Enzymatic rotating biosensor for cysteine and glutathione determination in a FIA system

The high sensitivity that can be attained using an enzymatic system and mediated by catechols has been verified by on-line interfacing of a rotating biosensor and continuous flow/stopped-flow/continuous-flow processing. Horseradish peroxidase, HRP, [EC 1.11.1.7], immobilized on a rotating disk, in presence of hydrogen peroxide catalyzed the oxidation of catechols, whose back electrochemical reduction was detected on glassy carbon electrode surface at −150 mV. Thus, when l-cysteine (Cys) or glutathione (GSH) was added to the solution, these thiol-containing compounds participate in Michael addition reactions with catechols to form the corresponding thioquinone derivatives, decreasing the peak current obtained proportionally to the increase of its concentration. Cys was used as the model thiol-containing compound for the study. The highest response for Cys was obtained around pH 7. This method could be used to determine Cys concentration in the range 0.05-90 μM (r = 0.998) and GSH concentration in the range 0.04-90 μM (r = 0.999). The determination of Cys and GSH were possible with a limit of detection of 0.7 and 0.3 nM, respectively, in the processing of as many as 25 samples per hour. Current response of the HRP-rotating biosensor is not affected by the oxidized form of GSH and Cys (glutathione disulfide, GSSG, and l-cystine, respectively), by sulfur-containing and alkyl-amino compounds such as methionine and lysine, respectively. The interferences from easily oxidizable species such as ascorbic acid and uric acid are lowest.     

Determination of total nitrogen in food by flow injection analysis (FIA) with a potentiometric differential detection system

A flow injection set-up based on potentiometric detection and gas diffusion device for the determination of total nitrogen in food is described. The detection system consisted of two ammonium-sensitive electrodes placed sequentially and each alternately operating as reference electrode. Tubular electrodes without an inner reference solution were prepared with a PVC membrane composed of nonactin in Tris (2-ethylhexyl) phosphate and potassium tetrakis (4-chlorophenyl) borate to reduce the membrane resistance. The food sample digests were inserted into the system, and the ammonium present was converted into ammonia gas. The gas diffused through a gas-permeable membrane to a buffer acceptor stream with a pH that ensured transformation to the ammonium cation, which was potentiometrically detected. Good agreement between FIA results and those provided by the reference procedure was obtained, with relative deviation errors below 5%. Using the proposed system, low reagent consumption is possible, a sampling rate of about 30 samples/h was achieved, as well as a good reproducibility for consecutive injections of the same sample (variation coefficient < 2%). Received: 8 October 1998 / Revised: 7 January 1999 / Accepted: 12 January 1999     

Determination of total nitrogen in food by flow injection analysis (FIA) with a potentiometric differential detection system

A flow injection set-up based on potentiometric detection and gas diffusion device for the determination of total nitrogen in food is described. The detection system consisted of two ammonium-sensitive electrodes placed sequentially and each alternately operating as reference electrode. Tubular electrodes without an inner reference solution were prepared with a PVC membrane composed of nonactin in Tris (2-ethylhexyl) phosphate and potassium tetrakis (4-chlorophenyl) borate to reduce the membrane resistance. The food sample digests were inserted into the system, and the ammonium present was converted into ammonia gas. The gas diffused through a gas-permeable membrane to a buffer acceptor stream with a pH that ensured transformation to the ammonium cation, which was potentiometrically detected. Good agreement between FIA results and those provided by the reference procedure was obtained, with relative deviation errors below 5%. Using the proposed system, low reagent consumption is possible, a sampling rate of about 30 samples/h was achieved, as well as a good reproducibility for consecutive injections of the same sample (variation coefficient < 2%).     

