Microsoft Word 郑菲菲.doc

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1 5 7 Vol. 5 No Journal of Food Safety and Quality Jul., 2014 α- 郑菲菲 1, 赖世云 1, 陶保华 1, 黄百芬 2, 任一平 (1., ; 2., ) 2* 摘要 : 目的 α- β- 方法,,, C 30, 4 结果 4, ; 70%~120%, 20% 结论,,,, α-, β- 100, 关键词 : ; ; α-; ; β- ; Simultaneous determination of vitamin A, E and carotenoids in human milk by high performance liquid chromatography ZHENG Fei-Fei 1, LAI Shi-Yun 1, TAO Bao-Hua 1, HUANG Bai-Fen 2, 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 method for simultaneous determination of retinoid, α-tocopherol, lutein and β-carotene in human milk by high performance liquid chromatography. Methods The human milk sample was saponified and extracted with a mixture of petroleum ether and diethyl ether. The sample was separated after concentration by a C 30 column and detected by the UV detector and FLD detector. Results The method showed a good linearity over with R 2 > The recoveries at three spiked levels were between 70%~120%. The relative standard deviation was less than 20%. Conclusion The purity of standard was analyzed using HPLC after standardization with UV spectrometer, thus the accuracy of the method was improved. The method validation showed a high accuracy, sensitivity and stability. This method could be used to simultaneous determination of retinoid, α-tocopherol, lutein and β-carotene in human milk. A preliminary database of the fat-soluble vitamin concentration in human milk was established with 100 mature human milk samples. KEY WORDS: human milk; retinoid; α-tocopherol; lutein; β-carotene; purity analysis of standards * 通讯作者 :,, renyiping@263.net *Corresponding author: REN Yi-Ping, Senior Engineer, Zhejiang Provincial Center for Disease Control and Prevention, NO.3399, Binsheng Road, Binjiang District, Hangzhou , China. renyiping@263.net

2 7, : α 引言 200,, [1],,, [2] A,,,, A,, [3] [4] A, [5] A [6] α- β- γ-, β-, β- A,,,,,, [7] E 8 : α- β- γ- δ- α- β- γ- δ-, α-, [8] E, [9],,,,,,,, [10] [11-18] [19-20] Liu [14] A,,,, Romeu-Nadal [15] HPLC ELSER E α- γ- ELSER,, Kamao [20],,,,,, β- [12], : 1., ; 2. α-,, 4 ; 3.,,, ;, α-, β-, 2 材料与方法 2.1 材料与试剂试剂 ( : 97.5%, Sigma); α-( : 97%, USP); ( : 96.7%, ); β- ( : 97%, Fluka); (, Merck); ( : 99%, Sigma); (, ); (, ); (, ); (, ); (, ); 2, 6- (BHT)(, )

3 样品采集,,,, 仪器与设备 Ultimate-3000RS,, ( DIONEX ); : YMC Carotenoid (4.6 mm 250 mm 3 μm)( YMC ); UV (); SHA-B ( ) 2.2 样品前处理 : 1 g( g) 15 ml 0.1 g, 2 ml 0.1%BHT, 0.5 ml 50%, 70±1 15 min, 提取 : 6 ml.. (1.. 1, v.. v), 90 s 4 ml,, 7000 r/min 1 min 10 ml (, 0.1 g h ), 35±2 1 ml 0.1%BHT :(1.. 1, v.. v), 0.22 μm, 2.3 标准溶液配制及校准标准储备液配制 : α- 10 mg, 10 ml, 1 mg/ml β- 10 mg, 0.1%BHT 25 ml, 400 μg/ml, 20 C 标准溶液浓度标定 : α-, β-, 3 μg/ml α- 100 μg/ml, 2 μg/ml β- 2 μg/ml,, A, X, 1 A 4 : X = 10 1% E1cm : Table 1 X: (μg/ml); E 1% 1cm : ; A: ; 10 4 : 表 1 标准品吸光值及测定条件 Absorption and determination conditions of standards (nm) (nm) β α 标准溶液纯度校准 : (2.3.2), α-, β-, 2.4, C 30,,, 2.4 分离检测参考条件 : YMC Carotenoid C 30 (4.6 mm 250 mm, 3 μm); : 30 ; : 20 μl; A: ; B: ; C: ; : 0~2.5 min A% 95% C% 5%, 2.5~5 min A% 95%~92% C% 5%, 5~6 min A% 92%~90% C% 5%~0%, 6~13 min A% 90%~70%, 13~14 min A% 70%~60% C% 0%, 14~20 min A% 60% C% 0%, 20~20.5 min A% 60%~95% C% 0%~5%, 20.5~27 min A% 95% C% 5%; : 1 ml/min; : 27 min: : 325 nm; β- : 450 nm; : α- : 295 nm, : 345 nm

