A Standard Method And Application For Determination Of Methyl-hippuric Acid In Urine

ObjectiveXylene is widely used to product chemical raw materials, solvents, thinners, etal. Whenprolonged exposure, xylene can be absorbed through the respiratory tract, digestive tract and skin,bringing serious damage to health. So it is of great significance to carry on biological monitoringfor it. The study showed that the urine methyl-hippuric acid was the special metabolite of xyleneinto the human body, and the correlation between the environmental exposure concentration andthe concentration in urine of methyl-hippuric acid was strongest. Therefore it can be used asspecific biomarkers of occupational exposure to xylene. The BEIs recommended by the ACGIHand BATs recommended by the DFG were1.5g/g creatine,2.0g/L respectively formethyl-hippuric acid in urine. As the biological exposure limits had not yet developed in China,,so Medical College in Wuhan University of Science and Technology and Wuhan MunicipalOccupational Disease Prevention Hospital jointly apply to the Professional Committee ofoccupational health standards belong to Ministry of Health in China for the subject of thebiological exposure limit values for MHA and its detection method, what,s more item numberwas20110206.Methyl-hippuric acid isomers can not be separated with national method. Thepurpose of this study was to set a standard method for determination of methyl-hippuric acid inurine and provide supports in the technology and method for the next research of bio-exposurelimits of urinary methyl-hippuric acid.MethodThe methyl-hippuric acid in the urine samples was extracted by liquid-liquid extractionwith ethyl acetate, flowing centrifugal process and water bath to dryness organic phase,anddetected by HPLC.A C18column was used to determine the methyl-hippuric acid. The mobilephase was: Methanol-0.1%acetic acid (33:67).The detector was performed at λ=230nm, at1.0ml/min. The qualitative and quantitative analyses were carried out by chromatography basedon the retention time and peak area respectively. According to demands of Guide for EstablishingOccupational Health Standards, the best determination conditions of HPLC were chosen and themethod was tested for LOD, accuracy, precision, and sample storage stability, anti-interferenceand field application capacity and performance. This method was applied for the57workersexposed to xylene and231residents.ResultsThe separation of4-MHA and3-MHA was best when the mobile phase was Methanol-0.1% acetic acid (33:67) and the detector was performed at λ=230nm, at1.0ml/min. Calibration curvesof three methyl-hippuric acids were liner in the range of25～1000μg/ml, with a correlationcoefficient more than0.999.The average recovery was in the range of91.4～101.4%and theaverage RSD was in the range of2.0%～9.6%. The Minimum detection concentration of2-MHA,3-MHA,4-MHA was respectively0.47mg/L,0.46mg/L,0.41mg/L. The resolution of4-methyl-hippuric acid and3-methyl-hippuric acid was up to1.44. It has been confirmed that thedetermination of methyl-hippuric acid by this method was not interfered by the metabolites ofcoexist compounds such as benzene, toluene and styrene in the workplace together with xylene.Stability test found that urine samples were stable for at least2weeks when stored at4℃. Theapplied result showed that the concentration of methyl-hippuric acid in urine had goodcorrelation with workplace air xylene concentration(R~2=0.6292). All of occupationalexposed-workers were detected with MHA.The detect rate of231people in two chemicalpolluted areas and one control area was12.17%、5.97%、4%respectively.ConclusionThe HPLC method proposed in this study was able to make3-methyl-hippuric acid and4-methyl-hippuric acid baseline separated, and the methodology indicators are in line with therelevant requirements of the method of determination of chemicals in the occupational healthstandard-setting Guide Part V with high sensitivity, good precision, high recovery, good stabilityand less interference. These results suggest that the method could be used as a standard methodfor biological monitoring of xylene.The applied result were corresponding to the actual situation.So this method could be used to determine methyl-hippuric acids in urine of occupationalexposed-workers and other people, both with providing supports in the technology and methodfor the next research of bio-exposure limits of urinary methyl-hippuric acid.