Amino Acid Analysis

Shane M. Rutherfurd1, G. Sarwar Gilani2

1 Riddet Institute, Massey University, Palmerston North, New Zealand, 2 Health Canada, Nutrition Research Division, Food Directorate, Health Products and Food Branch, Ottawa, Ontario, Canada
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 11.9
DOI:  10.1002/0471140864.ps1109s58
Online Posting Date:  November, 2009
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Amino acid analysis is used to determine the amino acid content of amino acid–, peptide‐ and protein‐containing samples. With minor exceptions, proteins are long linear polymers of amino acids connected to each other via peptide bonds. The first step of amino acid analysis involves hydrolyzing these peptide bonds. The liberated amino acids are then separated, detected, and quantified. The method was first developed by Moore, Stein and coworkers in the 1950s using HCl acid hydrolysis, and, despite considerable effort by many workers, the basic methodology remains relatively unchanged. This unit provides an overview and strategic planning for amino acid analysis, discussing a range of methodologies and issues. In addition, several common methods used for analysis of L‐amino acids are described in detail, including: HCl acid hydrolysis, performic acid oxidation for methionine and cysteine analysis, base hydrolysis for tryptophan analysis, analysis of free amino acids, and analysis of reactive lysine. Curr. Protoc. Protein Sci. 58:11.9.1‐11.9.37. © 2009 by John Wiley & Sons, Inc.

Keywords: amino acids; hydrolysis; derivatization; chromatography

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Acid Hydrolysis of Proteins and Peptides for Amino Acid Analysis
  • Basic Protocol 2: Performic Acid Oxidation of Proteins for Cysteine and Methionine Analysis
  • Basic Protocol 3: Base (LiOH) Hydrolysis of Proteins for Tryptophan Analysis
  • Basic Protocol 4: Analysis of Free Amino Acids by HPLC
  • Basic Protocol 5: Analysis of Reactive Lysine Content in Food Samples
  • Support Protocol 1: Preparation of Samples to be Subjected to Amino Acid Analysis
  • Reagents and Solutions
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Acid Hydrolysis of Proteins and Peptides for Amino Acid Analysis

  Materials
  • Sample (see protocol 6)
  • Constant boiling 6 M HCl containing 0.1% phenol (see recipe)
  • Internal standard (norleucine) solution (see recipe)
  • Buffer suitable for downstream applications
  • Hydrolysis tubes: 100 × 12–mm Schott Duran rimless test tubes (http://www.us.schott.com)
  • Glass blowing torch (Wale Apparatus; http://www.waleapparatus.com/; if the laboratory has a methane or natural gas tap, then use this and bottled oxygen to provide the fuel mix)
  • Tweezers
  • Chemically resistant, oil‐free PTFE diaphragm pump with a vacuum controller and vacuum gauge (e.g., Buchi V‐700 pump with V‐850 controller)
  • Rubber vacuum hose to attach tube to vacuum pump
  • 110°C forced air oven
  • Low‐speed centrifuge (capable of 2600 × g) with a rotor adapter that will accommodate 100 mm × 12 mm glass tubes
  • Glass‐etching pen
  • Glass rod
  • Savant Speedvac (centrifugal concentrator) or equivalent that will accommodate 100 mm × 12 mm glass tubes, resistant to acid
  • Sonication bath
  • 0.22‐µm syringe filters (13‐mm diameter)
  • 1‐ml luer‐lock syringes
  • HPLC vials (2 ml or 4 ml depending on the HPLC autosampler)
  • Vortex mixer
  • Additional reagents and equipment to prepare samples for amino acid analysis (see protocol 6)

Basic Protocol 2: Performic Acid Oxidation of Proteins for Cysteine and Methionine Analysis

  Materials
  • Sample (see protocol 6)
  • Performic acid (see recipe), freshly prepared and ice cold
  • 48% hydrobromic acid, ice‐cold
  • Savant Speedvac (centrifugal concentrator) or equivalent that will accommodate 100‐mm × 12‐mm glass tubes, resistant to formic acid
  • Additional reagents and equipment to prepare samples for amino acid analysis ( protocol 6) and hydrolysis of proteins/peptides for amino acid analysis ( protocol 1)

