Kinetic determination of aromatic amines at millimolar level

A kinetic method for the determination of aromatic amines is reported. The method involves the formation of an azo dye between 1-(4-hydroxy-6-methylpyrimidin-2-yl)-3-methylpyrazolin-5-one and a diazonium salt formed from the amine in the presence of nitrite in weakly acidic media. The reaction is monitored via the initial rate of change of the absorbance of the azo dye at 420 nm, because this is proportional to the aniline concentration.The optimum acidity and concentration of reagents were established. The concentration ranges for which the calibration lines are linear are quite large. Detection limits were estimated. The effect of several metal ions usually present in real samples, e.g. waste water, was examined to assess interference.     

Kinetic determination of zineb in agricultural samples by continuous addition of reagent

The technique of automatic continuous addition of reagent has been applied for the determination of zineb in pesticides. The method is based on photometric monitoring of the fast complexation reaction between zinc(II) and Zincon. The special feature of the technique is the direct kinetic measurement, which provides a highly selective determination of zineb at mug/ml levels. The sampling rate of 140 samples/hr makes the method very useful in routine analysis for this pesticide in vine and olive leaves.     

Automatic kinetic determination of oxazepam by the continuous addition of reagent technique

An automatic continuous-addition-of-reagent method for the routine determination of oxazepam based on the formation of a coupling product between diazotized 2-amine-5-chlorobenzophenone (the hydrolysis product of oxazepam) and 1-naphthol in a basic medium is proposed. The reaction is developed by continuously adding basic 1-naphthol to a reaction vessel containing the other ingredients and is monitored spectrophotometrically at 530 nm. The results obtained show the proposed kinetic method to surpass the performance of its conventional equilibrium counterpart as it permits distinguishing the intact drug from its hydrolytic degradation product and is suitable for the determination of oxazepam in dosage forms and urine samples.     

Liquid chromatographic-tandem mass spectrometric determination of selected sulphonamides in milk

Liquid chromatography–tandem mass spectrometry is used for the quantitative analysis of selected sulphonamides in milk. Ultrafiltration is the only sample pre-treatment technique which is required. Consequently, sample throughput is much higher than with conventional procedures, and analyte recoveries are high. As for quantification, both external standard and isotope dilution calibration yield satisfactory results. The method is fully validated for five sulphonamides with a maximum residue limit of 100 μg/kg, and which are included in the Dutch control programme on residues. Furthermore, results are presented on the applicability of the method to detect compounds at a much lower concentration level exemplified by a banned sulphonamide, dapsone, which has a provisional action limit of 5 μg/kg. The main conclusion is that the present, novel approach to the trace-level determination of veterinary drugs is simple and straightforward and has a wide-ranging application potential which is briefly exemplified by the analysis of selected benzimidazoles in milk by essentially the same procedure.     

Flow-injection spectrophotometric determination of arylamines and sulphonamides by diazotization and coupling in a micellar medium

In a sodium dodecyl sulphate (SDS) micellar solution, the rate of coupling of a diazonium ion with N-(1- naphthyl)ethylenediamine (NED) increases greatly, the protonation of the resulting azo dyes takes place at higher pH values and the dyes are more soluble. These favourable features were applied to the development of a simple flow-injection spectrophotometric procedure for the determination of diazotizable substances of pharmaceutical interest. Limits of detection in the range 0.2–0.5 μg ml^?1 (signal-to-noise ratio=3), with relative standard deviation of 0.7–3% (n=3) for 5 μg ml^?1 standards, were obtained.     

Improved supercritical fluid extraction of sulphonamides

Summary Different ways used for enhancing the yield of sulphonamides leached from solid supports are reported. Supercritical CO₂ and methanol-modified CO₂ were used as extractants of the target analytes and the impregnation of the solid sample with buffer, derivatization of the analytes and ion-pair formation were assessed. Only the sulphonamide/tetramethyl-ammonium ion-pairs are quantitatively extracted from the solid supports using pure supercritical CO₂, while the other modifications and the presence of a cosolvent lead to recoveries lower than 30% for most of the analytes. Individual separation/quantitation of the analytes was performed off-line using a liquid chromatograph.     

