Ctc pal autosampler

The HPLC system consisted of an Agilent 1260 Series HPLC system (Agilent Technologies, Palo Alto, CA, USA), a Shimadzu, System Controller, SCL-10A Vp, Pump, LC 10AD Vp Solvent Degasser (DGU14A, Shimadzu, Columbia, MD, USA), and an CTC PAL autosampler (Zwingen, Switzerland). The analytical column was XBridge C₁₈, 50 × 2.1 mm, 5µm (Waters Corp, Milford, MA, USA). The column temperature was maintained at 25 °C. Mobile phase A was water and mobile phase B was 0.1 % formic acid in acetonitrile. The elution pump (Agilent 1260) gradient was: 0–1.0 min 0.0% B at 0.4 mL min⁻¹ flow, 1.1–3.0 min from 50% to 90% B at 0.4 mL min⁻¹ flow rate, 3.1–7.0 min 90% B at 0.8 mL min⁻¹ flow rate and 7.1–10.0 min 0.0% B at 0.8 mL min⁻¹ flow rate. The makeup pump (Shimadzu) ran an isocratic gradient at 50:50 mobile phase A: mobile phase B and at flow rate of 0.2 mL min^-1. The switching valve program was: 0–1.8 min at position A (loading sample into column and desalting), 1.8–3.0 min at position B (elution to MS source) and 3.0 to 10.0 min at position A (column flushing and re-equalibration). Injection volume was 50 µL and total run time was 10.0 min.

Dissected corpus callosum samples were homogenized for 10 min in 500 μL of Folch solvent spiked with the internal standard cholesterol-d₇. Samples were spun down at 16,000 × g to remove particulate and the supernatant was transferred to a clean tube and dried under N₂. Samples were reconstituted in 100 μL of CHCl₃:MeOH (2:1) and injected (10 μL) onto a Luna C18(2) column (2 × 100, Phenomenex) at a flow rate of 0.45 mL/min. Analytes were separated using a Shimadzu chromatograph with a CTC PAL autosampler. Solvent A consisted of 1:1 H₂O:ACN with 0.1% formic acid and Solvent B consisted of 2:3 ACN/IPA with 0.1% formic acid. The gradient started at 65%B and increased to 100%B over 13 min. The column was held at 100%B for 3 min before returning to starting conditions for equilibration. Cholesterol (369.3→147.3) and cholesterol-d₇ (376.4→147.3) were analyzed by a Sciex 5000 triple quadrupole using selected reaction monitoring in positive ion mode. Peak area of cholesterol was normalized to the peak area of cholesterol-d₇ and tissue weight, and reported as a relative amount.