Dear Sir. Concerning your issue about the protocol for measuring 25-hydroxycholesterol (25-OHC) in cells.The measurement can be carried out as follows:  d6-25-Hydroxycholesterol was added to 500 μL of serum and CSF as an internal standard, and alkaline hydrolysis was performed in 2 mL of 1 N ethanolic KOH at 50°C for 2 h. The hydrolyzed sample was subsequently neutralized to pH 7 with 75 μL of phosphoric acid. The mixture was then centrifuged for 5 min at 1000 g and the clear supernatant was collected for subsequent solid phase extraction (SPE). A large amount of endogenous substance such as cholesterol, can give rise to poor chromatographic separation and ion suppression. Therefore, SPE was used for the pre-concentration of OHCs and clean-up of interferences such as cholesterol from the matrix. We modified the sample preparation using SPE based on previous research [38]. In our SPE process, cholesterol was monitored using the multiple reaction monitoring (MRM) mode to evaluate the removal of cholesterol from the matrix, and we found that most of the cholesterol was removed. As a result, serious effects on chromatographic separation (interference peaks) and ion suppression by cholesterol were not observed. It was also checked for validation and sample analysis. The sample was loaded onto an Oasis HLB extraction cartridge (150 mg, Waters Corporation, Massachusetts, USA) that had been preconditioned with 1 mL of hexane/2-propanol (50:50, v/v), 1 mL of methanol, and 2 mL of water. The extraction cartridge was washed with 4 mL of methanol/water (75/25, v/v) and briefly dried under vacuum. The analyte was then eluted with 3 mL of hexane/2-propanol (50:50, v/v) using gravity, and was evaporated to dryness with an evaporator (Rotavapor, Buchi, Flawil, Switzerland) at 35°C. For serum, the residue was dissolved in 100 μL of methanol, and 2 μL was injected into the LC-MS/MS system. In addition, several steps were added for the derivatization of CSF OHC (OHCCSF). For OHCCSF, cholesterol removed CSF was converted to picolinic acid derivatives and was evaluated as blank for calibration. As a result, exogenous 24-, 25- and 27-OHCs were not observed in the blank CSF. For PE, a reagent mixture was prepared with 2-methyl-6-nitrobenzoic anhydride (10 mg), 4-dimethylaminopyridine (3 mg), picolinic acid (8 mg), pyridine (150 μL), and triethylamine (20 μL). The freshly prepared reagent mixture (170 μL) was added to the dried residue and incubated at 80°C for 1 h to derivatize. After the addition of 1 mL of hexane, the mixture was vortexed and centrifuged for 5 min at 1000 g. The clear supernatant was collected and evaporated at 80°C under a steam of nitrogen gas. The residue was dissolved in 50 μL of methanol, and 2 μL were injected into the LC-MS/MS system. For more details, I think the following below links may help you in your analysis:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5152860/pdf/pone.0167819.pdf

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355309/#R33

Thanks

Thank you very much Isam Eldin Hussein Elgailani. ·