Fluorimetric determination of arsanilic acid by flow-injection analysis using on-line photo-oxidation

A flow-injection-fluorimetric method for the determination of arsanilic acid is proposed. The assay is based on the on-line decomposition of arsanilic acid in the presence of peroxydisulfate on irradiation with UV light. The arsenate generated in the photochemical reaction was reacted with molybdate in dilute nitric acid to form arsenomolybdic acid, which oxidised thiamine to thiochrome. The thiochrome was monitored fluorimetrically at 440 nm with excitation at 375 nm. The calibration graph was linear in the range 0.10-10.8 microg mL(-1) with a correlation coefficient of 0.999. The detection limit was 0.01 microg mL(-1) and the sample throughput was 55 samples h(-1). The applicability of the method was demonstrated by determining arsanilic acid in animal foodstuffs and water.     

Use of a continuous flow solid-phase spectroscopic sensor using two sensing zones: determination of thiamine and ascorbic acid

A simple, rapid, inexpensive, and automated flow-through solid-phase spectroscopic sensing device is proposed for the sequential determination of 2 vitamins: thiamine and ascorbic acid. The vitamins are concentrated on ion-exchange gels, thiamine on Sephadex SP C-25, and ascorbic acid on Sephadex QAE A-25; both solid supports are packed in 2 different flow cells. The absorbance is monitored directly on the solid phase with a double-beam spectrophotometer at 250 nm, without derivatization or additional elution. With the use of 2 carrier/self-eluting solutions (0.1 5M sodium acetate/acetic acid and 0.18M citric acid/K2HPO4) and a sample volume of 1000 microL, the sensor responds linearly in the range of 0.5-15 and 3-50 microg/mL with detection limits of 0.14 and 0.36 microg/mL for thiamine and ascorbic acid, respectively. When the method was applied to synthetic samples and pharmaceutical preparations, precise and accurate values were obtained.     

Flow injection determination of methamidophos using online photo-oxidation and fluorimetric detection

A photochemical method for the determination of methamidophos using a flow injection system is proposed. The method is based on the rapid decomposition of methamidophos in the presence of peroxydisulphate upon irradiation with UV light. The phosphate generated in the photochemical process is made to react with molybdate in dilute nitric acid to form phosphomolybdic acid, which oxidises thiamine to thiochrome. The thiochrome can then be fluorimetrically monitored at 440 nm with excitation at 375 nm. The method shows a linear range between 14 and 1400 ng ml(-1) with a limit of detection of 1.7 ng ml(-1). The repeatability was 0.3% expressed as relative standard deviation (n=10) and the reproducibility, studied on five different days, was +/-3%. The sample throughput was 70 injections per h. The reliability of the method for routine analysis of water and vegetable samples is demonstrated.     

停流—荧光反应速率仪及其应用于测定维生素B1

本文利用国产930型荧光光度计作为检测器,采用自已制作的流通池,配以蠕动泵及记录仪,组装成停流-荧光动力学分析装置,并用以进行了停流荧光反应速率法测定维生素B_1的研究,利用Hg~(2+)离子在碱性介质中将硫胺素(维生素B_1)氧化为硫胺荧的动力学反应,测定了维生素B_1片剂中维生素B_1的含量。结果表明,这套仪器用于动力学分析,操作简便、省时,维生素B_1的测定结果与标准值基本相符,相对标准偏差为1.28％,其准确度和重现性均令人满意。     

Studies on the distribution and concentration of thiamine in blood and urine

Studies are reported resulting in a reliable procedure for estimating the thiamine content in human blood and urine. For the determination in blood, heparinized blood is hemolyzed with 0.3 N hydrochloric acid at 100 °C. Cocarboxylase is then converted to free thiamine by means of wheat germ acid phosphatase at pH 5.0 in an acetate buffer. The liberated thiamine is adsorbed to a CG-50 (Rohm & Haas) carboxylic acid ion exchange acrylic resin column and then eluted with 1 N H₂SO₄. The thiamine is then oxidized to thiochrome and extracted with n-butyl alcohol, at pH 9.8–10.0, in the presence of disodium phosphate. Readout is by fluorometry at an excitation wavelength of 371 nm and an emission wavelength of 425 nm. The range found for thiamine in whole blood by this procedure on 18 normal adults was 1.9–3.9 μg/100 ml, with a mean value of 2.77 μg/100 ml of whole blood. The mean recovery of 12 recovery experiments was 94.1%. The same procedure is applicable to the determination of thiamine in urine. Conversion of cocarboxylase to free thiamine is not necessary since it was determined that practically all of the thiamine found in urine is not phosphorylated. Urine values were variable, the range for 11 healthy adults being 5.6–77.9 μg/100 ml with a mean value of 19.2 μg/100 ml. This corresponds to a value of 346 μg of thiamine/24 hours.     

