6495b qqq mass spectrometer

Two hundred microliter of the supernatant of each sample was dried under a gentle nitrogen gas flow. The residue was resuspended in 100 μl of a 13C3-cholesterol internal standard solution. Along with the sample preparation, serially diluted standard solutions of sterols were prepared in the same internal standard solution. 100 μl of each sample solution or each standard solution was mixed with 100 μl of 20 mM dansyl chloride solution and 100 μl of 25-mM dimethylaminopyridine solution. The mixture was allowed to react at 50 °C for 60 min. After reaction, the solution was dried in a speed-vac concentrator, and the residue was reconstituted in 400 μl of acetonitrile. 10 μl aliquots were injected onto a C18 UPLC column (2.1 × 50 cm, 1.7 μm) to run UPLC-MRM/MS on an Agilent 1290 UHPLC system coupled to an Agilent 6495B QQQ mass spectrometer operated in the positive-ion mode, using 0.1% formic acid in water acetonitrile/isopropanol (1:1) as the LC mobile phase for binary-solvent gradient elution at 0.35 ml/min and at 60 °C. Concentrations of individual sterols were calculated with the analyte-to-internal standard peak area ratios measured from injections of sample solutions to interpolate the constructed linear regression calibration curves of individual sterols in appropriate concentration ranges.