24-Hydroxycholesterol LC-MS Analysis

Samples were injected onto a SCIEX 6500 QTRAP Mass Spectrometer with an Eclipse XDB-C18 (3 × 100 mm, 3.5 μm) column using a gradient elution using mobile phase A of water with 0.1% formic acid and mobile phase B of methanol:acetonitrile (50:50, v:v) at a flow rate of 600 μL/min at 50°C. The gradient was maintained at 80% B for 0.01 min, then ramped linearly to 100% B in 3 min, then returned linearly after 3.3 min to 80% B in 4.30 min with a total runtime of 11 min. The reaction product 24-hydroxycholesterol was monitored (parent m/z 385.0 and daughter m/z 367.0) and quantified in comparison to d7-24-hydroxycholesterol standard. Dose response curves were generated by graphing 24-hydroxycholesterol levels vs. inhibitor concentrations.

Male CD1 mice (Vital River, Beijing, China) were housed in plastic cages with metal covers (GAU-2, Suzhou Fengshi Laboratory animal equipment Co., LTD, Suzhou, China) in groups of 5 under controlled conditions (temperature of 20−26°C and 12:12 light-dark cycle, lights on at 5:00 am) with a standard rodent diet and water available ad libitum. The mice were acclimated for 5 days under these conditions before testing. The animals were randomly assigned to experimental groups; each mouse was used only once. At the time of the experiment mice were 5–6 weeks old. The experiment was approved by the Institutional Animal Care and Use Committee of Shanghai ChemPartner Co., Ltd.

Under the assay conditions, both 24S and 24R-hydroxycholesterol were detected in a single peak and quantified as total 24-hydroxycholesterol. Total 24-HC was measured using ester hydrolysis, liquid-liquid extraction, and measurement by LC-MS/MS. Brain or plasma samples were first diluted with PBS at a ratio of 3 mL of PBS to 1 g of tissue. Tissue homogenate was prepared using a bead mill type homogenizer (Spex-certiprep Genogrinder).

The 50 μL sample with 5 μL of 24-hydroxycholesterol-d6 internal standard was added to 100 μL of 0.35 N KOH in methanol and heated to 80°C for 2 h. Hydrolyzed plasma or tissue homogenate (155 μL) was mixed with 200 μL of DI water and extracted with 1 mL of methyl-t-butyl ether. The organic layer was separated and taken to dryness under nitrogen at 50°C. The dry residue was reconstituted in 100 μL of 80% methanol in DI water. Reconstituted sample (5 μL) was injected on an ACE 3C18 0.5 mm × 1.0 mm HPLC column. Gradient elution was performed using an Eksigent LC200 HPLC system running a simple water:acetonitrile gradient from 70% acetonitrile to 95% acetonitrile over 4.75 min at a flow rate of 100 μL/min.

Calibrators and assay quality controls were made by spiking 24S-hydroxycholesterol-d7 into control mouse plasma or brain homogenate and preparing them as samples. Detection of 24S-HC was accomplished using a Sciex Qtrap 5500 mass spectrometer running selected reaction monitoring of the analyte and internal standard. Ions were formed using a TurboV electrospray ion source operated in the positive ion mode. Sample concentrations were determined using the peak area ratio of analyte to internal standard and the least squares linear regression equation from the standard curve.

Assay acceptance criteria for each LC-MS/MS run were ±20% accuracy compared to the nominal spiked concentration and ±20% CV. Endogenous quality controls made from control plasma or brain tissue were used to track longitudinal assay performance. Endogenous quality controls must reproduce within 3 SDs of the latest 20 measurements.