Determination of nucleotides, nucleosides and nucleobases in cells of different complexity by reversed-phase and ion-pair high-performance liquid chromatography

Procedures are presented for the analysis of profiles of purine and pyridine compounds in human and rabbit red blood cells by reversed-phase high-performance liquid chromatography and in Ehrlich ascites tumour cells of mouse by ion-pair high-performance liquid chromatography. These compounds are present in rabbit erythrocytes in higher concentrations than in human blood cells, and in rabbit reticulocytes the concentration of purine compounds is still higher. During glucose-free incubation, human red cells accumulate adenosine and adenine in the presence of coformycin owing to the inhibition of adenosine and AMP deamination. Ehrlich ascites tumour cells lose major portions of purine mono-, di- and triphosphates between the seventh and eleventh day after inoculation into mouse peritoneal cavities.     

Simultaneous determination of myocardial nucleotides, nucleosides, purine bases and creatine phosphate by ion-pair high-performance liquid chromatography.

An ion-pair reversed-phase high-performance liquid chromatographic method is described for the separation and quantification of myocardial nucleotides, nucleosides, their metabolites and creatine phosphate-related compounds in a single run. Separation of a standard mixture containing 21 compounds was achieved on a 5-microns Hypersil ODS column with a 5-min isocratic elution (buffer: 0.1 M NaH2PO4, pH 5.5, containing 5.9 mM tetrabutylammonium hydrogen-sulphate) followed by a slow linear gradient to 17% acetonitrile. The method was applied to extracts of freeze-clamped rat heart tissue samples as well as to extracts of neonatal rat heart cardiomyocytes, and it provided good resolution of high-energy phosphates, including creatine phosphate, as well as of their degradation products.     

Microbore packed-column anion-exchange liquid chromatography of nucleobases, nucleosides and nucleotides

Low-capacity anion-exchange resin was packed into a 45 cm X 0.19 mm I.D. fused-silica tubing and applied to micro-column liquid chromatography of nucleobases, nucleosides and isomers of nucleotides. The effects of the chromatographic conditions on the elution behaviour of these compounds were studied. The efficiency of the microbore packed column was comparable with that of the conventional size column.     

Separation of purine bases, nucleosides and nucleotides by a column-switching technique combining reversed-phase and anion-exchange high-performance liquid chromatography

An on-line two-stage column chromatographic technique is described which combines reversed-phase and anion-exchange chromatography for the separation of purine nucleic acid components. The elution program applied, consisting of two gradient programmes, provides a separation of bases and nucleosides on the octadecyl silica column and a separation of the nucleotides on the anion-exchange column to which they have been switched at the beginning of the elution. This method is easy to modify for special problems and can be used when establishing a complete profile of purines.     

Determination of 18 nucleobases, nucleosides and nucleotides in human peripheral blood mononuclear cells by isocratic solvent-generated ion-pair chromatography

The paper describes a method for the separation of 18 nucleotides, nucleosides and nucleobases by isocratic solvent-generated ion-pair chromatography in 55 min. It has been developed for pharmacodynamic monitoring of mycophenolic acid, the active metabolite of the immunosuppressant mycophenolate mofetil. The method was applied for the detection of mycophenolic acid-induced changes in inosine 5′-monophosphate dehydrogenase (IMPDH) activity in the lysate of human peripheral blood mononuclear cells.     

Simultaneous determination of cytosine arabinoside, its nucleotides and metabolites by ion-pair high-performance liquid chromatography

Currently available high-performance liquid chromatographic assays for cytosine arabinoside (ara-C) and its metabolites suffer from two major shortcomings: inability to resolve both ara-C and its nucleotides in a single chromatographic step and/or inadequate sensitivity to allow quantitation of intracellular cytosine arabinofuranoside-5'-triphosphate (ara-CTP) without the use of radiolabelled drug. In this paper, we describe a new ion-pairing high-performance liquid chromatographic assay for ara-C in biological samples that can separate ara-C from its nucleotides, metabolites, and naturally occurring ribonucleotides in a single chromatographic step with a lower limit of quantitation of 5 pmol for ara-C and 10 pmol for ara-CTP. Examples of the utility of this assay are shown in studies of intracellular pharmacokinetics of ara-C in cultured human breast cancer cells and in analysis of plasma nucleoside levels in patients receiving high-dose thymidine chemotherapy. We conclude that this assay provides a rapid and versatile system that can be applied to the study of both cellular and plasma nucleoside pharmacokinetics.     

