Anodic Polaroqraphic Determination of lsradipine in Pharmaceutical Formulations

The anodic behaviour of isradipine was studied using cyclic voltammetry (CV), direct current (DC,) and differential-pulse (DPP) polarography. lsradipine exhibited well-defined anodic polarographic waves over the pH range of 4-8 in Britton-Robinson buffer (BRb). At pH 6, the analytical pH, the diffusion-current constant (Id) was 2.12 + 0.42 (n = 4). The oxidation potential is 0.1 0 V vs AgIAgCI reference electrode with correlation coefficients of 0.9977 and 0.9948 in the DCt and DPP modes, respectively. The current-concentration plots were rectilinear over the ranges 5-14 pglml and 2.4-12 pglml using the DCt and DPP modes, respectively. The lower detection limit was 1 . I2 pglml (3.02 x M) adopting the DPP mode. The proposed method was applied to commercial tablets and capsules, the % recoveries were 100.52 rt 1.267 and 99.4 + 0.87 (n = 6) for the tablets and 101.3 + 1.57 and 100.7 + 2.10 (n = 6) for the capsules adopting both DCt and DPP modes, respectively. The number of electrons involved in the electrode process was accomplished and a proposal of the electrode reaction was presented.

lsradipine is the subject of monographs in both the British Pharmacopoeia, BP [2] and the United States Pharmacopoeia, USP [3].The BP [2] recommends spectrophotometric measurement for the raw material and liquid chromatography for the formulations.The USP [3] on the other hand, described liquid chromatography for both the drug and its formulations.
Reviewing the literature revealed that all the reported methods for isradipine in dosage forms and biological fluids rely on the use of chromatographic techniques such as thin layer chromatography (TLC) [4,5], GC  Although chromatographic methods offer a high degree of specificity, yet, sample clean-up and the instrumentation limitations preclude their use in routine clinical studies.The proposed method was developed as an alternative substitute to the chromatographic methods, and the results obtained were promising.The presence of the oxidizable dihydropyridine ring structure in the molecular formula of isradipine initiated the present study.Recently, a voltammetric method based on the reduction of furazayl ring of isradipine has been proposed for its determination [I 81.
The proposed method is characterized by being stability-indicating, as it is based on the presence of dihydropyridine ring and the oxidation of the latter is the major pathway for the degradation of this class of compounds [ I 91.

Reagents and Materials
lsradipine was kindly provided by Novartiz Pharma, Cork, Ireland, Batch # 19525 D and was used as received.Tablets and capsules containing isradipine were obtained from commercial sources in the local market.Lomir SRO capsules Batch # 61016 (5 mg of isradipine/capsule).Lomir tablets Batch # TI001 (2.5 mg of isradipine/tablet).Both are products of Novartis Pharma, AG, Basle, Switzerland.
Standard solutions: A stock solution of isradipine (1.0 mg ml-I) was prepared in methanol, and was further diluted with the same solvent to give the appropriate working standard solutions.The solutions are stable for one week if kept in the refrigerator.

Apparatus
The cyclic voltamograph (757 VA computrace) was obtained from Metrohm (Herisau, Switzerland).It consists of a hanging mercury drop electrode in the DPP mode, a saturated AgIAgCI reference electrode and a platinum wire as the auxiliary electrode.Nitrogen gas was used for deoxygenation.Scan rate was 2 mV1sec and mercury drop size was 0.2 mm2.Current range was 0-30 nA and potential range was -0.7 to -1.35 V.A computer driven HP Printer was used.
The polarographic study and the differential pulse polarographic (DPP) measurements (pulse amplitude 50 mV was applied) were carried out using the Polarecord E 506 Metrohm (Herisau, Switzerland).The drop-time of 1 second was electronically-controlled using a 505 Stand from the same company.The polarograms were recorded using a potential scan-rate of 10 mv.sec-'.A threeelectrode system composed of a Dropping Mercury Electrode (DME) as the working electrode, AgIAgCI reference electrode, and a platinum wire as the auxiliary electrode, was used.The solutions were purged with pure nitrogen gas for 5 min before being polarographed at room temperature.

