CN111307978B - 一种脂溶性维生素有关物质检测方法 - Google Patents
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Abstract
维生素是人和动物为维持正常的生理功能而必须从食物中获得的一类微量有机物质，在人体生长、代谢、发育过程中发挥着重要的作用。脂溶性维生素(fat‑solublevitamins)是不溶于水而溶于脂肪及非极性有机溶剂(如苯、乙醚及氯仿等)的一类维生素，包括维生素A、维生素D、维生素E、维生素K等。维生素在体内的含量很少，但不可或缺，对维生素D3进行有效的质量控制存在一定的难度且显得格外重要。其质量测定方法是用高效液相色谱一测多评的方法测定，10ug规格制剂中维生素D3含量不得低于90％，单个杂质含量不得大于0.5％。本发明的优点是方法简单，可以一次性测定制剂中维生素的含量及有关物质，同时也是首个在维生素类制剂中提出杂质控制的制剂品种。
Description
技术领域
本发明涉及医药技术领域，涉及一种或多种维生素制剂的质量检测方法，具体是一种补钙剂碳酸钙D3片的质量检测方法。
技术背景
维生素是人和动物为维持正常的生理功能而必须从食物中获得的一类微量有机物质，维生素在体内的含量很少，但不可或缺，在人体生长、代谢、发育过程中发挥着重要的作用。其中脂溶性维生素(fat-solublevitamins)是不溶于水而溶于脂肪及非极性有机溶剂（如苯、乙醚及氯仿等）的一类维生素，包括维生素A、维生素D、维生素E、维生素K等。
中国居民膳食指南（ 2016），美国卫生公共服务部、美国农业部，2015—2020美国居民膳食指南，第8版，推荐摄取含有维生素D食物，并适当补充维生素D，推荐维生素D摄入量每天不低于600UI；中华预防医学会推荐婴儿从出生开始，应当在医生指导下每天补充维生素D 400-800国际单位，中国营养学会推荐50岁以上人群每天摄入维生素D 10μg（但不要超过20μg）。
维生素D3是维生素D中生物代谢率最高的一种活性形式。每天需要补充约10ug维生素D3，维生素D3具有5个手性中心和一个顺反共轭结构，理论上具有63个异构体，实际维生素D3结构具有不稳定性，对光、热、紫外线都不稳定，所以控制维生素D3质量，显得格外重要。在成品制剂中每制剂单位含有维生素D3 10ug，又增加了质控难度。
国内生产维生素D3相关制剂企业众多，但国内外都没有制剂的杂质控制技术参考，无法保证临床应用效果，且会增加杂质带来的副作用。
英国药典2018版和中国药典2015版中收入了维生素D3原料的有关物质检测方法，采用正相色谱系统，硅胶色谱柱，以正己烷：正戊醇=997：3为流动相，进行洗脱。该方法可有效分离维生素D3，但维生素D3萃取繁琐，正己烷挥发快，对操作人员造成伤害，在色谱分析时，对高效液相色谱仪脱气系统、比例阀、密封垫加快老化，增加设备维护成本，不利于推广。文献“高效液相色谱法测定保健食品中维生素 D3的含量，食品安全质量检测学报，2017年5月，第8卷第5期”同样采用正相色谱系统检测含量。文献“RP—HPLC 法测定维 生素 D3制剂 中的有关物质，中国药科大学学报，2012，43（1）：55-59”采用正己烷萃取碳酸钙D3片中维生素D3，氮气吹干，甲醇复溶制备供试品溶液，采用美国Sepax公司C18色谱柱，流动相乙腈，柱温20℃，检测波长265nm，流速1.0ml/min，进样体积20uL对供试品进行洗脱。供试品溶液萃取、浓缩操作繁琐，以纯乙腈为流动相，等度洗脱，方法可行性有待考察。
发明内容
为解决上述技术问题，本发明的目的在于通过维生素D3有关物质进行定性定量分析，提供一种脂溶性维生素质控方法，所述脂溶性维生素是指维生素D、维生素A、生育酚及添加其他原料、辅料组成的维生素复方制剂，其中脂溶性维生素含量为5ppm~1000ppm；包括以下步骤：
