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Determination of mitoxantrone by flow injection analysis using an amperometric detector.

Mitoxantrone was determined by flow injection analysis using a flow cell modified in the laboratory and fitted with carbon paste as an amperometric detector. The sample solution (100 microliters, 5 x 10(-8)-1 x 10(-5) M) was injected into the carrier stream of 0.1 M perchloric acid (pH 1.12). Mitoxantrone was determined by oxidation at the carbon paste electrode (CPE) at +0.90 V. A 60-cm delay coil (0.5 mm i.d.) was incorporated just before the detector (a canal thin layer) and a flow rate of about 4 ml min-1 was used. The system was successfully applied to the determination of mitoxantrone in a pharmaceutical preparation; the method was fast and reproducible.

Electrodes

Fluorometric determination of urea by flow injection analysis.

Urea was determined using fluorometry with flow injection analysis. O-phthalaldehyde (OPA) reacts with enzymatically generated ammonia and sulfite in alkaline medium to give a highly fluorescent compound that has an excitation wavelength of 372 nm and an emission wavelength of about 430 nm. The method is more selective to ammonia than the one which uses mercaptoethanol in place of the sulfite. Urease was immobilized to a Pall Immunodyne membrane which is commercially available. The immobilization occurs through covalent bonding which results in a highly stable enzyme preparation. The enzymatic membrane was fitted in a 5 cm long, 0.125 inch o.d. Teflon tubing which served as the enzymatic reactor. The system is difficult to use for the analysis of urea in serum because some compounds normally present in serum fluoresce at the same wavelength. This results in higher values for urea. If the reaction system is to be used for the evaluation of urea in serum, a blank should be run so that urea concentration can be calculated by difference.

Calibration

Determination of aqueous fluoride with a helium microwave-induced plasma and flow injection analysis.

The determination of aqueous fluoride by flow injection analysis (FIA) with a helium microwave-induced plasma (He-MIP) is described. This system operates at 500 W and utilizes a modified TM010 resonator cavity with a demountable plasma torch. Both direct nebulization and FIA in conjunction with ultrasonic nebulization (USN) were investigated. FIA was found to be the most reliable method because extended nebulization of aqueous fluoride was found to cause memory effects. Detection limits for aqueous fluoride of 35 and 4 ppm were observed for FIA and direct USN, respectively. The interference effects of pH and selected elements were also studied.

Fluorides

Ionspray mass spectrometry of marine toxins. III. Analysis of paralytic shellfish poisoning toxins by flow-injection analysis, liquid chromatography/mass spectrometry and capillary electrophoresis/mass spectrometry.

Ionspray mass spectrometry has been used to monitor the purification of saxitoxin, the parent compound in the family of toxins responsible for paralytic shellfish poisoning (PSP), from a strain of the dinoflagellate Alexandrium excavatum. Quantitative results obtained by flow-injection analysis are compared to those obtained by high-performance liquid chromatography with post-column oxidation and fluorescence detection. The coupling of liquid chromatography and capillary electrophoresis with ionspray mass spectrometry is described for the separation of mixtures of PSP toxins and the highly potent pufferfish toxin tetrodotoxin. Tandem mass spectrometry is used to provide the structural information, and the ability to distinguish isomeric PSP toxins both chromatographically and mass spectrometrically is demonstrated.

Chromatography, High Pressure Liquid

[The adaptation of four protein determination methods to flow injection analysis (FIA)].

The adaptation of 4 manual methods to the flow-injection analysis is described for the determination of proteins (Biuret- and Exton-method, albumin and hemoglobin determination). The comparison of analytical results of FIA with the manual methods shows a very good agreement of values. Flow-injection-analysis appears not only as an excellent possibility to automate these 4 investigated methods but it also shows unlike the manual methods a considerable better precision and accuracy of the analytical results and a reduction of time and materials.

Blood Proteins

Determination of oxalate in urine by flow injection analysis.

