CN112578052A - 一种液相色谱串联质谱检测血清中药物含量的方法 - Google Patents
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Abstract
本发明公开了一种液相色谱串联质谱检测血清中药物含量的方法，首先进行色谱条件与系统适用性实验与进行质谱条件与系统适用性实验，再配置标准工作液，标准内标液；提取人或动物的血液的血清，添加标准内标液制备待测样品；对待测样品进行检测，得到检测数据，再建立校准曲线，代入所述检测数据，得到分析结果；本发明可以精确的检测药物在血清中的活性成分，具有稳定性和重现性好、可操作性强等优点；能将药物作为当成一个整体或者多个部分进行研究；定量准确，经济效益好，检测过程简便快速，实验成本较低。
Description
技术领域
本发明涉及一种液相色谱串联质谱检测血清中药物含量的方法，属于药物分析技术领域。
背景技术
血清药物化学研究显示，药物中虽含有诸多成分，但只有被吸收入血的成分才能产生作用，被认为药物活性成分；传统药物多为口服给药，口服给药后药物成分或经过消化道直接吸收入血，或经肝微粒体酶代谢后入血，其活性成分以血液为介质输送到靶点，从而产生作用；血清中含有的成分被认为是药物在体内直接起作用的物质，如今该如何精确的检测药物的活性成分已受到本领域研究者的关注。
发明内容
针对上述存在的技术问题，本发明的目的是：提出了一种液相色谱串联质谱检测血清中药物含量的方法，可以精确的检测药物在血清中的活性成分。
本发明的技术解决方案是这样实现的：一种液相色谱串联质谱检测血清中药物含量的方法，步骤如下：
步骤一：进行色谱条件与系统适用性实验，确定具体色谱条件和流动相梯度洗脱参数；
步骤二：进行质谱条件与系统适用性实验，确定具体质谱条件和质谱检测参数；
步骤三：配置标准工作液；
步骤四：配置标准内标液；
步骤五：对检测血液进行离心处理，静置后，取上层清液，上层清液为血清；
步骤六：将所述标准内标液与所述血清置于离心管中混合后进行离心处理；再在所述离心管中加入蛋白沉淀剂，涡旋混合后进行离心处理，静置后取上层清液，上层清液为待测样品；
步骤七：利用超高效液相色谱串联质谱仪和紫外检测器对待测样品进行检测，得到检测数据；
步骤八：将标准工作液与标准内标液比定为X轴，标准工作液与内标物峰面积比定为Y轴，建立校准曲线，代入所述检测数据，计算出血清中目标药物的浓度，得到分析结果。
在本发明的一个实施方案中，所述步骤三中标准工作液和步骤四中标准内标液均在-70℃至-75℃条件下保存。
在本发明的一个实施方案中，所述步骤五中血清置于-20℃条件下保存。
在本发明的一个实施方案中，所述步骤三中配置的标准工作液的浓度至少为八种。
在本发明的一个实施方案中，所述步骤五中检测血液为人或动物的血液。
在本发明的一个实施方案中，所述步骤六中蛋白沉淀剂为甲醇、5-磺基水杨酸与蒸馏水的质量比70:5:25的混合溶液。
由于上述技术方案的运用，本发明与现有技术相比具有下列优点：
本发明的一种液相色谱串联质谱检测血清中药物含量的方法，可以精确的检测药物在血清中的活性成分，具有稳定性和重现性好、可操作性强等优点；能将药物作为当成一个整体或者多个部分进行研究；定量准确，经济效益好，检测过程简便快速，实验成本较低。
附图说明
下面结合附图对本发明技术方案作进一步说明：
附图1为本发明的一种液相色谱串联质谱检测血清中药物含量的方法的实验步骤图。
具体实施方式
下面结合附图来说明本发明。
请参照图1，一种液相色谱串联质谱检测血清中药物含量的方法，步骤如下：
步骤一：进行色谱条件与系统适用性实验，确定具体色谱条件和流动相梯度洗脱参数;
流动相A为乙腈,流动相B为0.1％甲酸水溶液;
色谱柱填料内径为4μm的；柱温为25℃，流动相流速为1.3mL/min；
梯度洗脱条件如下：
0-10min A:B从10：90,v/v到20：80,v/v；
10-20min A:B从20：80,v/v到35：65,v/v；
20-30min A:B保持35：65,v/v；
30-40min A:B保持35：65,v/v；
40-70min A:B从35：60,v/v到45：55,v/v；
70-90min A:B到45：55,v/v；到70：30,v/v
85-90min A:B从70：30,v/v到80：20,v/v；
90-100min A:B从80：20,v/v到100：0,v/v；
步骤二：进行质谱条件与系统适用性实验，确定具体质谱条件和质谱检测参数；
质谱条件为：最佳激光强度180；最佳检测灵敏度9；设最高分子量为100000道尔顿(Da)，最佳状态从2500Da到25000Da 设Seldi数据收集参数检测点30-100(acquisitionparameters为30，delta4，transients per to 5 ending position to 100)；设预热位置用2个spots，激光强度200；
