CN112326820A - 一种艾地骨化醇血药浓度的高效液相色谱-质谱联用检测方法 - Google Patents
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Abstract
本发明涉及一种艾地骨化醇血药浓度的高效液相色谱‑质谱联用检测方法，属于药物分析领域。本发明所述方法如下：待测血浆前处理步骤：向待测血浆中加入内标液，再加入萃取剂涡旋、离心，然后取上清液浓缩至干，最后再复溶；待测血浆液质联用检测步骤：采用液质联用系统进行分析检测，色谱柱为C18色谱柱，流动相为：流动相A为挥发性铵盐的水溶液，流动相B为挥发性铵盐的甲醇溶液，洗脱方式为梯度洗脱，离子源类型为大气压化学电离源，电离方式为正离子，检测模式多反应监测。本发明采用液质联用方法对待测血浆中艾地骨化醇的浓度进行检测，不但显著地将检测下限提升至5pg/mL，为Cmax的1/20，完全满足了生物样本的检测需求。
Description
技术领域
本发明涉及一种艾地骨化醇血药浓度的高效液相色谱-质谱联用检测方法，属于药物分析领域。
背景技术
2012年，卫生部发布的《防治骨质疏松知识要点》指出：骨质疏松症是我国排名第4位的慢性疾病，也是中老年最常见的骨骼疾病。据国际骨质疏松基金会(IOF)统计，2020年，仅我国内地将有2.86亿人患骨密度过低或骨质疏松症，到2050年这一数字将上升到5.33亿人，且全球治疗骨质疏松症所产生的费用可高达1315亿美元。
艾地骨化醇由日本中外制药株式会社研发，规格为0.75μg和0.5μg，于2011年1月21日在厚生省获批上市，是继阿法骨化醇后又一新的用于治疗骨质疏松症的维生素D3类药物，具有改善骨代谢和促进骨吸收的双重作用。一项由1054例骨质疏松症患者参加历时3年的Ⅲ期临床试验数据表明，艾地骨化醇疗效优于阿法骨化醇，且安全性与阿法骨化醇相似，具有较好的应用前景。
艾地骨化醇作为最新一代抗骨质疏松类药物，安全性较高，不良反应较少、顺应性良好、用药限制少、与经CYP酶系统代谢的药物间很少产生相互作用，具有良好的市场前景。
然而，艾地骨化醇血药浓度的检测方法却鲜有报道。
中外制药株式会社曾公开报道健康成年男性单次口服给药0.75μg艾地骨化醇时，在投药后3.4±1.2小时内血清中艾地骨化醇浓度达到给药后血药浓度的最高值，即Cmax为99.8±12.7pg/mL。按照《化学药物制剂生物利用度和生物等效性研究技术指导原则》的要求，一般检测方法的定量下限应满足为Cmax的1/10-1/20。中外制药株式会社采用LC-MS/MS方法分析血清中艾地骨化醇的浓度，该方法的定量下限为0.025ng/mL，即25pg/mL，约为Cmax的1/4，不能完全满足生物样本定量分析方法的要求。
专利CN111272902A公开了一种艾地骨化醇血药浓度的检测方法，所述方法的定量下限为10pg/mL，不到Cmax的1/10，亦不能完全满足生物样本定量分析方法的要求。且所述方法采用的流动相A为不挥发性乙酸锂水溶液，易造成质谱的污染，需频繁清洗，不但会影响仪器寿命还会降低工作效率，影响产出。此外，所述方法采用价格昂贵的API6500+质谱仪作为检测仪器，无形中增加了企业经济负担，不利于检测技术的推广应用。
由于临床药代动力学检测时样本量常常很大，因此需要一种快速、灵敏且能完全满足生物样本定量分析要求的分析方法。
发明内容
本发明为检测艾地骨化醇血药浓度建立了一种新方法，该方法检测下限为5pg/mL，是Cmax的1/20，完全能够满足生物样本定量分析的要求，解决了上述的问题。