Basic Protocol 3: Base (LiOH) Hydrolysis of Proteins for Tryptophan Analysis

  Materials
  • Sample (see protocol 6)
  • Lysozyme from chicken egg white (Sigma, cat. no. L 7651)
  • 4.3 M lithium hydroxide (see recipe)
  • Internal standard (5‐methyltryptophan) solution (see recipe)
  • Constant boiling 6 M HCl containing 0.1% phenol (see recipe)
  • HPLC buffer for LiOH hydrolysis (see recipe)
  • 30‐ml screw‐cap Teflon containers (Nalge, cat. no. DS1630‐0001), thoroughly cleaned and rinsed with 18 MΩ deionized water.
  • Balance (accurate to 5 decimal places)
  • Nitrogen cylinder equipped with a flexible tube to which a Pasteur pipet can be attached
  • 110°C forced‐air oven
  • 25‐ml volumetric flask
  • 0.22‐µm syringe filters (13‐mm diameter)
  • 1‐ml luer‐lock syringes
  • HPLC vials (2‐ml or 4‐ml depending on the HPLC autosampler)
  • HPLC system with column heater
  • C 8 column (4.6 × 150 mm)
  • Peak integration software for HPLC

Basic Protocol 4: Analysis of Free Amino Acids by HPLC

  Materials
  • Sample (see protocol 6)
  • Internal standard (norleucine) solution (see recipe)
  • 2× HPLC loading buffer (see recipe)
  • Refrigerated microcentrifuge
  • Microcon tube (MWCO, 3000 Da; Millipore) or similar centrifugal ultrafiltration device
  • HPLC vials (2 ml or 4 ml depending on the HPLC autosampler)
  • Additional reagents and equipment for pre‐column derivitization (see )

Basic Protocol 5: Analysis of Reactive Lysine Content in Food Samples

  Materials
  • Sample (see protocol 6)
  • 0.6 M OMIU (see recipe)
  • 20°C shaking water bath
  • Savant Speedvac (centrifugal concentrator) or equivalent that will accommodate 100‐mm × 12‐mm glass tubes
  • Additional reagents and equipment to prepare samples for amino acid analysis ( protocol 6) and hydrolysis of proteins/peptides for amino acid analysis ( protocol 1)

Support Protocol 1: Preparation of Samples to be Subjected to Amino Acid Analysis

  Materials
  • Sample for analysis
  • Hydrolysis tubes: 100 × 12–mm Schott Duran rimless test tubes (http://www.us.schott.com/)
  • Muffle furnace (capable of attaining 500°C)
  • Freeze dryer
  • Grinder with mesh pore size ≤1 mm
  • Balance (accurate to 5 decimal places)
  • Savant Speedvac (centrifugal concentrator) or equivalent that will accommodate 100 × 12–mm glass tubes
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 11.9.1 Derivatization reactions for amino acid analysis.
  •   FigureFigure 11.9.2 Stretching the neck of the hydrolysis tube prior to degassing and sealing.
  •   FigureFigure 11.9.3 Degassing the hydrolysis tube using a vacuum pump.
  •   FigureFigure 11.9.4 Apparatus for the preparation of constant‐boiling HCl.

