An improved LC-MS/MS method for the quantification of alverine and para hydroxy alverine in human plasma for a bioequivalence study☆

Abstract

A highly sensitive and selective high performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of alverine (ALV) and its active metabolite, para hydroxy alverine (PHA), in human plasma. For sample preparation, solid phase extraction of analytes was performed on Phenomenex Strata-X cartridges using alverine-d5 as the internal standard. The analytes were separated on Symmetry Shield RP18 (150 mm×3.9 mm, 5 µm) column with a mobile phase consisting of acetonitrile and 10 mM ammonium formate (65:35, v/v). Detection and quantitation was done by electrospray ionization mass spectrometry in the positive mode using multiple reaction monitoring. The assay method was fully validated over the concentration range of 15.0–15,000 pg/mL for ALV and 30.0–15,000 pg/mL for PHA. The intra-day and inter-day accuracy and precision (% CV) ranged from 94.00% to 96.00% and 0.48% to 4.15% for both the analytes. The mean recovery obtained for ALV and PHA was 80.59% and 81.26%, respectively. Matrix effect, expressed as IS-normalized matrix factor ranged from 0.982 to 1.009 for both the analytes. The application of the method was demonstrated for the specific analysis of ALV and PHA for a bioequivalence study in 52 healthy subjects using 120 mg ALV capsules. The assay reproducibility was also verified by reanalysis of 175 incurred subject samples.

Single Dose Study Assessing the Pharmacokinetic and Metabolic Profile of Alverine Citrate in Healthy Volunteers

“…The study confirmed previous observations that pharmacokinetic variability of parent alverine far exceeds the expectations one would get upon review of literature data, albeit from studies conducted on Indian population (Gomes et al, 2009;Ghosh et al, 2010;Seelam et al, 2015;Rathod et al, 2017). The metabolic process most susceptible to outlying performance in Caucasian population was found to be hydroxylation to the active metabolite 4-hydroxy alverine.…”

“…Clinical efficacy data on alverine alone or in combination with simethicone is freely available (Wittmann et al, 2010;Martínez-Vázquez et al, 2012;Ducrotte et al, 2014). The sparse information regarding the metabolism of alverine available in scientific literature is solely centered on the pharmacokinetics of the parent compound and the active metabolite 4-hydroxy alverine (Gomes et al, 2009;Ghosh et al, 2010;Seelam et al, 2015;Rathod et al, 2017) and is only derived from studies conducted on Indian population, where intra-subject variability of the main pharmacokinetic parameters of the parent compound seems quite low based on the required sample size for successful demonstration of bioequivalence between distinct oral dosage forms.…”