MSACL 2017 EU Abstract

Simultaneous Quantification of Oxysterols and Bile Acids in Human Plasma by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry

Madlen Sander (Presenter)
Leipzig University

Bio: 2001 apprenticeship as a chemical lab assistant; awarded as the best trainee by the Leipzig Chamber of Commerce; 3-years continued training scholarship from the Federal Ministry of Education and Research; until 2008 chemical lab assistant and qualified trainer for in-job training and education of chemical lab assistants; 2008 Study chemistry at Leipzig University; 5-years advancement scholarship for outstanding students from the Federal Ministry of Education and Research; 2011 Bachelor of Science in Chemistry; 2012 6-months visiting scholar at Ohio University, funded by the German Academic Exchange Service; 2014 Master of Science in Chemistry; since 2014 Ph.D. student at the Medical Faculty, Leipzig University, in the group of Uta Ceglarek focused on mass spectrometry used in clinical applications

Authorship: M. Sander(1), J. Schröter(2), U. Ceglarek(1,3)
(1) Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Germany. (2) Institute for Medical Physics and Biophysics, Leipzig University, Germany. (3) LIFE –Leipzig Research Center for Civilization Diseases, Leipzig University, Germany.

Short Abstract

Bile acids and oxysterols are structurally related molecules derived from cholesterol. Despite evidence supporting a neuroprotective role in neurodegenerative disease, little is known about their molecular mechanisms or their physiological roles. The here described novel LC-ESI(+)MS/MS method combines the quantitation of 12 oxysterols and 17 free and conjugated bile acid without time consuming derivatization. The parameters for method validation: linear calibration range, inter-day coeffiecients of variation, lower limit of quantification, and autosampler storing stability were determined.

Long Abstract


In humans, the brain contains about 20% of the body's free cholesterol (CH), mostly synthesized de novo. CH itself cannot cross the blood-brain barrier (BBB). Bile acids (BA) as primary products of the CH catabolism and the oxidized CH metabolites collectively known as oxysterols are able to pass the BBB. Through their signaling function and activation of diverse signaling pathways, BAs and oxysterols can regulate lipid, energy and CH homeostasis (1-12). Little is known about the pathophysiological role of bile acids and oxysterols in neurodegenerative disease.

The study of BA and oxysterols functions requires methods which cover the complexity of this one the one hand structurally diverse and one the other hand highly isobaric group of molecules. In the last years many methods hab been establish to study the products of the cholesterol metabolism, oxysterols and bile acids, mainly separately by LC-MS or GC-MS with or without derivatization (13-20).

Here we describe a validated LC-MS/MS method for the quantification of 35 analytes of the cholesterol metabolism, including 12 oxysterols and 17 free and conjugated BAs in plasma without derivatization and enhanced ionization using the positive electrospray ionization mode.


Tandem mass spectrometric detection was performed on a SCIEX QTRAP 5500 hybrid triple quadrupole linear ion trap mass spectrometer with positive electrospray ionization. Protonated precursor and daughter ions of oxysterols and bile acids were studied by multiple reaction monitoring (MRM). Sample preparation of 45 µL human plasma or standard was carried out by simple protein precipitation in isopropyl alcohol containing 50 µg/mL butylated hydroxytoluene (BHT) and deuterated internal standards. Centrifugation was performed at 13,000g for 10 minutes at 4°C. 200 µL of the supernatant were injected into the HPLC system. An eluent gradient of water/acetonitrile/methanol 90:5:5 v/v/v and acetonitrile/water/methanol 90:5:5 v/v/v, containing 0.1 % formic acid each, with a flow gradient of 600 to 1000 µL/min was applied. Seperation was performed after on-line solid phase extraction on a StrataX (20x2.0 mm, 25 µm, Phenomenex) on a core shell C18 reversed phase column (ChromaNik, SunShell C18, 150x3.0 mm, 2.6 µm). An external calibration and deuterated internal standards had been used for quantification.


35 Analytes of the cholesterol metabolism, including 12 oxysterols (e.g. 24S-, 25-, 27-Hydroxycholesterol and Dihydroxycholesterol), 17 bile acids (e.g. the most abundant primary BAs cholic acid (CA) and chenodeoxycholic acid (CDCA) and the secondary BAs deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA) with the corresponding glycine and taurine conjugates), and 25 deuterated internal standards, were integrated in the developed method. Mass spectrometric parameters for ionization and analysis via MRM as well as chromatographic seperation within 23 minutes were optimized. Because of the great variablity of the expected concentrations in human plasma, different calibration ranges (1) 0.04 to 5 ng/mL, (2) 0.4 to 250 ng/mL, (3) 0.04 to 500 ng/mL and (4) 3 to 2000 µg/mL were established. In spiked human plasma, inter-day coeffiecients of variation (n=10) of 3 % - 26 % were determined for 35 analytes of the cholesterol metabolism, oxysterols and bile acid. The lower limit of quantification with S/N=10 was 0.2 to 10 ng/mL dependent on the analyte. We achived autosampler storing stability at 4°C for all analytes over 74 hours.

Conclusions & Discussion

The simultaneous analysis of 35 analytes of the cholesterol metabolism, including 12 oxysterols and 17 bile acids in human plasma can be accomplished applying the established novel liquid chromatography tandem mass spectrometric approach in an total run time of 23 min. This method was developed for human serum dealing with a simple protein precipitation without time comsuming derivatisation or extraction. Using the more intense and robust positive electrospray ionization mode, it is possible to overcome the poor ionization cross-section and to lower the limits of detection and quantification. This validated method may be applied to comparison studies of disease and healthy cohorts to get knowledge of the possible changes in cholesterol metabolism during the development of the disease. Further method development for the analysis of other sample matrizes e.g. liver or brain tissue is currently under investigation.

References & Acknowledgements:

This publication is supported by a grant of the Deutsche Forschungsgemeinschaft, Obesity Mechanisms (CRC 1052, A09 to UC), Bonn, the ROLAND ERNST STIFTUNG für Gesundheitswesen, Dresden and the LIFE - Leipzig Research Center for Civilization Diseases, Leipzig University. LIFE is funded by means of the European Union, by the European Regional Development Fund (ERDF) and by means of the Free State of Saxony within the framework of the excellence initiative.

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Financial Disclosure

GrantsyesROLAND ERNST STIFTUNG für Gesundheitswesen, Dresden
SalaryyesLeipzig University
Board Memberno

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above: