UPLC-MS/MS检测生物样本中西地那非及其代谢产物

UPLC-MS/MS Detecting Sildenafi l and Its Metabolites in Biological Samples

本文旨在建立血液和尿液中西地那非及3种主要代谢产物(N-去甲基西地那非、去乙烷西地那非和哌嗪-N-去烷基西地那非)的超高效液相色谱-质谱联用(UPLC-MS/MS)分析方法,并通过动物实验收集其血液和尿液作为待测样本对该方法加以验证。取血液或尿液200μL,以西地那非-d8为内标,加800μL乙腈沉淀蛋白。选用Waters BEH C18(2.1 mm×100 mm×1.7μm)色谱柱,流动相以0.01%甲酸水(2 mmol/L甲酸铵)为A相,乙腈为B相,梯度洗脱分离。质谱仪采用电喷雾离子源,正离子模式进行分析。该方法在血液和尿液中西地那非及3种主要代谢产物的检出限均可达到2 ng/mL;血添加样品中西地那非及其3种主要代谢物在5~1 000 ng/mL之间线性关系均良好(r≥0.997);尿添加样品中西地那非、N-去甲基西地那非和去乙烷西地那非在5~5 000 ng/mL之间线性关系良好(r≥0.997),哌嗪-N-去烷基西地那非在5~1 000ng/mL之间线性关系良好(r=0.999);血液和尿液中西地那非、N-去甲基西地那非、去乙烷西地那非和哌嗪-N-去烷基西地那非的提取回收率分别在102.27%~109.70%、77.16%~106.40%、84.22%~107.67%、92.02%~102.59%范围内;日内精密度分别在6.35%~11.07%、1.73%~9.81%、1.53%~11.19%、5.52%~12.51%范围内,日间精密度分别在8.19%~14.24%、1.79%~10.37%、1.71%~12.32%、6.41%~12.40%范围内;准确度分别在91.05%~103.27%、88.00%~110.20%、87.48%~111.31%、95.66%~112.73%范围内。用建立的分析方法对动物实验收集的血液和尿液样本进行定性定量分析,结果表明西地那非及其3种主要代谢物在血液和尿液样本中均可检出。该方法前处理操作简便,灵敏度高,线性范围宽,适用于血液和尿液中西地那非及其3种主要代谢产物的检验。

An ultra-performance liquid chromatography-mass spectrometry(UPLC-MS/MS)was to establish for analysis of sildenafil and its three major metabolites(N-demethylsildenafil,Descarbon Sildenafil and Sildenafil Impurity S)in human blood and urine,with its validation being carried through the collected blood and urine from experimental animals.The 200μL samples(blood or urine)were added with sildenafil-d8(the internal standard substance),successively precipitated of protein with acetonitrile(800μL)and then centrifuged to receive the supernatant.Afterfiltrated through membrane,the supernatant was eluted into Waters BEH C18 analytical column(2.1 mm×100 mm×1.7μm)that wasflowed with the gradient elution consisting of formic acid(0.01%)plus 2 mmol/L ammonium formate(mobile phase A)and acetonitrile(mobile phase B).Such the column-separated eluate was subjected to analysis of mass spectrometer into UPLC-MS/MS apparatus under multiple reaction monitoring(MRM)mode plus positive ionization(ESI+)by electrospray ionization.The results showed that limits of detection were up to 2 ng/mL for the sildenafil and its three major metabolites in blood and urine,with their linear ranges of the four chemicals being as 5–1000 ng/mL(all rendering an r higher than 0.997)in blood and as 5–5000 ng/mL(all rendering an r higher than 0.997)in urine for sildenafil,N-demethylsildenafil and Descarbon Sildenafil plus the remaining Sildenafil Impurity S as 5–1000 ng/mL(r=0.999).The extraction recovery was the respective 102.27%–109.70%,77.16%–106.40%,84.22%–107.67%,92.02%–102.59%for the sildenafil,N-demethylsildenafil,Descarbon Sildenafil and Sildenafil Impurity S in the analyzed biological samples,with the four chemicals showing their within-day precision being as the respective 6.35%–11.07%,1.73%–9.81%,1.53%–11.19%and 5.52%–12.51%,their inter-day’s as the 8.19%–14.24%,1.79%–10.37%,1.71%–12.32%and 6.41%–12.40%,and their accuracy as the individual 91.05%–103.27%,88.00%–110.20%,87.48%–111.31%and 95.66%–112.73%.Through qualitative and quantitative analysis into the blood and urine collected from experimental animals,the established method had been validated of ability to detect sildenafil and its three major metabolites in the tested samples,showing the concentrations of sildenafil,N-demethylsildenafil and Descarbon Sildenafil being as 27.52,9.97 and 11.04 ng/mL in blood yet Sildenafil Impurity S having not reached the limit of quantification,with the respective concentration of sildenafil,N-demethylsildenafil,Descarbon Sildenafil and Sildenafil Impurity S being as the 203.33,2963.84,3550.70 and 23.87 ng/mL in urine.This approach established here is of simple pretreatment,high extraction efficiency and wide linear range,hence capable of detection for sildenafil and its three major metabolites in blood and urine from suspected sildenafil poisoning case.