高效液相色谱-荧光检测法测定大鼠血小板内5-羟色胺的含量(1)
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[摘要] 目的:建立高效液相色谱-荧光检测测定大鼠血小板内5-羟色胺含量的方法。方法:色谱柱为Nucleosil-C18(4 mm×250 mm,10 μm),流动相:磷酸二氢钾(10 mmol/L,内含EDTA-Na2 0.3 mmol,pH=3.5)∶甲醇∶乙腈=85∶10∶5,流速:0.5 ml/min,荧光激发波长:254 nm,发射波长:360 nm,增益:32。血小板沉淀用300 μl纯水悬浮,加入15 μl内标溶液(30 μg/ml,α-甲基-5-HT水溶液),100 μl蛋白沉淀液,混匀,离心,取上清液20 μl,进样。结果:5-羟色胺在0.25～4.00 μg/ml浓度范围内,线性关系良好。5-羟色胺测定的批内和批间相对标准差(RSD)分别小于4.29%和4.44%,回收率为84.3%～88.7%。正常雄性SD大鼠血小板内5-羟色胺的平均含量分别为(1.65±0.14) μg/109 plts。结论:本法简便、准确、快捷,可用于大鼠血小板内5-羟色胺的测定。
[关键词] 高效液相色谱;荧光检测法;血小板;5-羟色胺
[中图分类号] R968[文献标识码] B[文章编号] 1673-7210(2010)05(a)-051-03
Determination of 5-Hydroxytryptamine in rat's platelets by HPLC-FD
YANG Lei, LI Weirong, WANG Ningsheng
(Institute of Clinical Pharmacology, Guangzhou University of Traditional Chinese Medicine, Guangzhou 510405, China)
[Abstract] Objective: To establish a method for the contend determination of serotonin in blood platelets by high performance liquid chromatography with fluorescence detector (HPLC-FD). Methods: Preparating platelets samples commonly with 2% EDTA-Na2 anticoagulated blood, we determinated them by HPLC. The nucleosil-C18 column (4 mm×250 mm,10 μm) was used. The mobile phases consisted of mobilphase of 0.01mol/L-potassium dihydrogen phosphate, acetonitrile and methanol (85∶5∶10), the flow rate was 0.5 ml/min and the detection wavelength was at 254 nm, the excitation wavelength was at 360 nm for emission, quantitating in internal standard method. Results: The results showed that the 5-Hydroxytryptamine peak was chromatographically resolved from the internal satandard; a good linearity range from 0.25-4.00 μg/ml; the RSD of within-batch and between-batch of the method was 4.29% and 4.44%; the average recovery was 84.3%-88.7%. The average contents of 5-Hydroxytryptamine in platelets of normal male Sprague-Dawley rats was (1.65±0.14) μg/109 plts. Conclusion: The method is simple, accurate, rapid, and can be used for the determination of 5-Hydroxytryptamine in rat's platelets.
[Key words] HPLC; Fluorescence detector; Platelets; 5-Hydroxytryptamine
5-羟色胺(5-hydroxytryptamine,5-HT)是一种重要的单胺类中枢神经递质,参与睡眠、摄食、体温、精神情感性疾病等多种生理病理活动的调节[1-3]。由于血小板摄取、贮存、释放5-HT的过程与中枢5-HT能神经元相似,且血小板获取容易,常作为中枢5-HT能神经元功能研究的外周模型[4] ......
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