Videos

Literature Cited

Literature Cited
   Anderson, G.H., Ashley, D.V.M., and Jones, J.D. 1976. Utilization of L‐methionine sulfoxide, L‐methionine sulfone and cysteic acid by the weanling rat. J. Nutr. 106:1108‐1114.
   AOAC. 1995. Official Methods of Analysis, 16th ed. AOAC International, Gaithersburg, Md.
   Bank, R.A., Jansen, E.J., Beekman, B., and te Koppele, J.M. 1996. Amino acid analysis by reverse‐phase high‐performance liquid chromatography: Improved derivatization and detection conditions with 9‐fluorenylmethyl chloroformate. Anal. Biochem. 240:167‐176.
   Bardelmeijer, H.A., Lingeman, H., de Ruiter, C., and Underberg, W.J.M. 1998. Derivatization in capillary electrophoresis. J. Chromatogr. A. 807:3‐36.
   Bensen, J.R. and Hare, P.E. 1975. o‐Phthalaldehyde: Fluorogenic detection of primary amines in the picomole range. Comparison with fluorescamine and ninhydrin. Proc. Nat. Acad. Sci. U.S.A. 72:619‐622.
   Bertrand, M., Chabin, A., Brack, A., and Westall, F. 2008. Separation of amino acid enantiomers via chiral derivatization and non‐chiral gas chromatography. J. Chromatogr. A 1180:131‐137.
   Bidlingmeyer, B.A., Cohen, S.A., and Tarvin, T.A. 1984. Rapid analysis of amino acids using pre‐column derivatization. J. Chromatogr. 336:93‐104.
   Booth, V.H. 1971. Problems with the determination of FDNB‐available lysine. J. Sci. Food Agric. 22:658‐666.
   Bos, C., Metges, C.C., Gaudichon, C., Petzke, K.J., Pueyo, M.E., Morens, C., Everwand, J., Benamouzig, R., and Tomé, D. 2003. Postprandial kinetics of dietary amino acids are the main determinant of their metabolism after soy or milk protein ingestion in humans. J. Nutr. 133:1308‐1315.
   Bosch, L., Alegriá, A., and Farré, R. 2006. Application of the 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate (AQC) reagent to the RP‐HPLC determination of amino acids in infant foods. J. Chromatogr. B 831:176‐183.
   Bruce, S.J., Tavazzi, I., Parisod, V., Rezzi, S., Kochhar, S., and Guy, P.A. 2009. Investigation of human blood plasma sample preparation for performing metabolomics using ultrahigh‐performance liquid chromatography/mass spectrometry. Anal. Chem. 81:3285‐3296.
   Brückner, H., Haasmann, S., Langer, M., Westhauser, T., and Wittner, R. 1994. Liquid chromatographic determination of D‐ and L‐amino acids by derivatization with o‐phthaldialdehyde and chiral thiols. J. Chromatogr. 666:259‐273.
   Bütikofer, U., Fuchs, D., Bosset, J.O., and Gmür, W. 1991. Automated HPLC‐amino acid determination of protein hydrolysates by precolumn derivatization with OPA and FMOC and comparison with classical ion exchange chromatography. Chromatographia 31:441‐447.
   Carpenter, K.J. and Bjarnason, J. 1968. Nutritional evaluation of proteins by chemical methods. In Evaluation of Novel Protein Products (A.E. Bender, R. Kihlberg, B. Löfqvist, and L. Munck, eds.) pp. 161. Pergamon Press, Oxford.
   Carraway, K.L. and Koshland, D.E. 1972. Carbodiimide modification of proteins. In Methods in Enzymology, Vol. 25. (C.H.W. Hirs, ed.) pp. 616‐623. Academic Press, New York.
   Chiou, S‐H. and Wang, K‐T. 1988. Peptide and protein hydrolysis by microwave irradiation. J. Chromatogr. 448:404‐410.
   Cohen, S.A. and Michaud, D.P. 1993. Synthesis of a fluorescent derivatizing reagent, 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high‐performance liquid chromatography. Anal. Biochem. 211:279‐287.
   Csapó, J., Schmidt, J., Csapó‐Kiss, Z., Holló, G., Holló, I., Wagner, L., Cenkvári, É., Varga‐Visi, É., Pohn, G., and Andrássy‐Baka, G. 2001. A new method for the quantitative determination of protein of bacterial origin on the basis of D‐aspartic acid and D‐glutamic acid content. Acta Aliment. 30:37‐52.
   Cuq, J‐L., Besancon, P., Chartier, L., and Cheftel, C. 1978. Oxidation of methionine residues of food proteins and nutritional availability of protein‐bound methionine sulfoxide. Food Chem. 3:85‐102.
   Darragh, A.J. and Moughan, P.J. 1998. The amino acid composition of human milk corrected for amino acid digestibility. Brit. J. Nutr. 80:25‐34.
   Darragh, A.J. and Moughan, P.J. 2005. The effect of hydrolysis time on amino acid analysis. J. AOAC Int. 88:888‐893.
