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

Abstract

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 × 2.1 mm,1.7 μm) 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 μL plasma and 50 μL urine were spiked with three deuterated internal standards, pretreated with 250 μL 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 μg/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.