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本文报道用HPLC-ED法测定家兔血小板和血浆中的5-HT,方法简便、精确、灵敏度高,适用于直接测定凝集血小板的5-HT 释放量,亦适用于研究凝集程度和释放量间的关系。实验用电化学检出器配有玻璃碳电极和Ag/AgCl参比电极,5μm Nucleosil C-18柱(15cm×4.6mm)反相层析。流动相为含17%甲醇的0.05 M 醋酸-枸橼酸盐缓冲液(pH 4.6)。实验温度37±1°,流速0.6ml/分。供试兔血浆的血小板量约为9×10~5/μl,以5-羟基-N-甲色胺(5-HMT)为内标。作者采用Shore 和Olin 方法,从血浆或血小板中提取5-HT。方法为,样品0.1ml 中加入0.1N 盐酸1ml,氯化钠1g,正丁醇3ml,充分混匀20分钟,离心,取正丁醇层2ml 于试管中,加入0.1N 盐酸0.5ml,正庚烷3ml,充分混匀5分钟,离心,弃去有机液层。取少量水层(5～10μl)注入HPLC-ED 系统中,
This article reports the determination of 5-HT in rabbit platelets and plasma by HPLC-ED method. The method is simple, accurate and sensitive. It is suitable for direct determination of 5-HT release of agglutinated platelets. It is also suitable for studying the relationship between agglutination and release Relationship. Experimental electrochemical detector with glassy carbon electrode and Ag / AgCl reference electrode, 5μm Nucleosil C-18 column (15cm × 4.6mm) reverse phase chromatography. The mobile phase was 0.05 M acetate-citrate buffer (pH 4.6) containing 17% methanol. Experimental temperature 37 ± 1 °, flow rate 0.6ml / min. The amount of platelet in the plasma of the test rabbits was about 9 × 10 ~ 5 / μl and the internal standard was 5-hydroxy-N-methyl-amine (5-HMT). The authors used Shore and Olin methods to extract 5-HT from plasma or platelets. The method comprises the following steps: 0.1 ml of hydrochloric acid, 1 ml of sodium chloride and 3 ml of n-butanol are added into 0.1 ml of sample, fully mixed for 20 minutes, centrifuged, 2 ml of n-butanol layer is added into a test tube, 0.5 ml of 0.1N hydrochloric acid, Alkane 3ml, mix well for 5 minutes, centrifuged, discard the organic layer. Take a small amount of water layer (5 ~ 10μl) into the HPLC-ED system,