Determination of bovine lactoferrin in milk and dairy products by ultra performance liquid chromatography-tandem mass spectrometry

doi:10.3724/SP.J.1123.2019.10025

Abstract

Abstract:

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established for the quantitative detection of bovine lactoferrin in dairy products. The samples were treated by degreasing and tryptic hydrolysis. Proteins of bovine lactoferrin and peptides were identified using ultra performance liquid chromatography-quadrupole/electrostatic orbitrap-high resolution mass spectrometry (UPLC-Q/Exactive-HRMS) and analyzed by Protein Pilot software. Eight species-specific marker peptides of bovine lactoferrin were identified by comparison of the basic local alignment search tool (BLAST) with the Uniprot database. Three markers with high response strength and stability were chosen for further quantitative research by UPLC-triple quadrupole mass spectrometry (QqQ-MS). The method showed a good linear relationship within its own range. The limits of detection and limits of quantification were 0.023-0.041 and 0.077-0.137 mg/kg, respectively. The observed recoveries were in the range of 93.8%-103.9%. The intra-day and inter-day RSDs were lower than 8.8% and 9.5%, respectively. This method presents various advantages such as strong anti-interference, high sensitivity and reproducibility, and it is suitable for the quantitative analysis of bovine lactoferrin in dairy products.

Key words: ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), marker peptide, bovine lactoferrin, milk and dairy products

CHEN Rouhan, GU Shuqing, ZHAO Chaomin, HUO Yihui, NIU Bing, DENG Xiaojun. Determination of bovine lactoferrin in milk and dairy products by ultra performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2020, 38(6): 663-671.

Figures/Tables 10

References

1	García-Montoya I A , Cendón T S , Arévalo-Gallegos S , et al. Bba-Gen Subjects, 2012, 1820 (3): 226
2	Gupta I , Sehgal R , Kanwar R K , et al. Nanomedicine, 2014, 10 (8): 1289
3	Legrand D , Elass E , Pierce A , et al. Biometals, 2004, 17 (3): 225
4	Iigo M , Alexander D B , Long N , et al. Biochimie, 2009, 91 (1): 86
5	Davoodi S H , Shahbazi R , Esmaeili S , et al. Iran J Pharm Res, 2016, 15 (3): 573
6	Iglesias-Figueroa B F , Espinoza-Sánchez E A , Siqueiros-Cendón T , et al. Inter Dairy J, 2019, 89: 37
7	GB 14880-2012
8	Wu C X , Liu Z N , Zhao Y , et al. Journal of Food Safety & Quality, 2018, 9 (4): 803
8	吴春香, 刘志楠, 赵源, et al. 食品安全质量检测学报, 2018, 9 (4): 803
9	Xing Z E , Wang J , Dai Y Q , et al. Food Science, 2011, 16: 274
9	邢志恩, 王军, 戴蕴青, et al. 食品科学, 2011, 16: 274
10	Huang J Y , He Z Y , Cao J , et al. Inter J Electro Sci, 2018, 13 (8): 7816
11	Cheng J , Gao X J , Liu X F , et al. Journal of Dairy Science and Technology, 2009 (1): 30
11	程静, 高学军, 刘晓飞, et al. 乳业科学与技术, 2009 (1): 30
12	Xia M , Ying T J . Food Science, 2010, 31 (2): 165
12	夏明, 应铁进. 食品科学, 2010, 31 (2): 165
13	Guan L , Dai J H , Jiang X , et al. Journal of Dairy Science and Technology, 2018, 41 (4): 21
13	管乐, 代江华, 江杏, et al. 乳业科学与技术, 2018, 41 (4): 21
14	Pochet S , Arnould C , Debornou P , et al. Food Chem, 2018, 259: 36
15	Zhang Y , Lou F , Wu W , et al. J AOCO Int, 2017, 100 (1): 133
16	Zhang J S , Lai S Y , Cai Z X , et al. Anal Chim Acta, 2014, 829: 33
17	Yuan M M , Feng C , Wang S Y , et al. PLoS One, 2017, 12 (9): 1
18	Chen H , Shi P , Fan F , et al. J Chromatogr B, 2018, 1084: 150
19	Montowska M , Fornal E , Piatek M , et al. Food Control, 2019, 104: 122
20	Gu S Q , Zhao C M , Cheng J , et al. Chinese Journal of Chromatography, 2016, 34 (7): 639
20	古淑青, 赵超敏, 程甲, et al. 色谱, 2016, 34 (7): 639
21	Meng J , Gu S Q , Fang Z , et al. Chinese Journal of Chromatography, 2019, 37 (7): 712
21	孟佳, 古淑青, 方真, et al. 色谱, 2019, 37 (7): 712
22	Sreedhara A , Flengsrud R , Prakash V , et al. Int Dairy J, 2010, 20: 487
23	Chang C , Gao Z , Ying Q , et al. Anal Chem, 2019, 91 (2): 1335
24	Fischer M, Muth T, Renard B Y. Mass Spectrometry of Proteins. New York: Humana Press, 2019: 159
25	Zhan L N , Chen Q , Gu S Q , et al. Chinese Journal of Chromatography, 2017, 35 (4): 405
25	詹丽娜, 陈沁, 古淑青, et al. 色谱, 2017, 35 (4): 405
26	Franco I , Castillo E , Pérez M D , et al. J Food Dairy Sci, 2010, 93 (10): 4480
27	Franco I , Pérez M D , Conesa C , et al. Food Res Int, 2018, 106: 173

