1. INTRODUCTION:

Diltiazem hydrochloride (DT-HCl) (Fig. 1), ¹⁾chemically, it is (2S,3S)-5-(2-Dimethylaminoethyl)-2,3,4,5-tetrahydro-2-(4-methoxyphenyl)-4-oxo-1,5-benzothiazepin-3-yl acetate hydrochloride. ²⁾ Medically, it is a benzothiazepine calcium-channel blocker. It is a peripheral and coronary vasodilator property. DT-HCl inhibits cardiac conduction, particularly at the sino-atrial and atrioventricular nodes.

DT-HCl is given by mouth in the management of angina pectoris and hypertension. DT-HCl is given by intravenous administration in the treatment of various cardiac arrhythmias

A number of methods are available for DT-HCl determination in various types of samples. These including high-performance liquid chromatography (HPLC)^3-5), gas chromatograph (GC)⁶⁾, high-performance thin layer chromatography (HPTLC)⁷⁾, capillary electrophoresis (CE)^8-10) and electrochemical methods^11-14). A few methods have been reported on the determination of DT-HCl as visible spectrophotometry^15-17). Several of these above mentioned methods require the use of hazardous and expensive chemicals which make the process not only a challenge for the environment but too much complicated, time consuming and expensive costly.

C₂₂H₂₆N₂O₄S, HCl =451.0

Fig. 1 Chemical structure of DT-HCl

Pipazethate hydrochloride (PZ-HCl) (Fig. 2), ¹⁾chemically, it is 2-(2-Piperidinoethoxy)ethyl pyrido[3,2-b][1,4]benzothiazine-10-carboxylate hydrochloride. ²⁾ Medically, it is a centrally acting cough suppressant which also has some peripheral actions in non-productive cough. It has a bronchodilator effect which reduces the increased resistance to expiration during paroxysms of cough.

C₂₁H₂₅N₃O₃S, HCl =436.0

Fig. 2 Chemical structure of PZ-HCl

In recent years, a very few analytical methods appeared in the literature for the determination of PZ-HCl in various types of samples such as high-performance liquid chromatography (HPLC)¹⁸⁾, visible spectrophotometry^19,20) and electrochemical method such as conductimetric method²¹⁾. These methods involve a time-consuming extraction procedures or heating and require strictly con trolled reaction conditions. Many of these methods are less sensitive.

As of our knowledge no reports have been mentioned in the literature for the quantitative determination of DT-HCl and PZ-HCl in various types of samples by UV spectrophotometric method.

Thus, the aim of the present work was to investigate economical, simple, precise, sensitive and environmental friendly three analytical methods (A─C) for the determination of DT-HCl and PZ-HCl using UV/VIS spectrophotometry. In method A, simpler direct spectrophotometric measurements in ultra violet region without any chemical reagents. Whereas the methods B and C involves the formation of intense yellow ion-association complex between drug(s) and either of phenol red (PR) or chlorophenol red (CPR) reagents followed by extraction with proper water-immiscible organic solvent. The results obtained from the proposed methods (A─C) also have been statistically compared using t- and F-tests to the reference methods. They also have the advantage of being cheaper than the reported methods.

2. EXPERIMENTAL:

2.1. Materials and Reagents:

DT-HCl and PZ-HCl standards were kindly supplied as a gift samples by Egyptian International Pharmaceutical Industries Co. Cairo, Egypt (E.I.P.I.CO.) and used without further puriﬁcation and purity was conﬁrmed by thin layer chromatography and by melting point measurements. Commercial tablets of DT-HCl such as Altiazem tablets containing 60 mg DT-HCl and Selgon tablets containing 20 mg PZ-HCl were purchased from local drug market. Phenol red (PR) (Fig. 3) and chlorophenol red (CPR) (Fig. 4) reagents from Merck chemicals. All other chemicals, solvents and reagents used were obtained from commercial sources and were of analytical reagent grade. Doubly distilled water was used throughout for final washings and preparations of all aqueous solutions. Freshly prepared solutions were always employed.

Fig. 3 Chemical Structure of PR

(Phenolsulfonaphthalein)

Fig. 4 Chemical Structure of CPR

(3´,3´´-Di-chlorophenolsulfonaphthalein)

2.2. Instruments and apparatus:

All spectrophotometric measurements were carried out by using UV-Visible Diode Array spectrophotometer (Hewlett Packard-Model 8452A), in 1.0 cm quartz cells, was connected to PC computer and Hewlett Packard DeskJet printer. The pHs of the prepared solutions were adjusted using Jenway pH–meter. Moreover, the doubly distilled water was obtained ELGA apparatus model, UHQ-II-MK3, UK. Temperature adjustment during experiments was carried out with controlled temperature Water Bath (MLW) Model, W11-TGL, GBR. Automatic Pipettes were used to measure the very small volumes whereas glass micropipettes and burettes were used to measure the large volumes.

2.3. Preparation of standard solutions:

In method A, standard stock solutions (100 mL, 500 μg/mL) of DT-HCl and PZ-HCl were freshly prepared by dissolving the appropriate weight of 50 mg of DT-HCl and PZ-HCl, respectively in least amount of 0.1M HCl solution then the solutions were made up to 100 mL with 0.1M HCl solution and kept in the dark and at ambient temperature for one month. Working standard solutions [2, 5, 10, 15, 20 and 25 µg/mL and 5, 10, 20, 30, 40, 50 and 60 µg/mL for DT-HCl and PZ-HCl, respectively] were daily prepared by successive dilutions for carrying out the subsequent studies.

For methods B and C, standard stock solutions 0.01 M of DT-HCl and PZ-HCl were freshly prepared by dissolving the appropriate weights of 1.1275 g (DT-HCl) and 1.09 g (PZ-HCl) in least amount of warm water then the solutions were made up to 100 mL with distilled water. Successive dilutions were prepared for carrying out the subsequent studies.

