Bioavailable Sulforaphane Quantitation in Plasma by LC-MS/MS Is Enhanced by Blocking Thiols

Quantifying sulforaphane(SFN) and its thiol metabolites in biologicalsamples using liquid chromatography-tandem mass spectrometryis complicated by SFN's electrophilic nature and the faciledissociation of SFN-thiol conjugates. SFN can be lost during samplepreparation due to conjugation with protein thiols, which are precipitatedand discarded. We observe that only 32 & PLUSMN; 3% of SFN is recovered2 h after spiking into fetal bovine serum. The SFN-glutathione conjugateprepared at 10 mM in 0.1% formic acid in water (pH 3) dissociatedby approximately 95% to free SFN, highlighting the difficulty in preparingthiol metabolite standards. We used the alkylating agent iodoacetamide(IAA) to both release SFN from protein thiols and force the dissociationof SFN metabolites. This thiol-blocking method increased SFN percentrecovery from serum from 32 to 94 & PLUSMN; 5%, with a 4.7 nM methodlimit of quantitation. Applying the method to clinical samples, SFNconcentrations were on average 6 times greater than when IAA was omitted.The IAA thiol-blocking method streamlines the analysis of bioavailableSFN in plasma samples.