论文部分内容阅读
目的建立呼出气冷凝液(exhaled breath condensate,EBC)中尿素的液相色谱-串联质谱(LC-MS-MS)测定方法。方法将100μl的EBC样品加入50.0μl的40 ng/ml Urea-~(15)N_2内标,于-70℃冷冻20 min后,置于真空冷冻浓缩装置,-56℃真空冷冻浓缩至干,加入20.0μl甲醇,振荡混匀。以HILIC Plus色谱柱(4.6 mm×100 mm,3.5μm)进行分离,以0.2%甲酸水溶液-甲醇(体积比为5∶95)为流动相,在正离子模式下,质谱多反应监测(MRM)模式检测,内标法定量。结果尿素在5.3~400 ng/ml的范围内,所得尿素的回归方程为y=0.008 82x+0.318,r=0.999 5,线性关系良好。该方法的检出限为1.6 ng/ml,定量下限为5.3 ng/ml,回收率在95.1%~102.3%之间,RSD<6.0%。结论该方法具有灵敏度高、稳定性好、所需样品量小的特点,适用于呼出气冷凝液中尿素的测定。
Objective To establish a method for the determination of urea in exhaled breath condensate (EBC) by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Methods 100 μl of EBC samples were added to 50 μl of 40 ng / ml Urea- (15) N 2 internal standard and frozen at -70 ° C for 20 min before being placed in a vacuum freeze-concentration apparatus and vacuum-concentrated at -56 ° C to dryness. 20.0μl methanol, shake and mix well. The separation was performed on a HILIC Plus column (4.6 mm × 100 mm, 3.5 μm) using mobile phase of 0.2% formic acid in methanol (5:95 by volume) as the mobile phase. In positive ion mode, Pattern detection, internal standard method. Results The regression equation of urea in the range of 5.3-400 ng / ml was y = 0.008 82x + 0.318, r = 0.999 5, with good linearity. The detection limit of this method was 1.6 ng / ml. The lower limit of quantitation was 5.3 ng / ml. The recoveries ranged from 95.1% to 102.3% with a RSD <6.0%. Conclusion The method has the characteristics of high sensitivity, good stability and small amount of sample required. It is suitable for the determination of urea in exhaled breath condensate.