5,5′-二硫硝基苯甲酸柱前衍生高效液相色谱法测定酶促反应液中的L-半胱氨酸  

Determination of L-Cysteine in Enzymatic Reaction Mixture with Pre-column Derivatization by High Performance Liquid Chromatography

作　　者：刘忠[1] 杨文博[1] 金永杰[1] 白钢[1] 

机构地区：[1]南开大学生命科学学院,天津300071

出　　处：《色谱》2004年第3期231-233,共3页Chinese Journal of Chromatography

摘　　要：以5,5′ 二硫硝基苯甲酸(DTNB)为柱前衍生试剂对酶促反应液进行衍生化,用C18色谱柱在室温下采用二元梯度洗脱,于330nm波长下检测,同时以2 巯基乙醇和二硫苏糖醇(DTT)为对照,对L 半胱氨酸的分离峰进行确认,并对酶促反应液中的L 半胱氨酸进行分离测定。L 半胱氨酸浓度为5～950μmol/L时,其浓度与峰面积呈显著的线性关系。高、中、低浓度水平的L 半胱氨酸加标回收率为99 7%～100 5%,相对标准偏差小于1 3%,检测限为0 8μmol/L。方法简便、快速、可靠。用于样品分析,结果令人满意。A new sensitive high performance liquid chromatographic method for the determination of L-cysteine in an enzymatic reaction mixture using ultra violet spectrometric detection was developed. The sample reacted with 5,5′-dithio-bis-nitrobenzoic acid (DTNB) and was　analyzed on a Shimadzu VP-ODS column at room temperature, using gradient elution with detection at 330 nm. The L-cysteine chromatographic peak was determined in comparison with derivatives of 2-mercapto ethanol and dithiothreitol. The linear range was 5-950 (μmol/L). The recoveries were 99.7%-100.5% and the relative standard deviations (RSDs) were less than 1.3%. The detection limit was 0.8 μmol/L. The method is simple and accurate.

关 键 词：高效液相色谱法 5 5′-二硫硝基苯甲酸 柱前衍生 L-半胱氨酸 酶促反应液 

分 类 号：O658[理学—分析化学]