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. 2020 Feb 13;25(4):829.
doi: 10.3390/molecules25040829.

Measuring Sulforaphane and Its Metabolites in Human Plasma: A High Throughput Method

Annie Langston-Cox  1 , Dovile Anderson  2 , Darren J Creek  2 , Kirsten Palmer  1 , Euan M Wallace  1 , Sarah A Marshall  1
Affiliations

Measuring Sulforaphane and Its Metabolites in Human Plasma: A High Throughput Method

Annie Langston-Cox et al. Molecules. .

Abstract

(1) Background: There is increasing understanding of the potential health benefits of cruciferous vegetables. In particular sulforaphane (SFN), found in broccoli, and its metabolites sulforaphane-glutathione (SFN-GSH), sulforaphane-cysteine (SFN-Cys), sulforaphane cysteine-glycine (SFN-CG) and sulforaphane-N-acetyl-cysteine (SFN-NAC) have potent antioxidant effects that may offer therapeutic value. Clinical investigation of sulforaphane as a therapeutic antioxidant requires a sensitive and high throughput process for quantification of sulforaphane and metabolites; (2) Methods: We collected plasma samples from healthy human volunteers before and for eight hours after consumption of a commercial broccoli extract supplement rich in sulforaphane. A rapid and sensitive method for quantification of sulforaphane and its metabolites in human plasma using Liquid Chromatography-Mass Spectrometry (LC-MS) has been developed; (3) Results: The LC-MS analytical method was validated at concentrations ranging between 3.9 nM and 1000 nM for SFN-GSH, SFN-CG, SFN-Cys and SFN-NAC and between 7.8 nM and 1000 nM in human plasma for SFN. The method displayed good accuracy (1.85%-14.8% bias) and reproducibility (below 9.53 %RSD) including low concentrations 3.9 nM and 7.8 nM. Four SFN metabolites quantitation was achieved using external standard calibration and in SFN quantitation, SFN-d8 internal standardization was used. The reported method can accurately quantify sulforaphane and its metabolites at low concentrations in plasma; (4) Conclusions: We have established a time- and cost-efficient method of measuring sulforaphane and its metabolites in human plasma suitable for high throughput application to clinical trials.

Keywords: Liquid Chromatography–Mass Spectrometry; pharmacokinetic; sulforaphane.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Molecular structures of sulforaphane and its metabolites: Sulforaphane (SFN) sulforaphane-glutathione (SFN-GSH), sulforaphane-cysteine (SFN-Cys), sulforaphane cysteine-glycine (SFN-CG) and sulforaphane-N-acetyl-cysteine (SFN-NAC).
Figure 2
Figure 2
Extracted ion chromatograms of 7.8 nM spiked extracted plasma. SFN and SFN-CG peaks were produced due to in-source fragmentation are marked with arrows. Transitions from the top to the bottom: SFN-Cys, SFN-d8, SFN-NAC-d8, SFN-CG, SFN-GSH, SFN, and SFN-NAC. Peaks produced by in source ionization marked with “ * ”.
Figure 3
Figure 3
Pharmacokinetic profiles of SFN and metabolites in plasma taken from participant one (dotted line) and participant two (solid line) over 8 h. Metabolites are: (a) SFN, (b) SFN-Cys, (c) SFN-GSH, (d) SFN-CG, (e) SFN-NAC and (f) Total all metabolites (combined value of all metabolites). Y-axis represents measured concentration in ng/ML.
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