| Objective As an important class of chemical feedstocks and organic solvents,volatile benzenes series are widely used in the fields of paints,pesticides,medicine,textile,as well as synthetic rubber,and are also used to improve the octane value of gasoline.Common volatile benzenes series comprise benzene,toluene,ethylbenzene,o-,m-,p-xylene,as well as styrene,among others,of which benzene is identified as a strong carcinogen by the International Agency for Research on Cancer.Volatile benzenes series in the workplace can be absorbed through the respiratory tract and skin,and after entering the body,a part is mainly distributed in fat rich tissues and organs,such as bone,brain,and other sites;the other is excreted in the liver and kidney by oxidative metabolism,and five volatile benzenes series are excreted in the urine in an unmetabolized form in approximately 1% of the inhaled dose.The American Conference of Governmental Industrial Hygienists(ACGIH)lists urinary unmetabolized crude toluene and styrene as biological monitoring indicators of occupational exposure and has established corresponding biological exposure limits:urinary toluene 30 μg/L and urinary styrene 40 μg/L,the biological limits and relevant detection methods for occupational exposure to unmetabolized prototypes in urine of five volatile benzenes series have not been established in China.This study aimed to construct a new method of headspace solid-phase microextraction gas chromatography tandem mass spectrometry for simultaneous determination of five volatile benzenes series in urine,and the established new method can be applied to practical work,which can provide technical support and methodological basis for biomonitoring of occupational exposure to five volatile benzenes series in China and has practical significance for protecting the health of occupationally exposed people.Method(1)Five volatile benzenes series in urine were extracted and adsorbed by headspace solid-phase microextraction on an HP-5MS column(30m×0.25mm×0.25μm)separation,mass spectrometer detection,external standard method quantification,the least squares method was used to fit the working curve,and the content of the target was calculated.(2)The method for optimization of chromatographic conditions contained two parts: qualitative and quantitative ions of five volatile benzenes series were screened by total ion current chromatogram of mixed standard solutions combined with the National Institute of Standards and Technology(NIST)spectral library;literature data combined with laboratory conditions,column temperature,temperature program as well as injection port temperature were selected.(3)The extraction fiber,extraction temperature,extraction time,salting out agent type and dosage of the headspace solid-phase microextraction technique were explored and optimized by single factor rotation method to determine the optimal test conditions for each studied factor.(4)On the basis of the results of single factor rotation experiment,the factors and levels that have great influence on the extraction efficiency were selected.Orthogonal table design was used to design the experiment,and polarity analysis was used to analyze the results of orthogonal design experiment to determine the optimal combination of headspace solid-phase microextraction technology.(5)The linear range,detection limit,lower limit of quantification,precision,accuracy,sample stability and other performance indicators of the method were explored,and the feasibility of the method was verified by urine samples from occupationally exposed people.(6)Identify the sources of uncertainty in the test process,quantify the relative standard uncertainty,judge the key links to introduce uncertainty,and control the quality of the sample detection process.Result(1)The determined chromatographic conditions are as follows: the temperature of the sample inlet is 250 ℃;The shunt ratio is 10: 1;Chromatographic Column HP-5MS(30m×0.25mm×0.25μm);Warming procedure: initial temperature40 ℃,keep for 2 min,then rise to 120 ℃ at 8 C/min for 3 min;Carrier gas(helium)flow rate: 1ml/min;The ion source temperature is 260 ℃.Quantitative analysis of samples was performed under the selected ion monitoring mode.(2)Optimized headspace solid-phase microextraction conditions: univariate rotation and orthogonal design results are consistent.The optimum experimental condition is to take 5.00 ml urine sample in a 20 ml top empty bottle,add 2.00 g sodium chloride,tighten the top empty cap of polytetrafluorethylene,seal the sealing membrane,and shake it oscillatically.Place the top empty bottle in a 60 ℃thermostatic water bath pan and insert the bottle with the extraction handle of the 75μm CAR/PDMS solid phase microextraction head exposes the extraction head and quickly inserts into the sample inlet of the gas chromatography mass spectrometer after 30 min of balance.(3)Methodological Performance Indicators: five volatile benzenes series in 0.10μg/L-80.00 μg/L was well linear over the concentration range.The correlation coefficient r was greater than 0.9950 and the detection limit was 0.025 μg/L to 0.042μg/L,Quantitative Lower Limit 0.083 μg/L to 0.141 μg/L;The intraday relative standard deviations of the method were 1.45%～7.81%,and the intraday relative standard deviations were 1.16%～4.09%.The average recoveries ranged from89.18%～102.30%.Samples were refrigerated at 4 ℃ and added one percentage concentrated hydrochloric acid for at least 7 days.(4)Following the established method,60 gas station workers’ urine samples were collected,and five volatile benzenes series were all detected in the urine of the occupational population,with concentrations of benzene ranging from 0.15 μg/L to1.24 μg/L,with concentrations of toluene ranging from 0.25 μg/L to 1.19 μg/L,and the concentrations of ethylbenzene were 0.24 μg/L to 0.60 μg/L,the concentrations of xylene were 0.50 μg/L to 1.06 μg/L,the concentrations of styrene were 0.20 μg/L to 0.34 μg/L;none of the five volatile benzenes series was detected in the urine samples of university students.(5)Measurement uncertainty analysis of the established new method showed that the influence on the method uncertainty sources ranged from large to small in the order of duplicate determination,preparation of standard solutions,fitting to the working curve,sample pretreatment,and weighing.Conclusion(1)A new method of headspace solid-phase microextraction gas chromatography-mass spectrometry for the simultaneous determination of five volatile benzenes series in urine was constructed with low method detection limit,high sensitivity,wide linear range,green environment protection and easy operation.(2)All performance measures of the new method met the requirements related to the general principles of biological monitoring methods for occupational populations(GBZ/T 295-2017),in which the detection limit of the new method was below the0.1-fold biological exposure limit established by the ACGIH.(3)The new method established was applied to the detection of occupational population,which verified the feasibility of the method and was suitable for the simultaneous determination of five volatile benzenes series in urine of occupationally exposed population,which may provide a reference basis and technical support for developing a standard determination method of five volatile benzenes series in urine. |
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