Rapid and reliable method for the analysis of nucleotide pools by reversed-phase high-performance liquid chromatography

A rapid and reliable protocol for the simultaneous separation of ribo-, deoxyribo- and cyclic nucleotides has been developed using high-performance liquid chromatography on a C18 microBondapak column and isocratic elution with ammonium phosphate buffer 0.2 m, pH 5.1). Resolution of deoxyribonucleotides has been confirmed by performing resolution before and after periodate oxidation. The general order of elution is ribonucleotides, deoxyribonucleotides and cyclic nucleotides. While periodate oxidation improved the clarity of separation of deoxyribonucleotides by eliminating ribonucleotides, incorporation of methanol in the eluent shortened the retention time of cyclic nucleotides. The application of this method to a complex biological system is reported.     

High-performance liquid chromatography and studies of neurophysin-neurohypophysial hormone pathways

A definitive method to determine adenine compounds simultaneously was established by introducing a new fluorescent reagent into high-performance liquid chromatography. Bromoacetaldehyde was the best reagent among the haloacetaldehydes examined. A quantitative reaction was obtained even for unstable ADP and ATP. A high resolution of adenine nucleotides was obtained using a column of Hitachi gel No. 3012-N. The method was applied to the measurement of cyclic AMP in urine, and ADP and ATP in brain and blood. Further, the sensitivity of the method was increased by a new fluorescence spectrophotometer constructed for micro-HPLC. Femtomole amounts of the adenine nucleotides were clearly separated.     

Analysis of Guanylate Cyclase Activity by High-Pressure Liquid Chromatography

Abstract

A method for the assay of guanylate cyclase which permits the correction of concurrent phosphatase and phosphodiesterase reactions has been developed using HPLC. The method, based on the conversion of tritium labelled guanosine triphosphate to tritium labelled cyclic guanosine monophosphate, uses [¹⁴C]-cGMP as the internal standard to account for the degradative and procedural-losses. Radiolabelled reaction products are isolated by high pressure liquid chromatography on a Partisil SAX column with a single step isocratic elution using 12.5 mM potassium phosphate buffer (pH 3.25). Since column recovery of the nucleotides is virtually quantitative and complete purification is achieved, the method possesses a high degree of accuracy and precision.     

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.     

The dielectric constant of a polar liquid by the simulation of microscopic drops

We report a new method of computing the static dielectric constant for a polar liquid using a natural system, a drop of liquid in its own vapour. Results for the Stockmayer potential agree with those computed for a homogeneous “cyclic” liquid with Ewald corrections. The method is quite general.     

Simultaneous determination of 5'-monophosphate nucleotides in infant formulas by HPLC-MS

A method was developed for simultaneous determination of 5'-monophosphate nucleotides, adenosine 5'-monophosphate, cytidine 5'-monophosphate, guanosine 5'-monophosphate, inosine 5'-monophosphate, and uridine 5'-monophosphate in infant formulas by high-performance liquid chromatography-mass spectrometry equipped with electrospray ionization source. The complete chromatographic separation of five nucleotides was achieved through a Symmetry C(18) column, after a binary gradient elution with water containing 0.1% formic acid and acetonitrile as mobile phase. The multi-reaction monitoring mode was applied for tandem mass spectrometry analysis. The established method was further validated by determining the linearity (R(2) > 0.999), recovery (92.0-105.0%), and precision (relative standard deviation ≤6.97%). To verify the applicability of the method, thirty commercially available infant formulas were randomly purchased from the supermarkets in Hangzhou, China, and then analyzed. The results showed that the developed method is validated, sensitive, and reliable for quantitation of nucleotides in infant formulas.     

