Simple unsegmented flow configurations for simultaneous kinetic determinations

Two unsegmented-flow manifolds were designed for simultaneous determinations based on differences in the kinetic behaviour of two analytes with the same reagent. The determination of Fe(III) and Co(II) based on displacement of Fe(III) and Co(II) from their EGTA complexes by PAR, was used as a model system. The displacement reactions were photometrically monitored at 510 nm.     

Fixed-time kinetic enthalpimetry: improved sensitivity for enthalpimetric enzyme activity determinations in homogeneous and heterogeneous systems

A fixed-time (integral) method is described for the enthalpimetric determination of enzyme activity. The method involves the determination of residual unreacted substrate after a fixed incubation time with the sample. Results are presented for the determination of cholinesterase in aqueous solution and in 0.1cm³ samples of reference sera using 30-min incubation periods. Results are correlated with a spectrophotometric procedure. A precision of 1.8% relative standard deviation is reported for serum assays. Preliminary data are also presented for the enthalpimetric determination of cholinesterase activity after immobilization onto non-porous glass beads by carbodiimide and glutaraldehyde coupling procedures.     

Cholesterol study: Reaction plateau and kinetic determinations

A study has been described of a comparison between reaction plateaus and kinetic modes for measuring cholesterol. A similar investigation of bilirubin reactions under the same procedural conditions was also carried out because this compound is a major interference which is frequently encountered in abnormal serums. The findings indicate that bilirubin is a more sensitive reactant than cholesterol by LB reaction but that its effect as an interference can be lessened by a kinetic approach providing that the effect of H₂O on reaction velocity and molar absorptivity can be minimized. An iron reaction even though more sensitive proved less workable in the kinetic mode owing to reaction velocity and the presence of air bubbles. However, its reaction plateau characteristics were superior to the LB reaction because of a favorable ratio of colors generated for the two reacting constituents. Based on this study, it is predictable that a kinetic approach is quite workable for the LB reaction and could be made more workable for the iron reaction if, in the latter case, conditions perhaps such as temperature and reaction media, could be altered to slow the rate of color formation.     

Computer control for simultaneous carbon,hydrogen, and nitrogen determinations in organic compounds

Summary The automation, via a digital computer, of CHN determinations in organic compounds by a manometric procedure is described. The accuracy of the method is expected to be ± 0.03%.

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Zusammenfassung Die Automation der manometrischen Bestimmung von C, H und N in organischen Verbindungen mit Hilfe eines Digitalzählers wurde beschrieben. Sie läßt als Genauigkeit der Methode ±0,03% erwarten.

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This work was performed under the auspices of the U. S. Atomic Energy Commission.     

A general standard addition method for kinetic substrate determinations in enzymatic analysis.

The influence of a preceding electrode process in the analytical use of a.c., differential pulse and other polarographic methods is discussed. Coupled chemical reactions, such as found with the Cr(III)/Fe(III) system, cause considerable interference in all the techniques examined, interference is readily recognized by a shift in peak potential and a change in the wave shape on addition of increasing concentrations of the more positively reduced species. With other systems, a depolarizer more negatively reduced than another can be determined over a wide range of conditions with three-electrode instrumentation. IR-drop problems with two-electrode systems place considerable restrictions on their use. With Zn/Cu and Cd/Cu and other systems, reliable determinations were possible only when the concentration of the more positively reduced species was less than 10^?2 M.     

Formation of two reaction zones in flow-injection systems for kinetic determinations of cobalt and nickel

The injection of a large sample volume (ca. 1 ml) into a single-channel flow-injection system was studied with a dye (to examine physical dispersion) and with chemical systems having easily-controlled reaction rates (to examine chemical kinetics). With the dye, the response curve has a central plateau caused by non-mixing of carrier and sample. When a chemical reaction takes place, two peaks are obtained with a central minimum corresponding to little or no mixing and reaction. Comparison of these two types of response provides relationships of analytical interest between response parameters and variables in te flow-injection system. The configuration is used for individual kinetic determinations of cobalt and nickel (2.5–30 μg ml^?1 based on the rate of their complex formation with 2-hydroxybenzaldehyde thiosemicarbazone. Sample injection rates were 15 h^?1 for cobalt and 40 h^?1 for nickel. Differential kinetic determinations of cobalt and nickel in mixtures are based on the increment in peak height (or area) between the two peaks obtained for each injection; sample throughput is 7 h^?.     

