| Objective Parathion is an organophosphate insecticide and acaricide that can enter the human body through the digestive tract,respiratory tract,skin,and mucous membranes.It inhibits the nervous system by inhibiting acetylcholinesterase,causing headaches,convulsions,poor vision,and vomiting,breathing difficulties,which may eventually lead to shock.Long-term contact sometimes causes obstructive pulmonary disease.The main metabolites of parathion in urine are diethyl thiophosphate,diethyl phosphate,paraoxon,parathion and p-nitrophenol.The American Association of Governmental Industrial Hygienists(ACGIH)requires that p-nitrophenol in urine be used as a monitoring indicator of parathion metabolites,and its biological exposure index(BEI)is 0.5 mg/g creatinine(0.6 mg/L),China has not yet established a detection method and biological exposure limit for p-nitrophenol in urine.Styrene can be used to produce styrene-butadiene rubber,polystyrene,etc.It mainly enters the human body through the respiratory tract.It is mainly manifested as central nervous system inhibitory symptoms.It has a stimulating effect on the respiratory tract.The main metabolites of styrene in the human body are phenylglycolic acid and phenylglyoxylic acid in urine.The ACGIH stipulates that the BEI of styrene with phenylglycolic acid plus phenylglyoxylic acid in urine are 400mg/g creatinine(480mg/L),China also stipulates that its BEI is the concentration of phenylglycolic acid plus phenylglyoxylic acid is 400mg/g creatinine(480mg/L).The detection limit of method in China is relatively high,and it is impossible to determine the concentration of phenylglycolic acid plus phenylglyoxylic acid in the urine of people with low exposure to styrene.The purpose of our studies is to establish a method for the determination of pnitrophenol in the urine of parathion metabolite by dispersive liquid-liquid microextraction(DLLME)coupled with high performance liquid chromatography(HPLC),and also to establish a method for the determination of mandelic acid and phenylglyoxylic acid in the urine of styrene by DLLME coupled with HPLC,then the methods can be used to meet the determination of urine metabolites in occupational exposure or low exposure parathion and styrene populations.Methods(1)The determination of p-nitrophenol,phenylglycolic acid and phenylglyoxylic acid in urine was carried out by the method of DLLME-HPLC.Urine sample,extractant,dispersant and salting out agent were successively added into the self-made centrifuge tube.After vortex extraction,they were centrifuged in the centrifuge,and the upper extraction liquid was taken out for HPLC detection.(2)Through the single-factor rotation experiment,the wavelength of the substance to be measured,the type and ratio of mobile phase,the type and amount of the extractant,the type and amount of the dispersant,the extraction time,the amount of salting-out,and the pH were sequentially explored in the experiment of determining p-nitrophenol in urine impact on experimental results.The wavelength of the substance to be measured,the type and ratio of mobile phase,the type and amount of the extractant,the type and amount of the dispersant,the extraction time,the amount of salting-out and hydrochloric acid were sequentially explored in the experiment of determining phenylglycolic acid and phenylglyoxylic acid in urine impact on experimental results.Then the optimal conditions were selected according to the experiment effect of experimental results.(3)Based on the method performance indicators such as linear range and detection limit,relative standard deviation,and spiked recovery,the feasibility of the method establishment,as well as the precision and accuracy of the experimental method,were explored.(4)The established detection method was applied to human urine samples and animal metabolized urine samples to verify the practicability of the method,and the uncertainty of the experimental process of detection of phenylglycolic acid and phenylglyoxylic acid is evaluated to determine the reliability of the experimental results.Results(1)The method for the determination of p-nitrophenol in urine: The liquid chromatography column was selected as C18(100×4.6mm,5?m),and the type and ratio of the mobile phase was methanol-ammonium acetate aqueous solution(20mmol/L)(50/50,v/v),and the wavelength of the UV detector was 317 nm for the conditions of HPLC.0.4mL of hydrochloric acid was put into 4.0mL urine,and they were boiled in the water bath for 1h.After cooling,placed the sample in a test tube,added 0.1mL noctanol,0.4mL methanol,0.32 g sodium chloride,adjust the pH to 6,and vortexed for 4min,then centrifuged at 2500 r / min for 5 min,the upper layer liquid was injected into HPLC for analysis and measurement.There is a good linear relationship between the p-nitrophenol concentration and the peak area response value in the concentration range of 0.0 ? 0.5mg/L.The standard curve equation is Y=867300x-112.8,the correlation coefficient is greater than 0.999,and the detection limit of the method was 0.002mg/L.The recovery rates were 80.2%?104.8%,the intraday RSDs of the method were 2.23% ? 5.12%,and the interday RSDs were 1.45% ? 4.17%.The SD rats were fed a 2.0 mg/L parathion water sample,and the p-nitrophenol content in the urine of the rats was 0.131 to 0.144 mg/L.(2)The method for the determination of phenylglycolic acid and phenylglyoxylic acid in urine: The liquid chromatography column was selected as RD-C18(4.6 × 250 mm,3 ?m),and the type and ratio of the mobile phase was methanol-0.3% acetic acid water(v/v: 55/45),and the wavelength of the UV detector was 225 nm for the conditions of HPLC.Took 4.5mL mixed standard series into a test tube,add 250?L hydrochloric acid(1mol/L),100?L n-octanol,400?L ethanol and 45.0mg sodium chloride,vortex for 4min,centrifuge at 2500 r / min for 8min,then the upper layer liquid was injected into HPLC for analysis and measurement.The linear equation of phenylglycolic acid in the concentration range of 0 ? 10.0mg/L is Y=45274x-2044.1,the correlation coefficient is greater than 0.999,and the detection limit of the method was 9.9?g/L.The recovery rates were 86.1%?101.6%,the intraday RSDs of the method were 1.07%?3.76 %,and the interday RSDs were 1.24%?3.33%.The uncertainty of 5.0 mg/L phenylglycolic acid sample was evaluated,and the expanded uncertainty was 0.298 mg/L.The linear equation of phenylglyoxylic acid in the concentration range of 0.0 ? 2.0mg/L is Y=330743x-7364.1,the correlation coefficient is greater than 0.999,and the detection limit of the method was 2.6?g/L.The recovery rates were 88.8%?100.3%,the intraday RSDs of the method were 1.02%? 3.17%,and the interday RSDs were 1.59%?2.41%.The uncertainty of 5.0 mg/L phenylglyoxylic acid was evaluated,and the expanded uncertainty was 0.0582mg/L.Conclusions(1)A DLLME-HPLC method was developed for the determination of p-nitrophenol in urine.The method has low detection limit,small amount of organic solution used,high enrichment ratio and easy operation,and it is suitable for determination of p-nitrophenol in urine of occupational exposure to parathion pesticides.(2)A DLLME-HPLC method was developed for the determination of phenylglycolic acid and phenylglyoxylic acid in urine.The method has low detection limit,high enrichment ratio and good sensitivity,and is suitable for determination of phenylglycolic acid and phenylglyoxylic acid in urine of occupational exposure to styrene.(3)Both methods meet the requirements of the GBZ/T 295-2017 standard for "General Methods for Biological Monitoring of Occupational Populations" in China.(4)The feasibility of the method is verified by the determination of actual samples and quality control samples,which has practical application value,and provides technical support for the establishment of detection methods of p-nitrophenol,phenylglyoxylic acid and phenylglycolic acid in urine in China. |
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