【Objective】To establish a method for the quantitative measurement of betaine and free choline in plasma simultaneously using liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and to study the stability of the two analytes in plasma. 【Methods】 Separation was achieved on a normal-phase silica column (2.1 mm × 100 mm

5 μm) with acetonitrile/15 mmol/L formic aicd (6:4v/v) under isocratic elution. A mass spectrometry was applied to measure the objective analytes. Acetonitrile

contained d9-choline and d9-betaine

as internal standards

was added into plasma to precipitate protein. 【Results】 For both betaine and choline

the lower limit of quantitation (LLOQ) was 0.62 μmol/L and the response was linerar over the range investigated (0.62 μmol/L-400.00 μmol/L

r2 > 0.999). The within-day relative standard devition (RSD) were below 8% and between-day below 15%. The recoveries were over 90%. For the stability tests

the concentration of the two analytes increased with the storage temperature and time. Choline was stable throughout three freeze-thawn cycles while concentration of betaine had a significantly rise after the first cycle. 【Conclusion】 This is a technically simple and accurate method that can be carried out with HPLC-MS/MS. Attention should be paid to the critical effects of storage temperature and time and freeze-thawn cycle on the measurement.