Fig. 4.

LC-MS separation of GP-derivatised cholestenoic acids, and mono- and dihydroxycholesterols in CSF. (A) RIC for the [M]⁺ ions of (upper panel) 7αH,3O-CA(25R/S) (564.3796 ± 5 ppm) found in CSF, and (lower panel) [²H₃]7αH,3O-CA(25R/S) (567.3984 ± 5 ppm). (B) RIC for the [M]⁺ ions of (upper panel) 7αH,3O-CA(25R/S) + 3β,7α-diHCA(25R/S) (569.4110 ± 5 ppm) found in CSF, and (lower panel) [²H₃]7αH,3O-CA(25R/S) (572.4298 ± 5 ppm). In (A) the derivatisation agent was [²H₀]GP and in (B) [²H₅]GP. (C) RIC for the [M]⁺ ions of diH,3O-CA isomers (585.4059 ± 5 ppm) found in CSF (upper panel), note the triHCA equivalents are absent. TIC for the MS³ fragmentation (585.4 → 501.3→) for diH,3O-CA isomers (2nd panel). MRM (585.4 → 501.3→427.3) targeting 7α,24-diH,3O-CA (3rd panel), and MRM (585.4 → 501.3→455.3) targeting 7α,25-diH,3O-CA (bottom panel). See Supplemental Figures S4P & S4Q for relevant fragmentation schemes. Chromatograms in (A–C) are from non-hydrolysed CSF. (D) RIC of the [M]⁺ ions of monohydroxycholesterols (539.4368 ± 3 ppm) found in CSF (upper panel). RIC (546.4807 ± 3 ppm) for [²H₇]24R/S-HC, [²H₇]7β-HC, [²H₇]7α-HC and dehydrated [²H₇]5α,6β-diHC (central panel). RIC (545.4744 ± 3 ppm) for [²H₆]25-HC and [²H₆](25R)26-HC (lower panel). (E) TIC for the MS³ fragmentations (555.4 → 471.4→) of 7α,25-diHC and 7α,(25R/S)26-diHC found in CSF (upper panel) and for the fragmentations (561.5 → 477.4→) of [²H₆]7α,25-diHC and [²H₆]7α,(25R/S)26-diHC. Chromatograms (D & E) are for hydrolysed CSF. Coloured dashed lines indicate the coincidence of peaks of the same oxysterol. All chromatograms were recorded over a 17 min gradient.