Dispersive liquid-liquid microextraction followed by spectrofluorimetry as a simple and accurate technique for determination of thiamine (vitamin B1)

Dispersive liquid-liquid microextraction (DLLME) combined with spectrofluorimetry was applied to the extraction, pre-concentration and analysis of thiamine (vitamin B₁). The procedure is based on (a) the oxidation of thiamine with ferricyanide to form fluorescent thiochrome (TC), (b) the trapping of TC into a microextraction solvent, and (c) spectrofluorometric determination. Microextraction solvent and disperser solvent are directly injected into an aqueous solution containing TC. After centrifuging, phase separation is performed by sedimenting the fine droplets of the microextraction solvent on the bottom of a test tube. The settled phase is transferred into a fluorometer for the determination of thiamine at excitation/emission wavelengths of 375/438 nm. Under the optimized experimental conditions, the method provides a linear dynamic range of 0.2–100 ng mL^?1, a detection limit of 0.06 ng mL^?1, and a relative standard deviation of 3.0%. The method was successfully applied to pharmaceutical formulations and human urine. The results were validated by recovery test and by comparison with other methods, and were found to be highly satisfactory.     

Native fluorescence flow-through optosensor for the fast determination of diphenhydramine in pharmaceuticals.

A single, rapid flow-through optosensor spectrofluorometric system is proposed for the determination of diphenhydramine in different pharmaceutical preparations. This sensor was developed in conjunction with a monochannel flow-injection analysis system with fluorometric solid-phase transduction. Diphenhydramine was directly injected into a carrier stream of ethanol/water, 50% (v:v), and transitorily retained on a sorption gel Sephadex G-15 placed in the detection area into the cell. The determination was carried out without any derivatization reaction by directly measuring the intrinsic fluorescence of the analyte and using the peak height as an analytical signal. The chemical and instrumental variables were optimized, and the influence of some foreign substances that can be found in typical pharmaceutical samples containing diphenhydramine was also investigated. Diphenhydramine could be determined in the concentration ranges of 0.5 - 8 microg ml(-1) and 0.1 - 1.2 microg ml(-1) with detection limits of 0.088 and 0.019 microg ml(-1) at sampling rates of 30 and 19 h(-1) for 200 and 800 microl of the sample volume, respectively.     

Application of an intensified diode array system to diagnosis of matrix effects and the rapid sequential determination of thiamine and riboflavin with fluorescence detection

The unique capabilities of a multiple wavelength spectrofluorometer based on an intensified diode array detector are used for diagnosis of matrix effects and for rapid sequential determination of two analytes. This system is used to investigate problems due to background fluorescence, blank reactions, and scattering for the determination of thiamine in cereal and urine samples by a fluorometric kinetic procedure. A novel determination of thiamine and riboflavin in vitamin pills is based on monitoring the native fluorescence of riboflavin over one wavelength region, and the rate of formation of fluorescent thiochrome from thiamine over another wavelength region, after a computer-controlled change in the pH of the reaction mixture.     

Determination of thiamine by high-performance liquid chromatography

High-performance liquid chromatographic methods for the determination of thiamine (vitamin B1) in foodstuffs or biological tissues and fluids are outlined and discussed. The methods are often similar and interchangeable, sample extraction and clean up procedures being the major difference. Most of the methods use either ultraviolet or fluorescence detection. Fluorescence detection requires either precolumn or postcolumn oxidation of thiamine to thiochrome. A number of methods are recommended and problems with standardization are emphasized.     

