Simultaneous determination of thiamine and its phosphate esters by a liquid chromatographic method based on post-column photolysis and chemiluminescence detection.

A sensitive method for the post-column detection of thiamine (T) and its phosphate esters is described. The procedure is based on the on-line photolysis of the analytes into photoproducts which have a strong enhancing effect on the CL permanganate-luminol reaction. The complete separation of the thiamines was obtained on a RP-amide C(16) column in isocratic elution with an analysis time of less than 7 min. Under the optimum conditions, analytical curves, based on standard solutions, were linear over the range 10-1000 nM for thiamine and 100-2000 nM for its mono- and di-phosphate esters. Intra- and inter-day precision values of less than 1.14% relative standard deviation (RSD) (n=10) and 1.86% RSD (n=15), respectively, were obtained. The method was successfully applied to the determination of the thiamines in pharmaceutical preparations and baby foods.

A sensitive post-column photochemical derivatization/fluorimetric detection system for HPLC determination of bisphosphonates.

A new reversed-phase ion-pair high-performance liquid chromatographic (HPLC) method has been developed for the determination of the following bisphosphonic acids: alendronic acid (ALEN), etidronic acid (ETID), ibandronic acid (IBAN) and risedronic acid (RISE). Separation was achieved on a C(18) column using a mixture of 50 mmol L(-1) borate buffer pH 9.0 containing 0.25 mmol L(-1) tetrabutylammonium chloride and 0.5 mmol L(-1) EDTA and acetonitrile (97:3) as the mobile phase. The sensitive detection of the above bisphosphonic acids was based on their oxidation to orthophosphate by the on-line peroxydisulfate-assisted photolysis followed by post-column reaction with molybdate to yield phosphomolybdate. This subsequently reacted with thiamine to generate thiochrome and, finally, the fluorescence of thiochrome was measured at 440 nm with excitation at 375 nm. The developed method is precise with a mean relative standard deviation of 1.3%, sensitive (with a detection limit at the nmol L(-1) level), accurate, specific, rapid (analysis time approximately 13 min) and inexpensive because to the low cost of the reagents. The assay was applied to the analysis of the four bisphosphonic acids in commercial dosage formulations, in which the excipients did not interfere with the determination. The method was also applied to the determination of etidronate, risedronate and ibandronate in human urine. Sample preparation involves precipitation of the analytes from urine along with endogenous phosphates such as calcium salts by addition of calcium chloride at alkaline pH and dissolution of the precipitate in 0.05 mol L(-1) ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid.

Simultaneous determination of amiodarone and its metabolite desethylamiodarone by high-performance liquid chromatography with chemiluminescent detection.

A novel method was developed for the determination of amiodarone and desethylamiodarone by high-performance liquid chromatography (HPLC) coupled with chemiluminescent (CL) detection. The procedure is based on the post-column photolysis of the analytes into photoproducts which are active in the tris(2,2'-bipyridyl)ruthenium(III) [Ru(bpy)(3)(3+)] CL system. Ru(bpy)(3)(3+) was on-line generated by photo-oxidation of the Ru(II) complex in the presence of peroxydisulfate. The separation was carried out on a Mediterranea C(18) column with isocratic elution using a mixture of methanol and 0.017 mol L(-1) ammonium sulfate buffer of pH 6.8. Under the optimum conditions, analytical curves, based on standard solutions, were linear over the range 0.1-50 microg mL(-1) for amiodarone and 0.5-25 microg mL(-1) for desethylamiodarone. The detection limits of amiodarone and desethylamiodarone were 0.02 and 0.11 microg mL(-1), respectively. Intra- and inter-day precision values of 0.9% relative standard deviation (R.S.D.) (n=10) and 1.6% R.S.D. (n=15), respectively, were obtained. The method was applied successfully to the determination of these compounds in serum and pharmaceutical formulations.

Chemiluminescent determination of EDTA and related compounds using tris(2,2'-bipyridyl)ruthenium(III) photogenerated on-line.

