Determination of 2,5-hexandione by high-performance liquid chromatography after derivatization with dansylhydrazine

A sensitive method for the determination of free and total urinary 2,5-hexandione (2,5-HD) using high-performance liquid chromatography with fluorescence detection was developed. After purification of urine with a disposable C18 cartridge, 2,5-HD was derivatized with dansylhydrazine; 1,3-diacetyl benzene (1,3-DAB) was added to the samples, as internal standard, prior to extraction. The resulting fluorescent adducts were separated on a reversed-phase column with a gradient mobile phase of 25 mM phosphate buffer (pH 6.4) and acetonitrile. The retention times of the 2,5-HD and 1,3-DAB derivatives were 9.4 and 13.7 min, respectively. The derivatives were detected by a fluorescence detector (excitation 340 nm, emission 525 nm). The mean recoveries of 2,5-HD and 1,3-DAB were 92.0 and 94.0%, respectively; the detection limit of 2,5-HD (signal-to-noise ratio of 3) was 5 micrograms/l in urine without hydrolysis and ca. 12 micrograms/l in hydrolyzed samples. The method was applied to 39 urine samples from workers exposed to n-hexane; the mean values were 2.597 mg/l (S.D. = +/- 0.758) for total 2,5-HD and 0.179 mg/l (S.D. = +/- 0.086) for free 2,5-HD. Urine samples of 22 non-exposed subjects showed a mean concentration of 0.437 mg/l (S.D. = +/- 0.109) and 0.022 mg/l (S.D. = +/- 0.011) for total and free 2,5-HD, respectively.