Modified Edge Plane Pyrolytic Graphite Research Articles

A novel and intelligent chemometric-electrochemical-enzymatic biosensing procedure and mimicking a clinical condition environment to trick the red blood cells for counting them under physiological conditions: A new connection among chemometry, electrochemistry and hematology

Here, a novel electrochemical biosensing procedure has been developed for determination of the number of red blood cells (RBCs) under physiochemical conditions based on chemometric modeling of hydrodynamic differential pulse voltammetric (HDPV), and amperometric data as responses of a modified edge plane pyrolytic graphite electrode (EPPGE). In order to obtain a good sensitivity from the EPPGE, its surface was modified with a thin layer of multiwalled carbon nanotubes-ionic liquid (MWCNTs-IL). Catalase (CAT) was immobilized onto the surface of MWCNTs-IL/EPPGE with help of nafion. The response of the biosensor was based on electrochemical reduction of oxygen of the blood samples which was enhanced by a trick based on addition of hydrogen peroxide (H2O2) to blood samples which can be reduced by the CAT to produce extra oxygen. Prior to experiments, the solution in electrochemical cell was bubbled with pure N2 to purge the oxygen in the solution, but in order to increase the selectivity of the biosensor towards detection of the oxygen obtained from the red blood cells, voltammetric responses of the biosensor were modeled by multivariate chemometric calibration methods with the help of radial basis function-partial least squares (RBF-PLS), least squares-support vector machines (LS-SVM), recursive weighted partial least squares (rPLS), ant colony optimization-mathematical pre-processing selection by genetic algorithm-sample selection through a distance-based procedure-partial least squares-1 (ACO-GA-SS-PLS1), and radial basis function-artificial neural networks (RBF-ANN) to select the best method for determination of the number of the RBCs. The results confirmed the amperometric methods modeled by RBF-ANN showed the best performance for supporting the biosensor in determination of the number of the RBCs with a performance which had an excellent compatibility with the results of a hemocytometer. The results of this study as the newest application of chemometric-electrochemical methods can make a strong connection among electrochemists, chemometricians and hematologists to expand their collaborations on determination of blood factors.

Development and first application of the edge plane pyrolytic graphite electrode modified with graphene nanoplatelets for highly sensitive voltammetric determination of oxolinic acid

In this paper, a graphene nanoplatelets (GNPs) were used as a modifier of an edge plane pyrolytic graphite electrode (EPPGE) surface, and the graphene nanoplatelets modified edge plane pyrolytic graphite electrode (GNPs–EPPGE) was prepared by simple drop casting procedure. The as-prepared GNPs–EPPGE was characterized by a cyclic voltammetry (CV), a scanning electron microscopy (SEM), and an atomic force microscopy (AFM). The electrochemical behavior of OxA on the EPPGE and the GNPs–EPPGE was performed using the cyclic voltammetry and an electrochemical impedance spectroscopy (EIS). Further, a sensitive and selective square–wave voltammetric (SWV) procedure using the GNPs–EPPGE was developed for the determination of a quinolone antibiotic oxolinic acid (OxA). An oxidation peak of OxA was observed in Britton–Robinson buffer solution with the highest response at pH 5.0 at potential c.a. +1.4 V vs. Ag/AgCl reference electrode. Compared to the unmodified EPPGE, the EPPGE modified GNPs exhibited a marked enhancement in the current response of OxA. Calibration curve on the GNPs–EPPGE was constructed in the concentration range of 3.0 × 10−7–9.1 × 10−6 mol L−1 with a correlation coefficient of 0.9997, a sensitivity of 1.39 μA L μmol−1, a limit of detection (LOD) of 5.2 × 10−8 mol L−1, and a limit of quantification (LOQ) of 1.7 × 10−7 mol L−1. The determination of OxA in the spiked human urine samples was carried out using the GNPs–EPPGE with the satisfactory recovery of 99.6%. The selectivity of the developed method for the determination of OxA using the GNPs–EPPGE was tested.

