Fully automated method based on on-line molecularly imprinted polymer solid-phase extraction for determination of lovastatin in dietary supplements containing red yeast rice

A fully automated method for the determination of lovastatin in dietary supplements containing red yeast rice has been developed. It uses a sequential injection analysis system combined with solid-phase extraction applying highly selective molecularly imprinted polymer sorbent. A miniaturized column for on-line extraction was prepared by packing 4.5 mg of the sorbent in a 5.0 × 2.5-mm-i.d. cartridge, which was used in the flow manifold. Sequential injection analysis manifold enabled all steps of lovastatin extraction and continuous spectrophotometric detection at 240 nm. A limit of detection of 60 μg g⁻¹, a limit of quantitation of 200 μg g⁻¹, and a linear calibration range of 200–2000 μg g⁻¹ were achieved. Intra-day and inter-day precision values (RSD) were ≤ 6.7% and ≤ 4.9%, respectively, and method recovery values of spiked red yeast rice extracts at 200, 1000, and 2000 μg g⁻¹ concentration levels were 82.9, 95.2, and 87.7%. Our method was used for determination of lovastatin lactone in four dietary supplements containing red yeast rice as a natural source of lovastatin, also known as monacolin K. The extracted samples were subsequently analyzed by the reference UHPLC-MS/MS method. Statistical comparison of results (F test, t test, α = 0.05) obtained by both methods did not reveal significant difference. A substantial advantage of the new automated approach is high sample throughput thanks to the analysis time of 7.5 min, miniaturization via down-scaling the extraction column, and smaller sample and solvent consumption, as well as reduced generation of waste.

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Highly reproducible capillary zone electrophoresis of humic acids in cyclodextrin- or oligosaccharide-modified background electrolytes

Capillary zone electrophoresis has been used for the characterization and separation of humic acids. It was found that addition of saccharides like alpha-, beta-, gamma-cyclodextrins, maltose, hydroxyethylcellulose or dextran sulfate in the background electrolyte (50 mM Na2 B4 O7, pH 9.6) yields better separation patterns and highly reproducible electropherograms. Electropherograms with higher numbers of peaks and high reproducibility were obtained with alpha- and beta-cyclodextrins or with a mixture of alpha- + gamma-cyclodextrin-modified background electrolytes. Separation was carried out with the cathode at the detector end of the column. Adsorption of humic acids to the capillary wall was diminished using an epoxy-coated capillary tube.     

Dediazoniations of Arene Diazonium Ions in Homogenous Solution. Part IV: Change-over from a heterolytic to a homolytic mechanism in 2,2,2-trifluoroethanol pyridine mixtures

There are only two dediazoniation products of benzenediazonium tetrafluoroborate in 2,2,2-trifluoroethanol (TFE), namely phenyl 2,2,2-trifluoroethyl ether ( 1 ) and fluorobenzene ( 2 ). The reaction kinetics are strictly first-order with respect to the diazonium salt. The addition of increasing amounts of pyridine to the system results in a gradual decrease in the yields of 1 and 2 and an increase in the yields of the homolytically formed products, benzene ( 3 ), biphenyl ( 4 ), isomeric phenylpyridines ( 5 ) and diazo tar ( 6 ). The reaction kinetics show that the rate of dediazoniation of the benzene diazonium salt increases with increasing amounts of pyridine. The reaction with added pyridine is no longer first-order with respect to the diazonium ion. The product analyses and the kinetic data are consistent with the view that in pure TFE this diazonium salt decomposes completely by a heterolytic mechanism. The addition of pyridine brings about a competitive homolytic mechanism which becomes increasingly dominant as the concentration of pyridine increases.     

FIA titrations of phenothiazine derivatives in aqueous micellar and non-aqueous media

New methods of flow injection analysis (FIA) neutralization titrations of phenothiazine derivatives in aqueous micellar medium of a cationic surfactant using potentiometric and spectrophotometric detection were proposed; titrations with a mixing gradient chamber and high-speed titrations were compared. The FIA titration method in non-aqueous media based on an official method of determination (titration with perchloric acid in anhydrous acetic acid) was also developed. Under optimized reaction conditions and flow-through parameters, the calibration range and equations, the sensitivity, and the repeatability of all methods were found and discussed. All titrations were assayed for medicinal forms.     

