CN115372511A - 一种脂溶性维生素的检测试剂盒及检测方法 - Google Patents
一种脂溶性维生素的检测试剂盒及检测方法 Download PDFInfo
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Abstract
本发明提供的一种脂溶性维生素的检测试剂盒，包括：标准品、内标溶液、液相色谱洗脱液、蛋白沉淀剂、复溶剂和耗材；标准品用于配置标准浓度的脂溶性维生素溶液，脂溶性维生素为维生素A、25‑羟基维生素D2、25‑羟基维生素D3、维生素E、维生素K1和维生素K2；标准品的外表面标记有用于配置的浓度和需加入复溶剂的体积，标准品内含有与标示浓度匹配的定量的脂溶性维生素的冻干粉，加入标示体积的复溶剂后即可配置为标示浓度的脂溶性维生素溶液；标准品至少有六个，标示浓度从低到高；复溶剂为不含待测脂溶性维生素的空白人血清基质。本发明提供的一种脂溶性维生素的检测试剂盒，能够降低检测误差，实现对维生素A、D2、D3、E、K1、K2的同时检测。
Description
技术领域
本发明属于维生素检测领域，特别涉及一种脂溶性维生素的检测试剂盒及检测方法。
背景技术
维生素(vitamin)是人和动物为维持正常的生理功能而必需从食物中获得的一类有机物质，在人体生长和代谢过程中发挥着极其重要的作用。机体缺乏维生素将引起代谢障碍，并发生各种疾病，这类疾病统称为维生素缺乏症。目前，根据溶解性可将维生素分为脂溶性维生素和水溶性维生素两大类，水溶性维生素包括B族维生素和维生素C等；脂溶性维生素是一种含有环结构和长脂肪族烃链的一组维生素，包括维生素A、D、E和K等。
维生素A具有调节表皮及角质层新陈代谢的功效，可以抗衰老，去皱纹，能减少皮脂溢出而使皮肤有弹性，同时淡化斑点，柔润肌肤；有助于保护表皮、黏膜不受细菌侵害，预防皮肤癌，预防夜盲症、视力衰退，治疗眼疾，还可以促进骨骼生长，帮助牙齿生长、再生，有效预防肥胖，保持女性苗条的身材，可有助于防治脱发。
25-羟基维生素D2、D3为固醇类衍生物，具抗佝偻病作用，又称抗佝偻病维生素。维生素D可维持血清钙磷浓度的稳定，血钙浓度低时，诱导甲状旁腺素分泌，将其释放至肾及骨细胞。维生素D促进怀孕及哺乳期输送钙到子体1位羧基化酶除受血清中钙磷浓度及膳食中钙磷供给量的影响外，还受激素的影响，停经后的妇女VD3浓度减低，易有骨质软化等症状。VD3对小肠作用为诱导合成cabp.1，与小肠细胞的受体形成复合体进入细胞核染色体上，促使cabp的信使mRNA的合成，此mRNA在胞浆内转录为cabp。骨的矿物化作用的机理尚未阐明，在现阶段中只了解到维生素D促进钙磷的吸收，又可将钙磷从骨中动员出来，使血浆钙、磷达到正常值，促使骨的矿物化，并不断更新。
维生素E能促进生殖。它能促进性激素分泌，使男子精子活力和数量增加；使女子雌性激素浓度增高，提高生育能力，预防流产。维生素E缺乏时会出现睾丸萎缩和上皮细胞变性，孕育异常，在临床上常用维生素E治疗先兆流产和习惯性流产。
维生素K1是叶绿基甲萘醌，主要用于因缺乏维生素K出现的出血症状。维生素K1的主要作用为促进血凝，促进肝脏中凝血酶原的合成，增加胃肠道的蠕动及分泌功能，还具有缓解支气管痉挛，止痛的作用。维生素K1主要用于凝血酶过低症、维生素K1缺乏症、新生儿自然出血症、防止慢性腹泻导致的出血、水杨酸钠导致的低凝血酶原血症。
目前，脂溶性维生素检测常用的方法为液相色谱法，但是液相色谱法由于检出限有限，主要用于维生素含量较高的样品测定，一般只能进行单组份或含量较高的几个组分的同时测定。目前的液相色谱-质谱法只能对一种或两种维生素做定性定量分析，由于血浆/血清成分复杂，且维生素含量较低，在进行定量分析时，杂质严重影响检测结果的准确性，因此，为了降低检测时的基质效应，使用者都是对不同的维生素逐个检测分析，存在检测成本高、操作复杂、耗费时间长和检测需要的血量多等缺陷。因此急需一种简单快捷、灵敏度高、准确性好的方法实现对维生素A、D、E、K1的同时检测。
