Bile acids/-conjugates determination - Von Eckardstein

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A sensitive and specific high-performance liquid chromatography (HPLC)–MS/MS method was developed for the differentiated quantification of unconjugated as well as glycine- and taurine-conjugated cholic, chenodeoxycholic (CDCA), deoxycholic (DCA), ursodeoxycholic (UDCA) and lithocholic acid (LCA) in serum samples. After solid-phase extraction and reversed-phase HPLC separation, detection of the conjugated bile acids was performed using electrospray ionization (ESI)- MS/MS and selected reaction monitoring mode, whereas unconjugated bile acids were determined by ESI-MS and selected ion monitoring mode. The within-day and between-day coefficients of variation were below 7% for all bile acids and the recovery rates of the extraction procedure were between 84.9 and 105%.

Analysed Matrices

human serum


  • 15 bile acids (+ 7 IS, BA-d4)


Sample Preparation

0.75 ml serum were mixed with 50 µl internal standard solution (containing 0.65µg of CA-d4, CDCA-d4, DCA-d4, UDCA-d4, LCA-d4, GCA-d4 and GCDCA-d4, respectively), followed by an addition of 0.75 ml 100mM ammonium carbonate buffer pH 9.3. The clean-up procedure was performed by solid-phase extraction. The Varian Bond Elut C18 cartridges (200 mg) were pre-conditioned with 2 ml methanol, 2 ml water and 2 ml 100mM ammonium carbonate buffer pH 9.3. The serum sample was then applied onto the cartridge and allowed to pass through the cartridge using gravity. Afterwards, the cartridge was washed with 2 ml water only using gravity and briefly dried under vacuum. The bile acids were desorbed with 3ml methanol with gravity elution. The eluted substances were dried at 30°C by evaporation (Rotavapor,Büchi, Flawil, Switzerland), and the residue dissolved in 100 µl methanol and mixed with 100 µl water. Thirty microlitres of the extracts were injected separately three times into the HPLC–MS system since unconjugated, glycine- and taurine-conjugated bile acids were quantified by three separate runs.

Analytical Method



  • Rheos 2000 pump (Flux Instruments, Basel, Switzerland)
  • HTC PAL autosampler(CTC Analytics, Zwingen, Switzerland).
  • columns: Uptisphere C18 ODB (5 µm,125 mm ×2 mm); guard column (10 mm×2 mm, Interchim)
  • mobile phase:

eluent A: 10mM ammonium acetate buffer (pH 4.5) containing 0.012% formic acid

eluent B: methanol containing 0.012% formic acid

gradient: linearly changed from 30% A and 70% B to 20% A and 80% B within 9 min (glycine- and taurine-conjugated bile acids) and within 12 min (unconjugated bile acids)This final ratio was maintained for 2 min (glycine- and taurine-conjugated bile acids) and for 5 min (unconjugated bile acids). Afterwards, the eluents were changed to 5%A and 95% B within 1 min and finally adjusted to the original ratio of 30% A and 70% B within 8 min in order to enable equilibration of the column.

  • total runtime (including the re-equilibration): 20 min (glycine- and taurine-conjugated bile acids); 26 min (unconjugated bile acids)
  • flow-rate: 200 µl/min

MS Thermo-Quest Finnigan TSQ 7000

  • split: 1:2.5
  • flow: 80 µl/min
  • triple quadrupole mass spectrometer
  • ESI ionization source
  • negative mode
  • capillary temperature: 280°C
  • spray voltage: 4.5 kV.
  • sheath gas pressure: 50 psi
  • single ion monitoring:

m/z 407.3 (CA), m/z 391.3 (CDCA, DCA and UDCA) and m/z 375.3 (LCA)

m/z 411.3 (CA-d4), m/z 395.3 (CDCA-d4, DCA-d4 and UDCA-d4), m/z 379.3 (LCA-d4)

  • selected reaction-monitoring:

m/z 464.4→73.9 (GCA), m/z 448.4→73.9 (GCDCA, GDCA, GUDCA), m/z 432.4→73.9 (GLCA) for the glycine-conjugated bile acids

m/z 514.4→79.8(TCA), m/z 498.4→79.8 (TCDCA, TDCA, TUDCA) and m/z 482.4→79.8 (TLCA) for the taurine-conjugated bile acids

m/z 468.4→73.9 (GCA-d4) and m/z 452.4→73.9 (GCDCA-d4) for the deuterated internal standards

  • collision energy: 40 eV (glycine derivatives); 75 eV (taurine derivatives)

Method Validation


0.001 - 0.062 µM


0.001 - 0.103 µM


0.12 - 60 µM

Internal Standard (IS)

CA-d4, CDCA-d4, DCA-d4, UDCA-d4, LCA-d4, GCA-d4, GCDCA-d4


  • Burkard, A. von Eckardstein, K. M. Rentsch J. Chrom.B 2005, 826, 147-159.

Institute of Clinical Chemistry; University Hospital Zurich; Rämistrasse 100; CH-8091 Zurich; Switzerland.

Tel.: +41 1 255 22 90; Fax: +41 1 255 45 90; e-mail:

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