   Darragh, A.J., Garrick, D.J., Moughan, P.J., and Hendriks, W.H. 1996. Correction for amino acid loss during acid hydrolysis of a purified protein. Anal. Biochem. 236:199.
   Davey, J.F. and Ersser, R.S. 1990. Amino acid analysis of physiological fluids by high‐performance liquid chromatography with phenylisothiocyanate derivatization and comparison with ion‐exchange chromatography. J. Chromatogr. 528:9‐23.
   Davies, M.G. and Thomas, A.J. 1973. An investigation of hydrolytic techniques for the amino acid analysis of foodstuffs. J. Sci. Food Agric. 24:1525‐1540.
   Davies, R.L., Baigent, D.R., Levitt, M.S., Mollah, Y., Rayner, C.J., and Frensham, A.B. 1992. Accuracy and precision in amino acid analysis. J. Sci. Food Agric. 59:423‐436.
   Desrosiers, T., Savoie, L., Bergeron, G., and Parent, G. 1989. Estimation of lysine damage in heated whey proteins by furosine determinations in conjunction with the digestion cell technique. J. Agric. Food Chem. 37:1385‐1391.
   Dorresteijn, R.C., Berwald, L.G., Zomer, G., De Gooijer, C.D., Wieten, G., and Beuvert, E.C. 1996. Determination of amino acids using o‐phthalaldehyde‐2‐mercaptoethanol derivatization effect of reaction conditions. J. Chromatogr. A 724:159‐167.
   Einarsson, S., Josefson, B., and Lagerkvist, S. 1983. Determination of amino acids with 9‐fluorenylmethylchloroformate and reversed‐phase high performance liquid chromatography. J. Chromatogr. 282:609‐618.
   Einarsson, S., Folestad, S., and Josefson, B. 1987. Separation of amino acid enantiomers using precolumn derivatization with o‐phthalaldehyde and 2,3,4,6‐tetra‐O‐acetyl‐1‐thio‐β‐glucopyranoside. J. Liq. Chromatogr. 10:1589‐1601.
   Elfakir, C. 2005. HPLC of amino acids without derivatization. In Quantitation of Amino Acids and Amines by Chromatography, Vol. 70. (I. Molnár‐Perl, ed.) pp. 120‐136. Elsevier, Amsterdam.
   Elias, R.J., McClements, J., and Decker, E.A. 2005. Antioxidant activity of cysteine, tryptophan, and methionine residues in continuous phase β‐lactoglobulin in oil‐in‐water emulsions. J. Agric. Food Chem. 53:10248‐10253.
   Ellinger, G.M. and Duncan, A. 1976. The determination of methionine in proteins by gas‐liquid chromatography. Biochem. J. 155:615‐621.
   Finley, J.W. 1985. Reducing variability in amino acid analysis. In Digestibility and Amino Acid Availability in Cereals and Oilseeds. (J.W. Finley and D.T. Hopkins, eds.) pp. 15‐30. American Association of Cereal Chemists, St. Paul, Minn.
   Finot, P.A., Bricout, J., Viani, R., and Mauron, J. 1968. Identification of a new lysine derivative obtained upon hydrolysis of heated milk. Experientia 24:1097‐1099.
   FAO/WHO. 1991. Protein Quality Evaluation: Report of a Joint FAO/WHO Expert Consultation. Paper No. 51. Food and Agriculture Organization of the United Nations, Rome.
   Fountoulakis, M. and Lahm, H‐W. 1998. Hydrolysis and amino acid composition analysis of proteins. J. Chromatogr. A 826:109‐134.
   Friedman, M. and Gumbmann, M.R. 1988. Nutritional value and safety of methionine derivatives, isomeric dipeptides and hydroxy analogs in mice. J. Nutr. 118:388‐397.
   Friedman, M., Pang, J., and Smith, G.A. 1984. Ninhydrin‐reactive lysine in food proteins. J. Food Sci. 49:10‐20.
   Garcia, S.E. and Baxter, J.H. 1992. Determination of tryptophan content in infant formulas and medical nutritionals. J. AOAC Int. 75:1112‐1119.
   Gehrke, C.W., Wall, L.L. Sr., Absheer, J.S., Kaiser, F.E., and Zumwalt, R.W. 1985. Sample preparation for chromatography of amino acids. Acid hydrolysis of proteins.. J. AOAC Int. 68:811‐821.
   Gilani, G.S., Xiao, C., and Lee, N. 2008. Need for accurate and standardized determination of amino acids and bioactive peptides for evaluating protein quality and potential health effects of foods and dietary supplements. J. AOAC Int. 91:894‐900.
   Gjøen, A.U. and Njaa, L.R. 1977. Methionine sulfoxide as a source of sulfur‐containing amino acids for the young rat. Br. J. Nutr. 37:93‐105.
   Golaz, O., Wilkins, M.R., Sanchez, J‐C., Appel, R.D., Hochstrasser, D.F., and Williams, K.L. 1996. Identification of proteins by their amino acid composition: An evaluation of the method. Electrophoresis 17:573‐579.