Standard stock solutions 0.01 M of PR and CPR were freshly prepared by dissolving the appropriate weights of 0.8860 g and 1.0582 g, respectively in least amount of methanol then the solutions were made up to 100 mL with distilled water. Successive dilutions were prepared for carrying out the subsequent studies.

2.4. Recommended Procedures for the determination of DT-HCl and PZ-HCl (calibration standards):

In method A, six different concentrations of DT-HCl as working standard solutions, chosen for the calibration curve (Fig. 7) were 2, 5, 10, 15, 20 and 25 µg/mL (n=5) in 0.1M HCl solution. Then the absorbance of these solutions was measured at 238 (Fig. 5), 0.1M HCl was used as a blank solution. In a similar way, seven different concentrations of PZ-HCl as working standard solutions, chosen for the calibration curve (Fig. 8) were5, 10, 20, 30, 40, 50 and 60 µg/mL (n=5) in 0.1M HCl solution. Then the absorbance of these solutions was measured at 253 (Fig. 6), 0.1M HCl was used as a blank solution. The calibration data of DT-HCl and PZ-HCl are presented in Table 1.

For methods B and C, 2.5 mL of 0.005 M (PR or CPR) were added in acid medium to a solution of DT-HCl using the concentration range of 11.28—112.75 µg/mL (PR) and 2.26—48.48 µg/mL (CPR) of DT-HCl (n=5) were transferred into a series of 125 mL separating funnels. Methylene chloride (10 mL) was added to each of the separating funnel, the contents were shaken well for two minutes and left at room temperature for a minute. The two phases were allowed to separate and the methylene chloride layer was passed through anhydrous sodium sulphate. The absorbances of the yellow ion-association complexes were measured at 390 and 402 nm for PR and CPR, respectively, against corresponding reagent blank. This blank was prepared in the same manner without the addition of DT-HCl. A calibration curves were plotted (Fig.23). In a similar way, 2.5 mL of 0.005 M PR or 0.001 M CPR were added in acid medium to a solution of PZ-HCl within concentration range of 8.72—104.64 µg/mL (PR) and 3.27—49.05 µg/mL (CPR) of PZ-HCl (n=5). Using the same procedures described for DT-HCl. The absorbance of the extract was measured at λ_max 393 and 405 nm for PR and CPR, respectively, against corresponding reagent blank. A calibration curves were plotted (Fig.24). The results of DT-HCl and PZ-HCl ion-associates with CPR or PR correlated to Beer’s law are presented in Table 8.

2.5. Determination of DT-HCl and PZ-HCl in tablets:

For methods (A─C), ten tablets of altiazem and selgon were accurately weighed separately and finely powdered and mixed. A portion of the powder equivalent to the average weight of one tablet was transferred into a 100 mL volumetric flask and 30 mL of 0.1 M HCl solution and distilled water for method A and methods (B─C), respectively were added. The content of the flask was sonicated for 15 min. and filtered through whatman No.41 filter paper to separate out the insoluble excipients. The residues were washed thoroughly with 0.1 M HCl solution and distilled water for method A and methods (B─C), respectively. Then take aliquot of the filtrate made up to 100 mL volume with 0.1 M HCl solution and distilled water for method A and methods (B─C), respectively in volumetric flask. Appropriate solutions were prepared by taking suitable aliquots of the clear supernatant and diluting them with 0.1M HCl solution and distilled water for method A and methods (B─C), respectively to give final concentrations. Then the absorbance of these solutions was measured against 0.1 M HCl and distilled water for method A and methods (B─C), respectively at λ_max. The amount of DT-HCl or PZ-HCl per tablet was calculated using the calibration curve method. A standard addition method was also used to confirm the accuracy and recoveries.

3. RESULTS AND DISCUSSION:

3.1. Method A:

The method A is based on the simple scanning of DT-HCl and PZ-HCl in 0.1M HCl solution using UV/visible spectrophotometer in ranges of 200─400 nm and their determination in the presence of various excipients.

Different media were investigated to develop a suitable UV-spectrophotometric method for the analysis of DT-HCl and PZ-HCl. For selection of media the criteria employed were sensitivity of the method, ease of sample preparation, solubility of the drug, cost of solvents and applicability of method to various purposes. Hydrochloric acid was investigated and the UV spectra of DT-HCl and PZ-HCl were measured. In hydrochloric acid solution, well defined peaks were obtained in 0.1 M HCl shows maximum absorbance at wavelength of 238 nm (Fig. 5) and 253 nm (Fig. 6) for DT-HCl and PZ-HCl, respectively. At the end of studies, 0.1 M HCl solution was chosen for the working solutions. These wavelengths 238 nm and 253 nm were used for the determination of DT-HCl and PZ-HCl, respectively.

Fig. 5 UV Absorption spectrum of 10 μg/mL

concentration of DT-HCl in 0.1 M HCl

Fig. 6 UV Absorption spectrum of 30 μg/mL

concentration of PZ-HCl in 0.1 M HCl

3.1.1. Analytical validation:

Validation is one of the most important steps in method development for analytical determination. The main validation parameters^22,23) such as stability, linearity of calibration curve, sensitivity (DL and QL), precision, specificity, selectivity, accuracy and recovery were evaluated in developed method.

3.1.2. Stability:

The standard stock solutions of DT-HCl or PZ-HCl were stored in two different conditions, at +4°C and at ambient temperature for one month. During this period, the solutions were analyzed with UV spectrophotometric method, the spectra were compared with the spectra of daily prepared standard solutions and no differences were obtained between them. Therefore, DT-HCl and PZ-HCl are highly stable in the mentioned conditions.

3.1.3 Linearity of calibration curves:

For the DT-HCl at λ_max of 238 nm, calibration curve (Fig. 7) was constructed after analysis of six different concentrations with each concentration was measured five times. Each point of the calibration graph corresponded to the mean value obtained from five independent measurements. Five level calibration series with seven analyses at each concentration level were measured for UV determination at λ_max of 253 nm. The standard calibration curve of PZ-HCl was constructed by plotting absorbance verses concentration (Fig. 8).