Flow injection fluorimetric determination of thiamine and copper based on the formation of thiochrome

The reaction, involving the oxidation of thiamine by copper(II) in basic solutions to fluorescent thiochrome, has been adapted to the determination of thiamine by flow-injection analysis. Linear calibration graphs are obtained between 0.30 and 6.02 mug/ml with a sampling rate of 50 samples/hr and a relative standard deviation of 0.53%. This reaction has also been adapted to the determination of copper(II) over the range 0.5-5.0 mug/ml. The applicability of both methods for determination of thiamine and copper is demonstrated by investigating the effect of potential interferences and by the analysis of real samples (pharmaceuticals for thiamine and ores and alloys for copper).     

Flow-through optical fiber sensor for automatic sulfide determination in waters by multisyringe flow injection analysis using solid-phase reflectometry

A software-controlled flow-through optical fiber diffuse reflectance sensor capitalized on the implementation of disk-based solid-phase pre-concentration schemes in a multisyringe flow injection analysis (MSFIA) set-up is proposed for the trace determination of sulfide in environmental waters and wastewaters. The fully automated flowing methodology is based on Fischer's coupling reaction of sulfide with N,N-dimethyl-p-phenylenediamine (DMPD) in the presence of Fe(iii) as oxidizing reagent in a 0.5 M HCl medium. The on-line generated methylene blue dye is subsequently delivered downstream to a dedicated optode cell furnished with an octadecyl-chemically modified (C(18)) disk, while continuously recording the diffuse reflectance spectrum of the pre-concentrated compound. A double regeneration protocol is finally executed to warrant minimum background noise and negligible baseline. Under the optimized chemical and hydrodynamic conditions, the optosensing MSFIA method features coefficients of variation better than 0.7%(n= 10) at 50 microg l(-1) concentration, a linear working range of 20-200 microg l(-1) sulfide, a 3sigma(blank) detection limit of 2.9 microg l(-1) sulfide and an injection throughput of 8 h(-1) for a pre-concentration sample volume of 2.9 ml. The interfacing of the robust and versatile multisyringe flow injection-based optode with a plug-in spectrophotometer furnished with a light emitting diode assures the miniaturization of the overall flow analyzer, which is, thus, readily adaptable to real-time monitoring schemes. The potential of the multisyringe flow method was assessed via the determination of sulfide traces in water samples of different complexity (namely, freshwater, seawater and wastewater).     

Liquid chromatographic analysis of propranolol enantiomers in human blood using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate.

A method for the determination of the R-(+) and S-(-) enantiomers of propranolol in blood was developed. After extraction with heptane-isopentanol and derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate, excess reagent was removed using solid-phase extraction. The enantiomers were separated on an achiral, reversed-phase, radially compressed column, and detected by fluorescence with excitation and emission wavelengths of 260 and 340 nm, respectively. The limit of quantification was 0.5 ng/ml. This method was used for pharmacokinetic analysis of propranolol enantiomers after administration of immediate-release (80 mg) or sustained-release (160 mg) racemic propranolol.     

Determination of salbutamol using on-line solid-phase extraction and sequential injection analysis. Comparison of chemiluminescence and fluorescence detection

Determination of salbutamol using sequential injection analysis (SIA) with chemiluminescence and fluorescence detection has been devised. The chemiluminescence signal was emitted during the oxidation of salbutamol by potassium permanganate in sulfuric acid medium. Sodium polyphosphate was used as chemiluminescence enhancer. The fluorescence signal (excitation wavelength 230 nm) was also measured in sulfuric acid medium. Both detection techniques were compared with respect to the application of the methods to the determination of salbutamol in biological materials. The sample pre-treatment takes place directly in the SIA system, when salbutamol is adsorbed on the solid-phase (Baker-carboxylic acid) microcolumn integrated into the system. Sulfuric acid serves both as the reagent and the eluent. The lab-made SIA system consisted of a 2.5-mL Cavro syringe pump, ten-port Vici Valco selection valve and Spectra-Physics FS 970 fluorescence detector, which was lab-modified for chemiluminescence detection. The system was controlled by a PC using originally compiled LabVIEW-supported software. Concentrations, volumes of reagents and flow rates were optimised by a simplex method. Salbutamol was determined in the linear range 0.05-10 microg mL(-1) (RSD 1.53%), with the detection limit (3 sigma) 0.03 microg mL(-1) and sample throughput of 42 samples per hour with chemiluminescence detection in standard solutions. The fluorescence detection enabled the determination of salbutamol in standard solutions in the linear range 0.5-100 microg mL(-1) (RSD 2.69%), with the detection limit 0.2 microg mL(-1) and sample throughput of 24 h(-1). The proposed methods were applied to the determination of salbutamol in human serum and urine. However, serum is a very complicated matrix and the SIA-SPE analysis did not provide satisfactory results. It was possible to determine salbutamol in human urine using this technique. Better recovery was achieved with fluorescence detection.     

