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Cathodic adsorptive stripping voltammetric determination of Ribavirin in pharmaceutical dosage form, urine and serum
⁎Corresponding author. Fax: +20 86 2363011. Salwa_kasem2003@yahoo.com (Salwa A. Ahmed)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
A sensitive, simple and rapid square-wave adsorptive stripping voltammetric method was developed and validated for the determination of Ribavirin in pharmaceutical formulations. The proposed method was based on the electrochemical reduction of Ribavirin at a hanging mercury drop electrode in Britton Robinson buffer at pH 10. A well-defined peak was observed at 880 mV with 30 s of accumulation time and 50 mV of accumulation potential. Under these optimized conditions, the square-wave adsorptive stripping voltammetric peak current showed a linear correlation on drug concentration over the range of 1 × 10−10–2 × 10−7 mol L−1 with a correlation coefficient of 0.9995 for the proposed method. The detection and quantitation limits for this method were 2.02 × 10−10 and 6.80 × 10−10 mol L−1, respectively. The results obtained for intra-day and inter-day precision (as RSD %) were between 0.447% and 1.024%. This method was applied successfully for the determination of Ribavirin in its pharmaceutical dosage forms with mean recoveries of 99.68 ± 0.13 with RSD % of 0.81% and 99.20 ± 0.24 with RSD % of 0.49% for two concentrations 5 × 10−9 and 5 × 10−8 mol L−1, respectively for 200 mg capsules. The results obtained from the developed square-wave adsorptive stripping voltammetric method were compared with those obtained by the analytical method reported in the literature.
Keywords
Ribavirin
Square-wave adsorptive stripping voltammetry
Validation
Pharmaceutical formulation
1 Introduction
Ribavirin (RBV), (1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide, also known as virazole) (Fig. 1), is a synthetic purine nucleoside analog first synthesized (Witkowski et al., 1972). It was reported to have broad-spectrum activity against a variety of DNA and RNA viruses (Sidwell et al., 1972; Tam et al., 2001). It has been used in treating respiratory syncytial virus infections in seriously ill pediatric patients, and against influenza A and B virus and Lassa fever virus infections (Gilbert and Knight, 1986) It has been found to be efficacious also in the treatment of hepatitis C virus (HCV) infections, in particular when combined with either interferon α-2a/2b or peginterferon α-2a/2b (Fried et al., 2002; Manns et al., 2001).
The structural formula of Ribavirin.
Studies of the therapeutic effect of the drug required the use of sensitive methods for its determination at trace levels. Several analytical methods for quantitating RBV have been developed; these include bioassay (Smee et al., 1981), radioimmunoassay (Austin et al., 1983), gas chromatography–mass spectrometry (Roboz and Suzuki, 1978), capillary electrophoresis (CE) (Breadmore et al., 2004.) and high performance liquid chromatography (HPLC) with UV (Breadmore et al., 2004; Larra et al., 2003; Granich et al., 1989; Svensson et al., 2000; Inoue et al., 2004) or with mass spectrometric (MS) (D’Avolio et al., 2006; Shou et al., 2002; Lin et al., 2002, Liu et al., 2006; Li et al., 2007) detection. There is no information about the voltammetric determination of RBV at a hanging mercury drop electrode (HMDE) and its analytical applications in the literature.
This work aimed to develop a new square-wave adsorptive stripping voltammetric (SWAdSV) method for the determination of RBV in bulk form, pharmaceutical formulations and biological samples. Furthermore, the determination of RBV using an electrochemical method, especially stripping analysis was not studied before.
