血浆游离氨基酸的柱前衍生反相高效液相色谱分析及其在临床检测中的应用

Pre-column Derivatization and RP-HPLC Determination of Free Amino Acids in Plasma and Its Application in Inborn Aminoacidopathies Sreening

用邻苯二甲醛(OPA)-9-芴基甲氧基羰酰氯(FMOC)柱前衍生反相高效液相色谱法测定了婴幼儿血浆中的游离氨基酸.衍生由自动进样器在线自动完成;氨基酸的衍生物用Hypersil ODS反相柱分离,荧光检测器检测;流动相A为10 mmol·L-1pH7.2的磷酸盐溶液(PB),含0.5%(ψ)四氢呋喃;流动相B为PB-甲醇-乙腈(体积比50:35:15);用二元线性梯度程序洗脱,0 min时A为100%,B为0%;25 min时A为0%,B为100%.本法在25 min内使22种氨基酸全部得到良好分离,分离度大于1.5,最小检出限为1 nmol·mL-1,在20～300nmol·mL-1浓度范围内,22种氨基酸的峰面积和浓度之间的线性相关系数在0.982～0.999之间,保留时间和峰面积的相对标准偏差分别为0.05%～0.50%和0.7%～2.9%,回收率为98%～101%.用本法在医院送检的血浆样品中检出了高甘氨酸血症、苯丙酮尿症、精氨琥珀酸血症、胍氨酸血症、同型半胱氨酸尿症、枫糖尿症和酪氨酸血症等氨基酸遗传代谢缺陷症.

Amino acids in human plasma were detemained by OPA - FMOC pre-column derivatization and RP- HPLC. The derivatization procedure was accomplished by an online auto-injector. The derivatives were separated on a Hypersil ODS and the signals were detected with a programmal）le fluorospectrophotometer detec- tor. Mobile phase A was 10 mmol·L^-1 pH 7.2 sodium phosphate buffer（PB） containing 0. 5% （ φ ） tetrahydro-furan, and B was PB - methanol - acetonitrile（50 ： 35 ： 15 by volume）. The elution progrmn was 100% A with 0% B at the start（0 min）, and 0% A with 100% B at the end（25 min） of the program. The 22 amino acids were separated satisfactorily in 25 min with R 〉 1.5 under above conditions. The lowest limit of detection was 1 pmol. The linear correlation coefficients of 22 amino acids were between 0. 982 and 0. 999 in the range of 20 to 300 pmol of each amino acid injected. The coefficients of variation of retention times and peak areas of 22 amino acids were 0.05% - 0.50% and 0.7% - 2.9% , respectively. The recoveries of the amino acids from spiked samples were 98% - 101%. The method was used to determine free mnino acids in plasma samples from hospitals and aminoacidopathies such as hyperglycinemia, phenylketonuria, algininosuccinic acidemia, citrullinemia, homocystinuria, maple syrup urine disease and tyrosinemia were detected.