4 7, : α 结果 3.1 标准品纯度校准,,,, 1, α- β min,,,,, 3.2 HPLC 流动相选择, α-, α-, 5%, 4, ( 2) 3.3 方法学验证,,,,, 线性, 4,, α-, β-, 0.1% BHT : (1 : 1, v : v), : μg/ml; α- : μg/ml; β-: μg/ml, 2.4,, 定量限的计算 4, : μg/ml, α-: 0.05 μg/ml, : μg/ml, β- : μg/ml, 1 4 Fig. 1 Chromatograms of 4 vitamin standards after storage

5 Fig. 2 2 Chromatograms of samples at different wavelength Table 2 表 2 回归方程, 相关系数和定量限 Calibrations, correlation coefficients (R 2 ) and limits of quantitations (μg/100 g) Y=1.1638X α- Y=10.665X Y=0.3470X β- Y=0.3292X 表 3 加标回收率和相对标准偏差 Table 3 Recoveries and RSDs (μg/100 g) (n=6) (n=6) % 17.50% % 12.37% % 4.00% % 19.38% ,,, 回收率,,, 3 2.2,, %~115%, (RSD) 20%, 2002/657/EC, 60%~115%, <22.44% 4,, β-, RSD 12.5%, RSD, RSD α % 5.72% % 8.11% % 8.49% % 8.89% % 4.12% % 14.74% β % 12.50% % 14.94% 仪器精密度, : 0.75 μg/ml, α- :7.5 μg/ml, : μg/ml, β- : 0.08 μg/ml, 5,, 5, 4 4

6 7, : α %, 日内精密度, ,, 日间精密度 2.2 5,, 4, 4 表 4 Table 4 仪器精密度, 日内精密度和日间精密度 Precision of the instrument, intraday and day to day α- β- (n=5) 0.83% 0.86% 0.65% 2.15% (n=11) 7.10% 8.10% 3.66% 6.34% (n=20) 12.40% 8.60% 8.00% 14.00% 3.4 样品测定 100, 2.2,,, 4 : 27.81~70 μg/100 g, α- 93.6~234 μg/100 g, 2.71~ 8.8 μg/100 g, β- 0~1.5 μg/100 g, 3 [21], 61 μg/100 g; α- 80 μg/100 g; β- 7 μg/100 g; α- β-,, β-, α- 3, α-, β- Fig. 3 Box plots of retinoid, α-tocopherol, lutein and β-carotene amounts in mature human milk 4 结论 α- β-,, HPLC,,, 100, 参考文献 [1] Ballard O, Morrow AL. Human milk composition nutrients and bioactive factors[j]. Pediatr Clin N Am, 2013, (60): [2],. [J]., 2002, 13(6): Xin WZ, Yuan Z. Human milk composition and function[j]. Foreign Med Sci, 2002, 13(6): [3] Olson JA. Vitamin A In the nutrition foundation, nutrition review s present knowledge in nutrition [M]. Washington D.C.:

2072 5 National Academy Press, 1984. [4] Clagett-Dame M, DeLuca HF. The role of vitamin A in ammalian reproduction and embryonic development[j]. Annuev Nutr, 2002, (22): 347 381. [5] Bates CJ.

Washington D.C.: National Academy Press, 1991. [7],,,. β- [J]., 2007, 26(3): 61 65. Hu XJ, Zhang LF, Wang XL, et al. β-carotene oxidation products and its inhibition on tumorous cells [J].