Determination of sulphonate dyes in water by ion-interaction high-performance liquid chromatography

An ion-interaction high-performance liquid chromatography method for quick separation and determination of the sulphonated dyeAcid Yellow 1, and the sulphonated azo dyes Acid Orange 7, Acid Orange 12, Acid Orange 52, Acid Red 2, Acid Red 26, Acid Red 27 and Acid Red 88 has been developed. An RP-ODS stationary phase is used, and the mobile phase contains an acetonitrile-phosphate buffer (27:73, v/v) mixture at pH 6.7, containing 2.4 mM butylamine as ion-interaction reagent. Good separations were obtained using isocratic elution and spectrophotometric detection at 460 nm. The detection limits for the eight dyes ranged from 7 to 28 microg/l for an injection volume of 100 microl. Spiked tap water samples (100 ml), containing different concentration levels (0.3-1.2 microg/l) of the dyes were analyzed after acidification (pH 3) and preconcentration in disposable solid-phase extraction C18 cartridges.     

High-performance liquid chromatographic assay of phosphate and organophosphorus pesticides using a post-column photochemical reaction and fluorimetric detection

A sensitive method for the post-column reaction detection of organophosphorus pesticides is described. The method relies on photolysis of the organocompounds by irradiation with a low-pressure mercury lamp (main spectral line, 254 nm) in the presence of peroxydisulfate. The resultant orthophosphate was reacted with molybdate to form molybdophosphoric acid, which subsequently reacted with thiamine to generate thiochrome. Finally, the fluorescence intensity of thiochrome was measured at 440 nm with excitation at 375 nm. Factors affecting the rate of these reactions were optimized so that its contribution to the total band-broadening was negligible.

This detection system was used for the determination of phosphate, acephate and methamidophos, which were separated on an ODS column by isocratic reversed phase chromatography with acetonitrile–water as the mobile phase. A linear relationship between analyte concentration and peak area was obtained within the range 0.016–7.0 μg ml⁻¹ with correlation coefficients greater than 0.9995 and detection limits between 4 and 12 ng ml⁻¹. Intra- and inter-day precision values of about 1.2% R.S.D. (n = 10) and 2.1% R.S.D. (n = 30), respectively, were obtained.

Pesticide residues below ng ml⁻¹ levels could be determined in environmental waters when a preconcentration device was coupled on-line with the HPLC system. Detection limits as low as 0.01 ng ml⁻¹ were achieved for only 250 ml of sample. In the analyses of vegetables and grains, the detection limit was about 1 μg kg⁻¹.     

Flow injection turbidimetric determination of thiamine in pharmaceutical formulations using silicotungstic acid as precipitant reagent

A simple flow injection system is proposed for the determination of thiamine in pharmaceutical formulations. The determination is based on the precipitation reaction of thiamine with silicotungstic acid in acidic medium to form a thiamine silicotungstate suspension that is measured at 420 nm. Adding 0.05% (w/v) poly(ethyleneglycol) in the carrier solution (0.5 mol l(-1) hydrochloric acid), an improvement in the sensitivity, repeatability and baseline stability of the flow injection system was obtained. The calibration graph was linear in the thiamine concentration range from 5.0x10(-5) to 3.0x10(-4) mol l(-1) with a detection limit of 1.0x10(-5) mol l(-1). The relative standard deviations for ten successive measurements of 1.0x10(-4) mol l(-1) and 2.5x10(-4) mol l(-1) thiamine were less than 1% and an analytical frequency of 90 h(-1) was obtained.     

