Determination of Natural Amino Acids in Functional Foods by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

Milk powder and rice flour samples were extracted with 0.1% (volume fraction, the same below) formic acid solution, proteins in which were removed by the addition of acetonitrile, and fats and other matrices were removed by n-hexane. The solution obtained was diluted 10 times with 0.1% formic acid solution and then passed through a Pall polytetrafluoroethylene syringe filter with pore size of 0.22 μm. The filtrate was determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Functional beverage samples were directly diluted 1 000 times with 0.1% formic acid solution and then determined by UHPLC-MS/MS. In the chromatographic analysis, an ACQUITY UPLC^® HSS T3 column was used as the stationary phase, and a mixture of 0.1% formic acid solution and methanol solution containing 0.1% formic acid at different volume ratios was used for gradient elution. In the mass spectrometry analysis, electrospray ionization positive ion mode was used for ionization, and multiple reaction monitoring mode was used for detection, with external standard method for quantification. It was shown that linear relationships of values of the mass concentrations and quantitative ion peak areas were kept in the ranges of 5-50 mg·L⁻¹ for glycine and 0.05-0.5 mg·L⁻¹ for other 19 amino acids, with detection limits (3S/N) of 7.50 mg·kg⁻¹ and 0.006 00-0.300 mg·kg⁻¹, respectively. Recovery tests at three concentration levels were conducted using two types of milk powder, one type of rice flour, and three types of functional beverage samples as matrices, giving recoveries in the range of 78.4%-136%, and RSDs (n=6) of the determined values in the range of 0.10%-14%. The proposed method was applied to the analysis of four types of milk powder, one type of rice flour, and three types of functional beverages, and no cysteine was detected in any of them, while the remaining amino acids were detected at varying levels.