Enzymatic determination of N-methylcarbamate pesticides at the nanomolar level by the stopped-flow technique

A sensitive and selective kinetic enzymatic method for the determination of N-methylcarbamate pesticides is presented. It is based on their inhibitory effect on electric eel acetylcholinesterase and the use of 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) as chromogenic reagent for the thiocholine released from the acetylthiocholine iodide substrate. The fast DTNB-thiocholine reaction is monitored photometrically by the stopped-flow technique. Carbaryl, propoxur and carbofuran can be determined at concentrations in the ranges 6.5-120, 2-15 and 0.1-5.0 ng/ml, respectively, by the proposed method. An interference study was also reported.     

Determination of sulphonamides in animal tissues by high performance liquid chromatography with pre-column derivatization of 9-fluorenylmethyl chloroformate

A novel approach for simultaneous determination of 12 sulphonamides (sulphadiazine, sulphamethazine, sulphathiazole, sulphadimethoxine, sulphamerazine, sulphapyridine, sulphamethoxazole, suphamethizole, sulphaquinoxaline, sulphameter, sulphamonomethoxine, and sulphachloropyridazine) in animal tissues (swine muscle and liver, chicken muscle, beef muscle) by HPLC with UV detection has been developed. A pre-column derivatization of the sulphonamide compounds with 9-fluorenylmethyl chloroformate (FMOC-Cl) has been proposed and the reaction conditions have been optimized. The FMOC-sulphonamide derivatives were purified by SPE with silica gel as solid support prior to HPLC separation. The limits of detection for the sulphonamide compounds were greatly improved after the derivatization and purification step for the derivatives. Sulphonamide residues in animal tissues were extracted by acetonitrile and purified by solid phase extraction with C(18) as the solid support. The method developed has high sensitivity and good repeatability, and the average recoveries for most of the sulphonamides at various spiking levels were above 70% with relative standard deviations below 13.7%. The limits of detection for most sulphonamides can reach 3-5 microg/kg.     

‘In situ’ preparation of nitrous acid solutions by photoreduction in a flow assembly for analysis of pharmaceuticals

The solutions of nitrite ion, or nitrous acid, are frequently used as reagent in different analytical procedures and in pharmaceutical analysis. The low stability of the aqueous solutions of this reagent implies the tedious work of freshly prepared solutions every day and being kept in the refrigerator. In this paper, a photoreactor formed by a low pressure mercury lamp and nesting in a continuous-flow manifold is studied, tested, and proposed for the in situ reduction of nitrate solution into nitrite. The conversion into nitrite is reproducible and it has been successfully applied to determination of different drugs, mostly from the sulphonamide family, in an FIA assembly.     

Kinetic control of reagent dissolution for the flow injection determination of iron at trace levels

A novel methodology for the determination of iron at the ppb level by spectrophotometric flow injection analysis is described. The method is based on the control of the flow dissolution of the colorimetric reagent 1,10-phenanthroline. This is achieved by means of the minimization of the area of contact between the carrier and the solid reagent, thus allowing the use of the fairly soluble organic compound without affecting the reactor lifetime. The reagent is melted inside an acrylic column (3.0 x 0.5 cm id) in such a way that a hollow space is left in the center after cooling. This new design improves some aspects of the performance of the classical solid-phase reactors as no problems related to the increase in the backpressure of the system are evidenced. Furthermore, the total reagent loading of the column is increased as no inert support is needed. A comparison between the performance of this novel methodology and that of the conventional packed reactor was performed and several advantages were observed: the use of higher flow rates, an increase in the reactor lifetime and a decrease in reagent consumption. A mathematical model to fit the concentration profiles of the dissolved reagent as a function of the residence time of the sample within the column is presented. The application of this strategy to the determination of Fe(II) improves the figures of merit in comparison to those obtained with a single-line homogeneous system: the limit of detection is 2 microg Fe L(-1) (3s) and the sensitivity is similar to that of the batch procedure. Results obtained for the determination of iron in natural waters are also presented.     

