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.     

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.     

Determination of thiamine in pharmaceutical preparations by sequential injection renewable surface solid-phase spectrofluorometry.

Fluorometric determination of thiamine requires the conversion of the analyte to fluorescent thiochrome by hexacyanoferrate(III) oxidation in alkaline solution and the isolation of the produced thiochrome from the reaction medium by solvent extraction. It was observed that thiochrome could be concentrated and separated from the reaction medium by solid-phase extraction. The thiochrome sorpted on the surface of octadecyl-alklylated poly[styrene/divinylbenzene] (C18-PS/DP) microbeads emitted strong fluorescence upon excitation, the maximum excitation and emission wavelengths being 385 nm and 433 nm, respectively. Based on this observation, a sequential injection renewable surface solid-phase spectrofluorometry was developed for the determination of thiamine. A sequential injection system on-line coupled to a chip-based flow-through cell was employed to handle the chemical reaction, bead injection and discharging, and adsorption of thiochrome. Solid-phase fluorometric detection was realized by coupling the chip-based flow-through cell to a spectrofluorometer with a multistrand bifurcated optical fiber. Under the optimized condition, a detection limit of 0.03 microg ml(-1) was achieved at the sample throughput of 30 h(-1) and consumption of 1 mg C18-PS/DP microbeads for each run. Eleven runs of a 2 microg ml(-1) thiamine standard solution gave a relative standard deviation of 1.0%. The developed approach was successfully applied for the determination of thiamine contents in pharmaceutical preparations.     

Development and validation of a dried blood spot test for thiamine deficiency among infants by HPLC–fluorimetry

Thiamine deficiency, if detected early in infancy, can be treated with thiamine supplementation and can prevent seizures, other disabilities and death. The dried blood spot (DBS) sampling technique is an attractive sample collection technique for infants. The present study reports the development and validation of a highly sensitive and precise method for quantification of thiamine diphosphate from DBS. The method utilizes full‐spot analysis of a volumetrically deposited 40 μl DBS. The analyte was extracted from the DBS using 50% methanol and then derivatized using potassium ferricyanide to thiochrome. Separation was achieved with the help of an Inertsil ODS C₁₈ column (5.0 μm, 250 × 4.6 mm) using 150 mm phosphate buffer pH 7–acetonitrile (90:10, % v/v) as the mobile phase. The use of a fluorimetric detector gave a good response to the thiochrome derivative offering good sensitivity for the method. The excitation and emission wavelengths were 367 and 435 nm, respectively. The limit of detection and lower limit of quantification were 5 and 10 ng/ml, respectively. Linearity was demonstrated from 10 to 1000 ng/ml, and precision (CV) was <12.08%, at all tested quality control levels. The method accuracy was 89.34–118.89% with recoveries >80%. Bland–Altman analysis of DBS sampling vs. whole blood demonstrated a mean bias of only 1.16 ng/ml, with a majority of the 60 investigated patient samples lying within 7.2% of the corresponding concentration measured in blood, thereby meeting the clinical desirable biological specification criterion and showing that the two methods are comparable.     

Rapid determination of sulphonamides in milk using liquid chromatographic separation and fluorescamine derivatization.

A simple and selective method is presented for the multiple residue determination of eight sulphonamides in consumers' milk. The drugs are sulphisomidine (ID), sulphadiazine (DZ), sulphamerazine, sulphadimidine, sulphamonomethoxine, sulphamethoxazole, sulphadimethoxine and sulphaquinoxaline (SQ). The milk sample was deproteinized with the same volume of 2 M hydrochloric acid and filtered. A 1-ml volume of the filtrate was mixed with 1 ml each of 1.25 M sodium acetate solution and a buffer (pH 3.0) for derivatization with 0.6 ml of 0.02% fluorescamine solution in acetone. A high-performance liquid chromatographic analysis was carried out on a C18 column with a mobile phase of acetonitrile-2% acetic acid (3:5) at 55 degrees C using a fluorescence detector at an excitation wavelength of 405 nm and an emission wavelength of 495 nm. Average recoveries at fortification levels of 2, 5 and 10 ng/ml were 114%, 109% and 106%, respectively. Relative standard deviations were 1-4% at 10 ng/ml for ID, 5 ng/ml for DZ and SQ and 2.5 ng/ml for the other five sulphonamides. The method was applied to 25 milk samples and all appeared to be free from the drugs.     

