Development and validation of bio analytical liquid chromatography and mass spectrometry method for quantification of upadacitinib in human plasma

Abstract

In the present investigation, a simple, rapid and sensitive liquid chromatography and mass spectrometry (LC/MS) method was developed for quantification of Upadacitinib (UCB) in spiked human plasma. The drug UCB was extracted by liquid –liquid extraction using diethyl ether and chloroform in the ratio of 75:25 (v/v). The drug Leflunomide (LFM) was used as an internal standard. The extracted drug mixture was followed by the liquid chromatography mass spectrometry (LC/MS) analysis and electrospray ionization interface. The chromatographic separation of UCB was carried out on Phenomenex Luna C18 column (100mm X 2.0 mm X 5μm) at ambient temperature followed by isocratic elution of 0.05% formic acid in methanol and acetonitrile in the ratio of 65:30(v/v). The method is concluded at a flow rate of 0.8 mL/min, 262 nm of UV detector wavelength. Protonated ions formed by electrospray ionization were recorded in the positive mode and were used to detect the analyte (UCB) with internal standard (LFM). The mass detection was made by monitoring the fragmentation of m/z 381.0 for UCB m/z 271.0 for LCM on mass spectrometer. The method was validated for accuracy, precision, linearity and recovery. The assay was linear over the entire range of calibration standards i.e 2.5-500ng/mL. The recoveries of the UCB after liquid-liquid extraction at 5, 20, 35 ng/mL were 100.7, 99.4 and 99.8. The lowest limit of the analytical method of UCB was 2 ng/mL in spiked human plasma. The developed method was successfully validated and can be used for determination of the pharmacokinetic parameters of the UCB in biological samples.