A Simple Sample Preparation with HPLC-UV Method for Estimation of Clomipramine from Plasma

Clomipramine is a tricyclic antidepressant. Different methods for determination of clomipramine hydrochloride in plasma have been described. Most of these procedures favor the use of acidic back-extraction in extraction procedure and HPLC as the analytical technique. In this study, the clomipramine extraction procedure was modified and a direct injection to the column was performed to shorten the time of sample preparation considerably. Furthermore, the method was applied in bioequivalence study of new formulations of clomipramine in comparison with reference tablets. The drug and internal standard were extracted from plasma with heptan : isoamyl alcohol (95:5) and re-extracted with 200 μL of orthophosphoric acid (0.3% v/v). The organic layer was discharged and analysis was performed on C(8) reverse phase ODS2 HPLC column with a mobile phase, acetonitrile : water (75:25) and UV detection set at 215 nm. Additionally, a single dose study was carried out with a two-sequence, crossover block-randomized design for bioequivalence study. Clomipramine tablets (3 × 25 mg) of either formulations (reference or test products) were administered separately in two occasions to 12 fasting healthy male volunteers. Blood samples were taken prior to and at 9 points within 48 h after dose administration. The retention time of internal standard (cisapride), clomipramine, and desmethyl clomipramine were 5.6 ± 0.2, 10.3 ± 0.3, and 9.5 ± 0.3 min, respectively. The standard curve covering the concentration ranges of 2.5-120 ng/mL was linear (r(2) = 0.9950 and 0.9979) for clomipramine and desmethyl clomipramine. The co-efficient of variation for intra-day and inter-day accuracy and precision was less than 18.3%. The pharmacokinetic parameters C(max) and T(max) were obtained directly from plasma clomipramine concentrations. K(el )was estimated by log-linear regression and AUC was calculated by the linear trapezoidal rule. The pharmacokinetic parameters AUC and C(max) were tested for equivalence after log-transformation of data. The 90% standard confidence intervals of the mean values for the test/reference ratios, AUC, and C(max) were within the acceptable bioequivalence limits of 0.80-1.20. These results indicated that the analytical method was linear and accurate. Test and reference formulations were found to be bioequivalent and therefore interchangeable.