M. Pintér-Szakács

Optimum yield of pyridosine and furosine originating from maillard reactions monitored by ion-exchange chromatography

Abstract An improved ion-exchange chromatographic method for rapid separation and quantitative estimation of deoxyfructosyllysines and their hydrolysis products (pyridosine and furosine) is described. The proposed elution procedure has good reproducibility; the optimum hydrolysis conditions for the highest molar ratio of pyridosine and furosine are given. The furosine/pyridosine molar ratios obtained by ⌈dhydrolysis” of model fructosyllysine as well as of natural protein matrices (soya beans, tomato powder, barley and malts) are in good agreement and without exception > 1. The smallest amount of pyridosine determined was 0.009% (w/w). The relationship between the compounds pyridosine, furosine, lysine and carbohydrate units present in the parent moiety is discussed.

High-performance liquid chromatography of tryptophan and other amino acids in hydrochloric acid hydrolysates

Abstract Tryptamine [3-(2-aminoethyl)indole] as an additive to 6 M HCl largely prevented the decomposition of tryptophan in proteins, and also in gas-phase hydrolysis, using a Pico-Tag Work Station, at 145°C for 4 h. This procedure proved to be excellent for amino acid analysis using conventional high-performance liquid chromatography with derivatization with phenyl isothiocyanate. The recovery of tryptophan from model solutions and from proteins was 80–98%. The results proved to be as good as any other obtained by the analysis of special alkaline or organic acid hydrolysates, most of which are suitable exclusively for the determination of tryptophan. The most important advantage of tryptamine was that it did not affect the quantitative recovery of other amino acids of proteins, as shown by the composition of lysozyme hydrolysates obtained in the absence and presence of various amounts of tryptamine. The reproducibility of the measurements was 3.7% (relative standard deviation) or less.

Gas-liquid chromatographic determination of the rafflnose family of oligosaccharides and their metabolites present in soy beans

Abstract The relative retention times and detector responses of trimethylsilyl-oxzime derivatives of the components of soluble soy saccharides, up to pentasaccharide, on SP-2250 liquid phase are reported. Arabinose, rhamnose, fructose, galactose, glucose, sucrose, cellobiose, galactobiose, melibiose, rafflnose, cellotriose, galactotriose, manninotriose, stachyose, verbascotetraose and verbascose were wll resolved. A gas—liquid chromatographic method for rapid separation and quantitation of the constituents of every member of the rafflnose oligosaccharides, present in soy beans, is described

Spectrophotometric determination of tryptophan in intact proteins by the acid ninhydrin method

The interaction of tryptophan, lysozyme and tyrosine with ninhydrin in strong acid media has been investigated at 20, 25, 30, and 35 degrees C by spectrophotometry. Second-order rate constants and molar absorptivity values have been evaluated from an analytical point of view. Optimum conditions for the selective estimation of tryptophan, tryptophan residues in intact proteins, and indoles--without the disturbing effect of tyrosine--have been given. Under optimum conditions, in the concentration range from 2.5 X 10(-8) to 3.0 X 10(-7)M, molar absorptivity values and reproducibility data for various reactants have been reported. Molar absorptivity values (Am X 10(-3)/M X cm) of tryptophan (21.35), lysozyme (19.33), bovine serum albumin (21.05), human serum albumin (21.00), casein (17.85), alpha-chymotrypsin (18.28), trypsin (14.43), indole (5.03), and indole-3-acetic acid (13.75) have been measured with a standard error of 2.3% or less for any particular reactant.

Modifications in the chemical derivatization of carboxylic acids for their gas chromatographic analysis

Abstract Gas chromatographic analysis of (i) C 1 C 20 fatty acids, (ii) C 2 C 16 aliphatic dicarboxylic acids, and (iii) in a single run, C 16 and C 18 fatty acids, C 2 C 7 aliphatic dicarboxylic acids, and aromatic di- and polycarboxylic acids, was made possible by derivatization with sulphuric acid-butanol. In addition, it has been shown that aqueous solutions of fatty acids, aliphatic dicarboxylic acids, aromatic polycarboxylic acids and aliphatic hydroxy acids can be esterified by sulphuric acid-butanol, with different yields, depending on the amount of water present. The efficiency of the esterification process in the presence of water has been extended by use of anhydrous sodium sulphate.

