液相色谱-质谱法快速检测4 种乳源低聚糖

doi:10.7506/spkx1002-6630-201614015

摘要/Abstract

摘要：

建立一种乳样经简单预处理后采用超高效液相色谱-串联质谱同时检测4 种重要的乳源低聚糖方法。牛乳及人乳经过脱脂、脱蛋白后经amide液相色谱柱用0.1%氨水-乙腈溶液梯度分离，采用质谱检测。2 种唾液酸乳糖标准品的标准曲线在0.78～50.00 mg/L范围内线性良好（R2＞0.990），2 种岩藻糖乳糖标准品的标准曲线在1.56～100.00 mg/L范围内线性良好（R2＞0.990）。加标回收率在80.00%～100.00%之间，重复性、精密度实验所得相对标准偏差均小于5.00%。

关键词: 超高效液相色谱-串联质谱, 低聚糖, 牛乳, 人乳

Abstract:

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was used to
rapidly and simultaneously detect 4 oligosaccharides in bovine milk and human milk. Samples were defatted, deproteined,
resolved on an ACQUITY BEH amide column using gradient elution with 0.1% ammonia water/acetonitrile as mobile
phase, detected by mass spectrometric specific ion monitoring and quantified. The standard curves of 2 fucosyllactose
oligosaccharides were linear within the range from 0.78 to 50.00 mg/L with R2 > 0.990. The standard curves of 2
sialyllactose were linear within the range from 1.56 to 100.00 mg/L with R2 > 0.990. The recoveries of spiked samples
ranged from 80.00% to 100.00%. The relative standard deviation (RSD) of repeatability and precision was less than 5.00%.

Key words: ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), oligosaccharides, bovine milk, human milk

中图分类号:

TS207.3

魏京华，陈历俊，赵军英，张毅，景萌娜，王品，姜铁民，乔为仓. 液相色谱-质谱法快速检测4 种乳源低聚糖[J]. 食品科学, doi: 10.7506/spkx1002-6630-201614015.

WEI Jinghua, CHEN Lijun, ZHAO Junying, ZHANG Yi, JING Mengna, WANG Pin, JIANG Tiemin, QIAO Weicang. Rapid and Simultaneous Detection of 4 Milk Oligosaccharides Using LC-MS[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201614015.

参考文献

[1] TAO N, de PETERS E J, GEMAN J B, et al. Variations in bovine milk

oligosaccharides during early and middle lactation stages analyzed by

high-performance liquid chromatography-chip/mass spectrometry[J].

Journal of Dairy Science, 2009, 92(7): 2991-3001.DOI:10.3168/

jds.2010-3156.

[2] CHATURVED P, WARREN D C, GUILLERMO M, et al.

Milk oligosaccharide profiles by reversed-phase HPLC of their

perbenzoylated derivatives[J]. Analytical Biochemistry, 1997, 251(1):

89-97. DOI:10.1006/abio.1997.2250.

[3] FABRIK I, ?MELÍK R, BOBÁ?OVÁ J. Analysis of free

oligosaccharides by negative-ion electrospray ion trap tandem mass

spectrometry in the presence of H2PO4-anions[J]. International

Journal of Mass Spectrometry, 2012, 309: 88-96. DOI:10.1016/

j.ijms.2011.09.002.

[4] UEMURA Y, ASAKUMA S, YON L, et al. Structural determination

of the oligosaccharides in the milk of an Asian elephant (Elephas

maximus)[J]. Comparative biochemistry and Physiology Part A,

Molecular and Integrative Physiology, 2006, 145(4): 468-478.

DOI:10.1016/j.cbpa.2006.08.001.

[5] BAO Yuanwu, CHEN Ceng, NEWBURG D S. Quantification of

neutral human milk oligosaccharides by graphitic carbon highperformance

liquid chromatography with tandem mass spectrometry[J].

Analytical Biochemistry, 2013, 433(1): 28-35. DOI:10.1016/

j.ab.2012.10.003.

[6] KUNZ C. Lactose-derived oligosaccharides in the milk of elephants:

comparison with human milk[J]. British Journal of Nutrition, 1999,

85(2): 391-399. DOI:10.1017/s0007114599001798.

[7] NINONUEVO M R, WARD R E, LOCASCIO R G, et al. Methods

for the quantitation of human milk oligosaccharides in bacterial

fermentation by mass spectrometry[J]. Analytical Biochemistry, 2007,

361(1): 15-23. DOI:10.1016/j.ab.2006.11.010.

[8] 吕玉泉. 人乳低聚糖的生物学功能[J]. 食品科学, 2002, 23(9): 144-

147. DOI:10.3321/j.issn:1002-6630.2002.09.042.

[9] MARTINEZFEREZ A, GUADIX A, GUADIX E. Recovery of

caprine milk oligosaccharides with ceramic membranes[J]. Journal

of Membrane Science, 2006, 276(1/2): 23-30. DOI:10.1016/

j.memsci.2005.09.027.

[10] CAPSL S, NAPOLI M A D, SEPE L, et al. Sialyloligosaccharides

content in colostrum and milk of two goat breeds[J]. Small

Ruminant Research, 2014, 121(1): 116-119. DOI:10.1016/

j.smallrumres.2013.12.024.

