Metabolites were analyzed using HPLC-ESI-TOF MS as previously described [28 (link)]. Briefly, their separation was conducted on a HPX-87H column with 8% cross-linkage (150-mm length, 7.8-mm inside diameter, and 9-μm particle size; Bio-Rad, Richmond, CA) using an Agilent Technologies 1100 Series HPLC system. Metabolites were eluted isocratically with a mobile-phase composition of 0.1% formic acid in water at a flow rate of 0.5 ml/min. Drying and nebulizing gases were set to 13 liters/min and 30 lb/in2, respectively, and a drying-gas temperature of 330°C was used throughout. ESI was conducted in the negative ion mode and using a capillary voltage of −3,500 V. Luteolin, chrysoeriol (ChromaDex, Inc., Irvine, CA), tricin (ChromaDex, Inc., Irvine, CA), and selgin were quantified via 8-point calibration curves of authentic standard compounds for which the R2 coefficients were ≥ 0.99. Stock solutions of metabolites used for enzymatic assays and standard curves were quantified spectrophometrically using published molar absorption coefficients: S-adenosylmethionine (ε = 15,400 L.mol-1.cm-1 at 254 nm) [29 (link)], Luteolin (ε = 14,790 L.mol-1.cm-1 at 350 nm) [30 (link)], chrysoeriol (ε = 15,400 L.mol-1.cm-1 at 347 nm) [30 (link)], and tricin (ε = 41,000 L.mol-1.cm-1 at 349 nm) [31 (link)].
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