A method is described for the determination of oxalate in urine using flow injection analysis and fluorimetry. Oxalate is precipitated with calcium chloride at pH 4.5, redissolved in H2SO4 and measured by flow injection analysis. The minimum detection limit is 6 mumol/l. The coefficient of variation is 7%. Results are in good accordance with normal values found with traditional oxalate analysis.

Adult

Determinations of lactate and lactate dehydrogenase activity in serum with the flow injection analysis system involving immobilized enzyme column and chemiluminescence.

The methods for the highly sensitive flow injection analysis of lactate and lactate dehydrogenase (LDH) activity in serum using immobilized enzymes in column form and chemiluminescence detection which does not require a blank correction are described. The methods were based on the determination of chemiluminescence formed by the reaction of a luminol-ferricyanide mixture with hydrogen peroxide. This hydrogen peroxide was produced by the lactate oxidase (LOD) reaction from lactate, which was in serum or was produced by the action of LDH in serum. The action of LDH in a flow injection analysis system was performed for 2 min in an incubation coil placed parallel to the substrate-buffer line between the LOD column and the LOD/catalase column. Endogenous lactate in serum was removed by an immobilized LOD/catalase column prior to the action of LDH. The present method gave perfect linearity of the data up to 5.6 mmol/liter for lactate and 1840 IU/liter for LDH activity with satisfactory precision, reproducibility, and accurate reaction recoveries. The results from the lactate and LDH activity correlated satisfactorily with those obtained by other well-established methods.

Autoanalysis

[Construction and application of atropine flow-through sensor in flow injection analysis].

A new kind of flow-through sensor for atropine has been studied. It exhibits Nernstian response for atropine with a slope of 54 +/- 1 mV/decade over the concentration range of 0.02-200 mmol/L at pH 5-8. The sensitivity coefficients of common compounds were determined. Only bromo-geramine, clonidine, strychnine and amantadine showed remarkable interference. Direct potentiometry for determination of atropine showed an average recovery of 99.2% and a relative standard deviation of 1.3%. It has been used in flow injection analysis (FIA) of atropine, anisodamine and scopolamine and belladonna preparations. Rate of analysis of as high as 60-100 samples/h was achieved.

Atropa belladonna

Flow injection analysis of inorganic cationic species in serum and urine.

This paper describes three flow injection analysis (FIA) systems for the automatic determination of sodium, potassium, lithium, calcium, magnesium, zinc, copper and iron in certain biological fluids and compares the results obtained to those of flame photometry and atomic absorption spectrometry. The set-ups were designed to allow the samples to be prepared in the same manner as that used for batch procedures with the same analytical instrument. For determinations requiring a high sample dilution, it was found of definite advantage to split the stream and pass large amounts of liquid through the detection systems. The comparison of the results obtained by FIA and conventional methods yielded correlation coefficients in the range 0.990 to 0.999. The proposed methodology show good precision, with variation coefficients between 0.5% to 5%.

Calcium

[Flow injection analysis for determination of choline-containing phospholipids by luminol chemiluminescence].

A sensitive flow injection analysis using luminol/peroxidase chemiluminescence was developed for the determination of choline-containing phospholipids in serum. Flow injection manifold was composed of two channel system with an enzyme column, in which phospholipase D was immobilized together with choline oxidase. The serum sample (5 microliters) was pretreated by Extrelut column (diatomite column) extraction with chloroform-methanol (95:5). The extract (20 microliters) was injected into a sample carrier at 38 degrees C and passed through the enzyme column, which converted phospholipid to choline and subsequently to hydrogen peroxide. Produced hydrogen peroxide was monitored by measuring the chemiluminescence intensity of luminol/peroxidase system at 5 degrees C. The response was linear against the amount of phospholipids ranging from 2 to 2000 pmol/test, and the relative standard deviation was less than 2%. In the determination of phospholipids in the serum, a correlation coefficient (r) between 4-aminoantipyrine/phenol and the proposed methods was found to be 0.983 (Y = 1.035X-6.2). The throughput rate was 15 samples/h.

Blood Chemical Analysis

The characterisation of immobilised lignin peroxidase by flow injection analysis.

