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.     

Indirect Speciation Analysis of Thallium in Plant Extracts by Anodic Stripping Voltammetry

A sensitive voltammetric method (DPASV) was developed for the determination of Tl(I) and Tl(III) in plant extracts. To limit the influence of the organic matrix on the measurements, UV irradiation and addition of Amberlite XAD‐7 resin was studied. The application of 0.5 g of the resin allowed defining thallium speciation in 10.0 mL of a solution containing 0.20 mL of Sinapis alba extract. The quantification limit of 0.5 ng mL^?1 Tl(I) was found for only 10 min of preconcentration, and is low enough to allow dilution of the sample before thallium determination. The procedure was validated using the recovery study and intermethod comparison with HPLC ICP MS.     

Rapid Quantitative Method for the Determination of Dextrose,Mannitol, and Sorbitol in Meat Products by Liquid Chromatography

Abstract

A liquid chromatographic (LC) method for the determination of dextrose, mannitol, and sorbitol in meat products was developed. Dextrose, mannitol, and sorbitol were extracted from comminuted meat products with 52% ethanol. After filtration, the extracts were purified by passing through a C₁₈ Sep-Pak cartridge and two ion exchange resin Econo-Columns in series. After concentration and filtration, extracts were analyzed by liquid chromatography using a cation exchange analytical column and a differential refractometer detector. Homogeneously ground samples of cooked and fresh sausages and ground beef were fortified with dextrose, mannitol, and sorbitol at four different concentrations. Average overall recovery for all three compounds at all four levels of fortification was greater than 80% with coefficients of variation less than 10%.     

The Rapid Determination of Neutral Sugars in Biological Samples by High-Performance Liquid Chromatography

Abstract

A procedure for the analysis of neutral sugars in biological specimens is described. The method entails acid hydrolysis of the sample to liberate monosaccharides, which are subsequently derivatized with dansyl hydrazine. The sugar-dansyl hydrazones are separated and quantitated by hplc on a 5μ C18 RadialPak column with a gradient of acetonitrile in 10mM ammonium sulfate at pH 7. Fluorescent detection of the derivatized sugars permits 100-fold increased sensitivity compared to previously published glc methods.

This procedure was applied to the neutral sugar analysis of a glycoprotein of known composition (thyroglobulin) and to hard keratin fibers. The latter substance served as a model to critically evaluate the method on a highly resistant biological matrix containing low concentrations of neutral sugars.     

Removal of Triton X-100 and Reduction of Tween 20 Concentration in Extracts of Fatty Oxidation Products

Abstract

A technique was developed using silica Sep-Paks to remove Triton X-100 and reduce the concentration of Tween 20 in fatty extracts. Tween 20 concentration was primarily reduced by using non polar solvents to lower solubility. Although each Sep-Pak was capable of adsorbing up to 75 mg of Triton X-100, separation of the detergent from sample extracts was most effective when using sample loads approximately one tenth that size.     

Preparative HPLC for the Facile Isolation of Drug Glucuronide Conjugates from Crude Extracts of Urine

Abstract

Reverse-phase preparative HPLC has been used to advantage for the isolation of crystalline quantities of drug glucuronide conjugates from crude urinary extracts. Following chronic administration of large doses of diazepam, levorphanol and hydroxyethylflurazepam to dogs, urine was collected and the water soluble drug conjugates adsorbed on a column of Amberlite XAD-2 resin. In each instance elution with methanol and solvent evaporation yielded a crude oil (approx. 3 g) which was chromatographed in one portion on either PrepPAK 500 C₁₈ (Waters) or Magnum 40 ODS-3 (Whatman) columns using aqueous methanol solvent systems. A greater than 90% purification was achieved in this single initial chromatographic step. Employing a combination of subsequent semi-preparative HPLC steps on either C₁₈ or silica gel columns, milligram quantities of the glucuronides of oxazepam, levorphanol and hydroxyethylflurazepam were isolated. The isolation procedures provide a general approach for obtaining milligram quantities of intact drug conjugates which may otherwise be difficult to obtain by chemical synthesis. Such conjugates can be used as authentic standards in the quantitation of certain drug metabolites in biological media during pharmacokinetic/biopharmaceutic studies.     

