A SENSITIVE, HIGH THROUGHPUT ESTIMATION OF METHOTREXATE IN HUMAN PLASMA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRYAbstract
A sensitive and high throughput high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method has been developed for estimation of methotrexate in human plasma. The sample extraction of methotrexate and methotrexate-d3 (Internal standard) was done from 200 µL of human plasma using solid phase extraction procedure. The chromatographic retention was done on Thermo Butyl Hypersil, 50 x 4.6 mm, 5µm column under isocratic conditions using acetonitrile, 10 mM ammonium formate and 0.5% Formic aid (70:15:15, v/v) as the mobile phase. The mass transition for methotrexate (m/z 455.3 → 308.2), and IS (m/z 458.3 → 311.2) were monitored in the positive ionization mode. The method was validated over a wide-ranging concentration of 0.500-1000 ng/mL. The extraction recovery for the analyte and internal standard was 85.6 %. Stability of methotrexate in plasma was examined under different storage conditions like bench top, wet extract, freeze-thaw and long-term stability. The validated method was used to support a bioequivalence study of 15 mg methotrexate tablet in 16 adult patients. The method reproducibility was proved by reanalysis of incurred samples.
V. Upadhyay*, M. Rajput, A. Sen, S. Suvarna and S. Dhanse
Bioanalytical Department - Clinical Research Services, Reliance Life Sciences Private Limited, Dhirubhai Ambani Life Sciences Centre (DALC), Thane - Belapur Road, Rabale, Navi Mumbai , Maharashtra, India
17 January, 2017
20 March, 2017
27 May, 2017
01 August, 2017