HPLC with fluorescence detection of urinary phenol, cresols and xylenols using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a fluorescence labeling reagent†

Abstract

A simple and sensitive HPLC method for the determination of phenolic compounds, i.e., phenol (Phe), cresols (Cres) and xylenols (Xyls), was developed. After a pre-column fluorescence derivatization of these compounds with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) at 60 °C for 30 min, 11 DIB derivatives were successfully separated within 50 min with an ODS column using CH₃CN–H₂O–CH₃OH (25 + 22 + 53, v/v) as the eluent. The detection limits of DIB derivatives at a signal-to-noise ratio of 3 ranged from 0.15 to 1.09 µM (0.2–1.6 pmol per 20 µl). The precision of the proposed method for both within- and between-day assays of free and total phenol related compounds was satisfactory (RSD <9.5% ). By the proposed method, Phe and p-Cre could be detected in normal urine samples, and the calculated concentrations of free Phe and p-Cre in unhydrolysed urine samples were 1.5 ± 1.3 and 23.9 ± 24.3 µM and those of total Phe and p-Cre in hydrolysed urine samples were 87.3 ± 81.2 and 200.7 ± 195.4 µM (n=21), respectively.

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