（1） 色谱条件：用反相色谱条件检测，以十八烷基硅烷键合硅胶柱对复方制剂中维生素进行杂质分析，单个杂质不得大于1%；
（2）洗脱方法：以乙腈和水作为流动相，采用梯度洗脱方法，流速0.9~1.5mL/min，乙腈在50分钟内由40%增加到100%。
优选地，所述梯度洗脱方法以水与乙腈的体积比按时间段均匀变化，具体为：0-15min，水由50%均匀变化至10%，乙腈由50%均匀变化至90%；15-40min，水由10%均匀变化至0%，乙腈由90%均匀变化至100%；40-55min，水保持0%，乙腈保持100%；55-57min，水保持50%，乙腈保持50%。具体如下表：
| 时间 | A | B |
| 0 | 50 | 50 |
| 15 | 10 | 90 |
| 40 | 0 | 100 |
| 55 | 0 | 100 |
| 55.01 | 50 | 50 |
| 57 | 50 | 50 |
优选地，所述的维生素复方制剂是指碳酸钙和维生素D3粉或维生素D3加适量辅料制成的碳酸钙D3片、颗粒、咀嚼片、泡腾片等固体制剂，具体包括碳酸钙D3片、碳酸钙D3咀嚼片、碳酸钙D3颗粒、碳酸钙D3多维片。
优选地，所述十八烷基硅烷键合硅胶柱为Ultimate Polar-RP，4.6*250mm，5um色谱柱。
本维生素质控方法是一种简单供试品处理方法，加上常规反相色谱系统，对维生素D3复方制剂进行有关物质测定，除前维生素D3（CAS：1173-13-3）外，其他单个杂质不得大于1%，总杂不得大于2%。其中已知杂质包括反式维生素D3（5,6-trans-Vitamin D3，CAS号：22350-41-0），原维生素D3（(3β)-7-Dehydro Cholesterol，CAS号：434-16-2），光甾醇（Lumisterol 3，CAS号：5226-01-7），异速甾醇（Isotachysterol3，CAS号：22350-43-2），速甾醇（Tachysterol3，CAS号：17592-07-3）。
色谱条件：以十八烷基硅烷键合硅胶柱（推荐Ultimate® Polar-RP，4.6*250mm，5um），A相为水，B相为乙腈；流速为0.9-1.5mL/min；色谱柱温为35℃；检测波长为265nm。
对照品溶液，分别精密称定维生素D3，反式维生素D3，原维生素D3，光甾醇，异速甾醇，速甾醇对照品，加含0.1%丁羟甲苯的甲醇溶解，制成每1ml分别含上述对照品1ug的溶液，作为对照品溶液。
系统适应性溶液，取维生素D3对照品适量，加甲醇稀释制成每 1mL 中约含10μg的溶液，量取适量溶液，密塞，90℃水浴加热45min，冷却。取适量溶液置1cm石英吸收池中，在2支8W主波长分别为254nm和265nm的紫外灯下，将石英吸收池斜放45°，并距灯管（5~6）cm，照射20分钟，作为系统适用性溶液。
供试品溶液，置50mL棕色容量瓶内，精密加95%甲醇溶液20ml溶解，于 37℃±5℃，频率180±10水浴振摇30-45分钟，取出，迅速冷却，37℃±5℃超声 10-30 分钟，超声完毕，迅速冷却至室温。取上清液作为供试品溶液，加95%甲醇溶液稀释100倍，作为对照溶液。
按相对保留时间计算，反式维生素D3 RT0.88~0.91，前维生素D3 RT0.92~0.93，异速甾醇RT0.93~0.95，速甾醇RT 0.97~0.99，维生素D3 RT1.0，光甾醇RT1.01~1.06，原维生素D3 RT1.26~1.34，前维生素D3峰与相邻峰分离度不得小于1.0，维生素D3与速甾醇分离度不得小于1.0，其他各相邻杂质峰的分离度不得小于1.5，理论塔板数按维生素D3峰计不得低于5000。
