呼出气冷凝液中尿素的高效液相色谱-串联质谱测定法

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,RSD6.0%。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。

Determination of urea in exhaled breath condensate by liquid chromatography-tandem mass spectrometry

Objective To develop a liquid chromatography-tandem mass spectrometry (LC-MS-MS)method for the determination of urea in exhaled breath condensate (EBC).Methods 100 μl EBC was lyophilized to dryness by vacuum freeze drying equipment.Urea was separated by HILIC Plus column (4.6 mm×100 mm,3.5 μm) with methanol and 0.2％ formic acid water (5∶95,volume) was used as the mobile phases and then the sample was quantitatively analyzed by liquid chromatography tandem mass spectrometry method in multiple reactions monitoring (MRM) mode with positive mode.Results The internal matrix standard solution was used for the quantitative determination.There was a good linear relationship with a linear equation of y=0.008 82x+0.318 in the range of 5.3-400 ng/ml with the correlation coefficient of 0.999 5.The limit of quantification (LOQ) was 5.3 ng/ml.The spiked recoveries ranged from 95.1％ to 102.3％.The relative standard deviations were less than 6.0％.Conclusion The method is sensitive,accurate,less sample consumption and applicable to the determination of urea in exhaled breath condensate.