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

doi:10.3724/SP.J.1123.2024.04017

Abstract

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

CLC Number:

O658

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.

Figures/Tables 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