反相高效液相色谱法测定血清中γ-氨基丁酸

• 作 者: 沈佐君 王治国 李小鹏 胡翠华 杨树德
• 机 构: 卫生部北京医院临床检验中心
• 出 处: 生物化学与生物物理进展
• 发表日期: 1998年06期
• 关键词: 最佳色谱条件 醋酸钠缓冲液 衍生化试剂 衍生化反应 批间变异系数 二元梯度洗脱 荧光检测 氯甲酸 批内变异系数 内标法

建立一种高灵敏度的氯甲酸芴甲酯柱前衍生荧光检测反相高效液相色谱法测定血清中γ氨基丁酸的方法．内标为己氨酸，固定相为ＳｈｉｍＰａｃｋＣＬＣＯＤＳ（Ｍ），４６ｍｍ×１５０ｍｍ，填充料为５μｍ；流动相采用二元梯度洗脱，Ａ相为００５ｍｏｌ／Ｌ醋酸钠缓冲液∶水∶四氢呋喃∶冰乙酸（２５０∶１００∶１５∶２２），Ｂ相为乙腈∶甲醇（４∶１）．系统研究了ｐＨ值、反应时间、离子强度及衍生化试剂用量对衍生化反应的影响，确定最佳反应条件和最佳色谱条件．方法的精密度为批内变异系数＜４６％，批间变异系数＜６１％；最低检出限（信噪比＝２）为３１ｎｍｏｌ／Ｌ；线性范围为５０～１０００ｎｍｏｌ／Ｌ，γ２＝０９９９２；平均回收率为９７１％．

To establish a method for the determination of γ-aminobutyric acid in serum by reversed-phase high performance liquid chromatography (HPLC) with pre-column derivatization and high sensitivity of flumethyl chloroformate. The internal standard was made of histidine, the stationary phase Shim PackCLC ODS (M), 4  6mm × 150mm, the filler was 5μm; mobile phase using binary gradient elution, A phase was 0  05mol / L sodium acetate buffer Solution: Water: Tetrahydrofuran: Glacial acetic acid (250: 100: 15: 2  2), Phase B acetonitrile: Methanol (4: 1). The effects of pH value, reaction time, ionic strength and the amount of derivatization reagent on the derivatization reaction were systematically investigated. The optimal reaction conditions and the best chromatographic conditions were determined. The precision of the method was: intra-assay CV <46%, inter-assay CV <61%; minimum detection limit (signal to noise ratio = 2) was 31nmol / L, γ2 = 09992; the average recovery was 971%.