Three-minute method for amino acid analysis by UHPLC and high-resolution quadrupole orbitrap mass spectrometry

• Published in: Amino acids Vol. 47; no. 11; pp. 2345 - 2357
• Main Authors: Nemkov, Travis, D’Alessandro, Angelo, Hansen, Kirk C
• Format: Journal Article
• Language: English
• Published: Vienna Springer Vienna 01.11.2015; Springer Nature B.V
• Subjects: 5-Fluorouracil; amino acid composition; Amino acids; Amino Acids - analysis; Amino Acids - metabolism; Analytical Chemistry; Animals; Biochemical Engineering; Biochemistry; Biological; Biomedical and Life Sciences; biomedical research; Bronchoalveolar Lavage; Cancer; carbon; Chromatography; Chromatography, High Pressure Liquid; Emergency medical care; Fluid dynamics; Fluid flow; fluorouracil; glutamic acid; Glutamine; Hemorrhage - metabolism; High fat diet; High performance liquid chromatography; High resolution; hypoxia-inducible factor 1; Hypoxia-Inducible Factor 1, alpha Subunit - metabolism; Hypoxia-inducible factor 1a; Hypoxia-inducible factors; Life Sciences; Linearity; Low fat diet; lymph; Lymph - metabolism; Lysine; Mass Spectrometry; Mass spectroscopy; Medical research; medicine; methods; Mice; muscle tissues; Muscles; neoplasm cells; Neurobiology; neurophysiology; Nutrient deficiency; Original Article; palps; Pancreatic cancer; pancreatic neoplasms; Proteomics; Quadrupoles; Quantitation; Rats; Rats, Sprague-Dawley; Scientific imaging; Sensitivity; stable isotopes; Taste buds; Taste Buds - metabolism; ultra-performance liquid chromatography; Tissue extract; Biofluid; Cell extract; QExactive; Amino acids; PLS-DA; Mass spectrometry
• ISSN: 0939-4451; 1438-2199; 1438-2199
• DOI: 10.1007/s00726-015-2019-9

Amino acid analysis is a powerful bioanalytical technique for many biomedical research endeavors, including cancer, emergency medicine, nutrition and neuroscience research. In the present study, we present a 3 min analytical method for underivatized amino acid analysis that employs ultra high-performance liquid chromatography and high-resolution quadrupole orbitrap mass spectrometry. This method has demonstrated linearity (mM to nM range), reproducibility (intra-day <5 %, inter-day <20 %), sensitivity (low fmol) and selectivity. Here, we illustrate the rapidity and accuracy of the method through comparison with conventional liquid chromatography–mass spectrometry methods. We further demonstrate the robustness and sensitivity of this method on a diverse range of biological matrices. Using this method we were able to selectively discriminate murine pancreatic cancer cells with and without knocked down expression of hypoxia-inducible factor 1α; plasma, lymph and bronchioalveolar lavage fluid samples from control versus hemorrhaged rats; and muscle tissue samples harvested from rats subjected to both low-fat and high-fat diets. Furthermore, we were able to exploit the sensitivity of the method to detect and quantify the release of glutamate from sparsely isolated murine taste buds. Spiked in light or heavy standards (¹³C₆-arginine, ¹³C₆-lysine, ¹³C ₅ ¹⁵ N₂-glutamine) or xenometabolites (5-fluorouracil) were used to determine coefficients of variation, confirm linearity of relative quantitation in four different matrices, and overcome matrix effects for absolute quantitation. The presented method enables high-throughput analysis of low-abundance samples requiring only one percent of the material extracted from 100,000 cells, 10 µl of biological fluid, or 2 mg of muscle tissue.