超高效液相色谱-串联质谱法测定乳与乳制品中牛乳铁蛋白

doi:10.3724/SP.J.1123.2019.10025

色谱 ›› 2020, Vol. 38 ›› Issue (6): 663-671.DOI: 10.3724/SP.J.1123.2019.10025

超高效液相色谱-串联质谱法测定乳与乳制品中牛乳铁蛋白

陈柔含¹, 古淑青²^,^*(), 赵超敏², 霍忆慧², 钮冰¹, 邓晓军²

¹ 上海大学生命科学学院食品工程系, 上海 200436
² 上海海关动植物与食品检验检疫技术中心, 上海 200135

收稿日期:2019-10-24 出版日期:2020-06-08 发布日期:2020-12-10
通讯作者: 古淑青
作者简介:古淑青.Tel:(021)38620526, E-mail:gushuqing@sina.cn
基金资助:
国家重点研发计划项目(2017YFF0211303);国家重点研发计划项目(2018YFC1603503);上海市农业科技项目(19391901500);长三角科技合作项目(19395810100);上海市技术标准专项项目(17DZ2201100)

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

CHEN Rouhan¹, GU Shuqing²^,^*(), ZHAO Chaomin², HUO Yihui², NIU Bing¹, DENG Xiaojun²

¹ Department of Food Engineering, School of Life Science, Shanghai University, Shanghai 200436, China
² Technical Center for Animal Plant and Food Inspection and Quarantine, Shanghai Customs, Shanghai 200135, China

Received:2019-10-24 Online:2020-06-08 Published:2020-12-10
Contact: GU Shuqing
Supported by:
National Key Research and Development Plan Project(2017YFF0211303);National Key Research and Development Plan Project(2018YFC1603503);Shanghai Agricultural Science and Technology Project(19391901500);Science and Technology Joint Project of the Yangzte River Delta(19395810100);Shanghai Technical Standard Project(17DZ2201100)

摘要/Abstract

摘要：

建立了超高效液相色谱-串联质谱技术定量检测乳制品中牛乳铁蛋白含量的方法。样品经脱脂、酶解后，利用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱（UPLC-Q/Exactive-HRMS）和Protein Pilot软件分析，实现了乳铁蛋白及其肽段的鉴定；通过初级局部序列搜索工具（basic local alignment search tool，BLAST）与Uniprot数据库对比分析筛选出牛乳铁蛋白的8个特征肽段；选择其中3个响应强度高、稳定性好的特征肽段进一步通过超高效液相色谱-三重四极杆质谱（UPLC-QqQ-MS）进行验证和多反应监测（MRM）定量研究，并考察了空白基质匹配的外标法和内标法对乳铁蛋白检测结果的差异。采用外标法定量时，3个肽段在各自范围内线性关系良好，检出限为0.023~0.041 mg/kg，定量限为0.077~0.137 mg/kg，平均回收率为93.8%~103.9%，日内精密度≤8.8%，日间精密度≤9.5%。该方法抗干扰能力强，灵敏度高，重复性好，适用于乳及乳制品中牛乳铁蛋白的含量测定。

关键词: 超高效液相色谱-串联质谱, 特征肽段, 牛乳铁蛋白, 乳及乳制品

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

陈柔含, 古淑青, 赵超敏, 霍忆慧, 钮冰, 邓晓军. 超高效液相色谱-串联质谱法测定乳与乳制品中牛乳铁蛋白[J]. 色谱, 2020, 38(6): 663-671.

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.

图/表 10

参考文献

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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

超高效液相色谱-串联质谱法测定乳与乳制品中牛乳铁蛋白

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

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Metrics

Sample	Detected contents/(mg/kg)
	ESM			ISM
	ESPQTHYYAVAVVK	SFQLFGSPPGQR	LRPVAAEIYGTK	SFQLFGSPPGQR
Detected contents: mean±SD.
1	171.72±2.14	189.85±5.55	215.97±3.85	188.97±4.37
2	218.77±4.46	242.27±3.60	256.52±2.97	246.62±2.49
3	323.95±4.67	308.60±4.30	312.05±3.44	310.73±3.67

Matrix	No.	Content/ (mg/kg)
UHT: ultra high temperature instantaneous sterilization.
Milk	1	255.72±3.74
powder	2	345.97±1.21
	3	223.13±1.59
	4	4299.07±0.71
	5	418.53±4.74
	6	266.75±2.64
	7	874.68±5.44
	8	283.27±3.71
	9	337.70±1.64
	10	376.38±2.46
	11	308.60±4.30
	12	678.55±1.35
Yogurt	1	9.10±1.64
	2	9.90±4.49
	3	7.27±1.75
	4	53.70±0.86
	5	7.14±1.61
Pasteurized	1	75.07±0.87
milk	2	86.81±0.75
	3	111.38±0.72
	4	116.16±0.89
UHT milk	1	62.65±4.32
	2	63.15±2.17
	3	59.45±1.02

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