| Part one Determination of Toluene in Workplace Air by Capillary Gas Chromatography BackgroundAt present, one of the most widely used organic solvent in the printing, painting, automotive, shoemaking, and speaker-manufacturing industries was toluene, which was of particular concern because recent research indicating that toluene exposure can result in several toxicities, such as mucous membrane irritation, decrements in central nervous system function, and endocrine disruption. Prevention of occupational exposure to toluene was essentially to be solved for occupational health. Many countries proposed the occupational exposure limit values of toluene to protect workers'health, and correspondently established biological toluene limit values for occupational exposure to toluene and relevant determination method for regularly environmental surveillance and biomonitoring to evaluate the exposure extent of toluene. In 2007, China promulgated the standard method of the GBZ/T160.42—2007—determination of aromatic hydrocarbons in workplace. The packed column was recommended for use, with filling liquid of PEG6000 polyethylene glycol or polyethylene glycol and 2 - the reaction product of terephthalic acid nitro FFAP) and phthalic acid, DNP, respectively. The detection limit of method for toluene was 1.8μg/ml, the lowest detected concentration was 1.2 mg/m3. In 2007, considering the neurotoxicity of toluene, ACGIH proposed the threshold limit (TLV) as 75 mg/m3 (20ppm) based on the large epidemiological and toxicology research data. In 2002, China also had changed the occupational exposure limit values for toluene from 100 mg/m3 to 50mg/m3. With the lower occupational exposure limit value for toluene, and serious health hazards caused by indoor air pollutions, it was necessary to establish sensitive detection methods corresponding to low concentration toluene in the air.ObjectiveTrying to use the capillary chromatographic column 30QC3/AC–1, exploring the appropriate condition for the chromatographic analysis of toluene, a method of detecting toluene in workplace air and indoor air by the capillary gas chromatographic simultaneously was developed and validated, so as to further improve the determination method for toluene in workplace air—the occupational health standards GBZ/T160.42—2007, which could better satisfy actual work.Methods1. This article related to the collection of toluene in the air of breathing zone on working days through individual sampling followed by carbon bisulfide resolving and analyzed by capillary gas chromatograph, in which 30QC3/AC—1 capillary was applied and the column oven temperature, injector temperature and detector temperature were set at 80℃, 150℃, and 160℃, respectively, using a split/splitless mode. Following qualitative and quantitative analysis of toluene concentration in workplace air, simultaneously drawing standard curve and establishing the linear regression equation; 2. Preparing toluene standard gas 100mg/m3 (twe times to the occupational exposure limit values of workplace air) for penetrate capacity testing to gain the sampling efficiency of active carbon, which we had used;3. 18 carbon adsorbent tubes were divided into three groups, and some content toluene was added to them in six hits and placed overnight, dissolved in 1ml carbon disulfide for desorption, and the analytic efficiency of sampling was calculated by detecting the content of toluene in the active carbon tubes.4. Interference experiment was carried out for testing possible distractions such as benzene, xylene.5. Field application was developed to detect pollution levels of toluene in the air of Dongfeng automobile company coating workshop by designated area sampling and personal sampling.Results1. There was a good linearity in a range from 0.4335μg/ml to 173.28μg/ml, with correlation coefficients (r) of 0.9995 and linear regression equation for toluene was Y=115.82X-2.4978. Relative standard deviation (RSD) was 3.90%, 1.67%, 7.43%, respectively, when monitoring the concentration of toluene in spiked samples at low, medium, high concentration levels. The detection of limit (LOD) of the method was 0.4μg/ml. The average recovery rate ranged from 93.63% to 97.06%.2.The average desorption efficiency of CS2, and the sampling efficiency were above 93% and 100%, respectively.3. Interference experiment demonstrated that toluene was not affected by benzene and xylene.4.Through field application, the determination datas of occupational exposure to toluene by air sampling showed that the average concentration of toluene in the coating workshop air ranged from 4.7mg/m3 to 4.9mg/m3, and the worker's time-weighted average concentration of individual sampling ranged from 1.8mg/m3 to 3.5mg/m3.ConclusionsThe method was found to have higher sensitivity with the minimum detection concentration of toluene for 0.267 mg/m3 after practical application for determination of toluene in workplace air, far below 1.2 mg/m3, the recommended minimum detection concentration of toluene in workplace air, namely T160.2 GBZ/T in 2007– the determination of the aromatic hydrocarbons toxic substances in workplace air by solvent desorption--gas chromatography. What's more, the method satisfied requirements of determination limits of indoor air quality standards for toluene (GB/T18883—2002). The desorption efficiency was more than 90%, correspondent to requirements of GBZ/T160.42-2007. Capillary column 30QC3 / AC—1 used was selective, and the separating effect was very good, with the optimization of peak shape. Furthermore it greatly reduced the detection limit and also shortened testing time. Benzene, toluene and xylene in the air had been completely separated in short time, and the total operation analysis time was controlled within 6min. This method could meet the inspection requirements of indoor air and in workplace air at the same time. Part two Studies on Determination of Standard Methods for Toluene's Metabolites -Hippuric Acid and o-Cresol in UrineBackgroundBiological monitoring had more obvious advantages than environment monitoring,the biological monitoring of toluene was therefore required for preventive care to exposed workers'health. As a class of sanitary standard, biological exposure limit values had its unique advantages, it was the external dose of body, and was relatively more objective than occupational exposure limit values for actually reflecting accurate exposure of workers. The studies on toluene-exposed humans had identified hippuric acid as the major metabolite, and was selected as long-term biomarker of exposure to toluene. The determination of the urinary hippuric acid level was accepted as effective measures for monitoring risk groups of workers. As far as the correlation of urinary hippuric acid (HA) levels and the concentrations of toluene in the air, there were many reports at home and abroad. Most believed that there existed a good dose—response(effect) relation between them as long as the sampling time and