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1 High sensitive determination of oxysterols in human plasma using gas chromatography-mass spectrometry (GC-MS) Saori Nakagawa and Susumu Yamato Oxysterols, cholesterol oxidation products, have been important markers for various diseases. For example, it is suggested that 24S-hydroxycholesterol, 27-hydroxycholesterol and 7-ketocholesterol can be used as a marker of Alzheimer's disease, cerebrotendinous xanthomatosis and diabetes mellitus, respectively. Therefore, it is necessary to determine the concentration levels of individual oxysterols in plasma. In this report, we have described a pretreatment procedure of human plasma for the simultaneous determination of oxysterols using gas chromatography-mass spectrometry (GC-MS). Pretreatments were carried out as follows; extraction from lipoprotein, saponification by hydrolyzing oxysterols esters, extraction with n-hexane, solid-phase extraction to remove the large levels of cholesterol in plasma, elution of oxysterols from their solid-phase, and their derivatization by trimethylsilyl reagent. Twelve oxysterols were simultaneously determined using this pretreatment procedure and a GC-MS analysis. The procedure is a simple and useful one for application to human plasma. Oxysterol, Plasma, GC-MS, Solid phase extraction, Biomarker
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3 GC QP2010 ( Shimadzu ) MS GCMS-QP2010 plus ( Shimadzu ) Column DB-5MS ( 30 m 0.25 mm, 0.25 m thickness ) Column oven 180 for 1 min 20/min /min 300 for 15 min Carrier gas Helium Flow rate 0.96 ml/min Injection temperature 250 Ion source temperature 250 Sample injection mode splitless Injection volume 5 L m/z Oxysterols Quantities ion Certificate ion 7 -hydroxycholesterol hydroxycholesterol hydroxycholesterol S- hydroxycholesterol , R- hydroxycholesterol , hydroxycholesterol S- hydroxycholesterol hydroxycholesterol , 327, hydroxycholesterol ketocholesterol , 382 cholesterol-5, 6 -epoxide , 459 cholesterol-5, 6 - epoxide , hydroxycholesterol -26, 26, 26,27, 27-d5 (I.S.) I.S., internal standard
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7 Liner regression equation Correlation coefficient 7 -hydroxycholesterol y x hydroxycholesterol y x hydroxycholesterol y x S- hydroxycholesterol y x R- hydroxycholesterol y x hydroxycholesterol y x S- hydroxycholesterol y x hydroxycholesterol y x hydroxycholesterol y x ketocholesterol y x Cholesterol-5, 6 -epoxide y x Cholesterol-5, 6 - epoxide y x Recovery represents the mean standard deviation (n=4). Sjövall Björkhem I, Reihnér E Björkhem I, Lütjohann D, Diczfalusy U, Ståhle L
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