Flavonoids in Selected Mediterranean Fruits: Extraction,Electrochemical Detection and Total Antioxidant Capacity Evaluation

Flavonoids are natural phenolic derivatives that, in low concentration, can provide health benefits by preventing biomolecules (proteins, nucleic acids, lipids, sugars) oxidative damage through free radical mediated reactions. The flavonoids, in selected Mediterranean seasonal fruits: apricot, sour cherry, plum, pomegranate, date, prickly pear (cactus fruit), and nectarine, by RP‐HPLC, coupled with photodiode array and electrochemical detectors, after microwave‐ultrasound assisted extraction, using flavonoid standards, were detected. The total antioxidant capacity in the lyophilized fruit extracts, by differential pulse voltammetry, (electrochemical index‐EI), integrated peak area, and chronoamperometry, was evaluated. In the lyophilized fruit extracts, and the catechin standard, the free radical scavenger efficient concentration (EC₅₀), using DPPH^. assay, was determined.     

Use of Millipore Norganic Resin for the Extraction of Proctolin and Other Pharmacologically Active Constituents from Cockroach Tissue Homogenates

Abstract

Millipore Norganic resin, a proprietary product marketed for the preparation of HPLC-grade water, can serve as an alternative to Sep-Pak C₁₈ cartridges for the solid phase extraction of proctolin and other physiologically active components from cockroach tissue homogenates. For large scale isolations of such substances, this resin is most useful for first stage purification. Retained substances can be further purified by application to Sep-Pak C₁₈ cartridges which are less hydrophobic than the Norganic resin. Results were evaluated by reversed-phase HPLC of extracts and bioassay of fractions. Typical recoveries of proctolin were in the range of 75–90%.     

Ultramicro Analysis of Reducing and Non-Reducing Sugars by Liquid Chromatography

Abstract

A post-column detection system for ultramicro amount of sugars has been developed using taurocyamine as the labelling reagent. Less than 10 pmol of reducing sugars were determined by this HPLC system. Non-reducing sugars were detected by the addition of periodate to the reagent.

Modification of the reaction reagent components made this detection system feasible to apply to various methods for separation of carbohydrates using pure water, acetonitrile/water mixtures, borate buffer or aqueous sodium hydroxide as the eluent.     

The Identification of Adenosine in Cacao Products

Abstract

Adenosine has been identified as a component of defatted chocolate liquor through HPLC of aqueous extracts. The nucleoside was identified by comparing its behavior with an authentic sample in a variety of mobile phases, column types, and absorbance ratios at 254 and 245 nm.     

Mixed-Bed Ion-Exchange Papers

Abstract

Retention of some cations and anions on ion-exchange resin-loaded disks has been reported to be less than quantitative. A mixed-bed resin-loaded paper was made in which mixed cation-exchange and anion-exchange resins were incorporated into the paper to take advantage of the ‘crossed equilibria’ effect and improve ion retention. The use of mixed-bed paper disks in analysis for cesium by X-ray fluorescence spectrometry is described.     

Novel tandem column method for the rapid isolation of radiostrontium from human urine

A method has been developed for the isolation of strontium from human urine for subsequent determination in sample volumes as low as 5–20 mL. This method involves the acidification of the sample using methanesulfonic acid and its decolorization using charcoal, treatment of the filtrate with Diphonix^® resin, and subsequent concentration of strontium on Sr resin. Data from retention model simulations provided the initial conditions which were then optimized by actual column separations. Diphonix^® resin was shown to be effective at removing alkali metal ions from the urine matrix under conditions that retain higher valence ions. The suggested processing method provides 99% recovery of Sr²⁺, a concentration factor of 50, and an expected per sample processing time of less than 1 h.     

