Introduction: Urinary Hippuric acid (HA) and Methylhippuric acid (MHA) are considered reliable biomarkers for monitoring exposure to toluene and xylenes. A simple and robust HPLC method for simultaneous determination of HA and MHA in urine samples was developed and validated as per Internation Conference on Harmonization (ICH) Q2 guidelines.


Methods: Mobile phase consisting of two solvents A & B, solvent A was 0.1% TFA in water, and solvent B was 0.1% TFA in acetonitrile.  Separation was performed in gradient elution mode and the time program was as follows - Time (min)/%B: 0/20, 4/20, 11/80, 11.1/90, 16/90, 16.1/20, 25/20. The detection wavelength was 205 nm.


Results: The limits of detection (LOD) and quantification (LOQ) of HA in urine were 0.3 and 1.0 mg/L, and for MHA were 0.25 mg/L and 1.0 mg/L respectively and the recovery for HA and MHA were 95% and 98.9% respectively. 


Conclusion: The developed method was successfully applied for the analysis of urine samples

Anupa Yadav

Pankaj Maurya

Pawan Kumar Maurya

International Journal of Occupational Safety and Health

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  Int. J. Occup. Safety Health,Volume 13, No 3 (2023), 331-337 https://www.nepjol.info/index.php/IJOSH 331   Original Article HPLC analysis of biomarkers of Toluene and Xylene in human urine samples Maurya PK1, Yadav A1, Maurya P2 1ICMR-Centre for Ageing & Mental Health, Block DP-1, Sector V, Salt Lake, Kolkata-700091, India 2Department of Environmental Sciences, Institute of Biosciences and Biotechnology, Chhatrapati Sahu Ji Maharaj University, Kanpur-208024, India ABSTRACT  Introduction: Urinary Hippuric acid (HA) and Methylhippuric acid (MHA) are considered reliable biomarkers for monitoring exposure to toluene and xylenes. A simple and robust HPLC method for simultaneous determination of HA and MHA in urine samples was developed and validated as per Internation Conference on Harmonization (ICH) Q2 guidelines. Methods: Mobile phase consisting of two solvents A & B, solvent A was 0.1% TFA in water, and solvent B was 0.1% TFA in acetonitrile.  Separation was performed in gradient elution mode and the time program was as follows - Time (min)/%B: 0/20, 4/20, 11/80, 11.1/90, 16/90, 16.1/20, 25/20. The detection wavelength was 205 nm. Results: The limits of detection (LOD) and quantification (LOQ) of HA in urine were 0.3 and 1.0 mg/L, and for MHA were 0.25 mg/L and 1.0 mg/L respectively and the recovery for HA and MHA were 95% and 98.9% respectively.   Conclusion: The developed method was successfully applied for the analysis of urine samples. Keywords: Biomarker, Hippuric acid, HPLC, Methylhippuric acid, Method validation  Introduction Smoking and combustion of fossil fuels are well-established sources of volatile organic compounds (VOCs) containing benzene and substituted benzenes such as toluene and xylenes (have three isomers (m-/p-/o-xylenes). Agency for Toxic Substances and Disease Registry (ATSDR) of the U.S. Department of Health and Human Services published toxicological profiles of toluene and xylenes. VOC exposure is associated with toxic effects on human health and may lead to various diseases but respiratory problems in the case of xylenes and central nervous system (CNS) problems in the case of toluene are most prevalent.1-4 Xylenes exposure may happen due to skin permeation as well as inhalation, whereas toluene exposure happens mainly by inhalation. Post-exposure xylene and toluene are absorbed into the blood. In humans major metabolic pathway of toluene proceeds primarily by oxidation to benzyl alcohol catalyzed by Cytochrome P450 enzymes followed by benzoic acid which further converts to hippuric acid (HA) whereas, in the case of xylenes oxidation to methyl benzoic acids followed by methyl hippuric acids (MHA) in liver. HA and MHA are considered primary exposure   Corresponding author:   Pawan Kumar Maurya ICMR-Centre for Ageing & Mental Health, Block DP-1, Sector V, Salt Lake, Kolkata-700091, India Telephone: +91-33-23674390 Email:maurya.pk@icmr.gov.in, maurya_pawan@yahoo.com ORCID ID:https://orcid.org/0000-0001-6135-6578  Date of submission: 29.07.2022 Date of acceptance: 15.03.2023 Date of publication: 01.07.2023  Conflicts of interest: None Supporting agencies: None DOI: