Chromatographic separation of (E)- and (Z)-isomers of entacapone and their simultaneous quantitation in human plasma by LC-ESI-MS/MS.
Author(s): Yadav M, Dixit P, Trivedi V, Gandhi A, Senger A, Guttikar S, Singhal P, Shrivastav PS
Affiliation(s): Bioanalytical Research Department, Veeda Clinical Research, Ambawadi, Ahmedabad 3800015, Gujarat, India.
Publication date & source: 2009-02-15, J Chromatogr B Analyt Technol Biomed Life Sci., 877(5-6):533-40. Epub 2009 Jan 13.
Publication type: Randomized Controlled Trial
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 500microL of human plasma. Both the analytes were chromatographically separated with a resolution factor of 3.0 on a Gemini C18 (50mmx4.6mm, 5microm particle size) analytical column using 1% formic acid and methanol (50:50, v/v) as the mobile phase. The selectivity factor (alpha) 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 24.3-6076ng/mL and 23.8-5960ng/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 200mg (E)-entacapone tablet formulation under fasting conditions.