7 National Academy Press, [4] Clagett-Dame M, DeLuca HF. The role of vitamin A in ammalian reproduction and embryonic development[j]. Annuev Nutr, 2002, (22): [5] Bates CJ. Vitamin A in pregnancy and lactation[j]. Nutr Sic, 1983, (42): [6] Committee on nutritional status during pregnancy and lactation, Institute of medicine. Nutrition during lactation[m]. Washington D.C.: National Academy Press, [7],,,. β- [J]., 2007, 26(3): Hu XJ, Zhang LF, Wang XL, et al. β-carotene oxidation products and its inhibition on tumorous cells [J]. J Food Sci Biotechnol, 2007, 26(3): [8] Ullrey DE. Vitamin E for swine [J]. J Animal Sci, 1981, (53): [9] Kolb E, Seehawer J. The development of the immune system and vitamin levels in the bovine fetus and neonate: a review including the effect of vitamins on the immune system[j]. Tiera rztl Umsch, 1998, (53): [10],. [J]., 2005, (5): Zhu HX, Zheng JX. A review: structure, distribution, properties and physiological functions of lutein [J]. China Food Addit, 2005, (5): [11],,,. 7 [J]., 2013, 29(1): Chen WQ, Wan J, Huang LY, et al. Simultaneous determination of seven fat soluble vitamins in infant formula using high performance liquid chromatography[j]. J Anal Sic, 2013, 29(1): [12],,. β- [J]., 2014, 5(1): Zheng FF, Tao BH, Lai SY, et al. Simultaneous determination of lutein and β-carotene in infant formulas by high pressure liquid chromatography[j]. J Food Safe Qual, 2014, 5(1): [13] Larsen E, Christensen LP. Simple saponification method for the quantitative determination of carotenoid in green vegetables[j]. J Agric Food Chem, 2005, 53(4): [14] Liu Y, Xu MJ, Canfield LM. Enzymatic hydrolysis, extraction, and quantitation of retinol and major carotenoids in mature human milk[j]. Nutr Biochem, 1998, (9): [15] Romeu-Nadal M, Morera-Pons S, Castellote AI, et al. Determination of γ- and α-tocopherols in human milk by a direct high-performance liquid chromatographic method with UV vis detection and comparison with evaporative light scattering detection[j]. Chromatogr A, 2006, (1114): [16],,. A [J]., 2001, 30(4): Zhang LJ, Bao JM, Chen HL. Determination result analysis of vitamin A in the human milk of Zhoushan Island [J]. J Hyg Res, 2001, 30(4): [17],. [J]., 1997, 4(3): Zheng MC, Yu JH. Fat-soluble vitamins in cord blood and colostrum in guilin and o saka[j]. Guangxi Sci, 1997, 4(3): [18],,. [J]., 1986, 8(1): Wang DK, Xie JW, Wang ZL. The mineral and vitamin contents of human milk[j]. Acta Nutrimenta Sinica, 1986, 8(1): [19] Plozza TV, Trenerry C, Caridi D. The simultaneous determination of vitamins A, E and β-carotene in bovine milk by high performance liquid chromatography ion trap mass spectrometry(hplc-msn) [J]. Food Chem, 2012, (134): [20] Kamaoa M, Tsugawa N, Suhara Y, et al. Quantification of fat-soluble vitamins in human breast milk by liquid chromatography tandem mass spectrometry[j]. Chromatogr B, 2007, (859): [21] USDA National Nutrient Database for Standard. Full report (ALL nutritions) 01107, Milk, human, mature, fluid [EB/OL]. &format=&count=&max=25&offset=&sort=&qlookup=milk. 作者简介 ( 责任编辑 : 邓伟 ) 郑菲菲, 助理工程师, 主要研究方向为食品检验技术 xiaodcd@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及食品安全检验技术 renyiping@263.net

Microsoft Word 郑菲菲.doc

Microsoft Word 郑菲菲.doc 5 1 Vol. 5 No. 1 2014 1 Journal of Food Safety and Quality Jan., 2014 β- 郑菲菲 1, 陶保华 1, 赖世云 1, 黄百芬 2, 任一平 (1., 310057; 2., 310051) 2* 摘要 : 目的 β- 方法 2 ml 50%, 70 15min, : (1:1,v/v),, C 30, 450 nm, 结果 β-