高效液相色谱法测定人脑脊液中γ-氨基丁酸和谷氨酸

Determination of γ Aminobutyric Acid and Glutamic Acid in Human Cerebrospinal Fluid by High Performance Liquid Chromatography

研究了丹酰氯柱前衍生反相高效液相色谱法测定人脑脊液中γ－氨基丁酸（Ｇａｂａ）和谷氨酸（Ｇｌｕ）的方法。检测波长为ＵＶ２５４ｎｍ；流动相Ａ：甲醇；流动相Ｂ：四氢呋喃－甲醇－０．０５ｍｏｌ／Ｌ醋酸钠（ｐＨ６．２）（５７５４２０，Ｖ／Ｖ）；流速为１ｍＬ／ｍｉｎ；梯度洗脱。讨论了有关测试条件。方法的线性范围分别为５～１０００μｍｏｌ／Ｌ（Ｇｌｕ）和１～６００μｍｏｌ／Ｌ（Ｇａｂａ）；最低检出限（μｍｏｌ／Ｌ）分别为０．００２（Ｇｌｕ）和０．００１（Ｇａｂａ）；Ｇｌｕ和Ｇａｂａ的回收率分别为９６．９％～９８．８％和９７．５％～９８．８％；３种浓度的Ｇｌｕ、Ｇａｂａ标准液的峰高测定（ｎ＝５）表明，其变异系数分别为２．２％～３．４％和３．８％～５．６％。方法简便、快速、可靠。

Е ? Aminobutyric acid (Gaba) and glutamic acid (Glu) are believed to be the major neurotransmitter. Levels in cerebrospinal fluid (CSF) may reflect their metabolism in various neurotic and psychiatric diseases. Measurements of Gaba and Glu in body fluids will help to elucidate their metabolic role and diagnostic value. In the present work, the concentrations of glutamic acid and γ aminobutyric acid in human cerebrospinal fluid were determined by reversed phase high performance liquid chromatography using pre column derivatized with dansyl chloride and UV detection at 254nm. The mobile phase was A:CH 3OH and B: THF/CH 3OH/0.05mol/L NaAc(pH 6.2)(5/75/420,V/V) with gradient elution. The flow rate was lmL/min CSF samples were deproteinizated with methanol. After centrifugation at 15 000 r/min for 10 min, the supernatant was introduced into a screw capped vial and evaporated to near dryness at 80℃. Derivatization was carried out by the addition of 250μL of bicarbonate solution (pH 9.8) and 250 μL of dansyl chloride solution (4g/L) followed by heating at 40℃ water bath for 30 min. Extraction was performed with 300μL of ethyl acetate and the organic layer separated was dried at room temperature under nitrogen. The dry residue was dissolved and injected into the HPLC system. The linear range of the method was 5 1 000μmol/L (r= 0.998 8 ) for Glu and 1 600μmol/L (r=0.998 4) for Gaba. The lower limit of detection were 0.002 μmol/L and 0.001 μmol/L respectively. The recoveries were 96.9% 98.8% and 97.5% 98.8% and the coefficients of variation of peak height measurements were 2.2% 3.4% and 3.8% 5.6% respectivery. The method enables a simple, rapid and reproducible quantification of Glu and Gaba neurotransmitter.