CN109633040A - 一种羧甲司坦含量测定方法 - Google Patents
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Abstract
本发明涉及体内药物的测定方法,特别涉及一种羧甲司坦含量测定方法,包括以下步骤:(1)标准曲线溶液的制备;(2)待测血浆样本的制备;(3)标准曲线的绘制;(4)待测血浆样本检测。本发明的检测方法可以对人体血浆样本中羧甲司坦含量进行准确定量分析,灵敏度高,检测限低,下限可达到30ng/mL;本发明的检测方法可高选择性的检测待测物产生的特定离子对,检测不受血浆中其它物质的干扰;本发明的检测方法步骤简单,快速准确,从样本接收到得到数据仅需30分钟。
Description
技术领域
本发明涉及体内药物的测定方法,特别涉及一种羧甲司坦含量测定方法。
背景技术
羧甲司坦(Carbocisteine)为黏液稀化剂,作用与溴己新相似,主要在细胞水平影响支气管腺体的分泌,可使黏液中黏蛋白的双硫键断裂,使低黏度的涎黏蛋白分泌增加,而高黏度的岩藻黏蛋白产生减少,从而使痰液的黏滞性降低,有利于痰液排出。口服起效快,服后4小时即可见明显疗效。广泛分布至肺组织中,最后以原型和代谢产物的形式经尿液排泄。
研究表明,羧甲司坦在不同个体相同剂量条件下,药物的吸收与代谢会有明显的差别,直接影响治疗效果。因此,通过测定人血浆中不同时间点羧甲司坦的含量,可以判断个体药物吸收情况,从而可以差别化指导用药,达到理想的治疗效果。
目前,已报道的羧甲司坦检测方法多是测定药物本身的含量,多采用液相色谱、紫外光谱进行不同制剂含量测定。方法灵敏度低,无法满足人体血浆中低含量,复杂基质条件下的药物浓度检测。
发明内容
本发明克服了现有技术中的不足,提供了一种采用微生物和化学还原相结合的处理含氮废水的方法,以解决上述问题。
为了解决上述技术问题,本发明是通过以下技术方案实现的:一种羧甲司坦含量测定方法,包括以下步骤:一种羧甲司坦含量测定方法,其特征在于,包括以下步骤:
(1)标准曲线溶液的制备:将羧甲司坦溶解后采用50%甲醇水溶液进行逐级稀释,稀释范围为600 ng/mL~80000 ng/mL,分别取稀释后的各级羧甲司坦溶液,加入空白血浆和同位素内标溶液,混合均匀,以14000 r/min离心15 min,取上清液得羧甲司坦标准曲线溶液;
(2)待测血浆样本的制备:取待测血浆样本50μL,加入250 μL含同位素内标的沉淀剂甲醇,涡旋1 min后,在4℃的离心机中,以14000 r/min离心15 min,取上清液在2~8℃冷藏备用;
(3)标准曲线的绘制:将步骤(1)制备的标准曲线溶液进行液相质谱检测,以羧甲司坦标准曲线溶液校正浓度理论值为横坐标,以羧甲司坦与同位素内标的面积比值为纵坐标进行线性回归分析,线性系数不小于0.99;
(4)待测血浆样本检测:将步骤(2)制备的待测血浆样本进行液相质谱检测,检测结果带入步骤(3)得到的标准曲线中,计算得到待测血浆样本中羧甲司坦的含量。
作为优选方案,所述标准曲线溶液与待测血浆样本中的内标液浓度相同,均为100ng/mL。
作为优选方案,所述步骤(1)和步骤(2)中,所述同位素内标液为羧甲司坦各构型对应的氘代物或13C3代物或15N代物中的一种。
作为优选方案,所述同位素内标液为羧甲司坦13C3。
作为优选方案,所述步骤(1)中,将羧甲司坦分别稀释为80000ng/mL、64000ng/mL、20000ng/mL、12000ng/mL、6000ng/mL、2000ng/mL、1200ng/mL、600ng/mL。
发明的有益效果是:本发明的检测方法可以对人体血浆样本中羧甲司坦含量进行准确定量分析,灵敏度高,检测限低,下限可达到30ng/mL;本发明的检测方法可高选择性的检测待测物产生的特定离子对,检测不受血浆中其它物质的干扰;本发明的检测方法步骤简单,快速准确,从样本接收到得到数据仅需30分钟。
附图说明
图1是本发明梯度洗脱中流动相和时间的关系图;
图2是本发明羧甲司坦的标准曲线溶液的线性回归图;
图3是本发明给药时间与人体血浆中羧甲司坦含量的关系。
具体实施方式
下面结合具体实施方式对本发明作进一步详细描述:
1.仪器与药品
高效液相色谱仪:岛津Nexera XR,
泵:LC-20AD XR,
自动进样器:SIL -20AC XR,
柱温箱:CTO-20A;
质谱仪:AB SCIEX API 4000;
离心机:Thermo SORVALL ST 8R。
羧甲司坦购自中检所,羧甲司坦13C3购自于TLC公司。
2. 色谱检测条件
液相质谱所用离子源为ESI(-),所用的离子对母离子为:178.0±0.5,子离子为:90.0±0.5;
色谱柱:Agela -Innoval ODS-2 2.1×100mm,3μm色谱柱,
预柱:Agela Durashell C18(L)(2.1*10mm, 3μm);
