A Reliable Methodology for Quantitative Extraction of Fruit and Vegetable Physiological Amino Acids and Their Subsequent Analysis with Commonly Available HPLC Systems


Many of the extraction and amino acid analysis methodologies currently employed do not provide complete analysis of all the physiological amino acids and biogenic amines. Extraction procedures frequently employ dilute acid which partially converts gln and asn to glu and asp. A commonly used pre-column derivatizing agent, o-phthalaldehyde, does not react with the imino acids, pro and hydroxypro. The purpose of this investigation was to integrate extraction and analysis procedures into a reliable method for measuring the complete physiological amino acid profiles of fruit and vegetables using HPLC instrumentation commonly available to most laboratories. Water extraction of ground, frozen-thawed tissues effected complete recovery of the physiological amino acids as demonstrated by spiking experiments and tissue combination experiments. HPLC of dabsyl derivatives of the free amino acids allowed their quantification in a selection of fruit and vegetables. Physiological amino acid levels were determined for peach, apple, potato, onion, tomato, bell pepper, broccoli, and seven types of cucurbits. The coefficient of variation for estimation of an amino acid level generally fell in the range of 5% to 7%. Because of marked variability in physiological amino acid content as a result of growing conditions, cultural practices, and inherent cultivar differences, comparisons of results with literature values were not possible.

