The health benefits associated with the consumption of cruciferous vegetables (Brassicaceae family) are largely attributed to glucoraphanin (GR), which must be enzymatically hydrolyzed into its bioactive form sulforaphane (SF). SF is absorbed and metabolized primarily in intestinal epithelial cells to sulforaphane-glutathione (SF-GSH), sulforaphane-cysteine (SF-CYS) and sulforaphane-N-acetyl-cysteine (SF-NAC); each have been shown to have therapeutic effects. Clinical investigations of GR, SF and SF metabolites however, require a robust, sensitive and high throughput method that can be applied to a range of biological tissues (plant, digesta, culture medium, cell lysates). Currently-available methods require sample pre-treatment with solid phase extraction (SPE); increasing analysis time and reducing sensitivity. To address this, a novel UHPLC-QQQ-MS/MS method without SPE pre-treatment was developed and used for the simultaneous determination of GR and SF metabolites in a range of biological materials. The LODs for this 9 min method ranged from 0.03 to 1.95 μg L-1 and the LOQs ranged from 0.06 to 3.90 μg L-1. The intra- and inter-day variation was ≤ 15 %. Linearity ranged from 3.90-1000 μg L-1 (GR), 0.06-500 μg L-1 (SF), 3.90-1000 μg L-1 (SF-NAC) and 31.25-1000 μg L-1 for SF-CYS and SF-GSH. The matrix recoveries ≥ 85 %. The method was validated by determining transport and metabolism of SF in Caco-2 monolayers and in broccoli seed powder. Results indicate that 70% of SF was absorbed, transported, and metabolized (SF-CYS, SF-GSH, SF-NAC) in the Caco-2 monolayers after 4 h incubation. This is a robust, time- and cost-efficient method for measuring GR, SF and its metabolites in biological samples and is suitable for high throughput application in clinical trials.

Novel and efficient UHPLC/MS/MS method for assessing glucoraphanin and sulforaphane bioavailability in biological samples

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