Quantitative Oxysterol Analysis via EADSA

We adopted a charge-tagging approach utilising “enzyme-assisted derivatisation for sterol analysis” (EADSA) to enhance liquid chromatography (LC) separation and mass spectrometry (MS) detection of oxysterols [17 (link),25 (link),26 (link)]. This involves the stereospecific enzymatic oxidation of the 3β-hydroxy-5-ene function in oxysterols (and sterols) to a 3-oxo-4-ene group and subsequent reaction with the cationic Girard P (GP) hydrazine reagent to give charged GP-hydrazones compatible with chromatographic separation using reversed phase solvents and high-sensitivity analysis by electrospray ionisation (ESI)-MS and MS with multistage fragmentation (MSⁿ) (Fig. S1). GP-derivatives give intense [M]⁺ ions in ESI and informative MS² and MS³ spectra. As some oxysterols naturally contain a oxo group they give GP-derivatives even in the absence of oxidising enzyme. However, oxysterols containing a native 3-oxo group are readily differentiated from oxysterols oxidised to contain one by dividing each sample in two and performing derivatisation without oxidising enzyme on one portion of the sample (Fraction B) and performing derivatisation with added enzyme on the second portion (Fraction A), and by exploiting differentially isotope labelled GP reagents to allow discrimination by mass (Fig. S1).
Unless otherwise indicated, materials and methods are as described by Griffiths and co-workers [17 (link),25 (link),26 (link)]. Quantification was achieved using isotope dilution mass spectrometry. No hydrolysis or solvolysis steps were performed. 3β,7α-Dihydroxycholest-5-en-(25S)26-oic acid was supplied as a mixture with 3β,7α-dihydroxycholest-5-en-(25R)26-oic acid (1:3, mole:mole) as the [3α,7β-²H₂] compounds by Avanti Polar Lipids (Alabaster, AL, USA).