柱前衍生-高效液相色谱法同时测定血清中氨基酸类及单胺类神经递质

以邻苯二甲醛(o-phthalaldehyde,OPA)为衍生试剂,建立了柱前衍生-高效液相色谱(HPLC)同时测定血清中氨基酸类神经递质牛磺酸(Tau)、谷氨酸(Glu)、甘氨酸(Gly)、γ-氨基丁酸(γ-GABA)和单胺类神经递质多巴胺(DA)含量的分析方法。血清与乙醇以1∶2的体积比混合,进行蛋白质沉淀后离心,取其上清液,氮吹至近干。前处理后的样品与OPA进行柱前衍生,衍生化产物采用Luna 5u C18色谱柱(250 mm×4.6 mm,5μm)分离,以柠檬酸-乙酸钠缓冲溶液(pH 3.73)为流动相A、乙腈为流动相B进行梯度洗脱,流速为1.0 mL/min,柱温为30℃,检测波长为338 nm。5种神经递质在各自范围内线性关系良好(r2≥0.9866),检出限为0.10~0.40μmol/L,不同加标水平下目标物的加标回收率为87.57%~115.31%,相对标准偏差均低于7.80%。方法操作简单,灵敏度高,精密度、线性关系和回收率等方法学指标较好,可实现血清中氨基酸类及单胺类神经递质的同时检测。

Simultaneous determination of amino acid and monoamine neurotransmitters in serum by high performance liquid chromatography coupled with precolumn derivatization

Using o-phthalaldehyde (OPA) as the derivatization reagent, a precolumn derivatized -high performance liquid chromatography (HPLC) method was developed for the simultaneous determination of amino acid neurotransmitters taurine (Tau), glutamic acid (Glu), glycine(Gly), and γ -aminobutyric acid (γ-GABA), as well as the monoamine neurotransmitter dopamine (DA), in serum samples. The samples and ethanol were mixed at a volume ratio of 1:2 (v/v) for protein precipitation. After centrifugation, the supernatant was withdrawn and blown to dryness using nitrogen. The residue was pre-column derivatized with OPA, and the derivatized product was isolated by gradient elution ona Luna 5u C18 column (250 mm×4.6 mm, 5 μm). Under the optimal experimental conditions, the five neurotransmitters showed good linearities (r² ≥ 0.9866). The limits of detection were between 0.10 and 0.40 μmol/L. The spiked recoveries at different spiked levels were 87.57%-115.31%, and the RSDs were below 7.80%. This method is simple, sensitive, and it can be promised for the simultaneous detection of amino acid and monoamine neurotransmitters.