Quantifying Pomalidomide in Human Plasma Employing UPLC-MS/MS

Abstract

The current work aims to create a simple, fast, clear and accurate chromatography-tandem mass spectrophotometric (UPLC-MS/MS). 50µl ISTD (250ng/ml concentration) and 250µl of 5mM ammonium formate buffer (pH - 2.5) were put into the samples just before liquid-liquid extraction (LLE) using 2.5ml of tertiary butyl methyl ether (TBME). Chromatographic fraction was done using Hypersil gold column (50 mmX4.65mm, 5µ) with use of 5mM ammonium formate buffer (pH - 2.5) and acetone in 20: 80 v/v (mobile phase). The rate of flow was around 0.50ml/min. Pomalidomide and Celecoxib were ionized into ions with polarity (positive) and were recognized using MRM. Quantitation was made by transitions m/z 274.43 precursor ion to m/z 201.15 for drug Pomalidomide and m/z 382.12 precursor ion to m/z 362.0 of celecoxib. The concentrations of eight standards exhibited linearity between 1.9900 to 199.8380ng/ml (r2 ≥ 0.9968). Separation of chromatogram was obtained in two minutes. The recovery values in an average for three QC’s was 51.49% of Pomalidomide which was in concordance with acceptance. The Coefficient of variation was ≤15% in intra and inter batch assay values. The %CV ruggedness ranges from 1.26 - 2.25. Short and long term stability percentages of stock solutions were found acceptable like 99.76 and 98.69. Results show that all the validation parameters were in acceptable limits.