采用前述方法所测定的供试品溶液，其维生素含量在5ug~30ug/mL。
优选地，所述供试品溶液使用90%~100%的甲醇溶液制备。
优选地，所述供试品溶液使用含丁羟甲苯0.1~2%的90%~100%的甲醇溶液制备。
优选地，所述供试品溶液使用90%~100%的乙醇溶液制备。
优选地，所述供试品溶液使用含丁羟甲苯0.1~2%的90%~100%的乙醇溶液制备。
本发明优点是方法简单，采用反相色谱条件，整个实验操作不使用正己烷等正相系统溶剂，可以完成对维生素D3复方制剂进行有关物质分析，使维生素D3制剂质量可控。
本发明还可以用于生育酚检测，取生育酚对照品，加乙醇稀释成每 1mL 中约含0.1mg 的溶液，作为对照品溶液。
附图说明
图1为系统适应性图谱。
图2为实施例一的对照图谱。
图3为实施例一的供试品图谱。
图4为实施例三的对照图谱。
图5为实施例三的供试品图谱。
图6为生育酚图谱。
具体实施方式
实施例1：碳酸钙D3片有关物质检测
样品：碳酸钙D3片，处方：碳酸钙1500mg，维生素D3 10ug，加适量辅料制成片剂。
仪器与试剂
仪器 Thermo U3000高效液相色谱仪，试药 碳酸钙D3片由申请人提供，乙腈、甲醇为色谱出，水为超纯水，维生素D3对照品有中国食品药品检定研究院提供。
色谱条件：以十八烷基硅烷键合硅胶柱（型号Ultimate® Polar-RP，4.6*250mm，5um），A相为水，B相为乙腈；流速为每分钟1.2mL；色谱柱温为35℃；检测波长为265nm。照下表梯度洗脱：
系统适应性取对照品适量，加甲醇稀释制成每 1mL 中约含10μg 的溶液，量取适量溶液，密塞，90℃水浴加热45min，冷却。取适量溶液置1cm石英吸收池中，在2支8W主波长分别为254nm和265nm的紫外灯下，将石英吸收池斜放45°，并距灯管5~6cm，照射20分钟，作为系统适用性溶液。精密量取系统适用性溶液100μL，注入液相色谱仪，进样5次，记录色谱图，如图1所示，以相对保留时间计算。
连续5针维生素D3峰面积RSD不得大于2.0%。前维生素D3峰与相邻峰分离度不得小于1.0，维生素D3与速甾醇分离度不得小于1.0，其他各相邻杂质峰的分离度不得小于1.5，理论塔板数按维生素D3峰计不得低于5000。
供试品制备：全程避光操作。取本品35g（约相当于200ug维生素D3），置50mL棕色容量瓶内，精密加含0.1%丁羟甲苯的95%甲醇溶液20ml溶解，于 40℃，频率150水浴振摇30分钟，取出，迅速冷却，37℃超声 10 分钟，超声完毕，迅速冷却至室温。取上清液作为供试品溶液，用100uL微量进样器量取供试品溶液100uL，置10mL棕色量瓶中，加95%甲醇溶液定容，作为对照溶液。
测定法：精密量取供试品溶液和对照品溶液各100uL，注入高效液相色谱仪，记录色谱图，如图2所示。
以自身对照计算自制碳酸钙D3片，在0天时，反式维生素D3含量0.09%，40℃、RH75%条件下加速试验6个月，反式维生素D3含量0.17%，光甾醇0.25%，其他杂质未检出。
实施例2：碳酸钙D3颗粒有关物质测定
样品：安徽东盛友邦实验室自制，批号20191022。
供试品溶液制备，全程避光操作。取碳酸钙D3颗粒，置50mL棕色容量瓶内，精密加95%的乙醇溶液20ml溶解，于 45℃，频率180水浴振摇30分钟，取出，迅速冷却，37℃超声 10分钟，超声完毕，迅速冷却至室温。取上清液作为供试品溶液，取供试品溶液用95%的乙醇稀释100倍，作为对照溶液。
采用实施例1的色谱法检测，测得碳酸钙D3颗粒中有关物质含量，光甾醇0.14%，最大未知单杂0.86%，总杂1.24%。
实施例3：市售品碳酸钙D3片(ll)有关物质检测
样品：碳酸钙D3片(ll)，规格：钙500mg、维生素D3 200国际单位，批号：20190116.