Multiplex inhibitor screening and kinetic constant determinations for yeast hexokinase using mass spectrometry based assays

An electrospray ionization mass spectrometry based assay was developed for kinetic measurements and inhibitor screening of yeast hexokinase. There is considerable discrepancy in the literature as to the accuracy of kinetic data obtained for hexokinase. In the assay described herein, the product, glucose 6-phosphate was directly monitored by ion trap mass spectrometry and quantified using an internal standard, 2 deoxy-glucose 6-phosphate. The kinetic parameters, K(M) and V(max) for the two substrates were determined without using a coupling enzyme as is normally employed in the traditional spectrophotometric assay for systems lacking a chromophore. In addition, hexokinase was successfully immobilized onto an amino-link gel, and a mock library was screened against the immobilized enzyme for the identification of possible inhibitors. After comparing the mass spectra of the library before and after incubation, trehalose 6-phosphate, ADP, and oxidized glutathione were differentiated from other weak or non-inhibitors. Inhibition behavior of ADP with respect to ATP was further evaluated with the ESI-MS assay and the value of K(i) was determined. This ESI-MS assay was demonstrated to be both accurate and precise for determining kinetic constants and for identifying enzyme inhibitors.     

停流流动注射-诱导动力学光度法同时测定微量铁和锑

依据诱导反应的基本原理, 提出了一种同时测定多元素的方法; 研究了Fe(II)、Sb(III)诱导的Cr(VI)-I^-氧化还原反应体系, 并据此建立了停流-诱导动力学光度法同时测定微量铁和锑的工作条件。线性范围分别为0~1.2μg.mL^-1Fe(II)和0~2.0μg.mL^-1Sb(III); 检测限为0.012μg/mL^-1Fe(II)和0.014μg.mL^-1Sb(III)。除Ti(III)、V(IV)及As(III)外, 其余共存离子不影响测定。用本法测定了锌标准物及模拟样品中的铁和锑含量, 结果满意。     

Mean centering of ratio kinetic profiles for the simultaneous kinetic determination of binary mixtures in electroanalytical methods

In this work, the applicability of mean centering (MC) of ratio kinetic profiles method to the kinetic voltammetry data is verified. For this purpose, a procedure is described for the determination of Sb(III) and Sb(V) by adsorptive linear sweep voltammetry using pyrogallol (py) as a complexing agent. The method is based on the differences between the rate of complexation of pyrogallol with Sb(V) and Sb(III) at pH 1.2. The results show that the mean centering of ratio kinetic profiles method is suitable for the speciation of antimony. Sb(III) and Sb(V) can be determined in the ranges of 3.0-120.0 and 10.0-240.0 ng mL⁻¹, respectively. Moreover, the solution is analyzed for any possible effects of foreign ions. The obtained results show that the method of MC in combination to electroanalytical techniques is a powerful method with high sensitivity and selectivity. The procedure is successfully applied to the speciation of antimony in pharmaceutical preparations.     

A sensitive high-throughput HPLC assay for simultaneous determination of everolimus and clobetasol propionate

A novel sensitive high-throughput high-performance liquid chromatography assay is developed and validated for the simultaneous determination of everolimus and clobetasol propionate in pharmaceutical formulations. The chromatographic separation is achieved on a Zorbax Eclipse XDB-C18 reversed-phase column using a gradient elution, with solvent A: ammonium acetate (pH 6.8; 0.01 M) and solvent B: acetonitrile. The mean recovery ranges from 95.1% to 100.0% for clobetasol propionate and from 97.9% to 103.7% for everolimus. The limit of quantitation for each analyte is 0.02 microg/mL. The percent relative standard deviations are less than 3% for intra- and inter-day analyses. The proposed method can be used for the routine quality control of everolimus and clobetasol propionate in complex pharmaceutical formulations, especially the drug-delivery systems with a low total drug-load.     

