体液中西地那非的测定及其代谢物的确认

Determination of Sildenafil in Human Blood and Confirmation of Its Metabolites in Urine

建立了高效液相色谱-电喷雾串联质谱(HPLC-ESI-MS/MS)测定血液中西地那非以及利用电场轨道阱回旋共振组合质谱仪(LTQ-Orbitrap)鉴定尿液中西地那非代谢产物的方法。用乙醚提取体液,采用UltraIBD色谱柱,以乙腈-20 mmol乙酸铵(含0.1%甲酸)缓冲液(70∶30,V/V)为流动相,恒流分离后,在LC-MS/MS多反应监测模式下进行定性与定量分析;代谢物分离鉴定选用PLRP-S聚合色谱柱,以0.1%甲酸溶液(A)和乙腈(B)为流动相,梯度洗脱分离后,在LTQ-Orbitrap全扫描模式下进行代谢物鉴定。本方法在5～1000μg/L内线性关系良好,回收率为81.3%～103.3%,日内与日间精密度RSD均小于10%,检出限为1.0μg/L;尿液中检出5种代谢产物,分别为N1,N4-去乙基代谢物(M1),N-去甲基代谢物(M2,M3),羟化代谢物(M4)和N-甲基氧化代谢物(M5),其中M3和M5在人体尿液中为第一次检测到。

A method was developed to determine sildenafil in human blood by high performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS） and identify metabolites of sildenafil in human urine by linear trap quadropole（LTQ）-Orbitrap.Body fluids were extracted with ethyl ether.Sildenafil in blood was separated using IBD column with a mobile phase consisting of acetonitrile and 20 mmol/L ammonium acetate buffer including 0.1% formic acid（70∶30,V/V）.The flow rate was 0.2 mL/min.Then the analyte was analyzed by LC-MS/MS in the positive ion mode;the incurred urine sample was separated by PLRP-S polymeric column.0.1% formic acid（A） and acetonitrile（B） were used as mobile phase with elution gradient.The metabolites of sildenafil were aN-Alyzed by LTQ-Orbitrap.Sildenafil showed a good linear relationship over the range of 5-1000 μg/L（r=0.9972）.The recovery of sildenafil was 81.3%-103.3% and the RSD of intra-day and inter-day precision was less than 10%.The detection limit was 1 μg/L.Five metabolites were identified in human urine： N-N-desethylsildenafil（M1）,demethysildenafil（M2,M3）,propyl-oxidated sildenafil（M4） and CH3 to COOH sildenafil（M5）.M3 and M5 were the first reported in the human body fluids.