Separation of synthetic cycloalkylated bases, nucleosides and nucleotides by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography was used to determine the elution profiles of a series of synthetic cycloalkylated bases, nucleosides, and their corresponding 5'-monophosphates. A 70% aqueous methanol solution proved to be the most efficient solvent system for the separation of a mixture of the bases, all of which were eluted in times ranging from 3.3 to 4.8 min at a flow-rate of 0.8 ml/min. Subsequently, the same percentage of methanol solvent, at 0.8 ml/min, eluted the nucleoside mixture as well, with retention times ranging from 3.3 to 5.0 min. Optimum separation and resolution were achieved with 70% methanol at a flow-rate of 0.6 ml/min for a mixture of the base and nucleoside series. A phosphate buffer, containing acetonitrile-tetrabutylammonium ion, was used to analyze the 5'-monophosphate derivatives. Elution times ranged from 2.6 to 6.1 min at a flow-rate of 1.0 ml/min. Three variables were considered in order to determine optimum conditions for separation and resolution: (a) the percentage of methanol in the solvent; (b) flow-rate of solvent; and (c) the size of the cycloalkylated group of each synthetic analogue. The procedures and conditions described herein have potential use as a monitoring system to detect modified nucleic acid derivative which are prevalent in the body fluids of patients with certain metabolic disorders.     

Nucleotide preparation from cells and determination of nucleotides by ion-pair high-performance liquid chromatography.

Procedures for the analysis of cellular purine and pyrimidine nucleotides are described. The commonly used perchloric acid and especially the trichloroacetic acid methods for nucleotide extraction interfere with ion-pair high-performance liquid chromatography, but we have developed such a system for the separation and determination of major cellular nucleotides in biological matrices, including tri-, di-, monophosphates, cAMP, cGMP, NAD, NADP, UDP-glucose and UDP-galactose. Compared with perchloric acid extraction, no degradation of the nucleotide standards used was observed with respect to triphosphates and other relatively unstable nucleotides. Cellular nucleotides were extracted by lysing cells in a hypotonic buffer containing an ion-pair reagent (tetrabutylammonium hydrogen-sulphate) to decrease enzymic degradation of nucleotides in combination with ultrafiltration of the cell lysate to remove compounds of higher molecular mass, for example enzymes. This method is a simple and reproducible procedure for investigating nucleotide pools in cells.     

Determination of purine nucleosides and their bases by high-performance liquid chromatography using co-immobilized enzyme reactors

A selective and sensitive assay of inosine, guanosine, hypoxanthine, guanine and xanthine by high-performance liquid chromatography with immobilized enzyme reactors was developed. The separation was achieved on a Capcell Pak C18 column (15 cm x 0.46 cm I.D.) with a mobile phase of 0.1 M phosphate buffer (pH 8.0) containing 7 mM sodium 1-hexanesulphonate and 0.1 mM p-hydroxyphenylacetic acid. The fluorimetric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of these compounds, which were oxidized to yield hydrogen peroxide in the presence of immobilized enzyme (purine nucleoside phosphorylase, guanase and xanthine oxidase). Enzyme reactions occurred sufficiently without post-column addition of reagents. Enzymes that catalysed the conversion of purine compounds were co-immobilized on aminopropyl controlled-pore glass packed in stainless-steel tubing. The detection limits were 30-200 pg per injection.     

Ion-pair reversed phase HPLC determination of nucleotides,nucleosides and nucleobases — Application to nucleotide metabolism in hepatocytes

Chromatographia - Three groups of metabolites were analyzed in extracts of rat hepatocytes by an HPLC method: (i) nucleotides (ATP, ADP, AMP, GTP, GDP, UTP, UDP, IMP, UMP), (ii) nucleosides and...     

Isocratic separation of some purine nucleotide, nucleoside, and base metabolites from biological extracts by high-performance liquid chromatography.

Separation of ATP, ADP, AMP, adenine, adenosine, cAMP, ITP, IDP, IMP, hypoxanthine, inosine, cIMP, the guanine series, NAD, NADPH, xanthine, 3-methylxanthine, theobromine, theophylline, and caffeine was accomplished using high-performance liquid chromatography with a microparticulate reversed-phase column. Under isocratic conditions all compounds could be eluted with reasonable resolution and retention time. Quantitation by peak height for several of the compounds was used to the 10-ng level.     

