Broccoli Glucosinolate Extraction and Analysis

The glucosinolate extraction, desulfation, and analysis was performed as previously described [15 (link),46 (link)]. Briefly, 10 mL of methanol:water (70:30, v/v), previously heated for 10 min at 70 °C, were added to 0.2 g of broccoli powder followed by 50 μL of a 3 mM solution of sinigrin as internal standard. Samples were vortexed and incubated at 70 °C for 30 min to ensure myrosinase inactivation. Afterward, extracts were left to cool at room temperature and centrifuged (3000× g, 5 min, 4 °C). Glucosinolates were desulfated and purified using disposable polypropylene columns (Thermo Fisher Scientific, Waltham, MA, USA) as previously described [15 (link),46 (link)]. Desulfoglucosinolates were analyzed by HPLC–DAD and HPLC–ESI–MSⁿ.
The identification and quantification of desulfoglucosinolates was performed with the chromatographic method previously described by Villarreal-García et al. [15 (link)]. Individual desulfoglucosinolates were identified based on retention time, ultraviolet (UV) spectra, and their mass-to-charge ratio as compared with authentic standards and previous reports [15 (link),17 (link)]. Individual glucosinolate concentrations were calculated using the response factor methodology to correct for absorbance differences between desulfosinigrin and the other desulfoglucosinolates [47 (link)].