Kinetic fluorimetric determination of organic peroxides and lipohydroperoxides at the nanomole level

A new kinetic fluorimetric method for determination of cumene hydroperoxide, tert-butyl hydroperoxide and lipohydroperoxides is reported. It is based on the manganese(II)-catalysed oxidation of 2-hydroxynaphthaldehyde thiosemicarbazone (HNTS) by a hydroperoxide. Measurements are made by the initial-rate method, which allows determination of as little as 0.1 nmole of peroxide and also permits establishment of the differences in reactivity between the hydroperoxides assayed. The method has been applied to the determination of lipohydroperoxides in six commercial oil samples (grape, corn, sunflower seed, cod-liver and linseed). The results obtained can be expressed as meq of peroxide per kg of oil, and are in close agreement with those obtained by the classical iodometric method.     

Fluorescence detection and identification of eight sulphonamides using capillary electrophoresis on released excipients in lake water

A simple and sensitive capillary electrophoresis method with fluorescence detection was developed for the determination of sulphanilamide, sulphamerazine, sulphacetamide and sulphanilic acid, sulphathiazole, Sulphisomidine, sulphadoxine and sulphadiazine in lake water. The sulphonamides were extracted from lake water, derivatized with fluorescamine and determination of sulphonamide was achieved using 20 mM borate buffer of pH 9.5 at an applied voltage of 25 kV. Detection was performed using UG-11 excitation filter of 405 nm and 495 nm emission filters. A fast, simple and sensitive method with limit of detection in the range 0.89–1.43 n mol L⁻¹ for all the eight sulphonamides with good recoveries of 80–110% is seen. Inter-day and intra-day validation of the separation method shows fairly good results. The detection and quantification limits for this newly developed method are too low to determine drug residues in lake water.     

Optimization and validation of a fast ultrahigh‐pressure liquid chromatographic method for simultaneous determination of selected sulphonamides in water samples using a fully porous sub‐2 μm column at elevated temperature

High temperature in HPLC is considered a valuable tool helping to overcome the increase in the column backpressure when using small packing particles such as sub‐2 μm, as it allows reduction in the mobile‐phase viscosity. In this study, a fast analytical method based on HPLC‐UV was developed using a sub‐2 μm column at elevated temperature for the simultaneous determination of nine sulphonamides. Owing to the lower viscosity of the mobile phase, the separation could be achieved in 3 min at 60°C for all analytes. The effect of temperature, the organic modifier percentage and the flow rate on the retention time was studied. The method developed was used for the determination of selected sulphonamides in surface and wastewater samples. Sample preparation was carried out by solid‐phase extraction on Oasis HLB cartridges. The method developed was validated based on the linearity, precision, accuracy, detection and quantification limits. The recovery ranged from 70.6 to 96 % with standard deviations not higher than 4.7%, except for sulphanilamide. Limits of detection ranged from 1 to 10 μg/L after optimization of all analytical steps. This method has the highest performance in terms of analytical speed compared with other published HPLC‐UV methods for the determination of sulphonamides in water.     

Derivative spectrophotometric determination of sulphonamides by the Bratton-Marshall reaction

A modified method for the determination of sulphonamides, based on the Bratton-Marshall reaction with derivative spectrophotometry, is proposed. Diverse sulphonamides are determined with excellent precision by using first- to fourth-order derivative spectra. The method was applied for the determination of sulphonamides in urine, honey and pharmaceutical formulations without pretreatment of the samples.     