本发明提供了一种艾地骨化醇血药浓度的高效液相色谱-质谱联用检测方法，所述方法包括如下步骤：待测血浆前处理步骤：向待测血浆中加入内标液，再加入萃取剂涡旋、离心，然后取上清液浓缩至干，最后再复溶；待测血浆液质联用检测步骤：采用液质联用系统进行分析检测；色谱柱为：C18色谱柱；流动相为：流动相A为挥发性铵盐的水溶液，流动相B为挥发性铵盐的甲醇溶液；洗脱方式为：梯度洗脱，梯度洗脱程序如下表1所示：
表1
离子源类型为：大气压化学电离源；电离方式为：正离子；检测模式：多反应监测。
本发明优选为所述内标液为：用30-70％甲醇水溶液或30-70％乙腈水溶液稀释的艾地骨化醇对照品溶液，使其浓度为1.0-5.0ng/mL；所述内标液加入量为待测血浆体积的1/16-1/4。
本发明优选为所述萃取剂为：甲基叔丁基醚与正己烷体积比1：9的混合液、甲基叔丁基醚与正己烷体积比3：7的混合液、甲基叔丁基醚与正己烷体积比7：3的混合液、甲基叔丁基醚与正己烷体积比9：1的混合液或甲基叔丁基醚；所述萃取剂加入量为待测血浆体积的7.5-12.5倍。
本发明优选为所述复溶用的溶液为：30-70％甲醇水溶液或30-70％乙腈水溶液。
本发明优选为所述色谱柱的规格为：内径2.1×长度50mm，填料粒径3.0-5.0μm。
本发明优选为自动进样器温度为：4-8℃，柱温为：25-50℃。
本发明优选为所述挥发性铵盐为乙酸铵，且含有0.1％甲酸。
本发明优选为所述挥发性铵盐的水溶液浓度为1-5mM，所述挥发性铵盐的甲醇溶液浓度为1-5mM。
本发明优选为进样量为：15-30μL。
本发明的有益效果为：
本发明采用液质联用方法对待测血浆中艾地骨化醇的浓度进行检测，以挥发性铵盐的水溶液和挥发性铵盐的甲醇溶液为流动相，不但显著地将检测下限提升至5pg/mL，为Cmax的1/20，完全满足了生物样本的检测需求。同时，以挥发性铵盐的水溶液和挥发性铵盐的甲醇溶液为流动相避免了质谱仪的频繁清洗程序，在延长仪器寿命的基础上，显著的提高了检测效率。
附图说明
本发明附图3幅，
图1为艾地骨化醇空白基质的质谱图；
图2为艾地骨化醇定量下限的质谱图，其中：左图为ED-71的质谱图，右图为ED-71-d6的质谱图；
图3为艾地骨化醇标准曲线图谱。
具体实施方式
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何方式限制本发明。
一种艾地骨化醇血药浓度的高效液相色谱-质谱联用检测方法，所述方法包括如下步骤：
待测血浆前处理步骤：向400μL待测血浆中加入内标液，所述内标液的加入量为25-100μL，优选内标液的加入量为25μL，再加入萃取剂，所述萃取剂的加入量为3-5mL，优选萃取剂的加入量为5mL，所述萃取剂为甲基叔丁基醚与正己烷体积比1：9的混合液、甲基叔丁基醚与正己烷体积比3：7的混合液、甲基叔丁基醚与正己烷体积比7：3的混合液、甲基叔丁基醚与正己烷体积比9：1的混合液或甲基叔丁基醚，优选萃取剂为甲基叔丁基醚与正己烷体积比1：9的混合液，以1600rpm涡旋5min、3000rpm离心5min，然后取上清液浓缩至干，最后再用100μL溶液复溶，所述复溶用的溶液为30-70％甲醇水溶液或30-70％乙腈水溶液，优选复溶用的溶液为30％甲醇水溶液；
待测血浆液质联用检测步骤：采用液质联用系统进行分析检测，再根据谱图和标准曲线计算待测血浆中艾地骨化醇的浓度；
