Environmentally friendly determination of urinary trans, trans-muconic acid for biological monitoring of benzene exposure by green high-performance liquid chromatography

An environmentally friendly, so-called green, high performance liquid chromatography method was developed and validated for the determination of trans, trans-muconic acid (t,t-MA) in human urine as a biomarker of benzene exposure. After urinary t,t-MA was extracted and enriched using solid-phase extraction, a MF-Ph1 SG80 (150 mm×2.0 mm I.D., 5 μm) column with a mobile phase of 10 mM KH₂PO₄ containing 0.1% H₃PO₄ was used for isocratic separation of t,t-MA with UV detection at 259 nm. The calibration curve was constructed in the range of 0.1-5.0 mg/L with good linearity (r²=0.9992). The intra-day and inter-day precision (as RSD) were 0.9-8.5% and 3.1-4.5%, respectively. The average recovery ranged from 97.5% to 101.7%. The green sample preparation and separation with no organic solvents were successfully achieved. The validated method would be suitable for the routine biological monitoring of benzene exposure in the occupational settings.