2 Experimental
2.1 Apparatus
Square-wave adsorptive stripping voltammetric studies were carried out using AMEL 433 TRACE ANALYSER involving a three-electrode system consisting of a hanging mercury dropping electrode (HMDE) as a working electrode, an Ag/AgCl with saturated KCl as a reference electrode and a platinum wire as a counter electrode. A magnetic stirrer and stirring bar provided the convective transport during pre-concentration. The peak heights were automatically or manually measured using the ‘tangent fit’ capability of the instrument. Cyclic voltammograms were recorded with the same instrument (scan rate 50 mV s−1). All measurements were performed at room temperature (25 ± 1 °C). The pH measurements were made with Accumet® model 825 pH meter.
2.2 Reagents and solutions
All chemicals used for the preparation of buffers and supporting electrolytes were of analytical grade (Merk or Sigma). Bidistilled water was used throughout all experiments. Pure-grade powder of RBV and the pharmaceutical product, namely “Virin® 200 mg” capsules were obtained from Sigma Co., Cairo, Egypt. A stock solution of 1 × 10−3 mol L−1 RBV was prepared by dissolving the required amount of this drug in bidistilled water. Three different buffer solutions, Britton-Robinson (B.R.), ammonia/ammonium chloride and boric acid/NaOH buffers were used.
2.3 General analytical procedure
10 mL of the Britton-Robinson (B.R.) at pH 10 including 0.01 M KCl was taken to the electrochemical cell and the solution was purged with pure nitrogen for 15 min. The required accumulation potential (Eacc. = 50 mV) was applied to the working electrode for a selected accumulation time (tacc. = 30 s), while the solution was stirred continuously at 400 rpm. The stirring was stopped and after equilibrium time of 10 s, a negative-going potential scan was initiated using the following parameters: frequency (f) = 100 Hz; pulse amplitude (Esw) = 100 mV; scan increment (ΔE) = 10 mV. After the voltammogram of supporting electrolyte had been recorded, aliquots of the RBV standard were introduced by micropipette and the square-wave voltammetric cycles were repeated using a new mercury drop. The SWAdSV scan and cyclic voltammetry were conducted from 600 to 1300 mV. All data were obtained at room temperature.
2.4 Analytical applications
2.4.1 Assay of drug in capsule
The whole content of one capsule Virin® (200 mg) was accurately weighed; the required amount from the capsule powder was transferred to a 100 mL volumetric flask and dissolved in bidistilled water. The solution was then filtrated and the residue was washed three times with water, the flask was completed to the mark with water. A suitable volume of the stock solution was pipetted into a 10 mL-measuring flask and the procedure was repeated as described above. The nominal content of the capsule was calculated using standard addition method.
2.4.2 Assay of drug in human urine
A urine sample (10 μL) taken from a healthy person was added to the voltammetric cell containing 10 mL of Britton-Robinson B.R. buffer at pH 10 including 0.01 M KCl, i.e. the dilution factor of the urine sample in the cell was 1:1000. The voltammogram was recorded, then 10 μL spikes of the standard solution of RBV were introduced into the cell and the voltammograms were recorded after each addition.
2.4.3 Assay of drug in spiked human plasma
Serum samples were obtained from a healthy volunteer and stored frozen until assay. After gently thawing, an aliquot volume of sample was fortified with RBV dissolved in bidistilled water to achieve appropriate concentration. The solution was centrifuged for 30 min at 3600 rpm to remove the precipitated serum protein and the supernatant was taken carefully. Appropriate volume of the supernatant liquor was transferred to the voltammetric cell.
3 Results and discussion
3.1 Square-wave stripping voltammetry
3.1.1 Effect of supporting electrolyte and pH
The voltammetric response of drugs is mainly dependent on the pH of the buffer. Therefore, the electrochemical behavior of RBV was evaluated over a pH range of 2.010 at HMDE using the SWAdSV method. RBV exhibited a cathodic peak in B.R. buffer over the pH range of 8.010 (Fig. 2). No peak was observed at pH values 2.07.0. The response was examined in the presence of other buffers, e.g. ammonia/ammonium chloride and boric acid/NaOH. The best result with respect to sensitivity (peak height), resolution (peak shape) and reproducibility was recorded in B.R. buffer solution of pH 10. Therefore, B.R. buffer at pH 10 was chosen as the supporting electrolyte for the optimization of other variables and for analysis of RBV (Fig. 3).