   Guitart, A., Hernández Orte, P., and Cacho, J. 1991. Stability of phenyl(thiocarbamoyl) amino acids and optimization of their separation by high‐performance liquid chromatography. Analyst 116:399‐403.
   Hanczkó, R., Kőrös, Á., Tóth, F., and Molnár‐Perl, I. 2005. Behavior and characteristics of biogenic amines, ornithine and lysine derivatized with the o‐phthalaldehyde‐ethanethiol‐fluorenylmethyl chloroformate reagent. J. Chromatogr. 1087:210‐222.
   Hanczkó, R., Jámbor, A., Perl, A., and Molnár‐Perl, I. 2007. Advances in the o‐phththalaldehyde derivatizations comeback to the o‐phththalaldehyde‐ethanethiol reagent. J. Chromatogr. 1163:24‐43.
   Hanko, V.P. and Rohrer, J.S. 2002. Direct determination of tryptophan using high‐performance anion‐exchange chromatography with integrated pulsed amperometric detection. Anal. Biochem. 30:204‐209.
   Hayashi, R. and Suzuki, F. 1985. Determination of methionine sulfoxide in protein and food by hydrolysis with p‐toluenesulfonic acid. Anal. Biochem. 149:521‐528.
   Hendriks, W.H., Moughan, P.J., Boer, H., and van der Poel, A.F.B. 1994. Effects of extrusion on the dye‐binding, fluorodinitrobenzene‐reactive and total lysine content of soyabean meal and peas. Anim. Feed Sci. Technol. 48:99‐109.
   Hendriks, W.H., Tarttelin, M.F. and Moughan, P.J. 1998. The amino acid composition of cat (Felis catus) hair. Anim. Sci. 67:165‐170.
   Hendriks, W.H., Rutherfurd, S.M., and Rutherfurd, K.J. 2001. Importance of sulfate, cysteine and methionine as precursors to felinine synthesis by domestic cats (Felis catus). Comp. Biochem. Physiol. C, Pharmacol. Toxicol. Endocrinol. 129C:211‐216.
   Higbee, A., Wong, S., and Henzel, W.J. 2003. Automated sample preparation using vapor‐phase hydrolysis for amino acid analysis. Anal. Biochem. 318:155‐158.
   Hurrell, R.F. and Carpenter, K.J. 1974. Mechanisms of heat damage in proteins. 4: The reactive lysine content of heat‐damaged material as measured in different ways. Br. J. Nutr. 32:589‐604.
   Hurrell, R.F. and Carpenter, K.J. 1981. The estimation of available lysine in foodstuffs after Maillard reactions. Prog. Food Nutr. Sci. 5:159‐176.
   Janssen, P.S.L., van Nispen, J.W., Melgers, P.A.T.A., van den Bogaart, H.W.M., Hamelinck, R.L.A.E., and Goverde, B.C. 1986. HPLC analysis of phenylthiocarbamyl (PTC) amino acids. I. Evaluation and optimization of the procedure. Chromatographia. 22:345‐350.
   Kirschbaum, J. and Luckas, B. 1994. Pre‐column derivatization of biogenic amines and amino acids with 9‐fluorenylmethyl chloroformate and heptylamine. J. Chromatogr. 661:193‐199.
   Kőrös, Á., Hanczkó, R., Jámbor, A., Qian, Y., Perl, A., and Molnár‐Perl, I. 2007. Analysis of amino acids and biogenic amines in biological tissues as their o‐phthalaldehyde/ethanediol/fluorenylmethyl chloroformate derivatives by high‐performance liquid chromatography A deproteinization study. J. Chromatogr. A 1149:46‐55.
   Kőrös, Á., Varga, I., and Molnár‐Perl, I. 2008. Simultaneous analysis of amino acids and amines as their o‐phthalaldehyde‐ethanethiol‐9‐fluorenylmethyl chloroformate derivatives in cheese by high‐performance liquid chromatography. J. Chromatogr. A 1203:146‐152.
   Kroll, J., Rawel, H., and Kröck, R. 1998. Microwave digestion of proteins. Z. Lebensm. Unters. Forsch. 207:202‐206.
   Landry, J. and Delhaye, S. 1992. Simplified procedure for the determination of tryptophan of foods and feedstuffs from barytic hydrolysis. J. Agric. Food Chem. 40:776‐779.
   Landry, J. and Delhaye, S. 1994. Tryptophan determination in feedstuffs: A critical examination of data from two collaborative studies through the evaluation of tryptophan recovery. J. Agric. Food Chem. 42:1717‐1721.
   Landry, J., Delhaye, S., and Jones, D.G. 1992. Determination of tryptophan in feedstuffs: Comparison of two methods of hydrolysis prior to HPLC analysis. J. Sci. Food Agric. 58:439‐441.
   Liardon, R. and Hurrell, R.F. 1983. Amino acid racemisation in heated and alkali‐treated proteins. J. Agric. Food Chem. 31:432‐437.