Fig. 7:Calibration curve of DT-HCl

against 0.1 M HCl as blank

Fig. 8 Calibration curve of PZ-HCl

against 0.1 M HCl as blank

For the DT-HCl, the linearity range was found to be 2─25 μg/mL at 238 nm. In PZ-HCl, the linearity range was found to be 5─60 μg/mL at 253 nm. Lower values of parameters like slope and intercept (Table 1) indicated high precision of the proposed method. Goodness of fit of regression equations was supported by high regression coefficient values. The molar absorpitiveties (ε) were calculated to be 1.837 x 10⁴ and 7.931 x 10³ L mol^-1 cm^-1for DT-HCl and PZ-HCl, respectively and also Sandell’s sensitivities were calculated to be 2.5 x 10^-2 and 5.5 x 10^-2 µg cm^-2 for DT-HCl and PZ-HCl, respectively.

Table 1. Features of the standard calibration curves of DT-HCl and PZ-HCl by the proposed UVspectrophotometric method (N═5)

*Features*	*UV spectrophotometric method* *(DT-HCl)*	*UV spectrophotometric method* *(PZ-HCl)*
λ max, nm	238	253
Number of data points	6	7
Beer’s law verification range, μg/mL	2─25	5─60
Molar absorpitivity (ε) [Lmol^-1cm^-1 ]	1.837 x 10⁴	7.931 x 10³
Sandell’s sensitivity [μg/mL cm^-2 ]	2.5 x 10^-2	5.5 x 10^-2
) Regression equation (Y^a	Y = a + bC	Y = a + bC
Slope (b)	0.0401	0.0180
Intercept (a)	0.004	0.0053
Correlation coefficient (r²)	0.9999	0.9998
RSD^b)(%)	0.4034─1.7349	0.4924─2.5659
Limit of Detection, LOD, μg/mL	0.136	0.123
Limit of Quantification, LOQ, μg/mL	0.412	0.373

a) Y = a + bC (where C is the concentration of analyte, μg/mL and Y is absorbance); b) Calculated from five determinations

Table 2. Summary of assay the intra-and inter-day precision obtained data from DT-HCl and PZ-HCl by the proposed UV spectrophotometric method (N=6)

Drug	Conc. (μg/mL)	N	Intra-day precision Absorbance at 238 or 253 nm X¯ ±S.D R.S.D (%)			Inter-day precision Absorbance at 238 or 253 nm X¯ ±S.D R.S.D (%)
DT-HCl	06.00	6	0.23958	0.005	2.223	0.23189	0.010	4.458
	12.00	6	0.47698	0.008	1.607	0.46350	0.017	3.567
	18.00	6	0.70929	0.021	3.002	0.69871	0.029	4.084
PZ-HCl	16.00	6	0.28431	0.008	2.865	0.27378	0.016	5.696
	32.00	6	0.57596	0.008	1.341	0.56384	0.018	3.123
	48.00	6	0.86348	0.015	1.701	0.85360	0.023	2.711

Table 3. Determination of DT-HCl and PZ-HCl in presence of excipients (each value is result of five separate determinations)

*Excipients*	*DT-HCl*		PZ-HCl
*Excipients*	Amount taken of DT-HCl μg/mL	(%) Recovery (±SD)	Amount taken of PZ-HCl μg/mL	(%) Recovery (±SD
Microcrystalline cellulose	10.00	100.02±0.47	30.00	99.99±0.27
Lactose	10.00	99.82±0.53	30.00	100.07±0.44
Povidone	10.00	99.68±1.01	30.00	99.89±0.25
Starch	10.00	100.02±0.89	30.00	100.18±0.33
Magnesium stearate	10.00	100.06±0.68	30.00	99.94±0.27
Sucrose	10.00	100.08±0.52	30.00	99.93±0.38
Glucose	10.00	99.79±0.66	30.00	100.02±0.69
Hydroxypropylmethylcellulose	10.00	99.74±0.65	30.00	99.96±0.46

The linear regression equations (with intercepts and slopes) and correlation coefficients of the mean of five consecutives calibration curves of DT-HCl and PZ-HCl are given in Table 1. The regression equations were Y = 0.0401 C + 0.004 and Y = 0.0180 C + 0.0053 for DT-HCl and PZ-HCl, respectively where Y is the absorbance and C is the concentration in μg/mL, with correlation coefficients of 0.9999 and 0.9998 for DT-HCl and PZ-HCl, respectively

3.1.4. Detection limit (DL) and quantitation limit (QL):

The DL and QL of DT-HCl and PZ-HCl, respectively were determined using standard calibration curves. For the DT-HCl, DL and QL were calculated to be 0.136 μg/mL and 0.412 μg/mL, respectively. Whereas PZ-HCl, DL and QL were calculated to be 0.123 μg/mL and 0.373 μg/mL, respectively. [DL and QL were calculated as 3.3 σ/S and 10 σ/S, respectively, where S is the slope of the calibration curve and σ is the standard deviation of y-intercept of regression equation²²⁾].

3.1.5. Precision:

The precision of a method is defined as the closeness of agreement between independent test results obtained under the experimental conditions. The precision of the proposed method for the determination of DT-HCl and PZ-HCl was investigated with respect to repeatability (% RSD). For intra-day precision, three different concentrations of DT-HCl in linear range (6, 12 and 18 µg/mL) and PZ-HCl in linear range (16, 32 and 48 µg/mL) were analyzed in six independent series at various time interval in the same day and % RSD was ranged from 1.61 to 3.00 and 1.34 to 2.87 for DT-HCl and PZ-HCl, respectively (Table 2). Repeatability results indicate the precision under the same operating conditions over a short interval of time. And the day-to-day precision was studied by taking the absorbance of the same three different concentrations at various consecutive days (inter-day precision) and the % RSD was calculated and ranged from 3.57 to 4.46 and 2.71 to 5.70 for DT-HCl and PZ-HCl, respectively (Table 2). The all R.S.D values for both intra- and inter-day precision were lower than 10%. Therefore, R.S.D values were within the acceptable range indicating that this method has excellent repeatability and intermediate precision.

