Analysis of dansyl amino acids by reverse-phase high-performance liquid chromatography

All 24 dansyl amino acids were separated by reverse-phase high-performance liquid chromatography on Develosil C8-5, using a linear gradient made from Tris-HCl buffer (pH 7.75) and methanol. A linear relationship between the amount of sample and peak area was found over the range of 6 to 300 ng (0.02–1 nmol) of dansyl derivatives. An application of this method to the NH₂-terminal analysis of lysozyme is described.     

The analysis of aflatoxins by high-performance liquid chromatography.

The potential of high-performance liquid chromatography as a technique for separating aflatoxins B₁ B₂, G₁, G₂, B_2a, Q₁, M₁, P₁, aflatoxicol, and a degradation product of aflatoxin B₁, 2,3-dihydrodiol, has been assessed. A microparticulate silica adsorption column used with a 1:1 chloroform -dichloromethane eluant provided good resolution of aflatoxins B₁, B₂, G₁, and G₂ but the addition of 1% propan-2-ol was necessary for the elution of aflatoxins M₁ and Q₁. By selecting appropriate solvent mixtures, good resolution of all of the aflatoxins studied was obtained using columns containing an octadecyl (C₁₈) reversed-phase bonded to a microparticulate support. Details are given for resolving: (1) aflatoxins B₁, B₂, G₁, and G₂ using a 5% tetrahydrofuran-15% dimethylformamide in water eluant and (2) aflatoxins B₁ B_2a, Q₁ M₁ P₁ aflatoxicol, and a product of aflatoxin B₁ 2,3-dihydrodiol treated with Tris-buffer, using either 15% dimethylformamide in water or 10% tetrahydrofuran in water as eluant.     

Assay of short-chain acyl coenzyme A intermediates in tissue extracts by high-pressure liquid chromatography

A high-pressure liquid chromatographic method for the measurement of short- and medium-chain-length acyl-CoA compounds is described. Compounds are separated on a reverse-phase μBondapak C₁₈ column with the order of elution based on differences in lipophilicity. The mobile phase consisted of variable mixtures of methanol and 50 mm KH₂PO₄, pH 5.3. Conditions are described that allow isocratic separation of groups of compounds of similar lipophilicity. With increasing methanol concentration, the more lipophilic compounds are eluted earlier. This has the effect of sharpening the peaks and improving quantitation. Detection of acyl-CoA intermediates is achieved using a uv detector and is based on the high absorbance of CoA-containing compounds at 254 nm. Neutralized perchloric acid extracts of tissues can thus be analyzed directly without further purification or derivatization. A mobile phase consisting of a 9:1 phosphate buffer-to-methanol mixture is used to separate CoASH, methylmalonyl-CoA, succinyl-CoA, β-hydroxy-β-methylglutaryl-CoA and acetyl-CoA. Increasing the methanol concentration to a 4:1 mixture allows separation of acetyl-CoA, propionyl-CoA, and isobutyryl-CoA, while with a 7:3 mixture of phosphate buffer to methanol, β-methylcrotonyl-CoA and isovaleryl-CoA are readily separated. Examples of results obtained using extracts from isolated hepatocytes, rat liver mitochondria, and perfused rat hearts incubated with α-ketoisocaproate, α-ketoisovalerate, or propionate are presented. In addition, methods and optimal conditions are presented for the analysis of malonyl-CoA, glutathione-CoA, dephospho-CoA, and oxidized CoA in tissue extracts.     

Analysis of porphyrin carboxylic acids in biological fluids by high-performance liquid chromatography

A method for the rapid and sensitive fluorometric analysis of porphyrin carboxylic acids by reverse-phase high-performance liquid chromatography is described. Separation of free porphyrin carboxylic acids was carried out with a microparticulate octadecylsilane column with elution by a gradient of methanol in phosphate buffer containing tetrabutylammonium hydroxide. Separation and quantitation of di-, tri-, tetra-, penta-, hexa-, hepta-, and octacar-boxylic porphyrins was achieved within 25 min at picomolar concentrations. The method is also capable of separating the type I and type III isomers of tetracarboxylic through hexacarboxylic porphyrins. By using a stopped flow technique, one can record fluorescence excitation and emission spectra of porphyrin carboxylic acids. This method is directly applicable to biological fluids such as urine, plasma, red cell lysates, or medium or extracts from cell culture.     