A Simple and Highly Selective Determination of Telmisartan at Sodium Dodecyl Sulfate Modified Pyrolytic Graphite Surface

AbstractA simple, facile, selective and cost effective electrochemical method is proposed for the determination of telmisartan (TMS); a drug used for hypertension. A sodium dodecyl sulfate (SDS) modified edge plane pyrolytic graphite (EPPG) is prepared by simple immersion of EPPG in SDS solution at concentration greater than critical micelle concentration (CMC). The modified sensor exhibited superior sensing properties towards the oxidation of TMS. The modified surface was characterized by using the Energy dispersive X‐ray analysis, Field Emission Scanning Electron Microscopy, Electrochemical Impedance Spectroscopy and cyclic voltammetry. The quantitative investigations of the TMS were performed by applying the square wave voltammetry. The micelles of SDS form a pseudo complex with cation radical of TMS and catalyse the oxidation. The proposed sensor shows the linear calibration plot in the concentration range of 5–100 μM with sensitivity 0.2983 μA/μM and the limit of the detection of the sensor was found to be 0.082 μM. The specificity of the developed sensor was also evaluated in the presence of commonly present interfering substances in biological samples. The amount of TMS excreted in urine of the patients undergoing treatment has also been determined. The proposed method can be effectively applied for the investigation of TMS in pharmaceutical formulations and biological samples.

A novel graphene and conductive polymer modified pyrolytic graphite sensor for determination of propranolol in biological fluids

A simple and sensitive method for the determination of propranolol using graphene and conductive polymer (poly-1,5-diaminonaphthalene) modified edge plane pyrolytic graphite (EPPG) sensor is presented. The electrochemical measurements and surface morphology of modified sensor was studied using square wave voltammetry, cyclic voltammetry, electrochemical impedance spectroscopy (EIS) and field emission scanning electron microscopy (FE-SEM). The modified sensor exhibited excellent catalytic activity toward electrochemical oxidation of propranolol in phosphate buffer of pH 7.2. The modified sensor facilitated the determination of propranolol in the concentration range 0.1–750μM and a detection limit and sensitivity of 20nM and 0.062μA/μM were achieved. The proposed method was successfully employed to detect propranolol in pharmaceutical samples, human urine and blood plasma samples. The method showed tremendous reproducibility and intra- and inter-day stability and has proved to be highly reliable for the analysis of propranolol in clinical samples.

A novel nanogold–single wall carbon nanotube modified sensor for the electrochemical determination of 8-hydroxyguanine, a diabetes risk biomarker

An electrochemical study of the oxidation of 8-hydroxyguanine (8-OH-Gua) at gold nanoparticles attached to single walled carbon nanotube modified edge plane pyrolytic graphite electrode (AuNP-SWCNT/EPPGE) has been carried out to develop a method for the self diagnosis of diabetes. The level of 8-OH-Gua, an important biomarker of oxidative DNA damage, is higher in urine of diabetic patients than control subjects. A detailed comparison has been made between the square wave voltammetric (SWV) response of SWCNT/EPPGE and AuNP-SWCNT/EPPGE towards the oxidation of 8-OH-Gua in respect of several essential analytical parameters viz. sensitivity, detection limit, peak current and peak potential. The AuNP-SWCNT/EPPGE exhibited a well defined anodic peak at potential of ~221mV for the oxidation of 8-OH-Gua as compared to ~312mV using SWCNT/EPPGE at pH=7.2. Under optimized conditions linear calibration curve for 8-OH-Gua is obtained over a concentration range of 0.01–10.0nM in phosphate buffer solution (PBS) of pH=7.2 with detection limit and sensitivity of 5.0 (±0.1) pM and 4.9 (±0.1) μAnM−1, respectively. The oxidation of 8-OH-Gua occurred in a pH dependent process and the electrode reaction followed adsorption controlled pathway. The electrode exhibited an efficient catalytic response with good reproducibility and stability. The method has been found selective and successfully implemented for the determination of 8-OH-Gua in urine samples of diabetic patients.

In vitro chloramphenicol detection in a Haemophilus influenza model using an aptamer-polymer based electrochemical biosensor

A sensitive and selective electrochemical biosensor is developed for the determination of chloramphenicol (CAP) exploring its direct electron transfer processes in in-vitro model and pharmaceutical samples. This biosensor exploits a selective binding of CAP with aptamer, immobilized onto the poly-(4-amino-3-hydroxynapthalene sulfonic acid) (p-AHNSA) modified edge plane pyrolytic graphite. The electrochemical reduction of CAP was observed in a well-defined peak. A quartz crystal microbalance (QCM) study is performed to confirm the interaction between the polymer film and the aptamer. Cyclic voltammetry (CV) and square wave voltammetry (SWV) were used to detect CAP. The in-vitro CAP detection is performed using the bacterial strain of Haemophilus influenza. A significant accumulation of CAP by the drug sensitive H. influenza strain is observed for the first time in this study using a biosensor. Various parameters affecting the CAP detection in standard solution and in in vitro detection are optimized. The detection of CAP is linear in the range of 0.1–2500nM with the detection limit and sensitivity of 0.02nM and 0.102µA/nM, respectively. CAP is also detected in the presence of other common antibiotics and proteins present in the real sample matrix, and negligible interference is observed.

