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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,5.0μm),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 m L·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/z 347.2→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·m L-1范围内线性关系良好(r>0.99),最低定量限为1.26ng·m L-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 a HPLC-MS / MS method for the determination of plasma corticosterone in rats and to study its pharmacodynamic effects in depression model rats. Methods: The plasma samples were spiked with appropriate amount of internal standard and analyzed by HPLC-MS / MS. The column was equipped with an Ultimate C18 column (150 mm × 2.1 mm, 5.0 μm) with a column temperature of 35 ° C. The mobile phase consisted of acetonitrile-5 mmol·L-1 ammonium acetate (90:10) with a flow rate of 0.5 m L · min-1. Quantitative analysis was performed by multiple reaction monitoring (MRM) using electrospray ionization (ESI). Cortisone and the internal standard of pioglitazone in the positive ion mode quantitative analysis of the ion pairs were m / z 347.2 → 121.1 and m / z 357.1 → 133.7. Chronic unpredictable mild stress (CUMS) rat model was established, and the concentrations of corticosterone in plasma of normal and depressed model rats before and after fluoxetine treatment were collected and measured respectively. Results: The linear range of corticosterone was 1.26 ~ 80.64 ng · m L-1 (r> 0.99), the lowest limit of quantification was 1.26ng · m L-1 and the recovery was 84.3% -90.9% , Daytime precision is not higher than 13.8%. Animal experiments showed that plasma corticosterone levels were significantly higher in depressed rats than in normal rats, but plasma corticosterone concentrations were significantly reduced after antidepressant treatment. Conclusion: The method is rapid, sensitive and accurate and provides a methodological basis for the clinical study of corticosterone and depression.