Gas chromatography of tryptophan together with other amino acids in hydrochloric acid hydrolysates

The classical hydrolysis of proteins with hydrochloric acid using tryptamine [3-(2-aminoethyl)indole] as additive revealed that tryptophan can be measured without destruction together with other amino acids by gas chromatography. An extensive study was made to establish the optimum conditions for protein hydrolysis (time and temperature of hydrolysis, amount of tryptamine) and for the derivatization of amino acids. The amino acid contents (including tryptophan) of standard proteins such as lysozyme, bovine and human albumin, human gamma-globulin, casein and alpha-chymotrypsin and protein matrices (meat and fish meals, sunflower) were determined, after hydrochloric acid hydrolysis (4 h, 145 degree C) in the presence of tryptamine. as N, O, (S)-trifluoroacetyl isobutyl esters with SE-30 as the stationary phase. The reproducibility of the measurements was 4.6% (relative standard deviation) or less.

Standardization of cation-exchange clean-up prior to gas chromatography of amino acids

Abstract The optimum conditions for the cation-exchange clean-up of amino acids, present in protein hydrolysates, prior to their gas chromatographic determination were investigated. The results of exchaustive study monitoring the amounts of amino acids as N,O(S)-trifluoroacetyl isobutyl esters, revealed that the recovery of amino acids from the column was affected appreciably by either the particle size or the divinylbenzene content of the resins. Quantitative recovery and reproducible determination of amino acids require (i) a 50-fold excess of resin (calculated as equivalent capacity relative to the amino acids present) and (ii) sufficient amounts of eluates: the volumes both of distilled water and of 7 M ammonia solution must be about six times the volume of the wet resin applied. Under optimum conditions the recoveries of alanine, glycine, threonine, serine, valine, leucine (isoleucine), proline, hydroxyproline, methionine, aspartic acid, phenylalanine, ornithine, glutamic acid, tyrosine, lysine, arginine and cystine, were quantitative, both without and after hydrolysis, and that of tryptophan was ca . 85%.

Gas chromatographic determination of isocitric and malic acid in the presence of a large excess of citric acid

Abstract Derivatization yields of different esters (methyl, ethyl, n -propyl, isopropyl, n -butyl and isobutyl) of citric, malic and isocitric acids with various acylating agents, such as acetic anhydride, propionic anhydride, trifluoroacetic anhydride and heptafluorobutyric anhydride, were investigated. The formation of acylated malic and isocitric acid esters is rapid and quantitative whereas the acylation of citric acid esters was slow and partial in most instances. It was found that selective analytical conditions can be achieved with the O -heptafluorobutyryl n -butyl esters. The analytical applicability of the separation and determination of the malic, isocitric and citric acid contents of model solutions and of pressed lemon juice is discussed. Concentrations of 1.2 × 10 −3 −9.0 × 10 −3 g per 100 g of isocitric acid and 4.2 × 10 −3 −3.5 × 10 −2 g per 100 g of malic acid in the presence of 9.3 × 10 −1 g per 100 g of citric acid were measured with relative standard deviations of 7.5 and 4.2%, respectively.

Dye-binding stoichiometry of AO 12, AB 10B and OG with etalon proteins, feed and feedingstuffs and its application for reactive lysine determination

Abstract Our recently developed method for the determination of the reactive lysine content of soya bean proteins with the dye, Orange G, has been extended. The interactions of several etalon proteins and high protein-containing food and feedstuffs with Orange G, Acid Orange 12 and Amido Black 10B have been studied. The stoichiometric investigations showed that the reactive lysine content can be quantitatively determined by dyebinding although total nitrogen content measurements are no more advantageous than by Kjeldahl. A probable mechanism for the interaction has been given.

Gas chromatographic analysis of different homologous series of acids esterified in aqueous solutions with butyl and propyl alcohols

Abstract The esterification of homologous series of fatty acids, aliphatic and aromatic di- and polycarboxylic acids, with aliphatic alcohols in the presence of water and a mineral acid as catalyst, is described. The efficiency of derivatization as well as the detector responses, referred to the organic carbon content of the derivatized products, obtained with two alcohols are compared to each other to establish the optimum conditions for various series of acids. The molar ratios of water/alcohol which yielded quantitative esterifications are given, as is the maximum water content of the esterifying mixture permitted in order for esterification to proceed to completion. The optimum conditions for a reasonable ester yield in the presence of anhydrous sodium sulphate are also presented.