基于亲水相互作用的超高效液相色谱-三重四极杆质谱法测定不同成熟阶段草莓中18种游离氨基酸

doi:10.3724/SP.J.1123.2024.04017

摘要/Abstract

摘要：

不同成熟时期草莓中游离氨基酸的组成和含量测定对草莓的品质评价和营养研究具有重要意义。本研究建立了基于亲水相互作用的超高效液相色谱-串联质谱技术快速直接测定不同成熟阶段草莓中18种游离氨基酸的分析方法。将经过研磨后的草莓样品用水提取并通过离心和膜过滤后,进行液相色谱-串联质谱分析。采用ACQUITY UPLC Glycan BEH Amide亲水相互作用液相色谱柱(150 mm×2.1 mm, 1.7 μm),以含有5 mmol/L甲酸铵和0.1%甲酸的乙腈水溶液为流动相进行梯度洗脱。采用三重四极杆质谱仪,在电喷雾离子源正离子扫描模式下,以基质匹配标准曲线法对氨基酸定量分析。结果表明,该方法在0.5~40.0μmol/L范围内线性关系良好(r²≥0.992)。方法的日内精密度为1.0%~14.8%,日间精密度为3.6%~17.6%;检出限在50~250 nmol/L范围内。在3个添加水平下,草莓基质中样品加标回收率为75.0%~114.6%,相对标准偏差为0.3%~13.5%。将建立的定量分析方法用于5个不同成熟时期草莓中氨基酸含量的测定,经统计分析后共筛选出7种差异性游离氨基酸(苯丙氨酸、异亮氨酸、谷氨酰胺、4-氨基丁酸、精氨酸、谷氨酸和天冬氨酸)。该方法快速、准确,具有良好的重复性和稳定性,可对草莓中游离氨基酸组成进行定量检测。

关键词: 超高效液相色谱, 亲水相互作用色谱, 串联质谱, 游离氨基酸, 草莓

Abstract:

The determination of free amino acids is important for quality evaluation and nutritional studies of strawberries. In this study, an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method using hydrophilic interaction chromatographic column was established for the simultaneous determination of 18 free amino acids in strawberries, and the pretreatment, chromatography, and mass spectrometry conditions were optimized. Specific pretreatment processes include grinding, extraction with water, centrifugation, and membrane filtration. The treated samples were then analyzed by hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry. The separation was performed on an ACQUITY UPLC Glycan BEH Amide column (150 mm×2.1 mm, 1.7 μm), with a water-acetonitrile system containing 5 mmol/L ammonium formate and 0.1% (v/v) formic acid as the mobile phase for gradient elution. A triple quadrupole mass spectrometer was used in positive-ion electrospray ionization (ESI) scanning mode, with target amino acids quantified using the matrix-matched standard-curve method. Eighteen free amino acids were determined with good linearities in the range of 0.5-40.0 μmol/L, along with r² greater than 0.992. The intra-day and inter-day precisions of the method were 1.0%-14.8% and 3.6%-17.6%, respectively. The limits of detection (LODs) were in the range of 50-250 nmol/L. The recoveries of the 18 amino acids were 75.0%-114.6% with relative standard deviations (RSDs) of 0.3%-13.5%. The contents of free amino acids in strawberries at different ripening stages were statistically analyzed, and a total of seven differentiated free amino acids (phenylalanine, isoleucine, glutamine, 4-aminobutyric acid, arginine, glutamic acid, and aspartic acid) were statistically screened. The method is rapid, accurate, reproducible and stable, and can quantitatively determine the content of amino acids in strawberries.

Key words: ultra performance liquid chromatography (UPLC), hydrophilic interaction chromatography (HILIC), tandem mass spectrometry (MS/MS), free amino acids, strawberry

中图分类号:

O658

鞠敏, 宋玉明, 赵金凤, 孙玉明, 周丽娜, 殷庆鑫, 王晨, 蔡蕊, 徐强, 万慧慧. 基于亲水相互作用的超高效液相色谱-三重四极杆质谱法测定不同成熟阶段草莓中18种游离氨基酸[J]. 色谱, 2025, 43(4): 372-381.

JU Min, SONG Yuming, ZHAO Jinfeng, SUN Yuming, ZHOU Lina, YIN Qingxin, WANG Chen, CAI Rui, XU Qiang, WAN Huihui. Determination of 18 free amino acids in strawberries at different ripening stages by ultra performance liquid chromatography-triple quadrupole mass spectrometry based on hydrophilic interaction[J]. Chinese Journal of Chromatography, 2025, 43(4): 372-381.