3.1.6. Specificity and selectivity (Interferences Study):

The UV spectra of DT-HCl (10 µg/mL) and PZ-HCl (30 µg/mL) solutions were not changed in the presence of excess of different additive and excipients (microcrystalline cellulose, lactose, povidone starch, magnesium stearate, sucrose, glucose and hydroxypropylmethycellulose). The spectra obtained from tablets and synthetic tablets solutions were identical with that obtained spectra from standard solutions containing an equivalent concentration of DT-HCl and PZ-HCl. Tablets solutions showed that the wavelength of maximum absorbance of DT-HCl and PZ-HCl did not change. It was concluded that the excipients did not interfere with quantification of compounds of DT-HCl and PZ-HCl in this method and the proposed method could be considered specific (Table 3).

3.1.7. Accuracy and recovery:

Recovery studies were performed to judge the accuracy of the proposed method. Three different concentrations of DT-HCl (6, 12 and 18 µg/mL) and PZ-HCl (16, 18 and 48 µg/mL) in linear range were analyzed in six independent series. [From the amount of drug found, percentage recovery was calculated and accuracy was assessed as the percentage relative error (Bias %) between the measured mean concentration and added concentration at the same concentration of DT-HCl or PZ-HCl] And then, the results of the accuracy and recovery studies of compounds of DT-HCl and PZ-HCl in their pure forms and their tablet dosage forms are summarized in Table 4.

To give additional support to accuracy of the developed assay method, standard addition method was done. In this study, three different concentrations of pure DT-HCl drug (3, 6 and 9 µg/mL) and pure PZ-HCl drug (8, 16 and 24 µg/mL) were added to a known pre-analyzed formulation sample (6 µg/mL of DT-HCl tablet) and (16 µg/mL of PZ-HCl tablet), respectively and the total concentrations were determined using the proposed method (n=5). [The percent recovery of the added pure drug was calculated as, % recovery = [(C_v–C_u)/C_a]× 100, where C_v is the total drug concentration measured after standard addition, C_u is drug concentration in the formulation and C_a is drug concentration added to formulation]. Therefore, the results of the analysis and recovery studies of DT-HCl and PZ-HCl are summarized in Table 5.

The results obtained from the analyses and recovery studies of DT-HCl and PZ-HCl drugs shows the excellent mean % recovery values (nearly 100 %) and their low standard deviation values (S.D. <1) represent accuracy.

3.1.8. Analysis of tablets:

The optimized spectrophotometric method was applied to the direct determination of pure DT-HCl and Altiazem 60 mg /tablet as well as for pure PZ-HCl and Selgon 20 mg/tablet using calibration curve method without any sample extraction or filtration. The average amount present was discussed in details (section 3.1.7). The results show that the proposed method was successfully applied for the assay of compounds of DT-HCl and PZ-HCl in their pure forms and their tablet dosage forms (Tables 4 and 5). This indicates that the interference of excipients matrix is insignificant in estimation of compounds of DT-HCl and PZ-HCl by proposed method.

Table 4. Accuracy and recovery data for the developed method (each value is result of six separate determinations)

Drug

Sample

Predicted conc. (μg/mL)^a

Range Mean (±S.D) % R.S.D

Mean % recovery (±S.D)

Accuracy (%)^b

DT-HCl

Pure

solution

05.96-06.10

11.95-12.20

17.96-18.20

06.02±0.06

12.05±0.09

18.03±0.09

0.95

0.77

0.49

100.36±0.95

100.54±0.67

100.18±0.49

0.36

0.40

0.18

Altiazem Tablets

05.92-06.05

11.89-12.10

17.88-18.01

05.97±0.05

12.01±0.07

17.95±0.04

0.81

0.59

0.25

99.56±0.81

100.06±0.59

99.72±0.25

-0.44

0.06

-0.28

PZ-HCl

Pure

solution

15.72-16.03

31.95-32.10

47.76-48.07

15.92±0.13

32.02±0.06

47.97±0.11

0.79

0.19

0.24

99.52±0.79

100.07±0.19

99.95±0.24

-0.48

0.07

-0.06

Selgon

Tablet

15.60-16.01

31.63-32.10

47.45-47.85

15.85±0.15

31.87±0.18

47.62±0.16

0.94

0.55

0.33

99.09±0.93

99.58±0.55

99.21±0.32

-0.92

-0.42

-0.79

^a Predicted concentration of DT-HCl and PZ-HCl is calculated by linear regression equation.

^b Accuracy is given in % relative error = 100 ×[(predicted concentration – nominal concentration) /(nominal concentration)].

Table 5. Results of standard addition method for DT-HCl and PZ-HCl (each value is result of five separate determinations)

Drug

Method

Concentration of drug

in formulations (μg/mL)

Concentration of pure drug added (μg/mL)

Total concentration

of drug found (μg/mL)

% Analytical Recovery

(±S.D)

DT-HCl

0.1M HCl

05.97

03.00

06.00

09.00

08.95

11.97

14.97

99.40±2.13

100.07±0.73

100.02±0.65

PZ-HCl

0.1M HCl

15.86

08.00

16.00

24.00

23.81

31.83

39.77

99.43±1.60

99.79±0.62

99.64±1.10

Table 6. Statistical evaluation of obtained data from DT-HCl and PZ-HCl (pure drugs) and pharmaceutical formulations (tablets) containing DT-HCl and PZ-HCl by the proposed and reference methods