https://doi.org/10.3126/ijosh.v13i3.46615   Copyright:This work is licensed under a CreativeCommons Attribution-NonCommercial 4.0 International License ISSN: 2091-0878 (Online) ISSN: 2738-9707 (Print) Maurya et al. HPLC analysis of biomarkers of Toluene and Xylene in human urine samples 332 biomarkers of toluene and xylenes which are excreted from the body in urine.3-5 According to the American Conference of Governmental Industrial Hygienists (ACGIH), the permissible exposure limit in workers after 1-day exposure is 1.6 w/w creatinine for urinary HA and 1.5 w/w creatinine for urinary MHA, respectively.6 Many chromatographic methods are reported for the determination of HA and MHA in urine samples such as gas chromatography (GC),7 capillary zone electrophoresis (CZE),8 ion chromatography (IC),9 liquid chromatography and each has its own merits and demerits.   There are various efforts to develop novel methods for HA and MHA. Recently, Takeuchi et. al. developed Direct methyl esterification with 2,2-dimethoxy propane for the simultaneous determination of urinary metabolites of toluene, xylene, styrene, and ethylbenzene by gas chromatography-mass spectrometry.7  Chiu et. al. reported the use of urinary hippuric acid and methyl hippuric acid to evaluate surgical smoke exposure in operating room healthcare personnel.10 Here we tried to develop and validate a simple high-performance liquid chromatography (HPLC) for analysis of HA and MHA as per ICH Q2 R(1) guidelines in human urine samples because HPLC methods are convenient as it is cost-effective and derivatization is not required.11   Methods Standards of urinary metabolites, HA and MHA were procured from Sigma Aldrich USA. Trifluoroacetic acid (TFA), HPLC grade acetonitrile was purchased from Merck. Ultrapure water obtained by Merck Synergy water purification system. The HPLC system consists of two LC-10AT vp pumps, a diode array detector (SPD-M10A vp), a column oven (CTO-10AS vp) and a system controller (SLC-10A vp) (all from Shimadzu, Kyoto, Japan), was used. Reversed-phase Thermo ODS-2 Hypersil, 250 x 4.6 mm, particle size: 5 m column was used for analysis. The chromatographic and the integrated data were recorded using the LG computer system using LC-Solution data acquiring software (Shimadzu, Kyoto, Japan). The mobile phase consisted of two solvents A & B, solvent A was 0.1% TFA in water and solvent B was 0.1% TFA in acetonitrile.  Separation was performed in gradient elution mode and the time program was as follows –Time (min)/%B: 0/20, 4/20, 11/80, 11.1/90, 16/90, 16.1/20, 25/20. The detection wavelength was 205 nm. Water and methanol were mixed (1:1 ) to form a diluent solution. solution: 10.0 mg of HA and MHA standard were taken separately into a 100.0 mL volumetric flask and dissolved in methanol up to mark to prepare 100ppm Stock HA and MHA individually. Working standard solutions of HA and MHA of concentration 1, 2, 3, 5, and 10 mg/L were prepared respectively by diluting 100 µL, 200 µL, 300 µL, 500 µL and 1000 µL of the 100 mg/L stock solution of HA and MHA to 10.0 mL with diluent. The reference solution of HA and MHA was prepared by diluting 500 µL of the 100 mg/L stock solution of HA and MHA to 10.0 mL with diluent. 2.5 g of disodium hydrogen orthophosphate, 2.5 g of sodium dihydrogen orthophosphate and 8.2 g of sodium chloride was dissolved in 950 mL of water. pH of the solution was adjusted to 6.4 with 1 M sodium hydroxide or 1 M hydrochloride acid, if necessary and diluted to 1000.0 mL to prepare phosphate buffer at pH 6.4.  This study was part of our previous “Consortium on Vulnerability to Externalizing Disorders and Addictions (cVEDA)” study and the HPLC method was developed for the analysis of volatile organic compounds (VOCs) in urine samples, is incorporated in cVEDA as a measure of exposure to environmental neurotoxins.12 Details of sample size and study recruitment centers can be found in this reference However, only three samples were selected randomly and used for method development purposes.  