Innovative approach to treating waste waters by a membrane capacitive deionisation system

The application of membrane capacitive deionisation was investigated for treating model water samples and real waste waters from the textile industry. For the pre-treatment of waste waters, nanofiltration was integrated in order to prevent scaling and fouling of membranes and electrodes during membrane capacitive deionisation. Different conditions were applied when treating water samples with membrane capacitive deionisation with the aim of optimising conditions for high desalination efficiency and, consequently, for conductivity reduction. The conductivity of waste waters with high salt concentrations was reduced to the required value, below 1.5 mS cm^?1. The desalination rates achieved as much as 95 %, depending on the initial conductivity and the different ions present in the water samples. In addition, chemometric characterisation of the samples was performed in order to determine the existence of significant correlations between the monitored parameters: the presence of various ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^?, Br^?, F^?, SO₄^2?, NO₃^?, desalination and water recovery, the duration of each phase and the flow of the solution during each phase. A model for desalination rate prediction was designed using multiple linear regression. It was established that the model values accorded well with the experimental values — the differences between model and experimental values were less than 1 %.     

Sequential injection analysis implementing multiple standard additions for As speciation by liquid chromatography and atomic fluorescence spectrometry (SIA-HPLC-AFS)

An analytical procedure for multiple standard additions of arsenic species using sequential injection analysis (SIA) is proposed for their quantification in seafood extracts. SIA presented flexibility for generating multiple specie standards at the ng mL⁻¹ concentration level by adding different volumes of As(III), As(V), monomethylarsonic (MMA) and dimethylarsinic (DMA) to the sample. The mixed sample plus standard solutions were delivered from SIA to fill the HPLC injection loop. Subsequently, As species were separated by HPLC and analyzed by atomic fluorescence spectrometry (AFS). The proposed system comprised two independently controlled modules, with the HPLC loop acting as the intermediary device. The analytical frequency was enhanced by combining the actions of both modules. While the added sample was flowing through the chromatographic column towards the detection system, the SIA program started performing the standard additions to another sample. The proposed method was applied to spoiled seafood extracts. Detection limits based on 3σ for As(III), As(V), MMA and DMA were 0.023, 0.39, 0.45 and 1.0 ng mL⁻¹, respectively.     

Use of Alkaline Degradation for the Analysis of Dihydrotriazines via High Pressure Liquid Chromatography

Abstract

An HPLC method is described for the determination of 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3′,4′-dichlorobenzyloxy)-1,3,5-triazine hydrochloride (WR 38839). The procedure required the isomerization of the drug sample by alkaline treatment with sodium hydroxide, as the parent compound was retained by the column. The reaction product of the drug was analyzed by HPLC using a strong cation exchange resin as the stationary phase and glycine buffer, pH 10.4 as the mobile phase. The product was isolated and identified by TLC, UV, IR, mass spectroscopy and elemental analysis. The postulated mechanism indicates that this would be a general analytical method for dihydrotriazine compounds. This technique, developed for the assay of the dihydrotriazine in an aqueous system, was successfully applied to rat urine samples spiked with the drug.     

Immunoassay for Highly Water-Soluble Nitenpyram: Evaluating the Analytical Performance of an Easy-to-Use Screening Method for Agricultural Samples

Abstract

Nitenpyram, a neonicotinoid insecticide, is highly soluble in water (>5.9?×?10⁵?mg/L). Specifically examining its physicochemical properties, we have established a simple and rapid residue analytical method for nitenpyram using an enzyme-linked immunosorbent assay (ELISA) which requires no organic solvents. The I₅₀ value found with this ELISA method using a selective monoclonal antibody toward nitenpyram was 4.8?ng/mL. A hand-shaking method using water was applied for ELISA analysis to extract nitenpyram from fruiting vegetable samples of four kinds. Matrix effects caused by interfering substances coexisting in aqueous sample extracts were reduced by dilution with water. No problems were found in the analytical values obtained using ELISA. From analyses of nitenpyram-incurred fruiting vegetables with the proposed ELISA and the reference HPLC protocols, high mutual correlation was found, which suggests that the measurement accuracy of the proposed ELISA method is comparable to the reference HPLC procedure.     

Statistical Model for Organic Chromatographic Trace Analysis of Complex Samples. A Case Study: Plant Extracts

Abstract

The use of pooled plant extracts is described in the estimation of matrix interference in HPLC (UV and EC) determinations of organic compounds in plant extracts. An extract from freeze dried leaves of 134 different plant species was used for this purpose. It was split in different subgroups with solid extraction clean-up procedures. UV, EC and chromatographic data of the subgroups were used in the calculation of minimum concentrations of organic compounds which are still accurately determinable in plant samples with HPLC methods. The UV and/or EC characteristics of the compound must be known. The contribution of the solid phase extraction procedures and of the analytical system to the selectivity of the method can be estimated. Information is also supplied which allows rapid comparison of the selectivity of the UV and EC (single, or dual parallel) detectors for the determination of a specified compound.     

