目的: 建立用于快速测定维生素D 滴剂、维生素AD 滴剂中维生素D 含量的柱切换二维液相色谱方法。方法: 样品用正己烷溶解后直接注入液相色谱系统, 在第一维色谱完成前维生素D3、维生素D3 的初步净化;以9 mL 大体积的收集流路收集含有前维生素D3、维生素D3 的组分, 在第二维色谱完成前维生素D3 与维生素D3 的分离和定量。本方法可以直接分析2 种制剂（维生素D 滴剂和维生素AD 滴剂）, 无需烦琐的样品制备程序。结果: 2 种制剂中维生素D3 在0. 100 8~0. 907 2 μg·mL-1 浓度范围内线性关系良好, r=0. 999 7;重复性试验的RSD 分别为0. 4%、0. 8%;平均回收率（RSD）分别为100. 5%（0. 78%）、100. 7%（1. 6%）;检测下限为1. 6 ng·mL-1, 定量下限均为5. 3 ng·mL-1;7 批维生素D 滴剂样品的维生素D3 含量范围在102. 6%~111. 8%, 11批维生素AD 滴剂样品的维生素D3 含量范围在97. 4%~104. 7%。结论: 本实验建立的方法灵敏、准确, 操作简单, 对于油性和脂溶性维生素D 制剂中维生素D3 的含量测定具有很好的应用前景。
Objective: To develop a rapid method for direct determination of vitamin D in vitamin D drops and vitamin A D drops using two-dimensional column switch liquid chromatography. Methods: The sample was dissolved in n-hexane and injected directly into the liquid chromatography system. Preliminary purification of pre-vitamin D3 and vitamin D3 were achieved in the first-dimensional liquid chromatography. The eluent containing pre-vitamin D3 and vitamin D3 from the first dimensional column was collected by a 9 mL collection pipe and then switched into the second dimensional column to complete quantification of pre-vitamin D3 and vitamin D3. Two kinds of liquid vitamin D supplements（vitamin D drops and vitamin A and D drops） were directly analyzed using this system without any complicated preparation procedure. Results: The linear range of pre-vitamin D3 and vitamin D3 in vitamin D drops and vitamin A and D drops was 0. 100 8-0. 907 2 μg·mL-1 with the correlation coefficient of 0. 999 7. The RSDs of repeatability were 0. 4% and 0. 8%. The average recoveries were 100. 5%（RSD=0. 78%）and 100. 7%（RSD=1. 6%）. The LOD was 1. 6 ng·mL-1 and LOQ was 5. 3 ng·mL-1. The contents of vitamin D3 in 7 batches of vitamin D drops ranged from 102. 6% to 111. 8%. The contents of vitamin D3 in 11 batches of vitamin A D drops ranged from 97. 4% to 104. 7%. Conclusion: The method is sensitive, accurate and easy to operate. It has a good application prospect for the determination of vitamin D3 in oily and fat soluble vitamin D preparations.
[1] WEAVER CM, ALEXANDER DD, BOUSHEY CJ, et al. Calcium plus vitamin D supplementation and risk of fractures: an updated meta-analysis from the National Osteoporosis Foundation[J]. Osteoporos Int, 2016, 27(1): 367
[2] FISCHER V, HAFFNERLUNTZER M, AMLING M, et al. Calcium and vitamin D in bone fracture healing and post-traumatic bone turnover[J]. Eur Cell Mater, 2018, 35: 365
[3] MIRHOSSEINI N, VATANPARAST H, MAZIDI M, et al. Vitamin D supplementation, glycemic control, and insulin resistance in prediabetics: A meta-analysis[J]. J Endocr Soc, 2018, 2(7): 687
[4] BOLCAS P, BRANDT E, ZHANG Z, e t a l. Vitamin D supplementation attenuates asthma development following trafficrelated particulate matter exposure[J]. J Aller Clin Immunol, 2018, 141(2): AB175
[5] STOKIĆ E, KUPUSINAC A, TOMICNAGLIC D, et al. Vitamin D and dysfunctional adipose tissue in obesity[J]. Angiology, 2015, 66(7): 613
[6] PILZ S, VERHEYEN N, GRÜBLER MR, et al. Vitamin D and cardiovascular disease prevention[J]. Nat Rev Cardiol, 2016, 13(7): 404
[7] 中华人民共和国药典2015年版. 四部总则: 维生素D测定[S]. 2015
ChP 2015. Vol IV General provisions 0722, Detemination of Vitamin DhP 2015. Vol IV General provisions 0722, Detemination of Vitamin D[S]. 2015
