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Immobilized reagent phase flow injection analysis can be configured as discrete reagent cells upstream of the sensor element or as an integral reagent/transduction system (flow injection analysis-biosensor). The former approach has attracted greater attention because several assays can be assembled with greater versatility in reagent column units employing a single sensor, than can be co-immobilized on the surface of a transducer.
An automated two-dye flow injection analysis system to quantitate DNA and RNA in crude extracts of tissues is described. The method uses the fluorochrome dyes ethidium bromide and Hoechst 33258. DNA concentration is determined directly from its fluorescence in Hoechst dye. RNA is estimated from fluorescence in ethidium bromide after subtraction of the fluorescence due to DNA. This method has several advantages: a simple extraction procedure, a low detection limit (0.01 micrograms DNA and 0.10 micrograms RNA), automation, and a high sample throughput.
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.
The use of flow-injection analysis for the direct determination of calcium and magnesium in blood serum and plasma is described. An inexpensive rotary valve is used to inject the serum sample (4 microliters) into a flowing non-segmented stream of reagent which carries the sample slug through a long narrow-bore coil--where it gradually disperses--and into the nebuliser of an atomic absorption spectrometer. This on-stream sample dilution removes the need for predilution of the sample. The resulting absorbance signals are recorded as peaks less than 40 seconds after sample injection. Analytical recoveries and precision are good for both elements and the results by flow-injection analysis compare well with established routine methods.
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.
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.
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.
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.
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.
A 3-channel flow-injection procedure was developed, which enables the simultaneous determination of protein, phosphorus, and calcium in a wide range of animal feeds from a single digestion. Samples are digested with a block digestor, diluted, and analyzed at a rate of 82 samples/h. Protein (nitrogen) as ammonia is determined colorimetrically by the indophenol method. Phosphorus and calcium are determined by measuring the absorbances of the molybdenum blue and calcium-cresolphthalein complexes at 660 and 580 nm, respectively. Protein is determined in the range from 0 to 75%, phosphorus in the range from 0 to 6%, and calcium in the range from 0 to 6%. The results obtained do not differ significantly from those obtained by proven manual methods, and considerable time, space, and reagents are saved.
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.
The flow infection analysis (FIA) described by Ruzicka and Hansen was adjusted for lactic acid determination by Rydevik et al. We were able to elaborate some other NAD or NADH-dependent enzyme reactions with the FIA system. The reliability of the alanine assay corresponds to that of the lactate FIA method. The coefficient of variation was on an average 2.8%; th sample rate was 60/h with consistent duplicates. The beta-OHB assay had almost the same validity. Pyruvate, however, was less reliable with higher coefficients of variation. ACAC FIA assay, which was suitable for the determination in acid solution after neutralization, could not yet be employed for the determination in serum. In comparison to the manual enzyme methods, the FIA assays described here indicated a higher sampling rate, a lower reagent consumption, a lower coefficient of variation, a better reproducibility, and a greater consistency of duplicates.
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.
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%.
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.
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.
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.
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.