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1 5 7 Vol. 5 No Journal of Food Safety and Quality Jul., 陶保华 1, 黄焘 1, 赖世云 1, 任一平 (1., ; 2., ) 2* 摘要 : 目的 B - 方法, UPLC HSS T3 (MRM), 结果 B 1 () B 2 () B 6 () (LOQ) 0.2 µg/100 g, B 3 () B 5 ( ) LOQ 0.3 µg/100 g 93.1%~101.1%, (RSD) 2.8%~6.2%5, R 结论 5 B -, ;, 关键词 : B ; ; ; Determination of thiamin, riboflavin, nicotinamide, pantothenic acid and pyridoxal in human milk by UPLC-MS/MS TAO Bao-Hua 1, HUANG Tao 1, LAI Shi-Yun 1, REN Yi-Ping 2* (1. Beingmate Baby & Child Food Co., Ltd, Hangzhou , China; 2. Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou , China) ABSTRACT: Objective To establish a rapid and sensitive quantitative method of several B-vitamins in human milk by ultra-pressure liquid-chromatography tandem mass spectrometry (UPLC-MS/MS). Methods The non-polar substances and protein were removed from samples by ether and methanol respectively. The sample extracts were separated by a UPLC HSS T3 column. The detection was performed with mass spectrometry under multiple reaction monitoring (MRM) mode with stable isotopedilution method. Results LOQs of thiamin, riboflavin, pyridoxalnicotinamide and pantothenic acid were between 0.2 and 0.3 µg/100 g. The spiked recoveries were 93.1%~101.1%. The precision RSD were below 6.2%.The lineal range had got high correlation coefficients (R 2 >0.99). Conclusion A simultaneous quantification of water-soluble vitamin B in human milk was developed by using a UHPLC-MS/MS method. The preparation of sample was simple, rapid with small volume of samples. Five stable isotope standards for each vitamin B were used which showed high precisive and accurate. The developed method is suitable for the study of vitamin B concentration in human milk and can provide * 通讯作者 :,,, [email protected] *Corresponding author: REN Yi-Ping, Senior Engineer, Zhejiang Provincial Center for Disease Control and Prevention, No.3399, Binsheng Road, Binjiang District, Hangzhou , China. [email protected]

2 the technical support for infant formula. KEY WORDS: water-soluble vitamins B; human milk; stable isotope dilution; ultra pressure liquid chromatography-mass spectrometry/mass spectrometry 1 引言,, [1] : B B,, [2] B, B, Stuetz [3] B 1 Roughed [4] (FAD) B 2, B 6 B, Sakurai [5] HPLC, ; Roughed [4] 5, UPLC-MS/MS, [6] [7,8] ; B [9,10] Lu [11] UPLC-MS/MS,,,, UPLC-MS/MS,, (MRM), 2 方法与材料 2.1 仪器设备 - : UPLC-XEVO TQ-S MS, (Waters, ); (Beckman, ); : 0.01 g g; Milli-Q (Millipore, ) 2.2 标准品与试剂 : ( : 99.9%) ( : 99.9%) ( : 99.9%)( : 99.9%) ( : 99.0%), Sigmaaldrich ; 13 C 4 - (98%) 13 C 4, 15 N 2 - (97%) 2 H 4 -(97%) 13 C 6, 15 N 2 -(97%) 2 H 3 - ( 98%), Poly Sciences : () (, 37%); () () ROE ; ()() Merck ; Milli-Q 2.3 储备液配制 13 C 4-2 H 4-13 C 6, 15 N 2-2 H 3-10 mg, 10 ml, 1 mg/ml 13 C 4, 15 N 2-1 mg, 5 mol/l, 10 ml, 0.1 mg/ml 2.4 样品采集,,,, 80

3 7, : 样品前处理, 0.15 g ( g), 50 μl 100 μl 1.2 ml, 2 min, 10 min 2 ml, 500 μl 500 μl, 2 min, 4 15 min 10 min, 0.22 µm, 2.6 分析检测条件 超高压液相色谱参考条件, : : UPLC HSS T3 (100 mm 2.1 mmi.d., 1.8 μm, Waters ), : 40 ; : 10 ; : 10 μl; A: 0.01% 10 mmol/l ; B: 5 mmol/l ; : 0~1.0 min, 99% A; 1~2.5 min, 99%~75% A; 2.5~3.5 min, 75%~50% A; 3.5~4.0 min, 50%~30%A; 4.0~4.5 min, 30% A; 4.5~4.8 min, 30%~5% A; 4.8~5.8 min, 5% A; 5.8~6.0 min, 5%~99% A 质谱参数参考条件 ESI +, : 3.0 kv, : 500, : 800 L/min, : 150 L/h, : mbar, 1:2.8 V, 1:15.2 V, 1:0.8; 2:2.8 V, 2:15.5 V, 2:0.9; : 150, : 3.0 V, : 0.5 V, : 0.5 V, : 1.0, 1: 表 1 质谱 MRM 参数 Table 1 Mass spectrometry parameters m/z V m/z ev C , C 15 4 N H C 6, 15 N H * * * * * * * * * * 12 : * Quantitative ions

