UPLC-MS/MS同时测定大鼠血浆12种神经递质和激素的方法学研究及对寒性中药的应用分析

Methodology study on simultaneous determination of 12 kinds of neurotransmitters and hormones in rat plasma by UPLC-MS/MS and its application in cold-natured Chinese medicine

目的:建立超高效液相色谱-串联质谱(UPLC-MS/MS)方法同时检测大鼠血浆中12种神经递质和激素的含量,包括去甲肾上腺素(NE)、肾上腺素(Epi)、L-甲状腺素(L-T4)、β-雌二醇(β-E2)、乙酰胆碱(Ach)、5-羟色胺(5-HT)、多巴胺(DA)、组胺(HA)、γ-氨基丁酸(GABA)、睾酮(T)、孕酮(P)、皮质醇(Cor),并应用该方法研究寒性中药对神经递质和激素的影响。方法:(1)正常大鼠血浆样品经甲醇-乙腈(1∶1)沉淀蛋白后,取上清液挥干,流动相复溶,2μl进样分析;采用BEH amide(2.1 mm×100 mm,1.7μm)色谱柱,以含0.2%甲酸的水溶液-乙腈作为流动相进行梯度洗脱,流速0.2 ml/min、柱温35℃,并进行方法学考察。(2)正常大鼠灌胃给予寒性中药后,于0.5、1.5、2.5和4.5 h分别采血,分离血浆,测定12种神经递质和激素的含量。结果:12种神经递质和激素检测的线性关系良好,低、中、高浓度的提取回收率RSD范围为1.31%~10.96%,基质效应RSD范围为1.68%~10.70%,批内、批间精密度RSD均小于10.99%。寒性药物可以显著降低大鼠血浆Epi、DA、T、5-HT、Cor水平,升高GABA、P、β-E2、Ach的含量。结论:该方法灵敏、快捷、准确、简便,适用于血浆中12种神经递质和激素含量的同时测定;寒性中药可诱导机体副交感神经功能亢进,但对交感神经-肾上腺髓质、皮质功能显示抑制效应。

Objective: To establish the UPLC-MS/MS method for simultaneous determination of 12 kinds of neurotransmitters and hormones in rat plasma,including norepinephrine( NE),epinephrine( Epi),L-thyroxine( L-T4),β-estradiol( β-E2),acetylcholine( Ach),5-hydroxytryptamine( 5-HT),dopamine( DA),histamine( HA),γ-aminobutyric acid( GABA),testosterone( T),progesterone( P) and cortisol( Cor),and study the effects of cold-natured Chinese medicine on the neurotransmitters and hormones.Methods:(1)The plasma samples from normal rats were precipitated by methanol-acetonitrile( 1: 1),then the supernatant was volatilized and re-dissolved with the mobile phase. Liquid chromatographic separation was performed by a BEH amide column( 2.1 mm×100 mm,1.7μm) with gradient elution of mobile phase that consisted of aqueous solution( containing 0.2% formic acid) and acetonitrile,the flow rate was 0.2 ml/min,the column temperature was 35 ℃,the injection volume was 2 μl. And the methodology study was performed.(2)The normal rats was treated with cold-natured Chinese medicine by intragastric administration. The blood samples were collected after administration for 0.5 h,1.5 h,2.5 h and 4.5 h,respectively. The plasma was separated and the levels of 12 kinds of neurotransmitters and hormones were determined. Results: The calibration curves of 12 kinds of neurotransmitters and hormones showed good linear relationships. The RSD on recovery rate of low-,medium-and high-concentration was 1.31%-10.96%. The RSD on matrix effects was1.68%-10.70%. The intra-day and inter-day RSD were less than 10. 99%. The cold-natured Chinese medicine could significantly reduce the plasma levels of Epi,DA,T,5-HT and Cor,and increase the levels of GABA,P,β-E2 and Ach. Conclusion: This method is sensitive,rapid,accurate and simple,and can be used for simultaneous determination of12 kinds of neurotransmitters and hormones in plasma. The cold-natured Chinese medicine can induce the hyperactivity of parasympathetic nerve function,but inhibit the activity of sympathetic-adrenal medulla and cortex function.