Aza-Arenophilic Gels as Supports for the Reversible Immobilization of Enzymes—Bioanalytical ApplicationsJogie, S.; Narinesingh, D.; Ngo, T. T.
doi: 10.1080/00032719808001860pmid: N/A
Abstract A method is described for the simple, reversible immobilization of enzymes to an aza-arenophilic support. The support is prepared by reacting Sepharose CL-4B first with 3,5-dichloro-2,4,6-trifluoropyridine and 4-dimethylaminopyridine followed with 2-mercaptoethanol. The enzymes were non-covalently adsorbed using a phosphate buffer (0.5M, pH 7.4) and readily desorbed using sodium acetate (0.1M, pH 2.9). The same solid support could be used many times to reversibly bind enzymes. Urease bioreactors prepared by this method were incorporated into a flow injection manifold and used for the quantitation of urea. Calibration curves were found to be linear up to 120 mgdL−1 urea with a detection limit of.0.08mgdL−1. Recovery yields varied between 98 – 103%. The within day RSD was <1.9% and the day to day RSD was < 2.8 for a 20mgdL−1 solution. There was no detectable decrease in bioreactor activity over a period of 34 days. Up to 18 urea samples can be analysed manually per hour.
A Fluorescence-Based Sensor for Ammonium and NitrateSasaki, Satoshi; Ando, Yoshihiro; Dejima, Makiko; Arikawa, Yoshiko; Karube, Isao
doi: 10.1080/00032719808001861pmid: N/A
Abstract Measurement of nitrite and nitrate in rain samples was performed using a fluorescence based sensor. Nitrite and nitrate were reduced to ammonia using Devarda's alloy, and the gas was then passed through a membrane and reacted with o-phthalaldehyde. A linear relationship between the nitrate concentration and the luminescence intensity was observed over the concentration range of 1 – 5 mg/1, with a relative standard deviation (RSD) of 2.3% at a nitrate concentration of 1 mg/1 (n = 5). The system did not show any response toward sulfate or chloride. Results obtained from the measurement of river water samples using the sensor showed good agreement with those obtained using a conventional method.
Employing Heterocyclic Dihydropolyazines for Amperometric Glucose SensingKulys, J.; Palaima, A.; Urbelis, G.
doi: 10.1080/00032719808001862pmid: N/A
Abstract Glucose sensors containing heterocyclic dihydropolyazines: 5,10-dihydro-5,10-dimethylphenazine (DHDMP) or 1,4-dihydro-1,3,4,6-tetraphenyl-s-tetrazine (DHTPT) as mediators and glucose oxidase adsorbed on graphite rods generated an anodic current at 0.10 (DHDMP) and 0.55 (DHTPT) V vs SCE in the presence of glucose at pH 7. The potential of the sensors correlated with the electrochemical conversion of the mediators. Under aerobic conditions calibration graphs for the sensors showed a slightly sigmoidal character with half-saturation concentrations of 58 ± 6 and 43 ± 9 mM glucose and Hill coefficients of 1.68 ± 0.07 and 1.88 ± 0.16, respectively. The calibration curves were hyperbolic over the whole glucose concentration in deaerated buffer solution and the sensitivities were 0.47 and 0.18 μA/mM for DHDMP and DHTPT containing electrodes, respectively. The sensor response increased in weakly alkaline medium. Calculated values of pKa, (7.0 ± 0.2), were similar for both type electrodes, were independent of buffer solution components and correlated with glucose oxidase reactivity (pKa = 7.3 ± 0.05) in homogeneous solution. The sensors action was explained by formation of the radical cation of the dihydropolyazines, their action with reduced glucose oxidase and oxidation of the electrochemical mediators. The potential of the electrodes depended on the formal redox potential of the mediator, whereas current was a result of the radical cation reactivity and solubility.
