Vol. 52 No. 4/2005, 915–922 on-line at: www.actabp.pl New

Two simple, sensitive and economical spectrophotometric methods were developed for the determination of nifedipine in pharmaceutical formulations. Method A is based on the reaction of the nitro group of the drug with potassium hydroxide in dimethyl sulphoxide (DMSO) medium to form a coloured product, which absorbs maximally at 430 nm. Method B uses oxidation of the drug with ammonium molybdate and subsequently reduced molybdenum blue is measured at 830 nm. Beer's law is obeyed in the concentration range of 5.0-50.0 and 2.5-45.0 microg ml(-1) with methods A and B, respectively. Both methods have been successfully applied for the assay of the drug in pharmaceutical formulations. No interference was observed from common pharmaceutical adjuvants. The reliability and the performance of the proposed methods are established by point and interval hypothesis tests and through recovery studies.

The estimation of nifedipine alone was carried out using second-order derivative spectra (Umapathi, 1994) of the compound in 0.1 M HCl whereas first derivative spectra were utilized for its assay in combined dosage forms (El-Walily, 1997).A methanolic solution of the drug reacts with 4-dimethylaminobenzaldehyde resulting in the formation of yellow-coloured product, which forms a basis for its determination at 380 nm (Mahadik et al., 1991).Two spectrophotometric methods have been recommended, one is based on the formation of blue-coloured complex with Folin Ciocalteau reagent (Sastry et al., 1997), and the second method involves the charge transfer complex formation with chloranil (Golcu & Serin, 1998).A kinetic spectrophotometric method has also been described based on the oxidation of the drug with KMnO 4 at neutral pH (Rahman & Azmi, 1999).Two other spectrophotometric methods were developed in which the -NO 2 group of nifedipine was reduced with Zn/NH 4 Cl and Zn/ HCl to hydroxylamino and primary aromatic amino derivatives, respectively (Karadi et al., 2000;Rahman & Hoda, 2002).The hydroxylamino derivative was reacted with 4-(methylamino)phenol and potassium dichromate to give a coloured chromophore, which absorbed maximally at 525 nm while the primary aromatic amino derivative formed Schiff's base with 3,4,5-trimethoxybenzaldehyde which was subsequently determined at 365 nm.Extractive spectrophotometric methods have also been reported for the estimation of the drug in pharmaceutical preparations which are based on the coloured complex of the drug with reagents like bromocresol green, bromophenol blue, bromothymol blue and eriochrome black-T (Rahman et al., 2004).
This paper describes two simple and sensitive spectrophotometric methods for the determination of nifedipine in pharmaceutical formulations.Method A is based on the reaction of the nitro group of the drug with potassium hydroxide in dimethyl sulphoxide to form a coloured product peaking at 430 nm.Method B uses the oxidation of the drug with ammonium molybdate, which subsequently reduced to molybdenum blue, is measured at 830 nm.The proposed methods are validated as per the guidelines of the International Conference on Harmonisation (ICH, 1995).