Detection of damage to DNA and antioxidative activity of yeast polysaccharides at the DNA-modified screen-printed electrode

A simple procedure for the voltammetric detection of the DNA damage and antioxidants protecting DNA from its damage using a disposable electrochemical DNA biosensor is reported. The carbon-based screen-printed electrode (SPE) modified by a surface layer of the calf thymus double stranded (ds) DNA was used as a working electrode in combination with a silver/silver chloride reference electrode and a separate platinum auxiliary electrode. The [Co(phen)(3)](3+) ion served as the dsDNA redox marker and the [Cu(phen)(2)](2+) and [Fe(EDTA)](-) complex compounds were used as the DNA cleavage agents under the reduction by a chemical reductant (ascorbic acid). Four yeast polysaccharides with different chemical structure were investigated as the antioxidants within the concentration range of 0.05-4 mg ml(-1) in the cleavage mixture. A remarkable antioxidative activity of polysaccharides in order mannan (Candida krusei)>extracellular glucomannan (Candida utilis)>mannan (Candida albicans)>glucomannan (C. utilis) was found which is in agreement with that refered to trolox (a structural derivative of alpha-tocopherol) and determined by photochemiluminescent method.     

Study of enzyme kinetics of phenol sulfotransferase by electrophoretically mediated microanalysis

Electrophoretically mediated microanalysis (EMMA) was applied for the study of the kinetic parameters of the enzymatic reaction of phenol sulfotransferase SULT1A1 isoenzyme with 4-nitrophenol as a substrate. The SULT1A1 activity was determined by the quantitation of the product, 4-nitrophenyl sulfate, at 274 nm by using different injection and separation steps. This new approach solved the problem of the presence of the very strong inhibitor, adenosine 3',5'-bisphosphate (PAP), in the co-substrate solution (adenosine 3'-phosphate 5'-phosphosulfate, PAPS) which is unstable at room temperature. The inhibitor PAP was electrophoretically separated from the co-substrate PAPS before the injection of enzyme and substrate inside the capillary (and thus before their in-capillary encountering). With the developed in-capillary SULT1A1 activity assay an average Michaelis constant (Km) for 4-nitrophenol was calculated to be 0.84 microM, a value which is consistent with a previously reported value. Strong substrate inhibition (above a 4-nitrophenol concentration of 2.5 microM) was observed, and this is also in accordance with literature values.     

Determination of gold using clay modified carbon paste electrode

Sorption of gold(III) chlorocomplexes was studied by means of a carbon paste electrode modified with montmorillonite. Anionic exchange behavior was found in chloride media with low ionic strength. Anionic sorption of [AuCl4]- can be used as a preconcentration step to the determination of Au(III). Linear calibration dependences were found in the concentration range 4.06 x 10(-6) - 1.22 x 10(-5) mol/L Au(III) after 5 min of sorption and in the range 8.12 x 10(-7) - 6. 1 x 10(-6) mol/L after 10 min of sorption. Interferences of several anions and cations were studied. Model samples of table water were analyzed.     

Properties of glucose biosensors based on dendrimer layers. Effect of enzyme immobilization

The properties of glucose biosensors based on dendrimer layers on a gold support, which depend on the method of immobilization of glucose oxidase (GOX), were studied by amperometry. The kinetic parameters of enzymatic reactions, response time, sensitivity, detection limit, linear range, and enzyme turnover were determined. We showed that a more stable and sensitive sensor was obtained when GOX was immobilized on the dendrimer by crosslinking with glutaraldehyde in vacuum. This biosensor was stable for at least eight weeks. The response time was approximately 1.3 min, the detection limit of glucose was 25 micro M, and the apparent Michaelis-Menten constant was relative low ( K(m)=1.1+/-0.1 mM) in comparison with that for GOX in solution. The reason for these differences is discussed. The example of the application of the developed biosensors for the detection of mercury is also presented. The inhibitory effect of mercury on GOX activity was observed at mercury concentration of 100 nM.     

Analyte and system eigenpeaks in nonaqueous capillary zone electrophoresis: theoretical description and experimental confirmation with methanol as solvent

A mathematical model developed for aqueous solutions and adapted to methanol as solvent was applied to predict the electromigration characteristics of analytes and background electrolytes in capillary zone electrophoresis. These characteristics are the effective mobility, and the tendency of the analyte to undergo peak-broadening due to electromigration dispersion. The input parameters for calculation like limiting mobilities and dissociation constants were experimentally determined or taken from the literature. By the aid of the model, the molar response for conductivity detection was calculated as well as the transfer ratio when indirect UV detection was used. They allow depicting the electropherogram by computer simulation. An additional important program output is the prediction of the occurrence of system- or eigenpeaks that mimic peaks of analytes or electroosmotic flow markers. The measured electropherograms were in agreement with those theoretically predicted. Deviations were attributed to ion pairing in methanolic solutions, which was not implemented in the model.     

Voltammetric determination of benzidine and its derivatives, at a glassy-carbon electrode

Conditions have been found for the determination of benzidine, o-tolidine, o-dianisidine, 3,3'-diaminobenzidine and 3,3'-dichlorobenzidine by classical and differential pulse voltammetry at a glassy-carbon stationary or rotating electrode, in the concentration range 10(-5)-10(-3)M. The mechanism of the oxidation is discussed and applications are presented for the determination of these chemical carcinogens and their mixtures either directly, or after preliminary separation by extraction or thin-layer chromatography.