Distance and Color Change Based Hydrogel Sensor for Visual Quantitative Determination of Buffer Concentrations.

We present here an innovative platform for the determination of pH buffer capacity based on FITC-dextran loaded hydrogels. Optical signals from the pH-sensitive hydrogels were analyzed by simple parameters including distance and color change. The methodology was validated on five different buffer systems and exhibited wide linearity (0.1 to 100 mM), good batch-to-batch reproducibility, high versatility, and resistance to background ionic strength changes. Experimental results also fit well with a theoretical model based on numerical simulation. Preliminary application in carbonate alkalinity determination of seawater proved very successful. This hydrogel buffer concentration sensor is fundamentally different from conventional acid-base titrations, brings minimum perturbation to samples, and shows great potential in real applications.

Contribution of tris buffer on xylitol enzymatic production.

Xylitol enzymatic production can be an alternative to chemical and microbial processes, because of advantages like higher conversion efficiency. However, for an adequate conversion, it is necessary to investigate the effect of many parameters, such as buffer initial concentration, pH, temperature, agitation, etc. In this context, the objective of this work was to evaluate xylitol enzymatic production under different Tris buffer initial concentrations in order to determine the best condition for this parameter to begin the reaction. The best results were obtained when Tris buffer initial concentration was 0.22 M, reaching 0.31 g L(-1) h(-1) xylitol volumetric productivity with 99% xylose-xylitol conversion efficiency. Although the increase in buffer concentration allowed better pH maintenance, it hindered the catalysis. The results demonstrate that this bioreaction is greatly influenced by involved ions concentrations.

Salt-assisted liquid-liquid microextraction with water-miscible organic solvents for the determination of carbonyl compounds by high-performance liquid chromatography.

A simple and rapid method has been reported for the determination of carbonyl compounds involving reaction with 2,4-dinitrophenylhydrazine and extraction of hydrazones with water-miscible organic solvent acetonitrile when the phase separation occurs by addition of ammonium sulphate, a process called salt-assisted liquid-liquid microextraction. The extract was analyzed by high-performance liquid chromatography with UV detection at 360 nm. The procedure has been optimized with respect to solvent suitable for extraction, salt for phase separation between water and organic solvent, reaction temperature and reaction time. The method has been validated when a linear dynamic range was obtained between the amount of analyte and peak area of hydrazones in the range 7 microg-15 mg L(-1), the correlation coefficient over 0.9964-0.9991, and the limit of detection in the range 0.58-3.2 microg L(-1). Spiked water samples have been analyzed with adequate accuracy, and application of the method has been demonstrated in the analysis of benzaldehyde formed as oxidation product in pharmaceutical preparation where benzyl alcohol is used as preservative, and for a keto drug dexketoprofen.

Identification of oxidative degradates of the TRIS salt of a 5,6,7,8-tetrahydro-1,8-naphthyridine derivative by LC/MS/MS and NMR spectroscopy--interactions between the active pharmaceutical ingredient and its counterion.

The Tris(hydroxymethyl)aminomethane (TRIS) salt of a substituted 5,6,7,8-tetrahydro-1,8-naphthyridine compound (I) in a mannitol-based formulation was stressed at various conditions. Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses of the stressed samples revealed that oxidation and dimerization were the primary degradation pathways for this compound. 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy were used to characterize the isolated dimers. The aromatized degradate, N-oxide, amide, and three dimeric products were all confirmed by either LC/MS using authentic standards or NMR spectroscopy. In general, the aromatized product was always the primary degradate produced under all stress conditions. When stressed at 80 degrees C, the TRIS counterion also underwent thermal degradation to yield formaldehyde in situ which reacted with the parent compound to form a unique methylene-bridged dimeric product and an N-formyl degradate. A minor condensation product between the compound I and the TRIS counterion was also detected in the 80 degrees C stressed samples. Under 40 degrees C/75% RH stress conditions, TRIS derived degradates were insignificant, while dimers formed by compound I became predominant. In addition, two hydroxylated products (7-OH and 5-OH) were also detected. Mechanisms for the formation of the oxidative and dimeric degradates were proposed.

A HILIC method for the analysis of tromethamine as the counter ion in an investigational pharmaceutical salt.

A hydrophilic interaction chromatography (HILIC) method using an aminopropyl stationary phase was developed and validated to determine the content of tromethamine as counter ion in an investigational drug substance. Tromethamine was a very polar compound without any chromophores, and could not be readily retained and detected by conventional reserved-phase HPLC-UV methods. Furthermore, the tromethamine salt of the drug compound also had limited solubility in aqueous solution. The method employed simple acetonitrile/water mobile phase (80/20, v/v) to provide sufficient retention for tromethamine. Meanwhile, the high acetonitrile content also helped to dissolve the drug substance sample. Refractive index (RI) was used for the detection of tromethamine. The method was found to be specific without interference from the drug compound and related impurities. The method was also validated for suitable precision, linearity and accuracy for the analysis of drug substance samples. The effects of various parameters, such as acetonitrile content, mobile phase salt concentration, and column temperature on HILIC separation were investigated.