Apparatus.
The absorbance measurements were made on a Spectronic 20D + spectrophotometer (Milton Roy, USA) with 1 cm matched glass cells.A water bath shaker was used to control the temperature for colour development.An Elico model Li-10 pH meter was used for pH measurements.
Reagents and standards.All chemicals used were of analytical or pharmaceutical grade.A 0.05 M KOH (Merck, India) solution was prepared in tertiary butyl alcohol (Qualigens, India).A 0.02 M ammonium molybdate (Merck, India) solution was prepared by dissolving 2.472 g of ammonium molybdate in 100 ml of 4 M sulphuric acid.Buffer solutions ranging from pH 2.4-5.6 were prepared by mixing 1.24-11.60ml of 0.2 M disodium hydrogen phosphate and 18.76-8.40ml of 0.1 M citric acid in 20 ml (Bri�on, 1942).Reference standard of pure nifedipine and a nifedipine nitrosophenyl pyridine analog were kindly provided by J.B. Chemicals and Pharmaceuticals Ltd. (Mumbai, India) and Novartis Pharmaceuticals Ltd. (Mumbai, India), respectively.Commercially formulated tablets such as Nicardia Retard (J.B.Chemicals), Calciguard (Torrent), and Adalat Retard (Bayer) were purchased from local market.Standard solutions of 0.1 and 0.05% nifedipine were prepared in DMSO and methanol, respectively, and kept in dark.
Preparation of degraded nifedipine.A standard solution of pure nifedipine (1.0 mg ml -1 ) was exposed to diffused sunlight (natural) for 2 h.A sample of 8.0 µl of this solution and simultaneously the same amount of the nifedipine nitrosophenyl pyridine analog reference standard were spo�ed on a thin layer chromatographic plate of silica gel G (Merck, India) which was then developed in a mobile phase using chloroform/ethyl acetate/cyclohexane (19:2:2, by vol.) and observed under UV lamp.Two spots were present having the same R f value (0.5), thus it can be suggested that the degraded product of nifedipine is nifedipine nitrosophenyl pyridine (Pi-e�a et al., 1981).
Method A: Recommended procedure for the determination of nifedipine.Aliquots of 0.05-0.5 ml of standard drug solution (0.1%) of nifedipine were pipe�ed into a series of 10.0 ml standard volumetric flasks.Then, 0.6 ml of 0.05 M KOH solution was added into each flask and diluted to 10.0 ml with DMSO.The contents of each flask was mixed well at room temperature (25 ± 1 o C) and the absorbance was measured at 430 nm against the reagent blank prepared similarly within the stability time period of 2 h.The concentration of nifedipine was calculated either from a calibration curve or regression equation.
Method B: Recommended procedure for the determination of nifedipine.Into a series of boiling test tubes, different volumes (0.1-0.9 ml) of 0.05% nifedipine were pipe�ed.To each test tube 1.4 ml of 0.02 M ammonium molybdate and 5.0 ml of phosphate/citric acid buffer solution (pH 4) were added, mixed well and heated in a water bath at 100 ± 1 o C for 20 min.A�er heating, the solutions were cooled at room temperature and transferred to 10.0 ml standard volumetric flasks and diluted to volume with doubly distilled water.The absorbance was measured within the stability time period of 4 h at 830 nm against the reagent blank treated similarly.The amount of the drug in a given sample can be calculated from a calibration graph or regression equation.
Procedure for the assay of nifedipine in pharmaceutical formulations.Two portions of powdered tablets equivalent to 50.0 and 25.0 mg of nifedipine were weighed accurately, and separately extracted into 50.0ml chloroform with shaking, and the residues were filtered using Whatmann No. 42 filter paper.The filtrates were evaporated to dryness under vacuum and the corresponding residues were dissolved in DMSO and methanol, respectively, and transferred to 50.0 ml standard volumetric flasks and diluted to volume with their corresponding solvents.The assay was completed following the recommended procedures for determination of nifedipine.

RESULTS AND DISCUSSION
Nitro compounds are known to give interesting colours with alkali in different polar media.It has been suggested that the reactions of nitro aromatic compounds with alkali in acetone, alcohol, N,N ' -dimethylforamide or DMSO (Porter, 1955;Maiti et al., 1982) yield nitroquinoid ions.Nifedipine contains a nitro group a�ached to the benzene ring, which reacts with KOH in DMSO medium to produce coloured nitroquinoid ion which absorbs maximally at 430 nm (Fig. 1A).The coloured chromophore formed was found to be negatively charged as it was adsorbed on anion exchange resin beads.Therefore, based on the literature background and our experimental findings the reaction mechanism was proposed and is given in Scheme 1.
Ammonium molybdate (Mo VI ) behaves as an oxidizing agent in acidic medium and is reduced to molybdenum blue (Mo V ) on treatment with reducing substances.This property is exploited for the spectrophotometric determination of nifedipine which reduces Mo VI to Mo V showing maximum absorbance at 830 nm (Fig. 1B).