[8] 黄芳, 吴惠勤, 杭义萍. 高效液相色谱-串联质谱法测定食品中维生素D2 和维生素D3[J]. 理化检验(化学分册), 2011, 47(5): 577
HUANG F, WU HQ, HANG YP. HPLC-MS/MS detexmination of vitam in D2 and vitam in D3 in food[J]. Phys Test Chem Anal (PartB: Chem Anah), 2011, 47(5): 577
[10] ADAMEC J, JANNASCH A, HUANG J, et al. Development and optimization of an LC-MS/MS-based method for simultaneous quantification of vitamin D2, vitamin D3, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3[J]. J Separation Sci, 2015, 34(1): 11
[11] KO JH, KWAK BM, AHN JH, et al. Development of vitamin D determination in infant formula by column-switching HPLC with UV Detector[J]. Kor J Food Sci Animal Res, 2012, 32(5): 571
[12] TAGUCHI K, FUKUSAKI E, BAMBA T. Simultaneous analysis for water-and fat-soluble vitamins by a novel single chromatography technique unifying supercritical fluid chromatography and liquid chromatography[J]. J Chromatogr A, 2014(1362): 270-277.
[13] FIRAS J, SARA L, SOFIA E, et al. A rapid method for the separation of vitamin D and its metabolites by ultra-high performance supercritical fluid chromatography-mass spectrometry[J]. J Chromatogr A, 2016, 1440: 191
[14] ANDRI B, LEBRUN P, DISPAS A, et al. Optimization and validation of a fast supercritical fluid chromatography method for the quantitative determination of vitamin D3 and its related impurities[J]. J Chromatogr A, 2017, 1491: 171
[15] 胡代花, 张嘉昕, 韩豪, 等. UPLC 法快速测定婴儿维生素D3 滴剂、维生素D3 片、维生素AD 滴剂及维生素D2 片中维生素D3 和维生素D2[J]. 药物分析杂志, 2016, 36(8): 1409
HU DH, ZHANG JX, HAN H, et al. Rapid determination of vitamin D3, D2 in baby vitamin D3 drops, vitamin D3 tablets, vitamin AD drops and vitamin D2 tablets by UPLC[J]. Chin J Pharm Anal, 2016, 36(8): 1409
[16] HAMADA NK, GUO YL, JI F, et al. Determination of vitamin D in oily drops using a column-switching system with an on-line clean-up by supercritical fluid chromatography[J]. Talanta, 2018, 190: 9
[17] 萧伟斌, 蹇阳, 李桦. 二维液相色谱在药物和毒物分析中的应用进展[J]. 分析化学, 2014, 42(12): 1851
XIAO WB, JIAN Y, LI H. Progress in the application of twodimensional liquid chromatography in the analysis of drugs and toxicants[J]. Chin J Anal Chem, 2014, 42(12): 1851
[18] COOPER AD, STUBBINGS GW, KELLY M, et al. Improved method for the on-line metal chelate affinity chromatography-highperformance liquid chromatographic determination of tetracycline antibiotics in animal products[J]. J Chromatography A, 1998, 812(1-2): 321
[19] ZHONG Q, QIU X, LIN C, et al. An automatic versatile system integrating solid-phase extraction with ultra-high performance liquid chromatography-tandem mass spectrometry using a dualdilution strategy for direct analysis of auxins in plant extracts[J]. J Chromatogr A, 2014, 1359: 131
[20] TETZNER NF, MANIERO MG, RODRIGUES-SILVA C, et al. On-line solid phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry as a powerful technique for the determination of sulfonamide residues in soils[J]. J Chromatogr A, 2016, 1452: 89
[21] ZHONG Q, SHEN L, LIU J, et al. Automatic on-line solid-phase extraction with ultra-high performance liquid chromatography and tandem mass spectrometry for the determination of ten antipsychotics in human plasma[J]. J Sep Sci, 2016, 39(11): 2129
[22] GB 5009. 82-2016 食品中维生素A、D、E 的测定[S]. 2016
GB 5009 82-2016 Determination of Vitamins A, D, E in Food[S]. 2016