色谱柱为：C18色谱柱，其规格为：内径2.1×长度50mm，填料粒径3.0-5.0μm，优选填料粒径3.0μm；
流动相为：流动相A为1-5mM乙酸铵(含有0.1％甲酸)的水溶液，优选流动相A为1mM乙酸铵(含有0.1％甲酸)的水溶液，流动相B为1-5mM乙酸铵(含有0.1％甲酸)的甲醇溶液，优选流动相B为5mM乙酸铵(含有0.1％甲酸)的甲醇溶液；
洗脱方式为：梯度洗脱，梯度洗脱程序如下表1所示：
表1
优选梯度洗脱程序如下表2所示：
表2
自动进样器温度为：4-8℃，优选自动进样器温度为5℃；
进样量为：15-30μL，优选进样量为15μL；
柱温为：25-50℃，优选柱温为27℃；
离子源类型为：大气压化学电离源；
电离方式为：正离子；
检测模式：多反应监测。
所述内标液的配制方法为：向有机溶剂中加入艾地骨化醇对照品(ED-71-d6)，所述有机溶剂为甲醇、乙腈或二甲基亚砜，优选有机溶剂为甲醇，使其浓度为1-5mg/mL，优选浓度为2mg/mL，再以30-70％甲醇水溶液或30-70％乙腈水溶液稀释至1.0-5.0ng/mL，优选以30％甲醇水溶液稀释至5.0ng/mL。
实施例1
定量下限检测方法：
使用有机溶剂配制浓度为5.0、10.0、20.0、50.0和120.0pg/mL的艾地骨化醇(ED-71)溶液，所述有机溶剂为甲醇、乙腈或二甲基亚砜，优选有机溶剂为甲醇，再加入到空白血浆中，配制成不同浓度的艾地骨化醇血浆溶液，然后按照上述待测血浆液质联用检测步骤和待测血浆液质联用检测步骤进行操作，并记录信噪比。图1和图2表明，浓度为5.0pg/mL时，信噪比良好，可满足生物样本检测灵敏度的需求。
实施例2
线性关系检测方法：
标准曲线工作液的配制：向有机溶剂中加入ED-71，所述有机溶剂为甲醇、乙腈或二甲基亚砜，优选有机溶剂为甲醇，使其浓度为1-5mg/mL，优选浓度为2mg/mL，再以30-70％甲醇水溶液或30-70％乙腈水溶液稀释至100、200、400、800、1600、2400、4800、7200和8000pg/mL，优选以30％甲醇水溶液稀释，得到标准曲线工作液。
标准曲线样品配制：取系列标准曲线工作液，用空白血浆稀释定容混匀，即得浓度为5.0、10.0、20.0、40.0、80.0、120.0、240.0、360.0和400.0pg/mL的标准曲线样品。
按照上述待测血浆液质联用检测步骤和待测血浆液质联用检测步骤对系列标准曲线样品进行操作。以艾地骨化醇系列标准曲线样品浓度为横坐标，艾地骨化醇和内标液峰面积比为纵坐标，用最小二乘法进行回归运算，求得直线回归方程即为标准曲线。计算其线性相关系数r＝0.9983，表明采用本发明所述的检测方法，血浆中艾地骨化醇在5-400pg/mL的浓度范围内线性关系良好。
实施例3
精密度和准确度测试：
为确保本发明所述检测方法的精密度和准确度，本实施例不仅测试了定量下限浓度、低质控浓度和高质控浓度，更在中质控浓度附近取了2个测试浓度，共5个浓度的质控样品。
质控工作液的配制：向有机溶剂中加入ED-71，所述有机溶剂为甲醇、乙腈或二甲基亚砜，优选有机溶剂为甲醇，使其浓度为1-5mg/mL，优选浓度为2mg/mL，再以30-70％甲醇水溶液或30-70％乙腈水溶液稀释至100、200、1000、2000和6400pg/mL，优选以30％甲醇水溶液稀释，得到质控工作液。
质控样品配制：取系列质控工作液，用空白血浆稀释定容混匀，即得浓度为5.0pg/mL(定量下限浓度，LLOQ)、10.0pg/mL(低质控浓度，LOQ)、50.0pg/mL(中质控浓度1，MOQ)、100pg/mL(中质控浓度2，GMOQ)和320pg/mL(高质控浓度，HOQ)的质控样品。
