LC-MS/MS法测定针刺穴位给药大鼠血清中氯贝胆碱的含量___

目的：建立能够灵敏、特异、准确、可靠的测定血清中氯贝胆碱浓度的分析方法。用于针刺穴位药物增效机理的研究。方法：优化氯贝胆碱及内标氘代乙酰胆碱离子化及断裂方式，确定相关液相色谱条件；开展方法学考察，以验证新建分析方法的准确性、精密度等，在此基础上将该方法用于分析大鼠给药后所得的实际血清样品，以验证新建分析方法的适用性。同时测定了大鼠穴位给药及静脉给药后血清样品中氯贝胆碱的含量。结果：ESI（+）为氯贝胆碱提供最佳的离子化条件，采用MRM工作模式，用m/z106.0101.9来检测氯贝胆碱，同时，以氘代乙酰胆碱-d9 m/z15587为内标化合物。氯贝胆碱及氘代乙酰胆碱-d9色谱保留时间分别为5.4 min和5.0 min。乙酰胆碱在50~300 ng•mL-1范围内线性良好，回归方程为Y=0.002397X+0.0266，r=0.9944，检测限为0.0025 ng•mL-1。高、中、低浓度的绝对回收率为96.4%~102.1%，方法回收率为100.6%~108.9%。日内精密度（n=6）为2.6%~5.7%，日间精密度（n=3）为4.1%~8.4%。结论：应用LC-MS/MS技术建立测定血清中氯贝胆碱的新方法灵敏可靠，为针药联合干预后血药浓度的比较研究以及针药联合干预后靶器官药物浓度的检测等研究工作打下了重要的分析方法学基础

Objective: To develop a sensitive and reliable method for determination of Bethanechol chloride in blood serum of rats. Method: The optimal ionization and fragmentation conditions，as well as liquid chromatographic ones, to detect Bethanechol chloride were developed and validated. The newly-developed analytical method was further applied in determination of Bethanechol chloride in blood serum after administration of Bethanechol chloride. Result: The detection was performed by MRM mode via electrospray ionization (ESI) source operating in the positive ionization mode. The precursor-to-product ion transitions of the analyte Bethanechol chloride is m/z106.0101.9 with internal standard (IS, Acetylcholine-d9) at m/z 15587. The method was linear over the concentration range of 50-300 ng•mL-1 ( r=0.9944). The limit of quantification is 0.0025 ng•mL-1. The absolute recovery was 96.4%-102.1%, the recovery of the method was 100.6%~108.9%. The intra- and inter-day precision values were 2.6%-5.7% (n=6) and 4.1%-8.4% (n=3), respectively. Conclusion：The fully validated LC–MS/MS method has been successfully applied to determination of Bethanechol chloride in blood serum of rats .