Micellar electrokinetic chromatography assay for the simultaneous quantification of urinary transferrin and albumin

A micellar electrokinetic chromatography (MEKC) method for simultaneous determination of urinary transferrin and albumin has been developed. The performance of the assay was evaluated. Under the optimum conditions, the linearity of transferrin and albumin is in the range of 10~1000 mg L^?1. The detection limits are 1.3 mg L^?1 for transferrin and 1.1 mg L^?1 for albumin, with relative standard deviations of less than 5.4%. The within-run and between-run coefficients of variation were <5.5% for transferrin and <8.2% for albumin, respectively. The recoveries of transferrin and albumin are 97.7~106.1%. The performance of the MEKC assay was compared with radioimmunoassay, and correlated well with this.     

A novel spectrophotometric method for the simultaneous kinetic analysis of ternary mixtures by mean centering of ratio kinetic profiles

In this paper, a novel and very simple method was developed for the simultaneous spectrophotometric determination of ternary mixtures, without prior separation steps. The method is based on the mean centering of ratio kinetic profiles. The mathematical explanation of the procedure is illustrated. In order to investigate the applicability of the proposed method, it was applied to the simultaneous spectrophotometric determination of hydrazine, phenylhydrazine and acetylhydrazine based on their condensation reactions with p-(dimethylamino)benzaldehyde (DAB) and p-nitrobenzaldehyde (NB) in micellar sodium dodecyl sulfate (SDS) media as a typical ternary mixture. The analytical characteristics of the method such as accuracy, precision, relative standard deviation (R.S.D.) and relative standard error (R.S.E.) were calculated.     

Localized surface plasmon resonance sensor for simultaneous kinetic determination of peroxyacetic acid and hydrogen peroxide

A new sensor for simultaneous determination of peroxyacetic acid and hydrogen peroxide using silver nanoparticles (Ag-NPs) as a chromogenic reagent is introduced. The silver nanoparticles have the catalytic ability for the decomposition of peroxyacetic acid and hydrogen peroxide; then the decomposition of them induces the degradation of silver nanoparticles. Hence, a remarkable change in the localized surface plasmon resonance absorbance strength could be observed. Spectra-kinetic approach and artificial neural network was applied for the simultaneous determination of peroxyacetic acid and hydrogen peroxide. Linear calibration graphs were obtained in the concentration range of (8.20 × 10⁻⁵ to 2.00 × 10⁻³ mol L⁻¹) for peroxyacetic acid and (2.00 × 10⁻⁵ to 4.80 × 10⁻³ mol L⁻¹) for hydrogen peroxide. The analytical performance of this sensor has been evaluated for the detection of simultaneous determination of peroxyacetic acid and hydrogen peroxide in real samples.     

The mutual catalytic effect in kinetic reactions and its application to simultaneous determination of binary components

The mutual catalytic effect between iron(II) and antimony(III) on the chromium(VI)-iodide kinetic reactions has been studied by stopped-flow FIA. The concept of mutual catalytic coefficient is defined. The deviations from additive principles of absorbance within certain concentration ranges in previous procedures can be compensated effectively, by introducing the mutual catalytic coefficient into the simultaneous determination of a binary mixture with stopped-flow-FIA method. The linear concentration ranges have been enlarged, 0–3.5 g/ml Fe(II) and 0–3.7 g/ml Sb(III) compared with 0–1.2 g/ml Fe(II) and 0–2.0 g/ml Sb(III) without use of the coefficient. Iron and antimony contents in wastewater, a simulated sample and a zinc standard were determined, the recoveries and relative standard deviations being, respectively, 99.8–101.3% and 2.7–3.6% for iron and 95.4–100.3% and 2.3–5.3% for antimony compared with 95.2–98% and 3.0–4% for iron and 96–104% and 4.5–4.8% for antimony, assuming additivity.     