图/表 9

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No.	Compound	Retention time/min	Parent ion (m/z)	Product ions (m/z)	Declustering potential/V	Collison energies/eV
1	Ala	5.68	90.0	44.0^*	20	12
2	Arg	7.37	175.2	70.2^*, 116.1	40	27, 20
3	Asn	6.37	133.1	74.2^*, 87.2	40	20, 12
4	Ile	4.64	132.1	69.2^*, 86.1	40	24, 14
5	Glu	6.15	148.1	83.9^*, 102.0	30	20, 15
6	His	7.49	156.1	110.0^*, 83.1	30	20, 32
7	GABA	5.08	104.0	87.0^*, 69.4	40	13, 20
8	Asp	6.75	133.9	88.0^*, 74.2	30	14, 19
9	Leu	4.47	132.1	86.2^*, 104.1	30	14, 11
10	Pro	5.12	116.1	70.0^*, 43.3	30	20, 38
11	Thr	5.91	119.9	74.0^*, 56.2	20	14, 20
12	Phe	4.36	166.2	120.2^*, 102.9	30	17, 38
13	Tyr	4.98	182.2	136.1^*, 165.1	20	19, 13
14	Ser	6.28	106.1	60.2^*, 42.2	20	13, 25
15	Val	5.04	118.1	72.3^*, 55.1	30	15, 27
16	Met	4.77	150.0	133.2^*, 104.2	30	13, 13
17	Gln	6.20	147.1	130.1^*, 84.1	40	13, 22
18	Lys	7.52	147.1	84.1^*, 130.1	30	21, 13

No.	Compound	Retention time/min	Parent ion (m/z)	Product ions (m/z)	Declustering potential/V	Collison energies/eV
1	Ala	5.68	90.0	44.0^*	20	12
2	Arg	7.37	175.2	70.2^*, 116.1	40	27, 20
3	Asn	6.37	133.1	74.2^*, 87.2	40	20, 12
4	Ile	4.64	132.1	69.2^*, 86.1	40	24, 14
5	Glu	6.15	148.1	83.9^*, 102.0	30	20, 15
6	His	7.49	156.1	110.0^*, 83.1	30	20, 32
7	GABA	5.08	104.0	87.0^*, 69.4	40	13, 20
8	Asp	6.75	133.9	88.0^*, 74.2	30	14, 19
9	Leu	4.47	132.1	86.2^*, 104.1	30	14, 11
10	Pro	5.12	116.1	70.0^*, 43.3	30	20, 38
11	Thr	5.91	119.9	74.0^*, 56.2	20	14, 20
12	Phe	4.36	166.2	120.2^*, 102.9	30	17, 38
13	Tyr	4.98	182.2	136.1^*, 165.1	20	19, 13
14	Ser	6.28	106.1	60.2^*, 42.2	20	13, 25
15	Val	5.04	118.1	72.3^*, 55.1	30	15, 27
16	Met	4.77	150.0	133.2^*, 104.2	30	13, 13
17	Gln	6.20	147.1	130.1^*, 84.1	40	13, 22
18	Lys	7.52	147.1	84.1^*, 130.1	30	21, 13

No.	Compound	Linear range/(μmol/L)	Linear equation	r²	LOD/(nmol/L)	ME/%
1	Ala	0.5-40.0	y=2325.5x+3762.8	0.996	100	105.40
2	Arg	0.5-40.0	y=413946.0x+277758.0	0.993	50	118.26
3	Asn	0.5-40.0	y=9726.9x+169119.0	0.997	100	101.83
4	Ile	0.5-40.0	y=52658.1x+5822.4	0.999	50	108.77
5	Glu	0.5-40.0	y=31522.2x+36703.0	0.999	100	93.86
6	His	0.5-40.0	y=218849.0x+49215.2	0.998	100	118.27
7	GABA	0.5-40.0	y=10913.9x+15204.0	0.998	100	95.25
8	Asp	0.5-40.0	y=6251.4x+6305.6	0.998	100	114.70
9	Leu	0.5-40.0	y=43221.3x+3021.3	0.999	50	96.87
10	Pro	0.5-40.0	y=57499.8x+12560.5	0.994	100	102.96
11	Thr	0.5-40.0	y=9086.8x+4237.1	0.997	250	100.83
12	Phe	0.5-40.0	y=180570.0x-8084.2	0.999	100	110.48
13	Tyr	0.5-40.0	y=32588.4x+1813.2	0.999	100	101.57
14	Ser	0.5-40.0	y=4438.0x+12601.9	0.998	50	100.29
15	Val	0.5-40.0	y=9584.1x+6998.3	0.997	100	104.75
16	Met	0.5-40.0	y=12958.6x+1561.3	0.997	100	95.76
17	Gln	0.5-40.0	y=2419.3x+56090.2	0.999	100	113.91
18	Lys	0.5-40.0	y=88302.4x+48543.0	0.992	100	109.18