Spot urine samples were collected from all subjects after washing their hands to avoid any contamination and collect the mid-stream urine sample in polythene bottles and samples were brought to the laboratory and kept under refrigerated conditions and preserved in a deep fridge at -20°C. All urine samples were obtained after approval from the Human Ethics Committee of the Institute. Before analysis, urine Maurya et al. HPLC analysis of biomarkers of Toluene and Xylene in human urine samples 333 samples were cleaned up by solid phase extraction (SPE) for removing matrix interferences. Strong anion exchange (SAX) SPE cartridges from Whatman  (500 mg per 3 mL cartridge) were used for this purpose., A vac-Elute vacuum elution system was used for SPE. SAX cartridges are a quaternary ammonium bonded phase of the silica matrix, positively charged in the full pH range, and have a strong anion exchange capacity. SAX cartridges are mainly used for extracting weakly acidic compounds such as carboxylic acids. Matrix interferences with strong negative charge bind strongly on SAX cartridges and thus help in eliminating matrix interferences. First, for conditioning, the SAX cartridge was washed with methanol (3 times), followed by washing with pH 6.4 phosphate buffer solution (elution rate was adjusted to between 2 to 3 mL. 5 mL urine sample and 5 mL of phosphate buffer solution was mixed thoroughly and passed through SAX column. The SAX column was washed by 1 mL water, followed by 1 mL phosphate buffer and 1 mL of 1.0% aqueous acetic acid. The column was dried in air and analytes were eluted with 5 mL of 10% aqueous acetic acid.2 Results During HPLC Method Validation following parameters were evaluated: Selectivity, Calibration Curve and Linearity, Accuracy and Precision, Limit of Detection (LOD) and Quantitation (LOQ), Robustness, and Application of the method to real samples. During Selectivity evaluation, no endogenous components extracted from blank urine were eluted at the retention time of the peak of HA and MHA (Fig.1 and 2). The developed method was found to be selective for the determination of HA and MHA without interference from the endogenous constituents of urine. Figure 1: Chromatogram for Blank (Diluent)          Figure 2: Chromatogram for HA and MHA under optimized conditions.  Maurya et al. HPLC analysis of biomarkers of Toluene and Xylene in human urine samples 334  Figure 3: Linearity chart for HA and MHA Analysis was based on the external standard method. Calibration curve and linearity studies were performed with calibration standards fresh on the day of analysis by diluting the appropriate working solutions with diluent. The standard calibration curve was constructed using blank urine samples spiked with HA and MHA at five different concentrations from 1, 2, 3, 5 and 10 mg/L. The data were subjected to statistical analysis using a linear regression model. The calibration curves were obtained by weighted linear regression (weighing factor 1/x2) using Microsoft Excel 2007 software. The suitability of the calibration model was confirmed by back-calculating the concentrations of the calibration standards. The developed method was linear over the tested concentrations with correlation coefficient r2 = 0.999 (for HA) and r2 = 0.999 (for MHA), the calibration curve (Fig. 3) was described by equations y = 76553x + 30207 (for HA) and y = 11738x + 57166 (for MHA). Accuracy and precision studies were performed with, 5 mg/L concentration of HA and MHA, in three replicates were used to validate the accuracy and precision of the developed method. The results showed that the intra- and inter-day accuracy (% bias) for the method ranged between −14.8% and 1.7%, respectively for HA (Table 1). The % CV of intra- and inter-day precision was <8.85% for HA. The results showed that the intra- and inter-day accuracy (% bias) for the method ranged between −16.7% and 14.2%, respectively for MHA (Table 2). The % CV of intra- and inter-day precision was <9.23% for MHA. The developed method was thus found to meet generally accepted requirements of accuracy and precision over the studied concentration ranges .      