Effect of pH of B.R. buffer on the SWAdCS voltammetric peak current (ip), for 5 × 10−7 mol L−1 RBV at Eacc. = −50 mV, tacc. = 30 s, rest time = 10, scan increment ΔE = 10 mV and pulse amplitude Esw = 100 mV.
Effect of different buffers at pH = 10 on the SWAdCS voltammetric peak current (ip), for 5 × 10−7 mol L−1 RBV. The other conditions were as those indicated in Fig. 2. (a) B.R., (b) ammonia/ammonium chloride and (c) boric acid/NaOH buffers.
The presence of KCl increases the ability of the analyte to adsorb on the electrode surface (Ali, 1999). Therefore the optimal conditions for studying the square-wave adsorptive stripping voltammetry of RBV involve 0.04 M B.R. buffer at pH 10 including 0.01 M KCl.
3.1.2 Optimization of the operating parameters
The dependence of adsorptive stripping peak current of RBV on the accumulation time tacc. was tested at four concentration levels, 2 × 10−6, 2 × 10−7, 2 × 10−8 and 5 × 10−9 mol L−1 RBV from 10 to 60 s at Eacc. = 50 mV (Fig. 4). When the tacc. was increased, a remarkable enhancement was observed for the peak current of RBV up to 30 s and then the peak current leveled off due to the saturation of the surface of the working electrode by the analyte. For further experiments an accumulation time of 30 s was selected as optimal because it provided relatively high peak current with adequate practical time.
Effect of the accumulation timetacc. on the SWAdCS voltammetry peak current (ip) for (a) 2 × 10−6 mol L−1, (b) 2 × 10−7 mol L−1, (c) 2 × 10−8 mol L−1 and (d) 5 × 10−9 mol L−1 RBV in B.R. buffer pH = 10 including 0.01 M KCl, and other operational parameters were as those indicated in Fig. 2.
The influence of Eacc. on the stripping voltammetric signal of 5 × 10−7 mol L−1 RBV was examined over the range of 0 to 600 mV at constant tacc. = 30 s in stirred solution (Fig. 5). The peak current increased steadily and reached its maximum value at 50 mV and then decreased sharply. Hence, Eacc. = 50 mV was chosen for optimal analytical sensitivity.
Effect of the accumulation potentialEacc. on the SWAdCS voltammetry peak current (ip) of 5 × 10−7 mol L−1 RBV in B.R. buffer pH = 10 including 0.01 M KCl, and other operational parameters were as those indicated in Fig. 2.
Study of the effect of scan increment (ΔE) on square-wave stripping peak current of the drug in B.R. at pH 10 including 0.01 M KCl revealed that, the peak current enhanced on the increase of scan increment (212 mV). A scan increment of 10 mV was preferable in the present study. At pulse amplitude(Esw of 100 mV), the peak was found to be much more sharp and defined. Hence, Esw = 100 mV was chosen for the determination of RBV. At 10 mV scan increment and 100 mV pulse amplitude, wave period varied from 50 to 120 ms. The highest peak current was found at 100 ms, which is used in the present study.
In order to obtain the maximum development of the SWAdCS voltammetry peak current, optimization of such variables was attempted. Frequency was found to be 100 Hz using accumulation potential (Eacc.) = −50 mV, tacc. = 30 s, scan increment (ΔE) = 10 mV, and pulse amplitude (Esw) of 100 mV.
3.2 Cyclic voltammetry
A cyclic voltammogram of 5 × 10−6 mol L−1 RBV in 0.04 M B.R. buffer at pH 10 including 0.01 M KCl was recorded. A single cathodic peak, corresponding to the reduction of the adsorbed drug, was observed at −880 mV. No peak was observed in the anodic branch, indicating that RBV reduction is an irreversible process and the desorption of the adsorbed product, inhibits the appearance of the oxidation product. A maximum developed peak current (ip) was achieved after accumulation of the drug onto the electrode surface for 30 s; this behavior confirmed the adsorptive character of the drug at the mercury surface.