Optimization of a FIA system with amperometric detection by means of a desirability function: determination of sulfadiazine, sulfamethazine and sulfamerazine in milk

A sensitive and cheap FIA, with amperometric detection, analytical procedure is developed in this paper to determine sulfadiazine, sulfamethazine and sulfamerazine in milk. A multicriteria optimization based on the use of a desirability function is used for optimizing two analytical responses (peak height and its variability) since single-objective optimizations lead to conflicting experimental conditions. In the optimum conditions, the determination of the three sulfonamides in milk samples is carried out, the analytical procedure being validated according to Commission Decision 2002/657/EC. The decision limit at 0 and 100 microg L(-1) (which is the maximum residue limit in milk) are 13.9 and 110.2, 9.5 and 107.1 and 9.1 and 107.1 microg L(-1) for sulfadiazine, sulfamethazine and sulfamerazine, respectively. Whereas the values of capability of detection, CCbeta, obtained at 0 and 100 microg L(-1) were 26.9 and 119.8, 18.2 and 113.6, and 17.5 and 113.7 microg L(-1) for sulfadiazine, sulfamethazine and sulfamerazine, respectively. Recovery values between 67.4% and 119.1% are found for milk test samples of two brands of milk. The accuracy of the method is confirmed. The ruggedness of the procedure is evaluated by means of a Plackett-Burman design. The relative errors were lower than 2.5% (n=7).     

Enzymatic methods for the determination of alpha-glycerophosphate and alpha-glycerophosphate oxidase with an automated FIA system

A procedure for the enzymatic determination of alpha-glycerophosphate (alpha-GP) has been developed, using an automated in-house FIA system, with immobilized glycerol-3-phosphate oxidase (GPO) on non-porous glass beads, following optimization of the immobilization and analytical parameters. Fabricated single bead string reactors (SBSR) were used in connection with the FIA system, following optimization of its parameters. The half-life of GPO-SBSR regarding reduction of the enzyme activity was found to be 110 days for its use in 20 triplicate measurements daily and storage at 4 degrees C in the appropriate buffer. The regression equation of the calibration graph for the determination of alpha-GP was: A(max)=(10+/-2)x10(-4)+(22 134+/-12)x10(-4) (mmol l(-1)alpha-GP). The lower limit of quantitation was 0.74 mumol l(-1)alpha-GP and the RSD of the method 0.05% (r=0.9999). The same FIA system and procedure can be also used for the determination of the GPO activity, with the alpha-GP as substrate. The regression equation for this calibration graph was: A(max)=(23+/-18)x10(-4)+(190+/-1)x10(-4) (mug ml(-1) GPO), the lower limit of quantitation was 0.782x10(-3) mg ml(-1) (0.782 ppm) GPO and the RSD of the method 0.53% (r=0.9999). Serum samples obtained from hospitalized patients were deproteinized by gel filtration and analyzed under pseudo-first order conditions, at various concentrations of alpha-GP. A kinetic study of the reduction of alpha-GP in serum versus time is given and an observed reaction rate constant k(ob)=106.5x10(-4) min(-1) was determined.     

Total determination of metal-binding carbohydrates in plant extracts by using FIA with electrochemical detection

FIA with pulsed amperometric detection (PAD) is used for the determination of metal-binding carbohydrates in plant extracts. Results of direct FIA measurements agree very well with those of corresponding HPLC-PAD analyses. The proposed method is used to determine total carbohydrates in isolated low-molecular-weight fractions of plant roots, which have been obtained after extraction at different pH values. The results are compared with those obtained by corresponding metal determinations (Ca, Mg, Zn, Mn) by AAS and also with constant potential amperometric detection at a copper working electrode. The latter detection mode is more sensitive, but less selective for carbohydrates. Received: 12 January 2000 / Revised: 2 March 2000 / Accepted: 4 March 2000     

Total determination of metal-binding carbohydrates in plant extracts by using FIA with electrochemical detection

FIA with pulsed amperometric detection (PAD) is used for the determination of metal-binding carbohydrates in plant extracts. Results of direct FIA measurements agree very well with those of corresponding HPLC-PAD analyses. The proposed method is used to determine total carbohydrates in isolated low-molecular-weight fractions of plant roots, which have been obtained after extraction at different pH values. The results are compared with those obtained by corresponding metal determinations (Ca, Mg, Zn, Mn) by AAS and also with constant potential amperometric detection at a copper working electrode. The latter detection mode is more sensitive, but less selective for carbohydrates.     

