Rapid Analysis of Nutritionally Important Free Amino Acids in Serum and Organs (Liver, Brain, and Heart) by Liquid Chromatography of Precolumn Phenylisothiocyanate Derivatives

1990 ◽  
Vol 73 (3) ◽  
pp. 470-475 ◽  
Author(s):  
Ghulam Sarwar ◽  
Herbert G Botting
Keyword(s):  
Amino Acids ◽  
Liquid Chromatography ◽  
Amino Acid ◽  
Ion Exchange ◽  
Free Amino Acids ◽  
Amino Acid Analysis ◽  
Free Amino ◽  
Rapid Analysis ◽  
Protein Hydrolysates ◽  
Modified Method

Abstract An amino acid analysis method for protein hydrolysates, using precolumn phenylisothiocyanate (PITC) derivatlzatlon and liquid chromatography, was modified for its application In rapid analysis of commonly occurring free amino acids in serum and other physiological samples. The modifications Included changes In column temperature (47.5°C compared to 25-35°C used In analyzing protein hydrolysates), method of preparing standard and test samples, and gradient conditions. By using a Waters Pico-Tag amino acid analysis 15 cm long column (which Is also used for analyzing protein hydrolysates), separation of 27 PTC-amino acids in human serum and rat liver, brain, or heart was completed in 20 mln by the modified method. The total time for analysis and equilibration was 30 mln. The modified method was much faster than the traditional ion-exchange methods (2-3 h) or the existing liquid chromatographic methods using PITC derivatlzatlon (66-80 mln) for determining nutritionally Important free amino acids In physiological fluids and tissues. Variability of the method (expressed as coefficients of variation) for the determination (Including deprotelnizatlon, derivatlzatlon, and liquid chromatography) of all amino acids was less than 5%, which compared favorably with the reproducibility of Ion-exchange methods

Chromatographic Separation of Free Amino Acids in Table Sirups

1971 ◽  
Vol 54 (1) ◽  
pp. 61-65
Author(s):  
Arthur Russell Johnson ◽  
Richard L Corliss ◽  
Enrique Fernandez-Flores
Keyword(s):  
Amino Acids ◽  
Quantitative Analysis ◽  
Amino Acid ◽  
Ion Exchange ◽  
Free Amino Acids ◽  
Free Amino ◽  
Amino Acid Mixture ◽  
Acid Mixture ◽  
Butyl Esters ◽  
Tlc Analysis

Abstract Qualitative chromatographic methods for the separation of free amino acids in table sirups are presented to aid in the development of chemical indices of composition which may be useful in establishing the identity of sirups and detecting their adulteration. Free amino acids in 2 table sirups were isolated on ion exchange columns and eluted with dilute ammonia. The concentrated amino acid mixture in the eluate was spotted directly on silica gel G plates for TLC analysis, or the amino acids were converted to their N-trifluoroacetyl n-butyl esters for GLC analysis. As many as 16 amino acids were qualitatively separated and identified and a potential for quantitative analysis was demonstrated.

ION EXCHANGE CHROMATOGRAPHY OF THE FREE AMINO ACIDS IN THE PLASMA OF INFANTS UNDER 2,500 GM AT BIRTH

PEDIATRICS ◽  
1970 ◽  
Vol 45 (4) ◽  
pp. 606-613
Author(s):  
Johanne C. Dickinson ◽  
Herman Rosenblum ◽  
Paul B. Hamilton
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ion Exchange ◽  
Free Amino Acids ◽  
Free Amino ◽  
High Sensitivity ◽  
Exchange Chromatography ◽  
Chromatographic Technique ◽  
The Individual ◽  
Amino Acid Concentrations

The free amino acids in the plasma of 46 infants who were under 2,500 gm at birth were determined by an ion exchange chromatographic technique of high sensitivity and resolution. Ninety-two plasma samples were collected from the 46 infants on different days after birth, and the data for 23 amino acids plus taurine and ethanolamine were summarized and compared with newborn, full-term and adult levels. In 16 cases tyrosine levels were high; these values are listed separately. With respect to the remaining amino acids, many showed marked changes during the first few postnatal days; but, by the end of the first week, stable patterns had developed. The decrease or increase of the individual amino acid concentrations in these infants compared to infants with birth weights over 2,500 gm and to the adult was not great and seemed to be characteristic for each amino acid. Attention was drawn to the technical details of preparing and analyzing physiological fluids which would minimize the changes in amino acid concentrations resulting from improper handling.