局部麻醉药的反相色谱安培法检测

本文系统研究了普鲁卡因,利多卡因,丁卡因等3种局部麻醉药的液相色谱安培法检测行法,并与液相色谱光度法检测结果进行了比较。     

固相萃取-液相色谱法测定婴幼儿配方乳粉中的游离核苷酸

该文建立了婴幼儿配方乳粉中5种游离核苷酸的检测方法。婴幼儿配方乳粉经乙二胺四乙酸二钠和氯化钠溶液提取,强阴离子（SAX）固相萃取柱净化,通过反相Atlantis T₃色谱柱分离,二极管阵列检测器检测,外标法定量。结果表明：方法抗干扰能力强、准确度高,尤其对羊奶粉净化效果好、分离度高。5种游离核苷酸在0.5~10 mg/L范围内线性关系良好（r≥0.999）;添加水平在0.05~0.50 g/kg时,回收率为91.1%~112%,相对标准偏差为2.3%~4.7%;检出限为0.0010~0.0015 g/kg,定量限为0.0030~0.0045 g/kg。对乳粉质控样品进行检测,结果与中位值比较的偏差在10%以内。该方法可为监管部门提供技术支持,为乳品行业健康发展提供保障。     

Ionic Liquids as Mobile Phase Additives for Separation of Nucleotides in High-Performance Liquid Chromatography

Ionic liquids are a type of salts that are liquid at low temperature (＜100℃). Because of their some special properties, they have been widely used as new “green solvents” for many chemical reactions and liquid-liquid extraction in the past several years. In this paper, a new method for the separation of nucleotides is developed and the essential feature of the method is that 1-alkyl-3-methylimidazolium salts are used as mobile phase additives, resulting in a baseline separation of nucleotides without need of gradient elution and need of organic solvent addition as currently used in RP-HPLC. This study shows the potential application of ionic liquids as mobile phase additives in reversed-phase liquid chromatograohy.     

Separation of nucleotides by hydrophilic interaction chromatography using the FRULIC-N column

A stationary phase composed of silica-bonded cyclofructan 6 (FRULIC-N) was evaluated for the separation of four cyclic nucleotides, six nucleoside monophosphates, four nucleoside diphosphates, and five nucleoside triphosphates via hydrophilic interaction chromatography (HILIC) in both isocratic and gradient conditions. The gradient conditions gave significantly better separations by narrowing peak widths. Sixteen out of nineteen nucleotides were baseline separated on the FRULIC-N column in one run. Unlike other known HILIC stationary phases, there can be dual-retention mechanisms unique to this media. Traditional hydrogen bonding/dipolar interactions can be supplemented by dynamic ion interaction effects for anionic analytes. This occurs because the FRULIC-N stationary phase is able to bind certain buffer cations. The extent of the ion interaction is tunable, in comparison to stationary phases with embedded charged groups, where the inherent ionic properties are fixed. The best mobile phase conditions were determined by varying the organic modifier (acetonitrile) content, as well as salt type/concentration and electrolyte pH. The thermodynamic characteristic of the FRULIC-N column was investigated by evaluating the column temperature effect on retention and utilizing van’t Hoff plots. This study shows that there is a greater entropic contribution for the retention of nucleotide di and triphosphates, whereas there is a greater enthalphic contribution for the cyclic nucleotides with the FRULIC-N column.     

Nucleotide pools and energy charges of smooth molluscan muscles analysed by reversed-phase,ion-pair,high-performance liquid chromatography

Summary A method is described for the analysis of the 5 mono-, di- and triphosphates of adenosine, guanosine, uridine and cytidine, as well as uridinediphosphate-glucose and cyclic AMP. Separation is achieved by reversed-phase ion-pair chromatography with linear gradient elution. Application of this method to the analysis of nucleotides in smooth molluscan muscles is described, including the determination of cAMP-levels and the calculation of energy charges for all of the four nucleotide systems.     

Kinetics of Cu2+ Reduction and Nanoparticle Nucleation at Micro-scale 1,2-Dichlorobenzene-water Interface Studied by Cyclic Voltammetry and Square-wave Voltammetry

Reduction and nanoparticle nucleation of Cu²⁺ by decamethylferrocene was studied with cyclic and square-wave voltammetry at a microscale liquid–liquid interface established at the tip of a micropipette. With square-wave voltammetry, two reduction steps could be distinguished as two separate current waves. Comparing the experimental results of cyclic voltammetry with finite element method simulations, particle growth could be observed as an increasing limiting current. Furthermore, kinetic parameters could be estimated with square-wave voltammetry simulations following Butler-Volmer kinetics.     