*Statistical values*	*DT-HCl* *Pure solution*	*Reference method*	*Altiazem* *Tablets*	*Reference method*	*PZ-HCl* *Pure solution*	*Reference method*	*Selgon* *Tablets*	Reference method
N	6	3	6	3	6	3	6	3
X¯, Recovery (%)	100.36	99.78	99.78	100.05	99.85	100.22	99.29	99.95
S.D	0.70	0.73	0.60	0.61	0.52	0.66	0.65	0.58
R.S.D (%)	0.70	0.73	0.60	0.61	0.52	0.66	0.66	0.58
F- value : F^c F^t	1.09 5.79		1.06 5.79		1.64 5.79		1.2681 5.79
t-value : t^c t^t	2.024 2.571		1.11 2.571		1.755 2.571		2.4834 2.571

N: number of determination, X¯: mean recovery, S.D: standard deviation, F-value and t-value are theoretical values at 95% confidence level, F^c: calculated F-value, F^t: tabulated F-value, t^c: calculated t-value, t^t: tabulated t-value

The results obtained for the proposed method was compared statistical with those obtained using the reference methods²⁴⁾ and summarized in Table 6. The calculated student's t-values and F-values²⁵⁾ did not exceed the theoretical ones at 95% confidence level. Therefore, there is no significant difference between the proposed method and reference methods. Based on the foregoing, the proposed method is highly sensitive, precise, simple and rapid and is successfully applied for the quality control of pure DT-HCl and PZ-HCl drugs and their pharmaceutical dosage forms (tablets).

The percent recovery of the added pure drug was calculated as, % recovery = [(C_v–C_u)/C_a] ×100, where C_v is the total drug concentration measured after standard addition; C_u, drug concentration in the formulation; C_a, drug concentration added to formulation

3.2. Methods B and C:

Both methods (B and C) involves the formation of intense yellow ion-association complex between drug(s) and either of PR or CPR reagents followed by extraction with methylene chloride. Many drugs are easy to be determined by spectrophotometry based on colour. Optimum reaction conditions for quantitative determination ion-association complexes of DT-HCl and PZ-HCl with PR and CPR reagents were established via a number of following preliminary experiments.

3.2.1. Selection of suitable wavelength:

The absorption spectra of the formed ion–association complexes were measured in the visible region within 300─700 nm wavelength range against blank reagent prepared in the same manner without the addition of the drug.

The DT-HCl ion-associates with PR and CPR reagents, λ_max of 390 and 402 nm have been obtained, respectively as shown in Fig. 9. For PZ-HCl ion-associates, λ_max of 393 and 405 nm with PR and CPR reagents have been obtained, respectively as shown in Fig. 10.

Fig. 9 Absorption spectra of DT-HCl

ion-associates with PR and CPR

Fig. 10 Absorption spectra of PZ-HC

ion-associates with PR and CPR

3.2.2. Effect of extracting solvents

The polarity of the solvent affects both extraction efficiency and absorpitivity of the ion-associates. Therefore, several water-immiscible organic solvents including n-hexane, petroleum ether, cyclohexane, carbon tetrachloride, toluene, benzene, diethyl ether, methylene chloride and chloroform were investigated.

The most convenient solvent for DT-HCl and PZ-HCl ion-associates which exhibit the maximum absorbance, high extraction power and stable colours is methylene chloride.

In all cases the aqueous to organic phase ratio of 1:1.5 was the most suitable for the ion-associate extraction. Complete extraction was attained by using single portion of 10 mL solvent upon using the above reagents. Figs. 11 and 12 summarize the effect of extracting solvents on the formed ion-associates.

Fig. 11 Effect of extracting solvents on DT-HCl

ion-associates with PR and CPR

Fig.12 Effect of extracting solvents on PZ-HCl

ion-associates with PR and CPR

3.2.3. Effect of pH:

To investigate the optimum medium conditions to determine DT-HCl and PZ-HCl, quantitatively the effect of pH was studied by using a series of solutions (HCl/NaOH) in the pH range of 1─14, for developing the best colour of drug–reagent ion-associates against the chosen reagents. In DT-HCl and PZ-HCl, the optimum pH range for complete formation of the ion-associates showed that highest absorbance values, at their respective λ_max were found to be in the ranges 2─6 with PR and 2─5 for CPR, as shown in Figs. 13 and 14.

At pHs less than 2 for DT-HCl and PZ-HCl, the absorbance decrease may be attributed to the formation of diprotonated species of the drug. In case of pH < 6 or pH < 5 the absorbance decrease due to the formation of free base of the drug(s) which are insoluble in water and precipitates during the mixing.

Fig. 13 Effect of pH on DT-HCl ion associates

with PR and CPR

Fig. 14 Effect of pH on PZ-HCl ion associates

with PR and CPR

3.2.4. Effect of reagent concentration

The effect of reagent concentration was tested by using varying amounts (1─6) mL of 0.005 M (PR or CPR) with 1 mL of 0.001 M (DT-HCl or PZ-HCl).

After implementing the optimum pH condition for DT-HCl and PZ-HCl, the formed ion-associate was completely extracted with single portion of 10 mL proper solvent. The mixture was shaked for 2 minutes. The results showed that 5 mL of 0.005 M (PR or CPR) were sufficient for good colour intensity with maximum absorption of the investigated ion-associates.

3.2.5. Effect of time:

Under the above mentioned conditions the effect of time on the formation of the ion-associates was studied by measuring absorbance of the extracted ion-associates with increasing time intervals. The results showed that the ion-associates are formed almost instantaneously.

The effect of time on the stability of the ion–associates of DT-HCl and PZ-HCl are represented graphically in Figs. 15 and 16, respectively. For DT-HCl, the developed colour remained stable for 18 and 21 hours for PR and CPR at λ_max of 390 and 402 nm, respectively. Similarly, the ion-associates of PZ-HCl are formed almost instantaneously. Moreover, the developed colour for PZ-HCl remained stable for 21 and 18 hours for PR and CPR, at λ_max of 393 and 405 nm, respectively. After these intervals, a decrease in colour intensity occurred in ion associated of DT-HCl and PZ-HCl.