Immobilised lignin peroxidase has been investigated using a flow system in the steady state and by flow injection analysis (FIA). In the steady state, the extreme sensitivity of the enzyme towards inactivation by H2O2 resulted in a stable response only in the presence of saturating levels of organic substrate and at very low (10 microM) peroxide concentrations. By contrast, the low contact time during FIA led to a stable response to injections of 100 microM H2O2. At higher peroxide concentrations a reproducible inactivation was observed, allowing a study of factors affecting both activity and stability. Lignin peroxidase substrates that undergo at least semi-reversible oxidation/reduction, including high-molecular-weight lignin fractions, could be detected by electrochemical reduction of the oxidation products. With this detection system it was possible to demonstrate the role of veratryl alcohol as mediator. This mediated oxidation of lignin functioned only when all components were present simultaneously, and was not observed when lignin was separated from the site of veratryl alcohol oxidation.

Acetonitriles

Lowry protein determination by automated flow injection analysis for bovine serum albumin and hepatitis B surface antigen.

The Lowry method for quantitation of protein was adapted to automated flow injection analysis. The procedure was developed using two different pure proteins: bovine serum albumin and hepatitis B surface antigen. The system was optimized for reagent concentration, pH, gain, temperature, sample volume, and output. The response of each protein was affected differently by temperature. The reaction slopes and absorbance values of the proteins were similar at 90 degrees C to allow quantitation of hepatitis surface antigen against bovine serum albumin. Advantages of the automated flow injection analysis Lowry procedure include: rapid analyses (90 samples/h), small sample volume (30 microliters, 100 microliters), fast response (20 s), reproducibility (less than or equal to 2% CV within an assay and 3 to 6% CV among assays), sensitivity (5 micrograms), and high correlation (99.8%) with manual assay. After a 30-min set-up period, the analyzer was available to assay protein on demand throughout the day, making it suitable for process and quality control testing.

Animals

[Fluorometric determination of pyridine and its derivatives by flow injection analysis].

A method for the fluorometric determination of pyridine and its derivatives has been developed by flow injection analysis using hydrogen peroxide at high temperature. The reaction system consists of two pumps to deliver reagent and carrier stream, sample injector, reaction coil (0.5 mm ID x 15 m, 150 degrees C), cooling coil (0.5 mm ID x 3 m, 30 degrees C), and cooling coil (0.5 mm ID x 20 cm, 0 degrees C). The wavelengths of the fluorometric spectrophotometer were set at Ex 305-350 nm and Em 380-410 nm, the flow rate of each solution was 1.0 ml/min. The carrier stream was deionized water. The reaction solution containing 10 mmol/L hydrogen peroxide in 0.2 mol/L phosphate buffer (pH 6.0) gave the maximum fluorescence intensity for pyridine and its derivatives. Linear calibration curves were obtained from 5 ng up to 100 ng of pyridine and its derivatives. The coefficient of variations for 2.5 ng (n = 10) and 25 ng (n = 10) of isonicotinic acid, isoniazide and acetylisoniazide were 1.8% and 1.1%, 1.6% and 1.2%, 2.1% and 1.4%, respectively. The detection limit (S/N = 3) was 250 pg for isonicotinic acid, 500 pg for isoniazide, acetylisoniazide, nicotinamide, isonicotinamide, nicotinic acid, and 2.5 ng for pyridine, nicotine, 2--picoline, 2--picolinamide and picolinic acid. The carrier stream containing organic solvent (methanol, ethanol or acetonitrile) decreased the fluorescence intensity, but in the case of acetonitrile there was less decrease than methanol or ethanol. This method allowed the analysis of 30 samples/h.

Flow Injection Analysis

Flow injection analysis of L-lactate with enzyme amplification and amperometric detection.

