Development and
validation of bio analytical liquid chromatography and mass spectrometry method
for quantification of upadacitinib in human plasma
Srinivasarao Pakalapati, Chidananda Swamy Rumalla, Raghu Babu Korupolu and Muralidharan
Kaliyaperumal
Res. J. Chem. Environ; Vol. 25(8); 7-16;
doi: https://doi.org/10.25303/258rjce0716; (2021)
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.
