Analysis of amino acids

Although conventional amino acid analysis is extremely reliable, it does not satisfy today s demands for both higher sensitivity (fmol range) and shorter analysis times (10 to 15 min) while maintaining the same high resolution. These requirements can only be met by HPLC methods that have since been developed. Applications involve a precolumn derivatization with either o-phthaldialdehyde (OPA) [14,15], fluorenylmethyl-oxycarbonylchloride (FMOC) [16,17], phenylthiohydantoin (PTH) [18-20], phenyliso- 

Many procedures are available for measuring amino acids in biological samples. In the context of diagnosing pathological disorders, three groups of tests for amino acid analysis are of importance  

Screening tests, including thin layer chromatography (TLC), urine color tests, and the Guthrie microbiological test. (Details of these tests are given in a previous edition of this textbook.)  

Tests that identify an unknown amino acid or metabolite. Quantitative Tests 

Traditionally, amino acids have been quantitatively measured in body fluids using gas chromatography (GC) liquid chromatography (LG), including and ion- 

Advantages of GC include small sample size, sensitivity, and speed, but a major limitation is the relatively low volatility 

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The automated amino acid analy2er depends on ion-exchange chromatography (117) and is now a routine tool for the analysis of amino acid mixtures (118). This most advanced machine can detect as Htde as 10 pmol in ninhydrin reaction analysis. One-half to two hours are required for each analysis. An analysis chart is shown in Figure 2. 

The purification and analysis of individual amino acids from complex mixtures was once a very difficult process. Today, however, the biochemist has a wide variety of methods available for the separation and analysis of amino acids, or for that matter, any of the other biological molecules and macromolecules we... 

FIGURE 5.2 The peptide bond is shown in its usnal trans conformation of carbonyl O and amide H. The atoms are the oi-carbons of two adjacent amino acids joined in peptide linkage. The dimensions and angles are the average valnes observed by crystallographic analysis of amino acids and small peptides. The peptide bond is the light gray bond between C and N. (Adapted from Ramachandran, G. A., ct ai, 1974. Biochimica Biophysica Acta 359 298-302.)... 

The great leap forward for chromatography was the seminal work of Martin and Synge (7) who in 1941 replaced countercurrent liquid-liquid extraction by partition chromatography for the analysis of amino acids from wool. Martin also realized that the mobile phase could be a gas rather than a liquid, and with James first developed (8) gas chromatography (GC) in 1951, following the gas-phase adsorption-chromatographic separations of Phillips (9). 

Another reagent, 2,4-dinitrofluorobenzene, also known as Sanders Reagent, has been used in a similar manner in the analysis of amino acids and amines (41). 

Applications of the oxalate-hydrogen peroxide chemiluminescence-based and fluorescence-based assays with NDA/CN derivatives to the analysis of amino acids and peptides are included. The sensitivity of the chemiluminescence and fluorescence methods is compared for several analytes. In general, peroxyoxalate chemiluminescence-based methods are 10 to 100 times more sensitive than their fluorescence-based counterparts. The chief limitation of chemiluminescence is that chemical excitation of the fluorophore apparently depends on its structure and oxidation potential. 

Van Klinken, GJ. 1991 Dating and Dietary Reconstruction by Isotopic Analysis of Amino Acids in Fossil Bone Collagen—with Special Reference to the Caribbean. Ph.D. dissertation. University of Groningen, The Netherlands. 

Borroto-Esoda K, Miller MD, Arterburn S (2007) Pooled analysis of amino acid changes in the HBV polymerase in patients from four major adefovir dipivoxil chnical trials, J Hepatol 47 492 98... 

The analysis of amino acids involves chromatographic issues similar to those encountered in analysis of simple amines. Underivatized amino acids have, with a few exceptions, weak UV absorbance and a strong tendency to interact with stationary phases in undesirable ways. Underivatized amino acids are normally separated with ion exchange chromatography, then visualized post-column by reaction with ninhydrin, o-phthaladehyde (OPA), or other agents. Underivatized tryptophan and the metabolites kynurenine, 3-hydroxykynurenine, kynurenic acid, and 3-hydroxyanthranilic acid, were separated on a Partisphere 5-p ODS column with fluorescent detection.121... 

Bartok, T., Szalai, G., Lorincz, Zs., Borcsok, G., and Sagi, F., High-speed RP-HPLC/FL analysis of amino acids after automated two-step derivatization with o-phthaldialdehyde/3-mercaptoproprionic acid and 9-fluorenylmethyl chloroformate, /. Liq. Chromatogr., 17, 4391, 1994. 

