TY - JOUR
T1 - Extraction and Analysis of Sterols in Biological Matrices by High Performance Liquid Chromatography Electrospray Ionization Mass Spectrometry
AU - McDonald, Jeffrey G.
AU - Thompson, Bonne M.
AU - McCrum, Erin C.
AU - Russell, David W.
PY - 2007
Y1 - 2007
N2 - We describe the development of a high performance liquid chromatography mass spectrometry (HPLC-MS) method that allows the identification and quantitation of sterols in mammalian cells and tissues. Bulk lipids are extracted from biological samples by a modified Bligh/Dyer procedure in the presence of eight deuterated sterol standards to allow subsequent quantitation and determination of extraction efficiency. Sterols and other lipids are resolved by HPLC on a reverse-phase C18 column using a binary gradient of methanol and water, both containing 5 mM ammonium acetate. Sterol identification is performed using an Applied Biosystems (Foster City, CA) 4000 QTRAP® mass spectrometer equipped with a TurboV electrospray ionization source and operated in the positive (+) selected reaction monitoring (SRM) mode. The total run time of the analysis is 30 min. Sterols are quantitated by comparison of the areas under the elution curves derived from the detection of endogenous compounds and isotopically labeled standards. The sensitivity of the method for sterol detection ranges between 10 and 2000 fmol on-column. Cultured RAW 264.7 mouse macrophages contain many different sterols, including the liver X receptor (LXR) ligand 24,25-epoxycholesterol. Tissues such as mouse brain also contain large numbers of sterols, including 24(s)-hydroxycholesterol, which is involved in cholesterol turnover in the brain. The extraction procedure described is flexible and can be tailored to sample type or information sought. The instrumental analysis method is similarly adaptable and offers high selectivity and sensitivity.
AB - We describe the development of a high performance liquid chromatography mass spectrometry (HPLC-MS) method that allows the identification and quantitation of sterols in mammalian cells and tissues. Bulk lipids are extracted from biological samples by a modified Bligh/Dyer procedure in the presence of eight deuterated sterol standards to allow subsequent quantitation and determination of extraction efficiency. Sterols and other lipids are resolved by HPLC on a reverse-phase C18 column using a binary gradient of methanol and water, both containing 5 mM ammonium acetate. Sterol identification is performed using an Applied Biosystems (Foster City, CA) 4000 QTRAP® mass spectrometer equipped with a TurboV electrospray ionization source and operated in the positive (+) selected reaction monitoring (SRM) mode. The total run time of the analysis is 30 min. Sterols are quantitated by comparison of the areas under the elution curves derived from the detection of endogenous compounds and isotopically labeled standards. The sensitivity of the method for sterol detection ranges between 10 and 2000 fmol on-column. Cultured RAW 264.7 mouse macrophages contain many different sterols, including the liver X receptor (LXR) ligand 24,25-epoxycholesterol. Tissues such as mouse brain also contain large numbers of sterols, including 24(s)-hydroxycholesterol, which is involved in cholesterol turnover in the brain. The extraction procedure described is flexible and can be tailored to sample type or information sought. The instrumental analysis method is similarly adaptable and offers high selectivity and sensitivity.
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U2 - 10.1016/S0076-6879(07)32006-5
DO - 10.1016/S0076-6879(07)32006-5
M3 - Article
C2 - 17954216
AN - SCOPUS:41249093188
SN - 0076-6879
VL - 432
SP - 145
EP - 170
JO - Methods in Enzymology
JF - Methods in Enzymology
ER -