The interfacial adsorptive character of RBV onto the HMDE was identified from the peak current (ip) dependence after preconcentration of the drug for 30 s upon the scan rate ν. The plot of log ip vs. log ν (Fig. 6), gave a straight line over the 20–200 mV s−1 following the equation: Log ip (μ A) = 0.77 + 1.03 log ν (mV s−1) (r = 0.998). The slope value of 1.03 is close to the theoretical value of 1.0 that is expected for an ideal reaction of surface species (Laviron, 2002).
log ν (scan rate) vs. log ip (peak current) for 5 × 10−6 mol L−1, RBV in B.R. buffer pH 10 including 0.01 M KCl and equilibrium time = 15 s and tacc. = 30 s.
The repeatative cyclic voltammogram, (Fig. 7) shows that peak current decreases sharply in the second and third cycles indicating the rapid desorption of drug species out of the mercury drop surface during the accumulation.
Repeatative cyclic voltammograms for 5 × 10−6 mol L−1 RBV in B.R. buffer pH 10 including 0.01 M KCl at scan rate 50 mV s−1, equilibrium time = 15 s, (a) first cycle, (b) second cycle, (c) third cycle and (d) forth cycle.
3.3 Validation of the proposed method
In the present work, quantification of RBV was based on the extend of the dependence of peak current upon its concentration in the analyzed solution under the optimal procedural conditions. Validation of the proposed SWAdCS voltammetric procedure for trace assay of RBV was examined via linearity and sensitivity, repeatability and intermediate precision, robustness, ruggedness, specificity and interference study.
3.3.1 Linearity and sensitivity of the proposed method
Calibration curves for RBV were attempted under the optimized procedure conditions and followed different accumulation time periods at −50 mV. The regression equation associated with the calibration curves, (Table 1) exhibited a good linearity that supported the proposed procedure. RBV limit of detection (LOD) and limit of quantification (LOQ) of bulk RBV were estimated from the following equation:
Parameter
tacc. = 15 s
tacc. = 30 s
Linearity range (M)
1 × 10−10–2 × 10−7
1 × 10−10–2 × 10−7
Regression equation (slope in μA/nM)
ip = 61.9 + 39.7 c
ip = 86.2 + 61.2 c
Correlation coefficient (r)
0.9994
0.9995
Determination coefficient (r2)
0.9988
0.9990
LOD (M)
2.24 × 10−10
2.02 × 10−10
LOQ(M)
6.13 × 10−10
6.80 × 10−10
LOD = 3.3 SDa/b LOQ = 10 SDa/b (where SDa is the standard deviation of the intercept, and b is the slope of the calibration graph). Both LOD and LOQ values in (Table 1) confirmed the sensitivity of the proposed method compared with those that calculated by liquid chromatography tandem mass spectrometry method.
3.3.2 Repeatability and intermediate precision
Repeatability and intermediate precision were examined by performing six successive measurements for the different concentrations 5 × 10−7, 5 × 10−8 and 5 × 10−9 mol L−1 of authentic RBV demonstrated the reproducibility of the results obtained by the proposed procedure. For intra-assay precision, recoveries were calculated during one day and for inter-assay precision, recoveries were calculated from the repeated analysis for five days over a period of one week. The RSD values of intra- and inter-day studies (Within and between days not more than 0.21% at low and high concentrations) were illustrated in (Table 2). Accuracy of the result expressed as bias %:
Sample
Added amount mol L−1
Amount found mol L−1
R %
Precision (RSD %)
Accuracy (bias %)
Intra-day precision
5 × 10−7
5.01 × 10−7
100.15
0.447
0.036
5 × 10−8
4.95 × 10−8
99.6
1.024
−0.21
5 × 10−9
4.99 × 10−9
99.39
0.969
−0.096
Inter-day precision
5 × 10−7
4.99 × 10−7
99.58
0.542
−0.0173
5 × 10−8
4.89 × 10−8
99.32
0.893
−0.155
5 × 10−9
4.94 × 10−9
99.11
0.793
−0.128
[bias % = (measured concentration–concentration taken) × 100/(concentration taken)].
