Development of Bioanalytical HPLC Method for Estimation of Milnacipran Hydrochloride in Rabbit Plasma Using Solid Phase Extraction Technique and its Application in Pharmacokinetic Investigation

Background: The availability of a low-cost bioanalytical method, easy to transfer and to set up, represents an advantage in therapeutic drug monitoring and industrial research. Objective: A simple, sensitive and rapid high performance liquid chromatographic (HPLC) method is developed and validated for quantitative determination of milnacipran hydrochloride in rabbit plasma. Method: Simple and effective solid phase extraction technique was performed for sample treatment and it has resulted in consistent and high recoveries (93-95%) at all concentrations studied. Efficient chromatographic separation has been performed on a LiChrospher (R) C18 column using a mobile phase consisting of a mixture of phosphate buffer, acetonitrile and methanol (65:25:10; v/v/v) at a flow rate of 0.8 mL/min. Results: The method has demonstrated linearity from 25 ng to 2000 ng/mL with a regression coefficient of 0.9998. The accuracy was found to be very high (99.31 to 101.44 %). %RSD values for inter-day and intra-day variation were not more than 3.58. The method has demonstrated high sensitivity with a lower limit of quantification of 25 ng/mL and excellent stability of milnacipran in rabbit plasma. The method was applied for pharmacokinetic investigation of immediate release and controlled release milnacipran tablets. The in-vivo study results indicated that developed HPLC method was sensitive to accurately quantify the concentration of milnacipran in plasma, which is an essential requirement for determination of pharmacokinetic parameters for drug formulation studies as well as future bioavailability or bioequivalence studies. Conclusion: A simple and sensitive bioanalytical HPLC method was developed, validated and applied for pharmacokinetic studies.