Validation of LC-MS/MS Method for Monitoring of Upadacitinib in Rat Plasma Samples

Abstract

Aim: Upadacitinib (UDC) is a potent, orally active and selective Janus kinase-1 inhibitor. Upadacitinib is used in development for the treatment of several autoimmune disorders. In the present investigation, a rapid, specific, selective and novel method has been optimized for evaluation
of Upadacitinib in plasma using Methotrexate (MTX) as an internal standard by LC-MS/MS.
Methods: The Upadacitinib and Methotrexate extracted from rat plasma using liquid-liquid extraction method using tert-Butyl methyl ether as
extraction solvent. The principle analytes were eluted with the conditions of mobile phase having the 0.1% Formic acid : Acetonitrile (20:80%,
v/v) using the Eclipse Plus C18, 4.6 mm × 150 mm, (5 μm) analytical column with the 0.6 ml/min flow rate and 10 µl sample volume using
CEM array detector. The retention times of Upadacitinib and Methotrexate were 1.65±0.05 min 0.58±0.05 min with the total run time of 4 min.
The curve indicates correlation coefficient (r2
) was superior by having the value nearer to 1.0 with linear range of 10.0 n.g/m.l to 500.0 n.g/m.l
Results: The correlation coefficient (r2
) for Upadacitinib was found 0.999. The LOQ and LOD for the Upadacitinib found 1.03 n.g/m.l and
0.34 n.g/m.l
Conclusion: The method was validated to comply with the United States Food and Drug Administration (FDA) rules by evaluating system
suitability, specificity, sensitivity, linearity, precision, precision, roughness, and stability. The verification parameter results were found to
be within acceptable limits. Thus the method which has been originally developed should be used for routine Upadacitinib measurement in
plasma samples.