Bile acids/-conjugates determination - Perwaiz et al.
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Abstract
A simple, sensitive, and specific liquid chromatography- electrospray tandem mass spectrometry (LC-MS/ MS) method for the determination of bile acids in human bile has been developed. The bile acids were extracted with a C18 (octadecyl) reversed-phase column and identified and quantified by simultaneous monitoring of their parent and daughter ions, using the multiple reaction monitoring mode. Identification and quantification of conjugated bile acids in bile was achieved in 5 min. The detection limit was 1 ng, and the determination was linear for concentrations up to 100 ng. The percent recovery of standards made of single conjugated (glycine and taurine) bile acid or of mixture of glycine- or taurine-conjugated cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and lithocholic acid averaged 71.73% to 95.92%. The LC-MS/MS method was also used for the analysis of serum, urine, and fecal bile acids. In conclusion, LC-MS/MS is a simple, sensitive, and rapid technique for the analysis of conjugated bile acids in bile and other biological samples.
Analysed Matrices
human bile, serum, urine, feces
Analytes
- 15 bile acids (+ 2 IS)
CA, CDCA, DCA, UDCA, LCA, GCA, GCDCA, GDCA, GUDCA, GLCA, TCA, TCDCA, TDCA, TUDCA, TLCA
Sample Preparation
The bile samples (20 µl each) were diluted 10-fold in HPLC grade water, and then 20 µl from each diluted bile sample was transferred to a glass tube to which 1µg of internal standard, 3,12-diol-7-one-5b-cholanic acid, was added. The internal standard was chosen on the basis of preliminary studies, in which this compound gave consistent and reproducible daughter ion (m/z123). The samples were dried under nitrogen, redissolved in 1 ml of HPLC-grade water, and then subjected to solid-phase extraction using a Bond-Elute C18 cartridge. The C18 cartridge was preconditioned prior to loading the samples with successive elutions of 2 ml of chloroform –methanol 2:1 (v/v), methanol, and HPLC-grade water solutions. After loading the samples the column was then washed with 2 ml of HPLC-grade water and n-hexane. The column was left for 10 min to remove any excess solvents. Bile acids were recovered from the cartridge by elution with methanol (5 ml). The eluents were then evaporated to dryness under nitrogen. The residue was dissolved in 1 ml of acetonitrile–water 1:1 (v/v).
Analytical Method
HPLC-MS-MS
HPLC
The LC system used was a Hewlett-Packard (Palo Alto, CA) HPLC (series 1100) equipped with an automatic sample injector. The HPLC system was operated isocratically at a 10-µl/min flow rate for the mobile phase (acetonitrile–water 1:1), at room temperature. Sample (10 µl) was injected in the LC-MS/MS by the automatic injector.
MS
Negative ion mass spectra of the eluents were recorded, using the multiple reaction-monitoring mode (MRM). In this mode the mass spectrometers MS1 and MS2 are used and are operated in static mode for single ions, which allows a higher sensitivity compared with the scan mode.
The molecular and daughter ions selected for MS1 and MS2 for each glycine-conjugated bile acid (tri-, di-, and monohydroxylated bile acids) were as follows: m/z 464.6 and 74; m/z 448.6 and 74; and m/z 432.6 and 74, respectively; for taurine-conjugated bile acids (tri-, di-, and monohydroxylated bile acids) they were m/z 514.6 and 124; m/z 498.6 and 124; and m/z 482.6 and 124, respectively; and for the internal standard (3,12-diol-7-one-5b-cholanic acid) they were m/z 405.6 and 123.
Method Validation
LOD
1 ng
LOQ
n.d.
Linearity
1 - 100 ng
Internal Standard (IS)
- 5ß-cholanic acid
- 3,12-diol-7-one-5b-cholanic acid
Reference
- S. Perwaiz, B. Tuchweber, D. Mignault, T.Gilat, I. M. Yousef J. Lipid Res. 2001, 42, 114-119.
Department of Pharmacology and Department of Nutrition; Université de Montréal; Montréal; Canada H3C 3J7.
e-mail: ibrahim.yousef@umontreal.ca
