Optimization of HPLC chromatographic conditions for determination of Transkarbam 12 and its degradation products.

This paper deals with searching of HPLC chromatographic conditions for determination and separation of Transkarbam 12 (T 12) and its two main degradation products (omega-aminocaproic acid and dodecylalcohol). T 12 is a new substance which belongs to the group of accelerators of transdermal penetration. Chromatographic separation was achieved using Separon SGX C18 analytical column (150 mm x 3 mm i.d.; 5 microm). Mobile phase contained acetonitrile and sodium acetate buffer pH 4.5 at the flow rate of 1 ml/min. Separation was carried out under the conditions of gradient elution. After the modification of the structure by derivatization reagent (3,5-dinitrobenzoyl chloride) detection at wavelength 230 nm was realized. The aim of this study was not only the optimization of the separation of derivatization reagent and derivatized T 12, Ak and D but also optimal derivatization processes for all three substances.

Analysis of 6-hydroxy-2-aminocaproic acid (HACA) as a specific marker of protein oxidation: the use of N(O,S)-ethoxycarbonyl trifluoroethyl ester derivatives and gas chromatography/mass spectrometry.

An alteration of low density lipoprotein (LDL) apolipoprotein (apo) B-100 structure by direct oxidative modification is an important mechanism involved in atherogenesis. There is difficulty in quantifying this type of modification because a lack of specific assays. The use of N(O,S)-ethoxycarbonyl trifluoroethyl amino acid esters for a rapid and sensitive determination of 6-hydroxy-2-aminocaproic acid (HACA), a highly specific marker of metal catalyzed protein oxidation, by using standard gas chromatography/electron impact mass spectrometry, is discussed. The derivatives are formed by the unlabored reaction of amino acids with ethylchloroformate plus trifluoroethanol plus pyridine. Femtomole levels of HACA can be reproducible measured in different LDL preparations subjected to oxidative damage in the presence of iron or copper. HACA determination compares well with the measurement of carbonyl groups that are generally accepted as a nonspecific index of protein oxidation. Thus, the method could prove to be a sensitive assay for studying specific apoB-100 modification.

Semisynthesis of Arg15, Glu15, Met15, and Nle15-aprotinin involving enzymatic peptide bond resynthesis.

The semisynthesis of homologues of aprotinin, the bovine pancreatic trypsin inhibitor, is described. The P1 lysine15 residue was replaced by two methods. The first procedure, which consisted of two enzymatic steps for the incorporation of other amino acids has previously been described. The second approach consisted of six steps of both enzymatic and chemical nature. The modified inhibitor, in which the lysine15-alanine16 peptide bond is hydrolyzed, was used as the starting material. All carboxyl groups of the modified inhibitor were esterified with methanol; the lysine15 methylester group was then selectively hydrolyzed. Afterward, lysine15 itself was split off. Arginine, glutamic acid, methionine, and L-2-aminohexanoic acid (norleucine, Nle) were incorporated using water-soluble carbodiimide combined with an acylation catalyst. The methylester group was used to prevent polymerization. The reactive-site peptide bonds were resynthesized using either chymotrypsin or trypsin.

Experimental febrile convulsions in epileptic chickens: the anticonvulsant effect of elevated gamma-aminobutyric acid concentrations.

The high seizure susceptibility in epileptic chickens is due to an autosomal recessive mutation. In 3-day-old chicks homozygous for the epilepsy gene (epileptics), elevation of body temperature using microwave diathermy evoked an initial febrile seizure resembling the clonic seizures evoked in epileptic chicks by photic stimulation. After complete recovery, this was followed by a clonic-tonic seizure. In nonepileptic heterozygote hatchmates (carriers) of the same age, only the latter seizure pattern was observed. In 16- to 17-day-old chicks of either phenotype, both seizure patterns were observed during hyperthermia. In all cases, the temperature at which seizures occurred was significantly lower in epileptic than in nonepileptic chicks, indicating a lower threshold for febrile seizures when there is an inherited predisposition to convulse. The occurrence of seizures was dependent on the body temperature and not on the rate of rise of temperature. Elevation of the brain gamma-aminobutyric acid (GABA) concentrations by administration of the GABA transaminase inhibitor gamma-vinyl GABA reduced the incidence of the initial febrile seizures and increased the latency in those birds that were not fully protected.

