High performance liquid chromatography of peptide bioregulators, their fragments and derivatives. IV. Chromatographic behavior and purification of oxytocin

Chromatographic behaviour of oxytocin has been studied. A simple and convenient technique has been developed for preparative purification of oxytocin using silica and ethanol. The product obtained contains not more than 3% impurities and has activity about 450 IU/mg.     

High performance liquid chromatography of peptide bioregulators, their fragments and derivatives. II. Sorption of peptides from aqueous-organic eluents on octadecylsilylsilica gel and unmodified silica gel

Dependence of the peptide retention upon the organic component concentration in eluent has been studied. A parabolic dependence has been found in a wide range of acetonitrile concentrations. The effect observed with ODS- and unmodified silica as stationary phases extends analytical and preparative potentialities of HPLC of peptides.     

High performance liquid chromatography of peptide bioregulators, their fragments and derivatives. III. Regularities of sorption, prediction of retention and analysis of peptides by a reversed phase HPLC method

Parameters of statistical models of fully or partially protected peptides' retention on Zorbax ODS and Silasorb C18 have been compared. The proposed model can be used for non-protected linear and cyclic peptides. Special increments have to be introduced in calculation of hydrophobicity of these peptides.     

High-performance liquid chromatography of methotrexate analogs containing terminal lysine or ornithine and their dansyl derivatives

A procedure utilizing a reverse-phase semipreparative high-performance liquid chromatography column and a binary solvent system consisting of trifluoroacetic acid and 1-propanol has been developed for the semipreparative scale purification and analytical identification of four newly synthesized analogs of methotrexate. The methotrexate analogs containing a lysine or an ornithine residue in place of a terminal glutamate residue together with their respective dansyl derivatives were purified in milligram quantities by the procedures described.     

High-performance liquid chromatography in the separation and measurement of di- and polyamines and their derivatives, and specific preparation of isomers of their monoacetyl derivatives

We present a method for separating and quantitating the di- and polyamines and many of their derivatives found in mammalian tissues by high-performance liquid chromatography using a cation-exchange resin with gradient elution. Three different solvent systems are described, each with special advantages. The nitrate anion is used in order to permit increasing the cation concentration without increasing buffering capacity or introducing halide ions, which are corrosive for stainless steel. The specific synthesis of two isomers of N-monoacetylspermidine is described.     

High-performance liquid chromatography of coproporphyrin isomers.

A reversed-phase system is described for the simultaneous isocratic separation of coproporphyrin I, II, III and IV isomers. The retention behaviour of coproporphyrin I and III is studied in detail. The method is suitable for both analytical and semi-preparative separation.     

High-performance liquid chromatography of iodine-labeled insulin and glucagon derivatives with on-line gamma-detection

Insulin and glucagon were labeled with iodine. The reaction products were analyzed by high-performance liquid chromatography. It is shown that the pH of the reaction medium has a large effect on the position and the degree of iodine substitution as well as on the oxidation of the Met-containing glucagon and, furthermore, that the molar ratio of iodine to polypeptide hormone used during the labeling procedure affects not only the amount of iodine incorporated but also the distribution of iodinated products. The results show that certain iodinated derivatives are separated from each other and from the respective unlabeled polypeptide and thus can be obtained in a pure state.     

Identification of enzymatically produced peptide fragments by liquid chromatography and mass spectrometry

The qualitative and quantitative determination of peptide fragments of angiotensin I generated by rat lung dipeptidyl carboxypeptidase (angiotensin converting enzyme, EC 3.4.15.1) is described. Enzymatically formed peptide fragments, after derivatization with fluorescamine, were separated and isolated by reverse-phase high-performance liquid chromatography. The recovered fluorescamine derivative of histidyl-leucine was then further identified by mass spectrometry. It is anticipated that this approach would be widely applicable to other enzyme systems.     

High-performance liquid chromatography measurement of hyperforin and its reduced derivatives in rodent plasma

A reverse-phase high-performance liquid chromatography method was developed for the determination of hyperforin and its reduced derivatives octahydrohyperforin and tetrahydrohyperforin in rodent plasma. The procedure includes solid-phase extraction from plasma using the Baker 3cc C8 cartridge, resolution on the Symmetry Shield RP8 column (150 mm x 4.6 mm, i.d. 3.5 microm) and UV absorbance detection at 300 nm. The assay was linear over a wide range, with an overall coefficient of variation less than 10% for all compounds. The precision and accuracy were within acceptable limits and the limit of quantitation was sufficient for studies preliminarily assessing the disposition of tetrahydrohyperforin and octahydrohyperforin in the mouse and rat.     

High-performance liquid chromatographic separation of renin-angiotensin system peptides and most of their metabolic fragments

We describe here a gradient HPLC procedure for the separation, and quantification by UV absorption of renin tri- and tetradecapeptide substrates, angiotensins I, II, III, IV and V, angiotensin-derived peptides, and peptidase inhibitors including amastatin, bestatin, pepstatin, lisinopril, a renin peptide inhibitor, Z-Pro-prolinal, N-[1-(R,S)-carboxy-2-phenylethyl]-L-Ala-L-Ala-L-Phe-p-aminobenzoate, and phosphoramidon. Most peptides and peptidase inhibitors were baseline-resolved within 32 min. The overall intra- and inter-assay precisions ranged from 0.8 to 5.9 (n=6) and 2 to 13% (n=6), respectively. There was a linear relationship (correlation coefficients> or =0.9660) between peak height and peptide amount injected. In conclusion, the present method when combined with a peptidase-inhibitor paradigm can lead to the identification of renin-angiotensin system metabolizing enzymes, and when combined with radioimmunoassay can enhance the specificity of angiotensin measurement.     

