Determination Of Naphthol And O-cresol In Urine By Microextraction Coupled With Liquid Chromatography

Objective Naphthalene and toluene are widely used in industry,both of them can cause damage to the human body after occupational exposure.After metabolism,naphthalene is mainly excreted in the urine with 1-naphthol（1-NAP）and 2-naphthol（2-NAP）and toluene can be metabolized hippuric acid and o-cresol by urine.The American Association of Governmental Industrial Hygienists（ACGIH）stipulate urinary naphthols（1-NAP and 2-NAP,NAPs:Non-quantitative）and o-cresol（0.54mg/L）are biological test indicators for occupational exposure to naphthalene and toluene respectively.However,The standards to detect NAPs and o-cresol in urine has not yet developed in our country.In our studies,NAPs in urine was detected by magnetic solid phase microextraction coupled with high performance liquid chromatography and o-cresol in urine was detected by dispersive liquid-liquid microextraction coupled with high performance liquid chromatography,which provides a method for the detection of NAPs and o-cresol in occupational exposure,and also provides the technical support for the establishment of the standard detection method and basis for formulating BEI of NAPs and o-cresol in urine.Methods NAPs and o-cresol in urine were detected by high performance liquid chromatography.The pretreatment method of naphthol was magnetic solid phase microextraction,and the o-cresol pretreatment method was dispersion liquid-liquid microextraction.1)Pretreatment method for naphthol in urine:Fe₃O₄ was synthesized by hydrothermal method,and Fe3O4 and graphitized carbon black were assembled into M-Fe3O4@C by physical milling method.The external morphology,main functional bond and magnetic strength of M-Fe₃O₄@C are characterized by characterization to determine the properties of the synthesized M-Fe3O4@C.The urine and a certain amount of magnetic adsorption material activated with acetone were added in the glass bottle.After fully adsorbing naphthol in the vortex mixer,the M-Fe₃O₄@C was separated from the solution by a magnet,and the NAPs were separated by eluent.The amount of M-Fe₃O₄@C eluent volume,eluent dosage,adsorption time,elution time,pH,icon were explored to optimization experiment condition.Under the optimal pretreatment conditions,samples were collected for liquid chromatography analysis and methodological indicators were explored to ensure the reliability of the experiment.2)Pretreatment method of o-cresol in urine:The urine sample,extractant and dispersant were added in the homemade disposable plastic dropper.O-cresol in the urine was extracted into the extractant under vortex conditions and extractant was collected by centrifugation.The amount of extractant and dispersant,the type of dispersant and the extraction time were explored to optimization experiment condition.Under the optimal pretreatment conditions,the accuracy,precision,and linearity of the method were explored to ensure that the experimental method was feasible.3)The established detection methods were applied to the actual urine sample detection of occupational exposure to naphthalene and toluene,and the accuracy,precision and practicability of the method are further verified.Results 1)The characterization results show that the synthesized Fe₃O₄ is spherical,Fe3O4 is closely connected with graphitized carbon black.The signal of C=C and Fe-O were detected,and the magnetic strength satisfies the separation of M-Fe3O4@C from solution.20mg M-Fe3O4@C was added in 10 mL urine.After vortex extraction for 6 min,100μL of acetone was vortexed for 6 min,the acetone was collected for liquid chromatography to detect.The linearity range of the proposed method was 20–5000μg/L with correlation coefficients（r）of 0.999.The limits of detection for the target 1-NAP and 2-NAP were 0.9μg/L and 1.1μg/L,and an acceptable reproducibility was achieved by evaluating the intra-and inter-day precisions with relative standard deviations less than12.9%and 10%,respectively.2)0.1mL N-octanol,0.6 mL ethanol and 3.5mL urine were added in a homemade centrifuged device,which was vortexed for 8 min,centrifuged at 3000 r/min for 5 min.The extraction was collected for liquid chromatography to detect.The correlation coefficient of the method was greater than 0.999 within the range of 1.0 to 50.0μg/L.The detection limit was 0.075μg/L,the recovery rates were 81.9%¹06.7%,the intra-and inter-day precisions were 5.57%⁷.47%and 5.33%⁸.18%,respectively.No o-cresol was detected in urine samples of college students.Conclusions 1)The novel methods of magnetic solid-phase extraction based on magnetic graphitized carbon black for the determination of 1-naphthol and 2-naphthol in urine was established.The method has the characteristics of low cost,high enrichment efficiency,fast analysis speed,and is suitable for the determination of NAPs in urine with the occupational people exposed to naphthalene.2)The method of dispersion liquid-liquid micro-extraction coupled with high performance liquid chromatography to detect urinary o-cresol was established.This method has the characteristics of good sensitivity,high precision,less use of organic solvents,and environmental protection.It is suitable for the detection of urinary o-cresol with the occupational people exposed to toluene.3)Indicators of the method meet China’s requirements for chemicals assays for biological materials.4)Through the detection of the actual urine and quality control,both of the methods were verified to be feasible,practical and valuable.The both can provide the standard measurement method for urinary naphthol and o-cresol respectively and provide technical support for setting determination standards and occupational biological exposure limits of the urine.