Determination of flavonoids in the flowers of <Emphasis Type="Italic">Paulownia tomentosa</Emphasis> by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) technique coupled with photodiode array (PDA) detection was developed for the simultaneous determination of four flavonoids, i.e. apigenin (AP), diplacone (DI), mimulone (MI) and 5,4′-dihydroxy-7,3′-dimethoxyflavanone (DDF) in extracts of the flowers of Paulownia tomentosa. The optimized method was proposed for the separation and detection of the selected constituents, using methanol-1% acetic acid as the mobile phase at a flow rate of 0.8 mL/min, 290 and 267 nm as the detection wavelengths. All the flavonoids showed good linearity in a relatively wide concentration range (r > 0.9999). The detection limits for the analytes ranged from 0.2 to 4.0 ng, at a signal-to-noise ratio of 3. Inter- and intra-assay accuracy and precision were all lower than 5.0%. The recovery of the method was 95.9–101.9%. Moreover, the optimized HPLC method was employed to analyze the flowers of Paulownia tomentosa.     

Determination of Vitamins D2 and D3 in Feedingstuffs by High Performance Liquid Chromatography

Abstract

A sensitive and reliable HPLC method for complete separation and quantitation of vitamins D₂ and D₃ in complicated biological mixtures such as feedingstuffs has been developed and described. The method has been applied to the quantitative determination of D₂ and D₃ in feedingstuffs and related matrices at both high premix levels and low feed levels ranging down to a detection limit near 100 IU/1b or 0.22 IU/g sample. The procedures consist of the initial step of sample preparation by extraction; four sample cleanup stages: Sep/Pak/Silica Cartridge Cleanup, Millipore-Teflon Cleanup, Gel Permeation/Sephadex LH-20 Column Cleanup, and HPLC/Partisil-PAC Column Cleanup; and the final step of Reverse-Phase HPLC Separation, Identification, and Quantitation. The analytical Column used was Rainin Accupak 20 cm-3 um C-18 & Guard Columns. The Waters Associates Model 440 Fixed Wavelength UV Detector at 254 nm was used for all measurements. All separations and quantitations were carried out isocratically at room temperature and under subdued lighting. By these procedures, the sensitivity for both D₂ and D₃ is about the same (20 ng), and the resolution is excellent. Normally, the D₂ peak eluted at 30–31 min. and the D₃ peak followed at 2–3 min. later. By using the standard calibration and standard addition methods, the percent recovery ranges from 90.0%–104.8% with the mean value of 97.4%, whereas the accuracy is from 85.3% to 108.9% with the average of 97.8%. The standard deviation is ±5.2% and the coefficient of variation is 5.3%.     

Separation of Sr in combination of ion exchange and Sr resin with alcohol-nitric acid solution and rapid determination of <Superscript>90</Superscript>Sr in wine and soil samples

This paper describes the procedures of isolating strontium from wine and soil samples which enable creating of procedure for rapid determination of ⁹⁰Sr. The method of determination of ⁹⁰Sr includes binding of Sr on the cationic exchanger IR-120 from the sample and simultaneous elution from the cation column and binding on the Sr column, separation of Sr on Sr resin with HNO₃ even in presence of alcohols and subsequent Cherenkov counting. Sr can be efficiently bind on Sr resin and separated from the other elements with lower acid concentrations in the presence of a low portion of alcohol, or even from a wine sample without the loss of Sr resin capacity. The binding strength of Sr on Sr resin decreases with the rising of HNO₃ concentration (1–5 M) in the presence of 13% of ethanol or methanol, and with the rising of the alcohol portion in constant concentration of HNO₃. Application of cation exchanger for Sr binding in phase of sample preparation decreases Sr column loading and improve Sr recovery. The method allows the determination of ⁹⁰Sr activities in wine and soil sample lower than 10 mBq in reasonable time.