Spectrophotometric determination of some calcium channel blockers using Sulfochlorophenol-S , Bromopyrogallol red , Eriochromecyanine-R and Pyrocatechoil violet

Four simple, sensitive and accurate spectrophotometric methods have been developed for the determination of some calcium channel blockers: Amlodipine besylate (ADB), Diltiazem hydrochloride (DTZ) and Verapamil hydrochloride (VPM) in pharmaceutical formulations. These methods based on formation ion pair complexes with Sulfochlorophenol-S (SCPS), Bromopyrogallol red (BPR), EriochromecyanineR (ECC) and Pyrocatechol violet (PCV) in acidic medium. The colored products are extracted with chloroform and measured spectrophotometrically at 462, 600, 440 and 442 for ECC, SCPS, BPR and PCV, respectively. Beer’s low was obeyed in the concentration range, for ECC: 25 175 μg·ml, 50 150 μg·ml or 100 250 μg·ml, for SCPS: 300 800 μg·ml, 200 700 μg·ml or 100 550 μg·ml, for BPR: 50 400 μg·ml, 200 700 μg·ml or 200 700 μg·ml for VPM, DTZ or ADB, respectively and for PCV: 50 250 μg·ml for VPM or 200 500 μg·ml for DTZ with molar absorptivity, for ECC: 2.2 × 10, 2.1 × 10, 1.6 × 10 L·mol·cm, for SCPS: 3.8 × 10, 5.6 × 10, 8.1 × 10 L·mol·cm, for BPR: 11 × 10, 4.8 × 10, 6.9 × 10 L·mol·cm for VPM, DTZ or ADB, respectively and for PCV: 19.5 × 10 L·mol·cm for VPM and 6.6 × 10 L·mol·cm for DTZ and relative standard deviation, for ECC: 0.76%, 0.86%, 0.46%, for SCPS: 0.94%, 0.96%, 0.86%, for BPR: 0.96%, 0.95%, 0.55% for VPM, DTZ or ADB, respectively and for PCV: 0.81% for VPM and 0.65% for DTZ. These methods have been successfully applied for the assay of drug in pharmaceutical formulations. No interference was observed from common pharmaceutical adjuvants. Statistical comparison of the results with the reference method shows excellent agreement and indicates no significant difference in accuracy and precision.


INTRODUCTION
The calcium channel blockers are divided into three chemical classes, each with different pharmacokinetic properties and clinical indications [1].

Diphenylamine Derivatives
Verapamil is the only member of this class.Verapamil is the least selective of any calcium channel blocker, and has significant effects on both cardiac and vascular smooth muscle cells.

Benzothiazepine Derivatives
Diltiazem is the only member of this class.Like verapamil, diltiazem affects both cardiac and vascular smooth muscle cells; however, it has a less pronounced negative intropic effect on the heart than does verapamil.
In this communication, four new spectrophotometric methods for the determination of amlodipine, diltiazem and verapamil have been discussed.The methods based on formation of chloroform-extractable ion-pair complexes with Sulfochlorophenol-S (SCPS), Bromopyrogallol red (BPR), Eriochromecyanine-R (ECC) and Pyrocatechol violet (PCV).

Apparatus
A spectronic TM Genesys TM , ultraviolet/visible spectrophotometer (Milton Roy Co., Westhaven, USA) was used for measure absorbance.
A Milwakee SM 101 pH meter, (Portugal) was used for pH measurements.

Chemicals and Reagents
Stock solutions containing 1 mg•ml −1 of each of the studied drugs were prepared.50 mg of verapamil and diltiazem were accurately weighed (Alexandria Co.For pharmaceuticals, Alexandria, Egypt), carefully transferred into 50 ml volumetric flasks, then dissolved in double distilled water, sonicated for about 2 min and completed to volume with the same solvent.For amlodipine 50 mg was accurately weighed, carefully transferred into 50-ml volumetric flasks, then dissolved in 2 ml methanol, sonicated for about 2 min and completed to volume with double distilled water.Working standard solutions containing 50 -500 µg•ml −1 were prepared by a series of dilutions of the stock solutions with water.ECC (Merck, Darmstadt, Germany) 0.2% w/v stock solution was prepared by dissolving 200 mg of the commercial preparation in double distilled water into 100-ml volumetric flask and then diluted to volume with the same solvent.SCPS (Sigma, St. Louis, MO, USA) 0.1% w/v stock solution was prepared by dissolving 100 mg of the commercial preparation in double distilled water into a 100ml volumetric flask and then diluted to volume with the same solvent.BPR (Sigma, St. Louis, MO, USA) 0.05% w/v stock solution was prepared by dissolving 50 mg of the commercial preparation in double distilled water into a 100-ml volumetric flask and then diluted to volume with the same solvent.PCV (Sigma, St. Louis, MO, USA) 0.1% w/v stock solution was prepared by dissolving 100 mg of the commercial preparation in double distilled water into a 100-ml volumetric flask and then diluted to volume with the same solvent.
Stock 1 M solutions of each of the following acids; acetic, hydrochloric, nitric, oxalic and sulfuric acids which obtained from El-Nasr Co. (for pharmaceuticals and chemicals, Egypt) were freshly prepared in double distilled water.Stock solutions of citric acid, 0.1 -5 M were freshly prepared in double distilled water.

