuracy were less than 15% and within ±15% for both analytes. The recovery for MTX and 7-OH-MTX was more than 90% and the matrix effect ranged from 97.90% to 117.60%. Conclusion The method was successfully developed and applied to the routine therapeutic drug monitoring of MTX and 7-OH-MTX in human plasma.
Highlights
It was estimated that there were 385,700 new cases and 199,700 deaths of non-Hodgkin lymphoma (NHL) occurred in 2012 worldwide [1]
We report a newly developed and validated UHPLC-MS/MS method for the simultaneous determination of MTX and 7-OH-MTX in human plasma and the application was completed on clinical samples from NHL patients
The results showed that the highest sensitivity could be obtained after a 5 times’ dilution with the 20% methanol aqueous solution to the supernatant and the sensitivity was enhanced to 5 ng/mL for MTX and 7-OH-MTX
Summary
It was estimated that there were 385,700 new cases and 199,700 deaths of non-Hodgkin lymphoma (NHL) occurred in 2012 worldwide [1]. The incidence of NHL increased in the majority of more developed countries up to 1990 and leveled off thereafter [2, 3]. Plus calcium folinate (CF) is one of the classic therapeutic regimens for the treatment of NHL. MTX is a folate analogue that competitively inhibits dihydrofolate reductase and subsequently blocks the formation of tetrahydrofolate from dihydrofolate. Tetrahydrofolate is a one-carbon donor for the nucleic acid synthesis [4]. MTX could suppress the polyamine formation and methylation of proteins
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