Simultaneous determination of sulforaphane and its major metabolites from biological matrices with liquid chromatography–tandem mass spectroscopy

A simple, sensitive and specific LC–MS/MS method for the simultaneous determination of sulforaphane (SFN) and its major metabolites, the glutathione (SFN–GSH) and N-acetyl cysteine conjugates (SFN–NAC) from biological matrices was developed and validated. The assay procedure involved solid-phase extraction of all three analytes from rat intestinal perfusate using C2 extraction cartridges, whereas from rat plasma, metabolites were extracted by solid-phase extraction and SFN was extracted by liquid–liquid extraction with ethyl acetate. Chromatographic separation of SFN, SFN–GSH and SFN–NAC was achieved on a C8 reverse phase column with a mobile phase gradient (Mobile Phase A: 10 mM ammonium acetate buffer, pH: 4.5 and Mobile Phase B: acetonitrile with 0.1% formic acid) at a flow rate of 0.3 mL/min. The Finnigan LCQ LC–MS/MS was operated under the selective reaction monitoring mode using the electrospray ionization technique in positive mode. The nominal retention times for SFN–GSH, SFN–NAC and SFN were 8.4, 11.0 and 28.2 min, respectively. The method was linear for SFN and its metabolites with correlation coefficients >0.998 for all analytes. The limit of quantification was 0.01–0.1 μM depending on analyte and matrix, whereas the mean recoveries from spiked plasma and perfusate samples were approximately 90%. The method was further validated according to U.S. Food and Drug Administration guidance in terms of accuracy and precision. Stability of compounds was established in a battery of stability studies, i.e., bench-top, auto-sampler and long-term storage stability as well as freeze/thaw cycles. The utility of the assay was confirmed by the analysis of intestinal perfusate and plasma samples from single-pass intestinal perfusion studies with mesenteric vein cannulation in rats.