br Analytical improvements Since our review of Oxysterol Met
Analytical improvements Since our review of “Oxysterol Metabolomes” in 2011 , there have been a number of improvements in analytical methods for the analysis of oxysterols and related compounds. McDonald and colleagues in Dallas have refined their LC-MS methods which are now applicable for analysis of more than 50 sterols and oxysterols from only 200 μL of glycerol phosphate with detection limits of 1 ng/mL , . In an effort to improve sensitivity, there has been a revival of interest of exploiting derivatisation chemistry to enhance LC-MS analysis of oxysterols. Our preference is for EADSA where oxysterols are first oxidised at C-3 to a 3-oxosteroid, usually by conversion of the 3β-hydroxy group to a 3-oxo with cholesterol oxidase, then reaction with the Girard P (GP) reagent to enhance ESI signal and direct fragmentation upon MS/MS or MSn (Fig. 6) , . A disadvantage of this method is that it requires duplicate analysis, (a) with, and (b) without, addition of cholesterol oxidase to differentiate between compounds that naturally contain a 3-oxo group and those that naturally contain a 3β-hydroxy group (see Fig. 6B). We have now introduced a [2H5]GP reagent which allows the (a) and (b) fractions to be analysed simultaneously by LC-MSn. Derivatisation with Girard reagents offers improved specificity and sensitivity and this has been exploited by other groups using mainly the Girard T (GT) reagent rather than GP , , . Impressively, using EADSA and GT derivatisation Roberg-Larsen et al. were able to show that 26-HC is elevated in exosomes from an ER+ breast cancer cell line derived from only 200,000 cells . This group use low flow-rate chromatography with ESI to enhance sensitivity and incorporate on-line solid-phase extraction for sample clean-up. Of the various other derivatisation methods, the one developed by Jiang et al.  where the free hydroxy group(s) is derivatised with N,N-dimethylglycine (DMG) has become popular for identification of NP-C disease (Fig. 6C) , . This method suffers from an inherent lack of specificity on account of the prevalence of hydroxy-containing molecules in nature. An alternative derivatisation targeted at hydroxy groups is derivatisation to picolinic acid esters  or nicotinic acid esters . Sidhu et al. have exploited the latter derivatisation to analyse oxysterols in CSF and plasma  (Fig. 6C). A new derivatisation for sterols in general is reaction with PTAD (Fig. 6A). Although this derivatisation is well known for vitamins D analysis, Liu et al. have shown that it can be used for unsaturated sterols in general . By careful choice of conditions, the reaction can be “tuned” to favour reaction with B-ring unsaturated sterols like cholesterol via an “ene” reaction, or with side-chain double bonds as in desmosterol, also via an “ene” reaction, or with conjugated dienes like 7-DHC in a Diels-Alder reaction. The PTAD derivatisation offers improvement in sensitivity and also provides a route to stabilising reactive dienes . Matrix-assisted laser desorption/ionisation (MALDI)-MS imaging is an exciting technology for determining the location of molecules in a tissue. By coating tissue, e.g. 10 μm thick coronal section of rodent brain, with matrix and mounting the tissue on a stage then moving the stage relative to the laser, it is possible to record mass spectra at discrete pixels and reconstruct an image of a specific ion over the tissue. However, as in all MS, only molecules that can be ionised will be observed. Cobice et al. have cleverly exploited GT derivatisation to image 7O-C in mouse brain . In the absence of GT derivatisation any signal from 7O-C is hidden by back-ground noise. Cobice et al. found that by using GT derivatisation a significant increase in 7O-C signal was observed in brain sections from hydroxysteroid dehydrogenase (HSD) 11B1 deficient mice . HSD11B1 is the enzyme which converts 7O-C to 7β-HC and in its absence 7O-C accumulates.