FEB is a non-hygroscopic, white crystalline powder that is soluble in dimethylformamide and dimethylsulfoxide; slightly soluble in acetonitrile and methanol; sparingly soluble in ethanol; and practically insoluble in water. Several FEB determination methods have been reported. These included UV spectrophotometric methods [2-8], RP-HPLC-UV[9-15], RP-HPLC-MS/MS [1618] and UPLC-tandem mass [19-21].

In our knowledge, no work was found in the literature for the voltammetric determination of FEB, which was characterized in a highly sensitive way, down to ng mL^-1, as described below.

Experimental

Apparatus

A Metrohm voltammeter model 693 VA processor and 694 VA stand (Swiss), equipped with a Ag/AgCl reference electrode stored in a 3.0 M KCl solution, and a platinum counter electrode, as auxiliary electrode, were employed. The hanging mercury drop electrode HMDE with a drop size of 0.30 mm2 was used as working electrode.

A spectrophotometer JASCO V-570 model was used. A Metrohm pH-meter model 691 with a combined electrode (glass-Ag/AgCl electrode) was used for pH measurements (Swiss).

Materials and reagents

All chemicals used were of analytical grade. Febuxostat, B.N EL-03/L017/15004 (98.3 %) was dedicated by Eva Pharma, Egypt. Donifoxate tablets (Eva Pharma, Egypt) were purchased from the local market in two dosage strengths, 40 and 80 mg per tablet.

Universal Britton-Robinson buffer solutions of 0.04 mol L^-1 boric acid; 0.04 mol L^-1 orthophosphoric acid, and 0.04 mol L^-1 acetic acid with pH values 7.0-12.0 were prepared by adjusting with suitable aliquots of 0.05 M sodium hydroxide. Double distilled water was used throughout all experiments.

Standard solutions

A stock solution of standard FEB was first prepared by dissolving 25 mg FEB in 5 mL of 0.05 mol L^-1 sodium hydroxide solution, then completed to 100 mL with distilled water and stored in a dark and cold place. Further dilution was achieved by transferring 1 mL of the stock solution to a 50 mL measuring flask, and completing to the mark with a 2.5 mol L^-1 NaOH solution.

Procedure

Aliquots of a FEB solution corresponding to a concentration range of 125 -2000 ng were sequentially added to a 10 mL BR buffer solution, pH 10, and de-aerated by purging nitrogen gas for 3 min, then pre-concentrated for 60 s at -100 mV, with stirring at 600 rpm. After resting for 10s, differential pulse DP or square wave pulse SQW was scanned from -100 to -600 mV with a scan rate of 60 mV s^-1, pulse amplitude of 50 mV, pulse duration of 40 ms, measurement time of 20 ms, and frequency of 30 Hz. The experiment was triplicate, and the average of corresponding peak current was taken. The concentration and current were plotted to calculate slope, intercept and correlation coefficient.

Validation of the analytical procedure

For the validation of studied methods, the regression parameters, precision, and accuracy were checked by essaying samples with different concentrations of FEB on the same day and different days (between days), over a period of three days, using slope and intercept, as mentioned before.

Tablet essay procedure

Contents of five coated-tablets were weighted, and then thoroughly grounded to a fine powder. A sufficient amount of the powder equivalent to one tablet was accurately weighted, dissolved in 5 mL of a 0.05 mol L^-1 NaOH solution, with a magnetic stirrer for 15 minutes, and then diluted with a sufficient amount of double distilled water, to obtain a final concentration of 400 μg mL^-1 FEB. Further dilution was achieved by transferring 1 mL of the previous solution to a 50 mL measuring flask, and completing to the mark with a 2.5 mol L^-1 NaOH solution. The aliquot of 50 μL of the final solution was transferred to a voltammetric vessel containing a 10 mL BR buffer solution, pH 10, to give a final concentration of 40 ng mL^-1. The experiment was repeated as described in the above voltammetric procedure.

Analysis of human plasma samples

A procedure to extract FEB from spiked human plasma was achieved as described by Monita et al. [14], which can be detailed as it follows: Drug-free plasma samples were obtained from healthy people, and stored frozen in the dark until the essay. 1 mL of the plasma sample spiked with 2.5 μg mL^-1 methanolic solution of FEB was placed in a glass tube of 15 mL capacity, and 5 mL diethylether was added. The contents of the tube were mixed by a vortex mixer for 2 min, shaken by a shaker at 100 strokes per min for 45 min, and then centrifuged for 15 min at 3000 rpm at 4 °C, to precipitate plasma protein residues. The organic layer was pipetted out into a 25 mL beaker, evaporated to dryness in air at room temperature, and re-dissolved in 5 mL 2.5 mol L^-1 NaOH. The solution was then filtered through a PTFE 0.45 μm filter. 1 mL of the filtrate was transferred into a 10 mL measuring flask, and completed to the mark with a BR buffer solution, pH 10. The voltammetric measurement was repeated as described above.

Results and discussion

Different chemical and electrochemical parameters were thoroughly investigated to study the voltammetric behavior of FEB.

Effect of pH

The cathodic peak was also shown on using 0.3 mol L^-1 KCl, but it gave less peak symmetry with a lower peak height, and so, a BR buffer solution of pH 10 was selected in the next studies. No peaks were observed in the anodic direction in the same electrolyte media, suggesting that the electrochemical process is irreversible. To evaluate the number of protons relative to the number of electrons, the following equation is applied:

where, E° is the standard peak potential in Volts, is the number of protons, n is the number of electrons transferred in the mechanism, R is the ideal gas constant (8.314 J K^-1 mol^-1), T is the absolute temperature (298 K), F is Faraday's constant (96485 coulomb mol^-1), [Ox] and [Red] are the equilibrium concentrations of reduced and oxidized species, respectively. The plot of the peak potential Ep versus pH showed one straight line between 8.0 and 11.0, with slope equal to 0.058, which is close to the Nernst theoretical value of 0.059 V pH [22]; the ratio of protons to electrons that participated in the mechanism was calculated as 0.986, indicating a participation of an equal number of protons and electrons in the FEB reduction [23].

Effect of scan rate

For this kind of irreversible mechanism, electron transfer coefficient α, and heterogeneous catalytic reaction rate constant Ks were determined using Laviron's equation [24, 25] as it follows:

Effect of accumulation time

The accumulation time of 60 s was selected.

Stirring effect

The stirring speed of 600 rpm was selected.

Effect of pulsation mode

Validation of the analytical procedure

It is clear that there is no significance difference between intra and inter days in terms of accuracy and precision, and the 95% confidence interval revealed that the source of error is likely random on determining FEB.

Application to dosage form

This is in agreement with that obtained by UV-spectrophotometric method [5] within the accuracy and precision limits.

Application to plasma form

The plasma protein binding of FEB is known to be approximately 99.2%, (primarily to albumin) [21], and in order to evaluate the applicability of the proposed method to extract and determine the bound FEB, the recovery was found to be in the range 70-73%, which is in accordance with that obtained by Monita et al. [14]. It is worth mentioning that FEB was not detectable on spiking the plasma with an aqueous solution of FEB in NaOH. This may be attributed to the formation of an ionic compound which was not extractable by diethylether. This could be solved by spiking with a methanolic solution of FEB.

Conclusions

The reduction of FEB is pH dependent, and it is an irreversible process in a 0.04 M Britton-Robinson buffer solution, involving 4 electrons transfer at HMDE interface, to convert nitrile into amine group. The differential pulse voltammetric method was applied to direct quantification of FEB in tablets and human plasma.

References