Maurya et al. HPLC analysis of biomarkers of Toluene and Xylene in human urine samples 335 Table 1: Accuracy, Precision and Recovery for HA  Limit of Detection (LOD) and Quantitation (LOQ) were performed after sample clean-up, and the extracts from spiked urine were injected into the chromatographic systems. The analysis was carried out at decreasing concentrations to determine the minimal concentration with a signal-to-noise ratio of 3:1. The LOD and LOQ for HA was 0.3 mg/L and 1.0 mg/L respectively and for MHA were 0.25 mg/L and 1.0 mg/L respectively. According to the ICH guideline Q2A, “robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and indicates its reliability during normal usage”.11 Flow rate and temperature of the optimized method was changed deliberately and no drastic deviation was found by these alterations and the developed method was considered robust.  The developed method was used for three batches of samples that were analyzed after sample preparation and the result has been tabulated below (Table 3)  𝐴𝑠𝑠𝑎𝑦 =  𝐴𝑟𝑒𝑎 𝑜𝑓 𝑎𝑛𝑎𝑙𝑦𝑡𝑒 𝑖𝑛 𝑠𝑎𝑚𝑝𝑙𝑒𝑀𝑒𝑎𝑛 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑× 𝐶𝑜𝑛𝑐. 𝑜𝑓𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑      Mean Std. Deviation %RSD  Standard Area  (Day-1)   503103 23611.28 4.7  476445  Sample 3-8 Area of spike sample 3-8  Area spike sample 3-8 corrected Conc. (mg/L) Recovery % 521385  118852 589554 470702 4.7 93.6 511478    600789 481937 4.8 95.8    586578 467726 4.6 93.0  Mean 4.7 94.1  Std. Deviation 0.07 1.49  % Relative Std. Deviation 1.58 1.58 Standard Area  (Day-2)   481664 6728.629 1.4  480863  Sample 3-8 Area of spike sample 3-8  Area spike sample 3-8 corrected Conc. (mg/L) Recovery % 488757  131479 592500 461021 4.8 95.7 475371    604001 472522 4.9 98.1    621307 489828 5.1 101.7  Mean 4.9 98.5  Std. Deviation  0.15 3.01  % Relative Std. Deviation  3.06 3.06 Standard Area  (Day-3)   537854 56275.77 10.5  540587  Sample 3-8 Area of spike sample 3-8  Area spike sample 3-8 corrected Conc. (mg/L) Recovery % 480262  139432 683837 544405 5.1 101.2 592714    597473 458041 4.3 85.2    626738 487306 4.5 90.6  Mean 4.6 92.3  Std. Deviation 0.41 8.17  % Relative Std. Deviation 8.85 8.85  Overall Recovery 95% Maurya et al. HPLC analysis of biomarkers of Toluene and Xylene in human urine samples 336 Table 2: Accuracy, Precision and Recovery for MHA    Mean Std. Deviation %RSD  Standard Area  (Day-1)  768895 36203.59 4.7  727214 Sample 3-8 Area of spike sample 3-8  Area spike sample 3-8 corrected Conc. (mg/L) Recovery % 792523 65648 855513 789865 5.1 102.7 786946   863773 798125 5.2 103.8   796417 730769 4.8 95.0 Mean 5.0 100.5 Std. Deviation  0.24 4.78 % Relative Std. Deviation 4.75 4.75 Standard Area  (Day-2)  739690 19424.47 2.6  724339 Sample 3-8 Area of spike sample 3-8  Area spike sample 3-8 corrected Conc. (mg/L) Recovery % 761528 51623 838496 786873 5.3 106.4 733204   753579 701956 4.7 94.9   896272 844649 5.7 114.2 Mean 5.3 105.2 Std. Deviation 0.49 9.70 % Relative Std. Deviation 9.23 9.23 Standard Area  (Day-3)  824454 64365.06 7.8  811230 Sample 3-8 Area of spike sample 3-8  Area spike sample 3-8 corrected Conc. (mg/L) Recovery % 767729 76950 851699 774749 4.7 94.0 894405   763767 686817 4.2 83.3   868881 791931 4.8 96.1 Mean  4.6 91.1 Std. Deviation  0.34 6.84 % Relative Std. Deviation  7.51 7.51 Overall Recovery 98.9%  Table 3: Assay for HA and MHA in three batches of real urine samples. Standard Run 1 Run 2 Run 3 Mean STDEV %RSD HA (5mg/L) 476445 521385 511478 503103 23611.28 4.7 MHA (5mg/L) 727214 792523 786946 768895 36203.59 4.7 HA Sample 1 Sample 2 Sample 3 826443 562865 118852 Conc. (mg/L) 8.2 5.6 1.2 MHA Sample 1 Sample 2 Sample 3 267520 90923 65648 Conc. (mg/L) 1.7 0.6 0.4  Conclusion A simple, rapid, and robust HPLC method for the determination of HA and MHA was developed. The experimental results concerning linearity, accuracy, precision, specificity and sensitivity demonstrate the reliability of the procedure for its intended application. The method was successfully applied to test real urine samples for the determination of HA and MHA. Maurya et al. HPLC analysis of biomarkers of Toluene and Xylene in human urine samples 337 Acknowledgments The authors wish to thank Dr. Amit Chakrabarti, Officer-in-Charge, I-CAM for encouragement and permission to communicate the manuscript for publication. References 1. Mølhave L. 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HPLC analysis of biomarkers of Toluene and Xylene in human urine samples

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HPLC analysis of biomarkers of Toluene and Xylene in human urine samples

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