Quantification of cyproheptadine in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry in a bioequivalence study

A rapid, sensitive and specific method to quantify cyproheptadine in human plasma using amitriptyline as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using a diethyl-ether/dichloromethane (70/30; v/v) solvent. After removing and drying the organic phase, the extracts were reconstituted with a fixed volume of acetonitrile/water (50/50 v/v)+0.1% of acetic acid. The extracts were analyzed by high performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC-MS/MS). Chromatography was performed isocratically using an Alltech Prevail C18 5 mu m analytical column, (150mm x 4.6mm I.D.). The method had a chromatographic run time of 4min and a linear calibration curve ranging from 0.05 to 10 ng/mL (r(2)>0.99). The limit of quantification was 0.05 ng/mL. This HPLC/MS/MS procedure was used to assess the bioequivalence of cyproheptadine in two cyproheptadine + cobamamide (4mg+1mg) tablet formulations (Cobactin (R) [cyproheptadine + cobamamide] test formulation supplied from Zambon Laboratorios Farmaceuticos Ltda. and Cobavital (R) from Solvay Farma (standard reference formulation)). A single 4 mg + 1mg [cyproheptadine + cobamamide] dose of each formulation was administered to healthy volunteers. The study was conducted using an open, randomized, two-period crossover design with a 1-week washout interval. Since the 90% CI for Cmax and AUCs ratios were all within the 80-125% bioequivalence limit proposed by the US Food and Drug Administration, it was concluded that the cyproheptadine test formulation (Cobactin (R)) is bioequivalent to the Cobavital (R) formulation for both the rate and the extent of absorption of cyproheptadine. Copyright (C) 2011 John Wiley & Sons, Ltd.