Liquid chromatographic determination of less polar ginsenosides in processed ginseng

A novel method for the determination of iodide by size exclusion chromatography was established. The method was simple and highly sensitive with good precision. Iodide was converted to iodine, then sequestered with starch, and separated from the matrix using a Shim-pack DIOL-150 (250 x 7.9 mm) size exclusion column with methanol-0.01 mol l(-1) aqueous phosphoric acid (10:90, v/v) as mobile phase at 1.2 ml min(-1) and UV detection at 224 nm. The calibration graph was linear from 1.0 ng ml(-1) to 100.0 ng ml(-1) for iodide with a correlation coefficient of 0.9992 (n=6). The detection limit was 0.2 ng ml(-1). The method was successfully applied to the determination of iodide in seawater and urine. The recovery was from 92% to 103% and the relative standard deviation was in the range of 1.5% to 3.7%.     

Fluorimetric differential-kinetic determination of silicate and phosphate in waters by flow-injection analysis

A flow-injection analysis (FIA) method for simultaneous determination of silicate and phosphate, based on the different rates of formation of their molybdate heteropoly acids is suggested. The fluorimetrically monitored product is thiochrome, formed by oxidation of thiamine by the heteropoly acid. The FIA configurations designed allow performance of two measurements at different times on each sample injected. The method permits the determination of these anions in the range 30-600 ng ml in ratios from 1:10 to 10:1 and can be applied to samples of running and bottled water with good results. The sampling frequency achievable is 60 hr .     

Determination of pyridoxal 5'-phosphate in human serum by reversed phase high performance liquid chromatography combined with spectrofluorimetric detection of 4-pyridoxic acid 5'-phosphate as a derivative

A very sensitive spectrofluorimetric method for the determination of pyridoxal 5'-phosphate (PLP) in human serum is described. The specificity is based on the selective oxidation of PLP to 4-pyridoxic acid 5'-phosphate with potassium cyanide. Separation of the highly fluorescent 4-pyridoxic acid 5'-phosphate is achieved by reversed-phase high-performance liquid chromatography. Specificity is improved by a careful choice of fluorescence filters, maximised at the excitation (325 nm) and emission (418 nm) wavelengths of 4-pyridoxic acid 5'-phosphate. The detection limit for the reaction is 0.22 ng ml(-1). For quantification, the serum is spiked with PLP before protein precipitation with 3.3% m/V trichloroacetic acid. The method can be used for the determination of PLP in serum, even in vitamin B6 deficient patients. The mean value for human serum PLP from 30 healthy adults was found to be 14.6 +/- 4.8 ng ml(-1) (mean +/- standard deviation).     

Spectrofluorimetric method for determination of aluminium with chromotropic acid and its application to water samples

A rapid and sensitive method for the trace level determination of aluminium based on the formation of a 1:1 complex with chromotropic acid (1,8-dihydroxynaphthlene-3,6-disulfonic acid) in an methanol medium is reported. The fluorescence intensity of the system was 50 times greater than that of the system without aluminium. This method is very sensitive and selective for the direct determination of aluminium ion. The fluorescence is excited at 346 nm and measured at 370 nm. The optimum conditions are a chromotropic acid concentration of 5.0 ml (1.0x10(-4) M) and pH 4.0+/-0.5 (acetic acid-sodium acetate buffer). The fluorescence intensity is a linear function of the concentration of Al(III) in the range 2-100 ng ml(-1) and the detection limit is 1.0 ng ml(-1). The method has been applied successfully to the determination of trace amount of Al(III) in tap, river and sea-water samples.     

Spectrophotometric determination of osmium based on its catalytic effect on the oxidation of carminic acid by hydrogen peroxide

A highly sensitive spectrophotometric method is described for the determination of trace amounts of osmium(VIII), based on its catalytic effect on the oxidation of carminic acid by hydrogen peroxide. The reaction was monitored spectrophotometrically by measuring the decrease in absorbance of carminic acid at 540 nm after 3 min of mixing the reagents. The optimum reaction conditions were 1x10(-4) mol l(-1) carminic acid, 0.013 mol l(-1) hydrogen peroxide and pH 10 at 25 degrees C. By using the recommended procedure, the calibration graph was linear from 0.1 to 1.5 ng ml(-1) of osmium; the detection limit was 0.02 ng ml(-1); the RSD for five replicate determinations of 0.2-1.4 ng ml(-1) was in the range of 1.8-4.7%. The influence of several foreign ions on osmium determination were studied and the effect of interfering ions were removed by extracting osmium into isobuthyl methyl ketone and back extracting into sodium hydroxide solution.     

