Bioanalytical method development and validation of alimemazine in human plasma by LC-MS/MS and its application in bioequivalence studies.

Background: The use of anti-histaminic agents has been increased significantly from last decades and till now no method is available for quantitation of ALZ in human plasma which can be applied in a bioequivalence study using LC-MS/MS.

Objective: The present study is concerned with the development and validation of ALZ in human plasma by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS).

Methods: Sample preparation involved the extraction with liquid-liquid extraction method by using ethyl acetate as an organic solvent. Chromatographic separation was performed on Atlantis® T3 5 μm 4.6 mm × 150 mm column with the mobile phase consisting of acetonitrile: (10 mm ammonium formate buffer: Formic acid: 99.9:00.1 v/v) 50:50 v/v. The interface used with the application programming interface 4000 LC-MS/MS was a turbo ion spray in which positive ions were measured in multiple reaction monitoring mode. The precursor to product ions transition of m/z 299.30 → 100.20 amu and 305.30 → 106.30 amu were used for ALZ and ALZ D6 respectively.

Results: The method was validated over the concentration range of 20.013-10006.551 pg/mL. The mean percent recovery of ALZ was found 77.771% with a precision of 7.71% and the lower limit of quantification was 20.013 pg/mL. The intra- and inter-day precision of the method at three concentrations was 0.98-4.50% and 1.57-5.72% while the intra- and inter-day % accuracy was 99.02-93.82% and 101.78-106.96%. Stability of compounds was established in a series of stability studies. The application of this method was demonstrated in the bioequivalence study and was found suitable in a study of sample size as big as 30 enrolled volunteers.

Conclusions: For the very first time, a sensitive, selective and robust Liquid Chromatography- Mass Spectrometry method for the determination of alimemazine (ALZ) in human plasma has been developed and validated using ALZ D6 as an internal standard.