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目的:建立能够灵敏、特异、准确、可靠地测定脑微透析液中乙酰胆碱浓度的分析方法,用于药物干预下脑神经递质变化的研究。方法:建立乙酰胆碱液质联用检测方法,条件是ESI(+)离子源,MRM扫描检测离子对为乙酰胆碱m/z146→87,氘代乙酰胆碱-d9(内标)m/z155→87。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脑微透析手术后的实际样品,以验证新建分析方法的适用性。结果:乙酰胆碱浓度在0.0292～1.46ng.mL-1范围内线性良好(r=0.9979),检测限为0.00292ng·mL-1;高、中、低浓度的准确度为96.1%～111.7%;日内精密度(n=6)为1.8%～7.8%,日间精密度(n=3)为2.0%～19.2%。结论:应用LC-MS/MS技术建立不使用胆碱酯酶抑制剂测定大鼠脑微透析液中乙酰胆碱的方法迅速、灵敏、可靠。
OBJECTIVE: To establish a sensitive, specific, accurate and reliable method for the determination of acetylcholine concentration in brain microdialysates for the study of neurotransmitter changes under the intervention of drugs. Methods: A method for the determination of acetylcholine (BSA) was established according to the conditions of ESI (+) ion source. The ion pairs detected by MRM were acetylcholine m / z146 → 87 and deutero acetylcholine-d9 (internal standard) m / z155 → 87. The methodological study was conducted to verify the accuracy and precision of the new analytical method. Based on this method, the method was applied to analyze the actual samples after rat brain microdialysis operation to verify the applicability of the new analytical method. Results: The linearity (r = 0.9979) of acetylcholine concentration in the range of 0.0292-1.46ng.mL-1 was obtained. The detection limit was 0.00292ng · mL-1. The accuracy of high, middle and low concentration ranged from 96.1% to 111.7% The precision (n = 6) was 1.8% -7.8%, and the daytime precision (n = 3) was 2.0% -19.2%. Conclusion: The method of LC-MS / MS for the determination of acetylcholine in rat brain microdialysate without using cholinesterase inhibitors is rapid, sensitive and reliable.