Peptide	Position	Retention time/min	Precursor ion (m/z) (charge state)	Product ion (m/z)	CE/eV
CE: collision energy; * quantitative ion.
ADAVTLDGGMVFEAGR	73-88	8.78	804.8880 (2+)	1038.4673^* (y10⁺)	28.7
				1252.5990 (y12⁺)	28.7
				303.1775 (y3⁺)	40.7
TESLEPLQGAVAK	158-170	7.24	671.8643 (2+)	783.4723^* (y8⁺)	23.4
				445.2769 (y5⁺)	29.4
				912.5149 (y9⁺)	20.4
PYLSWTESLEPLQGAVAK	153-170	10.16	995.0200 (2+)	783.4523^* (y8⁺)	42.3
				1241.6736 (y12⁺)	39.3
				1112.6310 (y11⁺)	33.3
LSWTESLEPLQGAVAK	155-170	9.52	864.9620 (2+)	783.4723^* (y8⁺)	31.1
				912.5149 (y9⁺)	28.1
				1112.6310 (y11⁺)	31.1
SLEPLQGAVAK	160-170	6.98	556.8191 (2+)	783.4723^* (y8⁺)	18.8
				445.2769 (y5⁺)	24.8
				912.5149 (y9⁺)	18.8
ESPQTHYYAVAVVK	105-118	6.77	531.2753 (3+)	586.3923^* (y6⁺)	15.8
				749.4556 (y7⁺)	21.8
				912.5189 (y8⁺)	21.8
SFQLFGSPPGQR	304-315	8.52	660.8384 (2+)	554.3045^* (y5⁺)	22.9
				698.3580 (y7⁺)	25.9
				845.4264 (y8⁺)	25.9
LRPVAAEIYGTK	93-104	6.72	439.9924 (3+)	468.2453^* (y4⁺)	15.4
				581.3293 (y5⁺)	15.4
				710.3719 (y6⁺)	15.4