Optimization of variables and method development
The concentration of different reagents used for method development was optimized by performing a series of experiments.

Method A: Effect of KOH
The influence of the volume of 0.05 M KOH on the intensity of the colour developed at constant nifedipine concentration (30.0 µg ml -1 ) was examined in the range 0.05-0.6 ml of 0.05 M KOH.It is clear from Fig. 2A that the maximum absorbance was a�ained with 0.4 ml of 0.05 M KOH; above this volume the absorbance remained unchanged.Therefore, 0.6 ml of 0.05 M KOH was used in all further measurements.

Method B: Effect of heating
To study the effect of heating time for the development of maximum colour for method B, 0.4 ml of 0.05% nifedipine was mixed with 1.5 ml of 0.02 M ammonium molybdate and 5 ml of buffer solution.The contents of the mixture were heated for up to 23 min at 100 ± 1°C.The intensity of the colour developed was measured at room temperature (25 ± 1°C) a�er dilution to 10.0 ml with doubly distilled water.It is apparent from this investigation that the maximum intensity of colour was obtained a�er 18 min of heating and remained constant up to 23 min.Therefore, the optimum heating time was fixed at 20 min.

Method B: Effect of the concentration of ammonium molybdate
The effect of the volume of 0.02 M ammonium molybdate on the colour development was investigated by adding different volumes (0.50-1.5 ml) of 0.02 M ammonium molybdate to 200 µg of nifedipine.It was found that the maximum absorbance of the blue colour was reached with 1.2 ml of the reagent, and remained constant with higher volumes (Fig. 2).Therefore, 1.5 ml of the reagent was used throughout the experimental investigations.

Method B: Effect of pH
The influence of pH on the development of colour was studied using disodium hydrogen phosphate/citric acid buffer.The maximum colour intensity was observed in the pH range of 3.4-4.6(Fig. 3)

Specificity
The specificity of the proposed methods were evaluated by determining the concentration of nifedipine in the presence of varying amounts of degraded product of nifedipine such as nifedipine nitrosophenyl pyridine.It was found that the degraded product did not react with either of the reagents utilized in methods A and B. There was no interference from the common excipients such as sodium stearyl fumarate, magnesium stearate, starch, lactose and talc present in tablets.

Solution stability
The solution stability of the reference drug and tablet solutions was monitored by keeping the solutions at room temperature (25 ± 1 o C) under darkness for several days and then recording the absorption spectra of the solutions and also by performing TLC analysis.There was no change in the absorption spectra of reference and sample solutions for at least seven days.The band corresponding to the degradation product of nifedipine was not observed under UV lamp.A single spot at R f value of 0.3 was obtained on TLC plate using silica gel G as stationary phase and chloroform/ethyl acetate/cyclohexane (19 : 2 : 2, by vol.) as mobile phase.

Robustness
Each operational parameter was closely examined and challenged for the robustness of the proposed methods.The operational parameters investigated were as follows: For Method A • 0.6 ml of 0.05 M KOH For Method B • 1.5 ml of 0.02 M ammonium molybdate • 5 ml of buffer solution of pH 4 • 20 min heating time • cooling at room temperature The robustness of the proposed methods relative to each operational parameter was evaluated by analyzing the contents of nifedipine tablets under variable experimental conditions.A sample solution containing 40 µg ml -1 of active drug (Nicardia retard-10) was assayed five times using both methods.The results showed a mean recovery ± relative standard deviation of 100.11 ± 0.16% and 100.06 ± 0.15% for methods A and B, respectively.Thus the operational conditions for the proposed methods to determine nifedipine in tablet formulations were found to be very robust.

