尿中高香草酸和香草扁桃酸测定的高效液相色谱-串联质谱法

Determination of HVA and VMA in urine using high performance liquid chromatography-tandem mass spectrometry

目的建立尿中高香草酸和香草扁桃酸测定的高效液相色谱-串联质谱（HPLC／MS／MS）法。方法取尿样200μl置于离心管中，分别加入20μl氯唑沙宗内标，混匀后加入100μl甲醇（含1％乙酸），涡旋震荡1min，13000r／min（半径8．2cm）离心5min，提取上清后，取10μl进样，利用HPLC／MS／MS法对高香草酸和香草扁桃酸进行定性定量分析。结果该方法测定高香草酸和香草扁桃酸的线性范围分别为20．10000和20～7500ng／ml，相关系数分别为0．9956和0．9970，检出限分别为4．69和2．33ng／m1，回收率分别为85．72％。117．95％和95．82％-113．78％。结论该方法操作简便、快速、准确、灵敏度高，可满足尿中高香草酸和香草扁桃酸测定的要求。

Objective To establish a method for the determination of homovanillie acid （HVA） and vanil mandelic acid （VMA） in urine, using high performance liquid chromatography-tandem mass spectrometry （HPLC/MS/MS）detection. Methods A 200 μl volume of urine sample added with 20 μl chlorzoxazone internal standard was mixed in a centrifuge tube, then added 100μL methanol （containing 1% acetic acid）, vortex mixed and centrifuged at 13 000 rpm for 1 min. A 10 μl volume of the above supernatant solution was injected into HPLC/MS/MS system for quantitative analysis. Results With internal standard method, the calibration curves of HVA and VMA were linear in the range of 20-10 000 ng/ml and 20-7 500 ng/ml, with correlation coefficient of 0.995 6 and 0.997 0, respectively. The LOQ of HVA and VMA was 4.69 ng/ml and 2.33 ng/ml, respectively. The method recoveries were in the range of 85.72%-117.95% for HVA, and 95.82%-113.78% for VMA. Conclusion This method is simple, rapid and characterized with acceptable sensitivity and accuracy to meet the requirements for the analysis of homovanillic acid and vanilla mandelic acid.