Procedure for Calibration Curve
Into 60-ml separating funnel: for ECC; 1 ml of 3 M citric acid was transferred, followed by 1 ml of working standard solution of either VPM, DTZ or ADB, in the general concentration ranges 25 -175 µg•ml −1 , 50 -150 µg•ml −1 or 100 -250 µg•ml −1 , respectively.Then suitable aliquot of corresponding dye was added to the resultant solution, 2 ml of 0.2% w/v ECC solution for DTZ, 1.5 ml of 0.2% w/v ECC solution for VPM and ADB.For SCPS; 2 ml of 0.5 M sulfuric acid for VPM, 1.5 ml of 0.3 M sulfuric acid for DTZ and ADB was transferred, followed by 1 ml of working standard solution of VPM, DTZ or ADB, in the general concentration ranges 300 -800 µg•ml −1 , 200 -700 µg•ml −1 or 100 -550 µg•ml −1 , respectively.Then suitable aliquot of corresponding dye was added to the resultant solution, 1.5 ml of 0.1% w/v SCPS solution.For BPR; 1.5 ml of 1 M sulfuric acid was transferred, followed by 1 ml of working standard solution of VPM, DTZ or ADB, in the general concentration ranges 50 -400 µg•ml −1 , 200 -700 µg•ml −1 or 200 -700 µg•ml −1 , respectively.Then suitable aliquot of corresponding dye was added to the resultant solution, 1.5 ml of 0.05% w/v BPR solution.For PCV; 0.5 ml of 1 M sulfuric acid was transferred, followed by 1 ml of working standard solution of either VPM or DTZ, in the general concentration ranges 50 -250 µg•ml −1 or 200 -500 µg•ml −1 , respectively.Then suitable aliquots of corresponding dye were added to the resultant solution, 1.5 ml of 0.1% w/v PCV solution.The contents were mixed well and shacked with three 3 ml portions of chloroform.Then the extracts were combined into 10-ml volumetric flask and completed to volume with chloroform.The combined extracts were filtered over 2 gm anhydrous sodium sulfate and first portion of the filtrate was rejected.The absorbance of the chloroform extract was measured at  max = 462, 600, 440 and 442 nm against a reagent blank treated similarly, respectively.

Procedure for the Assay of Drug in Dosage Forms
An accurately weight amount equivalent to 100.0 mg of each drug from composite of 20 powdered tablets was carefully transferred into 100 ml volumetric flask, followed by addition of about 50 ml double distilled water, and the mixture was shaken well, sonicated for about 10 min and then completed to volume with the same solvent.The resulting solution was filtered where the first portion of the filtrate was rejected [4].Appropriate volume was diluted quantitatively with the same solvent to prepare a working standard solution containing 50 -500 µg•ml −1 and then was subjected to analysis as explained under general procedure.

RESULTS AND DISCUSSION
Upon interaction of the studied drugs with these acidic dyes such as SCPS, ECC, BPR and PCV, highly colored ion pair complexes extractable with chloroform, were formed.Absorption spectra of diltiazem hydrochloride as a presentative example and the formed ion pair complexes with ECC, SCPS, BPR and PCV in chloroform, are shown in Figure 1, The reagent blanks prepared under similar conditions showed no absorption.

Composition and Formation Constant of Ion-Pair Complexes
Using job's method of continuous variation [35], the results revealed a 1:1 ratio between ECC and the studied drugs, Figure 2, 1:4 ratios between SCPS and the investigated drugs, Figure 3, 1:1 ratio between BPR and the studied drugs, Figure 4 and 1:1 ratio between PCV and the studied drugs, Figure 5.The formation constant (K f ) of the studied drugs in complex with ECC, SCPS, BPR and PCV were calculated also the Gibbs free energy change of the reaction were calculated [36] and the collected data are illustrated in Table 1.