Polymelamine modified edge plane pyrolytic graphite sensor for the electrochemical assay of serotonin

A sensitive and novel electrochemical method has been developed for the determination of an important neurotransmitter, serotonin, using a polymelamine modified edge plane pyrolytic graphite sensor (EPPGS). Melamine was used for the modification of sensor by electropolymerizing it at the surface of EPPGS in acidic medium to form a layer of conducting polymer. Field emission scanning electron microscopy (FE-SEM) and electrochemical impedance spectroscopy (EIS) were used for the characterization of the surface of polymer modified sensor. The electrochemical measurements were carried out using square wave voltammetry and cyclic voltammetry. The polymelamine modified sensor exhibited excellent electrocatalytic activity towards the electrochemical oxidation of serotonin, exhibiting a larger peak current and shift of peak potential to less positive potentials as compared to the unmodified sensor. The dynamic range for the serotonin determination was found between 1-100 µm and 0.1-100 µm with detection limit of 492 nM and 30 nM for unmodified and polymer modified sensors, respectively. The determination of serotonin in human blood serum and urine has been carried out. The common metabolites such as ascorbic acid, dopamine, xanthine and hypoxanthine do not interfere in the determination up to 10-fold concentration, revealing good selectivity of the proposed sensor.

Electrochemical mechanism and sensitive assay of antiretroviral drug Abacavir in biological sample using multiwalled carbon nanotube modified pyrolytic graphite electrode

Multiwalled carbon nanotube modified edge plane pyrolytic graphite electrode (MWCNT/EPPGE) was developed and implemented for the determination of antiretroviral drug Abacavir using differential pulse adsorptive stripping voltammetry (DP AdSV). Cyclic voltammogram showed two oxidation peaks located at+0.91V and+1.10V on (MWCNT/EPPGE). Dependency of peak potential and peak current on pH was investigated as details. The oxidation potentials affected by the pH indicating protons are involved in the electrochemical oxidation of Abacavir. Mass transport to the electrode surface was found as a combination of diffusion and adsorption which was concluded by studying the potential scan rate dependency of both peak currents. The oxidation mechanism was proposed using the obtained data and discussed. A large enhancement in both of the peak currents was observed with applying an accumulation step presenting the effect of adsorption. Peak currents showed a linear relation upon the Abacavir concentration within a range of 1×10−7–2×10−5M with (r=0.999) for both peak responses. The method was fully validated related with selectivity, sensitivity, precision and accuracy studies. Abacavir was also determined with the proposed method in its pharmaceutical dosage forms and human serum samples. No interferences from the excipients of the dosage form or endogenous substances from biological material were found.

Electrochemical Investigations of the Anticancer Drug Idarubicin Using Multiwalled Carbon Nanotubes Modified Glassy Carbon and Pyrolytic Graphite Electrodes

AbstractThe effect of surface modifications on the electrochemical behavior of the anticancer drug idarubicin was studied at multiwalled carbon nanotubes modified glassy carbon and edge plane pyrolytic graphite electrodes. The surface morphology of the modified electrodes was characterized by scanning electron microscopy. The modified electrodes were constructed for the determination of idarubicin using adsorptive stripping differential pulse voltammetry. The experimental parameters such as supporting electrolyte, pH, accumulation time and potential, amount of carbon nanotubes for the sensitive assay of idarubicin were studied as details. Under the optimized conditions, idarubicin gave a linear response in the range 9.36×10−8–1.87×10−6 M for modified glassy carbon and 9.36×10−8–9.36×10−7 M for modified edge plane pyrolytic graphite electrodes. The detection limits were found as 1.87×10−8 M and 3.75×10−8 M based on modified glassy carbon and edge plane pyrolytic graphite electrodes, respectively. Interfering species such as ascorbic acid, dopamine, and aspirin showed no interference with the selective determination of idarubicin. The analyzing method was fully validated and successfully applied for the determination of idarubicin in its pharmaceutical dosage form. The possible oxidation mechanism of idarubicin was also discussed. The results revealed that the modified electrodes showed an obvious electrocatalytic activity toward the oxidation of idarubicin by a remarkable enhancement in the current response compared with bare electrodes.