步骤三：配置八种浓度的标准工作液，每种浓度的标准工作液均在-75℃条件下保存；
所述标准工作液的浓度分别为0.4ug/ml、 0.8ug/ml、1.6ug/ml、3ug/ml、8ug/ml、15ug/ml、30ug/ml、50ug/ml。
步骤四：配置标准内标液，标准内标液在-75℃条件下保存；标准内标液浓度均为2μg/ml的储备液；
步骤五：准备检测血液，检测血液为人或动物的血液；对所述检测血液进行离心处理，静置后，取上层清液，上层清液为血清，血清-20℃条件下保存；
步骤六：将所述标准内标液与所述血清置于离心管中混合后进行离心处理；再在所述离心管中加入蛋白沉淀剂，涡旋混合后进行离心处理，静置后取上层清液，上层清液为待测样品；所述蛋白沉淀剂为甲醇、5-磺基水杨酸与蒸馏水的质量比70:5:25的混合溶液；
所述血清与标准内标液混合的过程中，二者之间的体积比为1:4。
步骤七：利用超高效液相色谱串联质谱仪和紫外检测器对待测样品进行检测，得到检测数据；
步骤八：将标准工作液与标准内标液比定为X轴，标准工作液与内标物峰面积比定为Y轴，建立校准曲线；当需要检测的药品为阿立哌唑时，校准曲线为y＝0.0048x+0.0035；代入所述检测数据，计算出血清中目标药物的浓度，得到分析结果。
本发明的一种液相色谱串联质谱检测血清中药物含量的方法，可以精确的检测药物在血清中的活性成分，具有稳定性和重现性好、可操作性强等优点；能将药物作为当成一个整体或者多个部分进行研究；定量准确，经济效益好，检测过程简便快速，实验成本较低。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并加以实施，并不能以此限制本发明的保护范围，凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围内。
Claims (6)
1.一种液相色谱串联质谱检测血清中药物含量的方法，其特征在于，步骤如下：
步骤一：进行色谱条件与系统适用性实验，确定具体色谱条件和流动相梯度洗脱参数；
步骤二：进行质谱条件与系统适用性实验，确定具体质谱条件和质谱检测参数；
步骤三：配置标准工作液；
步骤四：配置标准内标液；
步骤五：对检测血液进行离心处理，静置后，取上层清液，上层清液为血清；
步骤六：将所述标准内标液与所述血清置于离心管中混合后进行离心处理；再在所述离心管中加入蛋白沉淀剂，涡旋混合后进行离心处理，静置后取上层清液，上层清液为待测样品；
步骤七：利用超高效液相色谱串联质谱仪和紫外检测器对待测样品进行检测，得到检测数据；
步骤八：将标准工作液与标准内标液比定为X轴，标准工作液与内标物峰面积比定为Y轴，建立校准曲线，代入所述检测数据，计算出血清中目标药物的浓度，得到分析结果。
2.如权利要求1所述的一种液相色谱串联质谱检测血清中药物含量的方法，其特征在于：所述步骤三中标准工作液和步骤四中标准内标液均在-70℃至-75℃条件下保存。
3.如权利要求1所述的一种液相色谱串联质谱检测血清中药物含量的方法，其特征在于：所述步骤五中血清置于-20℃条件下保存。
4.如权利要求1所述的一种液相色谱串联质谱检测血清中药物含量的方法，其特征在于：所述步骤三中配置的标准工作液的浓度至少为八种。
5.如权利要求3所述的一种液相色谱串联质谱检测血清中药物含量的方法，其特征在于：所述步骤五中检测血液为人或动物的血液。
6.如权利要求1所述的一种液相色谱串联质谱检测血清中药物含量的方法，其特征在于：所述步骤六中蛋白沉淀剂为甲醇、5-磺基水杨酸与蒸馏水的质量比70:5:25的混合溶液。
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| CN109085263A (zh) * | 2018-08-03 | 2018-12-25 | 杭州佰勤医疗器械有限公司 | 液相色谱串联质谱法检测血清血浆中抗精神分裂药物的试剂盒及其应用 |
| CN110208450A (zh) * | 2019-07-12 | 2019-09-06 | 北京和合医学诊断技术股份有限公司 | 一种检测血液中阿立哌唑和去氢阿立哌唑的方法 |
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| CN109085263A (zh) * | 2018-08-03 | 2018-12-25 | 杭州佰勤医疗器械有限公司 | 液相色谱串联质谱法检测血清血浆中抗精神分裂药物的试剂盒及其应用 |
| CN110208450A (zh) * | 2019-07-12 | 2019-09-06 | 北京和合医学诊断技术股份有限公司 | 一种检测血液中阿立哌唑和去氢阿立哌唑的方法 |
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