4 结果与讨论 3.1 色谱条件的优化 色谱柱的选择, UPLC BEH C 18 ( mm, 1.7 µm, 100 Å) UPLC HSS T3( mm, 1.8 µm, 100 Å) 2, 2, HSS T3 C 18, BEH C 18, 2, 5 2,,, 5 mmol/l, 10 mmol/l, 4 5, 2, 2,,, ; A B 0.01%( 2 3),, A 0.01% 10 mmol/l, B 5 mmol/l 3.2 质谱条件的选择 1 Fig. 1 Standard chromatograms 流动相的选择,,,,,, 5 A 200 ng/ml ESI,,,,,, ESI + ( 1),,, ( 1) 3.3 线性关系与灵敏度 线性关系 0.01%10 mmol/l

5 7, : : A 0.1%, B 0.1 % ; 2: A 0.01 %, B 0.1 % ; 3: A 0.01 %, B ; 4: A B ; 2 5 Fig. 2 The chromatograms of five water-soluble vitamins separated by mobile phase with different formic acid concentration 5 : ng/ml; : ng/ml; : ng/ml; 5 13 C 4-2 H 3-5 ng/ml, 13 C 4, 15 N 2-2 H 4-13 C 6, 15 N 2-10 ng/ml 5, 5%, R 定量限计算,, S/N=10,,

6 LOQ, LOQ 0.2 µg/100 g; LOQ 0.3 µg/100 g 3.4 回收率和重复性实验,, µg/100 g, µg/100 g, µg/100 g, µg/100 g, µg/100 g, LC-MS/MS 93.1%~98.2%, RSD 2.8%~6.2% 92.6%~99.6%, RSD 2.9%~4.7% 94.5%~100.3%, RSD 3.8%~5.1% 96.1%~101.1%, RSD 3.7%~5.5% 93.4%~97.1%, RSD 4.3%~5.9%,, 实际样品检测 100,, LC-MS/MS 5 : 3.6 µg/100 g; : 4.2 µg/100 g ; : 26.3 µg/100 g; : µg/100 g; : 4.4 µg/100 g [12] (: 14 µg/100 g : 36 µg/100 g : 117 µg/100 g: 223 µg/100 g : 11 µg/100 g),,, ; 5 [13], Table 2 表 2 5 种水溶性维生素的加标回收率 (n=6) Recoveries and RSDs of five water-soluble vitamins in spiked samples (n=6) µg/100 g µg/100 g % %

7 7, : a. b. 3 5 Fig. 3 The box plot of five water-soluble vitamins content 4 结论 5 B UPLC-MS/MS,, ;, ; 4, ; MRM, 5,, 100, 参考文献 [1],,,. 4 [J]., 2003, 21(1): Li K, Wang HJ, Pan CH, et al. Simultaneous determination of four water soluble vitamins in multivitamin tablets by Ion-pair Reversed-Phase High Performance Liquid Chromatography[J]. Chin J Chromatogr, 2003, 21(1): [2]. [J]., 1983, 14(1): Zhao WD. Breast-feeding and breast milk nutrition research[j]. Prog Physiol Sci, 1983, 14(1): [3] Stuetz W, Carrara VI, McGready R, et al. Micronutrient status in lactating mothers before and after introduction of fortified flour: cross-sectional surveys in Maela refugee camp[j]. European J Nutr, 2012, 51(4): [4] Roughead KZ, McCormick DB. Flavin composition of human milk[j]. American Soc Clin Nutr, 1990, 52: [5] Sakurai T, Furukawa M, Asoh M. Fat-soluble and water-soluble vitamin contents of breast milk from Japanese women[j]. J Nutr Sci Vitaminol, 2005, 51(4): [6] Churchwell MI, Twaddle NC, Meeker LR, et al. Improving LC MS sensitivity through increases in chromatographic performance: Comparisons of UPLC ES/MS/MS to HPLC ESI/MS/MS [J]. J Chromatogr B, 2005, 825: [7],,. HPLC-MS/MS 19 [J]., 2007, 26: Wang HY, Hu P, Jiang J. Determination of 19 kinds of Free Amino Acids in human serum by HPLC-MS/MS[J]. J Instrum Anal, 2007, 26: [8],,,. UPLC-MS/MS 12 [J]., 2007, 28(7): Wang FM, Chen JH, Lin LM, et al. Determination of 12 macrolide antibiotics residues in products of animalorigin by ultra performance liquid chromatography coupled with tandem mass spectrometry[j]. J Instrum Anal, 2007, 28(7): [9] Brouwera VD, Storozhenkob S, Stovea CP, et al. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the sensitive determination of folates in rice[j]. J Chromatogr B, 2010, 878: [10] Chandra-Hioe MV, Bucknall MP, Arcot J. Folate analysis in foods by UPLC-MS/MS: development and validation of a novel, high throughput quantitative assay; folate levels determined in Australian fortified breads[j]. Anal Bioanal Chem, 2011, 401(3):

8 [11] Lu B, Ren Y, Huang B, et al. Simultaneous determination of four water-soluble vitamins in fortified infant foods by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry[j]. J Chromatogr Sci, 2008, 46(3): [12] United States Department of Agriculture.National Nutrient Database for Standard Reference [DB/OL]. [ ]. [13] Committee on nutritional status during pregnancy and lactation, Institute of medicine.nutrition during lactation[m]. Washington D.C.: National Academy Press, 作者简介 ( 责任编辑 : 邓伟 ) 陶保华, 本科, 主要研究方向为营养分析 tbh_ @163.com 任一平, 高级工程师, 主要研究方向为卫生理化检验技术, 食品安全检验技术 [email protected] 转基因食品检测与研究 专题征稿 [email protected] 食品安全质量检测学报 编辑部