High-Performance Liquid Chromatographic Analysis of Amino Acids in Edible Seaweeds after Derivatization with Phenyl Isothiocyanate

Summary A reversed-phase high-performance liquid-chromatographic method has been used for analysis of the amino acids in edible seaweed. Sample proteins were hydrolysed with hydrochloric acid and the amino acids produced were derivatized with phenyl isothiocyanate. The resulting phenylthiocarbamyl amino acids were chromatographed on an ODS2 column with UV detection at 254 nm. The mobile phase was a mixture of 0.14 M ammonium acetate buffer, pH 6.4, containing 0.05% triethylamine (A) and 60:40 (v/v) acetonitrile–water (B), at a flow rate of 1.1 mL min^–1; the elution gradient (min:A%) was: 0:90, 8:90, 10:70, 12:70, 18:52, 20:0, 25:0, 28:90, 35:90. Method precision for the different amino acids was between 1.33 and 3.88% (relative standard deviation); detection limits were between 6.9 and 14.3 ng mL^–1. The amino acid content of the algae analysed ranged from 22.4 ± 1.9 to 138.0 ± 5.6 mg g^–1 d.w. The amino acids present at highest concentrations were glutamic acid, alanine, and phenylalanine.     

Estimation of the hydration of polar groups of α-amino acids by differential scanning calorimetry

The heat capacity of hydration of zwitterions derived from aliphatic amino acids depends linearly on the surface area of the amino acid side radicals accessible to water molecules with the slopeb = 2.35±0.11 J mol^–1 K^–1 Å^–2 at 298 K. The linear correlation between hydration heat capacities of zwitterions of aliphatic amino acids and the corresponding aliphatic alcohols with a coefficient of approximately unity confirms the assumption that hydrophobic hydration does not depend on the nature of the surrounding groups. Using the assumption that the hydration of hydrocarbon radicals is independent of the neighboring groups, theb value has been used to calculate the contributions of polar groups. The contributions of OH, COON, and CONH groups of the side radicals in polar amino acids in the zwitterion form are close to zero; in the case of organic nonionic molecules, these contributions are negative. The increments for polar groups obtained for the zwitterions can be used for the calculation of the heat capacities of proteins and polypeptides incorporating charged amino acid residues. The difference between hydrophilic and hydrophobic hydration mechanisms is manifested not only as different magnitudes and signs of heat capacities and temperature coefficients but also in the fact that the neighboring polar (charged) groups have an effect on hydrophilic hydration but have no effect on hydrophobic hydration.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 2237–2242, September, 1996.     

Mixed-ligand fluorocuprates(II). Synthesis and characterisation of [CuF3L(H2O)2]− containing amino acids as coligands

Complex species of the type NH₄[CuF₃L(H₂O)₂] · nH₂O (L = glycine, n = 1; 1-alanine, n = 0) have been prepared by reacting hydrated copper(II) oxide, CuO · nH₂O, with the amino acids in the presence of bifluoride, HF₂ ^–, in aqueous solution. The order of addition of the ligands, fluoride and amino acids is crucial to the synthesis. A reaction solution pH lower than the isoelectric point of the respective amino acids seems to favour the formation of mixed-ligand fluoro complexes. Occurring as zwitterions, the amino acids in the complexes have been shown to be bonded to the copper(II) centre via the carboxyl (—COO^–) moiety representing an unexpected non-chelated form of the coligands. Magnetic moments and e.s.r. data are consistent with six-coordinate copper(II) complexes.     