Carbon nanotube-based electrochemical sensor for the determination of halobetasol propionate, a topical corticosteroid

Electrocatalytic reduction of halobetasol propionate (HBP) at single-walled carbon nanotube (SWCNT) modified edge plane pyrolytic graphite electrode (EPPGE) was performed by square wave voltammetry and cyclic voltammerty in phosphate buffer of pH 7.2. The surface morphology of SWCNT/EPPGE was characterized by field emission scanning electron microscopy. The enhanced peak current (ip) and lower reduction peak potential (Ep) at modified electrode as compared to bare electrode were obvious evidences for the electrocatalytic ability of SWCNT toward the reduction of HBP. The cathodic peak current varied linearly with concentration of HBP in the range 0.02 to 1 mM with sensitivity of 2.432 μA mM−1 and detection limit (3σ/slope) of 10 μM. The product of electrochemical reduction of HBP was characterized using FT-IR and 1H-NMR spectroscopic techniques. A tentative mechanism for the formation of product was suggested and it was found that reduction of >C=O occurred at position three. The proposed methodology was successfully applied to the detection of HBP in pharmaceutical preparations.

Electrochemical Sensor Based on Oxidation of 2,8‐Dihydroxyadenine to Monitor DNA Damage in Calf Thymus DNA

AbstractA novel and reliable direct electrochemical method has been established to monitor DNA damage in acid hydrolyzed calf thymus DNA, based on the determination of 2,8‐dihydroxyadenine (2,8‐DHA). A single‐wall carbon nanotubes (SWCNT) modified edge plane pyrolytic graphite electrode (EPPGE) has been used as a sensor to monitor the DNA damage. 2,8‐DHA the main in vivo adenine oxidation product undergoes oxidation at ∼395 mV at SWCNT modified EPPGE using square wave voltammetry (SWV). The sensor exhibits potent and persistent electron‐mediating behavior. A well‐defined oxidation peak for the oxidation of 2,8‐DHA was observed at modified electrode with lowering of peak potential and increase in peak current as compared to bare EPPGE. At optimal experimental conditions, the catalytic oxidative peak current was responsive with the 2,8‐DHA concentrations ranging from 0.05 nM to 100 nM. The detection limit was 3.8×10−11 M and limit of quantification was 1.27×10−10 M. The modified electrode exhibited high stability and reproducibility.

Application of modified pyrolytic graphite electrode as a sensor in the simultaneous assay of adenine and adenosine monophosphate

A simple and sensitive method based on square wave voltammetry (SWV) at single-walled carbon nanotube (SWNT) modified edge plane pyrolytic graphite electrode (EPPGE) is proposed for the simultaneous determination of adenine and adenosine-5′-monophosphate (5′-AMP). The modified electrode exhibits remarkable electrocatalytic properties towards adenine and 5′-AMP oxidation with a peak potential of ∼850 and 1165 mV respectively. Linear calibration curves are obtained over the concentration range of 5–100 nM for adenine and 10–100 nM for 5′-AMP with sensitivity of 677 and 476 nA nM −1 for adenine and 5′-AMP respectively. The limit of detection for adenine and 5′-AMP was found to be 37 × 10 −10 M and 76 × 10 −10 M, respectively. The effect of pH revealed that the oxidation of adenine and 5′-AMP at SWNT modified EPPGE involved equal number of electrons and protons. The modified electrode exhibited high stability and reproducibility.