A rapid and sensitive chemiluminescence method using flow-injection has been developed for the determination of EDTA. The method is based on the chemiluminescent reaction of EDTA with tris(2,2'-bipyridyl)ruthenium(III), which is generated on-line through the photooxidation of tris(2,2'-bipyridyl)ruthenium(II) with peroxydisulfate. After optimizing the different experimental parameters, a calibration graph was obtained over the concentration range of 7 x 10(-8) M to 3 x 10(-6) M, with a minimum detectability of 7.2 x 10(-9) M (S/N = 3) observed. The correlation coefficient was 0.9992 (n = 8). The repeatability was 0.88 % (for a level of 4 x 10(-7) M) expressed as the relative standard deviation (n = 10), and the reproducibility (studied on five consecutive days) was 1.5%. The method was successfully applied to the determination of EDTA in ophthalmic collyrium and sauce samples. The method is also useful for determining other aminopolycarboxylic acids, such as NTA, EGTA, DTPA, DCTA and EDDS.

High-performance liquid chromatography-post-column chemiluminescence determination of aminopolycarboxylic acids at low concentration levels using tris(2,2'-bipyridyl)ruthenium(III).

A simple, selective and sensitive method for the determination of aminopolycarboxylic acids [diethylenetriaminepentaacetic acid (DTPA), S,S-ethylenediamine N,N'-disuccinic acid (EDDS), ethylenediaminetetraacetate (EDTA) and nitrilotriacetate (NTA)] has been developed using high-performance liquid chromatography with chemiluminescence detection. The aminopolycarboxylic acids were separated on a C18 reversed-phase column with an aqueous sulfuric acid mobile phase at a pH lower than 1.5. The eluate was mixed with tris(2,2'-bipyridyl)ruthenium(III), which was on-line generated by photooxidation of the ruthenium(II) complex in the presence of peroxydisulfate, and the generated chemiluminescence was detected. Calibration graphs, based on standard solutions, were linear over the range 8x10(-9) to 4x10(-5) M. The detection limits at a signal-to-noise ratio of 3 ranged from 9x10(-10) to 8x10(-8) M. The relative standard deviations of intra- and inter-day precision were below 1.3% and 2.1%, respectively. This HPLC system was successfully applied to the determination of aminopolycarboxylic acids in three different types of water samples. The low pH of the mobile phase limits interference from metal ions in natural waters. When such interference occurs, a cation-exchange column can be used to suppress it. The lowest amounts measurable were: 75 pg for NTA, 0.7 ng for EDDS, 0.8 ng for DTPA and 12ng for EDTA. The method was also applied for the EDTA assay in canned foods.

Determination of N-methylcarbamate pesticides in environmental samples by an automated solid-phase extraction and liquid chromatographic method based on post-column photolysis and chemiluminescence detection.

An automated solid-phase extraction-high performance liquid chromatography method has been developed to determine trace concentration of N-methylcarbamate pesticides in water and fruits. The method is based on the post-column conversion of the pesticides into methylamine by irradiation with UV light. The resultant methylamine was subsequently detected by chemiluminescence using tris(2,2'-bipyridyl)ruthenium(III), which was on-line generated by photo-oxidation of the ruthenium(II) complex with peroxydisulfate. Factors affecting the rate of the reactions were optimized so that their contribution to the total band-broadening was negligible. This detection system was used to determine bendiocarb, carbaryl, promecarb and propoxur, which were separated on an ODS C(18) column. The mobile phase consisted of water and acetonitrile using a gradient elution. A linear relationship between peak area and concentration was obtained for all pesticides (r(2)>0.999). Intra- and inter-day precision values of about 0.64-1.3% RSD (n=10) and 2.2-2.8% RSD (n=15), respectively, were obtained. N-Methylcarbamate pesticide residues at ultratrace levels could be determined in environmental samples when an automated solid-phase extraction device was coupled on-line with the HPLC system. Detection limits were within the range 3.9-36.7 ngl(-1) for water samples and 0.5-4.7 microgkg(-1) for fruits.

Determination of propoxur in environmental samples by automated solid-phase extraction followed by flow-injection analysis with tris(2,2'-bipyridyl)ruthenium(II) chemiluminescence detection.

