Skip to content
Licensed Unlicensed Requires Authentication Published by De Gruyter November 22, 2021

Targeted profiling of 24 sulfated and non-sulfated bile acids in urine using two-dimensional isotope dilution UHPLC-MS/MS

Katharina Habler ORCID logo EMAIL logo , Bernhard Koeppl , Franz Bracher and Michael Vogeser



Bile acids serve as biomarkers for liver function and are indicators for cholestatic and hepatobiliary diseases like hepatitis, cirrhosis, and intrahepatic cholestasis of pregnancy (ICP). Sulfation and renal excretion of bile acids are important elimination steps. The power of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) allows specific profiling of primary and secondary bile acids as well as their sulfated counterparts.


Twenty-four sulfated and non-sulfated primary and secondary bile acids were quantified in urine with 15 corresponding stable isotope labeled internal standards by using two-dimensional UHPLC-MS/MS. The sample preparation was based on a simple dilution with a methanolic zinc sulfate solution followed by an automated online solid phase extraction clean up.


The validation results of the method fulfilled the criteria of the European Medicine Agency (EMA) “Guideline on bioanalytical method validation”. To verify fitness for purpose, 40 urine samples were analyzed which showed an average of 86% sulfation, 9.1% taurine-conjugation, 14% non-conjugation, and 77% glycine-conjugation rates.


Lossless one-pot sample preparation, automated sample purification, and high number of internal standards are major innovations of the presented profiling method, which may allow diagnostic application of BA profiling in the future.

Corresponding author: Dr. rer. nat. Katharina Habler, Institute of Laboratory Medicine, University Hospital, LMU, Marchioninistr. 15, 81377 Munich, Germany, Phone: +49 89 4400 76248, E-mail:

  1. Research funding: None declared.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Informed consent was obtained from all individuals included in this study.

  5. Ethical approval: The local Institutional Review Board deemed the study exempt from review.


1. Alnouti, Y. Bile acid sulfation: a pathway of bile acid elimination and detoxification. Toxicol Sci 2009;108:225–46. in Google Scholar

2. Griffiths, WJ, Sjövall, J. Bile acids: analysis in biological fluids and tissues. J Lipid Res 2010;51:23–41. in Google Scholar

3. Gressner, AM, Gressner, OA. Gallensäuren. In: Gressner, AM, Arndt, T, editors. Lexikon der Medizinischen Laboratoriumsdiagnostik. Berlin, Heidelberg: Springer Berlin Heidelberg; 2017.10.1007/978-3-662-49054-9Search in Google Scholar

4. Rassow, J. Biochemie: 50 Tabellen. 2., aktualisierte Aufl. Stuttgart: Thieme; 2008 (Duale Reihe).10.1055/b-002-85529Search in Google Scholar

5. Chiang, JY. Regulation of bile acid synthesis. Front Biosci 1998;3:176–93. in Google Scholar

6. Wess, G, Enhsen, A, Kramer, W. Gallensäuren: Wiederentdeckt. Nachr. Chem. Tech. Lab. 1995;43:1047–55. in Google Scholar

7. Mayo Clinic Laboratories. Test definition: BAFS: bile acids, fractioned and Tot, S; 2021. Available from: in Google Scholar

8. Shima, T, Tada, H, Morimoto, M, Nakagawa, Y, Obata, H, Sasaki, T, et al.. Serum total bile acid level as a sensitive indicator of hepatic histological improvement in chronic hepatitis C patients responding to interferon treatment. J Gastroenterol Hepatol 2000;15:294–9. in Google Scholar

9. Sawkat Anwer, M, Meyer, DJ. Bile acids in the diagnosis, pathology, and therapy of hepatobiliary disease. Vet Clin North Am Small Anim Pract 1995;25:503–17. in Google Scholar

10. Huang, WM, Tropper, P, Seubert, D, Donnelly, J, Liu, M, Javitt, N. Random urinary bile acids as a diagnostic method for intrahepatic cholestasis of pregnancy (ICP). Am J Obstet Gynecol 2006;195:S121. in Google Scholar

11. Marschall, H-U. Management of intrahepatic cholestasis of pregnancy. Expet Rev Gastroenterol Hepatol 2015;9:1273–9. in Google Scholar PubMed

12. Tribe, RM, Dann, AT, Kenyon, AP, Seed, P, Shennan, AH, Mallet, A. Longitudinal profiles of 15 serum bile acids in patients with intrahepatic cholestasis of pregnancy. Am J Gastroenterol 2010;105:585–95. in Google Scholar

13. Danese, E, Salvagno, GL, Negrini, D, Brocco, G, Montagnana, M, Lippi, G. Analytical evaluation of three enzymatic assays for measuring total bile acids in plasma using a fully-automated clinical chemistry platform. PLoS ONE 2017;12:e0179200. in Google Scholar

14. Liu, Y, Rong, Z, Xiang, D, Zhang, C, Liu, D. Detection technologies and metabolic profiling of bile acids: a comprehensive review. Lipids Health Dis 2018;17:121. in Google Scholar

15. Almé, B, Bremmelgaard, A, Sjövall, J, Thomassen, P. Analysis of metabolic profiles of bile acids in urine using a lipophilic anion exchanger and computerized gas-liquid chromatography-mass spectrometry. J Lipid Res 1977;18:339–62. in Google Scholar

16. Alnouti, Y, Csanaky, IL, Klaassen, CD. Quantitative-profiling of bile acids and their conjugates in mouse liver, bile, plasma, and urine using LC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci 2008;873:209–17. in Google Scholar

