Improvement in determination of isothiocyanates using high-temperature reversed-phase HPLC

The largely adopted reversed-phase HPLC analysis of the molecular species of isothiocyanates (ITCs) was performed and showed losses during the chromatographic run with eight ITCs. These losses, which obviously impact the accuracy of quantitative determinations, were due to precipitation in the chromatographic system. At 22°C, they ranged from 5.4% for sulforaphane (SFN) to 11.0% for benzyl-ITC when ITCs were injected at 80 μg mL(-1) , but they were up to three times higher at 1 mg mL(-1) reaching 31.9% for benzyl-ITC. The water solubility of the ITCs was a key determinant of the extent of the measured loss. When the column was heated at 60°C, losses in injected ITCs were reduced, in comparison with 22, 40, and 50°C, by two to ten times depending on the ITC considered. A reversed-phase HPLC method based on column heating was suggested and its quantitative performance was determined. It was then applied to the separation of methylene chloride extracts of various cruciferous vegetables. Ally-ITC, SFN, and iberin in cabbage; SFN and iberin in cauliflower; and allyl-ITC and phenylethyl-ITC in horseradish could be identified and quantified. The obtained results cast doubt on quantitative determinations of ITCs that are carried out at room temperature using reversed-phase HPLC.