Stocchi, V., Palma, F., Piccoli, G., Biagiarelli, B., Magnani, M., Masat, L., and Cucchiarini, L., Analysis of amino acids as DABS-derivatives with a sensitivity to the femtomole using RP-HPLC narrow-bore columns, Amino Acids, 3, 303, 1992. 

Benson, Jr., J. V. and Patterson, J. A., Accelerated automatic chromatographic analysis of amino acids on a spherical resin, Anal. Chem., 37, 1108, 1965. 

Murayama, K. and Shindo, N., Recommended method for the analysis of amino acids in biological materials, /. Chromatogr., 143, 137, 1977. 

Klinker CC, Bowser MT (2007) 4-Fluoro-7-nitro-2, 1, 3-benzoxadiazole as a fluorogenic labeling reagent for the in vivo analysis of amino acid neurotransmitters using online microdialysis-capillary electrophoresis. Anal Chem 79 8747-8754... 

M.R. Shilling, H.P. Khanjian, L.A.C. Souza, Gas chromatographic analysis of amino acids as ethyl chloroformate derivatives. Part 1 compositions of proteins associated with art objects and monuments, Journal of American Institute for Conservation, 35, 45 59 (1996). 

Mateo Castro R., Domenech Carbo M.T., Peris Martinez V., Gimeno Adelantado J.V., Bosch Reig F., Study of binding media in works of art by gas chromatographic analysis of amino acids and fatty acids derivatized with ethyl chloroformate, Journal of Chromatography A, 1997, 778, 373 381. 

A GC analysis of amino acids requires a derivatisation step to increase the volatility of the amino acids. Generally, norleucine and/or norvaline are the internal standards added to the hydrolysate to check the derivatisation yield. According to the experimental method applied, the limits of detection (LOD) vary in the range 10 100 pg for each amino acid. Regarding the chromatographic columns, as most of the derivatives are esters barely polar compounds the most commonly used are fused-silica capillary columns with a low... 

M.R. Schilling, H.P. Khanjian, Gas Chromatographic Analysis of Amino Acids as Ethyl Chloroformate Derivatives. II. Effects of Pigments and Accelerated Aging on the Identification of Proteinaceous Binding Media, Journal of the American Institute of Conservation, 35, 123 144 (1996). 

R.M. Castro, M.T. Domenech Carbo, V.P. Martinez, J.V. Gimeno Adelantado, F. Bosch Reig, Study of Binding Media in Works of Art by Gas Chromatographic Analysis of Amino Acids and Fatty Acids Derivatized with Ethyl Chloroformate, J. Chromatography, A, 778, 373 381 (1997). 

Fogel, M. L. and Tuross, N. (2003) Extending the limits of palaeodietary studies of humans with compound specific carbon isotope analysis of amino acids. Journal of Archaeological Science 30, 535 545. 

Macko, S. A., Uhle, M. E., Engel, M. H. and Andrusevich, V. (1997) Stable nitrogen isotope analysis of amino acid enantiomers by gas chromatography combustion/isotope ratio mass spectrometry. Analytical Chemistry 69, 926 929. 

McCullagh, J. S. O., Juchelka, D. and Hedges, R. E. M. (2006) Analysis of amino acid 13C abundance from human and faunal bone collagen using liquid chromatography/isotope ratio mass spectrometry. Rapid Communications in Mass Spectrometry 20, 2761 2768. 

Senes, A., Gerstein, M., and Engelman, D. M. (2000) Statistical analysis of amino acid patterns in transmembrane helices the GxxxG motif occurs frequently and in association with beta-branched residues at neighboring positions. J. Mol. Biol. 296,921-936. 

The column chromatography technique using Dowex 50 ion-exchange resin, introduced in 1951 (M2) and improved in 1954 (M3) by Moore and Stein, first made possible the precise quantitative analysis of amino acids liberated in the course of acid hydrolysis of urine. Similar results were also obtained by Muting in 1954 (M4), who used paper chromatography methods. In this procedure amino acids were quantitatively determined after staining on the paper and elution of the resulting spots. 

In the course of studies on other pathological amino acidurias, the accompanying peptiduria has also been observed by many authors. Rapp de Eston et al. (R2) observed a marked increase in the excretion of both free amino acids and peptides in patients with diffuse hepatic necrosis. Using a simplified chromatographic method adapted to clinical purposes and suitable for analysis of amino acids excreted with urine, Skarzynski et al. (S5) demonstrated a raised level of a certain peptide which is always present in normal urine in smaller quantities, as well as the appearance of some new peptides in cases of jaundice and liver cirrhosis. Some abnormal peptide spots were also detected on the chromatograms in cases of progressive muscular dystrophy (K4) and in patients irradiated with X-rays (S2). 

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