Procedures Recommended Procedure
Transfer aliquots of isradipine working standard solution into a set of 25-ml volumetric flasks, so that, the final concentration is in the range cited in Tab. 2. Add sufficient methanol so that its content should be always 20% (vlv).Complete to volume (25 ml) with BRb of pH 6. Pass nitrogen gas for 5 min.Record the current in the DC, and DPP modes within the range of -0.4 to + 0.4 V. Plot the produced current (in pA) in both the DCt and DPP modes versus the final concentration of the drug (in pglml) to get the calibration graph.Alternatively, derive the corresponding regression equation.

Analysis of Commercial Tablets and Capsules
Empty the contents of 10 capsules (average weight 410 mg) or weigh and pulverize 20 tablets (average weight 86 mg), then mix the powder.Weigh accurately a quantity of the powder equivalent to 25 mg of isradipine and transfer into a 50-ml volumetric flask.Add about 40 ml of methanol (in case of the tablets) or acetone (in case of the capsules) and sonicate for half an hour.Filter into a 50-ml volumetric flask.Wash the residue and flask with the same solvent and transfer the washing into the same volumetric flask and complete to the mark with the same solvent.Transfer aliquot volumes containing isradipine over the concentration range in Tab. 2, 3-10 pglml and 5-12 pglml in the DPP mode and DC, mode, respectively into a 25-ml volumetric flask.Adjust the volume to 5 ml with methanol.Complete to the mark with BRb of pH 6. Proceed as described under Recommended Procedure.Determine the nominal content of the tablets or capsules using either the calibration graph or the corresponding regression equation adopting both DC, and DPP modes.

Results and Discussion
The cyclic voltammogram of isradipine (1 r IO-~M) in BRb of pH 6 using a scan rate of 2.0 mvlsec.[ anodic peaks.The principal anodic peak (with peak potential of -0.98 V) is accompanied with a cathodic peak (with peak potential of -0.90 V).The difference between the peak potentials is 80 mV.As the oxidation of the dihydropyridine nucleus involves the transfer of 2 electrons, it is clear that, the oxidation process is completely irreversible.Logarithmic analysis of the anodic waves obtained in BRb of different pH values resulted in straight lines with different slopes.Assuming that the ratedetermining step involves the transfer of two electrons (a free-radical, one electron-transfer is not likely to occur).The values of the slopes suggest that the oxidation process is irreversible in nature.The aria values were calculated according to the treatment of Meites and Israel [21] and are listed in Tab. 1.The number of protons (ZH+) involved in the rate-determining step of the reduction process was also calculated according to the following formula [22].

Influence of pH on the Anodic Waves
AEx 1 ApH = -0.059Z(H+) I aria where a is the transfer coefficient and n, is the number of electrons transferred in the rate-determining step.The small figures obtained for Z(H+) (Tab. 1) point out to the irreversibility of the electrode process.

Study of the Wave Characteristics
Increasing the mercury reservoir height (h) resulted in a corresponding increase in waveheight (W); a plot of W vs h gave a straight line.A plot of Log W vs Log h gave a straight line with a slope of 0.69.Changing the buffer concentration over the range 0.01-0.06M (final concentration) resulted in a negligible increase in waveheight.These two characteristics point out to a diffusion-controlled process partially affected by adsorption phenomenon as indicated by the slightly high value of the slope (0.69).However, addition of methanol (20%, vlv) to the electrolysed solution decreases the effect of the adsorption process.The diffusion coefficient (D) of isradipine was determined in BRb of pH 6 according to llkovic equation [23] and was found to be 1.33 x cm2/sec.This small value may be attributed to the bulky nature of the molecule.

Number of Electrons Involved in the Oxidation Process
The number of electrons transferred during the electro-oxidation process was estimated through comparing the waveheight of isradipine with that obtained from an equimolar solution of an earlier studied compound of the same chemical group (1,4-dihydropyridine) and of nearly identical value of diffusion coefficient, that is nifedipine [24].In BRb of pH 6, both compounds gave one wave of the same height corresponding to the consumption of 2 electrons (nifedipine gave an additional illdefined anodically-shifted wave).The first wave of nifedipine corresponds to the consumption of 2 electrons.It is evident from the experimental results for isradipine that a slow electron-transfer reaction is involved in the oxidation process.Logarithmic analysis of the waves established that two electrons are involved in the rate-determining step of the anodic wave, and the shift in E x potentials with increasing pH indicates that two protons are consumed in this step.Based on these facts, and depending on the presence of the dihydropyridine ring structure, the following pathway for the electrode reaction may be postulated (Scheme 2): Scheme 2: Proposed pathway of the electrode oxidation reaction.