Ion-Exchange Separation of Amino Acids with Postcolumn Orthophthalaldehyde Detection

1987 ◽  
Vol 70 (2) ◽  
pp. 248-252 ◽  
Author(s):  
Raymond B Ashworth
Keyword(s):  
Amino Acids ◽  
Liquid Chromatography ◽  
Amino Acid ◽  
Ion Exchange ◽  
Amino Acid Analysis ◽  
Corrosion Damage ◽  
Essential Amino Acids ◽  
Detection Sensitivity ◽  
Poultry Products ◽  
Ion Exchange Separation

Abstract Moore's and Stein's classical ion-exchange separation of amino acids remains the standard by which all methods are judged. The adaptation of liquid chromatography (LC) equipment to amino acid analysis was inevitable because microprocessor control of gradients allowed almost infinite variation in gradient shape, producing superior resolution with only 2 buffers. The versatility of LC equipment allowed the instruments to be used for other assays. Adaptation of orthophthalaldehyde (OPA) to amino acid analysis increased detection sensitivity to the picomole range. A method for essential amino acids analysis in mechanically separated red meat and poultry products has been adapted to liquid chromatography using postcolumn hypochlorite oxidation, OPA derivatization, and fluorescence detection. Separation is achieved with 2 sequential concave exponential gradients combining ionic strength and pH increases with halidecontaining buffers. Hydroxyproline and proline are detected with increasing sensitivity through the use of 3-mercaptopropionic acid in a stabilized OPA reagent. Sample preparation is a critical part of the method. A defatting procedure removes fat and other nonprotein nitrogenous substances. The hydrolysis procedure is designed to protect tryptophan which can be routinely assayed in hydrolysates with a modified flow program. Corrosion damage to the equipment by halide buffers has brought about a search for alternative methodology.

Ion-Exchange Chromatography of Free Amino Acids in Human Intraocular Fluids

1971 ◽  
Vol 17 (4) ◽  
pp. 285-289 ◽  
Author(s):  
Davis G Durham ◽  
Johanne C Dickinson ◽  
Paul B Hamilton
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ion Exchange ◽  
Free Amino Acids ◽  
Free Amino ◽  
Subretinal Fluid ◽  
Aqueous Fluid ◽  
Exchange Chromatography ◽  
Chromatographic Technique ◽  
Order Of Magnitude

Abstract Free amino acids were determined in human aqueous, vitreous, and subretinal fluid (50 to 100 µl) from individual eyes by using an ion-exchange chromatographic technique of increased sensitivity. The concentrations of amino acids in aqueous fluid from intraocular malignant melanomas were compared with those found for normal eyes, and some differences were observed. Other pathological conditions investigated were mongolism, intraocular hemangioma, uveitis, Sturge—Weber’s syndrome, and Marfan’s syndrome; only the latter two appeared to have a normal amino acid pattern. Vitreous and subretinal fluid each had a distinctive pattern, with the amino acid concentrations generally lower than in aqueous fluid, except for glutamine, which appeared to be of the same order of magnitude in all intraocular fluids and plasma. Postmortem and eye-bank aqueous fluids were also analyzed, and showed considerable variability from normal. The presence of an additional 28 unknown compounds was demonstrated in aqueous fluid.

AMINO ACID COMPOSITION OF UREDOSPORES OF PUCCINIA GRAMINIS TRITICI

1965 ◽  
Vol 43 (6) ◽  
pp. 695-699 ◽  
Author(s):  
David Stefanye ◽  
Kenneth R. Bromfield
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ion Exchange ◽  
Free Amino Acids ◽  
Free Amino ◽  
Spore Wall ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Puccinia Graminis ◽  
Total Free Amino Acids

Uredospores of Puccinia graminis var. tritici (race 56) were analyzed quantitatively for total free amino acids and ninhydrin-positive substances by ion-exchange chromatography. Extracts of these substances were obtained by leaching the spores and by re-extracting leached spores with boiling water. Thirty-five ninhydrin-positive compounds were found and identified. The leach extract differed quantitatively from the extract obtained by boiling although both contained the same 35 substances. It is proposed that there are easily extractable ninhydrin-positive substances coating the spore wall and ninhydrin-positive substances in the protoplasm that can be extracted only with difficulty.