High-pressure liquid chromatography of polycyclic aromatic hydrocarbons and some of their derivatives.

The separation of polycyclic aromatic hydrocarbons and their derivatives by means of high-pressure liquid chromatography on Permaphase ODS is described. The method consists of the (isocratic) elution of compounds from the column with a methanol-water mixture of constant composition and is particularly suited to the identification of metabolic products of polycyclic hydrocarbons.     

High-performance liquid chromatography of type-III heptocarboxylic porphyrinogen isomers.

A reversed-phase h.p.l.c. system is described for the separation of the four type-III heptacarboxylic porphyrinogen isomers. The effects of buffer concentration, pH and type and proportion of organic modifier in the mobile phase on retention and resolution of isomers were studied. Optimum separation on an ODS-Hypersil column was by elution with a ternary mobile phase of acetonitrile, methanol and 1 M-ammonium acetate, pH 5.16 (7:3:90, by vol.). Isomer identification was based on a comparison of their retention times with those of authentic standards, and was further confirmed by h.p.l.c. analysis of the characteristic mixture of three pentacarboxylic porphyrins formed after partial decarboxylation of individual isomers in 0.3 M-HCl at 160 degrees C.     

Analysis of unsaturated fatty acids, and their hydroperoxy and hydroxy derivatives by high-performance liquid chromatography.

A simple procedure for the detection of endo-β-N-acetylglucosaminidase H activity is described. The method utilizes N-[¹⁴C]methylribonuclease B as substrate. This is prepared from ribonuclease B by reductive alkylation of free amine groups in the protein with [¹⁴C]formaldehyde. Because the carbohydrate moiety of ribonuclease B has α-mannosyl residues at nonreducing terminal positions, the radioactive molecule binds to Sepharose-concanavalin A. Endo-β-N-acetylglucosaminidase action releases this mannose-containing oligosaccharide by splitting the di-N-acetylchitobiosyl residue that links it with the peptide and thereby renders the radioactive portion of the molecule unreactive with Sepharose-concanavalin A. This forms the basis of a convenient assay for screening column fractions during the purification of the endoglycosidase. Although protease or α-mannosidase activity might also be detected by the procedure, no difficulties were presented by these enzymes when the assay was used for the preparation of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus.     

High-performance liquid chromatography of glucagon and related compounds

Glycerolpropylsilane bonded phases have been found to adsorb peptides and proteins via ionic interactions. In this paper high-performance liquid chromatography separation of glucagon and related compounds, using a Diol-silica matrix, is described. Crystalline, commercial preparations of glucagon, when analyzed on LiChrosorb Diol columns eluted with low-ionic-strength acidic buffers, contained up to four contaminant peaks, in different numbers and ratios. Three of these contaminants, called A, C, and D, were recovered and characterized. Contaminant A, representing a few percent of the total, was a mixture of mono- and didesamidoglucagon, as shown by treatment with bis(I,I-trifluoroacetoxy)iodobenzene, with which it is possible to differentiate between carboxamide and carboxylic acid residues. Contaminant C, ranging from 0 to about 30% of the total, was N-terminal degraded glucagon. Contaminant D, ranging from a few percent to about 25% of the total, was (Met27 sulfoxide) glucagon.     

Analysis of angiotensins I,II, III,and iodinated derivatives by high-performance liquid chromatography

Angiotensins I, II, and III were separated by reversed-phase high-performance liquid chromatography on an octadecylsilyl column. The peptides were isocratically eluted with 50 mm NaH₂PO₄-25% ($\text{v}\text{v}$) acetonitrile, pH 6.0. The retention times were 3.3, 6.0, and 9.6 min for angiotensin II, III, and I, respectively. ¹²⁵I-Angiotensins II, III, and I eluted with retention times of 5.4, 16.8, and 19.9 min, respectively, under the same chromatographic conditions used for the unlabeled angiotensins. The effect of iodination of the tyrosine residue on the retention time was also demonstrated by chromatographic comparison of tyrosine and diiodotyrosine. Saralasin (Sar¹, Ala⁸-angiotensin II), a partial agonist of angiotensin II, and des-Asp¹, Ile⁸-angiotensin II, an inhibitor of angiotensin III, eluted with retention times of 2.5 and 3.9 min, respectively.     

High-performance liquid chromatographic analysis of neutral glycosphingolipids as their per-O-benzoyl derivatives

Previous reports from this laboratory (1–4) described the perbenzoylation of neutral glycosphingolipids (GSL)¹ with benzoyl chloride in pyridine and analysis of the perbenzoylated derivatives by high performance liquid chromatography (hplc). A disadvantage of this procedure is that N-benzoylation occurs as well as the desired O-benzoylation. This does not permit recovery of the parent GSL after mild alkaline hydrolysis due to formation of a mixture of N-acylated and N-benzoylated GSLs(1). It has also been demonstrated that the benzoylation with benzoic anhydride in pyridine does not lead to the formation of N-benzoylated products. However, the anhydride reaction is sluggish and the benzoyl chloride method has been the preferred procedure.Gupta et al. (5) used N,N-dimethyl-4 amino pyridine (DMAP) as a catalyst in the acylation of phospholipids by the anhydrides of fatty acids. F. B. Jungalwala (private communication) has shown that this catalyst greatly accelerates the reaction of benzoic anhydride with sulfatides.In this communication we report the preparation and hplc analysis of per-O-benzoyl derivatives of GSLs by reaction with benzoic acid anhydride in the presence of DMAP as a catalyst. Reaction with these reagents avoids amide acylation, forms single products with satisfactory chromatographic properties and parent GSLs can be regenerated by mild alkaline hydrolysis.