Determination of l-threonate in human plasma and urine by high performance liquid chromatography-tandem mass spectrometry

A fast and selective HPLC-MS–MS method was established to determine l-threonate in human plasma and urine. Plasma and urine samples were extracted by protein precipitation and diluted with water, then chromatographed on an YMC JʹSphere C18 column with methanol–acetonitrile–10 mM ammonium acetate (20:5:75, v/v) as mobile phase, and at a flow rate of 0.2 ml/min. Detection was performed on a triple–quadrupole tandem mass spectrometer using negative electrospray ionization (ESI). Multiple reactions monitoring (MRM) was used and l-threonate was quantified by monitoring the ion transition of m/z 134.5 → 74.7. The linear calibration curves of l-threonate in plasma and urine were obtained over the concentration range of 0.25–50 μg/ml and 2.5–500 μg/ml, respectively. Lower limit of quantitation was 0.25 and 2.5 μg/ml, respectively. Accuracy was within 85–115%, and intra- and inter-batch precision (R.S.D.%) were within ±15%. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of l-threonate in Chinese healthy subjects.