| Objective N-butanol was a solvent for a variety of coatings and a raw material for the plasticizer dibutyl phthalate.It was also used in the manufacture of butyl acrylate,butyl acetate,and ethylene glycol butyl ether,as well as organic synthesis intermediates and biochemistry.The extractant of medicine was also used to make surfactants.As an important organic solvent,n-butanol was widely used in chemical production.Because n-butanol was poisonous,harmful and highly volatile,it would cause harm to the human body and pollution to the environment during production.N-butanol had irritant and anesthetic effects.It could cause typical symptoms of alcoholism ranging from moderate depression to anesthesia.The main symptoms were eye,nose,and throat irritation,leading to characteristic inflammation of the cornea,as well as headaches,dizziness,and drowsiness.The n-butanol that entered the human body could be metabolized to retain about 0.3% of the original shape in the urine after24 hours.A small amount of n-butanol in urine could be used as a biomarker of occupational exposure to n-butanol.Deutsche Forschungsgemeinschaft(DFG)developed the occupational exposure limits of n-butanol-the biological tolerance value(BAT)of n-butanol in urine at the end of the class was 10 mg/g creatinine.China had not yet formulated the biological limit index and detection methods for occupational exposure to n-butanol.The purpose of this study was to establish and standardize a method of Headspace Solid-Phase Microextraction-Gas Chromatography for the determination of n-butanol in urine,and to apply the established method to work.It would provide a methodological basis for China to formulate a standard test method for n-butanol in urine in the future and provide technical support for the formulation of a biological limit for occupational exposure to n-butanol in urine in China.Methods(1)The sample was pre-treated by Headspace Solid-Phase Microextraction(HS-SPME).The Polydimethylsiloxane/Divinylbenzene(PDMS/DVB)solid-phase micro-extraction head was used to extract the n-butanol in the urine in a constant temperature water bath,and then the extraction head was injected into the gas chromatograph for sample injection.The separation was performed on HP-5(30m×0.32mm×0.25μm)capillary column and detected with Flame Ionization Detector.The quantification was based on the external standard curve.(2)The single factor rotation method was used to explore experimental conditions such as the amount of salting out,extraction temperature,extraction time and desorption time.On the basis of the single factor rotation experiment,three factors that had a greater impact on the experiment were selected: the amount of salting out,the extraction temperature and the extraction time,and the orthogonal experiment was carried out by orthogonal table design.The best experimental conditions for the determination of n-butanol in urine by HS-SPME-GC were comprehensively selected.(3)The methodological performance indicators were as follows: linear range,detection limit,lower limit of quantification,accuracy,precision,stability.This method was used to detect urine samples of college student volunteers,and SD rats were selected to feed drinking water containing n-butanol,and the level of the original n-butanol retained in the urine after the metabolism of the rats was detected to verify the practicability of the method.(4)The uncertainty introduced during the experiment was evaluated to determine the key links that affect the test results.Results(1)The optimized pretreatment conditions were as follows: weighed 5.0g anhydrous sodium sulfate in the 20 ml empty bottle,added the 5.0ml urine sample,covered the top empty bottle cap with polytetrafluoroethylene pad,and fully vibrated.The sealed headspace bottle was put into a constant temperature water bath at 35 ℃and inserted into the PDMS/DVB solid phase microextraction head.After 30 min,the extraction head was extracted quickly,and the sample was injected into the gas chromatograph to desorb 4min.(2)The gas chromatograph conditions were as follows:HP-5 capillary column(30m×0.32mm×0.25μm);carrier gas(high purity nitrogen)flow rate 0.3ml/min,split ratio 20:1;The temperature of the inlet was 250°C,the temperature of the FID was 260°C,and the temperature program conditions were as follows: the initial temperature was 80°C,and the temperature rised to 100°C at a rate of 2°C/min.(3)The methodological performance indicators were as follows: the linear range is 0.04～3.0mg/L,the linear equation is y=51.32x+1.99,the correlation coefficient is r=0.9995,the detection limit is 0.04mg/L,and the lower limit of quantification is 0.13mg/L.The recovery rate of the method is 77.4%～102.8%,the intraday relative standard deviation is 3.67%～8.11%,and the intraday relative standard deviation is 4.94%～6.90%.The samples can be stored for at least 5 days in the refrigerator at 4℃.The n-butanol solution with the concentration of 1000mg/L was used to feed SD rats,and blank control group was also prepared.20 urine samples of the rats were collected for 10 days and measured on the same day.N-butanol was not detected in the control group.The original concentration of n-butanol in the urine of rats fed n-butanol ranged from 0.55 to 1.38 mg/L.(4)Using the established method to determine the concentration of n-butanol in a sample urine sample of 1.2 mg/L,the synthetic standard uncertainty was 0.034 mg/L,and the expanded uncertainty was0.068 mg/L.Conclusions(1)A new method for the determination of trace n-butanol in urine by Headspace Solid-phase Microextraction-Gas Chromatography was established.The method was simple to operate,green and environmentally friendly.It could also reduce the sample matrix effect,and no organic solvent was used in the enrichment process.(2)The detection limit of this new method was far lower than the occupational exposure biological limit for n-butanol established by DFG.It had high sensitivity,good enrichment efficiency,the precision and accuracy of the method all met the requirements of China’s 《Guidelines for the establishment of occupational health standards,Part 5:Methods for determination of chemical substances in biological materials》(GBZ/T 210.5-2008).(3)The method was applied to the determination of human and animal urine samples,which verified the feasibility of the method and had practical application value.It was suitable for the determination of n-butanol in urine samples of people who were professionally exposed to n-butanol.It can provide technical experience for the establishment of the standard for the determination of n-butanol in urine in China. |
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