3.3.3 Robustness and ruggedness
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in procedural parameters and provides an indication of its reliability during normal usage. The most important procedure variables at pH (10–11) included 0.01 M KCl, accumulation potential (Eacc.) (−50 to −55 mV), accumulation time (tacc.) (30 to 35 s) and a wave increment (ΔE) (10 to 11 mV). The results shown in (Table 3) indicated that none of these variables significantly affects the recovery of RBV. This provided an indication of the reliability of the proposed procedure for the assay of drug and it could be considered robust.
Variables
Conditions
(%) R ± S.D.a
Change in pH of the medium at pH = 11
tacc. = 30 s, Eacc. = −0.05 V and ΔE = 10 mV
99.92 ± 0.42
Accumulation time tacc. = 35 s
pH = 10, Eacc. = −0.05 V and ΔE = 10 mV
100.62 ± 0.63
Accumulation potential Eacc. = −55 mV
pH = 10, tacc. = 30 s, and ΔE = 10 mV
99.34 ± 0.93
Wave increment ΔE = 11 mV
pH = 10, Eacc. = −0.05 V and tacc. = 30 s
100.5 ± 0.82
Ruggedness was examined by applying the developed procedure to assay of drug using potentiostat instruments (potentiostats model 263 A (PAR) and AMEL-433A) under the same optimized experimental conditions at different elapsed times. The results obtained due to lab-to-lab and even day-to-day variations were found reproducible since there was no significant difference in the recovery or the standard deviation values obtained.
3.3.4 Specificity and interference study
The specificity of the proposed method was evaluated in the absence and presence of some organic and inorganic species e.g. starch, glucose, glycine, uric acid, sodium carbonate, ferric nitrate, magnesium perchlorate, and calcium nitrate as a common interference in pharmaceutical preparations. Assay of 5 × 10−7 mol L−1 RBV was examined by the additions of 1 × 10−6 mol L−1 up to 1 × 10−5 mol L−1 of (sodium carbonate, starch, glucose and calcium ions 1 × 10−6 mol L−1), Table 4 shows that there is no change in the peak current of the drug was observed, this indicate that is no significant interference. Thus, the procedure was able to assay RBV in the presence of interference and hence it can be considered specific. But by the addition of ferric nitrate, magnesium perchlorate, glycine, uric acid and calcium nitrate 1 × 10−5 mol L−1, a change in the peak current of this drug was observed. The influence of commonly used excipients (magnesium stearate, lactose and microcrystalline cellulose) was investigated before the determination of the drug in dosage forms. No interference was observed with the proposed method (no change in the peak current).
Interfering species
Concentration (mol L−1)
(%) R ± SDa
Sodium carbonate
1 × 10−5
99.55 ± 0.63
1 × 10−6
99.06 ± 0.23
Starch
1 × 10−5
100.11 ± 0.47
1 × 10−6
98.85 ± 0.75
Glucose
1 × 10−5
100.65 ± 1.09
1 × 10−6
99.85 ± 0.68
Calcium nitrate
1 × 10−6
99.7 ± 0.45
3.3.5 Assay of drug in a pharmaceutical
The proposed SWAdCS voltammetric procedure was successfully applied to the direct determination of RBV in capsule pharmaceutical formulations and the validity was assessed by applying the standard addition methods. On plotting of peak height vs. concentration of RBV, a straight line is obtained over a range of 1 × 10−9–7 × 10−8 mol L−1 for Virin® capsule. The average percentage recovery was 99.68 ± 0.81 and 99.2 ± 0.49 for two concentrations 5 × 10−9 and 5 × 10−8 mol L−1 respectively for 200 mg Virin® capsules, (Table 5). The obtained mean percentage recovery (R %) and the relative standard deviation (RSD %) based on the average of two replicate measurements were recorded. Recovery by the reference method, 99.6 ± 0.37 (n = 6). Theoretical value:t = 2.26 at the 95% confidence level. Theoretical value F = 5.19 at the 95% confidence level.