发明内容
针对现有技术中的缺陷，本发明提供一种一种脂溶性维生素的检测试剂盒及检测方法，能够降低检测误差，实现对维生素A、D2、D3、E、K1、K2的同时检测。
本发明提供的一种脂溶性维生素的检测试剂盒，包括：标准品、内标溶液、液相色谱洗脱液、蛋白沉淀剂、复溶剂和耗材；
所述标准品用于配置标准浓度的脂溶性维生素溶液，所述脂溶性维生素为维生素A、25-羟基维生素D2、25-羟基维生素D3、维生素E、维生素K1和维生素K2；
所述标准品的外表面标记有用于配置的浓度和需加入复溶剂的体积，所述标准品内含有与标示浓度匹配的定量的脂溶性维生素的冻干粉，加入标示体积的复溶剂后即可配置为标示浓度的脂溶性维生素溶液；所述标准品至少有六个，标示浓度从低到高；
所述复溶剂为不含待测脂溶性维生素的空白人血清基质。
本发明提供的一种脂溶性维生素的检测试剂盒的工作原理如下：本发明中，采用不含待测脂溶性维生素的空白人血清基质为复溶剂，从而尽量使标准组和检测组中除待测脂溶性维生素外的组分一致，降低检测误差，实现对维生素A、D2、D3、E、K1、K2的同时检测。
根据本发明提供的一实施方式，所述液相色谱洗脱液包括流动相A和流动相B，流动相A为标准浓度的甲酸水溶液，流动相B为标准浓度的甲酸甲醇溶液。
根据本发明提供的一实施方式，所述内标溶液由含有VA1-d6、VD3-d6、VD2-d6、VK1-d4和VE-d6的同位素内标溶液组成。
根据本发明提供的一实施方式，所述蛋白沉淀剂为乙腈和异丙醇的混合液，所述乙腈和异丙醇的体积比为1:1。
另一方面，本发明还提供了一种脂溶性维生素的检测方法，采用本发明提供的检测试剂盒进行检测，
分别向每个标准品中加入标示体积的复溶剂，从而配置成标示浓度的脂溶性维生素溶液；
分别向每个标准品内加入等量的内标溶液和蛋白沉淀剂；
随机抽取至少3个标准品使用液质联用仪检测，并拟合每一种脂溶性维生素所对应的标准曲线方程；
使用液质联用仪以及得到的标准曲线方程检测剩余标准品中脂溶性维生素的浓度，判断检测结果是否在预设的范围内，
如果是，对待测样品中的脂溶性维生素进行检测。
根据本发明提供的一实施方式，标准品和待测样品中加入的内标溶液的量相同。
附图说明
图1为一实施例提供的检测脂溶性维生素的步骤。
具体实施方式
下面将结合附图对本发明技术方案的实施例进行详细的描述。以下实施例仅用于更加清楚地说明本发明的技术方案，因此只作为示例，而不能以此来限制本发明的保护范围。
需要注意的是，除非另有说明，本申请使用的技术术语或者科学术语应当为本发明所属领域技术人员所理解的通常意义。
本实施例提供的一种脂溶性维生素的检测试剂盒，包括：标准品、内标溶液、液相色谱洗脱液、蛋白沉淀剂、复溶剂和耗材；
标准品用于配置标准浓度的脂溶性维生素溶液，脂溶性维生素为维生素A、25-羟基维生素D2、25-羟基维生素D3、维生素E、维生素K1和维生素K2；
标准品的外表面标记有用于配置的浓度和需加入复溶剂的体积，标准品内含有与标示浓度匹配的定量的脂溶性维生素的冻干粉，加入标示体积的复溶剂后即可配置为标示浓度的脂溶性维生素溶液；标准品至少有六个，标示浓度从低到高；
复溶剂为不含待测脂溶性维生素的空白人血清基质。
本实施例中，采用不含待测脂溶性维生素的空白人血清基质为复溶剂，从而尽量使标准组和检测组中除待测脂溶性维生素外的组分一致，降低检测误差，实现对维生素A、D2、D3、E、K1、K2的同时检测。
具体地，液相色谱洗脱液包括流动相A和流动相B，流动相A为标准浓度的甲酸水溶液，流动相B为标准浓度的甲酸甲醇溶液。流动相A用于配置0.1％的甲酸水溶液，流动相B用于配置0.1％的甲酸甲醇溶液。