Near infrared transflectance spectroscopy: determination of dexketoprofen in a hydrogel.

A new method for the determination of the active principle dexketoprofen (DKP) in a hydrogel using near infrared spectrometry was developed. A transflectance module allows the gel spectrum to be recorded with no sample pretreatment. Also, the PLS calibration method used allows low contents of the active principle in such a highly absorbing matrix as water/ethanol to be determined. The proposed method was validated and found to be an effective choice for the intended purpose.

Accelerated separation of random complex DNA patterns in gels: comparing the performance of discontinuous and continuous buffers.

Complex DNA fragment patterns produced by random amplified polymorphic DNA (RAPD) assays were separated in agarose as well as polyacrylamide gels using a continuous buffer and two discontinuous buffer systems, with and without urea, respectively. The effect of very high field strength/current levels on the duration of separation, the relative mobilities of the selected bands, and the appearance of the pattern was tested. The use of discontinuous buffer systems resulted in sharper bands and better resolution at 30 min duration of separation for DNA fragments in the size range of 200-2000 base pairs. These observations could help researchers in the rapid separation of band patterns produced by polymerase chain reaction (PCR)-based methods utilized in small- to mid-scale genome searches.

Separation of a range of cations by nonaqueous capillary electrophoresis using indirect and direct detection.

The use of nonaqueous media and indirect detection is reported for the separation and detection of a range of small cations. The novel applications involved separation of a range of metal ions, small nonchromophoric amines, cationic ion-pair reagents and cationic surfactants. Separations were achieved using acidified methanol containing imidazole as the UV co-ion for indirect detection. The methods produced different selectivity compared to aqueous methods using acidified aqueous imidazole solutions. Advantages of the methods include speed of analysis and prevention of sample micellerisation. The methods were shown to be quantitative and reproducible by their application to the determination of Tris content.

Determination of tromethamine in an eye-care pharmaceutical by capillary electrophoresis.

Tromethamine [tris(hydroxymethyl)aminomethane] is used as an emulsifying agent, alkaliser and buffering agent in pharmaceutical preparations such as eye-care solutions. A new method for the determination of this compound in an eye-care preparation was developed using capillary zone electrophoresis with indirect UV detection. The method displayed linearity between 2.5 and 25 mg l-1 with a correlation coefficient of 0.9992. The limit of detection was 2.5 mg l-1. Precision and accuracy were determined as relative standard deviation and % deviation, and were found to be 0.42% and 1.62%, respectively. Recovery studies of tromethamine in the pharmaceutical preparation gave a 102.98% recovery.

Validation of analytical capillary electrophoresis methods for use in a regulated environment.

Since its introduction into the analytical laboratory, CE has had to prove that it was capable of generating results comparable to HPLC or GC techniques in the six areas (specificity, precision, accuracy, linearity, ruggedness, and range) typically required of validated methods intended for submission to governmental agencies. This paper will showcase the development and validation of two analytical methods: one for specific identification of HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethane sulfonic acid]) and the other to quantitate millimolar concentrations of tris (tris[hydroxymethyl]aminomethane) in high electrolyte solutions. Utilizing neutral markers and internal standards, results for HEPES demonstrate that migration time reproducibility, expressed as %RSD of 2% or less on a variety of capillaries, is obtainable. Additionally, for quantitation of tris, the values obtained for accuracy (%relative mean bias), precision (%RSD), and linearity (r2) over multiple days and capillaries meet the rigorous standards we require of HPLC or GC methods.

Determination of diclofenac sodium, famotidine and ketorolac tromethamine by flow injection analysis using dichloronitrophenol.

Diclofenac sodium, famotidine and ketorolac tromethamine were determined by flow injection analysis (FIA) with spectrophotometric detection. The sample solutions (5-50 micrograms ml-1 of diclofenac sodium, 10-80 micrograms ml-1 of famotidine and 10-120 micrograms ml-1 of ketorolac tromethamine) in methanol were injected into a flow system containing 0.01% (w/v) of 2,4,dichloro-6-nitrophenol (DCNP) in methanol. The colour produced due to the formation of a charge transfer complex was measured with a spectrophotometric detector set at 450 nm. A sampling rate of 40 per hour was achieved with high reproducibility of measurements (RSD below 1.6%). The FIA method was applied to the determination of diclofenac sodium, famotidine and ketorolac tromethamine in pharmaceutical formulations.

