Simultaneous quantification of atenolol and chlorthalidone in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry.

A simple, sensitive and reproducible ultra-performance liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous determination of atenolol, a β-adrenergic receptor-blocker and chlorthalidone, a monosulfonamyl diuretic in human plasma, using atenolol-d7 and chlorthalidone-d4 as the internal standards (ISs). Following solid-phase extraction on Phenomenex Strata-X cartridges using 100 μL human plasma sample, the analytes and ISs were separated on an Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 µm) column using a mobile phase consisting of 0.1% formic acid-acetonitrile (25:75, v/v). A tandem mass spectrometer equipped with electrospray ionization was used as a detector in the positive ionization mode for both analytes. The linear concentration range was established as 0.50-500 ng/mL for atenolol and 0.25-150 ng/mL for chlorthalidone. Extraction recoveries were within 95-103% and ion suppression/enhancement, expressed as IS-normalized matrix factors, ranged from 0.95 to 1.06 for both the analytes. Intra-batch and inter-batch precision (CV) and accuracy values were 2.37-5.91 and 96.1-103.2%, respectively. Stability of analytes in plasma was evaluated under different conditions, such as bench-top, freeze-thaw, dry and wet extract and long-term. The developed method was superior to the existing methods for the simultaneous determination of atenolol and chlorthalidone in human plasma with respect to the sensitivity, chromatographic analysis time and plasma volume for processing. Further, it was successfully applied to support a bioequivalence study of 50 mg atenolol + 12.5 mg chlorthalidone in 28 healthy Indian subjects.