Analysis of Neuro-Oxysterols and Neuro-Sterols
Much of our understanding of the biochemistry of neuro-sterols and
neuro-oxysterols depends on their reliable detection and quantification
in brain tissue, CSF, plasma and in cells of the nervous systems. Both
gas chromatography – mass spectrometry (GC-MS) and liquid
chromatography (LC)-MS methods have been extensively applied. Gold
standard GC-MS methods are based on the classic paper by Dzeletovic et
al describing the analysis of oxysterols in plasma (Dzeletovic, Breuer,
Lund & Diczfalusy, 1995; Heverin et al., 2004; Schott & Lutjohann,
2015). There are many LC-MS method, with and without the use of
derivatisation. A major study using LC-tandem mass spectrometry (MS/MS)
has been performed by Russell and colleagues in Dallas in which they
analysed over 3,000 serum samples (Stiles, Kozlitina, Thompson,
McDonald, King & Russell, 2014). Their analytical method relied on
multiple reaction monitoring (MRM) exploiting a tandem quadrupole to
achieve maximum sensitivity and with use of multiple isotope labelled
standards for quantification (McDonald, Smith, Stiles & Russell, 2012).
To achieve high sensitivity others have exploited derivatisation in
combination with LC-MS/MS (Honda et al., 2009; Roberg-Larsen et al.,
2014; Sidhu et al., 2015; Xu, Korade, Rosado, Liu, Lamberson & Porter,
2011).
Our preference is to use the Girard P (GP) derivatisation reagent in
combination with enzymatic oxidation in a methodology called
“enzyme-assisted derivatisation for sterol analysis” EADSA (Crick et
al., 2015; Griffiths et al., 2013). The method is applicable to any
sterol/oxysterol with an oxo group or with a hydroxy group amenable to
enzymatic oxidation to a carbonyl. The most commonly used enzyme is
cholesterol oxidase which converts 3β-hydroxy-5-ene sterols to
3-oxo-4-enes and 3β-hydroxy-5α-hydrogen structures to 3-ones (Karu et
al., 2007). The oxo group is then reacted with the GP hydrazine reagent
to give a GP hydrazone which effectively tags the sterol/oxysterol with
a positive charge (Figure 5). The consequence of charge-tagging is a
major improvement in LC-MS sensitivity. Besides enhancing signal, the
charge-tag directs fragmentation in MS/MS or in MS with
multistage-fragmentation (MSn), improving confidence
for identification and allowing the identification of unexpected
sterols/oxysterols (Abdel-Khalik et al., 2018; Griffiths et al., 2019a;
Ogundare et al., 2010). We find the use of MS3 in
ion-trap mass spectrometers particularly valuable for structural
elucidation, where in a first step the GP-derivative
[M]+ ion is selected (MS1), in a
second step the [M]+ ion is fragmented
(MS2), the major fragmentation route is the loss of
pyridine (Py) from the derivative to give a
[M-Py]+ ion, which is then fragmented in the
MS3 step to reveal structurally informative
fragment-ions (Figure 5). To augment MS3 we like to
combine fragmentation information with exact mass information available
on Fourier transform hybrid and tribrid mass spectrometers. We have
extensively utilised this method to identify and quantify neuro-sterols
and neuro-oxysterols in brain and in CSF and to monitor the import and
export of these molecules into and out of brain (Iuliano et al., 2015).
We have now extended this method to on-surface analysis of tissue slices
to localise neuro-oxysterols and neurosterols in mouse brain (Yutuc et
al., 2020).