Quantification of Glutamate and Aspartate by Ultra-High Performance Liquid Chromatography

Abstract

Glutamic and aspartic acid fulfil numerous functions in organisms. They are proteinogenic amino acids, they function as neurotransmitters, and glutamic acid links the citrate cycle with amino acid metabolism. In addition, glutamic acid is a precursor for many bioactive molecules like γ-aminobutyric acid (GABA). In tomatoes, glutamic acid accumulates in ripening fruits. Here we present a simple and rapid method for quantification of glutamate and aspartate in tomatoes. A cleared extract is prepared and 2-aminoadipic acid added as internal standard. Subsequently, the amino acids are derivatised with 2,4-dinitro-1-fluorobenzene under alkaline conditions. The derivatives are separated by ultra-high performance liquid chromatography using a phenyl-hexyl column and 50 mM N-methylmorpholine/acetate buffer pH 7.4 containing 12% acetonitrile as eluent and detected by UV absorption at 363 nm. The whole analysis time including separation and column equilibration takes less than 2.8 min with a flow rate of 1 mL/min and less than 1.6 min with a flow rate of 2 mL/min, making this method suitable for high-throughput applications. The method shows excellent reproducibility with intra- and inter-day SDs of approximately 4% for both aspartic and glutamic acid. Using this method we show that the glutamate/aspartate ratio changes significantly during fruit ripening.

Chemico-Biological Characterization of Torpedino Di Fondi® Tomato Fruits: A Comparison with San Marzano Cultivar at Two Ripeness Stages

“…Glutamic acid, aspartate, tryptophan, and alanine rose upon the fruit ripening; asparagine and phenylalanine turned out to be constant; tyrosine, isoleucine, valine, threonine, GABA, and glutamine content decreased from pink to red SM fruits. These findings reflect data reported in literature about the analyses of SM [ 2 , 4 ] and other cultivars [ 3 , 45 , 47 , 48 , 49 , 50 , 51 , 52 ].…”

“…According to NMR scrutiny, TF and SM samples showed some/several similarities ( Figure 2 A): glutamine was found to be the most abundant amino acid in both cultivars at the pink stage, followed by GABA and glutamic acid, whereas, at the red stage, glutamic acid increased, becoming the most abundant amino acid. The pattern of SM developmental changes in free amino acid content was in agreement with literature data, being glutamic acid characterized by a remarkable increase in all ripe fruits [ 2 , 3 , 4 , 45 , 47 , 48 , 49 , 50 , 51 , 52 ]. Glutamic acid, aspartate, tryptophan, and alanine rose upon the fruit ripening; asparagine and phenylalanine turned out to be constant; tyrosine, isoleucine, valine, threonine, GABA, and glutamine content decreased from pink to red SM fruits.…”

GC-MS Based Metabolite Profiling to Monitor Ripening-Specific Metabolites in Pineapple (Ananas comosus)

“…Its level was known to be varied among the fruits. In banana and Vasconcellea quercifolia, the level of aspartic acid was found to be decreased along with the ripening progress, while mature or ripe tomato contained more aspartic acid that brings out the umami taste [49,51,52]. Other than the source of umami taste, the level of aspartic acid might also connect with the free auxin level in fruits that affect its ripening [53].…”

New Hybrid Tomato Cultivars: An NMR-Based Chemical Characterization

“…The amino acid profiles (Figure 2A) of the five cultivars showed some common aspects: GABA, alanine, glutamine, and glutamic acid were always the most abundant amino acids [6,[26][27][28]. In particular, glutamic acid was the most abundant amino acid in all cultivars.…”