   Llames, C.R. and Fontaine, J. 1994. Determination of amino acids in feeds: Collaborative study. J. AOAC. Int. 77:1362‐1402.
   Lozanov, V., Petrov, S., and Mitev, V. 2004. Simultaneous analysis of amino acid and biogenic polyamines by high‐performance liquid chromatography after pre‐column derivatization with N‐(9‐fluorenylmethoxycarbonyloxy)succinimide. J. Chromatogr. A. 1025:201‐208.
   Malmer, M.F. and Schroeder, L.A. 1990. Amino acid analysis by high‐performance liquid chromatography with methanesulfonic acid hydrolysis and 9‐fluorenylmethylchloromate derivatization. J. Chromatogr. 514:227‐239.
   Mao, L‐C., Lee, K‐H., and Erbersbobler, H.F. 1993. Effects of heat treatment on lysine in soya protein. J. Sci. Food Agric. 62:307‐309.
   Marfey, P. 1984. Determination of D‐amino acids. II. Use of a bifunctional reagent, 1,5‐difluoro‐2,4‐dinitrobenzene. Carlsberg Res. Commun. 49:591‐596.
   Mauron, J. and Bujard, E. 1964. Guanidination, an alternative approach to the determination of available lysine in foods. Proc. 6th Int. Nutr. Congr. Edinburgh.
   Mauron, J., Mottu, F., Bujard, E., and Eggli, R.H. 1955. The availability of lysine, methionine and tryptophan in condensed milk and milk powder: In Vitro digestion studies. Arch. Biochem. Biophys. 59:433‐451.
   McDonald, J.L., Krueger, M.W., and Keller, J.H. 1985. Oxidation and hydrolysis determination of sulfur amino acids in food and feed ingredients: Collaborative study. J. Assoc. Off. Anal. Chem. 68:826‐829.
   Meltzer, N.M., Tous, G.I., Gruber, S., and Stein, S. 1987. Gas‐phase hydrolysis of proteins and peptides. Anal. Biochem. 160:356‐361.
   Melucci, D., Xie, M., Reschiglian, P., and Torsi, G. 1999. FMOC‐Cl as derivatizing agent for the analysis of amino acids and dipeptides by the absolute analysis method. Chromatographia 49:317‐320.
   Messia, M.C., Di Falco, T., Panfili, G., and Marconi, E. 2008. Rapid determination of collagen in meat‐based foods by microwave hydrolysis of proteins and HPAEC‐PAD analysis of 4‐hydrolxyproline. Meat Sci. 80:401‐409.
   Molnár‐Perl, I. 1997. Tryptophan analysis in peptides and proteins, mainly by liquid chromatography. J. Chromatogr. A 763:1‐10.
   Molnár‐Perl, I. and Khalifa, M. 1992. Tryptophan analysis simultaneously with other amino acids in gas phase hydrochloric acid hydrolyzates using the Pico‐Tag Work Station. Chromatographia 36:43‐46.
   Molnár‐Perl, I., Pintér‐Szakács, M., and Khalifa, M. 1993. High‐performance liquid chromatography of tryptophan and other amino acids in hydrochloric acid hydrolysates. J. Chromatogr. A 632:57‐61.
   Moore, S. and Stein, W.H. 1948. Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 176:367‐388.
   Moore, S. and Stein, W.H. 1951. Chromatography of amino acids on sulfonated polystyrene resins. J. Biol. Chem. 192:663‐681.
   Moughan, P.J. and Rutherfurd, S.M. 1996. A new method for determining digestible reactive lysine in foods. J. Agric. Food Chem. 44:2202‐2209.
   Muramoto, K. and Kamiya, H. 1990. Recovery of tryptophan in peptides and proteins by high‐temperature and short‐term acid hydrolysis in the presence of phenol. Anal. Biochem. 189:223‐230.
   Nagaraja, P., Yathirajan, H.S., and Vasantha, R.A. 2003. Highly sensitive reaction of tryptophan with p‐phenylenediamine. Anal. Biochem. 312:157‐161.
   Nakagawa, S. and Fukuda, T. 1989. Direct amino acid analysis of proteins electroblotted onto polyvinylidene difluoride membrane from sodium dodecyl sulfate‐polyacrylamide gel. Anal. Biochem. 181:75‐78.
   Nakamura, A., Higuchi, M., Iwami, K., and Iwai, K. 1983. Digestion and absorption of oxidised casein. Agric. Biol. Chem. 47:2395‐2396.
   Nakazawa, M. and Manabe, K. 1992. The direct hydrolysis of proteins containing tryptophan on polyvinylidene difluoride membranes by mercaptoethanesulfonic acid in vapour phase. Anal. Biochem. 206:105‐108.
   Ng, L.T., Pascaud, A., and Pascaud, M. 1987. Hydrochloric acid hydrolysis of protein and determination of tryptophan by reverse‐phase‐high performance liquid chromatography. Anal. Biochem. 167:47‐52.
