体液中西地那非的测定及其代谢物的确认

摘要

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 bv 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 a N-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. 37 and the RSD of intra-day and interday precision was less than 107％. The detection limit was 1 μ　g/L. Five metabolites were identified in human urine : N-N-desethylsildenafil ( M1 ) , demethysildenafil ( M2 , M3) , propyl-oxidated sildenafil (M4) and CH3 to COOH sildenafiI(M5). M3 and M5 were the first reported in the human body fluids.%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Ultra IBD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5～1000 μg/L内线性关系良好,回收率为81.3%～103.3%旧内与日间精密度RSD均小于10%,检出限为1.0 μg/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(MS),其中M3和M5在人体尿液中为第一次检测到.