Multidata treatment applied to the simultaneous resolution of catechol-resorcinol mixtures by kinetic enzymatic processes

This paper reports a multicomponent least-squares regression method based on the use of multiple standards for the simultaneous resolution of substrates involved in kinetic enzymatic processes. The proposed method was applied to the determination of catechol-resorcinol mixtures by oxidation with hydrogen peroxide in the presence of the enzyme peroxidase (EC 1. 11.1.7) in a stopped-flow reversed flow injection system. The mathematical algorithm used is superior to other alternatives as it is not affected by side reactions between the oxidation products. The proposed method allows the simultaneous resolution of 50-150,muM catechol and 30-180,muM resorcinol over the wavelength range 340-500 nm. The concentration ranges can be modified by varying the injected amount of enzyme. The mathematical treatment is applied to the spectra of several standards and those of the samples, which are recorded 6 sec after the flow is stopped. This redounds to a high sampling frequency (up to 60/hr).     

A real-time fluorogenic phospholipase A(2) assay for biochemical and cellular activity measurements

A fluorogenic analog of the PLA(2) substrate PC, named Dabcyl-BODIPY-PC, or simply DBPC, was synthesized with a fluorescence quencher (Dabcyl, 4-[(4-[N,N-dimethylamino]phenyl)azo]benzoic acid) in the sn-1 acyl chain and a BODIPY fluor in the sn-2 acyl chain. DBPC was recognized by sPLA(2) from each of the four sources examined (bee venom, human synovial fluid, cobra venom, and bovine pancreas). A dramatic and quantifiable fluorescence enhancement of DBPC occurred upon phospholipase digestion both in the presence and absence of excess PC. Both real-time and endpoint assays for PLA(2) were sensitive, consistent, and rapid. Thus, DBPC can be used as a sensitive fluorogenic probe for in vitro high-throughput screening assays for PLA(2) activation and inhibition and would expedite studies of PLA(2) in cellular signaling, in vitro screening for drug discovery, and subcellular localization of enzyme activity.     

Solid-phase microextraction with on-fiber silylation for simultaneous determinations of endocrine disrupting chemicals and steroid hormones by gas chromatography-mass spectrometry

Based on solid-phase microextraction (SPME) and on-fiber silylation, a method for simultaneous determinations of exogenous endocrine disrupting chemicals (EDCs) and endogenous steroid hormones in environmental aqueous and biological samples by gas chromatography-mass spectrometry (GC-MS) was developed. The selected target compounds were: octylphenol (OP), technical grade nonylphenol (t-NP), diethylstilbestrol (DES), dehydroisoandrosterone (DEHA), estrone (E1), 17beta-estradiol (E2), testosterone (T) and pregnenolone (PREG). The optimization of operating conditions influencing the performances of SPME and derivatization were studied in detail. The average correlation coefficient of the calibration curves of the target compounds was 0.9968 and the linear ranges of most compounds spanned over three orders of magnitude. The LOD/(LOQ) values of the target compounds in river water and blood serum were in the range of 0.002-0.378/(0.008-1.261) microg L(-1) and 0.004-0.474/(0.013-1.579) microg L(-1), respectively, which were a bit higher than those in the pure water due to matrix effects. The developed method was applied to the determinations of target compounds in real samples. Exogenous OP, t-NP and DES were at 0.15, 4.67 and 0.02 microg L(-1) in river water and 3.21, 12.17 and 0.15 microg L(-1) in fish blood serum. Natural steroid hormones E1, E2 and T were at 0.18, 0.10 and 5.55 microg L(-1) in river water, and in female fish serum, E1, E2 and PREG were at 1.61, 1.08 and 4.58 microg L(-1), respectively. The proposed SPME method was compared with traditional SPE procedure and the results found using both methods were in the same order of magnitude and both are quite agreeable.     

Liquid chromatographic-mass spectrometric assay for simultaneous pyrimethamine and sulfadoxine determination in human plasma samples

We present a liquid chromatographic-mass spectrometric assay for the simultaneous determination of sulfadoxine and pyrimethamine in human plasma samples. Sample clean-up was achieved by adding acetonitrile for protein precipitation. Gradient elution in only 10 min resulted in high throughput capability. Tandem mass spectrometric detection in multiple reaction monitoring was used for quantification. The developed analytical approach was successfully validated and was applied in the pharmacokinetic evaluation of the bioavailability between two sulfadoxine/pyrimethamine formulations available on the Eastern African market, using a cross-over design.