A new simple and reliable in-situ mercury film sensor coupled with affinity differential pulse stripping voltammetry (ADSPV) or affinity cyclic voltammetry (ACV) was investigated. The interaction of fenoprofen with bovine serum albumin (BSA) onto the proposed electrochemical sensor was studied. The nature of the electrochemical process of fenoprofen by cyclic voltammetry was depicted. Reproducibility of the proposed method was checked giving a precision of 0.073 standard deviation. The limit of detection and limit of quantification were 7.0 and 22.0 nmol/L, respectively. Fenoprofen was interacted with BSA by 1:1 stoichiometry to form electroinactive supramolecular complex. The binding constant was precisely estimated by non-linear regression analysis based on the shifting of analyte peak potentials. The proposed experiments and data analysis could be used to investigate the drug-protein binding constant within a short analysis time compared to other chromatographic techniques.
Fenoprofen is a nonsteroidal antiinflammatory drug that is effective for treating the fever, pain, and swelling caused by inflammation. Fenoprofen was approved by the Food and Drug Administration (FDA) in March 1976. Fenoprofen blocks the enzymes that make prostaglandins (cyclooxygenases), resulting in lower concentrations of prostaglandins. As a consequence, inflammation, swelling, pain and fever are reduced. It is a propionic acid derivative (
hydroxypropyl derivatives of cyclodextrin are very often used in pharmacy due to their better complexation ability than that of natural cyclodextrin and they increase the solubility and bioavailability more than natural cyclodextrin [
Interaction of drugs with protein has recently aroused great interest to understand their bioavailability and has considered one of the essential steps of drug discovery [
Only one paper was dealt with the calculation of fenoprofen-protein binding constants by separation approaches [
In the present study, a new simple, sensitive and reliable ADPSV method for the determination of fenoprofen in aqueous media at in-situ mercury film sensor was investigated. The precise binding constant of fenoprofen with BSA was calculated by coupling ACV or ADSPV with non linear regression analysis based on the changes of electrochemical responses of analyte onto in-situ mercury film sensor.
All voltammetric investigations were performed in a 10.0 mL glass voltammetric cell using commercial available electrode stand (Metrohm, Switzerland). The electrode was connected via IME-663 module (Netherlands). Potentials were controlled using a 3-electrode configuration comprising a rotating glassy carbon disc working electrode (3 mm diameter, Metrohm), a Ag/AgCl (3.00 M KCl) reference electrode and a platinum wire counter electrode. Turbidity was obtained by using turbidity and chlorine benchtop meter, LaMotte LTC-3000we, 0 - 4000 NTU & 0 - 10.0 µg/mL. The pH’s were measured using the Fischer Scientific pH meter model 810 equipped with a combined glass electrode, which was calibrated regularly with buffer solutions (pH = 4.0 and 7.0) at 22˚C ± 2˚C.
Highly purified L-fenoprofen was produced by Western pharmaceutical industries, Egypt. A fresh solution of fenoprofen was prepared daily in 10.0 mL of 0.25 mM hydroxypropyl-β-cyclodextrine (HPβCD) and was diluted as required for quantitative analysis. Bovine serum albumin (BSA, 99%, Sigma) was used as received without further purification. The 1.0 g/L stock solution of BSA was prepared by dissolving it directly in twice distilled water and was stored at 4.0˚C. The working solutions were obtained by diluting the stock solution with phosphate buffer. A physiological concentration of 67.0 mmol/L phosphate buffer solution was used to control the pH of the solutions tested. It was prepared by mixing definite weights of Na2HPO4 (Sigma) and NaH2PO4 (sigma) at each desired pH value. Stock solution of mercuric ion (10−2 mol/L) was prepared by dissolving the required weight of basic nitrate (May & Baker Ltd., Dagenham, UK) in twice distilled water. All other reagents were of analytical reagents grade.
The glassy carbon electrode (GCE) was polished at the beginning of the experiments with 0.05 µm aluminum oxide (particle size = 0.10 µm, Metrohm, Switzerland) and was rinsed thoroughly with water to obtain a clean and renewed the electrode surface. The electrode was connected to the potentiostat and placed in the buffer solution; the potential was cycled 50X between −0.8 to +0.8 V using cyclic voltammetry at a scanning rate of 0.1 V/s. The electrochemical pretreatment was repeated daily, the polishing only when damage of the electrode surface was suspected. The in-situ mercury film sensor was prepared by pippeting 10.0 mL of the phosphate buffer at physiological pH into the cell followed by the simultaneous depositing of 30.0 µmol/L mercury (II) and fenoprofenHPβCD complex on GCE. Oxygen was removed by purging 5.0 min with nitrogen and then deposition was carried out for 60.0 s at −0.5 V, whilst the electrode was rotated at 700.0 rpm. Subsequently after 10.0 s equilibrium time the stripping step was cathodically performed from −0.5 to −1.35 V using a pulse height 50.0 mV. Each scan was preceded by an electrochemical cleaning step to remove the previous film by applying constant potential at 0.5 V for 30.0 s. All measurements were performed under ambient conditions.
