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

doi:10.3724/SP.J.1123.2019.12029

Abstract

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 After centrifugation, the supernatant was withdrawn and blown to dryness using nitrogen.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

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.

Figures/Tables 7

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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

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

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

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

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