Determination of amino acids by separation of their ion pairs with dodecyl sulphate.

The ion pairs of amino acids with dodecyl sulphate were separated on a reversed-phase column (Beckman Ultrasphere I.P.) using a sequence of eluents that are prepared by mixing 0.2 M phosphoric acid (containing 10 mM sodium dodecyl sulphate), 0.2 M sodium acetate (pH 4.50; containing 10 mM sodium dodecyl sulphate) and methanol. the amino acids commonly occurring in tissues, except taurine and related weakly basic amino acids, can be analysed at a rate of 95 min per sample at a sensitivity of less than 50 pmol per amino acid. Elution modes for specific amino acids (alpha-difluoromethylornithine, gamma-vinyl-4-aminobutyric acid, 4-aminobutyric acid, putreanine) and non-essential amino acids that allow higher separation rates are reported. The method is suitable for fully automated routine amino acid analysis.

Determination of the R(-)- and S(+)-enantiomers of gamma-vinyl-gamma-aminobutyric acid in human body fluids by gas chromatography--mass spectrometry.

An analytical procedure, which allows the determination and quantitation of the R(-)- and S(+)-enantiomers of gamma-vinyl-gamma-aminobutyric acid (gamma-vinyl-GABA; MDL 71.754) in body fluids was developed. The method is based on a combined gas chromatographic--mass spectrometric technique. A glass capillary column coated with a chiral phase enabled the separation of the enantiomers of gamma-vinyl-GABA as their N-trifluoroacetyl-O-methyl ester derivatives. This was followed by quantitation using a selected ion monitoring technique in the electron-impact mode of ionization. The internal standard, gamma-acetylenic GABA, was included throughout the work-up of the samples. The assay was shown to be reproducible, specific and sensitive. No interferences were encountered from plasma, urine or cerebrospinal fluid constituents. The method was applied to the analysis of plasma samples obtained from a human volunteer who had received racemic gamma-vinyl-GABA. Significant differences in the plasma concentrations and plasma half-lives of the two enantiomers were seen, clearly illustrating the need for a specific assay technique capable of distinguishing between the enantiomers of this drug.

Gas-chromatographic determination of an antifibrinolytic drug, epsilon-aminocaproic acid.

We describe a modified method for assay of epsilon-aminocaproic acid. Serum or cerebrospinal fluid is deproteinized, followed by cation-exchange column-chromatography, and N-trifluoroacetyl-n-butyl derivatives of amino acids are formed and separated by gas chromatography. Tranexamic acid, a nonprotein amino acid, was used as an internal standard. The assay is sensitive and precise, and results correlate adequately with those obtained with an automated amino acid analyzer (ion-exchange chromatography).

Purification of a lysinonorleucine cross-linked peptide fraction from porcine aorta elastin.

Highly purified elastin from porcine aorta was submitted to elastase digestion. The enzymic products were subjected to gel filtration on Sephadex G 25. The excluded fraction was then submitted to thermolysin digestion and gel filtration. The excluded fraction was submitted to partial acid hydrolysis and gel filtration. Several sub-fractions were obtained. The F3 subfraction containing cross-linking agents (desmosines and lysinonorleucine) was finally subjected to ion exchange chromatography. A highly enriched peptide fraction containing lysinomorleucine was obtained and then purified by preparative electrophoresis. The ratio of enrichment passed from 2 residues of lysinonorleucine (expressed as lysine) from starting material (elastin) to 178 out of 1,000 residues in the final step. In this peptide fraction, if we express in molar ratio and consider the amount of lysinonorleucine is one residue, the following amino-acid composition is Pro:3, Gly:1, Ala:2, LNL:1, Lys:2. No traces of desmosines are detected. The role of lysinonorleucine in bridging functions in elastin is discussed.