No.	Compound	Linear range/(μmol/L)	Linear equation	r²	LOD/(nmol/L)	ME/%
1	Ala	0.5-40.0	y=2325.5x+3762.8	0.996	100	105.40
2	Arg	0.5-40.0	y=413946.0x+277758.0	0.993	50	118.26
3	Asn	0.5-40.0	y=9726.9x+169119.0	0.997	100	101.83
4	Ile	0.5-40.0	y=52658.1x+5822.4	0.999	50	108.77
5	Glu	0.5-40.0	y=31522.2x+36703.0	0.999	100	93.86
6	His	0.5-40.0	y=218849.0x+49215.2	0.998	100	118.27
7	GABA	0.5-40.0	y=10913.9x+15204.0	0.998	100	95.25
8	Asp	0.5-40.0	y=6251.4x+6305.6	0.998	100	114.70
9	Leu	0.5-40.0	y=43221.3x+3021.3	0.999	50	96.87
10	Pro	0.5-40.0	y=57499.8x+12560.5	0.994	100	102.96
11	Thr	0.5-40.0	y=9086.8x+4237.1	0.997	250	100.83
12	Phe	0.5-40.0	y=180570.0x-8084.2	0.999	100	110.48
13	Tyr	0.5-40.0	y=32588.4x+1813.2	0.999	100	101.57
14	Ser	0.5-40.0	y=4438.0x+12601.9	0.998	50	100.29
15	Val	0.5-40.0	y=9584.1x+6998.3	0.997	100	104.75
16	Met	0.5-40.0	y=12958.6x+1561.3	0.997	100	95.76
17	Gln	0.5-40.0	y=2419.3x+56090.2	0.999	100	113.91
18	Lys	0.5-40.0	y=88302.4x+48543.0	0.992	100	109.18

No.	AA	Added/ (μmol/L)	Recovery/ %	RSD/ %	Intra-day precision/%	Inter-day precision/%	No.	AA	Added/ (μmol/L)	Recovery/ %	RSD/ %	Intra-day precision/%	Inter-day precision/%
1	Ala	1.0	88.4	11.8	4.9	11.0	10	Pro	1.0	93.2	3.2	7.8	3.9
		10.0	97.6	1.2					10.0	101.1	0.8
		25.0	96.1	3.7					25.0	106.1	2.1
2	Arg	1.0	94.6	6.2	4.7	3.6	11	Thr	1.0	97.3	9.0	4.6	6.1
		10.0	97.9	2.1					10.0	92.6	6.6
		25.0	100.3	3.0					25.0	106.6	3.4
3	Asn	1.0	82.0	13.1	1.0	7.2	12	Phe	1.0	111.1	1.8	14.8	6.5
		10.0	78.7	3.8					10.0	102.2	1.0
		25.0	79.6	2.9					25.0	114.6	1.8
4	Ile	1.0	87.7	2.4	5.9	6.1	13	Tyr	1.0	97.5	4.6	4.6	10.0
		10.0	87.4	4.9					10.0	90.2	2.6
		25.0	107.2	5.1					25.0	107.8	2.5
5	Glu	1.0	92.3	4.3	3.7	9.3	14	Ser	1.0	75.0	11.9	2.9	16.8
		10.0	96.8	2.3					10.0	83.1	3.7
		25.0	98.0	1.4					25.0	82.6	2.9
6	His	1.0	103.0	2.3	1.9	5.0	15	Val	1.0	94.5	3.7	6.6	4.8
		10.0	99.1	0.3					10.0	85.7	1.2
		25.0	101.1	1.7					25.0	96.2	2.3
7	GABA	1.0	93.0	4.4	8.3	4.7	16	Met	1.0	93.0	4.8	10.1	17.6
		10.0	82.1	5.4					10.0	88.8	3.5
		25.0	102.2	3.0					25.0	106.2	3.2
8	Asp	1.0	91.1	13.5	4.5	6.9	17	Gln	5.0	81.7	7.6	2.2	17.5
		10.0	85.1	6.2					10.0	95.2	6.5
		25.0	97.4	7.7					20.0	97.2	4.2
9	Leu	1.0	88.5	2.7	6.7	4.8	18	Lys	1.0	97.1	4.9	1.8	5.3
		10.0	88.3	4.2					10.0	96.3	1.7
		25.0	102.0	7.0					25.0	101.6	3.1