Simultaneous determination of epinephrine and norepinephrine in human blood plasma and urine samples using nanotubes modified edge plane pyrolytic graphite electrode

The simultaneous determination of catecholamines – epinephrine and norepinephrine by square wave voltammetry (SWV) at physiological pH 7.2 is reported using multi-walled carbon nanotubes modified edge plane pyrolytic graphite electrode (MWNT/EPPGE). A broad bump at ∼250 mV is appeared for the oxidation of epinephrine (EP) and norepinephrine (NE) at bare EPPGE whereas at MWNT/EPPGE two well-separated peaks at ∼150 and ∼215 mV are appeared for the oxidation of EP and NE, respectively. The oxidation peak current of both the neurotransmitters also increased significantly along with the negative shift of peak potentials using MWNT/EPPGE. The oxidation of both compounds occurred in a pH dependent, 2e and 2H + process and the electrode reaction followed diffusion controlled pathway. Linear calibration curves were obtained for epinephrine and norepinephrine in the range 0.5–100 nM with limits of detection 0.15 × 10 −9 and 0.90 × 10 −10 M, respectively. The developed protocol is implemented for the simultaneous determination of epinephrine and norepinephrine in blood plasma and urine samples of smokers as well as in athletes.

A Sensitive Voltammetric Sensor for Detecting Betamethasone in Biological Fluids

A rapid and sensitive voltammetric sensor based on reduction of betamethasone has been developed using single wall carbon nanotube modified edge plane pyrolytic graphite electrode (SWNT/EPPGE). The reduction of betamethasone occurred in a well-defined, pH dependent peak. Linear calibration curve was obtained in the range 1 to 25 nM in 1.0 M phosphate buffer solution (PBS) of pH 7.2 with the limit of detection (3sigma / slope) as 0.50 nM. The analytical utility of the developed method has been demonstarted by sensing the drug in human body fluids and for the determination of betamethasone content in several commercially available pharmaceutical preparations. Interfering effect of some common metabolites including ascorbic acid, uric acid, albumin and hypoxanthine has also been evaluated. A comparison of the observed results of proposed method with HPLC clearly indicates that the results of both methods are essentially similar.

Sensitive voltammetric sensor for the determination of oxidative DNA damage in calf thymus DNA

A simple and reliable method based on voltammetry is proposed for the determination of oxidative DNA damage by the simultaneous determination of guanine and 8-hydroxygunine using single-walled carbon nanotubes modified edge plane pyrolytic graphite electrode (SWNT/EPPGE). In acid-hydrolyzed calf thymus DNA, two well-defined peaks at ∼312 and ∼502 mV corresponding to the oxidation of 8-hydroxyguanine and guanine, respectively in phosphate buffer solution (PBS) of pH 7.2 were observed in square wave voltammetry. Remarkable enhancement in the oxidation peak current of both the compounds was observed along with the negative shift of peak potentials using SWNT/EPPGE as compared to bare EPPGE. The detection limits of guanine and 8-hydroxyguanine were calculated to be 0.05 × 10 −9 and 0.01 × 10 −9 mol L −1, respectively. The limits of quantification were found as 0.17 × 10 −9 and 0.34 × 10 −10 mol L −1 for guanine and 8-hydroxyguanine, respectively.

Voltammetric determination of amlodipine besylate in human urine and pharmaceuticals

Electrochemical determination of amlodipine besylate (ADB) using single and multi-walled carbon nanotubes modified edge plane pyrolytic graphite electrodes (SWNT/EPPGE and MWNT/EPPGE) is described by using cyclic and square wave voltammetries at physiological pH 7.2. An increased peak current with a shift of peak potential to less positive value was observed using carbon nanotubes modified EPPGE as compared to bare electrode. The effect of pH, scan rate and analyte concentration has been examined. Under the optimum conditions the peak current was linear to the concentration of amlodipine in the range 5.0 × 10 − 9 to 1.0 × 10 − 6 mol L − 1 for SWNT/EPPGE and the detection limit was found to be 1.0 × 10 − 9 mol L − 1 whereas, for MWNT/EPPGE the detection limit was found to be 5.0 × 10 − 9 mol L − 1 . The method was successfully used to determine the content of amlodipine in the pharmaceutical preparations and human urine samples of angina patients undergoing treatment with amlodipine. A comparison of electrocatalytic activities of SWNT and MWNT modified electrodes indicated that SWNT modified EPPGE is ∼ 1.8 times more sensitive in comparison to MWNT/EPPGE. Biological relevance of the developed method has been described by the determination of amlodipine in human body fluids. Amlodipine can be determined without any interference from common urine metabolites such as uric acid, ascorbic acid and xanthine.

