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

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.