Quantitative-profiling of bile acids and their conjugates in mouse liver, bile, plasma, and urine using LC-MS/MS

Abstract

The differences among individual bile acids (BAs) in eliciting different physiological and pathological responses are largely unknown because of the lack of valid and simple analytical methods for the quantification of individual BAs and their taurine and glycine conjugates. Therefore, a simple and sensitive LC-MS/MS method for the simultaneous quantification of 6 major BAs, their glycine, and taurine conjugates in mouse liver, bile, plasma, and urine was developed and validated. One-step sample preparation using solid-phase extraction (for bile and urine) or protein precipitation (for plasma and liver) was used to extract BAs. This method is valid and sensitive with a limit of quantification ranging from 10 to 40 ng/ml for the various analytes, has a large dynamic range (2500), and a short run time (20 min). Detailed BA profiles were obtained from mouse liver, plasma, bile, and urine using this method. Muricholic acid (MCA) and cholic acid (CA) taurine conjugates constituted more than 90% of BAs in liver and bile. BA concentrations in liver were about 300-fold higher than in plasma, and about 180-fold higher in bile than in liver. In summary, a reliable and simple LC-MS/MS method to quantify major BAs and their metabolites was developed and applied to quantify BAs in mouse tissues and fluids.

Figure 1

A) Effect of mobile phase pH and composition on the retention time and signal sensitivity. Ammonium formate was used at pH 3 and 5, ammonium acetate at pH 4 and 6, ammonium bicarbonate at pH 7 and 8, and ammonium carbonate at pH 9 and 10, at 7.5 mM each. B) The solvent gradient profile of the HPLC pumps at the final chromatography conditions. The mobile phase consisted of 5% acetonitrile (ACN) in methanol (MeOH) (mobile phase A) and 7.5 mM ammonium acetate adjusted to pH 4 using 10 M acetic acid (mobile phase B) at a total flow rate of 0.3 ml/min.

Figure 2

A) Backbone and side chain structures of the 6 major BAs, as well as their glycine and taurine conjugates in mice. B) Parent and fragment masses used for quantification of BAs, their conjugate metabolites, and internal standards using tandem MS.

Figure 3

Representative chromatogram at the lowest limit of quantification of a mixure of G-BAs (10 ng/ml) T-BAs (40 ng/ml), unconjugated BAs (2 0ng/ml), and the 2 ISs (+IS= 0.4 µg/ml, −IS= 4 µg/ml) under the final chromatography and detection conditions.

Figure 4

Representative chromatogram of BAs in mouse bile.

Figure 5

BA concentrations in mouse liver. The results are shown as mean (N=5) ± S.E.M.

Figure 6

BA excretion in mouse bile. The results are shown as mean (N=5) ± S.E.M.

Figure 7

Concentration of bile acids in plasma. The results are shown as mean (N=5) ± S.E.M.