Determination of hexafluoroisopropanol, a sevoflurane urinary metabolite, by 9-fluorenylmethyl chloroformate derivatization

Abstract

A reversed-phase HPLC method with fluorescence detection for the quantification of hexafluoroisopropanol (HFIP) in urine is presented. HFIP, a metabolite of the inhalation anesthetic sevoflurane, is excreted mainly in urine as glucuronic acid conjugate. After enzymatic hydrolysis of the glucuronate, primary amino groups of interferent urinary compounds are blocked by reaction with o-phthalic dicarboxaldehyde and 3-mercaptopropionic acid, followed by labeling of HFIP with 9-fluorenylmethyl chloroformate. The derivatization reaction proceeds in a water–acetonitrile (1:1) solution at room temperature with a borate buffer of pH 12.5 as a catalyst. A stable fluorescent derivative of HFIP is formed within 5 min. The HFIP–FMOC derivative is separated by reversed-phase chromatography with isocratic elution on an octadecyl silyl column (33×4.6 mm, 3 μm) and guard column (20×4.0 mm, 40 μm), at 35 °C, and detected by fluorescence detection at an excitation wavelength of 265 nm and an emission wavelength of 311 nm. The method detection limit is 40 pg, per 10-μl injection volume, corresponding to 16 μg/l of HFIP in urine. The among-series relative standard deviation is <6% at 200 μg/l ( n=6). As a preliminary application, the method was used to detect HFIP concentration in the urine of two volunteers exposed for 3 h to an airborne concentration of sevoflurane in the order of 2 ppm.