A liquid chromatography-tandem mass spectrometry-based method for the simultaneous determination of hydroxy sterols and bile acids

• Published in: Journal of Chromatography A Vol. 1371; pp. 184 - 195
• Main Authors: John, Clara, Werner, Philipp, Worthmann, Anna, Wegner, Katrin, Tödter, Klaus, Scheja, Ludger, Rohn, Sascha, Heeren, Joerg, Fischer, Markus
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 05.12.2014; Elsevier
• Subjects: adipose tissue; Analytical, structural and metabolic biochemistry; Animals; Bile acids; Bile Acids and Salts - analysis; Biological and medical sciences; blood plasma; Body Fluids - chemistry; Calibration; cholesterol; Chromatography, High Pressure Liquid - methods; derivatization; diet; energy; feces; Feces - chemistry; Fundamental and applied biological sciences. Psychology; gall bladder; HPLC-ESI-QqQ-MS/MS; Hydroxy sterols; inflammation; Investigative techniques, diagnostic techniques (general aspects); Limit of Detection; liquid chromatography; liver; Liver - chemistry; Male; Medical sciences; Mice; Mice, Inbred C57BL; Miscellaneous. Technology; Other biological molecules; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Polar embedded stationary phase; Solid Phase Extraction; Sterol profiling in biological matrices lipid metabolism; Sterols - analysis; tandem mass spectrometry; Tandem Mass Spectrometry - methods; taurine; Terpenes, steroids. Hormones; urine; Bile acids; HPLC-ESI-QqQ-MS/MS; Hydroxy sterols; Polar embedded stationary phase; Sterol profiling in biological matrices lipid metabolism; Steroid; Solid phase extraction; Biological fluid; Tandem mass spectrometry; Chemical analysis; Adipose tissue; Liver; HPLC chromatography; Lipids; Electrospray; Biological material; Chemical enrichment; Tissue; Sample preparation; Bile acid; Clinical biology; Feces; Quantitative analysis; Human; Validation; Healthy subject; Coupled method; Rodentia; Metabolism; Vertebrata; Mammalia; Position isomer; Mouse; Animal; Simultaneous measurement; Sterol
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2014.10.064

•Simultaneous quantification of 34 hydroxy sterols and bile acids via LC-ESI-MS/MS.•Rapid and simple sample clean-up without complex derivatization techniques or solid phase extraction.•Validated for six different biological matrices: plasma, liver, adipose tissue, urine, gall bladder and feces.•Chromatographic baseline separation of multiple isobaric compounds using a novel polar embedded stationary phase.•Good method application was illustrated taking the example of a mice feeding study. Recently, hydroxy sterols and bile acids have gained growing interest as they are important regulators of energy homoeostasis and inflammation. The high number of different hydroxy sterols and bile acid species requires powerful analytical tools to quantify these structurally and chemically similar analytes. Here, we introduce a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for rapid quantification of 34 sterols (hydroxy sterols, primary, secondary bile acids as well as their taurine and glycine conjugates). Chromatographic baseline separation of isomeric hydroxy sterols and bile acids is obtained using a rugged amide embedded C18 (polar embedded) stationary phase. The current method features a simple extraction protocol validated for blood plasma, urine, gall bladder, liver, feces, and adipose tissue avoiding solid phase extraction as well as derivatization procedures. The total extraction recovery for representative analytes ranged between 58-86% in plasma, 85% in urine, 79–92% in liver, 76–98% in adipose tissue, 93–104% in feces and 62–79% in gall bladder. The validation procedure demonstrated that the calibration curves were linear over the selected concentration ranges for 97% of the analytes, with calculated coefficients of determination (R2) of greater than 0.99. A feeding study in wild type mice with a standard chow and a cholesterol-enriched Western type diet illustrated that the protocol described here provides a powerful tool to simultaneously quantify cholesterol derivatives and bile acids in metabolically active tissues and to follow the enterohepatic circulation.