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Year : 2019  |  Volume : 4  |  Issue : 1  |  Page : 17-22

Method for estimation of hippuric acid as a biomarker of toluene exposure in urine by high-performance liquid chromatography after extraction with ethyl acetate

Department of Industrial Hygiene, ICMR-Regional Occupational Health Centre (Eastern), Kolkata, West Bengal, India

Date of Submission06-Dec-2018
Date of Acceptance27-Feb-2019
Date of Web Publication9-Apr-2019

Correspondence Address:
Miss. Anupa Yadav
Department of Industrial Hygiene, ICMR-Regional Occupational Health Centre (Eastern), Block – DP, Sector – V, Salt Lake, Kolkata - 700 091, West Bengal
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ed.ed_22_18

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Aim: This study aimed to establish liquid–liquid extraction (LLE) for estimation of hippuric acid (HA) in urine as a biomarker of the toluene exposure by high-performance liquid chromatography equipped with photodiode array detector (HPLC-PDAD).
Method: HA in urine was extracted by LLE and determined by HPLC-PDAD. The operating conditions with HPLC were ODS-2 hypersil column (250 mm × 4.6 mm, 5 μm), 0.1% trifluoro acetic acid (TFA) in acetonitrile and 0.1% TFA in water as mobile phase, 1 ml/min flow rate, and wavelength of 205 nm. The validity of the present method was tested by the estimation of HA in urine samples, collected from toluene-exposed (shoe workers) and unexposed or control subjects.
Results: Binary gradient system was used to achieve optimum separation. The analytical curve prepared for HA in aqueous solution in the range of 0.5–10 μg/ml showed determination coefficient value (R2) 0.998. Limit of detection and quantification (LOQ) were 0.46 and 1.53 μg/ml, respectively. The coefficients of variance for intraday precision were 1.4% for HA standard (5 μg/ml) and 1.1% for pooled urine, whereas inter-day precision values were 3.2% and 4.9% for HA standard and pooled urine, respectively. Method recovery obtained was 96%–120% for HA solutions containing 2, 3, and 5 μg/ml, demonstrating that precision and recovery of method were satisfactory. Compared to unexposed group, exposed group had significantly more HA. It was found significantly (P < 0.05) higher in urine of exposed workers (32.52 ± 10.91) than unexposed group (16.21 ± 10.14).
Conclusion: Sample preparation by LLE is simple and cost-effective for the determination of HA as a biomarker of toluene exposure by HPLC-PDAD. It can be used to detect HA in urine for population exposed to toluene.

Keywords: High-performance liquid chromatography, hippuric acid, liquid–liquid extraction, photodiode array detector, toluene exposure, urine

How to cite this article:
Yadav A, Basu A, Chakarbarti A. Method for estimation of hippuric acid as a biomarker of toluene exposure in urine by high-performance liquid chromatography after extraction with ethyl acetate. Environ Dis 2019;4:17-22

How to cite this URL:
Yadav A, Basu A, Chakarbarti A. Method for estimation of hippuric acid as a biomarker of toluene exposure in urine by high-performance liquid chromatography after extraction with ethyl acetate. Environ Dis [serial online] 2019 [cited 2023 Mar 30];4:17-22. Available from: http://www.environmentmed.org/text.asp?2019/4/1/17/255736

  Introduction Top

Toluene is an aromatic hydrocarbon, also known as methylbenzene or phenylmethane. It is a clear and water-insoluble liquid with a sweet smell derived from benzene compound. Use of this chemical is quite extensive in paint industry, rubber, cosmetics, adhesive, and printing ink.[1] Major sources of toluene in indoor environment are paint, thinner, together with tobacco smoke.[2] There are three main routes of toluene exposure, namely inhalation, ingestion, and dermal contact. It is a highly volatile compound; therefore, inhalation is the most important exposure pathway to be considered. Moreover, once it enters into the human body, it gets deposited and accumulated very easily in the vital organs such as the brain, liver, kidneys, and other organs.[3] Chronic exposure can cause health issues such as eye/skin irritation, dizziness, respiratory problems, impaired function of liver, kidney, and central nervous system (CNS).[3],[4]

Once toluene is absorbed in the body via respiratory tract, then it is oxidized in liver microsomes mainly (68%) to hippuric acid (HA).[5] HA is normally a main component of nonprotein nitrogen in urine. It is an important metabolic end product for protein or nucleic acid. It is also an established urinary biomarker in biological monitoring of people exposed to toluene.[6],[7] HA is excreted in urine with a half-life of 2–3 h.[8] Metabolic process of toluene is given in [Figure 1].[6]
Figure 1: Metabolism pathway of toluene

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Urinary HA has been extensively studied as an occupational biomarker for toluene exposure. This compound presents a good correlation with the degree of exposure and is one of the recommended biomarkers to assess exposure to toluene.[9],[10],[11] Estimation of this biomarker in urine is one of the most useful ways for biomonitoring of occupational exposure to toluene.[12] Studies reported that the lowest concentration of toluene in breathing environment is 30 ppm at which urinary HA increased to a measurable level.[13],[14] Various methods have been developed for the estimation of HA in urine such as colorimetric,[15] spectrophotometric, and[16] by high-performance liquid chromatography (HPLC).[17],[18],[19],[20] However, all these methods are expensive and require a long sample preparation process. The objective of this work was to develop a simple and cost-effective technique for the measurement of HA in urine, using liquid–liquid extraction (LLE) and HPLC with ODS-2 Hypersil column and photodiode array detector.