Assays of cyclic nucleotides

Cyclic nucleotides are recognized as important second messenger molecules, and many assay formats exist for their quantification. This article critically reviews these different approaches. For measurement of cAMP or cGMP in biological fluids or tissue extracts, immunoassay is effective. For other purposes, such as measurement of cyclic nucleotide phosphodiesterase activity, methods that separate nucleotides from their cyclic counterparts are best, and offer a variety of means of detection.     

高效液相色谱/电喷雾飞行时间质谱分析太子参中环肽类化合物

采用高效液相色谱/电喷雾飞行时间质谱联用方法(HPLC/ESI-TOFMS)分析太子参中的环肽类化合物。实验采用反相C18色谱柱,二元线性梯度洗脱,分离并检测了太子参中6种环肽类化合物;通过与电喷雾飞行时间质谱联用获得了这几种化合物的准确分子量信息,由于ESI-TOFMS具有高分辨率,能够测定化合物精确的分子质量而不降低灵敏度,对6种环肽类化合物成分进行了定性鉴定。该方法简便、快速、准确。     

A Comparison of the Efficiency of Nucleotide Extraction by Several Procedures and the Analysis of Nucleotides from Extracts of Liver and Isolated Hepatocytes by HPLC

Abstract

A high pressure liquid chromatography (HPLC) system was developed, capable of resolving most 5′-nucleotides and nucleotide-sugars present in liver tissue. Using three different extraction procedures, the recovery of the twelve major 5′-ribonucleotides from solutions of nucleotide standards, or liver samples, or isolated hepatocytes was compared. Nucleotides were obtained from acid extracts for HPLC analysis by adsorbing the nucleotides on charcoal, or precipitating the acid (perchloric acid) by KOH, or extracting the acid with alamine/ freon. The last two procedures were found to be superior to the charcoal adsorption procedure for recovering nucleotides from acid extracts. The recovery of nucleotides from either liver samples or isolated hepatocytes by these two procedures was similar; however, the recovery of nucleotides from standard solutions was slightly higher for the alamine/freon procedure than the KOH precipitation procedure.     

Rapid determination of adenine nucleotides in brain tissue by ion-paired reversed-phase column liquid chromatography under isocratic conditions

A rapid method for analysis of adenine nucleotides (AMP, ADP and ATP) in nervous tissue based on ion-paired reversed-phase column liquid chromatography under isocratic conditions is described. An optimal composition of elution buffer was 25 mM potassium phosphate and 4% triethylamine adjusted to pH 6.5 with phosphoric acid. Typical separation time did not exceed 10 min with a 10-cm long compact glass cartridge packed with 5-microns silica C18. The method was employed to determine ATP, ADP and AMP concentrations in rat brain extracts and values thus obtained were compared with those published elsewhere.     

Synthesis, photochemistry and application of (7-methoxycoumarin-4-yl)methyl-caged 8-bromoadenosine cyclic 3',5'-monophosphate and 8-bromoguanosine cyclic 3',5'-monophosphate photolyzed in the nanosecond time region

New caged derivatives of hydrolysis-resistant 8-bromoadenosine cyclic 3',5'-monophosphate (8-Br-cAMP) and 8-bromoguanosine cyclic 3',5'-monophosphate (8-Br-cGMP) are described. The compounds are the axial and equatorial isomers of the (7-methoxycoumarin-4-yl)methyl (MCM) esters of cyclic nucleotides. Synthesis is accomplished by treatment of 4-bromomethyl-7-methoxycoumarin with the tetra-n-butylammonium salts of the 8-bromo-substituted cyclic nucleotides or with the free acids of 8-Br-cAMP and 8-Br-cGMP in the presence of silver(I) oxide. MCM-caged 8-Br-cAMP and MCM-caged 8-Br-cGMP liberate 8-Br-cAMP and 8-Br-cGMP during irradiation with ultraviolet light within a few nanoseconds. They show favorable absorption properties and quantum yields and are resistant to hydrolysis in aqueous buffer solutions. The moderate fluorescence properties of the caged compounds in comparison with the strongly fluorescent 4-hydroxymethyl-7-methoxycoumarin (MCM-OH) photoproduct allow the indirect estimation of the amount of photolytically released cyclic nucleotides in aqueous buffer solutions using fluorescence measurements. Their usefulness for physiological studies has been examined in a mammalian cell line expressing the cyclic nucleotide-gated ion channel of bovine olfactory sensory neurons using the patch-clamp technique and confocal laser scanning microscopy. The caged compounds serve as efficient and rapid intracellular sources of 8-Br-cAMP and 8-Br-cGMP. However, at least in HEK 293 cells, fluorescence signals cannot be used to monitor the photolysis of MCM-caged 8-Br-cAMP and 8-Br-cGMP, due to quenching of the fluorescence of MCM-OH.     