Optimization of the Reaction Conditions
The optimum conditions for quantitative estimation of the drug were established via number of preliminary experiments.

Effect of Extracting Solvent
Preliminary experiments were carried out without adjusting variables.The absorption intensities of the formed complexes were greatly affected by different immiscible solvents, it was observed that the highest absorption intensity was obtained on using chloroform as an extracting solvent; thus it was selected for all the subsequent work.

Effect of Acid Type
This study revealed that the use of acids led to higher absorption intensities than those obtained upon using buffers.Among different acids, citric acid gave the maximum color intensity than others with ECC but sulfuric acid gave the maximum color intensity with SCPS, BPR and PCV.

Effect of Acid Concentration and Volume
As shown in Figures 5-8, 3 M citric acid gave the maximum color intensity with the highest stability in case of using ECC; 1 M sulfuric acid gave the maximum color intensity with the highest stability in case of using BPR and PCV; 0.5 M sulfuric acid gave the maximum color intensity with the highest stability in case of using SCPS with VPM.0.3 M sulfuric acid gave the maximum color intensity with the highest stability in case of using SCPS with ADB and DTZ.
The optimum volume was also studied for each acid where; in case of using 0.5 -3 ml of 3 M citric acid; 1.5 ml gave the maximum color intensity with the highest stability as indicated in Figure 9 For ECC.In case of using 0.5 -3 ml of 0.3 M sulfuric acid 1.5 ml gave the maximum color intensity with the highest stability in        case of ADB and DTZ, respectively.2 ml of 0.5 M sulfuric acid in case of VPM gave the maximum color intensity with the highest stability as indicated in Figure 10.For SCPS, in case of using 0.5 -2.5 ml of 1 M sulfuric acid 1.5 ml gave the maximum color intensity with the highest stability as indicated in Figure 11.For BPR, in case of using 0.1 -1.5 ml of 1 M sulfuric acid 0.5 ml gave the maximum color intensity with the highest stability as indicated in Figure 12 For PCV.

Effect of Dye Concentration
The absorption intensities of the formed complexes were dependent upon the dye concentration; for ECC dye, the optimum range was 0.1% -0.3% w/v, thus 0.2% w/v was selected, Figure 13.For SCPS dye, the optimum range was 0.05% -0.2% w/v, thus 0.1% w/v was selected, Figure 14.For BPR dye, the optimum range was     For PCV dye the optimum range was 0.07% -0.2% w/v, thus 0.1% w/v was selected, Figure 16.Then the formed colored complex was extracted using different volumes of dyes.For ECC dye, optimum range was 0.5 -3 ml, thus 2 ml for DTZ and 1.5 ml for both ADB and VPM was selected, Figure 17.For SCPS and BPR dyes, the optimum range were 0.25 -2 ml and 0.5 -2 ml respectively, thus 1.5 ml for each drug was selected, Figures 18 and 19.For PCV dye, the optimum range was 0.5 -2.5 ml, thus 1.5 ml in case DTZ and 2 ml in case of VPM was selected, Figure 20 and 21.

Analytical Data
Standard calibration graphs for the studied drugs with each dye were constructed by analyzing a series of concentrations of the drugs.Beer's plots showed linear cor-       relations over a wide range of drug concentrations with good correlation coefficients.Data of such studies are illustrated in Table 2.The slopes (b) of the calibration curves indicated the high sensitivity of the proposed method while the small intercept reflected the low interfering background [37].
The accuracy of the proposed method was demonstrated by recovery experiments, which were carried out by adding a fixed amount of pure drug to pre-analyzed dosage forms.The analytical results obtained were summarized in Table 3.The percentage of RSDs can be considered to be very satisfactory.
The performance of the proposed methods was statistically compared with those of reported ones [38][39][40] using student's t-test and the variance ratio F-test [41], Table 4.This comparison did not show any significance differences indicating good accuracy and high sensitivity of the proposed procedure.
The effect of the presence common excipients such as: starch, lactose, sucrose, glucose, magnesium stearate, gum acacia and talc was studied.The excellent recoveries, obtained in Table 5 for ECC as representative example, indicated that the presence of these excipients did not interfere with the proposed method.This could be explained on the basis that all these additives are natural and almost non extractable by organic solvents as well as very or non basic at all and so they do not contribute in the reaction.
The Precision [42] of the proposed method was checked by replicate analysis of five separate standard solutions of each compound at three concentration levels.The calculated relative standard deviation values were below 2% indicating good repeatability and reliability of the proposed method [37,43].The results and their statistical analysis were summarized in Table 6.for ECC as representative example.