17. Batta, A, Kumar, SG. Gas chromatography of bile acids. J Chromatogr B 1999;723:1–16. in Google Scholar

18. Amplatz, B, Zöhrer, E, Haas, C, Schäffer, M, Stojakovic, T, Jahnel, J, et al.. Bile acid preparation and comprehensive analysis by high performance liquid chromatography-high-resolution mass spectrometry. Clin Chim Acta 2017;464:85–92. in Google Scholar PubMed

19. Luo, L, Aubrecht, J, Li, D, Warner, RL, Johnson, KJ, Kenny, J, et al.. Assessment of serum bile acid profiles as biomarkers of liver injury and liver disease in humans. PLoS ONE 2018;13:1–17. in Google Scholar PubMed PubMed Central

20. Sarafian, MH, Lewis, MR, Pechlivanis, A, Ralphs, S, McPhail, MJW, Patel, VC, et al.. Bile acid profiling and quantification in biofluids using ultra-performance liquid chromatography tandem mass spectrometry. Anal Chem 2015;87:9662–70. in Google Scholar PubMed

21. Scherer, M, Gnewuch, C, Schmitz, G, Liebisch, G. Rapid quantification of bile acids and their conjugates in serum by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2009;877:3920–5. in Google Scholar PubMed

22. Burkard, I, Eckardstein, Avon, Rentsch, KM. Differentiated quantification of human bile acids in serum by high-performance liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2005;826:147–59. in Google Scholar

23. Huang, J, Bathena, SPR, Csanaky, IL, Alnouti, Y. Simultaneous characterization of bile acids and their sulfate metabolites in mouse liver, plasma, bile, and urine using LC-MS/MS. J Pharm Biomed 2011;55:1111–9. in Google Scholar

24. Ducroq, DH, Morton, MS, Shadi, N, Fraser, HL, Strevens, C, Morris, J, et al.. Analysis of serum bile acids by isotope dilution-mass spectrometry to assess the performance of routine total bile acid methods. Ann Clin Biochem 2010;47:535–40. in Google Scholar

25. Sangaraju, D, Shi, Y, van Parys, M, Ray, A, Walker, A, Caminiti, R, et al.. Robust and comprehensive targeted metabolomics method for quantification of 50 different primary, secondary, and sulfated bile acids in multiple biological species (human, monkey, rabbit, dog, and rat) and matrices (plasma and urine) using liquid chromatography high resolution mass spectrometry (LC-HRMS) analysis. J Am Soc Mass Spectrom 2021;32:2033–49. in Google Scholar

26. Wielders, JPM, Mink, JK. Analysis of vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid in human urine by high-performance liquid chromatography and fluorometry. J Chromatogr 1984;310:379–85. in Google Scholar

27. European Medicines Agency. Guideline bioanalytical method validation. London, UK: European Medicines Agency; 2011. Available from: GB/document library/Scientific guideline/2011/08/WC500109686.pdf.Search in Google Scholar

28. Bonfiglio, R, King, RC, Olah, TV, Merkle, K. The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds. Rapid Commun Mass Spectrom 1999;13:1175–85.<1175::aid-rcm639>;2-0.10.1002/(SICI)1097-0231(19990630)13:12<1175::AID-RCM639>3.0.CO;2-0Search in Google Scholar

29. Belyakova, LA, Besarab, LN, Roik, NV, Lyashenko, DY, Vlasova, NN, Golovkova, LP, et al.. Designing of the centers for adsorption of bile acids on a silica surface. J Colloid Interface Sci 2006;294:11–20. in Google Scholar

30. Belyakova, LA, Vlasova, NN, Golovkova, LP, Varvarin, AM, Lyashenko, DY, Svezhentsova, AA, et al.. Role of surface nature of functional silicas in adsorption of monocarboxylic and bile acids. J Colloid Interface Sci 2003;258:1–9. in Google Scholar

31. Tagliacozzi, D. Quantitative analysis of bile acids in human plasma by liquid chromatography-electrospray. Clin Chem Lab Med 2003;41:1633–41. in Google Scholar

32. Bathena, SPR, Mukherjee, S, Olivera, M, Alnouti, Y. The profile of bile acids and their sulfate metabolites in human urine and serum. J Chromatogr B Analyt Technol Biomed Life Sci 2013;942-943:53–62. in Google Scholar

33. Perwaiz, S, Tuchweber, B, Mignault, D, Gilat, T, Yousef, IM. Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry. J Lipid Res 2001;42:114–9. in Google Scholar

34. Simko, V, Michael, S, Kelley, RE. Predictive value of random sample urine bile acids corrected by creatinine in liver disease. Hepatology 1987;7:115–21. in Google Scholar PubMed

35. Bathena, SPR, Thakare, R, Gautam, N, Mukherjee, S, Olivera, M, Meza, J, et al.. Urinary bile acids as biomarkers for liver diseases I. Stability of the baseline profile in healthy subjects. Toxicol Sci 2015;143:296–307. in Google Scholar PubMed

36. Jahnel, J, Zöhrer, E, Scharnagl, H, Erwa, W, Fauler, G, Stojakovic, T. Reference ranges of serum bile acids in children and adolescents. Clin Chem Lab Med 2015;53:1807–13. in Google Scholar PubMed

Supplementary Material

The online version of this article offers supplementary material (

Received: 2021-08-12
Accepted: 2021-11-09
Published Online: 2021-11-22
Published in Print: 2022-01-27

© 2021 Walter de Gruyter GmbH, Berlin/Boston

Downloaded on 28.11.2022 from
Scroll Up Arrow