Simultaneous determination of carbaryl and azinphos-methyl in water by first-derivative synchronous spectrofluorimetry

A method is proposed for the simultaneous determination of the pesticides carbaryl (CBL) and azinphos-methyl (AZM) in water by first-derivative synchronous spectrofluorimetry. It is based on the alkaline hydrolysis of both pesticides to their metabolites 1-naphthol (from CBL) and anthranilic acid (from AZM). The constant wavelength difference chosen to optimize the determination is Δλ=λ_em?λ_ex=103 nm. CBL is measured at 302/405 nm and AZM at 333/436 nm. The calibration graphs are linear between 2.0 and 500.0 ng/ml for CBL and between 1.2 and 500.0 ng/ml for AZM with detection limits of 0.62 ng/ml and 0.35 ng/ml, respectively. The precision of the method (RSD) is 2.4% at the 80.0 ng/ml level for CBL and 2.5% at the 80.0 ng/ml level for AZM. The method is applied to the determination of both analytes in samples of natural waters.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition.

A high-performance liquid chromatographic method for the measurement of bumetanide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C18 column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C18 Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol-water-glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5-2000 ng/ml.     

Resonance Rayleigh-scattering method for the determination of vitamin B1 with methyl orange.

In a pH 2.3-3.0 acid medium, the resonance Rayleigh scattering (RRS) intensity is greatly enhanced when vitamin B, reacts with Methyl Orange to form an ion-association complex. The maximum RRS peak appears at 588 nm, another higher peak is at 403 nm and there are two smaller RRS peaks at 800 nm and 288 nm. The RRS intensity is directly proportional to the concentration of vitamin B, in the range of 0-400 ng ml(-1). The method has high sensitivity and the detection limit (3sigma) for vitamin B1 is 7.2 ng ml(-1). The effects of coexisting substances on the determination of vitamin B, were investigated, and the results show that this method has good selectivity and can be applied to the direct determination of vitamin B1 in composite vitamin B tablets and multivitamin tablet samples.     

Solvent extraction-spectrophotometric determination of phosphate with molybdate and malachite green in river water and sea-water

Molybdophosphate, formed between orthophosphate and molybdate in sulphuric acid solution, is extracted into a mixture of toluene and 4-methylpentan-2-one (1:3 v v ) with Malachite Green as counter-ion. A single extraction with equal phase volumes gives an apparent molar absorptivity for phosphate of 2.3 x 10(5) l.mole(-1).cm(-1) at 630 nm; the absorbance of the reagent blank is 0.03. With an organic to aqueous phase-volume ratio of 1:10, the molar absorptivity is 2.5 x 10(5) l.mole(-1).cm(-1) and the absorbance of the reagent blank 0.08. By the proposed method, ng ml levels of phosphorus can be determined, and the detection limit is about 0.1 ng ml . The standard deviation and relative standard deviation for the determination of phosphorus in tap water (4.3 ng ml ) are 0.05 ng ml and 1.1%, respectively. The method can also be applied to the determination of phosphorus in river water and sea-water.     

Low-level mercury determination with thiamine by fluorescence optosensing

A sensitive fluorescence optosensing method for the determination of Hg(II) in water samples is described. The method, using a flow injection technique, is based on the immobilization on a non-ionic-exchanger solid support (packed in a flow cell placed in a conventional fluorimeter) of the thiochrome formed by the oxidation of thiamine with Hg(II) in a continuous flow carrier at pH 8.1. Experimental parameters such as the solid support, the carrier pH, the thiamine concentration and the flow-rate were investigated to select the optimum operating conditions. The proposed optosensor showed a relative standard deviation of + 3.0% for ten replicates analysis of 100 ng ml(-1) of mercury(II). A detection limit of 3 ng ml(-1) for mercury(II) was achieved for 4-ml sample injections. A detailed study of interferences (possible elements present in natural waters) demonstrated that this optosensing method is virtually specific for this metal, because it allows the determination of mercury in the presence of relatively large amounts of other heavy metals and compounds present in natural waters, such as Mg(II) or Ca(II). The method was successfully applied to the determination of Hg(II) in spiked samples of mineral, tap and sea water.     

Determination of sialic acids in human serum by reversed-phase liquid chromatography with fluorimetric detection.

A simple, rapid and highly sensitive reversed-phase liquid chromatographic method has been developed for the determination of sialic acids in human serum. The sialic acids, released by hydrolysis of serum, are converted in borate buffer with malononitrile to highly fluorescent compounds. The reaction mixture is separated isocratically within 5 min using an octadecyl-bonded silica column and a mobile phase of methanol and ammonium acetate buffer (15:85, v/v; pH 5.5). Measurement of the fluorescence intensity of the reaction mixture at 434 nm with irradiation at 357 nm allowed determination of 30-1000 ng/ml of sialic acids with high reproducibility. The limit of detection was 2 ng/ml. Intra-day and inter-day coefficients of variation for assaying 300 ng/ml N-acetylneuraminic acid (NANA) were 1.5% (n = 9) and 2.6% (n = 7), respectively. The recoveries of NANA were 98.5-101.1% for serum. The method has been used for clinical determinations.     