Analytical Applications
DC, polarograms of isradipine in BRb of pH 6 exhibit well-defined anodic waves.No polarographic maxima were developed; therefore, no maximum suppressor was needed.The current is mainly diffusion-controlled and proportional to the concentration of the analyte over the concentration range 5-14 pglml and 2.4-12 pglml for DCt and DPP modes, respectively.At that pH value, the DC, wave was the steepest and the peak (in the DPP mode) had the least half-peak width (Wx) as shown in Tab. 1. Solutions of isradipine in methanol were found to be stable for at least one week if kept away from light in the refrigerator.In BRb of pH 6 (the analytical pH) the solutions were found to be stable for at least three hours.The relation between each of the limiting diffusion-current (id) in the DC, mode and the peak current (ip) in the DPP mode, and the concentration of isradipine (pglml) is rectilinear over the ranges indicated in Tab. 2. Data on the analytical performance of the proposed method (parameters of the regression equations, correlation coefficients, minimum detectability (taking SIN = 2), and diffusion-current constant, Id) are compiled in the same table .Statistical analysis of the two regression equations was presented in terms of the standard deviation of the residuals (Sdy), standard deviation of the slope (Sb), and standard deviation of the intercept (Sa) gave the values cited in Tab. 2. The small values of all these parameters point out to the high precision of the method [25].The good linearity of the calibration graph and the negligible scatter of the experimental points are clearly evident by the correlation coefficients (close to 1 in both cases).Toestablish the reproducibility of the electrode response, six replicate concentrations were tested at isradipine concentrations of 6, 8 and 10 pglml adopting the DCt mode.Mean current values of 0.1844 + 0.004; 0.2000 + 0.0042; and 0.2678 + 0.0089 pA, respectively were obtained.The precision of these measurements is expressed by the relative standard deviations (RSD) of 2.17; 2.10, and 3.32, respectively.These small values indicate a highly precise electrode response.
Both DCt and DPP modes were successfully applied to the determination of isradipine in commercial tablets and capsules.The percentage recoveries based on 6 separate determinations are abridged in Tab. 3. The results are in good agreement with the label claim.Both DCt and DPP modes proved to be equally useful; however, the DPP mode is more convenient.
The proposed method can be considered as a stability-indicating assay for isradipine as it is based on the presence of the dihydropyridine ring structure, the latter is subjected to atmospheric oxidation and loss of activity [I 91.

Conclusion
A simple and reliable stability-indicating polarographic method was developed for the determination of isradipine in dosage forms.The lower detection limit (3.02 x M) is comparable to that reported by chromatographic methods.
As applied to tablets or capsules, the method is very simple and time saving.The proposed method can be considered as stability-indicating assay of the drug.
Tab. **Each result is the average of 6 separate determinations.
Reference method (1 8) The theoretical values of t-and F-test at 95.0% confidence limit are 2.20 and 5.05, respectively.

Fig. 2 Fig. 2 .
Fig.2shows typical DCt and DPP polarograms of isradipine in BRb of pH 6.The effect of pH on the development of the anodic waves is shown in Fig.3.Welldefined anodic waves were obtained over the pH range 4-8.Below pH 4 and above pH 8 no waves were developed.The waves were positively shifted upon increasing the pH of the medium.The relation between the half-wave potentials (Ex) of the anodic waves and pH is expressed by the following regression equation:

Fig. 3 .
Fig. 3. Effect of pH on the development of the anodic weaves of isradipine (14 pglml) in BRb.Potential scan rate 10 mVlsec and drop time: 1 sec.
3. Application of the proposed polarographic method to the analysis of commercial tablets and capsules.*Both tablets and capsules are products of Novartis Pharma, AG, Basle, Switzerland.