A flow injection analysis method for the determination of the lactate anion with enzyme amplification and amperometric detection is described. The system utilizes an oxygen electrode for measurement of changes in the oxygen concentration in the flow stream. Two enzymes, lactate oxidase and lactate dehydrogenase, were randomly coimmobilized on aminopropyl controlled-pore glass (AMP-CPG) and packed into a reactor. beta-NADH was used as a coenzyme for the regeneration of lactate from pyruvate. The experimental conditions for the determination of the lactate anion were studied for this system by the simplex and the univariant methods. The results obtained under these two conditions were compared. The simplex experimental condition yielded a calibration curve whose linear portion had a slope that was 1.2 times greater than that of the linear portion of the curve obtained under univariant conditions. The limit of detection under simplex condition was 1.19 x 10(-7) M vs 3.29 x 10(-7) M lactate under univariant conditions. The relative standard deviation obtained for this system at 6 x 10(-6) M lactate (n = 10) was about 2.5% under simplex conditions and 3.6% under univariant maximization conditions.

Anions

Liquid chromatographic and flow injection analysis of tetracycline using sensitized europium (III) luminescence detection.

Europium (III) can be used as a luminescent chromophore for detection in the liquid chromatographic and flow injection analysis of tetracycline. Detection is dependent upon an intramolecular energy transfer from the tetracycline to Eu (III). In liquid chromatography, the Eu (III) is added post-column as a complex with ethylenediaminetetraacetic acid. The post-column phase also serves to adjust the pH for optimum sensitivity. The method is highly selective for tetracycline since few compounds are capable of transferring energy to Eu (III). Fluorescent impurities that would otherwise interfere in flow injection analysis can be eliminated through the use of a delay time between the source pulse and the start of data acquisition. The detection limits for tetracycline using sensitized Eu (III) luminescence are better than those obtained using ultraviolet detection. The method is applied to the analysis of tetracycline in urine, blood serum, and gingival crevice fluid.

Chromatography, High Pressure Liquid

[Berberine-electrochemical detector for the determination of berberine-type alkaloids in various Chinese patent medicines by flow injection analysis].

A description is given for the preparation of flow through sensor and of the incorporation of the sensor into a flow injection analysis system. The parameters affecting the measurement are discussed. An accurate, convenient and rapid method is proposed for the determination of berberine-type alkaloids in various Chinese patent medicines (Coptis chinensis Franch., Phellodendron chinesis Schneid., xianglian wan, zuojin wan, ermiao wan and sanmiao wan). The slope is 54-60 mV/decade over the concentration range of 10(-3)-3 x 10(-6) mol/L berberine at pH 2-9.5. Direct potentiometry determination of berberine in various samples showed an average recovery of 99.5-103.5% and a relative standard deviation of 1.3-3.5% at a sampling rate of 120/h.

Berberine

Flow injection analysis of pharmaceuticals.

An overview of the most representative problems solved by flow injection analysis (FIA) in drug analysis is presented. Different aspects of this technique which can be manipulated with specific purposes are discussed and special emphasis is placed on the possibilities of FIA in dissolution test control.

Chemistry, Pharmaceutical

Determination of glutathione in biological material by flow-injection analysis using an enzymatic recycling reaction.

A sensitive and specific assay for glutathione using a recycling reaction followed by spectrophotometric detection in a flow-injection analysis system is presented. The proposed method provides specific amplification of the response to glutathione by combined use of the enzyme GSSG reductase and the chromogenic reagent 5,5'-dithiobis(2-nitrobenzoic acid). Both oxidized (GSSG) and reduced (GSH) glutathione are detected, so that GSSG must be determined separately after alkylation of the GSH with N-ethylmaleimide. The sensitivity is controlled by the number of times the cycle occurs and therefore by the residence time of the sample in the reactor. This time depends on the reactor length and the flow rate. The influence of residence time, temperature, and enzyme concentration on the response has been studied and the optimum reaction conditions have been selected. The sample throughput is as high as 30 h(-1) and the detection limit is 1 pmol GSH at a signal-to-noise ratio of 3. The method has been evaluated by the quantification of GSH and GSSG in isolated hepatocytes. A high correlation between the new flow-injection analysis method and the original spectrophotometric batch assay has been found (slope = 1.039, intercept = 0.6, n = 216, r = 0.977). The main advantages of the proposed method are high sample throughout, high sensitivity, and good reproducibility.

Animals