Spectrophotometric determination of manganese by using redox reaction of tris(2,2'-bipyridine)osmium(II) with Mn7+.

This paper reports on the analytical application of the oxidation reaction of [Os(bpy)3]2+ by Mn7+ (MnO4-). The present study developed a very simple, sensitive and selective spectrophotometric method for the determination of manganese in an acidic medium. Three analytical wavelengths were employed in the UV and visible regions at 290, 315 and 480 nm. Beer's law was obeyed up to a concentration of 330 ng ml(-1) of Mn7+ at different wavelengths with regression equations 0.001 - 0.0042C(Mn), 0.001 + 0.0021C(Mn), and 0.001 - 9.34 x 10(-4)C(Mn) at 290, 315 and 480 nm, respectively. Under the optimum conditions, the achieved detection limits were 0.72, 1.37 and 3.32 ng ml(-1) at 290, 315, and 480 nm, respectively. In addition to the high sensitivity of the method (0.24 ng cm(-2) at 290 nm, 0.45 ng cm(-2) at 315 nm and 1.0 ng cm(-2) at 480 nm), it can be used for the determination of manganese in the presence of a large number of anions and cations, since it tolerates most of the potential interferents. The relative standard deviation was 0.5% (n = 11) for 90 ng ml(-1) manganese. The method was successfully applied to the determination of manganese in water from different resources.     

Fast determination of ochratoxin A in serum by liquid chromatography: comparison with enzymic spectrofluorimetric method.

A rapid high-performance liquid chromatographic method for the determination of low concentrations of ochratoxin A in serum is described. The extraction procedure was simple and short, and liquid chromatographic analysis was carried out isocratically on a reversed-phase C18 column, with methanol-water-acetic acid (30:70:1) as mobile phase and fluorescence detection (excitation at 336 nm, emission at 465 nm). The examined concentration range, 5-50 ng/ml ochratoxin, the recovery method was 87-94%, compared with 62-67% for the enzymic spectrofluorimetric method. The high-performance liquid chromatographic method was faster because the extraction procedure was shorter, and more sensitive so that small sample volumes could be used.     

Bioanalysis of diclofenac as its fluorescent carbazole acetic acid derivative by a post-column photoderivatization high-performance liquid chromatographic method

A sensitive and selective bioanalytical liquid chromatographic method for diclofenac is described. The drug was detected as a flourescent derivative, which was demonstrated by 1H NMR and mass spectrometric studies to be carbazole acetic acid. Diclofenac was derivatized by UV irradiation of the substance performed as a post-column photoreaction. The reactor was a PTFE capillary wound around a 254-nm UV lamp. Diclofenac was isolated from the plasma samples by precipitation of the proteins with acetonitrile. A 50-microliters volume of the supernatant was injected onto a Nucleosil C18 column. The mobile phase was 32% acetonitrile in pH 6.6 buffer. Carbazole acetic acid was detected by a fluorescence detector using an excitation wavelength of 288 nm and an emission wavelength of 360 nm. The recovery was 92%, the standard curve was linear in the range 10-5500 ng diclofenac per ml plasma, and the relative standard deviation at 10 and 5000 ng of diclofenac per ml plasma was 9.0% and 3.3%, respectively. The limit of detection was 6 ng/ml at an injection volume of 50 microliters. Chromatograms of human and rat plasma containing diclofenac are shown.     

FI-on line photochemical reaction for direct chemiluminescence determination of photodegradated chloramphenicol

A new, simple, clean and selective flow injection strategy based on the tandem photochemical reaction-chemiluminescence detection was applied to the determination of chloramphenicol. The determination is based on the on-line photodegradation of the drug in a glycine buffer at pH 8.8 by using a photoreactor consisting of 697 cmx0.5 mm PTFE tubing helically coiled around an 8 W low-pressure mercury lamp. Photodegradated chloramphenicol is detected by direct chemiluminescence of resulting photo-fragments and their subsequent reaction with potassium permanganate in sulfuric acid medium as oxidant. The method allows the chemiluminescence determination of compounds which do not exhibit native chemiluminescence. The calibration graph was linear up to 14 mug ml(-1) chloramphenicol, the limit of detection was 30 ng ml(-1), the relative standard deviation was 2.4% for 7 mug ml(-1) of the drug and the sample throughput was 60 h(-1). Taking into account the importance of the medium of photodegradation on the mechanism of photodegradation a comparative study in terms of selective was performed for different chemical media employed in the procedure of photodegradation. The proposed method was applied to the determination of chloramphenicol in commercially available pharmaceutical formulations.