Kinetic fluorimetric determination of carbamazepine in serum by the stopped-flow technique

Summary A simple, fast method for the determination of carbamazepine in serum is reported for the first time. It is based on the fluorescent reaction of this drug with cerium(IV) in an acid medium. A stopped-flow mixing module coupled to a conventional spectrofluorimeter is used for this purpose. The linear range of the proposed method is 0.04–140 g ml^–1 of carbamazepine and the detection limit is 0.01 g ml^–1. The within- and between-assay precision data and selectivity results show the method to be adequate for the determination of carbamazepine in serum by including a preliminary extraction step with dichloromethane. Analytical recoveries from different serum samples are reported.     

Kinetic spectrophotometric determination of certain cephalosporins using oxidized quercetin reagent

A simple, precise and accurate kinetic spectrophotometric method for determination of cefoperazone sodium, cefazolin sodium and ceftriaxone sodium in bulk and in pharmaceutical formulations has been developed. The method is based upon a kinetic investigation of the reaction of the drug with oxidized quercetin reagent at room temperature for a fixed time of 30 min. The decrease in absorbance after the addition of the drug was measured at 510 nm. The absorbance concentration plot was rectilinear over the range 80–400 μg mL⁻¹ for all studied drugs. The concentration of the studied drugs was calculated using the corresponding calibration equation for the fixed time method. The determination of the studied drugs by initial rate, variable time and rate-constant methods was feasible with the calibration equations obtained but the fixed time method has been found to be more applicable. The analytical performance of the method, in terms of accuracy and precision, was statistically validated; the results were satisfactory. The method has been successfully applied to the determination of the studied drugs in commercial pharmaceutical formulations. Statistical comparison of the results with a well established reported method showed excellent agreement and proved that there is no significant difference in the accuracy and precision.     

Indirect flow-injection determination of methadone by atomic absorption spectrometry

A simple continuous redox flow system coupled on-line with an atomic absorption spectrometer for the determination of methadone at the ng ml^?1 level is described. It is based on the reduction of the keto group of the analyte in a camium or zinc microcolumn and determination of the metal ions formed. The drug can be determined in the range 5–50 ng ml^?1 with a relative standard deviation between 1.8 and 2.5% at a sampling frequency of 150 h^?1. The selectivity of the method allows the analyte to be determined in the presence of other drugs.     

Kinetic photometric determination of copper at the nanograms per millilitre level in real samples

A highly sensitive and selective kinetic method is proposed for the determination of nanogram amounts of copper(II) based on its catalytic effect on the oxidation of 3-hydroxybenzaldehyde azine by potassium peroxydisulphase in an ammoniacal medium. The rate of the reaction is monitored spectrophotometrically at 465 nm and the method allows the determination of copper concentrations in the range 0.5–18 ng ml^?1 with a relative standard deviation of about 2%. The proposed method is virtually specific to copper and has been applied satisfactorily to its determination in a variety of food and industrial samples. A possible mechanism for the catalysed reaction is proposed.     

Kinetic enzymatic determination of ethanol/methanol mixtures by the stopped-flow technique

The analytical use of the enzymatic oxidation of alcohols by alcohol oxidase was assessed applying the stopped-flow technique for mixing sample and reagents, and the DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)/L-cysteine system as a new chromogenic reagent. The oxidation reaction was monitored by measuring the rate of absorbance decrease at 412 nm, the wavelength of maximum absorption of the reduced form of DTNB. The calibration graphs for the individual determination of the alcohols were linear over the range 1.0 x 10(-6)-1.0 x 10(-5)M, and the precision ranged between 2.1 and 4.8%. A differential rate principle was applied to the determination of ethanol/methanol mixtures involving mutual kinetic effects which allowed mixtures in molar ratios between 25:1.0 and 1.0:1.0 to be accurately resolved with good precision (r.s.d. less than 9 and 5%, respectively). Compared to the flow-injection analysis method, the proposed approach offers higher sensitivity and sample throughput, as well as the wider concentration ratio range for mixture resolution.