It is well known that oxysterols (OHCs), the oxygenated derivatives of cholesterol, such as 22(R)-OHC, 24(S)-OHC and 27-OHC, regulate lipid and cholesterol metabolism, by acting as natural ligands of Liver X nuclear receptor (LXR). In 2011, we discovered that a dihydroxycholesterol (di-OHC), 7a,25-OHC, is the natural ligand for a GPCR (EBI2) and its concentration gradient directs B-cell migration [Nature, 2011, 475, 519-523]. In working with oxysterols, it is often required to separate structurally very similar isomers (e.g., 7a25, 7�25, 7a27, 7�27), and we have developed various methods to separate the isomers in their native forms, using gas chromatography, normal- and reversed-phase liquid chromatography, as well as supercritical fluid chromatography.Di-OHCs usually exist in low concentrations in biological systems, due to their high affinity to receptors (sub-nM); And, because there is no nitrogen atom in oxysterols, their electrospray ionization efficiencies are really low, which makes the quantification of di-OHCs rather difficult by the usual LC-MS/MS method. To solve this problem, we have adopted a derivatization method developed by Wang and Griffiths [e.g., Biochimica et Biophysica Acta 2011, 1811, 784-799] using Girard Reagent P (GP) to add a permanent positive charge to the oxysterols which results in dramatically enhanced electrospray signals. Here we will report an rpHPLC method to separate the GP-labeled di-OHCs which allows their quantification by MS/MS. To our knowledge, such a separation method has not been reported previously; and we have used the combined derivation-LC-MS/MS method successfully in de-orphanizing another important receptor responsible for many immune disorders. In addition, we use the method routinely in-house in pharmacokinetics studies.

Separation and quantification of dihydroxycholesterol derivatives by rpHPLC-MS in biological samples

247th National Meeting (2014)

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