Trennung von Nucleotiden, Nucleosiden, cyclischen Nucleotiden, Dinucleotid-Pyridin-Coenzymen und Enzymsubstraten mittels Reversed-Phase-Chromatographie

Ohne Zusammenfassung

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Separation of nucleotides, nucleosides, cyclic nucleotides, dinucleotide-pyridine-coenzymes and enzyme substrates by reversed-phase chromatography

     

Assay of human erythrocyte pyrimidine and deoxypyrimidine 5'-nucleotidase by isocratic reversed-phase high-performance liquid chromatography

We report a rapid and reproducible assay for activity of human erythrocyte pyrimidine 5'-nucleotidase and deoxypyrimidine 5'-nucleotidase. The nucleotides CMP, UMP, dUMP, dCMP or dTMP are individually incubated 30 min at 37 degrees C with erythrocyte hemolysate and 4 mM magnesium chloride in Tris, pH 7.5. Data are provided for standardization of the reaction with each substrate. Individual nucleoside products are assayed in less than 10 min by reversed-phase high-performance liquid chromatography at 280 nm with 0-14% methanol in 0.01 M potassium dihydrogen phosphate. This is the first report of a high-performance liquid chromatographic assay system which allows quantitation of the activity of pyrimidine 5'-nucleotidase isozymes using five individual pyrimidine and deoxypyrimidine nucleotides as the substrates.     

Simultaneous monitoring of monoamines,amino acids,nucleotides and neuropeptides by liquid chromatography‐tandem mass spectrometry and its application to neurosecretion in bovine chromaffin cells

The primary functions of adrenal medullary chromaffin cells are the synthesis and storage in their chromaffin vesicles of the catecholamines noradrenaline (NA) and adrenaline (AD), and their subsequent release into the bloodstream by Ca²⁺‐dependent exocytosis under conditions of fear or stress (fight or flight response). Several monoamines, nucleotides and opiates, such as leucine‐enkephalin (LENK) and methionine‐enkephalin (MENK), are also co‐stored and co‐released with the catecholamines. However, other neurotransmitters have not been studied in depth. Here, we present a novel high‐resolution liquid chromatography‐tandem mass spectrometry approach for the simultaneous monitoring of 14 compounds stored and released in bovine chromaffin cells (BCCs). We validated the analytical method according to the recommendations of the EMA and FDA by testing matrix effect, selectivity, sensitivity, precision, accuracy, stability and carry‐over. After testing on six batches of BCCs from different cultures, the method enabled simultaneous quantitative determination of monoamines (AD, NA, dopamine, serotonin, 5‐hydroxyindoleacetic acid, histamine and metanephrine), amino acids (L‐glutamic acid, γ‐aminobutyric acid), nucleotides (adenosine 5′‐diphosphate, adenosine 5′‐monophosphate, cyclic adenosine 5′‐monophosphate) and neuropeptides (LENK and MENK) in the intracellular content, basal secretion and acetylcholine induced secretion of BBCs. The high‐resolution approach used here enabled us to determine the levels of 14 compounds in the same BCC batch in only 16 min. This novel approach will make it possible to study the regulatory mechanisms of Ca²⁺ signaling, exocytosis and endocytosis using different neurotrophic factors and/or secretagogues as stimuli in primary BCC cultures. Our method is actually being applied to human plasma samples of different therapeutic areas where sympathoadrenal axis is involved in stress situations such as Alzheimer's disease, migraine or cirrhosis, to improve diagnosis and clinical practice. Copyright © 2016 John Wiley & Sons, Ltd.