Hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method for the simultaneous determination of l‐valine, l‐leucine, l‐isoleucine, l‐phenylalanine, and l‐tyrosine in human serum

l-Valine, l-leucine, l-isoleucine, l-phenylalanine, and l-tyrosine are important proposed biomarkers for the early detection and diagnosis of type 2 diabetes. A simple and selective hydrophilic interaction chromatography with tandem mass spectrometry method was developed for the simultaneous determination of these amino acids in human serum, using stable isotope-labeled amino acids as internal standards. Chromatographic separation was carried out on a Syncronis HILIC column (150 mm × 2.1 mm, 5 μm) with the column temperature of 35°C and a mobile phase consisted of acetonitrile/120 mM ammonium acetate (89:11, v/v), and the run time was 11.0 min. The mass spectrometric analysis was performed using a QTRAP 5500 mass spectrometer coupled with an electrospray ionization source in positive ion mode. As these five amino acids are endogenous compounds in serum, we used the corresponding stable isotope-labeled amino acids to evaluate the matrix effect and recovery in serum. The matrix effect was 98.7-107.3%, and the recovery was 92.7-102.3%. Calibration curves spiked unlabeled amino acids in water were linear over the range of 0.200-100 μg/mL. The accuracy, inter-, and intraday precision were below 10.2%. Analytes were stable during the study. This assay method has been validated and applied to the early diagnosis research of type 2 diabetes.