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

TY - JOUR

T1 - A liquid chromatography-tandem mass spectrometry-based method for the simultaneous determination of hydroxy sterols and bile acids

AU - John, Clara

AU - Werner, Philipp

AU - Worthmann, Anna

AU - Wegner, Katrin

AU - Tödter, Klaus

AU - Scheja, Ludger

AU - Rohn, Sascha

AU - Heeren, Joerg

AU - Fischer, Markus

N1 - Copyright © 2014 Elsevier B.V. All rights reserved.

PY - 2014/12/5

Y1 - 2014/12/5

N2 - 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.

AB - 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.

U2 - 10.1016/j.chroma.2014.10.064

DO - 10.1016/j.chroma.2014.10.064

M3 - SCORING: Journal article

C2 - 25456597

VL - 1371

SP - 184

EP - 195

JO - J CHROMATOGR A

JF - J CHROMATOGR A

SN - 0021-9673

ER -