condition were strictly controlled. What's more, the urinary hippuric acid continued to be applied in many countries such as America and Japan, which was the major metabolite of toluene, broadly used as the biomarker of occupational exposure to toluene for many years. In addition, it could still be used as a marker for environmental toluene exposure. Our research on biomarkers of exposure to toluene was just at preliminary stage, the study of domestic biological limit values of urinary hippuric acid was based on the original occupational exposure limits. We had already revised biological limit values of toluene in workplace air in 2002, and had not amended biological limit values of hippuric acid in urine yet. Due to the tendency for air environmental limit values to be lowered continuously, hippuric acid was lack of specificity when used as biomarker for occupational exposure to low level concentration of toluene, and lack of specificity to be proposed as the biological exposure index of toluene accordingly. Relevant research reports showed that o–cresol was hopeful to be alternative as biological monitoring index of toluene exposure, and had higher value than hippuric acid, which had higher background interferences. Germany and America had formulated the biological exposure limit values for o-cresol respectively, Germany recommended BAT of o-cresol in the urine for 3.0 mg/L (50ppm MAK) after long-term exposure at the end of the shift or after exposure, and for the United States, BEI of o-cresol in the urine at the end of the shift was 0.5 mg/L, but China had not established relevant determination method and biological exposure limit values for o-cresol in urine.ObjectiveDue to the strong separation ability of high performance liquid chromatographic, HPLC had been popularly used in many countries. At present, foreign organization like NIOSH recommended HPLC/UV method for determination hippuric acid in urine. In early 1996 China had recommended the sanitary standard WS/T53-1996,the HPLC method of determination hippuric acid and methyl hippuric acid in the urine for occupational exposure to toluene and xylene. In accordance with the relevant provisions, health standards should be revised every five years, but the original detection method was still in use in China. There also had reported that choosing provided conditions could not reach completely separation requirements according to the standard, furthermore the operating procedures were complicated and overloaded, and the dry procedures were time-consuming. Currently research institutions abroad mainly recommended SPME - HS - GC/MS to monitor o-cresol in urine, however, China had still no related determination methods and standards. This study intended to establish the method of determining hippuric acid and o-cresol in urine to provide the experimental HPLC/UV method for the biological limit values of urinary hippuric acid and o-cresol, according to physical and chemical properties of hippuric acid and o-cresol,referring to the related testing methods and technical standards, for regularly biomonitoring laborers exposed to toluene so as to assess the exposure extent, which would fill the blank of domestic standards, and provide reasonable and effective detection means for further developing biological exposure limit values of the occupational exposure to toluene of our own countries.MethodsStudents of no occupational exposure to toluene and non-smokers were chosen to be ground substances of urinary samples, preparing the appropriate concentration of the standard solutions with hippuric acid and o–cresol for analytical purpose. With the appropriate sample pretreatment methods and ODS column being selected, column temperature was setting at 35 degrees, while selected 220nm UV detection wavelength and mixed mobile phase to identify the HA and o-cresol in urinary samples, and the contents of HA and o-cresol in urine were quantitatively analyzed. Constituting different concentration's of HA and o-cresol criterion series and manufacturing criterion curve, we developed methods to test the appropriate concentration linearity range of HA and o-cresol. High, medium and low concentration of three kinds of HA and o-cresol in urine being confected and the recovery rates being tested, the average recovery rates of this method were calculated. Testing the HA and o-cresol in parallel urinary samples at different time, the degrees of precision of this method were evaluated. Meanwhile the urinary samples were placed in different temperature and at different time, and its stability was estimated, and the method was applied in field application so as to assess it's validity.Results1. The HPLC/UV analytical method of determination urinary HA showed a good linearity in the range from 1.0μg/ml to 100.0μg/ml, and the established regression equations for urinary HA concentration was Y=27593X-16709, with the value of the correlation coefficient (r=0.9999) and the limit of detection was 0.5μg/ml, while the average recovery rate was 96.29%, and the average intra-day and inter-day precision (RSD) for the analysis were 2.25% and 3.96%, respectively.2.The HPLC/UV analytical method of determination urinary o-cresol showed a good linearity in the range from 2.0μg/ml to 200.0μg/ml and the limit of detection was 0.8μg/ml, while the average recovery rate was 95.02%, and the average intra-day and inter-day precision (RSD) for the analysis were 5.45% and 6.09%, respectively.3. The biological monitoring for occupational exposure to toluene by field application showed that the mean concentration of HA in the urine of 20 workers was expressed for 0.65μg/ml.4. Stability test confirmed that the urine placed in the 4℃refrigerator could be stable at least for 7 days, and the variation coefficients for hippuric acid and o-cresol in urine were 4.48% and 8.57%, respectively.Conclusions1. Chromatographic conditions for the HPLC method for determination urinary HA and o–cresol: A C18 column was used for separating. The column temperature was set at 35 degrees, with methanol and water to yield 40:60 (V/V) mixing ratio as mobile phase, adding phosphoric acid to adjust pH to 2.78, setting velocity for 1ml/min, the best detection UV wavelength for 220nm.2. The HPLC/UV method could simultaneously detect the contents of hippuric acid and o-cresol in urine. The method was well in agreement with the correspondent requirements of determination of biological materials in occupational health standard references of GBZT210.5—2008. This also could provide the experimental method for the biological limit values of urinary hippuric acid and o-cresol—occupational biomarkers of exposure to toluene.3. According to China's current environmental air exposure limit values to toluene of 50mg/m3 (PC-TWA) and in combination with PBPK model, the biological exposure limit values for urinary HA and o-cresol in China were proposed as 400mg/L and 1mg/L, respectively.
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