High Performance Liquid Chromatography And Application Research Of S-phenylmercapturic Acid In Urine For Occupational Exposure To Benzene

BackgroundBenzene is widely used industrial chemical and organic menstruum. Exposure to low level benzene chronically may damage hematopoietic system and even result in leucocythemia. Biological monitoring can roundly figure out the fact exposure value to people. For effectively distinguishing and evaluating benzene exposure level for different job, America, Germany, Singapore and other developed country have already constitute biology limit criterion basing on native entironment limit criterion for benzene successively. Benzene's biologic urinary metabolites include phenol, t,t-MA and S-phenylmercapturic acid (S-PMA) and so on. S-PMA is one of specific urinary metabolites and is recognized for benzene's biomarker. For protecting people exposuring to benzene, benzene's PC-TWA is emended to 6mg/m3 at present in my country. According as occupational exposure limit, developing test method of biological exposure value and biological exposure limit criterion according with the situation of our country has been present to the order of the day.ObjectiveAccording as the basal analysis principle of HPLC and physical chemistry character of SPMA, the urine sample pretreatment method of using HPLC to testing SPMA in urine and idiographic choice of chromatogram condition for chromatogram column type, column temperature, mobile phase, detection wavelengh and so on are discussed, which provides methodology basis for developing benzene's profession exposure biologic monitor. Basing on methodology of using HPLC to testing S-PMA in urine, the S-PMA biological exposure limit and application condition for profession benzene exposure biologic monitor of our country are put forward according to analysing benzene's entironment monitor data and the testing S-PMA concentration in urine and combining the environment exposure limit(PC-TWA=6mg/m3)to benzene of our country at present.MethodChoosing the students of no occupational exposure to benzene and non-smokers for research object of urine sample, adding SPMA liquor at corresponding concentration into urine sample according to testing analysis object and using appropriate pretreatment method, the S-PMA in samples is qualitatively analysed at the chromatogram condition of Shim-pack VP-ODS chromatogram column, 35℃column temperature, 205nm UV detection wavelengh and mixed mobile phase and so on. Confecting different concentration's S-PMA criterion series and facturing criterion curve, we develop method to test the concentration linearity range of S-PMA. Confecting three kinds of concentration's S-PMA urine sample and recovery test, we calculate the average recovery rate of this method. Testing the S-PMA in parallel urine at different time, we calculate the exactitude degree of this method. Putting the urine sample in different temperature environment and testing the S-PMA in urine sample at 3 days,7 days and 14 days before and after the experiment, we calculate its stability.32 occupational benzene-exposed workers are chosed for research object. Research object's entironment exposure level to benzene is samplinged. The urine samples were collected at the end of the work shift. S-PMA concentration in urine samples is tested by HPLC. And entironment benzene concentration and S-PMA concentration in urine samples are regression analysed. Then combining the entironment exposure limit(6mg/m3)to benzene of our country at present S-PMA biological exposure limit value according with the situation of our country can be calculated.ResultSPMA can be reserved about 30.5min. The linearity range of calibration curve is significantly correlated at the S-PMA concentration of 40～2000μg/L. The detection limit of the method was 40μg/L. The average recovery rate ranges from 92.64% to 103.66%. The intra-day and inter-day precisions (RSD) for the analysis are 2.4%～14.5% and 3.8%～9.2%. Relative standard deviation for S-PMA in urine samples are 6.60% and 2.40% at 4℃and -20℃before the samples are put in refrigeratory and 3 days ,7days and 14 days after the samples are put in refrigeratory.It is shown that benzene average concentration in air is 41.16.±34.74 mg/m3 from the samples test data for 32 occupational benzene-exposed workers. The S-PMA concentration in urine for 32 occupational benzene-exposed workers is 464.04.±400.79μg/g Cr. The linear regression equation for S-PMA in urine at the end of the work shift and body benzene-exposed level is Y(μg/g Cr)=10.134 X(mg/m3)+47.104,(n=32,r=0.7712,P<0.01). S-PMA biological exposure limit value is 107.89μg/g Cr for the entironment exposure limit(6mg/m3)to benzene.Conclusion1. Chromatogram condition for test S-PMA in urine by HPLC: An C18 column is used for separating. Acetonitrile is used for mobile phase A. Acetonitrile- methanol- 3,ethylamine is used for mobile phase B. Velocity of flow is at 2ml/min. Column temperature maintainde is at 35℃. UV detection wavelengh is at 205 nm.2. Chromatogram behavior in urine, lowest detection limit, test linear range, exactitude degree and preciseness degree by HPLC can reach the basic demand for testing occupational benzene-exposed biologic limit value.3. It is suggested that the urine for HPLC test should be reserved in about 14 days in the condition of 4℃and -20℃.4. The SPMA concentration in urine for occupational benzene-exposed public tested by HPLC and entironment exposure concentration in air are greatly correlative. The S-PMA concentration in urine can be used as a biomarker to evaluation the occupational exposure to benzene. 5. It is suggested that S-PMA biological limit criterion should be 107.89μg/g Cr according as the entironment exposure limit(6mg/m3)to benzene in our country at present.