Analytical data
Under the optimized experimental conditions, calibration graphs were constructed by plo�ing the absorbance against the concentration of nifedipine.Beer's law was obeyed in the concentration range 5.0-50.0 and 2.5-45.0µg ml -1 with molar absorption coefficients of 1.108 × 10 4 and 1.455 × 10 4 l mol -1 cm -1 for methods A and B, respectively.Table 1 summarizes the optical characteristics and the results of statistical analysis of the experimental data such as linear regression equations for methods A and B along with correlation coefficient, standard deviation of slope (S b ) and intercept (S a ), confidence interval of slope (tS b ) and intercept (tS a ), detection limit and quantitation limit.The limit of detection (LOD) and quantitation (LOQ) were calculated using the following relation (Ermer, 2001) LOD = 3.3 × S 0 /b, and LOQ = 10 × S 0 /b where S 0 is the standard deviation of the calibration curve and b is the slope.The small value of variance suggested negligible sca�er of experimental data points around the line of regression.

Spectrophotometric determination of nifedipine
The accuracy and precision of the proposed methods (A and B) was evaluated by performing five replicate determinations of nifedipine in pure forms at three different concentrations (10, 30 and 45 µg ml -1 ) by short term (intra day) and daily (inter day) precisions (Table 2).The standard analytical errors, relative standard deviations and recoveries obtained in the intra day and inter day analyses for methods A and B were found to be acceptable.Thus the proposed methods are effective for the determination of nifedipine.
The accuracy of the proposed methods was also checked by performing recovery experiments through standard addition technique.For this purpose, a known amount of pure nifedipine was added to pre-analyzed dosage forms and then determined by the recommended procedures.The results (Table 3) showed that the mean recovery and relative standard deviation were in the range of 99.97-100.17and 0.15-0.52%for method A and 100.03-100.10 and 0.12-0.24%for method B, respectively.No interference from the common excipients was observed.
Table 4 shows a comparison of the performance of the proposed methods with that of other existing UV-visible spectrophotometric methods.It is clear from the table that the proposed methods are sensitive with acceptable values of relative standard deviations.Thus the proposed methods can compete with other existing methods in the determination of the drug at lower concentrations.
The proposed methods were tested on tablet formulations and the results are presented in Table 5.The results (Table 5) of the proposed methods (A and B) were compared with those of the reference method (Rahman & Hoda, 2002) using point hypothesis tests.The results in the table show that the calculated paired t-and F-values are less than the theoretical ones (Christian, 1994) confirming no significant difference between the performance of the proposed methods and the reference method at 95% confidence level.The interval hypothesis tests (Hartmann et al., 1955) were also performed to judge the performance of the proposed methods and the results are summarized in Table 6.The Canadian

Spectrophotometric determination of nifedipine
Health Protection Branch has recommended that a bias of ± 2% (θ L = 0.98 and θ L = 1.02) based on recovery experiments (Canadian Health Protection Branch Guideline, 1992) is acceptable for pharmaceutical analysis.It is clear from the table that the true bias of all samples is less than ± 2%.

CONCLUSIONS
The proposed methods are compared with other existing spectrophotometric methods and are found to be more sensitive with low values of relative standard deviations.The proposed methods do not require any pretreatment of the drug and tedious extraction procedure prior to its analysis.The newly developed methods are sensitive enough to enable quantitation of the drug at low concentrations.These advantages encourage the application of the proposed methods in routine quality control analysis of nifedipine in pharmaceutical formulations.

Figure 2 .
Figure 2. Effect of the volume of (A) 0.05 M KOH (method A) and (B) 0.02 M ammonium molybdate (method B).

Table 5 . Comparison of the proposed methods using point hypothesis tests with the reference method at 95% confi- dence level
a Mean for five independent analyses; b Theoretical t-value (ν = 8) and F-value (ν = 4, 4) at 95% confidence level are 2.306 and 6.39, respectively

Table 3 . Determination of nifedipine in pharmaceutical formulations by standard addition technique
Mean for five independent analyses; b SAE, standard analytical error; c C.L., confidence limit at 95% confidence level and four degrees of freedom (t = 2.776) a

Table 6 . Comparison of the proposed methods using in- terval hypothesis tests with the reference method at 95% confidence level
A bias, based on recovery experiments, of ±2% (θ L = 0.98 and θ U = 1.02) is acceptable in pharmaceutical analysis. a