A sensitive method for the analysis of propoxur in environmental samples has been developed. It involves an automated solid-phase extraction (SPE) procedure using a Gilson Aspec XLi and flow-injection analysis (FI) with chemiluminescence (CL) detection. The FI-CL system relies on the photolysis of propoxur by irradiation using a low-pressure mercury lamp (main spectral line 254 nm). The resultant methylamine is subsequently detected by CL using tris(2,2'-bipyridyl)ruthenium(III), which is on-line generated by photo-oxidation of the ruthenium(II) complex in the presence of peroxydisulfate. The linear concentration range of application was 0.05-5 microg mL(-1) of propoxur, with a detection limit of 5 ng mL(-1). The repeatability was 0.82% expressed as relative standard deviation (n=10) and the reproducibility, studied on 5 consecutive days, was 2.1%. The sample throughput was 160 injection per hour. Propoxur residues below ng mL(-1) levels could be determined in environmental water samples when an SPE preconcentration device was coupled on-line with the FI system. This SPE-FI-CL arrangement provides a detection limit as low as 5 ng L(-1) using only 500 mL of sample. In the analysis of fruits and vegetables, the detection limit was about 10 microg kg(-1).

High-performance liquid chromatography: photochemical reduction in aerobic conditions for determination of K vitamins using fluorescence detection.

The development and application of a post-column detection system for K vitamins based on their photoreduction to the hydroquinone form is reported. The photoreduction yield is practically quantitative and occurs in a PTFE tubing coiled around a 6-W low-pressure mercury lamp in the presence of sodium dodecylsulfate and methanol. Factors affecting the rate of the photochemical reaction were optimised so that its contribution to the total broadening was negligible. The enhanced fluorescence and stability of the K vitamins reduced in micellar medium has permitted the use of a very sensitive photochemical detection system, which can work in aerobic conditions. Separations were carried out by reverse-phase chromatography using pure methanol as eluent. The determination of phylloquinone, menaquinone-4 and menadione in several real samples illustrates the potential of the photochemical detection system.

Development and validation of a capillary electrophoresis method with laser-induced fluorescence detection for the determination of captopril in human urine and pharmaceutical preparations.

This study describes the development of a CE method for the analysis of the antihypertensive drug captopril using LIF detection. The method is based on the derivatization of captopril with the fluorescent label 5-iodoacetamidofluorescein. The optimization of the electrophoretic electrolyte composition together with other variables, such as applied voltage and injection time, resulted in a solution of 20 mM phosphate buffer adjusted to pH 12.0. The calibration curve for the fluorescent captopril derivative was linear in the concentration range 3.5-6000 ng/mL with a detection limit of 0.5 ng/mL. Intra- and interday precision (at a concentration of about 100 times the LOD) were less than 0.86 and 1.16%, respectively, both expressed as RSD. The assay was successfully used for quantification of captopril in some marketed pharmaceutical preparations and urine samples.

Flow injection chemiluminescent determination of amiodarone in pharmaceutical preparations using photogenerated tris(2,2'-bipyridyl)ruthenium(III).

A flow injection configuration was developed and evaluated for the chemiluminescent determination of amiodarone. The method is based on the reaction of the drug with tris(2,2'-bipyridyl)ruthenium(III), which was generated through the on-line photo-oxidation of tris(2,2'-bipyridyl)ruthenium(II) with peroxydisulfate. Under the optimum experimental conditions, a linear calibration graph was obtained over the range 3.0-60.0 microg ml(-1) with a detection limit of 0.28 microg ml(-1). The proposed method allows 120 injections h(-1) with excellent repeatability and precision (R.S.D. less than 0.5% and 2.8%, respectively) and a reagent consumption of only 0.37 micromol (0.27 mg) of Ru(bpy)(3)Cl(2) x 6H(2)O per determination. The method was successfully applied to the determination of amiodarone in commercial pharmaceutical formulations.

Determination of omeprazole, hydroxyomeprazole and omeprazole sulfone using automated solid phase extraction and micellar electrokinetic capillary chromatography.

A sensitive method for the determination of omeprazole and its metabolites has been developed. It involves an automated solid phase extraction (SPE) procedure and capillary electrophoresis with UV detection. Omeprazole, hydroxyomeprazole and omeprazole sulfone could be separated by micellar electrokinetic capillary chromatography using a background electrolyte composed of 20 mM borate buffer and 30 mM sodium dodecyl sulfate, pH 9.5. The isolation of omeprazole and its metabolites from plasma was automatically accomplished with an original SPE procedure using surface-modified styrene-divinylbenzene polymer cartridges. Good recovery data and satisfactory precision values were obtained. Responses were linear for the three analytes, from 0.08 to 2.0 microg/mL of plasma. Intra- and inter-day precision values of about 1.6% R.S.D. (n=10) and 2.5% R.S.D. (n=36), respectively, were obtained. The method is highly robust and no breakdown of the current or capillary blockages were observed during several weeks of operation. The validated method was applied to the determination of omeprazole in pharmaceutical preparations and for the analysis of plasma samples obtained from three volunteers who received oral doses of omeprazole.

