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

doi:10.3724/SP.J.1123.2019.12029

摘要/Abstract

摘要：

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，5 μm）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0.10~0.40 μmol/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。

关键词: 高效液相色谱, 柱前衍生, 氨基酸类神经递质, 单胺类神经递质, 邻苯二甲醛, 血清

Abstract:

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 After centrifugation, the supernatant was withdrawn and blown to dryness using nitrogen.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.

Key words: high performance liquid chromatography (HPLC), precolumn derivatization, amino acid neurotransmitter, monoamine neurotransmitters, o-phthalaldehyde (OPA), serum

白洁, 王妲, 刘泽平, 张佳琪, 刘丽艳, 韩艳梅. 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[J]. 色谱, 2020, 38(8): 923-928.

BAI Jie, WANG Da, LIU Zeping, ZHANG Jiaqi, LIU Liyan, HAN Yanmei. Simultaneous determination of amino acid and monoamine neurotransmitters in serum by high performance liquid chromatography coupled with precolumn derivatization[J]. Chinese Journal of Chromatography, 2020, 38(8): 923-928.

图/表 7

表1 梯度洗脱程序

Table 1 Program of gradient elution

Time/min	φ(A)/%	φ(B)/%
A: 50 mmol/L citric acid-50 mmol/L sodium acetate buffer; B: acetonitrile.
0	68	32
6.50	68	32
6.51	35	65
21.00	35	65
21.01	68	32
35.00	68	32

图1 神经递质与OPA物质的量之比对神经递质峰面积的影响(n=3)

Fig. 1 Effect of the amount of substance ratio of neurotransmitters to o-phthalaldehyde (OPA) on the peak area of neurotransmitters (n=3) Tau: taurine; Glu: glutamic acid; Gly: glycine; γ-GABA: γ-aminobutyric acid; DA: dopamine.

图2 蛋白质沉淀剂对神经递质峰面积的影响(n=3)

Fig. 2 Effect of protein precipitants on the peak area of neurotransmitters (n=3)

图3 样品与乙醇的体积比对神经递质峰面积的影响(n=3)

Fig. 3 Effect of volume ratio of the sample to ethanol on the peak area of neurotransmitters (n=3)

图4 5种神经递质衍生后的色谱图

Fig. 4 Chromatograms of the five neurotransmitters after derivatization a. standard solution; b. real sample; c. spiked sample.

表2 5种神经递质的线性方程、线性范围、相关系数及检出限

Table 2 Linear equations, linear ranges, correlation coefficients (r2) and limits of detection (LODs) of the five neurotransmitters

Neurotransmitter	Linear equation	Linear range/(μmol/L)	r²	LOD/(μmol/L)
Y: peak area; X: concentration, μmol/L.
Tau	Y=8.78×10⁹X-1.50×10³	0.50-30.00	0.9966	0.10
Glu	Y=7.84×10⁹X-5.10×10³	0.50-30.00	0.9866	0.20
Gly	Y=7.86×10⁹X-0.60×10³	1.00-30.00	0.9987	0.40
γ-GABA	Y=8.12×10⁹X-0.66×10³	0.25-30.00	0.9979	0.10
DA	Y=5.53×10⁹X-2.13×10³	0.50-30.00	0.9973	0.10

表3 在血清样品中5种神经递质的加标回收率(n=3

Table 3 Spiked recoveries of the five neurotransmitters in serum samples (n=3)

Neurotransmitter	Background/(μmol/L)	Added/(μmol/L)	Found/(μmol/L)	Recovery/%	RSD/%
Tau	58.58	50.00	107.96	98.76	2.27
	58.58	60.00	124.21	109.38	7.80
	58.58	70.00	121.23	89.35	1.67
Glu	66.32	50.00	123.82	115.31	2.20
	66.32	60.00	129.18	104.77	7.17
	66.32	70.00	128.97	89.50	6.81
Gly	228.98	175.00	392.73	93.62	2.51
	228.98	210.00	412.88	87.57	1.02
	228.98	280.00	488.53	92.73	1.13
γ-GABA	3.42	50.00	57.08	107.34	0.90
	3.42	60.00	68.36	108.23	1.60
	3.42	70.00	73.96	100.79	0.49
DA	0	50.00	54.92	109.84	2.15
	0	60.00	54.60	91.00	6.63
	0	70.00	69.66	99.52	4.05

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