Determination of Free and Total Underivatized Amino Acids Including L-Canavanine in Bitter Vetch Seeds Using Hydrophilic Interaction Liquid Chromatography

A new hydrophilic interaction liquid chromatography method coupled with diode-array detector was developed for the determination of 17 underivatized amino acids including L-canavanine in bitter vetch [Vicia ervilia (L.) Willd.] seeds. Amino acids were extracted as free as well as total extracts after acid hydrolysis, followed by chromatographic separation on a Zorbax Rx-SIL column with a mobile phase of acetonitrile/potassium phosphate buffer (12.5?mM; pH 3.0) using gradient elution and detection at 190?nm. The method is characterized by a wide linear range (0.01–200?µg/mL, r?>?0.9987), sufficient accuracy (relative error 86.3–109.1%), and suitable precision for the results (relative standard deviation <4.9% in the case of intra-day and <9.8% in the case of inter-day precision). The limits of detection and quantification for free amino acids ranged from 0.01 to 0.24?mg/g and 0.03 to 0.72?mg/g, respectively, whereas the total amino acids ranged from 0.02 to 0.47?mg/g and 0.07 to 1.43?mg/g, respectively. The mean recoveries of free and total amino acids in spiked samples exceeded 70.3% for most amino acids. The mean total content of free and total amino acids in bitter vetch seeds was 1.71 and 14.88?g/100?g seed, whereas the corresponding values for canavanine were 0.07 and 0.19?g/100?g seed, respectively.     

Fruits of two seabuckthorn varieties

Fruits of two varieties of Hippophae rhamnoides L. collected in Kyrgyzstan (I) and Uzbekistan (II) were investigated. Differences in their morphological and biochemical properties were demonstrated. Titrable acids, ascorbic acid, and protein dominated in the fruits of I. Pulp oil of II contained more free fatty acids (acid number 2.9 mg KOH) and carotinoids (419.3 mg%). The principal pulp acid was 16:1. Among the rarely encountered acids of pulp oil, an insignificant quantity of 16:2 was detected (1.2–4.6%). Seed oil of the studied specimens contained >40% of the essential acids 18:2 and 18:3 (vitamin F).Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 435–437, November–December, 2004.This revised version was published online in April 2005 with a corrected cover date.     

PVC Membrane electrode for trinitrobenzenesulfonate and its use for indirect determination of amino acids

A PVC membrane electrode of the conventional type was prepared which is selective for trinitrobenzenesulfonate (TNBS^–). It showed a rapid Nernstian response within the TNBS^– concentration range 1.0 × 10^–5 to 1.0 × 10^–2 M at 25 ± 0.1°C. The electrode was selective, precise (RSD < 0.87% for TNBS^– and < 1.32% for amino acids) and usable within the pH range 2.5–12. The standard electrode potentials,E°, were determined at different temperatures and used to calculate the isothermal temperature coefficient of the cell. Selectivity coefficients for numerous compounds are given. The electrode has been used for the direct determination of trinitrobenzenesulfonic acid in aqueous solutions either by the standard additions method or by potentiometric titration against cetyltrimethylammonium bromide (CTABr) at pH 7. The electrode has been also applied successfully for the indirect microdetermination of neutral, acidic and basic amino acids separately, and as binary or ternary mixtures of neutral amino-acids. Total amino acids were determined in urine samples.     

Determination of total free amino acids with o-phthalaldehyde and N-acetyl- -cysteine

A spectrophotometric procedure is proposed for the determination of total free amino acids after reaction with o-phthalaldehyde and N-acetyl- -cysteine, using isoleucine as the reference. The procedure was applied to the analysis of five samples of widely different composition. Recoveries were 98–105%.     

Food engineering residues: amino acid composition of hydrolysates and application for the decontamination of metal polluted soils

Several residues of the brewing industry and slaughtering offals were investigated in order to evaluate their potential as raw materials for the hydrolytic preparation of amino acid containing solutions, applicable as extractants in amelioration processes for metal polluted soils. The residues were hydrolysed with 6 mol/L hydrochloric acid and the hydrolysates were analysed for their total nitrogen, TOC, amino acid and heavy metal contents. Then, the leaching capacities of the hydrolysates were examined in a series of batch tests with a contaminated soil.High amino acid yields in relation to the weight of the air-dried raw materials were achieved with blood meal (72.5%) and poultry feather meal (56.6%). The portion of the detected amino acids of the total organic carbon content of the hydrolysates ranged from 38.9% (brewer's spent grain) to 93.6% (blood meal). In extraction tests with hydrolysates adjusted to a total amino acid concentration of 60 mmol/L and to a pH value of 7.0, maximum extraction yields of 50.3% for copper (soil content 279 mg kg^–1) and 38.7% for nickel (soil content 54 mg kg^–1) were reached. An increase of the hydrolysate concentration and of the pH of an amino acid mixture resulted in higher solubilisation of the metals.Dedicated to Prof. Dr. Dieter Klockow on the occasion of his 60th birthday     