A simplified assay of furosemide in plasma and urine by high-pressure liquid chromatography

A simplified high-pressure liquid chromatographic method for determination of furose-mide in plasma and urine has been developed using a fluorometric detector directly coupled to the column effluent. The method includes an ether extraction from acidified biologic samples. The mobile phase used for chromatography on a reversed-phase column (C₁₈ hydro-carbon permanently bonded to silica particles) is sufficiently acidic to induce fluorescence of furosemide. The methylester of furosemide is employed as an internal standard. The sensitivity is 0.1 and 0.25 μg per ml plasma and urine, respectively. The applicability to pharmacokinetic studies of furosemide is shown.     

The analysis of acyl-coenzyme A derivatives by reverse-phase high-performance liquid chromatography

A method for determining tissue levels of Coenzyme A and various short-chain-length acyl-CoA derivatives using high-performance liquid chromatography is presented. Separation of the various compounds was accomplished using a reverse-phase Spherisorb ODS II, 5-microns C18 column. Mobile-phase solvents were (a) potassium phosphate, 220 mM; thiodiglycol (2,2-thiodiethanol), 0.05% (v/v), pH 4.0 and (b) methanol, 98%; chloroform; 2% (v/v). The various acyl-CoA derivatives were detected by monitoring the column effluent at 254 nm. Nearly baseline separation was obtained for a standard mixture of free CoASH, methylmalonyl-CoA, beta-hydroxy-beta-methylglutaryl-CoA, succinyl-CoA, acetoacetyl-CoA, acetyl-CoA, propionyl-CoA, isobutyryl-CoA, beta-methyl-crotonyl-CoA, and isovaleryl-CoA. CoA derivative profiles were determined in neutralized perchloric acid extracts of perfused rat hearts and livers and of isolated rat liver mitochondria to demonstrate the utility of this method for assessing the levels of CoA derivatives in biological samples.     

Determination of tissue UDP-glucuronic acid levels by high-pressure liquid chromatography

A new and sensitive method to measure UDP-glucuronic acid extracted from as little as 25 mg wet weight tissue has been developed. This procedure employs high-pressure liquid chromatography and liquid scintillation spectrophotometry to measure p-[¹⁴C]nitrophenylglucuronide generated enzymatically from p-[¹⁴C]nitrophenol and UDP-glucuronic acid. The reaction was catalyzed by UDP-glucuronyltransferase obtained from rat liver microsomes. The tissue levels of UDP-glucuronic acid assayed were 2 to 20 μmol/100 g wet wt, which are well below the levels detectable by the widely used spectrophotometric method.     

Separation of hemoglobin types by cation-exchange high-performance liquid chromatography

The use of a recently developed cation-exchange HPLC packing material for the separation of hemoglobin types in human blood has been investigated. Adult and newborn hemolysates from normal individuals and from subjects with hemoglobin disorders were analyzed using a weak cation carboxymethyl-bonded phase on 5-micron-particle-size silica. Elution was accomplished using a Bistris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1, 3-propanediol) gradient. Seven well-resolved HbA1 fractions eluted before the major HbA peak. Hbs A1a, A1b, A1c and an HbA1 fraction that increased with aging of the hemolysates were separately eluted. HbF when present or when added to the hemolysates eluted as a distinct peak. HbA was followed by Hbs A2, S, and C when present. An early-eluting peak corresponding to Hb Bart's was identified in newborn hemolysates. It is concluded that cation-exchange HPLC provides a new tool for the reliable separation of minor hemoglobin components.     