Fig. 15 Effect of time on the stability of DT-HCl

ion-associates with PR and CPR

Fig. 16 Effect of time on the stability of PZ-HCl

ion-associates with PR and CPR

3.2.6. Effect of temperature:

Under the afore mentioned conditions (solvents, pH, reagent concentration and time), the effect of temperature on the formation of the ion-associates was studied by measuring the absorbance of the extracted ion-associates at a temperature range of 25─90°C.

For DT-HCl, the results showed that the ion-associates are formed almost instantaneously in all cases at room temperature 25+5°C and remain constant up to 40°C and 45°C for PR and CPR, respectively as represented by its absorptivity at the recommended (λ_max). Similarly, the ion-associates of PZ-HCl are formed instantaneously with all reagents at room temperature 25+5°C and remain constant up to 40°C and 55°C for PR and CPR, respectively. The effect of temperature on the stability of ion–associates of DT-HCl and PZ-HCl are shown in Figs. 17 and 18, respectively.

Fig.17 Effect of temperature on the stability of

DT-HC ion-associates with PR and CPR

Fig. 18 Effect of temperature on the stability of

PZ-HCl ion-associates with PR and CPR

3.2.7. The stoichiometry of the ion-associates:

Aided by spectrophotometeric measurements, the stoichiometries of the ion-associates of DT-HCl and PZ-HCl with selected reagents were investigated by the aid of the following spectrophotometeric.

3.2.7.1. The molar ratio method:

The molar ratio method was described by Yoe and Jones²⁶⁾. At the optimum conditions described earlier for DT-HCl and PZ-HCl ion-associates with their proper reagents, a series of solutions were prepared in which the reagent contents was kept constant, while that of the drug regularly varied. The absorbancies of the resultant extracts were measured at the corresponding λ_max of the ion-associates. The absorbance values were plotted against the molar ratio of drug/reagent as shown in Figs. 19 and 20, respectively.

Two straight lines were intersecting at the molar ratio of 1 in case of PR and CPR which reflects the formation of 1:1 ratio of (drug: reagent) for all ion-associates.

Fig. 19 Molar ratio of DT-HCl ion-associates

with PR and CPR [D = drugs and R = reagents]

Fig. 20 Molar ratio of PZ-HCl ion-associates

with PR and CPR [D = drugs and R = reagents]

3.2.7.2 The continuous variation method

The modification of Job’s²⁷⁾ continuous variation method performed by Vosburgh and Cooper²⁸⁾ was utilized for investigating the reaction between drug and reagent. A series of solutions was prepared by mixing equimolar solutions of the drug and reagent in varying proportions while keeping the total molar concentration constant. The absorbance spectra of the resultant extracts were measured at the respective λ_max of the ion-associates to determine the absorbance. Then a plot of the absorbance against the mole fraction of the drug was constructed and presented graphically in Figs. 21 and 22, respectively.

The curves exhibit a maximum at mole fraction 0.5 with PR and CPR indicating the formation of 1:1 (drug: reagent) for the proposed ion-associates.

Fig. 21 Continuous variation of DT-HCl ion-

associates with PR and CPR

Fig. 22 Continuous variation of PZ-HCl ion-

associates with PR and CPR

3.2.8. Probable Reaction Mechanism for the Formation of Ion-Association Complexes

In the ﬁrst, aided by Chem Draw Ultra (Cambridge Soft Chem. Office, Ultra 2006 Versions 10.0), equipped with additional GAMES software^29-31) the structures of positive protonated nitrogen atom of DT-HCl and PZ-HCl compounds were proposed

In the second, the nature of the binding of reagents to each drug in the presence of equal amount of PR or CPR was determined by the molar ratio²⁶⁾ and the continuous variation methods^27,28). The results indicated that a 1:1 ratio of (drug: reagent) for all ion-associates are formed as shown in Figs. 19-22.

Found that DT-HCl and PZ-HCl reacts with PR or CPR forming ion-associated compounds through the electrostatic attraction between positive protonated nitrogen atom of DTH⁺ and PZH⁺and PR^- or CPR^- anions. Charts 1 and 2 summarize probable reaction mechanism for the formation of ion-association complexes of DT-HCl and PZ-HCl with PR or CPR.

Chart 1. Probable Reaction Mechanism for the Formation of Ion-Association Complexes of DTH⁺ with PR^- and CPR

Chart 2. Probable Reaction Mechanism for the Formation of Ion-Association Complexes of PZH⁺ with PR^- and CPR

Table 7. Optimal condition for the extraction of DT-HCl and PZ-HCl ion-associates with PR and CPR

Parameters	DT-HCl		PZ-HCl
Parameters	DT-PR	DT-CPR	PZ-PR	PZ-CPR
λ max, nm	390	402	393	405
Extracting solvents	Cl₂CH₂	Cl₂CH₂	Cl₂CH₂	Cl₂CH₂
Colour of extract	Yellow	Yellow	Yellow	Yellow
pH range	2–6	2–5	2–6	2–5
Stability of extracts, h.	18	21	21	18
Temperature on the stability, °C	40	45	40	55
The stoichiometry of the ion-associates	1:1	1:1	1:1	1:1

3.2.9. Validity of the Beer's Lambert Law:

The spectrophotometric determination of compounds of DT-HCl and PZ-HCl with PR and CPR were carried out using appropriate concentration range to ensure the obedience to Beer’s law (Figs 23 and 24).

Fig. 23 Calibration curves of DT-HCl ion-

associates with PR and CPR

Fig. 24 Calibration curves of PZ-HCl ion-

associates with PR and CPR

After optimization, the systems obeyed Beer’s law in the concentration range of 11.28—112.75 and 2.26—48.48 μg/mL for DT-HCl and 8.72—104.64 and 3.27—49.05 μg/mL for PZ-HCl with PR and CPR, respectively. The apparent molar absorptivities (ε) were found to be 4.086 x10³ and 9.919x10³ L mol^-1 cm^-1 for DT-HCl and 4.212 x 10³ and 9.624 x 10³ L mol^-1cm^-1 for PZ-HCl with PR and CPR, respectively and also Sandell’s sensitivities were calculated to be 1.10 x 10^-2 and 4.4 x 10^-2 µg cm^-2 for DT-HCl and 10.40 x 10^-2 and 4.5 x 10^-2 µg cm^-2 for PZ-HCl with PR and CPR reagents, respectively.

