Author(s): Konsam Hamlet Meetei, Eswaran. K, P. R. Vijaianand, Sam Solomon and R. Venkatanaryanan
A sensitive and specific ultra-performance liquid chromatography combined with electrospray ionization (ESI) tandem mass spectrometry (UPLC-MS/MS) method, operating in the positive ionization mode, for the determination of Colchicine in human plasma using Colchicine D6 as internal standard (IS) was developed and validated. The analyte and IS were extracted by simple liquid-liquid extraction with a mixture of nHexane: Dichloromethane: Isopropyl Alcohol (60:30:10, v/v/v). The chromatographic separation was performed on a Purospher RP 18 (100×4.6 mm, 5μm) analytical column under isocratic conditions using a mixture of Acetonitrile: 0.05% Ammonia (90:10, v/v) as mobile phase at a flow rate of 0.500 mL/min. Total chromatographic run time was 3.0 min. Detection was performed on a XEVO TQ-S mass spectrometer by Waters. Quantitation was performed using multiple reaction monitoring (MRM) mode to study parent product ion transitions of m/z 400.1?358.2 for Colchicine and m/z 406.1?362.2 for IS respectively. The method was validated over the concentration range 0.04-7.56 ng/mL, with a coefficient of determination (r 2 ) of 0.9968. Limit of detection and limit of quantification were found 40.85 pg and 113.01 pg respectively. The retention time for Colchicine and IS were 2.12 min and 2.11 min respectively and overall chromatography run time was 3.0 minutes. The mean recovery of Colchicine and internal standard is 57.11% and 55.39% respectively. The method was validated for linearity, accuracy, precision, specificity, limit of detection, limit of quantification and robustness. The intra- and inter-day precision and accuracy values were found to be within the assay variability limits as per the FDA guidelines.