[11] GALEOTTI F, COPPA G V, ZAMPINI L, et al. On-line highperformance

liquid chromatography-fluorescence detectionelectrospray

ionization-mass spectrometry profiling of human milk

oligosaccharides derivatized with 2-aminoacridone[J]. Analytical

Biochemistry, 2012, 430(1): 97-104. DOI:10.1016/j.ab.2012.07.027.

[12] CHATURVEDI P, WARREN C D, ALTAYE M, et al. Fucosylated

human milk oligosaccharides vary between individuals and over

the course of lactation[J]. Glycobiology, 2001, 11(5): 365-372.

DOI:10.1093/glycob/11.5.365.

[13] MORROW A L, RUIZ-PALACIOS G M, ALTAYE M, et al. Human

milk oligosaccharides are associated with protection against diarrhea

in breast-fed infants[J]. Journal of Pediatrics, 2004, 145(3): 297-303.

DOI:10.1016/j.jpeds.2004.04.054.

[14] LEO F, ASAKUMA S, FUKUDA K, et al. Determination of sialyl

and neutral oligosaccharide levels in transition and mature milks of

Samoan women, using anthranilic derivatization followed by reverse

phase high performance liquid chromatography[J]. Bioscience,

Biotechnology, and Biochemistry, 2010, 74(2): 298-303. DOI:10.1271/

bbb.90614.

[15] FIAME L, SADAKI A, TADASHI N, et al. Improved determination

of milk oligosaccharides using a single derivatization with anthranilic

acid and separation by reversed-phase high-performance liquid

chromatography[J]. Journal of Chromatography A, 2009, 1216(9):

1520-1523. DOI:10.1016/j.chroma.2009.01.015.

[16] WU Shuai, RUDOLF G, BRUCE J G, et al. Annotation and structural

analysis of sialylated human milk oligosaccharides[J]. Journal of

Proteome Research, 2010, 10(2): 856-868. DOI:10.1021/pr101006u.

[17] WU S, TAO N, GERMAN J B, et al. Development of an annotated

library of neutral human milk oligosaccharides[J]. Journal of Proteome

Research, 2010, 9(8): 4138-4151. DOI:10.1021/pr100362f.

[18] LOCASCIO R G, NIÑONUEVO M R, KRONEWITTER S

R, et al. A versatile and scalable strategy for glycoprofiling

bifidobacterial consumption of human milk oligosaccharides[J].

Microbial Biotechnology, 2009, 2(3): 333-342. DOI:10.1111/j.1751-

7915.2008.00072.x.

[19] NIÑONUEVO M R, AN H, YIN H, et al. Nanoliquid chromatographymass

spectrometry of oligosaccharides employing graphitized

carbon chromatography on microchip with a high-accuracy mass

analyzer[J]. Electrophoresis, 2005, 26(19): 3641-3649. DOI:10.1002/

elps.200500246.

[20] HONG Q, RUHAAK L R, TOTTEN S M. Label-free absolute

quantitation of oligosaccharides using multiple reaction monitoring[J].

Analytical Chemistry, 2014, 86(5): 2640-2647. DOI:10.1021/

ac404006z.

[21] GUERRERO A, LEBRILLA C B. New strategies for resolving

oligosaccharide isomers by exploiting mechanistic and thermochemical

aspects of fragment ion formation[J]. International Journal of Mass

Spectrometry, 2013, 354/355: 19-25. DOI:10.1016/j.ijms.2013.05.002.

[22] SHEN Zuojun, WARREN C D, NEWBURG D S, et al. Highperformance

capillary electrophoresis of sialylated oligosaccharides

of human milk[J]. Analytical Biochemistry, 2000, 279(1): 37-45.

DOI:10.1006/abio.1999.4448

[23] BAO Yuanwu, ZHU Libin, NEWBURG D S, et al. Simultaneous

quantification of sialyloligosaccharides from human milk by capillary

electrophoresis[J]. Analytical Biochemistry, 2007, 370(2): 206-214.

DOI:10.1016/j.ab.2007.07.004.

[24] ALBRECHT S, SCHOLS H A, HEUVEL E, et al. CE-LIF-MSn

profiling of oligosaccharides in human milk and feces of breast-fed

babies[J]. Electrophoresis, 2010, 31(7): 1264-1273. DOI:10.1002/

elps.200900646.

[25] ALBRECHT S, SCHOLS H A, van den HEUVEL E G, et al.

Occurrence of oligosaccharides in feces of breast-fed babies in

their first six months of life and the corresponding breast milk[J].

Carbohydrate Research, 2011, 346(16): 2540-2550. DOI:10.1016/

j.carres.2011.08.009.

[26] ALBRECHT S, SCHOLS H A, van ZOEREN D, et al.

Oligosaccharides in feces of breast- and formula-fed babies[J].

Carbohydrate Research, 2011, 346(14): 2173-2181. DOI:10.1016/

j.carres.2011.06.034.

[27] TOTTEN S M, WU L D, PARKER E A, et al. Rapid-throughput

glycomics applied to human milk oligosaccharide profiling for large

human studies[J]. Analytical and Bioanalytical Chemistry, 2014,

406(30): 7925-7935. DOI:10.1007/s00216-014-8261-2.

[28] LIU Zhiqian, PETER M, BEN C, et al. Simple liquid chromatographymass

spectrometry method for quantification of major free oligosacch

arides in bovine milk[J]. Journal of Agricultural and Food Chemistry,

2014, 62(47): 11568-11574. DOI:10.1021/jf5037849.