高效液相色谱-荧光检测法测定尿中1-羟基芘

Determination of 1-Hydroxypyrene in Urine by High Performance Liquid Chromatography with Fluorescence Detection

[目的]通过C18固相萃取小柱洗脱液浓缩与不浓缩试验对比,改进尿样中1-羟基芘的高效液相色谱-荧光检测器测定方法。[方法]取2组相同尿样,经酶避光水解后,通过C18固相萃取小柱进行富集,甲醇洗脱。一组对洗脱液进行氮吹浓缩后定容至1.5 ml,另一组直接洗脱定容至1.5 ml,然后,使用高效液相色谱-荧光检测器检测。以V乙腈∶V水=75∶25为流动相,等度洗脱,流速为1 ml/min,荧光检测器λex=275 nm,λem=430 nm。[结果]浓缩后测定的1-羟基芘的值为不浓缩测定值的72.9%～80.6%。利用不浓缩的酶水解高效液相色谱-荧光检测器法测定尿样中的1-羟基芘,检出限为0.02 ng/ml,在给定浓度范围内,1-羟基芘的峰面积与浓度有良好的线性关系,线性回归方程为y=2.404 1x-0.083(r=0.999 9),加标回收率为94.8%～108.7%,平均相对标准偏差为2.7%。[结论]不浓缩的酶水解高效液相色谱-荧光检测器法可用于测定尿样中的1-羟基芘,且该方法具有操作简单,灵敏度、精密度高,重现性、回收率好的特点。

[Objective] The aim was to improve the measuring method of 1-hydroxypyrene in urine by high performance liquid chromatography with fluorescence detection by comparing concentrated and non-concentrated eluate of C18 solid phase extraction.[Method] Take two same urine samples,hydrolyzed by enzyme first,then enriched by C18 solid phase extraction,elution by methanol,one nitrogen dry to 1.5 ml,another direct volume to 1.5 ml,then were determined with HPLC/FLD.With Vacetonitrile ∶ Vwater=75∶ 25 as mobile phase and 1 ml/min of flow rate,the excitation（λex） and emission wavelengths（λem） were 275 and 430 nm,respectively.[Result] The determined values of urinary 1-hydroxypyrene which after concentrating were 72.9%-80.6% of which non-concentrating.When use the method of non-condensed enzymatic hydrolysis high performance liquid chromatography with fluorescence detection,the limit of detection was 0.02 ng/ml,the calibration curve was linear at a given concentration range,the correlation coefficient of linear regression equation was y=2.404 1x-0.083（r=0.999 9）,the recoveries were from 94.8% to 108.7%,and the average RSD was 2.7%.[Conclusion] The method of non-condensed enzymatic hydrolysis high performance liquid chromatography with fluorescence detection can be used to determine the urine of 1-hydroyxpyrene,and it is simple,have high sensitivity and precision,good recoveries and reproducibility.