分散液-液微萃取-高效液相色谱法测定尿中苯巯基尿酸

Determination of S-phenylmercapturic acid in urine by dispersive liquid-liquid microextraction-high performance liquid chromatography

目的建立分散液-液微萃取-高效液相色谱测定尿液中苯巯基尿酸的方法。方法采用5 ml尿样,用盐酸调节pH值为1,快速加入200μl三氯甲烷(萃取剂)和800μl异丙醇(分散剂)的混合溶剂,混匀后室温静置5 min,再以12 000 r/min离心5 min,分离出下层有机相,50℃经氮气吹干,残渣用甲醇溶解,经C_(18)柱(C_(18)柱:250 mm×4.6 mm,4μm)分离,采用高效液相色谱-紫外检测器检测,以保留时间定性,峰面积定量。结果本法苯巯基尿酸在0.050 mg/L^1.0 mg/L浓度时呈线性,相关系数为0.999 3,以3倍噪音值估算方法检出限为0.003 mg/L。在0.050 mg/L^1.0 mg/L时添加低、中、高3个浓度水平,样品加标回收率为96.9%~104.9%;批内精密度为2.50%~5.74%(n=6),批间精密度为3.52%~7.86%(n=6)。结论本法简单实用,有机试剂用量少,灵敏度高,可以满足尿样中苯巯基尿酸检测的要求。

Objective To establish a method for the determination of S-phenylmercapturic acid（ S-PMA） in urine by dispersive liquid-liquid microextraction-high performance liquid chromatography（ DLLME-HPLC）. Methods Hcl was added into 5. 0 ml urine samples to adjust pH to 1. 0. Then 200 μl of chloroform（ extraction solvents） and 800 μl of isopropanol（ disperser solvents） were injected into the samples,and set at room temperature for 5 min. After centrifuged at 12 000 r/min for 5 min,the extract redissolved in methanol after being dried under nitrogen blowing at 50 ℃. Thereafter the S-phenylmercapturic acid was separated by EC-C_（18） column（ 150 mm × 4. 6 mm,4 μm） and detected by high-performance liquid chromatography-UV detector. The retention time was used for qualitative analysis and the peak area was for quantification. Results The correlation coefficient was 0. 999 3 and good linearity was obtained when the concentration of S-phenylmercapturic acid in urine was within 0. 050 mg/L-1. 0 mg/L（ S/N = 3）. The average recovery of S-phenylmercapturic acid was within 96. 9%-104. 9% at three spiked levels（ high,medium and low）. The intra-day and inter-day precisions（ RSD） for the analysis were within 2. 50%-5. 74%（ n = 6） and 3. 52%-7. 86%（ n = 6）,respectively. Conclusion The method was proved simple,practical,sensitive,with less organic reagent consumption,which could meet the determination request of S-phenylmercapturic acid in urine.