Fig. 2.

LC-MS separation of GP-derivatised monohydroxycholesterols (HC). (A) Upper panel, RIC of the [M]⁺ ions of monohydroxycholesterols (539.4368 ± 5 ppm) found in plasma. Lower panel, MRM 539.4 → 455.4→353.3 characteristic of 24R/S-HC. The red dashed line indicates the coincidence of 24S-HC in the upper and lower panels and the black dashed lines indicates where 24R–HC partially overlaps (in time, but not in MRM) with (25R)26-HC. (B) Upper panel, RICs for [²H₇]-labelled monohydroxycholesterols (546.4807 ± 5 ppm). The green arrow indicated the distortion in the [²H₇]24R–HC chromatographic peak as a consequence of the co-eluting and mass spectrometrically-unresolved [M+1]⁺ ion of [²H₆](25R)26-HC (m/z 546.4777). Lower panel, MRM 546.5 → 462.4→353.3 characteristic of [²H₇]24R/S-HC. Note the fragment ion at m/z 353.3 is also evident in MS³ spectra of [²H₇]22R/S–HCO. Coloured dashed lines indicate the coincidence of peaks of the same oxysterol. (C) Upper panel, RICs for [²H₆]-labelled monohydroxycholesterols (545.4744 ± 5 ppm). Lower panel total ion chromatogram (TIC) 545.5 → 461.4→ for [²H₆]-labelled monohydroxycholesterols. (D) RIC for monohydroxycholesterols in plasma (upper panel) and [²H₇]-labelled standards (lower panel) recorded on a shorter chromatographic time scale. (E) RIC for monohydroxycholestenones in plasma (534.4054 ± 5 ppm) and [²H₇]-labelled standards (541.4493 ± 5 ppm). Note in all chromatograms the deuterium labelled oxysterols elute slightly earlier than their non-labelled analogues. Relevant MS³ spectra are presented in Supplemental Figs. S3 and S5. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)