供试品制备，取碳酸钙D3片(ll) 20片，研碎，置50mL棕色容量瓶内，精密加95%的甲醇溶液20ml溶解，于 45℃，频率180水浴振摇30分钟，取出，迅速冷却，37℃超声 10 分钟，超声完毕，迅速冷却至室温。取上清液作为供试品溶液，取供试品溶液用95%的乙醇稀释100倍，作为对照溶液。
采用实施例1的色谱法检测，测得碳酸钙D3片(ll)中速甾醇含量2.78%，光甾醇0.56%，最大未知单杂0.64%，总杂5.02%。
Claims (8)
1.一种脂溶性维生素有关物质检测方法，所述脂溶性维生素是指维生素D、维生素A、生育酚及添加其他原料、辅料组成的维生素复方制剂，其中脂溶性维生素含量为5ppm~1000ppm；
其特征在于：
用反相色谱条件检测，以十八烷基硅烷键合硅胶柱对复方制剂中维生素进行杂质分析，单个杂质不得大于1%，所述杂质包括反式维生素D3，CAS号：22350-41-0；原维生素D3，CAS号：434-16-2；光甾醇，CAS号：5226-01-7；异速甾醇，CAS号：22350-43-2；速甾醇，CAS号：17592-07-3；
以乙腈和水作为流动相，采用梯度洗脱方法，流速0.9~1.5mL/min，色谱柱温为35℃；检测波长为265nm，梯度洗脱方法具体为：0-15min，水由50%均匀变化至10%，乙腈由50%均匀变化至90%；15-40min，水由10%均匀变化至0%，乙腈由90%均匀变化至100%；40-55min，水保持0%，乙腈保持100%；55-57min，水保持50%，乙腈保持50%。
2.根据权利要求1所述的一种脂溶性维生素有关物质检测方法，其特征在于：所述的维生素复方制剂为碳酸钙D3片、碳酸钙D3咀嚼片、碳酸钙D3颗粒、碳酸钙D3多维片。
3.根据权利要求1所述的一种脂溶性维生素有关物质检测方法，其特征在于：所述十八烷基硅烷键合硅胶柱为Ultimate Polar-RP，4.6*250mm，5um色谱柱。
4.根据权利要求1所述的一种脂溶性维生素有关物质检测方法，其特征在于：供试品溶液的维生素含量为5ug~30ug/mL。
5.根据权利要求4所述的一种脂溶性维生素有关物质检测方法，其特征在于：所述供试品溶液使用90%~100%的甲醇溶液制备。
6.根据权利要求4所述的一种脂溶性维生素有关物质检测方法，其特征在于：所述供试品溶液使用含丁羟甲苯0.1~2%的90%~100%的甲醇溶液制备。
7.根据权利要求4所述的一种脂溶性维生素有关物质检测方法，其特征在于：所述供试品溶液使用90%~100%的乙醇溶液制备。
8.根据权利要求4所述的一种脂溶性维生素有关物质检测方法，其特征在于：所述供试品溶液使用含丁羟甲苯0.1~2%的90%~100%的乙醇溶液制备。
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Denomination of invention: A method for detecting lipid soluble vitamin related substances Granted publication date: 20220823 Pledgee: Huainan Branch of Postal Savings Bank of China Pledgor: ANHUI DONGSHENG YOUBANG PHARMACEUTICAL CO.,LTD. Registration number: Y2024980001565 |