| Objective: A method for the determination of cholesterol in A549 cells by high performance liquid chromatography（HPLC）and a method for the determination of 27-hydroxyl cholesterol in the lung tissue by gas chromatography tandem mass spectrometry（GC-MS）were established.The detecting results were used as indicators to identify the degree of tissue cell foaming,and the specific tissue cell foam was used to provide technical support for assessing the effect of CYP27 pathway on cholesterol metabolism.Methods: A549 cells were homogenized on ice by sonication,and the triglycerides and proteins in the cell lysate were removed by KOH ethanol solution and trichloroacetic acid respectively.The intracellular cholesterol was extracted with the mixed solvent of n-hexane-isopropanol.The mobile phase consisted of acetonitrile-isopropanol（67:33）at a flow rate of 1 mL / min and the detection wavelength of 206 nm.The cholesterol content was determined by HPLC.The column was ZORBAX SB-C18（4.6 mm × 150 mm,5 μm）Taking lung tissues to high-speed homogenate,got homogeneous slurry.The homogenate was Saponified with ethanolic KOH and extracted with ether as a homogenate: Chloroform（3: 5）.The organic phase was washed with water until neutral,and the solvent was removed under reduced pressure.Chloroform complex solution was derived with a solution of pyridine / hexamethyldisilazane / trimethylchlorosilane（3: 2: 1）at 60 ℃ for 60 min.,The mixture was heated to dryness in a nitrogen atmosphere,dissolved with chloroform,and taken into the GC / MS for analyzing.The column is HP-5MS quartz capillary column（30mm’0.25mm’0.25mm）,ionization mode EI,injection volume 2.0ml,carrier gas is high purity helium.Results: The linear range of the method is 5 ng · μL-1-100 ng · μL-1 which was excellent.（R=0.999 9）The peak time of the intracellular cholesterol was consistent with the peak time of the standard solution,and the peak was at 8.2 min.The RSDs were lower than 3.0%.The average recovery was 94.89% and the RSD was 2.86%（n = 6）.The accuracy of the assay was 94.89% and RSD was 2.86%（n = 6）.The total ion current of 27 hydroxyl cholesterol in lung tissue was peaked at 55.39 min,and the mass-to-charge ratio of the fragments were m / z 456 and m / z 546.The method can be qualitative measured.But the recoveries of the samples were out of the range of specified range,from 85% to 110%.Conclusion: The established method of high performance liquid chromatography（HPLC）for detecting intracellular cholesterol is simple,accurate,reproducible and with high precision.The recovery rate is also within the range of the requirements.The method can be used to batch detection for analyzing the intracellular cholesterol horizontal.And it can be used to lay the foundation about identifying the degree of foam cells,the relationship of specific tissue with cell foam cells and the study of tissue damage,to explore the abnormal lipid metabolism and organ damage and further study of the relationship between cholesterol metabolism and lung injury.Gas chromatography-tandem mass spectrometry was used to detect 27-hydroxy cholesterol in lung tissue.The method was effective in detecting 27-hydroxy cholesterol in tissues.However,at the stage of the methodological verification,the calibration indexes are not required,and the stability and repeatability need to be improved. |
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