The Methylviologen-Doped Zeolite Modified Electrode as a New Detector for Suppressor Free Ion ChromatographyWalcarius, A.; Lamberts, L.
doi: 10.1080/00032719808001863pmid: N/A
Abstract A methylviologen-doped zeolite modified carbon paste electrode was applied to the amperometric detection of alkali metal cations in ion chromatography. The detection principle is based on the ion exchange of methylviologen by the target cationic analyte, followed by the reduction of methylviologen at the electrode surface at an operating potential of −0.85 V (vs. SCE). When using a solution of tetrabutylammonium bromide as the mobile phase, the background response was very low because of the size exclusion of tetrabutylammonium ions from the zeolite framework, and the modified electrode was only sensitive to non size-excluded cations. In a large volume wall-jet cell configuration, the sensor allowed the analysis of non reducible non size-excluded cations after their separation on an ion exchange column, in both the presence and the absence of dissolved oxygen. Use of a mobile phase with cations too large to be detected by the zeolite modified electrode did not require any further suppressor technique.
Simultaneous Determination of Five Caffeine Metabolites in Human Urine with Reverse-Phase HPLC MethodLu, J. F.; Cao, X. M.; Yi, T.; Zhuo, H. T.; Ling, S. S
doi: 10.1080/00032719808001865pmid: N/A
Abstract Caffeine metabolism via the 3-demethylation pathway is sequentially catalyzed by the enzymes cytochrome P4501A2 (CYP1A2), xanthine oxidase (XO) and N-acetyltransferase (NAT2). The activities of the three enzymes can be estimated from urinary metabolic ratios of five caffeine metabolites, 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 1-methylxanthine (1MX), 1-methyluric acid (1MU), 1,7-dimethylxanthine (1,7DMX), and 1,7-dimethyluric acid (17DMU), after the ingestion of caffeine. A HPLC method for simultaneous determination of the five metabolites in human urine was developed, with all the thirteen metabolites of caffeine having good separation. The recoveries of the five metabolites were all above 87.5%, and the interday and intraday coefficients of variation were less than 3%. The method was employed successfully in a population study of 120 healthy volunteers for the assessment of CYP1A2, NAT2, and XO enzymes.
Stability-Indicating Method for the Determination of Bromazepam via Its Mercury(II), Silver(I) and Zirconium(IV) ChelatesEl Khateeb, Sonia Z.; Amer, Mohammed M.; Abdel Razek., Sawsan A.
doi: 10.1080/00032719808001867pmid: N/A
Abstract An accurate method is investigated for the selective determination of intact bromazepam (Ia) in the presence of its acid – induced degradation products, 2-amino-5-bromobenzoylpyridine (Ib) and glycine. The method is based on the chelation of bromazepam with mercury (II), silver (I) or zirconium (IV) to form chelates that absorb at 363 nm, 287 nm and 289 nm, respectively. The three proposed procedures determine 16 – 80 μg ml−1, 8 – 48 μg ml−1 and 6.4 – 38 μg ml−1 of bromazepam, with mean accuracies of 100.1 ± 0.8%, 99.7 ± 0.7% and 100.3 ± 0.7%, respectively.
HPLC Impurity Profile Analyses of Pharmaceutical Substances Using UV Photodiode Array DEtectionRyan, Timothy W.
doi: 10.1080/00032719808001868pmid: N/A
Abstract High performance liquid chromatography (HPLC) is routinely used for determination of both assay and impurities in both bulk active and formulated drug products. Impurity profile analyses are required to demonstrate the ability to detect a wide range of impurities which may occur in pharmaceuticals. However, most impurity profile methods do not address the potential of co-elution of impurities with product peaks. UV photodiode array detection (PDA) evaluates the UV and/or the UV/VIS spectrum of an eluting species to determine spectral homogeneity. If variations in the spectrum are observed, the possibility of a co-eluting impurity must be addressed. By establishing consistent evaluations HPLC-PDA can be used to more accurately assess impurity levels and provide analyses with much higher informational content.