Development of an FIA system with amperometric detection for determination of bentazone in estuarine waters

On the basis of its electrochemical behaviour a new flow-injection analysis (FIA) method with amperometric detection has been developed for quantification of the herbicide bentazone (BTZ) in estuarine waters. Standard solutions and samples (200 microL) were injected into a water carrier stream and both pH and ionic strength were automatically adjusted inside the manifold. Optimization of critical FIA conditions indicated that the best analytical results were obtained at an oxidation potential of 1.10 V, pH 4.5, and an overall flow-rate of 2.4 mL min(-1). Analysis of real samples was performed by means of calibration curves over the concentration range 2.5x10(-6) to 5.0x10(-5) mol L(-1), and results were compared with those obtained by use of an independent method (HPLC). The accuracy of the amperometric determinations was ascertained; errors relative to the comparison method were below 4% and sampling rates were approximately 100 samples h(-1). The repeatability of the proposed method was calculated by assessing the relative standard deviation (%) of ten consecutive determinations of one sample; the value obtained was 2.1%.     

Automated determination of detection limits and selectivity coefficients of ion-selective electrodes by using a microcomputer-controlled potentiometric system

The detection limit and the potentiometric selectivity coefficients of ion-selective electrodes are determined automatically with a microcomputer-controlled potentiometric system. Measurements of these parameters for three commercially available electrodes of the liquid membrane type (chloride, nitrate and calcium electrodes) gave results in good agreement with those reported in the literature. The non-linear least-squares fit evaluation of data (potential activities) and the selection of the appropriate transfer functions are described. The reproducibility of the results is discussed.     

Enzymatic reverse FIA method for total phenols determination in urine samples

A reverse flow injection spectrophotometric enzymatic method is proposed to quantify total phenols in urine samples. The polyphenol oxidase (PPO; EC 1.14.18.1) obtained as a crude extract from sweet potato root (Ipomoea batatas) was used as enzymatic catalyze. The detection limit, the sample throughput and relative standard deviation were 7.7 mg l⁻¹ of total phenols, 49 h⁻¹ and 0.9%, respectively. The method was applied to real samples and a recovery study was carried out in order to its validation.     

Enzymatic flow-injection determination of urea in blood serum using potentiometric gas sensor with internal nonactin based ISE

Ion-selective electrode with cellulose triacetate membrane containing nonactin is employed for the potentiometric detection of ammonia produced in biocatalytic reaction in flow-injection system with enzyme reactor. The elimination of interferences occurring in the presence of alkali metal ions was achieved by covering a nonactin membrane with outer hydrophobic gas permeable membrane. The obtained flow-injection response to ammonia indicates a possibility of ammonia determination down to 10 microM ammonia. In the flow-injection system for urea determination 200 microl of 10-fold diluted blood serum sample was injected into carrier stream of distilled water merged with TRIS buffer, passed through the urease flow-through reactor and then after merging with NaOH stream delivered to the detector. It was found in several series of natural blood serum samples, that the correction for endogenous ammonia in such a determination is not indispensable.     

The determination of reducing carbohydrates using a cation-exchange column and potentiometric detection with a metallic copper electrode

Summary A metallic copper electrode is employed as a potentiometric detector for reducing carbohydrates after their separation on a cation-exchange column. A post-column reactant solution comprising 1 mM copper (II) and 35 mM ammonia is mixed with the column effluent and the metallic copper electrode provides a steady baseline potential by responding in a Nernstian manner to the level of copper ions present. This in turn is affected by the presence of eluted reducing carbohydrates, leading to an indirect detection method for these species. The method is applied to the detection of maltose, lactose, xylose, glucose, sorbose, fructose and arabinose. The electrode response mechanism is discussed and detection limits in the low nanomole range are reported. The potentiometric detector is shown to be more sensitive than refractive index detection.     