更具体地，内标溶液由含有VA1-d6、VD3-d6、VD2-d6、VK1-d4和VE-d6的同位素内标溶液组成。VA1-d6为维生素A的内标，VD3-d6为维生素D3的内标，VD2-d6为维生素D2的内标，VK1-d4为维生素K1和维生素K2的内标，VE-d6为维生素E的内标。
更具体地，蛋白沉淀剂为乙腈和异丙醇的混合液，乙腈和异丙醇的体积比为1:1。人血清中的脂溶性维生素，维生素A和E的含量较高，维生素D2/D3/K1/K2含量较少。维生素A、维生素E的含量是维生素D2/D3/K1/K2的近十倍。
通过实验测量峰面积，当乙腈和异丙醇的体积比为1:1时，能够极大的提高维生素D2/D3/K1/K2的峰面积，且对维生素A和E的影响较小，从而获得最优峰面积。
参照图1，采用上述检测试剂盒进行检测的方法如下：
1、配置标准液：分别向每个标准品中加入标示体积的复溶剂，从而配置成标示浓度的脂溶性维生素溶液；
2、提取维生素：分别向每个标准品内加入等量的内标溶液和蛋白沉淀剂；
3、绘制标准曲线方程：随机抽取至少3个标准品使用液质联用仪检测，并拟合每一种脂溶性维生素所对应的标准曲线方程；
4、判断方程准确性：使用液质联用仪以及得到的标准曲线方程检测剩余标准品中脂溶性维生素的浓度，判断检测结果是否在预设的范围内，如果是，说明可进行样品检测；
5、样品检测：对待测样品中的脂溶性维生素进行检测。其中，标准品和待测样品中加入的内标溶液的量相同。
本发明的说明书中，说明了大量具体细节。然而，能够理解，本发明的实施例可以在没有这些具体细节的情况下实践。在一些实例中，并未详细示出公知的方法、结构和技术，以便不模糊对本说明书的理解。
在本说明书的描述中，具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围，其均应涵盖在本发明的权利要求和说明书的范围当中。
Claims (6)
1.一种脂溶性维生素的检测试剂盒，其特征在于，包括：标准品、内标溶液、液相色谱洗脱液、蛋白沉淀剂、复溶剂和耗材；
所述标准品用于配置标准浓度的脂溶性维生素溶液，所述脂溶性维生素为维生素A、25-羟基维生素D2、25-羟基维生素D3、维生素E、维生素K1和维生素K2；
所述标准品的外表面标记有用于配置的浓度和需加入复溶剂的体积，所述标准品内含有与标示浓度匹配的定量的脂溶性维生素的冻干粉，加入标示体积的复溶剂后即可配置为标示浓度的脂溶性维生素溶液；所述标准品至少有六个，标示浓度从低到高；
所述复溶剂为不含待测脂溶性维生素的空白人血清基质。
2.根据权利要求1所述的一种脂溶性维生素的检测试剂盒，其特征在于，所述液相色谱洗脱液包括流动相A和流动相B，流动相A为标准浓度的甲酸水溶液，流动相B为标准浓度的甲酸甲醇溶液。
3.根据权利要求1所述的一种脂溶性维生素的检测试剂盒，其特征在于，所述内标溶液由含有VA1-d6、VD3-d6、VD2-d6、VK1-d4和VE-d6的同位素内标溶液组成。
4.根据权利要求1所述的一种脂溶性维生素的检测试剂盒，其特征在于，所述蛋白沉淀剂为乙腈和异丙醇的混合液，所述乙腈和异丙醇的体积比为1:1。
5.一种脂溶性维生素的检测方法，其特征在于，采用如权利要求1-3任一项所述的检测试剂盒进行检测，
分别向每个标准品中加入标示体积的复溶剂，从而配置成标示浓度的脂溶性维生素溶液；
分别向上述配置的每份脂溶性维生素溶液中加入等量的内标溶液和蛋白沉淀剂；
随机抽取至少3个标准品使用液质联用仪检测，并拟合每一种脂溶性维生素所对应的标准曲线方程；
使用液质联用仪以及得到的标准曲线方程检测剩余标准品中脂溶性维生素的浓度，判断检测结果是否在预设的范围内，
如果是，对待测样品中的脂溶性维生素进行检测。
6.根据权利要求5所述的一种脂溶性维生素的检测方法，其特征在于，标准品和待测样品中加入的内标溶液的量相同。
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