Objective  To establish an effective method of ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for quantifying serum free choline, betaine, trimethylamine (TMA) and trimethylamine N-oxide (TMAO) simultaneously.

  Methods  Serum samples were acidified and the proteins were precipitated with acetonitrile, then separated with a hydrop interaction liquid chromatography (HILIC) column. The adopted internal standard materials were d9-choline chloride, d9-TMAO and d9-TMA·hydrochloric acid (HCl); the moving phase A was 10 mmol/L ammonium formate – 0.1% formate and the moving phase B was mixture of 0.1% formic acid and acetonitrile. The samples were detected with mass spectrometry after gradient elution.

  Results  Serum choline, betaine, TMA and TMAO were effectively separated. The detection linearity ranged 0.391 – 100 μmol/L for choline, 0.782 – 200 μmol/L for betaine, 0.009 – 2.50 μmol/L for TMA, and 0.391 – 100 μmol/L for TMAO, respectively, with the four linear correlation coefficients of (R²) ≥ 0.998 3. The intra batch and inter batch coefficients of variance were within 6% and 10% and the recovery rates ranged from 90% – 115%, respectively, for the detections of the four compounds. Stability study revealed that under the storage temperature of 4 ℃, variations in concentrations of the four compounds ranged between 1.9% and 12.6%; while the variations in the concentration of choline and betaine exceeded 15% at the temperature of 25℃. The variations in concentrations of choline, betaine, and TMAO were within 15% after repeated freeze-thawing process but the variation exceeded 15% for TMA.

  Conclusion  The established UPLC-MS/MS method could detect serum free choline, betaine, trimethylamine and trimethylamine N-oxide simultaneously and result in accurate and precise detections.