Simultaneous and rapid determination of 6 primary bile acids in plasma by high-performance liquid chromatography-tandem mass spectrometry

  Objective  To develop a rapid and accurate high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of 6 primary bile acids in plasma.

  Methods  Plasma was separated on a Thermo Accucore C₁₈ column (100 mm × 4.6 mm; 2.6 μm) after being extracted by protein precipitation using methanol. The 6 primary bile acids were separated by using the mobile phase of methanol-water (0.1% formic acid + 5 mmol/L ammonium acetate) running in gradient elution. The mass spectrometer was operated on an electrospray ionization source in negative ion mode for the multiple reaction monitoring analysis. Plasma from 6 healthy adults and 6 adult Sprague-Dawley (SD) rats was detected.

  Results  Baseline separation of 6 primary bile acids was achieved within 6 min. The linearity of the calibration curves of the 6 primary bile acids was excellent in the range of 5 – 2000 μg/L with a correlation coefficient higher than 0.995. The limits of detection and limits of quantitation were in the range of 0.25 – 0.45 μg/L and 0.84 – 1.49 μg/L, respectively. The recoveries of intra-day and inter-day were 86.3% – 111.5% and 87.7% – 104.9%, with relative standard deviations of 5.2% – 11.9% and 6.6% – 14.3%, respectively. The concentrations of primary bile acids in plasma of healthy adults and adult SD rats were significantly different.

  Conclusion  This method is simple, rapid, sensitive, and accurate, and can meet the needs for rapid detection of primary bile acids in clinical tests and animal experiments.