Automatic determination of phylloquinone in vegetables and fruits using on-line photochemical reduction and fluorescence detection via solid phase extraction and flow injection.

A very simple, rapid and highly sensitive flow injection fluorimetric method was developed for the determination of phylloquinone. The assay was based on the on-line reduction of phylloquinone in dodecylsulfate micelles after irradiation with UV light. The micellar medium enhanced the fluorescence and stability of the reduced phylloquinone. Under optimum experimental conditions, the range of application of the technique was between 0.09 and 45.0 microg mL(-1) and the detection limit was 0.05 microg mL(-1). The sample throughput was 90 injections per hour. The reliability of the method for the routine analysis of phylloquinone in vegetables and fruits is demonstrated. Extractions were made with hexane, and an automated solid phase extraction system was used to purify the sample extracts prior to injection into the flow injection manifold.

Automated solid-phase extraction and high-performance liquid chromatographic determination of nitrosamines using post-column photolysis and tris(2,2'-bipyridyl) ruthenium(III) chemiluminescence.

A sensitive and selective post-column detection system for nitrosamines is described. The principle upon which the detector works is that UV irradiation of aqueous solutions of nitrosamines leads to cleavage of the N-NO bond. The amine generated is subsequent detected by chemiluminescence using tris(2,2'-bipyridyl) ruthenium(III), which is on-line generated by photo-oxidation of the ruthenium(II) complex in the presence of peroxydisulfate. Factors affecting the photochemical and chemiluminescent reactions were optimized to minimise their contribution to the total band-broadening. This detection system was tested for N-nitrosodimethylamine, N-nitroso-diethylamine, N-nitrosomorpholine, N-nitrosopiperidine and N-nitrosopyrrolidine, which were separated on an ODS column by isocratic reversed-phase chromatography with acetonitrile-water containing 5 mM acetate buffer at pH 4.0. A linear relationship between analyte concentration and peak area was obtained within the range 0.13-500 microg l(-1) with correlation coefficients greater than 0.9995 and detection limits of between 0.03 and 0.76 microg l(-1). Intra- and inter-day precision values of about 1.2% RSD (n = 11) and 2.5% RSD (n = 10), respectively, were obtained. The sensitivity may increase from 9 to 280 times with respect to UV detection, depending on the nitrosamine in question. An automated solid-phase extraction (SPE) system was used in conjunction with HPLC to determine nitrosamine residues in waters. Detection limits within the range 0.10-3.0 ng l(-1) were achieved for only 250 ml of sample.

An ultrasensitive method for the determination of thiouracil and phenylthiouracil using capillary zone electrophoresis and laser-induced fluorescence detection.

This paper reports the development of a method based on capillary electrophoresis with laser-induced fluorescence detection for the simultaneous determination of thiouracil (TU) and phenylthiouracil (PhTU) with high sensitivity (nanomolar range, i.e., attomoles detected). After derivatization with 5-iodoacetamidofluorescein, the analytes were separated by capillary zone electrophoresis using 20 mM phosphate buffer (pH 10.0) and quantified by fluorescence detection. The linearity range, precision, recovery, and detection limits were determined, and the method was shown to be applicable for the determination of TU and PhTU in spiked feed samples and urine.

Migration behaviour and separation of acetaminophen and p-aminophenol in capillary zone electrophoresis: analysis of drugs based on acetaminophen.

The migration behaviour of acetaminophen and p-aminophenol was investigated by capillary electrophoresis. The influence of different parameters (pH, nature and concentration of the running buffer and applied voltage) on the migration time, peak symmetry, efficiency and resolution was systematically investigated. The two analytes can be well separated within 4 min in a 57 cm fused-silica capillary at a separation voltage of 18 kV in a 50mM borate buffer adjusted to pH 9.5. Correlation coefficients for calibration curves in the range 0.2-200 microg ml-1 for acetaminophen and 0.3-3 microg ml-1 for p-aminophenol were higher than 0.999. The sensitivity of detection is 4.2 ng ml-1 for acetaminophen and 11.2 ng ml-1 for p-aminophenol. The method was applied to the analysis of various commercially available acetaminophen dosage forms with recoveries of 98.4-100.7%.