Multienzymatic-rotating biosensor for total cholesterol determination in a FIA system

Fabrication of an amperometric-rotating biosensor for the enzymatic determination of cholesterol is reported. The assay utilizes a combination of three enzymes: cholesterol esterase (ChE), cholesterol oxidase (ChOx) and peroxidase (HRP); which were co-immobilizing on a rotatory disk. The method is developed by the use of a glassy carbon electrode as detector versus Ag/AgCl/3 M NaCl in conjunction with a soluble-redox mediator 4-tert-butylcatechol (TBC). ChE converts esterified cholesterol to free cholesterol, which is then oxidized by ChOx with hydrogen peroxide as product. TBC is converted to 4-tert-butylbenzoquinone (TBB) by hydrogen peroxide, catalyzed by HRP, and the glassy carbon electrode responds to the TBB concentration. The system has integrated a micro packed-column with immobilized ascorbate oxidase (AAOx) that works as prereactor to eliminate l-ascorbic acid (AA) interference. This method could be used to determine total cholesterol concentration in the range 1.2 μM-1 mM (r = 0.999). A fast response time of 2 min has been observed with this amperometric-rotating biosensor. Lifetime is up to 25 days of use. The calculated detection limits was 11.9 nM. Reproducibility assays were made using repetitive standards solutions (n = 5) and the percentage standard error was less than 4%.     

Parabens determination with a hybrid FIA/HPLC system with ultra-short monolithic column

The combination of an ultra-short C18 monolithic column (5 mm long) with a flow injection analysis (FIA) scheme results in a versatile and efficient system that has been used for the chromatographic determination of four preservatives — methylparaben (MP), ethylparaben (EP), propylparaben (PP) and butylparaben (BP). The separation is carried out by using two carriers, A and B, consisting of a mixture of ACN: water in different proportions. The described procedure is able to separate the analytes in only 150 s. The applicable concentration range, detection limit and the relative standard deviation were the following: for MP from 1.6 × 10^-5 to 1.1 × 10^-3 M; 4.8 × 10^-4 M; 0.65%; for EP between 3.7 × 10^-5 and 2.0 × 10^-3 M; 1.2 × 10^-5 M; 1.2%; for PP between 3.9 × 10^-5 and 2.0 × 10^-3 M; 1.2 × 10^-5 M; 1.2%; and for BT between 6.0 × 10^-5 and 2.0 × 10^-3 M; 1.8 × 10^-5 M; 1.8%. The method was applied and validated satisfactorily for the determination of these parabens in commercial cosmetics samples, comparing the results with those obtained by HPLC reference method.     

Use of a solid-phase extraction disk module in a FI system for the automated preconcentration and determination of surfactants using potentiometric detection

The global determination of anionic surfactants is proposed by using flow injection potentiometry, and by employing specifically developed tubular flow-through ion selective electrodes (ISEs). The low concentration requirements needed for the environmental application are obtained with an on-line preconcentration stage embedded in the flow system, which has as its goal the unattended monitoring of anionic surfactants in surface waters. The on-line preconcentration is achieved by employing an octadecylsilica extraction disk in the FIA system. This stage performs the solid phase extraction (SPE) for the enrichment and purification of the target analytes from common interfering anions. The outlined procedure improves the detection limit of a direct injection system, which is decreased from 10 to 0.25 μM by using a preconcentration volume of 3.0 mL and 50 μL of 75% acetonitrile in water as the eluent. Precision was estimated as 2.9% relative standard deviation (n = 20) for a 0.25 μM (0.070 mg·L^− 1) sodium docecylsulfate standard.     