Peptide	Position	Retention time/min	Precursor ion (m/z) (charge state)	Product ion (m/z)	CE/eV
CE: collision energy; * quantitative ion.
ADAVTLDGGMVFEAGR	73-88	8.78	804.8880 (2+)	1038.4673^* (y10⁺)	28.7
				1252.5990 (y12⁺)	28.7
				303.1775 (y3⁺)	40.7
TESLEPLQGAVAK	158-170	7.24	671.8643 (2+)	783.4723^* (y8⁺)	23.4
				445.2769 (y5⁺)	29.4
				912.5149 (y9⁺)	20.4
PYLSWTESLEPLQGAVAK	153-170	10.16	995.0200 (2+)	783.4523^* (y8⁺)	42.3
				1241.6736 (y12⁺)	39.3
				1112.6310 (y11⁺)	33.3
LSWTESLEPLQGAVAK	155-170	9.52	864.9620 (2+)	783.4723^* (y8⁺)	31.1
				912.5149 (y9⁺)	28.1
				1112.6310 (y11⁺)	31.1
SLEPLQGAVAK	160-170	6.98	556.8191 (2+)	783.4723^* (y8⁺)	18.8
				445.2769 (y5⁺)	24.8
				912.5149 (y9⁺)	18.8
ESPQTHYYAVAVVK	105-118	6.77	531.2753 (3+)	586.3923^* (y6⁺)	15.8
				749.4556 (y7⁺)	21.8
				912.5189 (y8⁺)	21.8
SFQLFGSPPGQR	304-315	8.52	660.8384 (2+)	554.3045^* (y5⁺)	22.9
				698.3580 (y7⁺)	25.9
				845.4264 (y8⁺)	25.9
LRPVAAEIYGTK	93-104	6.72	439.9924 (3+)	468.2453^* (y4⁺)	15.4
				581.3293 (y5⁺)	15.4
				710.3719 (y6⁺)	15.4

Method	Peptide	Linear range/(mg/L)	Regression equation	R²	LOD/(mg/kg)	LOQ/(mg/kg)
Y: peak area; X: mass concentration, mg/L; Y′: peak area ratio of maker peptide to internal standard peptide.
ESM	ESPQTHYYAVAVVK	0.15-75	Y=5.29×10³X+6.63×10²	0.9989	0.041	0.137
	SFQLFGSPPGQR	0.10-75	Y=2.11×10⁴X+6.05×10²	0.9992	0.023	0.077
	LRPVAAEIYGTK	0.10-75	Y=2.18×10⁴X-5.97×10²	0.9988	0.027	0.090
ISM	SFQLFGSPPGQR	0.10-75	Y′=3.31×10^-2X+1.20×10^-2	0.9993	0.019	0.063

Method	Peptide	Linear range/(mg/L)	Regression equation	R²	LOD/(mg/kg)	LOQ/(mg/kg)
Y: peak area; X: mass concentration, mg/L; Y′: peak area ratio of maker peptide to internal standard peptide.
ESM	ESPQTHYYAVAVVK	0.15-75	Y=5.29×10³X+6.63×10²	0.9989	0.041	0.137
	SFQLFGSPPGQR	0.10-75	Y=2.11×10⁴X+6.05×10²	0.9992	0.023	0.077
	LRPVAAEIYGTK	0.10-75	Y=2.18×10⁴X-5.97×10²	0.9988	0.027	0.090
ISM	SFQLFGSPPGQR	0.10-75	Y′=3.31×10^-2X+1.20×10^-2	0.9993	0.019	0.063

Method	Peptide	Added/ (mg/kg)	Average recoveries (n=6)/%	Intra-day RSD (n=3)/%	Inter-day RSD (n=3)/%
ESM	ESPQTHYYAVAVVK	0.1	97.9	7.1	7.6
		1.0	101.9	7.3	7.0
		10	104.6	4.2	5.7
	SFQLFGSPPGQR	0.1	99.3	3.4	3.9
		1.0	93.8	8.8	9.5
		10	103.9	1.2	8.2
	LRPVAAEIYGTK	0.1	101.9	4.9	4.3
		1.0	102.9	2.4	4.4
		10	100.9	1.2	3.8
ISM	SFQLFGSPPGQR	0.1	98.5	3.6	3.9
		1.0	102.7	5.0	10.9
		10	102.7	7.7	8.8