Chromatographic separation of ( E)- and ( Z)-isomers of entacapone and their simultaneous quantitation in human plasma by LC–ESI-MS/MS

Abstract

A selective, sensitive and high throughput liquid chromatography–tandem mass spectrometry (LC–ESI-MS/MS) method has been developed and validated for the chromatographic separation and quantitation of ( E)-entacapone and ( Z)-entacapone in human plasma. Sample clean-up involved liquid–liquid extraction (LLE) of both the isomers and carbamazepine used as internal standard from 500 μL of human plasma. Both the analytes were chromatographically separated with a resolution factor of 3.0 on a Gemini C18 (50 mm × 4.6 mm, 5 μm particle size) analytical column using 1% formic acid and methanol (50:50, v/v) as the mobile phase. The selectivity factor ( α) of the column for the separation was 2.0, based on the capacity factors of 2.6 and 1.3 for ( E)- and ( Z)-isomers respectively. The parent → product ion transitions for both the isomers ( m/ z 306.1 → 233.0) and IS ( m/ z 237.3 → 194.2) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was validated over the concentration range of 24.3–6076 ng/mL and 23.8–5960 ng/mL for ( E)-entacapone and ( Z)-entacapone respectively. Matrix effect was assessed by post-column analyte infusion experiment and the process/extraction efficiency found was 94.3% and 89.3% for ( E)- and ( Z)-isomers respectively. The method was successfully applied to a pivotal bioequivalence study in 36 healthy human subjects after oral administration of 200 mg ( E)-entacapone tablet formulation under fasting conditions.