High-Performance Liquid Chromatography-Based Determination of Total Isothiocyanate Levels in Human Plasma: Application to Studies with 2-Phenethyl Isothiocyanate

Abstract

Dietary and pharmacologic isothiocyanates (ITCs) may play a role in reducing the risk of certain cancers. The quantification of ITCs in humans is important both for epidemiological and pharmacokinetic studies. We describe a modification of an HPLC-based assay of urinary ITCs for use with human plasma. The assay utilizes the cyclocondensation reaction of 1,2-benzenedithiol with ITCs present in human plasma, followed by a two-step hexane extraction and analysis by HPLC using UV detection at 365 nm. The method shows linearity and reproducibility with human plasma over a range of 49–3003 nM phenethyl isothiocyanate (PEITC) ( r 2 = 0.996 ± 0.003). A similar degree of linearity was seen with two other biologically occurring conjugates of PEITC: PEITC– N-acetylcysteine (PEITC– NAC) and PEITC–glutathione (PEITC–GSH). The recovery of PEITC assessed on multiple days was 96.6 ± 1.5% and was 100% for PEITC–GSH and PEITC– NAC. The reproducibility of the assay on multiday samplings showed a mean %CV of 6.5 ± 0.3% for PEITC, 6.4 ± 4.3 for PEITC– NAC and 12.3 ± 3.9 for PEITC–GSH. In clinical studies, mean plasma ITC level of 413 ± 193 nM PEITC equivalents was determined for a non-dietary-controlled group of 23 subjects. Multiday analysis data from pharmacokinetic plasma sets of 3 subjects taking a single dose of PEITC at 40 mg showed a good CV (range: 16–21%). The applicability of the methodology to pharmacokinetic studies of PEITC in humans is demonstrated.