Changes of nucleotide patterns in liver, muscle and blood during the growth of Ehrlich ascites cells: application of the reversed-phase and ion-pair reversed-phase high-performance liquid chromatography with radial compression column

The pool of purine compounds was analysed in liver, skeletal muscle and blood of mice during the growth of Ehrlich ascites tumour cells. Three fast isocratic high-performance liquid chromatographic methods were used. (1) Determination of nucleotides by an isocratic ion-pair reversed-phase chromatography with a 10 mM ammonium phosphate buffer containing acetonitrile and tetrabutylammonium phosphate. (2) Separation of nucleosides and nucleobases in cell extracts by a reversed-phase system with methanol and 50 mM potassium phosphate buffer as eluent. (3) Nucleosides and nucleobases in body fluids were analysed by a reversed-phase system with 10 mM potassium phosphate containing methanol. These methods allow the rapid determination of purine compounds in small biological samples from various cell types and body fluids, with high accuracy and sensitivity. The pool of cellular nucleotides increased during the exponential phase of tumour growth. Adenosine accumulated significantly in all tissues in the stationary phase of tumour growth.     

Separation of nucleotides by reversed-phase high-performance liquid chromatography: Advantages and limitations

Summary The determination of nucleotides by reversedphase high-performance liquid chromatography with 0.1 M (NH₄)₃PO₄ as an elution buffer is described. Effects of pH, type of RP packing and column efficiency are discussed. Currently used columns with efficiencies corresponding to 7,000–15,000 theoretical plates, containing 15–20% of bound carbon, have been shown not to be sufficient for the separation of nucleotides from tissue samples in the ionsuppression mode, but they provide excellent separation of 2-, 3-, and 5-monophosphates, deoxytriphosphates or synthetic derivatives of adenosine and guanosine.

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Trennung von Nucleotiden mit der RP-Hochleistungsflüssigkeits-Chromatographie: Vorteile und Beschränkungen

Zusammenfassung Die Bestimmung von Nucleotiden mit reversed-phase HPLC mit 0,1 M (NH₄)₃PO₄ als Elutionspuffer wird beschrieben. Der Einfluß des pH sowie der Art der RP-Belegung wird diskutiert, Säulen mit einer theroretischen Bodenzahl von 7000–15000, die 15–20% gebundenen Kohlenstoff enthalten, erwiesen sich als nicht geeignet für die Trennung von Nucleotiden aus Gewebeproben mittels Ionen-Verdrängung. Sie ergeben aber eine ausgezeichnete Trennung von 2-, 3- und 5-Monophosphaten, Desoxytriphosphaten oder synthetischen Derivaten von Adenosin und Guanosin.

     

Simultaneous determination of cyromazine,melamine and their biodegradation products by ion-pair high-performance liquid chromatography

An ion-pair high-performance liquid chromatography with ultraviolet detection method for the determination of cyromazine, melamine and its biodegradation products (ammeline, ammelide, cyanuric acid and biuret) was developed. C18 column was utilised to separate the six analytes with a mobile phase consisting of perchloric acid-ammonia solution and acetonitrile, under gradient elution and variable flow rate. The detection wavelengths were 205 nm for cyanuric acid and biuret and 222 nm for cyromazine, melamine, ammeline and ammelide. For analysis of sediment samples, the extraction solution containing acetonitrile, ammonia and water (80:10:10 by volume) was used to extract the analytes from sediment matrix. Using the extraction method for the spiked sediment sample, high linearity of matrix-matched standard curve could be obtained for the six analytes. The method detection limit was 0.1 μg g^?1 for melamine and cyromazine, 0.2 μg g^?1 for ammeline and ammelide, 1.2 μg g^?1 for cyanuric acid and 1.0 μg g^?1 for biuret in sediment matrix. The recoveries of these compounds were 70.1–98.3% and the relative standard deviations were 0.5–4.4%. Finally, the proposed method was successfully applied to the analysis of the sediment sample near the wastewater outlet of a melamine-producing factory.     

Optimization of the ion-pair high-performance liquid chromatographic separation of purine derivatives in erythrocytes, thymocytes and liver mitochondria

Various methods are described for the analysis of purine derivatives in biological samples by ion-pair high-performance liquid chromatography (HPLC) with both gradient and isocratic systems. A new approach is proposed that is suitable for the separation of nuclei acid constituents in different cells with a specific enzymatic activity pattern. The ion-pair HPLC methods were developed for the analysis of erythrocytes, lymphocytes and mitochondria acid-soluble fractions in clinical and experimental studies of normal and altered nucleotide metabolism. The results of studies of purine metabolite redistribution in mouse liver mitochondria during a 30-min incubation at 37 degrees C and data on purine metabolic alterations in mouse thymocytes during hepatoma growth are discussed.