Samples
Added conc. (mol L−1)
Found conc. (mol L−1)
Mean recoverya (R %)
RSD %
Virin® 200 mg
5 × 10−9
4.98 × 10−9
99.68 ± 0.808, t = 0.204, F = 1.75
0.81
5 × 10−8
4.96 × 10−8
99.20 ± 0.489, t = −1.5 , F = 4.8
0.49
Urine samples
8 × 10−9
8.01 × 10−9
100.12 ± 0.358
0.358
8 × 10−8
8.01 × 10−8
100.16 ± 0.5549
0.554
Serum samples
2 × 10−9
1.99 × 10−9
99.9 ± 0.742
0.743
2.5 × 10−8
2.499 × 10−8
99.99 ± 0.498
0.498
The results were compared statistically with the reference method (Chilukuri et al., 1998). The t- and F-values did not exceed the theoretical ones (Table 5), indicating no significant difference.
3.3.6 Assay of biological samples
3.3.6.1 Urine
RBV was successfully determined in spiked human urine samples by applying the optimized procedure without any prior extraction steps. The peak current vs. drug concentration for samples a and b, respectively was presented by a straight line followed by the equation;
ip (μA) = 0.46 C(M/10−8) + 0.367, ip (μA) = 0.41 C(M/10−8) + 3.35 with a correlation coefficient of 0.9993 and 0.9998. The percentage recoveries of RBV, based on average of five replicate measurements, were found to be 100.12 ± 0.358 and 100.16 ± 0.554 for the two samples (a and b) (Table 5).
3.3.6.2 Serum
The optimized procedures were successfully applied for the determination of RBV in protein free spiked human serum samples. No extraction steps other than the centrifugal protein separation were required prior to the assay of drug. The peak current vs. drug concentration for samples a and b respectively was presented by straight line followed by the equation;
ip(μA) = 1.25 C(M/10−8) + 0.25, ip (μA) = 1.28 C(M/10−8) + 2.55 with a correlation coefficient of 0.9999 and 0.9988. The percentage recoveries of RBV, based on average of five replicate measurements, were found to be 99.9 ± 0.743 and 99.99 ± 0.498 for the two samples (a and b) (Table 5 and Fig. 8).
SWAdCS voltammetry for RBV spiked with human serum samples in B.R. buffer pH = 10 including 0.01 M KCl, at Eacc. = −50 and other operational parameters were as those indicated in Fig. 2. (a) sample, (b) s + 0.5 × 10−8 mol L−1, (c) s + 1.0 × 10−8 mol L−1, (d) s + 1.5 × 10−8 mol L−1, (e) s + 2.0 × 10−8 mol L−1, (f) s + 2.5 × 10−8 mol L−1 RBV authentic.
4 Conclusion
A simple, fast, sensitive and precise SWAdSV method was developed for the determination of RBV in pharmaceutical formulations and biological samples. This method was based on the reduction of RBV at HMDE.
The sensitivity of the method significantly enhanced adsorption of the drug on the electrode surface and after careful choice of the operating parameters; extremely low LOD and LOQ values could be reached. The method is simpler and requires less expensive equipment than other methods.
It was concluded that the proposed method could be successfully and reliably applied to the analysis of RBV in bulk form and pharmaceutical formulations.
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