Into a 10.00 mL volumetric cell, 0.5 mL of 100.0 mmol/L fenoprofen dissolved in HPβCD and an appropriate amount of BSA were added. The mixture was diluted with 67.0 mmol/L phosphate buffer, pH = 7.4 and mixed homogeneously by rotation for 5.0 s. Subsequently, the differential or cyclic voltammetric curves were recorded to show the electrochemical changes of the reaction system. As well, such interaction was studied by titration of different fenoprofen concentrations in the presence of fixed concentration of BSA.
Fenoprofen is freely soluble in alcohol and slightly soluble in water causing a limitation to study its interaction with biomolecules such as protein. Macromolecules such as cyclodextrines could use to solve this limitation. In the present work, HPβCD was used to enhance the solubility of fenoprofen due to its high solubility in aqueous medium and low cost. The optimization of HPβCD amount was simply achieved by turbidimetry. An amount of fenoprofen (0.05 mmol/L) was added to 67.0 mmol/L phosphate buffer solution (pH = 7.4) containing varied concentrations of HPβCD in the range of 0.01 - 1.0 mmol/L. The suspensions were shaken under ambient conditions. Consequently, the solubility was followed by measuring the turbidity value indicating that the solubility of fenoprofen increased linearly with increasing concentrations of HPβCD. Solubility of fenoprofen was enhanced from 2.7% with 0.01 mmol/L HPβCD to 100.00% with 0.3 mmol/L HPβCD which was used as an optimal solvent for further experiments. This could be due to the possibility of information of inclusion complex between hydrophobic cavity of HPβCD and fenoprofen as well as a hydrogen bonding between carboxylate group and hydroxyl group on the outer hydrophilic shell of HPβCD (ACA). This concentration of solubilizer is sufficient low to dissolve completely fenoprofen without making any electrochemical interference to the main peak of analyte. Therefore, this inclusion complex could be used for the indirect determination of fenoprofen in aqueous media.
The effect of in-situ mercury film sensor on the determination of fenoprofen was tested by varying the concentration of mercuric ion from 0.3 to 30.0 µmol/L in the presence of 1.0 µmol/L fenoprofen. The effect of thickness of proposed mercury film on the sensitivity and reproducibility of the drug was studied. It was found that the excess mercuric ion concentration (30.0 µmol/L) gave the best thickness of 42.0 nm. Other instrumental parameters were optimized as indicated in the experimental section.
Under the foregoing optimal parameters, the preconcentration of fenoprofen at the proposed sensor was tested. A large response after 60.0 s accumulation time for an essay concentration of 0.02 µmol/L fenoprofen was obtained greater than the direct response (t = 0 s) as indicated in
situ mercury film sensor. Therefore, the proposed sensor could be applied for high throughput analysis of pharmaceuticals within few minutes.
Voltammetric studies were devoted to study the redox behavior of fenoprofen under complete aqueous conditions in the pH range (2.0 - 12.0) at in-situ mercury film sensor. Strong acidic media (below pH = 2.0) were avoided due to the easy hydrolysis of cyclodextrins that yield a series of mixture of oligosaccharides ranging from an opened ring down to glucose. Cyclic voltammetry of 5.0 µmol/L fenoprofen was scanned from −0.5 to −1.35 V with 100.0 mV/s scanning rate at pH = 2.0. Broad cathodic and anodic peaks were appeared in the first scan and the current sharply decreased with repetitive scans. This could be due to the protonation of fenoprofen in strong acidic medium leading to its good solubility. With increasing pH values, the peak current decreased gradually up to pH = 4.0 with the formation of precipitate. This is due to the weak solubility of fenoprofen after its conversion from protonated form to neutral form. Therefore, we modified the solubility of fenoprofen to be in 0.3 mM HPβCD. The nature of the electrochemical process in the presence of HPβCD as solubiliser was carried out by applying CV at different pH values. In order to increase the adsorbed amount of fenoprofen, an accumulation time of 60.0 s was applied. Fenoprofen molecules exhibited quasi-reversible peaks and then peaks disappeared completely in strong alkaline media. The sensitivity of reductive peak is higher than oxidative peak. All peak potentials were slightly shifted by increasing pH values. The effect of scan rate (10.0 - 200.0 mV/s) was tested (
ranged between 0.96 to 0.98 µAsmV−1 that tended to unity and the correlation coefficient was varied between 0.998 and 0.999. This behavior indicated the possibility for a charge transfer controlled electrochemical process. The effect of pH on the cathodic peak current of fenoprofen (5.0 µmol/L) was also studied using differential pulse cathodic voltammetry. The study indicated the best sensitivity of fenoprofen was achieved at pH = 7.4 in the presence of 67.0 mmol/L phosphate buffer. Therefore, further experiments were studied under the physiological conditions.
