Bioassay-directed fractionation for discovery of bioactive neutral lipids guided by relative mass defect filtering and multiplexed collision-induced dissociation

We report a synergistic method using bioassay-directed liquid chromatography fractionation and time-of-flight mass spectrometry to guide and accelerate bioactive compound discovery. To steer purification and assays toward anticipated neutral lipid activators of a constitutive androstane receptor splice variant, a relative mass defect filter was calculated, based on the ratio of the mass defect to the measured ion mass, and used to reduce the number of candidate ion masses. Mass measurements often lack sufficient accuracy to provide unambiguous assignments of elemental compositions, and since the relative mass defect reflects fractional hydrogen content of ions, this value is largely determined by the hydrogen content of a compound's biosynthetic precursors. A relative mass defect window ranging from 600-1000 ppm, consistent with an assortment of lipids, was chosen to assess the number of candidate ions in fractions of fetal bovine serum. This filter reduced the number of candidate ion m/z values from 1345 to 892, which was further reduced to 21 by intensity and isotope filtering. Accurate mass measurements from time-of-flight mass spectrometry and fragment ion masses generated using nonselective collision-induced dissociation suggested dioctyl phthalate as one of few neutral lipid constituents in the active fraction. The identity of this compound was determined to be di(2-ethylhexyl) phthalate using GC/MS, and it was ranked as a promising candidate for reporter assay screening.