  Materials and Methods Top


HA (98%) as standard was procured from Merck, Germany. All the chemicals and solvents such as hydrochloric acid, sodium chloride, ethyl acetate, acetonitrile, trifluoroacetic acid, and water were of HPLC grade.


HPLC (Shimadzu, Japan) consisted of a system controller (Model: SLC-10 A, VP-Series), connected with an automated liquid sampler (Model: LC-10AT, VP series), spectrophotometer (Model: SPD-M 10A, Diode Array Detector), column oven, and a monitor. The column used was ODS-2 Hypersil, 250 mm, 4.6 mm, particle size 5 μm (Thermo).

Chromatographic conditions

The pump was operated at a flow rate of 1 ml/min; UV detection wavelength was set at 205 nm. The mobile phase used was 0.1% trifluoro acetic acid (TFA) in acetonitrile and 0.1% TFA in HPLC grade water with flow of 1 ml/min, sample injection volume was 20 μl, reverse phase analytical column ODS-2 Hypersil (250 mm, 4.6 mm, and 5 μm), and oven temperature 25°C was used for chromatographic system. Under these conditions, HA was eluted and detected in around 18.90 min.

Sample preparation procedure

Well-mixed urine sample (1 ml) was taken in centrifuge tube, added 80 μl of 6N HCL, 0.3 g sodium chloride, and 4 ml ethyl acetate. Then, all the contents of the centrifuge tube were mixed for 2 min by rotation and followed by centrifugation at 1100 rpm for 5 min. Then, 200 μl of the upper (organic) layer was transferred to HPLC vial and evaporated to dryness using a heated (30°C) water bath and a gentle stream of nitrogen. The dried residue was re-dissolved in 200 μl of distilled water. For analysis, 20 μl of it was injected into the HPLC.

Method validation


Selectivity of the method was evaluated by comparing the analyte retention time (RT) and peak purity obtained for the standard solution and samples.


To obtain linearity, working standards containing HA in concentration range 0.5–10 μg/ml were prepared in aqueous solution and analyzed at optimized chromatographic conditions to obtain determination coefficient (R2) from calibration curve of analyte.

Limit of detection and quantification

Limit of detection (LOD) and limit of quantification (LOQ) for HA were determined based on standard deviation (σ) and slope (S) of calibration curve as per the guidelines of International Conference on Harmonization 2005 (LOD = 3 × σ/S; LOQ = 10 × σ/S).[21]


Intraday and interday precision was measured using the coefficient of variation (CV) obtained for repetition of sample preparation to injection onto the chromatographic column. For intraday precision, samples were analyzed for three times within the same day, whereas for interday, test samples were analyzed for 3 consecutive days. Values of up to 20% were normally considered acceptable for analytical methods applied to environmental samples with complex matrices.[22],[23]


Accuracy was determined using the percentage recovery of HA added to urine at concentrations 2, 3, and 5 μg/ml, with three replicates.[22]

Accuracy (%) =100 × (Cspiked × Csample)/Cstd

where Cspiked denotes concentration of HA added to urine sample, Csample denotes concentration of HA in urine sample determined from calibration curve, and Cstd denotes concentration of HA standard.

Matrix effects

Interference caused by the presence of the biological matrix was assessed by comparing the coefficient of determination obtained for analytical curve of HA prepared using aqueous solution and urine sample. The LLE was performed under the optimized conditions.

Method validity

In order to validate the present method, biological monitoring was done in urine (five samples of each exposed and unexposed samples used for this work). Urine samples used for this method validation were collected under institutional intramural project, and the project was approved by the Institutional Ethics Committee. Postshift urine samples were collected from toluene-exposed workers and also collected from unexposed individuals used as control. The values of unexposed and exposed individuals were compared by Student's t-test, and P value was determined.

