HPLC-MS/MS测定抑郁模型大鼠血浆中皮质酮的浓度

目的: 建立HPLC-MS/MS法测定大鼠血浆中皮质酮的浓度,并用于抑郁模型大鼠的药效研究。方法: 血浆样本加入适量内标,经甲醇直接沉淀蛋白后采用HPLC-MS/MS进行分析。色谱柱采用Ultimate C₁₈柱(150 mm×2.1 mm,5.0 μm),柱温35℃,流动相由乙腈-5 mmol·L^-1醋酸铵(含0.1%甲酸)(90:10)组成,流速0.5 mL·min^-1;采用电喷雾离子源(ESI),以多反应监测方式(MRM)进行定量分析。皮质酮和内标吡格列酮在正离子模式下定量分析离子对分别为m/z347.2→121.1和m/z 357.1→133.7。建立慢性不可预知温和应激(CUMS)大鼠模型,分别采集并测定氟西汀治疗前后正常和抑郁模型大鼠血浆中的皮质酮的浓度。结果: 皮质酮质量浓度在1.26~80.64 ng·mL^-1范围内线性关系良好(r>0.99),最低定量限为1.26 ng·mL^-1,提取回收率为84.3%~90.9%,日内、日间精密度均不高于13.8%。动物试验结果表明,抑郁大鼠血浆中皮质酮的含量显著性高于正常大鼠,而经过抗抑郁药物治疗后,血浆中皮质酮的质量浓度显著性降低。结论: 该方法分析速度快,灵敏、准确,为临床检测皮质酮和抑郁症的研究提供了方法学依据。

Objective: To establish an HPLC-MS/MS method for the determination of corticosterone in rat plasma,and promote the application of depression rat model in pharmacological study. Methods: In order to precipitate protein,methanol was added to the sample after addition of internal standard. Then,the samples were analyzed by API 4000 LC-MS/MS system.All separations were carried out on an Ultimate C₁₈ column (150 mm×2.1 mm,5.0 μm).The mobile phase was composed of acetonitrile-5 mmol·L^-1ammonium acetate(containing 0.1% formic acid) (90:10) and pumped at a flow rate of 0.5 mL·min^-1 under 35℃.Detection was performed with multiple reactions monitoring (MRM) using electrospray ionization (ESI).The precursor/product ion transitions were monitored at m/z 347.2→121.1 (+ion-mode) for corticosterone and m/z 357.1→133.7(+ion-mode) for pioglitazone(internal standard).A chronic unpredictable mild stress (CUMS) model was established and the rat plasma were collected before and after treatment with fluoxetine in the normal rat and CUMS model.The concentrations of corticosterone in plasma were determined. Results: A good linearity of corticosterone was obtained in the range of 1.26-80.64 ng·mL^-1.The correlation coefficient was more than 0.99 and the lower limit of quantification was 1.26 ng·mL^-1.The extraction recoveries were in the range of 84.3%-90.9%.The precision of intra-day and inter-day were all below 13.8%.The results of animal experiment showed that the concentration of corticosterone in CUMS model was significantly higher than in normal rats;and after antidepressant treatment,the plasma concentration of corticosterone significantly decreased. Conclusion: The established method is fast,sensitive,accurate,which provides an analytical basis for the determination of corticosterone in clinical and the study of depression.