Neutral lipids of the seeds ofPhlomis oreophilla andPh. regelii

The lipid and fatty acid compositions of the seeds ofPhlomis oreophilla andPh. regelii (familyLabiatae) have been studied. The lipids ofPh. oreophilla are enriched with linoleic acid and those ofPh. regelii with oleic acid. Laballenic acid is present in the total acids of the triacylglycerides and the free fatty acids of both species, being concentrated in the free acids. The epoxy acids ofPh. oreophilla are represented by 9,11-epoxystearic, vernolic, coronaric, and 15,16-epoxylinoleic acids.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 561–565, September–October, 1983.     

Chiral discrimination in biliverdin (S)-amino acids,II: Structural variations of the amino acid functional groups

The Biliverdin-(S)-amino acid derivatives2–21 have been synthesized, and are subject to a thorough c. d. and u. v.-vis. electronic absorption analysis in the bilatriene chromophoric region. It is shown that the extent of chiral discrimination of the bilatriene helices is particularly sensitive towards structural variations of the amino acids bound to the propionic side chains. Thus, a pronounced decrease of chiral induction occurs if hydrogen bonding between one of the two essential coordination sites of the amino acid entity and the bilatriene backbone is disturbed. Accordingly, derivatives of (S)-amino acidt-butyl esters (3,5,7,16 and17) andN-substituted (S)-amino acids (8–10, 20 and21) generally display weak c. d. spectra. If additional polar groups are present in bis(amino acid) derivatives mutual interferences of the adjacent side chains must be taken into account. The attenuations of -values observed for the bis(serine) and bis(aspartic acid) compounds14 and15 thus are mainly due to intramolecular interchain interactions. The results provide evidence in support of the proposed mechanism of chiral discrimination in biliverdin amino acids.Dedicated to Prof. Dr.Kurt L. Komarek on the occasion of his 60th birthday.     

Ternary complexes involving copper(II) and amino acids,peptides and DNA constituents. The kinetics of hydrolysis of α-amino acid esters

Binary and ternary complexes of copper(II) involving picolylamine (Pic) and amino acids, peptides (HL) or DNA constituents have been investigated. Ternary complexes of amino acids or peptides are formed by simultaneous reactions. Amino acids form the Cu(Pic)L complex, whereas peptides form Cu(Pic)L and Cu(Pic)(LH_–1). The ternary complexes of copper(II) with picolylamine and DNA are formed in a stepwise process, whereby binding of copper(II) to picolylamine is followed by ligation of the DNA components. The stability of the ternary complexes is compared with the stabilities of the corresponding binary complexes. The hydrolysis of glycine methyl ester (MeGly) is catalysed by the Cu(pic)²⁺ complex. The kinetic data is fitted assuming that the hydrolysis proceeds in two steps. The first step, involving coordination of the amino acid ester by the amino and carbonyl groups, is followed by rate-determining attack by the OH^– ion. The second step involves equilibrium formation of the hydroxo-complex, Cu(pic)(MeGly)(OH), followed by intramolecular attack.     

Investigation of kernel oils of <Emphasis Type="Italic">Quercus robur</Emphasis> and <Emphasis Type="Italic">Quercus cerris</Emphasis>

The kernel oils of Quercus robur and Quercus cerris were obtained by Soxhlet extraction using petroleum ether. Oil yields were found to be 5.2–5.6% and 4.3–4.8% for Q. robur and Q. cerris kernel, respectively (expressed in g per 100 g of dried plant material). The physical and chemical constants, unsaponifiable matter and total fatty acids were determined. The total fatty acid composition of oils was determined by GC in the methyl ester form. Considering the composition and content of fatty acids, the examined kernel oils were very similar. Seven fatty acid components were identified in both oils: palmitic, stearic, arachidic, palmitoleic, oleic, linoleic, and -linolenic. In Q. robur and Q. cerris kernel oils the principal acids were oleic (44.3% and 43.0%, respectively) and linoleic (37.2% and 32.6%, respectively), followed by a significant amount of palmitic acid.Published in Khimiya Prirodnykh Soedinenii, No. 5, pp. 347–348, September–October, 2004.     

