8 5 Vol. 8 No. 5 2017 5 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 (, 410001) 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 3 0.297~9.506 μg/ml, 1, 100.55%~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 410001, 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 100.55%~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 :,, E-mail: panxiaohong56781@163.com *Corresponding author: AN Xiao-Hong, Researcher Assistant, Hunan Institute for Food and Drug Control, No. 60, Bayi Road, Furong District, Changsha 410001, China. E-mail: panxiaohong56781@163.com
5, : D 3 1623 1 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 5413.9 [8],, 2,,,, [9] D 3,,, D 3 [10] D 3, GB 5413.9 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-907506, 98.2% 2.2 实验方法 2.2.1 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 2.2.2 标准溶液的配制 D 3 10 mg, 50 ml, 40 ml,, 1 min,,, (1) 5 ml, 50 ml,,,,, 0, (2) 2.2.3 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 1.25~3.5 μg,,, B 3 结果与分析 3.1 提取条件的选择 D 3, [10],,,, 130 90 min, D 3,,, D 3,
1624 8 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 5413.9, D 3 D 3, D 3, D 3 D 3, D 3, D 3 D 3, D 3 GB 5413.9 D 3,,,, A, D 3, D 3 [14] D 3 D 3,, 2 3.2 对照品的选择 3.2.1 对照品的校正 D 3,, D 3 D 3, 3 ml, 264 nm, D 3 462 [8], D 3 3.2.2 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)
5, : D 3 1625 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 3 3.3 方法学考察 3.3.1 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,,
1626 8 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 1 2 3 4 5 RSD/% D 3 339.10284 348.4043 343.36053 342.88489 342.06537 0.98 D 3 119.55872 116.30859 118.01784 118.80979 118.24834 1.02 D 3 2151.28687 2127.56274 2130.28491 2129.75537 2095.68237 0.94 D 3 55.77407 55.06005 55.41803 55.65182 55.49505 0.49 D 3 2.0%; ( D 3 0.5 min) D 3 ( D 3 0. 6 min) D 3 D 3 ( D 3 1.1 min) 1.0 [10] 1, 4 3.3.2 线性结果分析 HPLC 5, 0.297~9.506 μg/ml Y=27.443X-0.1267, r 2 =1 3.3.3 精密度考察, 6, RSD 0.93% 3.3.4 回收率考察 3, 100.58%, RSD% 0.72; 100.57%, RSD% 0.44; 100.55%, RSD% 0.45,, 2 4 Fig. 4 System suitability testing chromatograms
5, : D 3 1627 表 2 回收率结果 Table 2 Results of recovery rates (g) (μg) (μg) (ug) (%) (%) RSD(%) 1.0001 17.0017 14.2586 31.4342 101.22 1.0029 17.0493 14.2586 31.4106 100.72 1.0048 17.0816 14.2586 31.3131 99.81 100.58 0.72 1.004 17.068 142.5864 159.9681 100.22 0.9914 16.8538 142.5864 160.9516 101.06 1.0034 17.0578 142.5864 160.2431 100.42 100.57 0.44 1.0033 17.0561 475.288 494.0076 100.35 1.0024 17.0408 475.288 497.4144 101.07 1.0068 17.1156 475.288 493.5443 100.24 100.55 0.45 4 结论 5 D 3 Fig. 5 Standard curve of cholecalciferol 3.4 样品测定结果分析 D 3, GB 5413. 9 D 3,, D 3, GB 5413.9, D 3 D 3, 6 6 Fig. 6 Chromatogram of samples D 3, D 3 D 3, GB 5413.9 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): 1409 1414. 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): 1409 1414. [2],. [M]. :, 2003. Zhou JL, Liu QP. Practical vitamin mineral supplements the manual [M]. Beijing: China Light Industry Press, 2003. [3]. D D 2 D 3 [J]., 2007, 10(2): 58 61. 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): 58 61. [4]. D 3 [D]. :, 2016. Wang BL. Effect of vitamin D on postprandial hyperglycemia inflammatory factors in patients with hypertensive [D]. Taiyuan: Shanxi Medical University, 2016. [5],,,. [J].,2017, 23(1): 87 92. Lin ZM, Zhong JX, Jia HH, et al The effect of Hugu capsule s on calcium
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