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1 8 5 Vol. 8 No Journal of Food Safety and Quality May., 2017 D 3 * 刘玉玲, 姜成君, 龙凌云, 桂升华, 潘小红 (, ) 摘要 : 目的 D 3 方法,,, A,, - (997:3, V:V), 1.0 ml/min, Waters Nova-Pak Silia, 254 nm, 结果 D ~9.506 μg/ml, 1, %~100.58%, 0.44%~0.72% 结论, D 3 关键词 : D 3 ; ; Determination of cholecalciferol in health food by high performance liquid chromatography LIU Yu-Ling, JIANG Cheng-Jun, LONG Ling-Yun, GUI Sheng-Hua, PAN Xiao-Hong * (Hunan Institute for Drug Control, Changsha , China) ABSTRACT: Objective To establish a method for the determination of cholecalciferol in health food, which included purification with semi-preparative reversed liquid chromatography (RPLC) system, and separation with silica gel column of positive phase high performance liquid chromatography (HPLC). Methods Samples were saponified to exclude the interfering substances, extracted with ether, and then concentrated. Subsequently, the concentrated test solution was injected into RPLC machine to separate and purify, so that the impurities could be removed, meanwhile, the interference brought by vitamin A would be eliminated. Finally, positive phase high performance liquid chromatography was employed for quantitative analysis, using hexane-pentanol (997:3, V:V) as mobile phase at the flow rate of 1.0 ml/min, separated by Waters Nova-Pak Silia column, and determined by ultraviolet detector or diode array detector at the wavelength of 254 nm with external standard method for quantification. Results Cholecalciferol had a good linear relationship in the range of 0.297~9.506 μg/ml with the correlation coefficient of 1. The average recovery and relative standard deviation (RSD) were %~100.58% and 0.44%~0.72%, respectively. Conclusion The method is rapid and accurate, which can be used for the determination of cholecalciferol in health food. KEY WORDS: cholecalciferol; high performance liquid chromatography; health food 基金项目 : (2016JJ6079) Fund: Supported by the Natural Science Foundation of Hunan Province (2016JJ6079) * 通讯作者 :,, panxiaohong56781@163.com *Corresponding author: AN Xiao-Hong, Researcher Assistant, Hunan Institute for Food and Drug Control, No. 60, Bayi Road, Furong District, Changsha , China. panxiaohong56781@163.com

2 5, : D 引言 D 3 (cholecalciferol, ),,,, D 3, 4~6 h, D 3,,, D, D 3, [1] D 3,,,, ; [2-5], D 3 [6,7], D 3, D 3 GB [8],, 2,,,, [9] D 3,,, D 3 [10] D 3, GB D 3, D 3, D 3 2 材料与方法 2.1 仪器与试剂 D 3 DIONEXU3000 ( ); Agilent 1260 ( ); UV-2550 ( SHIMADZU ); SBZS-12D ( ); TB-215D ( Denver ) D 3 : Stanford, CLF , 98.2% 2.2 实验方法色谱条件 : Waters Nova-Pak Silia(3.9 mm 150 mm, 4 μm); : : (997:3, V:V); : 1.0 ml/min; : 190~400 nm; : 254 nm; : 30 ; : 100 μl 标准溶液的配制 D 3 10 mg, 50 ml, 40 ml,, 1 min,,, (1) 5 ml, 50 ml,,,,, 0, (2) 样品前处理 [5], ( D 15 μg, 2.0 g),, 30 ml C 0.2 g 50% 3 ml, 30 min,, 10 ml,, 100 ml,, 3, 1 60 ml, 2 40 ml,,, 100 ml,,,,,,,,,, 5 ml,, 3 ml,,,,, A A, D A 500 μl,, - (90:10, V:V), 254 nm,, D 3 D 3, A D 3 D 3,,, 10 ml, ml D ~3.5 μg,,, B 3 结果与分析 3.1 提取条件的选择 D 3, [10],,,, min, D 3,,, D 3,

3 D 3, D 3 [11],,, D [12,13] 3 D 3 ( 1), D 3 D 3, A, B 1,,,, D 3 D 3, D 3 D 3 A, A D 3,, D 3 GB , D 3 D 3, D 3, D 3 D 3, D 3, D 3 D 3, D 3 GB D 3,,,, A, D 3, D 3 [14] D 3 D 3,, 对照品的选择对照品的校正 D 3,, D 3 D 3, 3 ml, 264 nm, D [8], D 校正因子的测定,, D 3 D 3, D 3, D [10] 3, D 3 D 3 D 3, D, D, D,, D (2)5 ml, 50 ml,,, ; 10 μl,, 3A, D f [10] 1 f 1 = c 1 /A 1 c 1 D 3, mg/ml; A 1 D 3 A. B. Fig. 1 1 (A) (B) Spectrograms of reference substance solution (A) and raw product solution (B)

4 5, : D A. D 3 B. D 3 C. D 3 D. D 3 Fig. 2 2 D 3 D 3 Spectrograms of previtamin D 3 and vitamin D 3 in reference substance solution and solution after purification (2)5 ml, 50 ml, 2,6-1,,, 90 : 1.5 h,,,,, ; 10 μl,, 3B, D3 f [10] 2 f 2 =(c 1 -f 1 A 1 )/A 2 c 1 f 1 D 3, μg/ml; f 1 D 3 ; A 1 D 3 ; A 2 D 方法学考察系统适用性 D 3 (l)5 ml,,, 90 1 h,,, 5 ml,, l cm, 2 8 W 254 nm 365 nm, 45% 5.6 cm, 5 min, D 3 D 3 D 3 D 3 ;, 5,,

