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 3³ 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.

中文翻译：

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基于实验的 LC-MS/MS 方法开发设计，用于同时测定大鼠血浆中那格列奈和盐酸二甲双胍的含量


本研究旨在开发和验证一种生物分析方法，用于使用 LC-MS/MS 在大鼠血浆中同时估计抗糖尿病组合。那格列奈和盐酸二甲双胍是临床治疗 2 型糖尿病的常用组合。因此，血浆中的同步测定对于药代动力学研究样品的快速分析至关重要。通过实验设计（DoE）（Design Expert Version 11，Stat Ease Inc.，USA）方法对液相色谱（LC）参数和质谱（MS）参数进行统计优化。采用3× ³全因子设计优化LC参数；选择%甲醇、%甲酸和流速作为自变量，而峰面积和拖尾因子被视为两种药物的因变量。采用 Box-Behnken 设计优化 MS 参数，包括干燥气体流速、雾化气体流速、DL 温度、热块温度和正电压作为独立因素，选择的响应为 [M + H] ⁺那格列奈和二甲双胍的强度盐酸盐。那格列奈和盐酸二甲双胍优化方法的[M+H] ⁺强度分别为2,462,838和11,873,826。该模型对于优化那格列奈和盐酸二甲双胍的 LC 和 MS 参数具有重要意义， p < 0.05。优化后的方法按照ICH-M10指南进行验证，准确、精密、选择性强。该方法经济高效，能够同时定量那格列奈和盐酸二甲双胍的皮克水平浓度。