Design of experiment-based LC-MS/MS method development for simultaneous estimation of nateglinide and metformin hydrochloride in rat plasma.

This research aims to develop and validate a bioanalytical method for simultaneous estimation of an antidiabetic combination using LC-MS/MS in rat plasma. Nateglinide and metformin hydrochloride are commonly used combination for clinical management of Type 2 diabetes. Hence, simultaneous determination in plasma is essential for the rapid analysis of samples from the pharmacokinetic studies. Statistical optimization was carried out for liquid chromatography (LC) parameters and mass spectroscopic (MS) parameters by design of experiment (DoE) (Design Expert Version 11, Stat Ease Inc., USA) approach. A 3 full factorial design was used for optimization of LC parameters; %methanol, %formic acid, and flow rate were selected as independent variables, whereas peak area and tailing factor were considered as dependent variables for both drugs. Box-Behnken design was used to optimize MS parameters including drying gas flow rate, nebulizing gas flow rate, DL temperature, heat block temperature, and positive voltage as independent factors, and responses selected were [M + H] intensity of nateglinide and metformin hydrochloride. The [M + H] intensity of the optimized method for nateglinide and metformin hydrochloride were 2,462,838 and 11,873,826, respectively. The model was found significant for optimizing LC and MS parameters with p < 0.05 for both nateglinide and metformin hydrochloride. The optimized method was validated as per the ICH-M10 guideline, which was accurate, precise, and selective. The method was cost-effective and capable of quantitating concentrations in picogram levels for nateglinide and metformin hydrochloride simultaneously.