Development and Validation of Novel Analytical LC-MS Method for Simultaneous Quantification of Calcitonin Gene-Related Peptide Receptor Antagonists Ubrogepant and Atogepant in Human Plasma

Abstract

A simple, highly sensitive and selective liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for separation and simultaneous assay of approved calcitonin gene-related peptide receptor antagonists ubrogepant and atogepant in human plasma using frovatriptan as internal standards. Analytes were extracted using protein precipitation induced by acetonitrile followed by liquid-liquid extraction using dichloromethane. RP-HPLC analysis was carried using Xbridge C₁₈ column (50 mm × 4.6 mm, 5 μm) with a simple isocratic mobile phase composed of 0.01% NH₃ in 2 mM ammonium formate at pH 6.4 and acetonitrile at 45:55 (v/v) as mobile phase at 0.4 mL/min flow rate. Detection was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring (MRM), with the transitions of m/z 550→58, m/z 604→147, m/z and m/z 244→156 for ubrogepant, atogepant and frovatriptan, respectively, in the positive ion mode. The analysis was carried out within 5 min over a linear concentration range of 15-600 ng/mL for both ubrogepant and atogepant. The method was validated in accordance with the FDA guidelines for bioanalytical method and the results were within the acceptable limit for both the analytes. The method is useful for the analysis of ubrogepant and atogepant in human plasma and may also applicable for the pharmacokinetic study.