   Nissen, S. 1992. Amino acid analysis in food and physiological samples. In Modern Methods in Protein Nutrition and Metabolism (S. Nissen, ed.) pp. 1‐8. Academic Press, San Diego.
   Ozols, J. 1990. Amino acid analysis. Methods Enzymol. 182:587‐601.
   Peace, R.W. and Gilani, G.S. 2005. Chromatographic determination of amino acids in foods. J. AOAC Int. 88:877‐887.
   Petritis, K., Elfakir, C., and Dreux, M. 2002. A comparative study of commercial liquid chromatographic detectors for the analysis of underivatized amino acids. J. Chromatogr. A 961:9‐21.
   Puchala, R., Piór, H., von Keyserlingk, M., Shelford, J.A., and Barej, W. 1994. Determination of methionine sulfoxide in biological materials using HPLC and its degradability in the rumen of cattle. Anim. Feed Sci. Technol. 48:121‐130.
   Rao, S.R., Carter, F.L., and Frampton, V.L. 1963. Determination of available lysine in oilseed meal proteins. Anal. Chem. 35:1927‐1930.
   Rayner, C.J. 1985. Protein hydrolysis of animal feeds for amino acid content. J. Agric. Food Chem. 33:722‐725.
   Robel, E.J. and Crane, A.B. 1972. An accurate method for correcting unknown amino acid losses from protein hydrolysates. Anal. Biochem. 48:233‐246.
   Rodríguez, C., Frías, J., Vidal‐Valverde, C., and Hernández, A. 2007. Total chemically available (free and intrachain) lysine and furosine in pea, bean, and lentil sprouts. J. Agric. Food Chem. 55:10275‐10280.
   Rogers, C.J., Kimmel, J.R., Hutchin, M.E., and Harper, H.A. 1954. A hydroxyproline method of analysis for a modified gelatin in plasma and urine. J. Biol. Chem. 206:553‐559.
   Roth, M. and Hampai, A. 1973. Column chromatography of amino acids with florescence detection. J. Chromatogr. 83:353‐356.
   Rowan, A.M., Moughan, P.J., and Wilson, M.N. 1989. Alkaline hydrolysis for the determination of tryptophan in biological samples. Proc. Nutr. Soc. 14:169‐172.
   Rowan, A.M., Moughan, P.J., and Wilson, M.N. 1992. Effect of hydrolysis time on the determination of the amino acid composition of diet, ileal digesta, and feces samples and on the determination of dietary amino acid digestibility coefficients. J. Agric. Food Chem. 40:981‐985.
   Rutherfurd, S.M. 2008. Accurate determination of the amino acid content of selected feedstuffs. Int. J. Food Sci. Nutr. 22:1‐10.
   Rutherfurd, S.M. and Moughan, P.J. 1993. Use of free or bound amino acid molecular weights in the determination of amino acid compositions. In Manipulating Pig Production IV (E.S. Batterham. ed.) p. 229. Australasian Pig Science Association, Attwood, Victoria, Australia.
   Rutherfurd, S.M. and Moughan, P.J. 2000. Developments in the determination of protein and amino acids. In Feed Evaluation: Principles and Practice. (P.J. Moughan, M.W.A. Verstegen, and M.I. Visser Reyneveld, eds.) pp. 45‐56. Wageningen Academic Publishers, Wageningen, The Netherlands.
   Rutherfurd, S.M. and Moughan, P.J. 2007. Development of a novel bioassay for determining the available lysine contents of foods and feedstuffs. Nutr. Res. Rev. 20:3‐16.
   Rutherfurd, S.M. and Moughan, P.J. 2008. The determination of sulfur amino acids in foods as related to bioavailability. J. AOAC Int. 91:907‐913.
   Rutherfurd, S.M., Moughan, P.J., and van Osch, L. 1997. Digestible reactive lysine in processed feedstuffs: Application of a new bioassay. J. Agric. Food Chem. 45:1189‐1194.
   Rutherfurd, S.M., Schneuwly, A., and Moughan, P.J. 2007a. Analyzing sulphur amino acids in selected feedstuffs using least squares non‐linear regression. J. Agric. Food Chem. 55:8019‐8024.
   Rutherfurd, S.M., Kitson, T.M., Woolhouse, A.D., and Hendriks, W.H. 2007b. Felinine stability in the presence of selected urine compounds. Amino Acids 32:235‐242.
   Rutherfurd, S.M., Moughan, P.J., Lowry, D., and Prosser, C.G. 2008. An accurate determination of the amino acid content of three goat milk powders using multiple hydrolysis times. Int. J. Food Sci. Nutr. 59:679‐690.
   Sarwar, G., Christensen, D.A., Finlayson, A.J., Friedman, M., Hackler, L.R., Mackenzie, S.L., Pellett, P.L., and Tkachuk, R. 1983. Inter‐ and intra‐laboratory variations in amino acid analysis of food proteins. J. Food Sci. 48:526‐531.