CONCLUSION
The proposed methods are advantageous in comparison to other existing spectrophotometric methods because the detection limits are low.The molar absorptivities are also comparable with low RSDs.No interference from common excipients was encountered.Thus the proposed methods are simple, sensitive, accurate, precise and easy to use and might be preferred to other published methods for assay of the drug in clinical and quality control laboratories.

Table 1 .
Calculated stability constants and molar absorptivities of the formed chelates between the studied drugs and the four dyes.Parameter Drug Dye Formation constant (K f ) Log (K f ) ΔG ε (L −1 •mol −1

Figure 2 .
Figure 2. Job's plots method of continuous variation between the studied drugs and ECC ion pair complex.

Figure 3 .
Figure 3. Job's plots method of continuous variation between the studied drugs and SCPS ion pair complex.

Figure 4 .
Figure 4. Job's plots method of continuous variation between the studied drugs and BPR ion pair complex.

Figure 5 .
Figure 5. Job's plots method of continuous variation between the studied drugs and PCV ion pair complex.

Figure 6 .
Figure 6.Effect of citric acid morality on the absorption spectra of formed ion pair complex using 15 µg•ml −1 from each studied drug and 0.2% w/v ECC.

Figure 7 .
Figure 7. Effect of sulfuric acid morality on the absorption spectra of formed ion pair complex using 50 µg•ml −1 of each studied drug and 0.1% w/v SCPS.

Figure 8 .
Figure8.Effect of sulfuric acid morality on the absorption spectra of formed ion pair complex using 30 µg•ml −1 of each studied drug and 0.05% w/v BPR complex.

Figure 10 .
Figure 10.Effect of citric acid volume on the absorption spectra of the formed ion pair complex using 15 µg•ml −1 of each studied drug and 0.2% w/v ECC.

Figure 11 .
Figure 11.Effect of sulfuric acid volume on the absorption spectra of the formed ion pair complex using 50 µg•ml −1 of each studied drug and 0.1% w/v SCPS.

Figure 12 .
Figure 12.Effect of sulfuric acid volume on the absorption spectra of the formed ion pair complex using 30 µg•ml −1 of each studied drug and 0.05% w/v BPR.

Figure 14 .
Figure 14.Effect of eriochrome cyanine concentration on the absorption spectra of the formed ion pair complex using 15 µg•ml −1 of each studied drug.0.03% -0.07%w/v, thus 0.05% w/v was selected, Figure 15.For PCV dye the optimum range was 0.07% -0.2% w/v, thus 0.1% w/v was selected, Figure16.Then the formed colored complex was extracted using different volumes of dyes.For ECC dye, optimum range was 0.5 -3 ml, thus 2 ml for DTZ and 1.5 ml for both ADB and VPM was selected, Figure17.For SCPS and BPR dyes, the optimum range were 0.25 -2 ml and 0.5 -2 ml respectively, thus 1.5 ml for each drug was selected, Figures18 and 19.For PCV dye, the optimum range was 0.5 -2.5 ml, thus 1.5 ml in case DTZ and 2 ml in case of VPM was selected, Figure20and 21.

Figure 15 .
Figure 15.Effect of Sulfochlorophenol-S concentration on the absorption spectra of the formed ion pair complex using 50 µg•ml −1 of each studied drug.

Figure 16 .
Figure 16.Effect of Bromopyrogallol red concentration on the absorption spectra of the formed ion pair complex using 30 µg•ml −1 of each studied drug.

Figure 18 .
Figure 18.Effect of eriochrome cyanine concentration on the absorption spectra of the formed ion pair complex using 15 µg•ml −1 of each studied drug.

Figure 19 .
Figure19.Effect of 0.1% w/v Sulfochlorophenol-S volume on the absorption spectra of the formed ion pair complex using 50 µg•ml −1 of each studied drug.

Figure 20 .
Figure 20.Effect of 0.05% w/v Bromopyrogallol red volume on the absorption spectra of the formed ion pair complex using 30 µg•ml −1 of each studied drug.

Table 2 .
Quantitative parameters for proposed method.

Table 3 .
Analysis of pharmaceutical preparations using ECC, SCPS, BPR and PCV methods, applying standard addition technique.

Table 4 .
Statistical comparison between the proposed and reported methods for determination of studied drugs in their pharmaceutical formulations.

Table 5 .
Assay of the studied drugs in presence of some common excipients using eriochrome cyanine method.