Determination of tris(hydroxymethyl)aminomethane (tromethamine) in human plasma and urine by high-performance liquid chromatography with fluorescence detection.

Determination of tromethamine (2-amino-2-hydroxymethyl-1,3-propanediol) in human plasma and urine has been developed and validated. The method utilizes reversed-phase high-performance liquid chromatographic separation and fluorescence detection of a pre-column derivative. The assay was linear in the ranges of 1-200 micrograms/ml plasma and 5-500 micrograms/ml urine with accuracies (mean percent recovery) all within 10% of 100% and precisions (coefficient of variation) all less than 10%.

High-performance liquid chromatographic method for the determination of tris(hydroxymethyl)aminomethane (tromethamine) in human plasma.

Determination of tris(hydroxymethyl)aminomethane (tromethamine) in human plasma involved derivatization of the amino and hydroxyl groups with a ultraviolet-absorbing chromophore followed by extraction into an organic phase. Reversed-phase high-performance liquid chromatography with gradient elution was used for the separation of the analyte from the internal standard (2,3-butanediol). The assay was linear in the range 1.0-1000.0 micrograms/ml of plasma and the coefficient of variation varied between 9.6 and 16.3% whereas the accuracy varied between 90 and 108%. The limit of detection for the assay was 0.282 micrograms/ml. Stability of tris(hydroxymethyl)aminomethane in human plasma frozen at -20 degrees C was studied over a period of three month and the data indicated no significant change.

Kinetics and mechanism of zinc ion-mediated degradation of cephalosporins in tromethamine solution.

Earlier studies of the hydrolysis and aminolysis of penicillin, in the presence of zinc ion and tromethamine (Tris), revealed a very rapid catalysis mediated by a ternary complex in which the metal ion brought the reactants into close proximity in a suitable configuration for reaction. In the present work similar studies with a group of cephalosporins show not only much slower rates of reaction but a different mechanism in which the zinc ion-tromethamine complex functions as a nucleophile in a bimolecular reaction. Evidence for the differences in mechanism includes not only the different dependence of rate upon tromethamine concentration, but comparable rates of reaction of methyl esters of a penicillin and a cephalosporin and the reaction products observed by high-performance liquid chromatography.

Analysis of the variance components in a pharmaceutical aerosol product: lodoxamide tromethamine.

The contributions of several components to the variance in lodoxamide delivery from lodoxamide tromethamine metered-dose aerosol containers have been estimated. Two aerosol lots, manufactured with mean diameters of 2.3 and 7.2 micron, exhibited approximately equal variances. The variance was apportioned to the following components: container-to-container, 27%; mouthpiece-to-mouthpiece 18%; valve-to-valve, 11%; assay, 6%. The largest single contribution to the variance (38%) is attributed to unassignable variations which include within-container variations in the dose; quality improvement efforts should concentrate on this area. Little effort should be expended to minimize the assay, valve delivery, or mouthpiece variation as their contribution to lodoxamide dose variation is small. Likewise, the bulk drug particle size did not contribute appreciably to within-lot dose variation.

tris(Hydroxymethyl)aminomethane: an artifact detected as an aminotetrose by gas chromatography/mass spectrometry.

The monosaccharide content of the ethanol-soluble moiety of crude polysaccharide fractions of the tapeworm Hymenolepis diminuta and the blood fluke Schistosoma mansoni was investigated by gas-liquid chromatography of alditol acetate derivatives. A prominent, unusual peak was found, and mass spectrometry suggested identification as an aminotetrose, a four-carbon aminosugar reported only once in the past 15 years [D. A. Cumming, C. G. Hellerquist, and O. Touster (1981) J. Biol. Chem. 256, 7723-7726]. The unknown molecule was found to be tris(hydroxymethyl)aminomethane (Tris), whose acetylated derivative has a mass spectrum identical to that of an aminotetrose. The source of the Tris was traced to buffers used in conjunction with polysaccharide isolation.

The Schotten-Baumann reaction as an aid to the analysis of polar compounds: application to the determination of tris(hydroxymethyl)aminomethane (THAM).

The separation of polar, low molecular weight, water-soluble compounds from specimens prior to quantitation has long been a problem in analytical toxicology. Others have successfully used in situ derivatization, prior to extraction, to resolve this problem (1,3). An application of the Schotten-Baumann reaction leading to a simple extraction of tris(hydroxymethyl)aminomethane (THAM) followed by HPLC is presented. This may be a generic approach to the analysis of other polyhydroxy, water-soluble compounds.