5 Fig. 3 3 Chromatograms of reference substance solution (A) and the mixed reference substance solution (B) Table 1 表 1 系统适用性标准偏差表 The standard deviation table of system suitability testing RSD/% D D D D D 3 2.0%; ( D min) D 3 ( D min) D 3 D 3 ( D min) 1.0 [10] 1, 线性结果分析 HPLC 5, 0.297~9.506 μg/ml Y=27.443X , r 2 = 精密度考察, 6, RSD 0.93% 回收率考察 3, %, RSD% 0.72; %, RSD% 0.44; %, RSD% 0.45,, 2 4 Fig. 4 System suitability testing chromatograms

6 5, : D 表 2 回收率结果 Table 2 Results of recovery rates (g) (μg) (μg) (ug) (%) (%) RSD(%) 结论 5 D 3 Fig. 5 Standard curve of cholecalciferol 3.4 样品测定结果分析 D 3, GB D 3,, D 3, GB , D 3 D 3, 6 6 Fig. 6 Chromatogram of samples D 3, D 3 D 3, GB D 3 D 3,,, D 3,,,, D 3 参考文献 [1],,,. UPLC D 3 D 3 AD D 2 D 3 D 2 [J]., 2016, 36(8): Hu DH, Zhang JX, Han H, et al. Rapid determination of vitamin D 3,D 2 in baby vitamin D 3 drops, vitamin D 3 tablets, vitamin AD drops and vitamin D 2 tablets by UPLC [J]. Chin J Pharm Anal, 2016, 36(8): [2],. [M]. :, Zhou JL, Liu QP. Practical vitamin mineral supplements the manual [M]. Beijing: China Light Industry Press, [3]. D D 2 D 3 [J]., 2007, 10(2): Liang XY. High performance liquid chromatography method for determination of vitamin D2 and vitamin D3 in Calcim Magnesium D [J]. J Guangxi Tradit Chin Med Univ, 2007, 10(2): [4]. D 3 [D]. :, Wang BL. Effect of vitamin D on postprandial hyperglycemia inflammatory factors in patients with hypertensive [D]. Taiyuan: Shanxi Medical University, [5],,,. [J].,2017, 23(1): Lin ZM, Zhong JX, Jia HH, et al The effect of Hugu capsule s on calcium

1628 8 relate d hormone s and sex hormones in ovariectomized rats [J]. Chin J Osteoporosis, 2017, 23(1): 87 92. [6],,,. D 3 1~4 D m NA [J]., 2014, 26(7): 1760 1768. Wang BW, Wang D, Ge WH, et al.

7 relate d hormone s and sex hormones in ovariectomized rats [J]. Chin J Osteoporosis, 2017, 23(1): [6],,,. D 3 1~4 D m NA [J]., 2014, 26(7): Wang BW, Wang D, Ge WH, et al. Effects of Vitamin D 3 on serum biochemical parameters, immune function and vitamin D receptor mrna expression level of Qingnonghui geese aged from 1 to 4 weeks [J]. Chine J Anim Nutr, 2014, 26(7): [7],,, - D[J],2008,26 (1):113. Zhao R, Xue Y, Wu GH, et al. Setermination of vitamin D in calcium fortified foods using solid-phase extraction-high performance liquid chromatography [J]. Chin J Chromatogr, 2008, 26(1): 113. [8] GB A D E [S] GB National Food Safety Standard Determination of vitamin A, D, E in foods for infants and young children, milk and milk products [S]. [9] Perales S, Alegria, Barbera R, et al Review: determination of vitamin d in dairy products by high performance liquid chromatography [J]. Food Sci Technol Int, 2005, 11(6): 451. [10]. [M]. :, Chinese Pharmacopoeia Commission. Chinese pharmacopoeia [M]. Beijing: Chinese Medical Science and Technology Press, [11]. D 3 DMSO [D]. :, Jin XL. Thermal transformation of cholecalciferol in DMSO and the autooxidation products of isotachysterol [D]. Lanzhou: Lanzhou University, [12] Heudi O, Trisconi MJ, Blake CJ. et al. Simultaneous quantification of vitamins A, D 3 and E in fortified infant formulae by liquid chromatography-mass spectrometry [J]. J Chromatogr A, 2004, 1022: [13] Donald L, Gilliland, Charles K, et al Simultaneous determination of vitamins D 2 and D 3 by LC-MS/MS in infant formula and adult nutritionals [J]. J AOAC Int, 2012, 95(3): 1 5. [14],,,. 3 [J]., 2015, 33(3): Zhang YH, Qibulehasi, Jin Y, et al Simultaneous determination of vitamins, D 3 and E in infant formula and adult nutritions by online two-dimensional liquid chromatography [J]. Chin J Chromatogr, 2015, 33(3): 作者简介 ( 责任编辑 : 姚菲 ) 刘玉玲, 药师, 主要研究方向为药品食品保健食品及化妆品的检验及研发 lyl @126.com 潘小红, 助理研究员, 主要研究方向为食品药品保健食品及化妆品的检验及研发 panxiaohong56781@163.com

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min 25 4 2013 8 监测技术 5 1 2 1 ( 1. 江苏省疾病预防控制中心, 江苏南京 210009; 2. 南京医科大学第二附属医院, 江苏南京 210029) 5 Phenomenex kinetex C 18 2. 6 μm 4. 6 mm 150 mm 10 mmol /L ph 2. 5 + 60 40 DAD 280 nm 5 20. 0 mg /L ~ 100 mg /L 0.