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

⿢Extraction procedures for 7α-hydroxy-4-cholesten-3-one determination were compared.⿢Solid phase extraction is not needed for 7α-hydroxy-4-cholesten-3-one quantification.⿢Acetonitrile precipitation offers good performance with minimal time demands.

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.