流动相A相为10mmol/L 乙酸铵水溶液,B相为乙腈,采用梯度洗脱,具体梯度见图1,流速为0.3~0.4mL/min,柱温40℃,进样体积5μl。
3.标准溶液的制备
羧甲司坦储备液的制备:称量羧甲司坦10.0mg,用电动移液器加入50%甲醇水溶液使有效浓度为1.00 mg/mL,标记为stock 1。
羧甲司坦13C3储备液的制备:称量羧甲司坦13C3 1.00mg,用电动移液器加入50%甲醇水溶液使有效浓度为1.00 mg/mL,标记为stock 2。
内标溶液的制备:取羧甲司坦13C储备液 100μL,加入100mL容量瓶中,用甲醇定容到100mL,羧甲司坦13C含量为100ng/mL。
4.标准曲线溶液的制备
将储备液stock 1用50%甲醇水逐级稀释到80000ng/mL,64000ng/mL,20000ng/mL,12000ng/mL,6000ng/mL,2000ng/mL,1200ng/mL,600ng/mL,分别取上述各浓度溶液20μl,加入380μL空白血浆,加入含同位素内标液的沉淀剂(含内标100ng/mL),混合均匀,以14000r/min离心15 min,取上清液,即为羧甲司坦的标准曲线溶液,备用;计算得到标准曲线校正浓度理论值为4000ng/mL,3200ng/mL,1000ng/mL,600ng/mL,300ng/mL,100ng/mL,60ng/mL,30ng/mL 。
5.待测血浆样本的制备
取待测血浆样本50μL,加入250 μL含同位素内标的沉淀剂甲醇(含内标100ng/mL),涡旋约1 min后,在4℃离心机中,以14000 r/min离心15 min,取上清液,得待测血浆样本。
6.线性分析
取步骤4获得的羧甲司坦的标准曲线溶液进行液相质谱检测,采用液相色谱-质谱仪连用进行检测,羧甲司坦的色谱检测条件为步骤2所述条件;以标准曲线中校正浓度理论值为横坐标,以羧甲司坦与同位素内标的面积比值为纵坐标进行线性回归分析,得到回归曲线方程为y=0.00204x+0.00198(r=0.9991),权重系数为1/x2,见图2。
7.待测血浆样本检测
于给药前(0h)及给药后0.25h、0.5h、0.75h、1.0h、1.25h、1.5h、1.75h、2.0h、2.25h、2.5h、2.75h、3.0h、4.0h、6.0h、8.0h、12.0h、24.0h采集静脉血4ml,共18个时间点,采用步骤5方法制备血浆样本,转移至 96 孔板(未能及时检测的样品需保存在自动进样器中或 2~8℃冰箱中,以待下次检测),采用液相色谱-质谱仪连用进行检测,检测结果带入步骤6的标准曲线方程,得到血浆样本中羧甲司坦含量,见图3。
本发明未经描述的技术特征可以通过或采用现有技术实现,在此不再赘述,当然,上述具体实施方式并非是对本发明的限制,本发明也并不仅限于上述具体实施方式,本技术领域的普通技术人员在本发明的实质范围内所做出的变化、改型、添加或替换,也应落入本发明的保护范围内。
Claims (5)
1.一种羧甲司坦含量测定方法,其特征在于,包括以下步骤:
(1)标准曲线溶液的制备:将羧甲司坦溶解后采用50%甲醇水溶液进行逐级稀释,稀释范围为600 ng/mL~80000 ng/mL,分别取稀释后的各级羧甲司坦溶液,加入空白血浆和同位素内标溶液,混合均匀,以14000 r/min离心15 min,取上清液得羧甲司坦标准曲线溶液;
(2)待测血浆样本的制备:取待测血浆样本50μL,加入250 μL含同位素内标的沉淀剂甲醇,涡旋1 min后,在4℃的离心机中,以14000 r/min离心15 min,取上清液在2~8℃冷藏备用;
(3)标准曲线的绘制:将步骤(1)制备的标准曲线溶液进行液相质谱检测,以羧甲司坦标准曲线溶液校正浓度理论值为横坐标,以羧甲司坦与同位素内标的面积比值为纵坐标进行线性回归分析,线性系数不小于0.99;
(4)待测血浆样本检测:将步骤(2)制备的待测血浆样本进行液相质谱检测,检测结果带入步骤(3)得到的标准曲线中,计算得到待测血浆样本中羧甲司坦的含量。
2.根据权利要求1所述的羧甲司坦含量测定方法,其特征在于:所述标准曲线溶液与待测血浆样本中的内标液浓度相同,均为100ng/mL。
3.根据权利要求1所述的羧甲司坦含量测定方法,其特征在于:所述步骤(1)和步骤(2)中,所述同位素内标液为羧甲司坦各构型对应的氘代物或13C3代物或15N代物中的一种。
4.根据权利要求3所述的羧甲司坦含量测定方法,其特征在于:所述同位素内标液为羧甲司坦13C3。
5.根据权利要求1所述的羧甲司坦含量测定方法,其特征在于:所述步骤(1)中,将羧甲司坦分别稀释为80000ng/mL、64000ng/mL、20000ng/mL、12000ng/mL、6000ng/mL、2000ng/mL、1200ng/mL、600ng/mL。
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