按照上述待测血浆液质联用检测步骤和待测血浆液质联用检测步骤对系列质控样品进行操作。每个质控样品有4个，以评估批内的精密度和准确度。通过计算检测浓度的变异系数(％CV)评估精密度，计算检测浓度的平均值和偏差以评估准确度，结果详见表3。根据现行版《中国药典》四部通则9012生物样品定量分析方法验证指导原则，批内准确度均值一般应在质控样品标示值的±15％之内，定量下限准确度应在标示值的±20％之内；批内精密度变异系数一般不得超过15％，定量下限的变异系数不得超过20％。由表3可知，各质控浓度样品的精密度和准确度均在指导原则要求的范围内，表明本发明所述检测方法具有良好的精密度和准确度。
表3
综上，本发明所述检测方法高效、灵敏，且精密度和准确度均符合相关指导原则的要求，可用于血浆中艾地骨化醇浓度的检测。且本发明所述检测方法的定量下限为5pg/mL，完全能够满足生物样本的检测需求，优于现有检测技术。
Claims (9)
1.一种艾地骨化醇血药浓度的高效液相色谱-质谱联用检测方法，其特征在于：所述方法包括如下步骤：
待测血浆前处理步骤：向待测血浆中加入内标液，再加入萃取剂涡旋、离心，然后取上清液浓缩至干，最后再复溶；
待测血浆液质联用检测步骤：采用液质联用系统进行分析检测；
色谱柱为：C18色谱柱；
流动相为：流动相A为挥发性铵盐的水溶液，流动相B为挥发性铵盐的甲醇溶液；
洗脱方式为：梯度洗脱，梯度洗脱程序如下表1所示：
表1
离子源类型为：大气压化学电离源；
电离方式为：正离子；
检测模式：多反应监测。
2.根据权利要求1所述艾地骨化醇血药浓度的高效液相色谱-质谱联用检测方法，其特征在于：所述内标液为：用30-70％甲醇水溶液或30-70％乙腈水溶液稀释的艾地骨化醇对照品溶液，使其浓度为1.0-5.0ng/mL；
所述内标液加入量为待测血浆体积的1/16-1/4。
3.根据权利要求2所述艾地骨化醇血药浓度的高效液相色谱-质谱联用检测方法，其特征在于：所述萃取剂为：甲基叔丁基醚与正己烷体积比1：9的混合液、甲基叔丁基醚与正己烷体积比3：7的混合液、甲基叔丁基醚与正己烷体积比7：3的混合液、甲基叔丁基醚与正己烷体积比9：1的混合液或甲基叔丁基醚；
所述萃取剂加入量为待测血浆体积的7.5-12.5倍。
4.根据权利要求3所述艾地骨化醇血药浓度的高效液相色谱-质谱联用检测方法，其特征在于：所述复溶用的溶液为：30-70％甲醇水溶液或30-70％乙腈水溶液。
5.根据权利要求4所述艾地骨化醇血药浓度的高效液相色谱-质谱联用检测方法，其特征在于：所述色谱柱的规格为：内径2.1×长度50mm，填料粒径3.0-5.0μm。
6.根据权利要求5所述艾地骨化醇血药浓度的高效液相色谱-质谱联用检测方法，其特征在于：自动进样器温度为：4-8℃，柱温为：25-50℃。
7.根据权利要求6所述艾地骨化醇血药浓度的高效液相色谱-质谱联用检测方法，其特征在于：所述挥发性铵盐为乙酸铵，且含有0.1％甲酸。
8.根据权利要求7所述艾地骨化醇血药浓度的高效液相色谱-质谱联用检测方法，其特征在于：所述挥发性铵盐的水溶液浓度为1-5mM，所述挥发性铵盐的甲醇溶液浓度为1-5mM。
9.根据权利要求8所述艾地骨化醇血药浓度的高效液相色谱-质谱联用检测方法，其特征在于：进样量为：15-30μL。
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