Comparison of enantiomer separation on two chiral stationary phases derived from (+)-18-crown-6-2,3,11,12-tetracarboxylic acid of the same chiral selector

Liquid chromatographic comparisons for enantiomer resolution of α-amino acids and chiral primary amino compounds were made using chiral stationary phases (CSPs) prepared by covalently bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TA) of the same chiral selector. The resolution of all α-amino acids on CSP 1 developed in our group was found to be better than that on CSP 2 reported by Machida et al. All α-amino acids examined in this study were well enantioseparated on CSP 1 (α=1.22–2.47), while four analytes were not resolved or all the other analytes were poorly resolved on CSP 2 than on CSP 1. However, in resolving the primary amino compounds without a carbonyl group, CSP 1 was comparable with CSP 2. Although (+)-18-C-6-TA of the same chiral selector was used to prepare CSP 1 and CSP 2, this study showed that different connecting methods for the CSPs might influence their ability to resolve the analytes depending on their structures related to the chiral recognition mechanism.     

Comparative investigation of the molecular structures of the species-specific proteins of the seeds ofGossypium hirsutum andG. barbadense

The N-terminal amino acids and the amino acid compositions of four species-specific proteins of two species of the cotton plant —Gossypium hirsutum andG. barbadense — have been determined. Peptide maps of tryptic hydrolysates of these proteins have been obtained and compared. Individual tryptic peptides have been isolated and purified. The N-ends, amino acid compositions, and amino acid sequences of individual peptides have been determined. The N-terminal sections of the proteins ofG. hirsutum andG. barbadense as far as the 16th amino acid residue have been sequenced. A comparative analysis has been made of the peptides and proteins of these cottonplant species.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 40 64 75. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 858–864, November–December, 1997.     

Composition of the floral waxes ofRosa gallica, Jasminum grandiflorumm, andViola odorata

The chemical compositions of the waxes of the flowers ofRosa gallica L., Jasminum grandiflorum L., andViola odorata L. have been characterized. The waxes of these plants mainly contained hydrocarbons and fatty and cyclic alcohols and their esters with fatty acids. The highest level of free fatty acids (12.4%) was found in theJ. grandiflorum wax. Depending on the plant species, the above-mentioned groups of substances differed in composition and amount, but hydrocarbons predominated.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (371) 120 64 75. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 327–333, May–June, 1999.     

Fatty Acids from the Sponge <Emphasis Type="Italic">Tedania dirhaphis</Emphasis>

The fatty acid (FA) composition of total lipids from the marine sponge Tedania dirhaphis from the Sea of Okhotsk was studied. GC and GC-MS identified 50 acids, particular attention being paid to components with 14–22 C atoms. Acids 16-Me-19:0, 10,14-Me₂-15:1(Δ6), 18:1(Δ6), 18:1(Δ8), and 22:1(Δ16) were observed and identified for the first time in sponges. The main FA in lipids from T. dirhaphis was 28:3(Δ5,9,21), the relative content of which reached 63.3%.__________Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 233–236, May–June, 2005.     

The Free-Amino-Acid Content in Six Potatoes Cultivars through Storage

Potatoes of six cultivars (Solanum tuberosum L.) with red, purple, and yellow flesh were stored at 2 and 5 °C for 3 and 6 months, and the influence of these factors on the content of free amino acids was determined. The potato cultivar and storage time had the greatest impact on the free amino acid content. The tubers of red-fleshed (Rote Emma) and purple-fleshed (Blue Congo) potatoes contained over 28 mg/g DM of free amino acids, and the Blaue Annelise cultivar with purple flesh had over 18 mg/g DM. After 6 months, the highest increase in their content (by 36%) was recorded in tubers of the Fresco cultivar (yellow-fleshed). In the analysed potatoes, the content of alanine, proline, serine, γ-aminobutyric acid, and α-aminoadipic acid increased, while that of asparagine, aspartic acid, and glutamine decreased. Asparagine decreased to the greatest extent in “Blaue Annelise” potatoes (by 24%) and that of glutamine in tubers of Rote Emma and Vineta by 18%.     

