Liquid-chromatographic determination of serotonin and tryptophan in whole blood and plasma.
Liquid-chromatographic determination of serotonin and tryptophan in whole blood and plasma.
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DOI：
10.1093/clinchem/27.5.775
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发表时间：
1981-05
影响因子：
9.3
通讯作者：
George M. Anderson;Young Jg;D. Cohen;K. Schlicht;Nirav Patel
中科院分区：
文献类型：
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作者：
George M. Anderson;Young Jg;D. Cohen;K. Schlicht;Nirav Patel
本文介绍了一种快速测定全血和血浆中5-羟色胺(5-羟色胺)和色氨酸(Trp)的方法。它简单，有选择性，而且敏感。作为标准的5-羟色胺、5-羟色氨酸和色氨酸来自密苏里州圣路易斯的西格玛化学公司，邮编：63178。所有其他化学物质都是试剂级的，除了甲醇，它是“在玻璃中蒸馏”的(Burdick和Jackson Labs，Muskegon，MI 49440)。在去蛋白后，样品在埃彭多夫微型离心机(布林克曼仪器，韦斯特伯里，纽约11590)中离心。该液相色谱系统类似于前面描述的(1)：它包括一个Altex 110A泵、一个Rheodyne 71-25进样阀(均来自Rainen Instrument Co.，Bright ton，MA 02135)、ZBondapak C18反相柱(Waters Associates，Milford，MA 01757)和一个改进型Aminco荧光监测仪(American Instrument Co.，Silver Spring，MD 20907)。其他使用的荧光计，具有可比较的结果，是204型和650-10LC型(均来自帕金埃尔默公司，诺沃克，CT 06856)。当使用Perkin-Ehner荧光计时，激发波长和发射波长(10 nm带通)分别设置在285和345am。以乙酸钠(pH 4.0，10 mm ol/L)和甲醇(50 M L)为溶剂体系，流速2.0 m L/m in，采血于含乙二胺四乙酸乙二钠的真空容器中，温和倒置，室温保存2 h后制备。血浆在160×g的温度下0~4℃离心法获得，样品的制备一般为：将250毫升全血或血浆放入1.5毫升的聚丙烯微量离心管中，加入50毫升的1.5摩尔/L抗坏血酸，旋流搅拌2~3秒，加入50毫升的3.4摩尔高氯酸。立即将试管盖上盖子，将内含物涡流混合155次，然后将试管放入冰块中10-15 mm。然后将标本(S)在-43000xG下离心10-15min。将上清液转移到1.5毫升的聚丙烯试管中，在-13 000×g的条件下进行最后一次快速(-1 mm)离心，然后将10-50毫升的上清液直接注入色谱仪。这些化合物从峰高开始定量；每四到五个样品之间注入5 ng的5-羟色胺和100 ng的色氨酸标准品。
We describe here a rapid liquidchromatographic/fluorometric(LC-F) methodfor determining serotonin (5-HT) and tryptophan(TRP) in as little as 20MLof whole blood and plasma. It is simple, selective, and sensitive. Serotonin, 5-hydroxytryptophan, and tryptophan used as standards were from Sigma Chemical Corp., St. Louis, MO 63178. All other chemicals were reagent grade, except the methanol, which was “distilled in glass” grade (Burdick and Jackson Labs, Muskegon, MI 49440). After deproteinization, samples were centrifuged in an Eppendorf micro centrifuge(Brinkmann Instruments, Westbury, NY 11590). The liquid-chromatographicsystem was similar to that previously described (1): it consisted of an Altex 11OA pump, a Rheodyne 71-25 injection valve (both from Rainen Instrument Co., Brighton, MA 02135), a zBondapak C18 reversed-phase column (Waters Associates, Milford, MA 01757), and a modified Aminco Fluoromonitor(American Instrument Co., Silver Spring, MD 20907). Other fluorometers used, with comparableresults, were the Model 204 and the Model 650-1OLC (both from Perkin-Elmer Corp., Norwalk, CT 06856). Excitation and emission wave-lengths (10-nm bandpass) were set at 285 and 345 am, respectively, when the Perkin-Ehner fluorometers were used. The solvent system, 950 mL of sodium acetate (pH 4.0, 10 mmol/L) and 50 mL of methanol, was delivered at a flow rate of 2.0 mL/min.Blood was collected in EDTA-contaming Vacutainer Tubes, mixed by gentle inversion, and kept at room temperature(for up to 2 h) before preparation. Plasma was obtained by centrifugingat 160 X g at 0-4#{176} C. Samples were usuallyprepared by placing 250 ML ofwhole blood or plasma in a 1.5-mL polypropylene micro centrifuge tube, adding 50 ML of 1.5 mol/L ascorbic acid, vortex-mixing for 2-3s, and adding 50 MLof 3.4 molfL perchloric acid. Immediately, the tube was capped and its contents were vortex-mixed for 15 5; then the tube was placed on ice for 10-15 mm. The sample (s) were then centri-fuged at-43 000 xg for 10-l5min. The supernate was transferred to a 1.5-mL polypropylene tube, which was given a final quick (-‘1 mm) centrifugation at-13 000 X g before 10-50 ML of the supernate was directly injected into the chromatograph. The compounds were quantitated from the peak height; 5 ng of 5-HT and 100 ng of TRP standards were injected between every four or five samples.