Development and validation of a hydrophilic interaction ultra-high-performance liquid chromatography-tandem mass spectrometry method for rapid simultaneous determination of 19 free amino acids in rat plasma and urine

Determination of amino acids in biofluids is a challenging task because of difficulties deriving from their high polarity and matrix interference. A simple, reliable and high-throughput hydrophilic interaction UHPLC-MS/MS method was developed and validated for the rapid simultaneous determination of 19 free amino acids in rat plasma and urine samples in this paper. Hydrophilic method with a Waters Acquity UPLC BEH Amide column (100 x 2.1 mm,1.7 mu m) was used with a gradient mobile phase system of acetonitrile and water both containing 0.2% formic acid. The analysis was performed on a positive electrospray ionization mass spectrometer via multiple reaction monitoring. Samples of 10 mu L plasma and 50 mu L urine were spiked with three deuterated internal standards, pretreated with 250 mu L acetonitrile for one-step protein precipitation and a final dilution of urine samples. Good linearities (r > 0.99) were obtained for all of the analytes with the lower limit of quantification from 0.1 to 1.2 mu g/mL. The relative standard deviation of the intra-day and inter-day precisions were within 15.0% and the accuracy ranged from -12.8 to 12.7%. The hydrophilic interaction UHPLC-MS/MS method was rapid, accurate and high-throughput and exhibited better chromatography behaviors than the regular RPLC methods. It was further successfully applied to detect 19 free amino acids in biological matrix.