Simultaneous reactive extraction separation of amino acids from water with D2EHPA in hollow fiber contactors

Reactive extraction separation of binary amino acids from water using a microporous hollow fiber has been studied, in which the acidic extractant di(2-ethylhexyl)phosphoric acid (D2EHPA) was selected as an active carrier dissolved in kerosene. l-Phenylalanine (Phe) was extracted from an aqueous solution through the shell side of module to the organic phase through the lumen of fiber in the extraction module, in which l-Phe was then back-extracted to stripping phase in stripping module. Experiments were conducted as a function of the initial feed concentration of equimolar Phe and l-aspartic acid (l-Asp) (5 mol/m³), feed pH (3–5), the carrier concentration (0.1–0.5 mol/dm³), and stripping acidity (0.1–2 mol/dm³). The effect of process variables on the separation factor of Phe/Asp and the possible transport resistances including aqueous-layer diffusion, membrane diffusion, organic-layer, and interfacial chemical reaction were quantitatively studied and discussed. The high separation factor (β) of Phe/Asp was obtained to be 18.5 at feed pH 5 and 2 mol/dm³ of strip solution (HCl). The extraction and stripping processes appear to rely on pH dependence of the distribution coefficient of amino acids in reactive extraction system. The separation factor (β) was enhanced in hollow fiber membrane (HFM) process compared with conventional solvent process, which was a result of the counter transport of hydrogen ions.     

The Effect of Fermentation with Kefir Grains on the Physicochemical and Antioxidant Properties of Beverages from Blue Lupin (Lupinus angustifolius L.) Seeds

Plant derived fermented beverages have recently gained consumers’ interest, particularly due to their intrinsic functional properties and presence of beneficial microorganisms. Three variants containing 5%, 10%, and 15% (w/w) of sweet blue lupin (Lupinus angustifolius L. cv. “Boregine”) seeds were inoculated with kefir grains and incubated at 25 °C for 24 h. After processing, beverages were stored in refrigerated conditions (6 °C) for 21 days. Changes in microbial population, pH, bioactive compounds (polyphenolics, flavonoids, ascorbic acid), reducing sugars, and free amino acids were estimated. Additionally, viscosity, firmness, color, and free radicals scavenging properties were determined. Results showed that lactic acid bacteria as well as yeast were capable of growing well in the lupin matrix without any supplementation. During the process of refrigeration, the viability of the microorganisms was over the recommended minimum level for kefir products. Hydrolysis of polysaccharides as well as increase of free amino acids was observed. As a result of fermentation, the beverages showed excellent DPPH, ABTS^+·, ^·OH, and O₂⁻ radicals scavenging activities with a potential when considering diseases associated with oxidative stress. This beverages could be used as a new, non-dairy vehicle for beneficial microflora consumption, especially by vegans and lactose-intolerant consumers.     

The Complexes of Holmium with Methylenediphosphonate and 1-Hydroxyethylidenephosphonate

The composition and stability of holmium methylenediphosphonate (MDP) and 1-hydroxyethylidenephosphonate (HEDP) complexes were studied by potentiometric titration methods in 0.1M NaCl at 25 °C. It was found that besides L^4– anions the protonated H _n L^(4–n)– species (n = 1–3 for MDP and n = 1–4 for HEDP) are present in the pH region 3 to 10. The presence of the undissociated acids (H₄L) has not been unambiguously proved for MDP. The complexes of the composition HoH _n L (n varies from 1 to –2 for MDP and from 1 to –1 for HEDP) have been found if the concentration of the ligand is higher than the concentration of holmium. The protonation constants of both acids and the stability constants of the complexes discussed were determined and the comparison with literature data of analogical complexes of other lanthanides was performed.