The linear regression equations (with intercepts and slopes) and correlation coefficients of the mean of five consecutives calibration curves of DT-HCl and PZ-HCl with PR and CPR reagents, respectively are given in Table 8.

The regression equation (Y = a + bC where Y = absorbance, a = intercept, b = slope and C = concentration in µg/mL), calculated from the calibration graphs (N=5) using Kalied graph program, were evaluated and recorded in Table 8 for DT-HCl and PZ-HCl, respectively. The intercepts of the lines were very small indicating that there is no systematic difference between the determined and expected concentrations within the investigated range using the current methods.

The all RSD values from DT-HCl and PZ-HCl were evaluated and recorded in Table 8. Theses data indicated that the developed methods have a good repeatability (were lower than 10%).

LOD and LOQ of DT-HCl and PZ-HCl with PR and CPR reagents, respectively were determined using standard calibration curves and recorded in Table 8

3.2.10. Interferences study:

To study the potential interference problems from the commonly used excipients and other additives which may be present in the pharmaceutical preparations such as microcrystalline cellulose, lactose, povidone, starch, magnesium stearate, sucrose and hydroxypropylmethylcellulose, recovery studies were out. Under the experimental conditions employed, excipients in different concentrations were added to a known amount of 20 and 10 µg/mL for DT-HCl with PR and CPR, respectively and to a known amount of 20 and 9 µg/mL for PZ-HCl with PR and CPR, respectively and analyzed according to recommended procedures described earlier (section 2.4).

Results of the recovery studies of DT-HCl and PZ-HCl drugs and the above mentioned excipients are presented in Table 9. It was concluded that the excipients did not interfere with quantification of DT-HCl and PZ-HCl drugs in these methods and the proposed methods could be considered specific. In addition, the recoveries in most cases were around 100% and the lower values of the RSD indicate the good precision of the method, thus the procedures was able to determination of DT-HCl and PZ-HCl drugs in the presence of excipients. In the proposed methods, there were no needs for pre-separation and only centrifugation was applied to make the solution clear.

Table 8. Features of the calibration curves of DT-HCl and PZ-HCl ion-associates with PR and CPR (N═5)

Features	*DT-HCl*		*PZ-HCl*
Features	Values for DT-PR	Values for DT-CPR	Values for PZ-PR	Values for PZ-CPR
Number of data points	9	7	7	6
Beer's law verification range, µg /mL	3.27–49.05	8.72–104.64	2.26–48.48	11.28–112.75
Molar absorpitivity (ε) [L mol^-1cm^-1 ]	9.624 x 10³	4.212 x 10³	9.919 x 10³	4.086 x 10³
Sandell’s sensitivity [µg cm^-2 ]	4.5 x 10^-2	10.40 x 10^-2	4.4 x 10^-2	1.10 x 10^-2
) Regression equation (Y^a	Y = a + bC	Y = a + bC	Y = a + bC	Y = a + bC
Slope (b)	0.0214	0.0097	0.0217	0.0088
Intercept (a)	0.007	-0.002	0.006	0.011
Correlation coefficient (r²)	0.9998	0.9995	0.9997	0.9998
RSD^b)(%)	0.69–3.09	0.20–2.55	0.36–3.45	0.30–3.0
Limit of Detection, LOD, µg /mL	0.38	0.75	0.32	0.91
Limit of Quantification, LOQ, µg /mL	1.15	2.28	0.96	2.77

a) Y=a+bC (where C is the concentration of analyte, µg /mL and Y is absorbance).; b) Calculated from five determinations.

Table 9. Determination of compounds of DT-HCl and PZ-HCl with PR and CPR in presence of excipients (each value is result of five separate determinations)

*Excipients*	*DT-HCl*		PZ-HCl
*Excipients*	Amount taken 20μg/mL of DT-HCl % Recovery ±SD PR	Amount taken 10μg/mL of DT-HCl % Recovery ±SD CPR	Amount taken 20μg/mL of PZ-HCl % Recovery ±SD PR	Amount taken 9μg/mL of PZ-HCl % Recovery ±SD CPR
Microcrystalline cellulose	100.23±1.23	100.20±0.95	99.33±0.93	100.18±0.95
Lactose	99.88±0.86	100.13±1.25	100.03±1.57	99.70±1.079
Povidone	99.93±1.30	99.20±0.66	99.95±1.05	100.15±0.84
Starch	99.73±1.47	100.17±1.05	99.97±1.42	100.15±0.74
Magnesium stearate	100.17±0.73	100.47±1.34	99.32±0.67	99.33±0.55
Sucrose	100.37±1.33	100.30±1.31	100.13±0.60	100.18±1.11
Glucose	99.95±0.70	99.27±1.12	99.72±0.86	99.70±0.74
Hydroxypropyl methylcellulose	100.50±0.98	100.67±1.10	99.55±0.70	99.93±1.11

Table 10. Accuracy and recovery data for the developed methods (each value is result of five separate determinations)

Accuracy (%)^b

Mean % recovery (±S.D)

