高效液相色谱/荧光法测定血浆、红细胞与尿液中的硫胺素及其磷酸酯  

Determination of Thiamine and Its Phosphate Esters in Human Plasma,Erythrocytes and Urine by High Performance Liquid Chromatography-Fluorimetric Method

作　　者：董爱军[1] 张英春[1] 杨鑫[1] 张华[1] 井晶[1] 

机构地区：[1]哈尔滨工业大学食品科学与工程学院,黑龙江哈尔滨150090

出　　处：《分析测试学报》2014年第8期934-939,共6页Journal of Instrumental Analysis

摘　　要：建立了同时测定血浆、红细胞、尿液中硫胺素（T）、一磷酸硫胺素（TMP）、二磷酸硫胺素（TDP）的柱前衍生/反相高效液相色谱-荧光检测法.样品经高氯酸除蛋白,铁氰化钾衍生后,采用反相色谱柱分离,荧光检测器测定,外标法定量.色谱柱为C18柱（250 mm×4.6 mm,5μm i.d.）,流动相为0.2 mol/L KH2PO4溶液（pH 7.0,含0.000 3 mol/L四丁基氢氧化铵）-甲醇和甲醇-水,采用梯度程序进行洗脱,流速为0.8mL/min.荧光检测器激发波长为365 nm,发射波长为435 nm.结果表明：T,TMP在0.5～ 20.0μg/L,TDP在5.0 ～ 200.0 μg/L质量浓度范围内呈良好的线性关系,相关系数分别为0.99,0.98,0.99.T,TMP,TDP的平均回收率为86.8％～110.2％,相对标准偏差（RSDs）为2.5％～10.9％.3种化合物在血浆及红细胞中的检出限（LODs）为0.03～0.35 μg/L,定量下限（LOQs）为0.09～1.18 μg/L.T在尿液中的检出限（LODs）为1.58μg/L,定量下限（LOQs）为5.26 μg/L.该方法的准确度和精密度均较高,能够满足临床检测和科研需要.A reversed phase high performance liquid chromatography-fluorescence detection （RP-HPLC/FLD） method was developed for the determination of thiamine （T） and thiamine monophosphate（TMP）,thiamine diphosphate（TDP） in human plasma,erythrocytes and urine.After deproteinization with perchloric acid and derivation with potassium ferricyanide,the sample was separated on a C18 column（250 mm ×4.6 mm,5 μm i.d.） at 20 ℃.A gradient programme was carride out at flow rate of 0.8 mL/min using 0.2 mol/L KH2PO4 （including 0.000 3 mol/L TBAH,pH 7.0）-methyl alcohol and methyl alcohol-water as eluent.The excitation and emission wavelengths were set at 365 nm and 435 nm,respectively.The results showed that T,TMP and TDP could be completely separated within 18 min.The calibration curves were linear in the range of 0.5-20.0 μg/L for T and TMP,and 5.0-200.0 μg/L for TDP,with correlation coefficients of 0.99,0.98,0.99,respectively.The spiked recoveries of three compounds in plasma,erythrocytes and urine were in the range of 86.8％-110.2％ with relative standard deviations（RSDs） of 2.5％-10.9％.The limits of detection（LODs） and limits of quantitation（LOQs） for T,TMP,TDP in plasma and erythrocytes were in the range of 0.03-0.35 μg/L,and 0.09-1.18 μg/L,respectively.The LODs and LOQs of T in urine were 1.58 μg/L and 5.26 μg/L,respectively.The developed method was sensitive and precise,and could meet the demands for clinical application.

关 键 词：反相高效液相色谱 荧光法 硫胺素及其磷酸酯 血浆 红细胞 尿液 

分 类 号：O657.72[理学—分析化学] Q563.1[理学—化学]