CN115326993A - 适用于干血斑样本中维生素d的质谱检测试剂盒及检测方法 - Google Patents
适用于干血斑样本中维生素d的质谱检测试剂盒及检测方法 Download PDFInfo
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Abstract
本发明提供的适用于干血斑样本中维生素D的质谱检测试剂盒,包括:包括干血斑取样片和质谱检测套件;所述干血斑取样片包括能够打开的外壳和放置在所述外壳内的维生素D冻干片;所述质谱检测套件包括: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维生素D溶液;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维生素D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维生素D溶液。本发明提供的适用于干血斑样本中维生素D的质谱检测试剂盒,具有良好的生物稳定性和安全性,适用于边远地区样本采集、储存与运输。
Description
技术领域
本发明属于维生素检测领域,特别涉及适用于干血斑样本中维生素D的质谱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。
其中,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。维生素D能促进钙磷的吸收,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。
因此,对维生素D的检测具有重要意义,但是目前的检测方法通常为对血清/血浆的质谱检测,当样本运输距离远,时间长时,血清/血浆容易失效。
发明内容
针对现有技术中的缺陷,本发明提供一种适用于干血斑样本中维生素D的质谱检测试剂盒及检测方法,具有良好的生物稳定性和安全性,适用于边远地区样本采集、储存与运输。
本发明提供的适用于干血斑样本中维生素D的质谱检测试剂盒,包括:干血斑取样片和质谱检测套件;
所述干血斑取样片包括能够打开的外壳和放置在所述外壳内的维生素D冻干片,所述维生素D冻干片中的维生素D的含量为标准值,所述维生素D冻干片的上表面为凹面,且为多孔蜂窝结构;
所述质谱检测套件包括: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维生素D溶液;
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维生素D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维生素D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维生素D的空白人血清基质。
本发明提供的适用于干血斑样本中维生素D的质谱检测试剂盒的工作原理如下:本发明中,采用维生素D冻干片作为采样载体,从而保障对干血斑中维生素D的提取率,采用不含待测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维生素外的组分一致,降低检测误差。
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VD3-d6、VD2-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干血斑样本中维生素D的检测方法,采用本发明提供的检测试剂盒进行检测,
步骤1制备干血斑样本:打开外壳,将全血滴加在维生素D冻干片的上表面,自然干燥后关闭外壳;
步骤2拟合标准曲线方程: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维生素D溶液;
分别向上述配置的每份维生素D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,并拟合每一种维生素D溶液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维生素D溶液的浓度,判断检测结果是否在预设的范围内,如果是,则标准曲线方程有效;
步骤3检测样品:取出含有干血斑样本中维生素D冻干片,使用复溶剂对维生素D冻干片进行复溶制备成待测溶液,使用液质联用仪以及步骤2中得到的标准曲线方程对待测溶液中的维生素D溶液进行检测。
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为一实施例中维生素D冻干片的结构示意图;
图2为一实施例提供的检测干血斑样本中维生素D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。
本实施例提供的适用于干血斑样本中维生素D的质谱检测试剂盒,包括:干血斑取样片和质谱检测套件;
所述干血斑取样片包括能够打开的外壳和放置在所述外壳内的维生素D冻干片,所述维生素D冻干片中的维生素D的含量为标准值,所述维生素D冻干片的上表面为凹面,且为多孔蜂窝结构;
所述质谱检测套件包括: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维生素D溶液;
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维生素D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维生素D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维生素D的空白人血清基质。
如果使用滤纸进行采样制备干血斑样本,当提取干血斑中的维生素D时,不可避免的会有部分血斑样本残留在滤纸上,降低提取率。
本实施例中,采用维生素D冻干片作为采样载体,检测时,可通过复溶剂对含有干血斑样本中维生素D冻干片进行复溶,从而实现对干血斑的完全提取,从而提高了对干血斑中维生素D的提取率。此外采用不含待测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维生素外的组分一致,降低检测误差。
具体的,参照图1,维生素D冻干片的上表面为凹面,可防止采样时血滴外溢损耗,维生素D冻干片的上表面还为多孔蜂窝状,加快了对液态血滴样本的吸收速度,外壳能保障其内部的干燥环境,防止样本受潮变质。
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VD3-d6、VD2-d6的同位素内标溶液组成。VD3-d6为维生素D3的内标,VD2-d6为维生素D2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。
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的峰面积,对维生素D的提取率最高。
参照图2,采用上述检测试剂盒对维生素D进行检测的方法如下:
步骤1制备干血斑样本:打开外壳,将全血滴加在维生素D冻干片的上表面,自然干燥后关闭外壳;
步骤2拟合标准曲线方程: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维生素D溶液;
分别向上述配置的每份维生素D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,并拟合每一种维生素D溶液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维生素D溶液的浓度,判断检测结果是否在预设的范围内,如果是,则标准曲线方程有效;
步骤3检测样品:取出含有干血斑样本中维生素D冻干片,使用复溶剂对维生素D冻干片进行复溶制备成待测溶液,使用液质联用仪以及步骤2中得到的标准曲线方程对待测溶液中的维生素D溶液进行检测。
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.适用于干血斑样本中维生素D的质谱检测试剂盒,其特征在于,包括干血斑取样片和质谱检测套件;
所述干血斑取样片包括能够打开的外壳和放置在所述外壳内的维生素D冻干片,所述维生素D冻干片中的维生素D的含量为标准值,所述维生素D冻干片的上表面为凹面,且为多孔蜂窝结构;
所述质谱检测套件包括: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维生素D溶液;
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维生素D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维生素D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维生素D的空白人血清基质。
2.根据权利要求1所述的适用于干血斑样本中维生素D的质谱检测试剂盒,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所述的适用于干血斑样本中维生素D的质谱检测试剂盒,其特征在于,所述内标溶液由含有VD3-d6和VD2-d6的同位素内标溶液组成。
4.根据权利要求1所述的适用于干血斑样本中维生素D的质谱检测试剂盒,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。
5.一种干血斑样本中维生素D的检测方法,其特征在于,采用如权利要求1-4任一项所述的检测试剂盒进行检测,
步骤1制备干血斑样本:打开外壳,将全血滴加在维生素D冻干片的上表面,自然干燥后关闭外壳;
步骤2拟合标准曲线方程: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维生素D溶液;
分别向上述配置的每份维生素D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,并拟合每一种维生素D溶液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维生素D溶液的浓度,判断检测结果是否在预设的范围内,如果是,则标准曲线方程有效;
步骤3检测样品:取出含有干血斑样本中维生素D冻干片,使用复溶剂对维生素D冻干片进行复溶制备成待测溶液,使用液质联用仪以及步骤2中得到的标准曲线方程对待测溶液中的维生素D溶液进行检测。
6.根据权利要求5所述的一种干血斑样本中维生素D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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