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W. Fish, "A Reliable Methodology for Quantitative Extraction of Fruit and Vegetable Physiological Amino Acids and Their Subsequent Analysis with Commonly Available HPLC Systems," Food and Nutrition Sciences, Vol. 3 No. 6, 2012, pp. 863-871. doi: 10.4236/fns.2012.36115.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] D. H. Spackman, W. H. Stein and S. Moore, “Automatic Recording Apparatus for Use in the Chromatography of Amino Acids,” Analytical Chemistry, Vol. 30, No. 7, 1958, pp. 1190-1206. doi:10.1021/ac60139a006
[2] J. V. Benson Jr. and J. A. Patterson, “Accelerated Chromatographic Analysis of Amino Acids Commonly Found in Physiological Fluids on a Spherical Resin of Specific Design,” Analytical Biochemistry, Vol. 13, No. 2, 1965, pp. 265-280. doi:10.1016/0003-2697(65)90196-X
[3] K. L. Woo and D. S. Lee, “Capillary Gas Chromatographic Determination of Proteins and Biological Amino Acids as N(O)-tert.-Butyldimethylsilyl Derivatives,” Journal of Chromatography B, Vol. 665, No. 1, 1995, pp. 15-25. doi:10.1016/0378-4347(94)00515-7
[4] P. Lindroth and K. Mopper, “High-Performance Liquid Chromatographic Determination of Subpicomole Amounts of Amino Acids by Precolumn Fluorescence Derivatization with o-Pthaldialdehyde,” Analytical Chemistry, Vol. 51, No. 11, 1979, pp.1667-1674. doi:10.1021/ac50047a019
[5] R. Schuster, “Determination of Free Amino Acids by High Performance Liquid Chromatography,” Analytical Chemistry, Vol. 52, No. 4, 1980, pp. 617-620. doi:10.1021/ac50054a005
[6] S. Weiner and A. Tishbee, “Separation of Dns-Amino Acids Using Reversed-Phase High-Performance Liquid Chromatography: A Sensitive Method for Determining N-Termini of Peptides and Proteins,” Journal of Chromatography, Vol. 213, No. 3, 1981, pp. 501-506. doi:10.1016/S0021-9673(00)80501-4
[7] S. Einersson, B. Josefsson and S. Lagerkvist, “Determination of Amino Acids with 9-Fluorenylmethyl Chloroformate and Reversed-Phase High-Performance Liquid Chromatography,” Journal of Chromatography, Vol. 282, 1983, pp. 609-618. doi:10.1016/S0021-9673(00)91638-8
[8] K. Shimbo, T. Oonuki, A. Yahashi, K. Hirayama and H. Miyano, “Precolumn Derivatization Reagents for HighSpeed Analysis of Amines and Amino Acids in Biological Fluid Using Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry,” Rapid Communications in Mass Spectrometry, Vol. 23, No. 10, 2009, pp. 1483-1492. doi:10.1002/rcm.4026
[9] J.-Y. Chang, R. Knecht and D. G. Braun, “Amino Acid Analysis in the Picomole Range by Precolumn Derivatiztion and High-Performance Liquid Chromatography,” Methods in Enzymology, Vol. 91, 1983, pp. 41-48. doi:10.1016/S0076-6879(83)91009-1
[10] S. J. Leach and H. Lindley, “The Kinetics of Hydrolysis of the Amide Group in Proteins and Peptides. Part 1. The Acid Hydrolysis of L-Asparagine and L-Asparaginylglycine,” Transactions of the Faraday Society, Vol. 49, 1953, pp. 915-920. doi:10.1039/tf9534900915
[11] J.-Y. Chang, R. Knecht and D. G. Braun, “Amino Acid Analysis at the Picomole Level. Application to the C-Terminal Sequence Analysis of Polypeptides,” Biochemical Journal, Vol. 199, 1981, pp. 547-555.
[12] I. Krause, A. Bockhardt, H. Neckermann, T. Henle and H. Klostermeyer, “Simultaneous Determination of Amino Acids and Biogenic Amines by Reversed-Phase HighPerformance Liquid Chromatography of the Dabsyl Derivatives,” Journal of Chromatography A, Vol. 715, No. 1, 1995, pp. 67-79. doi:10.1016/0021-9673(95)00578-B
[13] R. Sethuraman, T. L. Lee and S. Tachibana, “Simple Quantitative HPLC Method for Measuring Physiologic Amino Acids in Cerebrospinal Fluid without Pretreatment,” Clinical Chemistry, Vol. 50, No. 3, 2004, pp. 665-669. doi:10.1373/clinchem.2003.026195
[14] H.-J. Schneider, “Amino Acid Analysis Using DABSCl,” Chromatographia, Vol. 28, No. 1, 1989, pp. 45-48. doi:10.1007/BF02290382
[15] W. W. Fish and B. D. Bruton, “Quantification of L-citrulline and Other Physiologic Amino Acids in Watermelon and Selected Cucurbits,” Cucurbitaceae, 2010, pp. 152-154.
[16] A. R. Davis, C. L. Weber III, W. W. Fish, T. C. Wehner, S. King and P. Perkins-Veazie, “L-citrulline Levels in Watermelon Cultigens Tested in Two Environments,” HortScience Vol. 46, No. 12, 2011, pp. 1572-1575.
[17] A. Kovács, K. Ganzier and L. Simon-Sarkadi, “Microwave-Assisted Extraction of Free Amino Acids From Foods,” Zeitschrift fuer Lebensmittel-Untersuchung und, Forschung A, Vol. 207, No. 1, 1998, pp. 26-30.
[18] F. Zhu, Y.-Z, Cai and H. Corke, “Compositions of Phenolic Compounds, Amino Acids and Reducing Sugars in Commercial Potato Varieties and Their Effects on Acrylamide Formation,” Journal of the Science of Food and Agriculture, Vol. 90, No. 13, 2010, pp. 2254-2262. doi:10.1002/jsfa.4079
[19] M. H. Gomes and E. Rosa, “Free Amino Acid Composition in Primary and Secondary Inflorescences of 11 Broccoli (Brassica oleracea var Italica) Cultivars and Its Variation Between Growing Seasons,” Journal of the Science of Food and Agriculture, Vol. 81, No. 3, 2001, pp. 295-299. doi:10.1002/1097-0010(200102)81:3<295::AID-JSFA811>3.0.CO;2-#
[20] J. Lee, J. W. Finley and J. M. Harnly, “Effect of Selenium Fertilizer on Free Amino Acid Composition of Broccoli (Brassica oleracea Cv. Majestic) Determined by Gas Chromatography with Flame Ionization and Mass Selective Detection,” Journal of Agricultural and Food Chemistry, Vol. 53, No. 23, 2005, pp. 9105-9111. doi:10.1021/jf051221x

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