  Results Top

Urinary HA is extensively used as an exposure biomarker of toluene as suggested by statutory bodies.[9],[11] In individuals exposed to toluene vapors such as workers of industries such as shoe making, painters, printing press, and cosmetics, HA in urine of exposed individuals can be found in higher concentrations than unexposed individuals. However, the expected amount of HA in exposed individuals may be 1.5 g/g creatinine as recommended by the American Conference on Hygiene of Governmental Industrial Hygienists.[11] For this reason, it is advantageous to have a simple and easy method that allows the evaluation of this compound in the urine samples. Human urine is a complex matrix that contains proteins, hormones, salts, drugs, and a variety of other compounds. Therefore, to obtain maximum elution of HA from urine, the samples were extracted by LLE method and analyzed at optimized analytical conditions. Under these optimized chromatographic conditions, HA peak for standard solution and urine sample was detected in around 18.90 min at wavelength 205 nm as shown in [Figure 2]. Method selectivity was confirmed by comparing the RT of HA peak in the standard and sample chromatogram (A and B), under the same chromatographic conditions, and degree of purity of the eluted peak was determined using the LC solutions software, version 1.25 (Shimadzu, Japan). For peak purity, the evaluation was made from the absorption spectra at the RTs corresponding to the up slope, top, and down slope regions of the chromatographic peak, comparing them in order to identify any possibility of co-elution of other substance.
Figure 2: Chromatograms obtained for the separation of hippuric acid at optimized chromatographic conditions. (a) Standard solution of hippuric acid (5 μg/ml); (b) Urine sample

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[Figure 3] presents the calibration curve with coefficient of determination (R2) value exceeding 0.9986 prepared using aqueous HA standards in the concentration range of 0.5–10 μg/ml. LOD and LOQ of the method were calculated using standard deviation (σ) and slope (S) of calibration curve, as described previously. The values obtained were 0.46 μg/ml (LOD) and 1.53 μg/ml (LOQ). In [Table 1], CV was calculated in order to determine the precision of the method. For intraday study, HA standards and urine sample were analyzed in three replicates, and inter-day study was performed in the same way for 3 consecutive days. The highest values of CV obtained were 1.4% and 8.1% for intra- and inter-day experiments, respectively, demonstrating that the method provided good precision. The results also showed that the precision was independent of the concentration of HA in sample. The accuracy of the method was determined using the recovery of HA standards 2, 3, and 5 μg/ml added to the urine. The concentration of HA in unfortified urine was 16 μg/ml, calculated from analytical curve prepared using aqueous standards. Accuracy obtained was 96%–120% as shown in [Table 2].
Figure 3: Calibration curve of hippuric acid with concentration range 0.5–10 μg/ml

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Table 1: Intra-day and Inter-day precision studies for determination of hippuric acid by high-performance liquid chromatography

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Table 2: Recovery (%) results of hippuric acid in urine

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As the method is developed for biological fluids like urine, it is important to evaluate the matrix effect. In our results, the presence of matrix effect was found as shown in [Figure 4]. Slope of analytical curve prepared in urine was slightly higher than the analytical curve prepared in aqueous solution. Quantification of results was done with analytical curve prepared in urine to nullify the matrix effect. Displacement of analytical curve for urine and of aqueous solution was due to the presence of HA in urine, which has also been observed in other studies.[5],[24],[25] The present method is validated by evaluating urinary HA concentration, in urine of exposed (shoe workers) and controls. When analytical results of exposed (n = 5) and unexposed (n = 5) groups were compared, it was found that exposed individuals had significantly more HA [Table 3], statistically these results were significant (P < 0.05), suggesting higher level of toluene exposure among workers.
Figure 4: Analytical curve of hippuric acid prepared in aqueous solution (blue dots) and in urine (red squares)

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Table 3: Results of hippuric acid biomonitoring among exposed individuals and controls

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  Discussion Top

Workers engaged in shoe making, printing/painting work, cosmetic shops/saloons, petrochemical work, etc., have exposure to toluene.[1],[2] Chronic exposure to this volatile solvent can cause various health issues such as impaired function of liver, kidney, and CNS.[4],[3] Air monitoring of toluene is very time consuming, costly, and laborious. Therefore, it becomes necessary to develop a simple, easy, and cost-effective method for biomonitoring of this solvent. Urinary HA is recommended as an exposure biomarker for toluene.[11] Estimation of urinary HA, with the present method, is simple. This method has multiple advantages, and one of them is that it has excellent linearity in the working range, precise LOD and LOQ, with adequate accuracy and precision. For spiked urine sample (5 μg HA/ml), recovery value was found higher, possibly because HA was already present in urine (16 μg/ml) in higher quantities. Ideally, the recovery study should be done with urine containing lower levels of analyte. In case of analytic methods involving complex matrix like urine, recovery values in the range 80%–120% are acceptable.[23],[26],[27]

The second advantage of the method is that urine used for exposure monitoring can be easily obtained and is noninvasive, unlike blood samples. Sample preparation is easy and cost effective than other methods described in the literature. Although the present method is good enough to use, ethyl acetate is used as an extraction solvent, it may cause irritation to eye, skin, and respiratory tract if its vapors are inhaled by laboratory workers. To check the validity of method, urine samples of exposed and unexposed groups were analyzed, and it was found that exposed individuals had significantly more HA. These results were statistically significant (P < 0.05), suggesting higher level of toluene exposure among workers. Our findings were similar to as reported by Duydu et al. (1999).[28] Therefore, this method can be used in any laboratory having HPLC and can be used for routine bio-monitoring of toluene-exposed workers.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2], [Table 3]

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