Complete Amino Acid Analysis in Hydrolysates of Foods and Feces by Liquid Chromatography of Precolumn Phenylisothiocyanate Derivatives

1988 ◽  
Vol 71 (6) ◽  
pp. 1172-1175
Author(s):  
Ghulam Sarwar ◽  
Herbert G Botting ◽  
Robert W Peace
Keyword(s):  
Amino Acids ◽  
Liquid Chromatography ◽  
Amino Acid ◽  
Ion Exchange ◽  
Amino Acid Analysis ◽  
Accurate Determination ◽  
Cysteic Acid ◽  
Simple Liquid ◽  
Liquid Chromatographic

Abstract The amino acid analysis method using precolumn phenylisothiocyanate (PITC) derivatization and liquid chromatography was modified for accurate determination of methionine (as methionine sulfone), cysteine/cystine (as cysteic acid), and all other amino acids, except tryptophan, in hydrolyzed samples of foods and feces. A simple liquid chromatographic method (requiring no derivatization) for the determination of tryptophan in alkaline hydrolysates of foods and feces was also developed. Separation of all amino acids by liquid chromatography was completed in 12 min compared with 60-90 min by ion-exchange chromatography. Variation expressed as coefficients of variation (CV) for the determination of most amino acids in the food and feces samples was not more than 4%, which compared favorably with the reproducibility of ion-exchange methods. Data for amino acids and recoveries of amino acid nitrogen obtained by liquid chromatographic methods were also similar to those obtained by conventional ion-exchange procedures.

Challenging the status quo: A comparison of ion exchange chromatography with liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry methods for the measurement of amino acids in human plasma

2020 ◽  
Vol 57 (4) ◽  
pp. 277-290
Author(s):  
Rachel S Carling ◽  
Benjamin AC McDonald ◽  
Donna Austin ◽  
Deborah Burden ◽  
Joana Correia ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Liquid Chromatography ◽  
Amino Acid ◽  
Ion Exchange ◽  
Amino Acid Analysis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Analytical Performance ◽  
Gold Standard Method

Background Plasma amino acid analysis is key to the diagnosis and monitoring of inherited disorders of amino acid synthesis, catabolism and transport. Ion exchange chromatography (IEC) is widely accepted as the gold standard method of analysis, but with the introduction of liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography mass spectrometry (LC-MS) methods, this should now be questioned. Methods The analytical performance of three commercially available reagent kits, Waters AccQ Tag™ ULTRA LC-MS, SpOtOn Amino Acids LC-MS/MS and Chromsystems MassChrom® Amino Acid Analysis LC-MS/MS, were evaluated and compared with Biochrom Physiological Amino Acids ion exchange chromatography. Correlation with IEC was assessed by Passing-Bablok regression, concordance correlation coefficients (CCC) and Bland-Altman analysis for 21 common amino acids. Calculation of the total error from imprecision and bias was also used to benchmark performance. Results The MassChrom® and SpOtOn kits demonstrated acceptable inter-batch imprecision (CV < 10%) and accuracy (mean bias < 10%), whereas the AccQ Tag™ ULTRA kit did not. Good correlation (CCC > 0.95) with Biochrom IEC was demonstrated for 10/21 analytes in both the MassChrom® and SpOtOn kits and 6/21 in the AccQ Tag™ ULTRA kit. Conclusions The LC-MS assay demonstrated variable analytical performance and correlated poorly with ion exchange chromatography. Both LC-MS/MS assays demonstrated comparable analytical performance and reasonable correlation with ion exchange chromatography. They also confer practical advantages which cannot be realized by ion exchange chromatography, superior specificity and significantly faster analysis time, suggesting that ion exchange chromatography should no longer be described as the gold standard method for plasma amino acid analysis.

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