尿中马尿酸、甲基马尿酸测定的乙腈萃取-高效液相色谱法

Determination of hippuric acid and methylhippuric acid in urine by high performance liquid chromatography after extracted with acetonitrile

目的 建立乙腈萃取前处理方法和高效液相色谱（HPLC）法测定尿中马尿酸（HA）、甲基马尿酸（MHA）.方法 尿中HA、MHA在酸性条件下用乙腈萃取,高效液相-二极管阵列检测器（HPLC-DAD）检测,色谱条件：C18柱（150 mm× 4.6 mm,5μm）,以甲醇：0.20％乙酸水溶液（含6.5 mmol/L磷酸二氢钾）（25：75,V/V）为流动相,流速为1 ml/min,波长为254 nm.结果 HA、2-甲基马尿酸（2-MHA）、3-甲基马尿酸（3-MHA）[4-甲基马尿酸（4-MHA）]分别在9.91～2 974.20 μg/ml、1.91～573.60μg/ml、2.00～598.65μg/ml线性关系良好,相关系数分别为0.999 98、0.999 84、0.999 85;HA、2-MHA、3-MHA （4-MHA）回收率分别为96.38％～98.01％、83.17％～94.05％、103.22％～ 104.45％,批内精密度相对标准偏差（RSD）分别为0.50％～1.20％、0.51％～ 1.59％、0.49％～0.95％,批间精密度RSD分别为1.70％～3.20％、1.30％～2.67％、0.86％～2.74％,检出限分别为0.18、0.46、0.12 μg/ml,最低检出浓度分别为0.36、0.92、0.24 μg/ml（以尿样1 ml计）;样品在4℃冰箱中可保存15d.结论 乙腈萃取尿中HA、MHA前处理方法简单易行,符合GBZ/T 210.5-2008《职业卫生标准制定指南第5部分：生物材料中化学物质测定方法》的相关要求,可用于职业接触甲苯、二甲苯人群尿中HA、MHA的测定.

Objective To establish pretreatment conditions of hippuric acid （HA） and methyl-hippuric acid （MHA） in urine and HPLC conditions.Methods HA and MHA in urine were extracted with acetonitrile under acid condition and determinated by HPLC-DAD.The operating conditions by HPLC were C18 column （150 mm× 4.6 mm,5 μm）,methanol-0.2% acetic acid （contained 6.5 mmol/L potassium dihydrogen phosphate） （25：75,V/V） as mobile phase,1 ml/min as flow rate and wavelength was at 254 nm.Results The standard curves for HA,2-MHA and 3-MHA （4-MHA） showed good linearity between 9.91～2 974.20 μg/ml （r=0.999 98）,1.91～ 573.60 μg/ml （r=0.999 84） and 2.00～598.65 μg/ml （r=0.999 85）,respectively.The mean recoveries were 96.38%～98.01%,83.17%～94.05%,103.22%～104.45%,respectively.The within-run precision were 0.50%～ 1.20%,0.51%～1.59%,0.49%～0.95%,respectively,and the between-run precision were 1.70%～3.20%,1.30%～ 2.67%,0.86%～2.74%,respectively.The detection limit of HA,2-MHA and 3-MHA （4-MHA） were 0.18 μg/ml,0.46 μg/ml and 0.12 μg/ml,and the low determination concentrations of the method were 0.36 μg/ml,0.92 μg/ml and 0.24 μg/ml （1 ml urine）.The urine can be kept 15 days at 4 ℃ refrigerator without significantly loss.Conclusion This method with simply pretreatment conforms to the relevant requirements of guide for establishing occupational health standards-part 5：determination methods of chemicals in biological materials.It can be used to detect HA and MHA in urine for occupational population exposure to toluene and xylene.