LC-MS/MS法测定食蟹猴血浆皮质醇及应激监测

目的 建立食蟹猴血浆皮质醇测定的LC-MS/MS分析方法并考察运输、禁食及猴椅保定对血浆皮质醇水平的影响.方法 7只雄性食蟹猴经过8h汽车和飞机运输后,在第1、7、14、20及28日采血.6只雄性食蟹猴禁食0、8、16及24 h后采血.6只雄性食蟹猴经历2 h/次,单次和5次猴椅保定后采血.采用液相色谱-质谱联用仪进行测定血浆皮质醇.结果 血浆皮质醇在运输当日显著升高,1周后基本恢复至正常,第20日（约3周）趋于稳定.不同时间的禁食均可引起雄性食蟹猴血浆皮质醇含量显著升高.单次和5次猴椅保定后,食蟹猴血浆皮质醇水平均极显著升高,但是在5次猴椅保定后,皮质醇浓度上升幅度小于单次.本方法在62.4～2000.0 μg/L范围内线性关系良好,日内及日间精密度分别小于4.94％和5.69％,准确度分别小于10.04％和5.69％,绝对回收率为70.3％ ～ 74.7％,-70℃冻融3次及进样器中20℃放置至少10h稳定.结论 本文建立的血浆皮质醇LC-MS/MS测定方法,快速、准确,适合应激后食蟹猴血浆皮质醇检测.运输、禁食及猴椅保定均可引起食蟹猴应激.

Determination of Cortisol in Plasma of Cynomolgus Monkeys by LC-MS/MS and Stress Monitoring

Objective To establish a liquid chromatography tandem mass spectrometry （LC-MS/MS） method to determine cortisol levels in the plasma of cynomolgus monkeys and evaluate the influence of transportation, fasting and chair restraint on its levels in plasma. Methods Seven male monkeys were transported for eight hours by airplane and truck, and the blood samples were collected on day 1, 7, 14, 20 and 28 after transportation. The blood samples in six male monkeys were collected after the animals were fasted for 0, 8, 16 and 24 hours, and after one or five times of two-hour chair restraint. Cortisol was determined by using LC-MS/MS. Results The cortisol levels in plasma samples increased significantly after transportation, and then decreased gradually, and reached a stable level three weeks later. The cortisol levels in plasma samples were significantly increased after 8, 16 and 24 hours of fasting, but no differences were observed among the three types of fasting. The cortisol levels in plasma samples increased significantly after one and five times of chair restraint, but the increased extent of the latter was lower than the former. The calibration curves were linear over the concentration range of 20. 0-2000.0 ng/ml. The intra- and inter-day precision was less than 4.94% and 5.69%, respectively, and the accuracy （RE） was less than 10.04% and 5.69%, respectively. The extraction recovery was between 70.3% and 74.7%. The analyte proved to be stable within three freeze-thaw tests and in the autosampler for ten hours at 20℃. Conclusion This method is rapid and specific and suited to cortisol determination in plasma. All of the stimuluses such as transportation, fasting and chair restraint were stressful for the monkeys.