Flow-injection system with ion-exchange separation and potentiometric detection for the determination of three halides

The use of ion exchangers in flow-injection systems is reviewed briefly. In the method described, halides are separated on a short column of a strongly basic ion-exchange resin (Dowex 1-X8) placed in the flow-injection conduit, with a laboratory-made tubular silver/silver halide ion-selective electrode as potentiometric sensor. The response capabilities of the different halide-selective electrodes to a wide concentration range (20-5000 mg dm^?3) of single and mixed halide solutions with and without the incorporated ion-exchange column are compared. By careful selection of suitable concentrations of the potassium nitrate carrier/eluent stream to satisfy the requirements of both the ion-exchange column and the halide-selective electrode, it is possible to separate and determine chloride, bromide and iodide in mixed halide solutions with a detection limit of 5 mg dm^?3. The bromide-selective electrode is the most satisfactory detector.     

Approaches to the liquid chromatographic determination of carboxylic acids using potentiometric detection with a metallic copper electrode

Summary A metallic copper electrode is evaluated as a potentiometric detector for carboxylic acids. The application of this device to ion-exchange chromatography is illustrated by the determination of oxalate in urine. Oxalate was selectively detected in the presence of a 100-fold excess of sulphate after separation on a low-capacity methacrylate anion-exchange column using 0.7mM potassium hydrogen phthalate at pH 7.1 as eluent. Calibration plots were linear up to 50ppm of oxalate. Potentiometric detection has also been applied to ion-exclusion chromatography using 0.005% phosphoric acid as eluent. With this method detection limits of 0.2, 2.1, 5.0 and 5.3μg were obtained for formic, acetic, propionic and iso-butyric acids, respectively.     

Single interface flow system with potentiometric detection for the determination of nitrate in water and vegetables

In this work a single interface flow system (SIFA) with potentiometric detection was for the first time implemented and applied to the determination of nitrate in waters and plant extracts. The analytical potential of the SIFA system was exploited not only to transport the sample towards detection but also to carry out, in a reproducible and automated way, the tasks associated with sample pre-treatment, namely ionic strength, pH adjustment and interfering species suppression. The advantageous aspects of combining a SIFA system with potentiometry with enhanced simplicity, ease of implementation and automation were further discussed and emphasised.The obtained results showed relative deviations lower than 5%, for both types of samples, with sampling rates of about 40 h⁻¹.In addition, an innovative and straightforward process for constructing plastic membrane ion selective electrodes with a tubular configuration able to be coupled to flow-based analytical systems is also proposed. The developed approach, consisting of assembling the electrode inside a flow tubing connector is very simple to implement, robust, particularly adequate to be combined with flow methodologies and maintains all dynamic and analytical characteristics exhibited by previous assembling processes.     

Simultaneous determination of fermented milk aroma compounds by a potentiometric sensor array

The paper reports on the application of an electronic tongue for simultaneous determination of ethanol, acetaldehyde, diacetyl, lactic acid, acetic acid and citric acid content in probiotic fermented milk. The αAstree electronic tongue by Alpha M.O.S. was employed. The sensor array comprised of seven non-specific, cross-sensitive sensors developed especially for food analysis coupled with a reference Ag/AgCl electrode. Samples of plain, strawberry, apple-pear and forest-fruit flavored probiotic fermented milk were analyzed both by standard methods and by the potentiometric sensor array. The results obtained by these methods were used for the development of neural network models for rapid estimation of aroma compounds content in probiotic fermented milk.The highest correlation (0.967) and lowest standard deviation of error for the training (0.585), selection (0.503) and testing (0.571) subset was obtained for the estimation of ethanol content. The lowest correlation (0.669) was obtained for the estimation of acetaldehyde content. The model exhibited poor performance in average error and standard deviations of errors in all subsets which could be explained by low sensitivity of the sensor array to the compound. The obtained results indicate that the potentiometric electronic tongue coupled with artificial neural networks can be applied as a rapid method for the determination of aroma compounds in probiotic fermented milk.