Voltammetric biosensors for the determination of paracetamol at carbon nanotube modified pyrolytic graphite electrode

The voltammetric oxidation of paracetamol on single-walled carbon nanotubes (SWNT) modified edge plane pyrolytic graphite electrode (EPPGE) was explored in phosphate buffer solution by using square wave voltammetry. Cyclic and square wave voltammetry studies indicated the oxidation of paracetamol at the electrode surface through a two-electron reversible step and fundamentally controlled by adsorption. Besides semi-infinite planar diffusion, the role of thin layer diffusion at nanotube modified electrodes is also suggested. The sensitivity at SWNT modified EPPGE is ∼2 times more than that at MWNT modified EPPGE. Paracetamol gave a sensitive oxidation peak at ∼187 mV at pH 7.2 ( μ = 0.5 M) which was used to quantitate the drug in the range of 5–1000 nM with a detection limit of 2.9 × 10 −9 M at SWNT modified EPPGE. The interfering effect of physiologically common interferents on the current response of paracetamol has been reported. The procedure was successfully applied for the assay of paracetamol in pharmaceutical formulations. The applicability of the developed method to determine the drug in human urine samples obtained after 4 h of administration of paracetamol is illustrated.

Effect of single walled carbon nanotube–cetyltrimethyl ammonium bromide nanocomposite film modified pyrolytic graphite on the determination of betamethasone in human urine

A sensitive voltammetric method has been described for the determination of betamethasone sodium phosphate (BSP) using edge plane pyrolytic graphite electrode (EPPGE) modified with single walled carbon nanotubes–cetyltrimethyl ammonium bromide nanocomposite film. The reduction of BSP occurred in a well-defined peak having E p −1062 mV in phosphate buffer solution (PBS) of pH 7.2. The voltammetric response of betamethasone sodium phosphate enhanced effectively using cationic surfactant cetyltrimethyl ammonium bromide (CTAB) as electrode surface modifier. The nanotubes-surfactant modified edge plane pyrolytic graphite electrode shows great improvement in peak current and shift of the reduction potential towards less negative potential. The role of cetyltrimethylammonium bromide on electrocatalytic property is discussed. The limit of detection (3 σ/slope) was found to be 0.25 × 10 −9 M and limit of quantification was 0.86 × 10 −9 M. The analytical utility of the proposed method is demonstrated by the direct assay of betamethasone in urine samples of pregnant women.

Electrochemical investigations of corticosteroid isomers—testosterone and epitestosterone and their simultaneous determination in human urine

Voltammetric investigation of two corticoid isomers—testosterone and epitestosterone has been carried out at bare and single-wall carbon nanotubes (SWNT)-modified edge plane pyrolytic graphite electrode (EPPGE). Square wave voltammetry (OSWV) has been used for the simultaneous determination of isomeric steroids. The reduction of the two isomers occurred in a pH dependent, 2e, 2H + process and well-defined voltammetric peaks were observed. Under the optimum experimental conditions, linear calibration curves are obtained within the concentration range 5–1000 nM for both the steroids with the limit of detection 2.8 × 10 −9 and 4.1 × 10 −9 M for testosterone and epitestosterone respectively. The developed protocol is successfully implemented for the analysis of both the compounds in the urine samples of normal subjects as well as in patients undergoing treatment with testosterone. The results obtained from the proposed voltammetric method were also compared with HPLC analysis and found to be similar.

A single-wall carbon nanotubes modified edge plane pyrolytic graphite sensor for determination of methylprednisolone in biological fluids

An electrochemical protocol based on reduction is developed to determine methylprednisolone using single-wall carbon nanotubes (SWNTs) modified edge plane pyrolytic graphite electrode (EPPGE). To obtain a good sensitivity, instrumental variables were studied using Square Wave Voltammetry (SWV). The voltammetric results indicate that SWNTs modified EPPGE remarkably enhances the reduction of methylprednisolone which leads to considerable improvement of peak current with shift of peak potential to less negative values. The voltammetric current showed a linear response for methylprednisolone concentration in the range 5–500 nM with a sensitivity of 98 nA nM −1. The limit of detection was estimated to be 4.5 × 10 −9 M. The developed method is used for the determination of methylprednisolone in pharmaceutical dosages and human blood plasma samples of patients undergoing treatment with methylprednisolone. The major metabolites present in blood plasma did not interfere with the present investigation as they did not exhibit reduction peak in the experimental range used. A comparison of results with high performance liquid chromatography (HPLC) indicates a good agreement.