Comparison of simple extraction procedures in liquid chromatography-mass spectrometry based determination of serum 7α-hydroxy-4-cholesten-3-one, a surrogate marker of bile acid synthesis.
The serum concentration of 7α-hydroxy-4-cholesten-3-one (C4), a marker of cholesterol 7α-hydroxylase activity, has recently become an attractive diagnostic tool for researchers interested in cholesterol and bile acid metabolism. The rapidly increasing demand of C4 measurement led to the development of various fast, mostly mass spectrometry-based analytical methods. Our aim was to compare four simple (i.e., not requiring solid phase extraction) extraction procedures (two "one-phase", and two "two-phase") in terms of basic analytical performance and their labouriousness. All methods exhibited comparable extraction recoveries (ranging from 88 to 97%) and intra-assay precision (variation coefficients below 10%), and failed in the removal of phospholipids. Although marked differences were observed in desalting and deproteination, all methods can be considered satisfactory. Simple acetonitrile precipitation can be recommended if a fast extraction and minimal hands-on time is preferred; while two-phase ammonium sulphate:acetonitrile extraction should be chosen when maximal deproteination is required.
Institute of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University in Prague, Kateſinská 32, 12108 Prague, Czech Republic. Electronic address: martin.lenicek@lf1.cuni.cz.
Martin
M
LeníĿek
Institute of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University in Prague, Kateſinská 32, 12108 Prague, Czech Republic; 4th Department of Internal Medicine, 1st Faculty of Medicine, Charles University in Prague, Kateſinská 32, 12108 Prague, Czech Republic.
Marek
M
Vecka
Institute of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University in Prague, Kateſinská 32, 12108 Prague, Czech Republic.
Kateſina
K
Žížalová
Institute of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University in Prague, Kateſinská 32, 12108 Prague, Czech Republic; 4th Department of Internal Medicine, 1st Faculty of Medicine, Charles University in Prague, Kateſinská 32, 12108 Prague, Czech Republic.
Libor
L
Vítek
D001647:Bile Acids and Salts / Q000096:biosynthesis*; D015415:Biomarkers / Q000097:blood; D002783:Cholestenones / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010743:Phospholipids / Q000302:isolation & purification
D001647:Bile Acids and Salts; D015415:Biomarkers; D002783:Cholestenones; D010743:Phospholipids; C002656:7 alpha-hydroxy-4-cholesten-3-one
7α-Hydroxy-4-cholesten-3-one; Bile acids; Cholesterol 7α-hydroxylase; Mass spectrometry