2.9. LC-MS/MS analysis

Standards (10μl) and samples (20μl) were injected into a NUCLEOSIL C18 column (100-5 125/2) fitted with a guard column for the liquid chromatography (LC) analysis. Our previously developed solvent composition in Dias et al., 2018 was used for separation of oxysterols. Solvent A is 70% MEOH, 10% water, 0.1% formic acid and solvent B is 90% isopropanol, 10% MEOH and 0.1% formic acid. A multistep solvent gradient was applied using ACQUITY Ultra performance liquid chromatography (UPLC) quaternary system purchased from Waters. The LC method used here was developed previously by Dias et al., 2018, and was set to 0 minutes 16% B- 7 minutes 16% B; 7 minutes 16% B-11 minutes 24% B; 11 minutes 24%B-25 minutes 100%B; 25 minutes 100%B-30 minutes 100% B; 30 minutes 100% B- 32 minutes 16% B and was held at 16% B up to 48 minutes. Mass spectrometry analysis was performed using a Xevo TQ-S Triple Quadrupole Mass Spectrometer (Waters) and operated with the positive electrospray ionisation (ESI) mode. Nitrogen gas is used for desolvation at 500 °C and a flow rate of 900 L/hour⁻¹ and collision gas argon at a flow rate of 0.15 ml/minute⁻¹ for collision. MRM transitions were set up using authentic oxysterol standards (Avanti Polar) lipids. Standards include- 24(S) hydroxycholesterol (700061P), 25-hydroxycholesterol (700019P), 27-hydroxycholesterol (700021P), 7α-hydroxycholesterol (700034P), 7 ketocholesterol (700015P), 7α,27-dihydroxycholesterol (700136P), 7α,24 (R/S)-dihydroxycholesterol (700119P), 7α,25-dihydroxycholesterol (700078P). The mass spectrometry method was set up with the MRM transitions for 9 analytes using the Intellistart feature in Xevo and are detailed in Table 1. The two best MRM transitions were identified for each analyte-the most abundant MRM as the quantifier and the second most abundant was used as the qualifier. Authentic oxysterols were dissolved in 50% methanol, 0.1% formic acid and working stocks were prepared in a range of 10ng.μl⁻¹ to 50ng.μl⁻¹. Analytes were infused directly into the MS system and transitions were determined manually by inspection of the chemistry of the analyte and optimisation of the ionisation parameters using the inbuilt Intellistart feature of the Xevo TQs system. Infusions were performed at 10–20μl min⁻¹ and the cone voltage and collision energy were optimised in order to obtain the 5 best MRM transitions. 7α,24Sdihydroxychoelsterol (24SdiOHC) and 7α,25Sdihydroxychoelsterol (25diOHC) had identical MS/MS transitions and co-eluted with identical retention times. So, these two analytes were analysed together as 24S25diOHC for detection and quantification. For every batch of standards and samples, an unprocessed standard mixture and pooled samples were used as the quality control for the analyses.

MRM transitions for oxysterols.