Determination of 14 Amino Acids in Blood Plasma from Patients with Coronary Artery Heart Disease by High Performance Liquid Chromatography Tandem Mass Spectrometry

The blood plasma sample (2 mL) was taken, and 4 mL of a mixture of acetonitrile, water, and formic acid at the volume ratio of 89:10:1 was added. After vortex and centrifugation, 4 mL of the supernatant was taken, and purified with 200 mg of C₁₈ and 100 mg of ammonia propyl powder. After vortex and settling, 2 mL of the supernatant was taken, mixed well with 4 mL of methanol, and blown to near dryness by nitrogen. The residue was dissolved in 0.5 mL of a mixture of 1.5% (φ) formic acid solution and acetonitrile at the volume ratio of 80:20. After shaking and filtration, 14 amino acids(proline, tryptophan, kynurenine, alanine, valine, glycine, threonine, arginine, leucine, isoleucine, phenylalanine, tyrosine, asparagine and aspartic acid) in the filtrate were determined by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). In chromatographic analysis, InertSustain AQ-C₁₈ column was used as the stationary phase, and a mixture of water, acetonitrile, and formic acid at different volume ratios was used as the mobile phase for gradient elution. Positive ion mode of electrospray ion source (ESI⁺) and multiple reaction monitoring (MRM) mode were used in mass spectrometry analysis. As shown by the results, linear relationships between values of peak area and mass concentration of 14 amino acids were found in definite ranges, with lower limits of determination (10S/N) in the range of 0.50-2.50 μg·L^-1. Test for recovery was made on the blood plasma samples at three concentration levels by standard addition method, giving results in the range of 82.3%-102%, with RSDs (n=6) of the determined values in the range of 3.3%-12%.