Desalting of nucleotides by reverse-phase high-performance liquid chromatography

Chromatography of AMP, NAD⁺, or NADH on a reverse-phase C₁₈ Porasil B column rapidly removes ammonium formate or potassium phosphate from 90% of the nucleotide. Earlier reports showed these salts could not be separated from nucleotides by conventional desalting using gel filtration.     

The use of high-pressure liquid chromatography in the preparation of tritium-labeled chloramphenicol and its analogs.

The 1-hydroxy epimers of chloramphenicol and thiamphenicol formed from the reduction of the respective 1-oxo derivatives with [³H]NaBH₄ have been separated preparatively by high-pressure liquid chromatography on a μBondapak C₁₈ column. This separation procedure permits the facile and rapid preparation of the 1-³H-labeled derivatives of chloramphenicol and its analogs.     

Measurement of nanogram quantities of protein by hydrolysis followed by reaction with orthophthalaldehyde or determination of glutamate.

Two methods are described for determining protein by measurement of amino acids released by acid or base hydrolysis. One achieves sufficient sensitivity to measure as little as 3 ng of protein by reaction of the amino acids with orthophthalaldehyde and mercaptoethanol in a small volume, followed by dilution in 0.5 n NaOH for fluorometric measurement of the product. The other method is somewhat more sensitive. Released l-glutamate is determined enzymatically by reaction with glutamic dehydrogenase (EC 1.4.1.2) and NAD⁺; the NADH produced is amplified by enzymatic cycling. To avoid the use of sealed tubes and minimize dangers of contamination, hydrolysis is carried out in small droplets in “oil wells.”     

Partial purification of egg development neurosecretory hormone with reverse-phase liquid chromatographic techniques

We report the use of reverse-phase liquid chromatographic techniques for the isolation of a steroidogenic neuropeptide (EDNH) from mosquito heads. Activity of fractions was assayed by measuring the ability of ovaries to produce ecdysteroid $\text{in}$$\text{vitro}$. Dose response profiles using crude head extracts or partially purified EDNH were nearly identical, indicating that the methods of preparation did not alter biological activity. EDNH activity eluted from a reverse-phase HPLC (RP-HPLC) column primarily near 35 percent acetonitrile using a linear gradient. Methods developed with an analytical RP-HPLC column were successfully adapted for preparative work. Active fractions from the preparative RP-HPLC were further purified on a second analytical column under isocratic conditions at 30% acetonitrile. Two adjacent UV absorbing peaks were found, each with EDNH activity. Activity was sensitive to proteolysis.     

Characterization of proteins and peptides by high-performance liquid chromatography and fluorescence monitoring of their tryptic digests.

Proteins and peptides can be characterized and compared at the subnanomole level by treatment with trypsin followed by high-performance liquid chromatography on reverse-phase partition columns. A fluorescamine monitoring system automatically analyzes a portion of the column effluent while the remainder can be collected for further studies. The method has already been used for characterization of rat β-endorphin and a protein which cross-reacts with antiserum prepared against prolyl hydroxylase.     

Selective formation of microparticles by homopolyribonucleotides and proteinoids rich in individual amino acids

The formation of phase-separated microparticles following the mixing of solutions of homopolyribonucleotides with solutions of several basic thermal proteinoids, each rich in an individual amino acid, has been studied. Three of the 4 proteinoids studied yielded results consistent with a matrix of anticodonicity; the fourth did not. The meaning of these results, and others, relative to a postulated matrix for the genetic coding mechanism is discussed.     

IV. Determination of aromatic L-amino acid decarboxylase in serum of various animals by high-performance liquid chromatography with electrochemical detection

This paper describes the distribution of aromatic L-amino acid decarboxylase (AADC), using both L-DOPA and L-5-hydroxytryptophan (L-5-HTP) as substrates, in serum of various animals. The ratio of the activities of the enzyme towards both substrates was also determined in the same serum. AADC activity was discovered in serum using our new and highly sensitive assay for AADC activity by high-performance liquid chromatography (HPLC) with electrochemical detection (ED) with L-DOPA and L-5-HTP as substrates. It was found that among the species used, guinea pig serum had the highest activity, and we made a systematic study on guinea pig serum AADC.     