Well-defined reduction peak was obtained by increasing fenoprofen concentrations in the presence of 67.0 mmol/L phosphate buffer, pH = 7.4. It was found that peak currents increased linearly with increasing their concentrations from 0.02 to 8.0 µmol/L. The linearity equation was found to be: Conc. (nmol/L) = 0.002 Current (µA) + 0.073 with correlation coefficient (r2) = 0.998 and standard deviation (SD) = 0.005. Reproducibility was checked by twenty one measurements within three consecutive days of 2.0 µmol/L fenoprofen under optimal conditions; the standard deviation of 0.073 was obtained. The limit of detection (LOD) was calculated by using the equation LOD = 3.3 SD/a [
The accuracy of the proposed method was determined by applying the optimized analytical approaches with three spiking replicates at three concentration levels covering the linearity range. The obtained mean recoveries of data collected by replicating the procedure within three consecutive days were ranged from 98.3% to 104.2%.
The selectivity was checked by adding the possible interfering ions or organic compounds, which are of great significance in biological matrices up to 200-fold of fenoprofen concentration. The effect of inorganic cations and anions like Na+, K+, Fe3+, Zn2+, Ca2+, , Cl−, and were studied. The presence of cations and anions had no influence on the peak of the investigated compounds. Gelatin and other surfactants like SDS, triton X-100 and CTAB, which can affect on the drug determination had no influence till their concentrations exceed 100-fold fenoprofen. Therefore, the proposed method could be used for the simple, rapid and precise determination of fenoprofen in aqueous media.
After appropriate experimental conditions were established, several practical considerations were investigated. The most effective parameters were the protein’s solubility in the supporting electrolyte, the electrochemical response of protein and the saturation of the drug-protein complex. For the protein’s solubility in phosphate buffer under physiological conditions, there was no problem when working with BSA below the solubility limit of the protein (100.0 µmol/L). The electrochemical response of protein was a second practical item that affects the affinity experiments. It was tested by scanning the cyclic voltammetry of 30.0 µmol/L BSA under optimal conditions. It was found that BSA had a quasi-irreversible electrochemical behavior with potentials far from the potential of drug.
The interaction of BSA macromolecule with fenoprofen was studied by adding several BSA concentrations to definite concentration of the drug and running voltammograms in the cathodic potentials under the optimal conditions as described in the experimental section. Some typical voltammograms obtained with the studied system at different protein concentrations were shown in
There may be three different explanations for the de-
crease of the reductive peak currents after the reaction of fenoprofen with BSA [
The stoichiometry of fenoprofen-BSA supramolecular complex was achieved by using molar ratio method. In such experiments, different concentrations of drug were added to a constant concentration of protein. Results of molar ratio graph investigated that fenoprofen interacts with BSA by 1:1 stoichiometry to form supramolecular complex.
As higher concentrations of protein were used, the extent of drug—protein binding increased and a shift in potential was observed. These shifts in potential and their relation to binding affinity made such studies useful in the determination of drug—protein binding constant by affinity voltammetry. The analysis of data for the calculation of binding constants was achieved by using four mathematical plotting models [
Pf is the peak potential of drug in the absence of protein; Pi is the peak potential of drug at a definite protein concentration; Pc is the peak potential of drug at the saturated protein concentration; c(L) is the micro-molar concentration of protein.
between free fenoprofen and free BSA. It is well observed from the data cited in
A new simple and reliable analytical method as an application of electrochemical in-situ mercury film sensor was used for the determination of binding constants between fenoprofen and BSA. The current paper demonstrated how affinity cyclic voltammetry (ACV) and/or affinity differential pulse voltammetry (ADPV) combined with the non-linear regression method on the proposed electrochemical sensor can be successfully used for the high throughput of drug-protein binding constants.