Flow injection determination of formaldehyde by its catalytic effect on the oxidation of sulfonazo III by bromate with spectrophotometric detection.

A simple and sensitive flow injection method with spectrophotometric detection was developed for the determination of formaldehyde. The method is based on the catalytic effect of formaldehyde on the oxidation of sulfonazo III with bromate in acidic media. The decrease in absorbance of the reaction mixture was measured at 566 nm. The calibration graph was linear in the range of 0.005 to 2.80 microg ml(-1) formaldehyde at a rate of 38 +/- 4 samples h(-1). The limit of detection was 4 ng ml(-1). The relative standard deviations for ten replicate measurements of 0.20, 0.50 and 1.00 microg ml(-1) formaldehyde were 1.3, 0.8 and 0.7%, respectively. The method was applied to the determination of formaldehyde in river water, shampoo and melamine-formaldehyde resin.     

Spectrophotometric, spectrofluorometric and HPLC determination of desloratadine in dosage forms and human plasma

Four sensitive, simple and specific methods were developed for the determination of desloratadine (DSL), a new antihistaminic drug in pharmaceutical preparations and biological fluids. Methods I and II are based on coupling DSL with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in borate buffer of pH 7.6 where a yellow colored reaction product was obtained and measured spectrophotometrically at 485 nm (Method I). The same product could be measured spectrofluorometrically at 538 nm after excitation at 480 nm (Method II). Methods III and IV, on the other hand, involved derivatization of DSL with 2,4-dinitrofluorobenzene (DNFB) in borate buffer of pH 9.0 producing a yellow colored product that absorbs maximally at 375 nm (Method III). The same derivative was determined after separation adopting HPLC (Method IV). The separation was performed on a column packed with cyanopropyl bonded stationary phase equilibrated with a mobile phase composed of acetonitrile-water (60 : 40, v/v) at a flow rate of 1.0 ml min(-1) with UV detection at 375 nm. The calibration curves were linear over the concentration ranges of 0.5-6, 0.02-0.4, 1-10 and 1-30 microg ml(-1) for Methods I, II, III and IV, respectively. The lower detection limits (LOD) were 0.112, 0.004, 0.172 and 0.290 microg ml(-1), respectively, for the four methods. The limits of quantification (LOQ) were 0.340, 0.012, 0.522 and 0.890 microg ml(-1) for Methods I, II, III and IV, respectively. The proposed methods were applied to the determination of desloratadine in its tablets and the results were in agreement with those obtained using a reference method. Furthermore, the spectrofluorometric method (Method II) was extended to the in-vitro determination of the drug in spiked human plasma, with a mean percentage recovery (n=4) of 99.7+/-3.54. Interference arising from endogenous amino acids has been overcome using solid phase extraction. The proposed methods are highly specific for determination of DSL in the presence of the parent drug loratadine. A proposal for the reaction pathways is postulated.     

Spectrophotometric determination of urea in dermatologic formulations and cosmetics

A rapid, relatively sensitive, and low-cost method for the determination of water-soluble urea content in dermatological therapy products and cosmetics is proposed using a new spectrophotometric assay with water as the only extraction solvent. Spectrophotometric methods involve addition of a known excess of bromate to urea in an acid medium, followed by the determination of residual bromine and chlorine reacting with methyl orange and measurement of absorbance at 505 nm. The absorbance increases linearly with urea concentration (r = 0.9998). The systems obey Beer's law for 6 - 90 microg ml(-1). The calculated apparent molar absorbance values are found to be 4.537 x 10(3) dm(3) mol(-1) cm(-1) and the Sandell's sensitivity is 0.013 microg cm(-2). The variables affecting the rate of the reaction were investigated. The relative standard deviation for five-replication determination of 60 microg ml(-1) urea was 2.1% and the detection limit of the method is 0.34 ng ml(-1).     

A Novel Spectrofluorimetric Method for the Determination of Thiamine with Iron(Iii)-Tetrasulfonato-Phthalocyanine as a Catalyst

Spectrofluorimetric determination of thiamine by means of the so-called thiochrome (TC) method remains an interesting area for analytical chemists.