The analysis of the betaine homarine by high pressure liquid chromatography

A procedure is presented which is suitable for the qualitative and quantitative analysis of the betaine homarine in aqueous tissue extracts. After preliminary purification of the extract by gel permeation chromatography on Sephadex G-25, quantitative analysis of the homarine content is performed by high pressure liquid chromatography on a 1-m column of Corasil II.     

High-pressure liquid chromatography of neurotoxic phenylphosphonothioate esters and related compounds

It has been suggested that perfluorooctanoic acid occurs in human plasma; however, no method of analysis for this compound in biological samples has been published to date. A method is presented for the analysis of perfluorooctanoic acid in plasma, urine, and liver tissue based on conversion of the acid to its methyl ester followed by separation and quantitation by gas chromatography.     

Chemotaxis toward carbohydrates and amino acids in Physarum polycephalum

A method is described for assaying chemotaxis in the acellular slime mold Physarum polycephalum. It consists of measuring the amount of plasmodium that moves on a strip of nitrocellulose membrane filter Millipore in response to a gradient of an attractant. Time course of chemotactic response of the slime mold is described. Different factors that affect chemotaxis in the slime mold such as: culture care and stage of growth of microplasmodia, substratum used for cell movement, nature of the gradient, effect of salts, pH and temperature are described. From concentration-response curves for different attractants several parameters of the chemotactic effect, such as threshold concentration, half maximal concentration, and maximal effective concentration can be determined. As a group, sugars are more effective chemotactic agents than amino acids. Glucose and galactose, which support the growth of the slime mold, are shown to have high positive chemotactic effect. 3-O-Methyl- -glucose and 2-deoxy- -glucose are two sugars that do not support growth but are very effective attractants. Conversely, fructose which supports slime mold growth is at best a weak attractant. The results support the view that the chemotactic effects of different sugars are not dependent on their growth-supporting value.     

Application of high-performance liquid chromatography to competitive labeling studies: The chemical properties of functional groups of glucaton

A competitive-labeling study of glucagon was carried out using [³H]- and [¹⁴C]-1-fluoro-2,4-dinitrobenzene to determine simultaneously the chemical properties of the α-amino and imidazole groups of the N-terminal histidine residue, and the lysine and tyrosine residues, under conditions where glucagon is in its physiologically active monomer form. The dinitrophenyl derivatives of these groups were purified by high-performance liquid chromatography which greatly simplified the separation steps of the procedure. The results showed the α-amino and tyrosine groups to have relatively normal behavior, with pK values of 7.98 and 10.22, respectively, while the lysine had a low pK of 8.46. The imidazole function had an apparent pK of 7.84, substantially higher than previous estimates. This difference may be accounted for by the effect of the charged form of the adjacent α-amino group on the nucleophilicity of the imidazole group.     

Separation and identification of pteroylpolyglutamates by polyacrylamide gel chromatography.

A simplified procedure for the determination of the glutamate chain lengths of labeled and endogenous tissue folate is described. Pteroylpoly-γ-glutamates in tissue extracts were reductively cleaved at the C,9N,10 bond to p-aminobenzoylpolyglutamates, which were converted to azo dyes by coupling their diazonium salts with naphthylethylene diamine. The azo dyes were well resolved, according to glutamate chain length, by gel chromatography on Bio-Gel P4. Unlabeled tissue folates were detected by the absorbance of their azo dye derivatives. The major endogenous pteroylpolyglutamate in rat liver, identified colorimetrically using 0,5 g tissue, was the pentaglutamate. The major labeled folates in Lactobacillus casei and Streptococcus faecalis, after incubating these bacteria with labeled folic acid, were identified as the octa- and tetraglutamates, respectively. Reductive cleavage of 10-formylfolate and 5.10-methenyltetrahydrofolate resulted in a mixture of N-substituted and unsubstituted p-amino-benzoylpolyglutamates. Methods are described for the complete cleavage of these formyl derivatives to unsubstituted p-aminobenzoylpolyglutamates.