Successive determination of thiamine and ascorbic acid in pharmaceuticals by flow injection analysis.

A simple and rapid fluorimetric method for the determination of mixtures of thiamine and ascorbic acid is proposed. The procedure is based on the oxidation with mercury(II) of the B1 and C vitamins to form thiochrome (TC) and quinoxaline derivate, respectively. Both reaction products exhibit fluorescence at the same wavelengths (lambdaex = 356 and lambdaem = 440 nm). The procedure is optimised in a flow injection (FI) system and applied with excellent results in the determination of B1 and C vitamins in commercial pharmaceutical preparations. The calibration graphs were linear over the range 2-100 microg ml(-1) for thiamine and 5-100 microg ml(-1) for ascorbic acid. The throughput was 25 samples per hour.

Development and validation of a quantitative assay for raloxifene by capillary electrophoresis.

The migration behavior of raloxifene was investigated by capillary electrophoresis (CE). The influence of different parameters (nature and concentration of the running buffer, pH and applied voltage) on migration time, peak symmetry and efficiency was systematically investigated. A buffer consisting of 20mM acetate buffer of pH 4.5 was found to provide a very efficient and stable electrophoretic system for the analysis of raloxifene. The optimized method was validated with respect to precision, linearity, limits of detection and quantification, accuracy and robustness. The applicability of the assay was demonstrated by analyzing this drug in human plasma and pharmaceutical preparations.

High-performance liquid chromatographic separation and quantification of citric, lactic, malic, oxalic and tartaric acids using a post-column photochemical reaction and chemiluminescence detection.

An HPLC method was developed for the determination of citric, lactic, malic, oxalic and tartaric acids by chemiluminescent detection following online irradiation with visible light. The organic acids were irradiated with visible light in the presence of Fe3+ and UO2(2+) to generate Fe2+, which was determined by measuring the chemiluminescence intensity in a luminol system in the absence of added oxidant. Factors affecting the photochemical and chemiluminescence reactions were optimised so that their contribution to the total band-broadening was negligible. The chromatographic separation was performed on a C18 column under isocratic reversed-phase conditions using 0.005 M H2SO4 mobile phase. The optimised method was validated with respect to linearity, precision, limits of detection and quantification, accuracy specificity and robustness. The applicability of the assay was demonstrated by analysing these compounds in real samples such as milk, fruit juices, soft drinks, wine and beer.

Development of a capillary electrophoresis method for the determination of allopurinol and its active metabolite oxypurinol.

A simple and sensitive capillary zone electrophoresis method with UV absorbance detection is described for the quantitation of allopurinol and its metabolite oxypurinol in aqueous solution. The influence of different parameters on migration times, peak symmetry, efficiency and resolution was systematically investigated; these parameters included the nature and concentration of the separation buffer, pH and applied voltage. A buffer consisting of 15 mM 2-[N-cyclohexylamino]ethanesulfonic acid (CHES) adjusted to pH 8.8 was found to provide a very efficient and stable electrophoretic system for the analysis of these compounds. The optimized method was validated with respect to precision, linearity, limits of detection and quantification, accuracy and robustness. The applicability of the assay was demonstrated by analyzing these compounds in serum and allopurinol in commercial pharmaceutical preparations.

Determination of ATP via the photochemical generation of hydrogen peroxide using flow injection luminol chemiluminescence detection.

The determination of ATP using the coupling between a photochemical reaction and a chemiluminescence reaction in a flow injection (FI) system is described. The method is based on the reaction of glucose with ATP catalyzed by hexokinase and Mg(2+) ions. The glucose that is not consumed by ATP is subsequently photooxidized using 9,10-anthraquinone-2,6-disulfonate as a sensitizer. The hydrogen peroxide produced in the photochemical reaction is monitored through the chemiluminescence reaction with luminol catalyzed by hematine. There is a linear relationship between the decrease in the chemiluminescence response and the ATP concentration at a constant concentration of glucose. Under the optimum conditions, the calibration graph is linear in the range 0.20-50.5 mg L(-1) with a throughput of 25 samples per hour and relative standard deviations between +/-0.62 and +/-1.42%. The limit of detection is 0.07 mg L(-1). The method was used for the determination of ATP in pharmaceuticals, milk, and soils.