   Sarwar, G., Blair, R., Friedman, M., Gumbmann, M.R., Hackler, L.R., Pellet, P.L., and Smith, T.K. 1985. Comparison of interlaboratory variation in amino acid analysis and rat growth assays for evaluating protein quality. J. AOAC Int. 68:52‐56.
   Sarwar, G., Botting, H.G., and Peace, R.W. 1988. Complete amino acid analysis in hydrolysates of foods and feeds by liquid chromatography of precolumn phenylisothiocyanate derivatives. J. Assoc. Off. Anal. Chem. 71:1172‐1175.
   Sedgwick, G.W., Fenton, T.W., and Thompson, J.R. 1991. Effects of protein precipitating reagents on the recovery of plasma free amino acids. Can. J. Anim. Sci. 71:953‐957.
   Shindo, N., Fujimura, T., Nojima‐Kazuno, S., Mineki, R., Furusawa, S., Sasaki, K‐I., and Murayama, K. 1998. Identification of multidrug resistant protein 1 of mouse leukemia P388 cells on a PVDF membrane using 6‐aminoquinolyl‐carbamyl (AQC)‐amino acid analysis and World Wide Web (WWW)‐accessible tools. Anal. Biochem. 264:251‐258.
   Sjøgerg, L.B. and Bostrøm, S.L. 1977. Studies in rats on the nutritional value of hydrogen peroxide‐treated fish protein and the utilization of oxidised sulphur‐amino acids. Br. J. Nutr. 38:189‐205
   Smith, J.T. 1997. Developments in amino acid analysis using capillary electrophoresis. Electrophoresis 18:2377‐2392.
   Soby, L.M. and Johnson, P. 1981. Determination of asparagine and glutamine in polypeptides using bis(1,1‐trifluoroacetoxy)iodobenzene. Anal. Biochem. 113:149‐153.
   Sochaski, M.A., Jenkins, A.J., Lyons, T.J., Thorpe, S.R., and Baynes, J.W. 2001. Isotope dilution gas chromatography/mass spectrometry method for the determination of methionine sulfoxide in protein. Anal. Chem. 73:4662‐4667.
   Stein, W.H. and Moore, S. 1954. The free amino acids of human blood plasma. J. Biol. Chem. 211:915‐926.
   Steinhart, H. 1984. Summary of the workshop on “tryptophan analysis”. In Proc. VI Int. Symp. Amino Acids. (T. Zebrowska, L. Buraczewska, S. Buraczewski, J. Kowalczyk, and B. Pastuszewska. eds.) pp. 434‐447. Polish Scientific Publishers, Warsaw.
   Todd, J.M., Marable, N.L., and Kehrberg, N.L. 1984. Methionine sulfoxide determination after alkaline hydrolysis of amino acid mixtures, model protein systems, soy products and infant formulas. J. Food Sci. 49:1547‐1551.
   Torbatinejad, N.M., Rutherfurd, S.M., and Moughan, P.J. 2005. Total and reactive lysine contents in selected cereal‐based food products. J. Agric. Food Chem. 53:4454‐4458.
   Tower, D.B. 1967. Enzymatic determination of glutamine and asparagine. In Methods in Enzymology, Vol. 11 (C.H.W. Hirs, ed.) pp. 77‐93. Academic Press, New York.
   van der Meer, J.M. 1990. Amino acid analysis of feeds in the Netherlands: Four‐year proficiency study. J. AOAC Int. 73:394‐398.
   Vigo, M.S., Malec, L.S., Gomez, R.G., and Llosa, R.A. 1992. Spectrophotometric assay using o‐phthaldialdehyde for determination of reactive lysine in dairy products. Food Chem. 44:363‐365.
   Weiss, M., Manneberg, M., Juranville, J‐F., Lahm, H‐W., and Fountoulakis, M. 1998. Effect of the hydrolysis method on the determination of the amino acid composition of proteins. J. Chromatogr. A 795:263‐275.
   Wilcox, P.E. 1967. Determination of amide residues by chemical analysis. In Methods in Enzymology, Vol. 11 (C.H.W. Hirs ed.) pp. 63‐76. Academic Press, New York.
   Wong, W.S.D., Osuga, D.T., Burcham, T.S., and Feeney, R.E. 1984. Determination of tryptophan as the reduced derivative by acid hydrolysis and chromatography. Anal. Biochem. 143:62‐70.
   Woodward, C. and Henderson, J.W. Jr. 2007. High‐speed amino acid analysis (AAA) on 1.8‐µm reversed‐phase (RP) columns. Technical note. Agilent Technologies, Wilmington, Delaware.
   Yamada, H., Moriya, H., and Tsugita, A. 1991. Development of an acid hydrolysis method with high recoveries of tryptophan and cysteine for microquantities of protein. Anal. Biochem. 198:1‐5.
   Yeung, K.K. and Lucy, C.A. 1999. Ultrahigh‐resolution capillary electrophoretic separation with indirect ultraviolet detection: Isotopic separation of [14N] and [15N]ammonium. Electrophoresis 20:2554‐2559.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library