Predicted con. (µg/mL)^a

Range Mean (±S.D) R.S.D

Method

Sample

Drug

0.04

-0.65

-0.42

100.04±1.14

99.35±0.83

99.58±0.63

1.14

0.83

0.63

20.01±0.23

49.68±0.41

89.62±0.57

19.73-20.11

49.25-50.14

88.86-90.25

Pure

solution

DT-HCl

0.72

-0.24

0.02

100.72±1.41

99.76±1.40

100.02±0.69

1.40

0.69

10.07±0.14

19.95±0.28

35.01±0.24

09.86-10.25

19.62-20.30

34.71-35.30

CPR

-0.52

-1.00

-0.90

99.48±1.03

99±1.14

99.098±0.81

1.03

1.15

0.82

19.90±0.21

49.50±0.57

89.19±0.73

19.62-20.11

48.86-50.15

88.45-90.16

Altiazem Tablets

-0.42

-0.35

-0.57

99.58±1.85

99.65±0.86

99.43±0.65

1.86

0.86

0.65

09.96±0.19

19.93±0.17

34.8±0.23

09.78-10.20

19.69-20.11

34.55-35.10

CPR

-0.27

-0.02

-0.05

99.73±0.93

99.98±0.79

99.95±0.31

0.94

0.79

0.31

19.95±0.19

39.99±0.31

79.96±0.25

19.73-20.20

39.49-40.31

79.66-80.22

Pure

solution

PZ-HCl

0.20

-0.14

-0.22

100.20±1.76

99.86±1.03

99.78±0.75

1.75

1.04

0.75

9.02±0.16

17.97±0.19

35.92±0.27

08.85-09.25

17.77-18.22

35.55-36.16

CPR

-0.45

-0.57

-0.38

99.55±1.19

99.44±0.96

99.62±0.36

1.20

0.97

0.37

19.91±0.24

39.77±0.38

79.69±0.29

19.65-20.20

39.29-40.21

79.31-80.05

Selgon

Tablets

-0.16

-0.56

-0.71

99.84±1.38

99.45±1.07

99.29±0.72

1.38

1.08

0.73

08.99±0.12

17.90±0.19

35.74±0.26

08.85-09.16

17.59-18.1

35.48-36.14

CPR

^a Predicted concentration of DT-HCl and PZ-HCl with PR and CPR were calculated by linear regression equations

^b Accuracy is given in % relative error = 100× [(predicted concentration – nominal concentration)/(nominal concentration)].

Table 11. Results of standard addition method for DT-HCl and PZ-HCl with PR and CPR (each value is result of five separate determinations)

% Analytical recovery (±S.D)

Total concentration of drug found (µg/mL)

Concentration of pure drug added (µg/mL)

Concentration of drug in tablets (µg/mL)

Method

Drug

99.59±1.06

99.35±0.65

99.42±0.51

39.78

69.54

109.29

20.00

50.00

90.00

19.86

DT-HCl

99.56±1.96

99.48±1.66

99.76±1.09

19.88

29.82

44.84

10.00

20.00

35.00

09.92

CPR

99.54±1.28

99.74±0.86

99.68±0.81

39.77

59.75

99.51

20.00

40.00

80.00

19.86

PZ-HCl

99.58±1.51

99.22±1.61

99.38±0.86

17.90

26.82

44.72

09.00

18.00

36.00

08.94

CPR

The percent recovery of the added pure drug was calculated as, % recovery = [(C_v–C_u)/C_a]×100, where C_v is the total drug concentration measured after standard addition; C_u, drug concentration in the formulation; C_a, drug concentration added to formulation

Table 12. Statistical evaluations of obtained data from DT-HCl and PZ-HCl (pure drugs) and pharmaceutical formulations (tablets) containing DT-HCl and PZ-HCl by the proposed and reference methods

Statistical values	*DT-HCl Pure* *solution*		*Reference method*	*Altiazem* *Tablets*		*Reference method*	*PZ-HCl Pure* *solution*		*Reference method*	*Selgon* *Tablets*		*Reference method*
Statistical values	PR	CPR	*Reference method*	PR	CPR	*Reference method*	PR	CPR	*Reference method*	PR	CPR	*Reference method*
N	5	5	3	5	5	3	5	5	3	5	5	3
X¯, Recovery (%)	99.53	99.54	100.22	99.95	99.89	100.05	99.55	99.21	99.78	100.17	99.66	99.95
S.D.	1.04	0.85	0.66	1.177	0.68	0.61	1.15	0.97	0.73	1.20	0.88	0.58
R.S.D (%)	1.04	0.85	0.66	1.177	0.68	0.61	1.16	0.98	0.73	1.20	0.88	0.58
F- value: F^c F^t	3.22 6.94	2.14 6.94		3.16 6.94	1.07 6.94		3.51 6.94	2.49 6.94		2.68 6.94	1.43 6.94
t-value: t^c t^t	0.90 2.776	1.08 2.776		0.51 2.776	1.08 2.776		0.97 2.776	1.94 2.776		0.73 2.776	0.31 2.776

The percent recovery of the added pure drug was calculated as, % recovery = [(C_v–C_u)/C_a]×100, where C_v is the total drug concentration measured after standard addition; C_u, drug concentration in the formulation; C_a, drug concentration added to formulation

4. CONCLUSIONS:

The proposed methods A, B and C are simple, rapid, precise and sensitive compared to the reported methods. The utility of the proposed methods for the determination of compounds of DT-HCl and PZ-HCl in their pure forms and their tablet dosage forms have been well demonstrated. The assay methods did not involve any stringent experimental conditions, and were also free from interference by common excipients. The mean values obtained and the calculated standard deviations are compared with those obtained by the reference methods, by applying the t- and F-tests. The results presented herein for DT-HCl and PZ-HCl express excellent agreement and considered significant with those obtained using reference methods

Hence, the proposed methods could be used for routine quality control. Thus, it clear that the present methods are of high accuracy, precision, speed and selectivity, beside being of low cost and easily applied for the determination of the drugs under investigation in pure form and tablet dosage form depend on simpler direct spectrophotometric measurements in ultra violet region without any chemical reagents and simpler spectrophotometric measurements in visible region using chemical reagents which are available.

5. REFERENCES:

1. The Merck Index, an Encyclopedia of Chemicals, Drugs and Biologicals Merck, N.J. Rahway, USA, 13^th ed., 2001.

2. K .Parfitt, ed. Martindale, "The Extra Pharmacopoeia", the Pharmaceutical Press, London, 36^th ed., 2009.

3. M.A. Garcia, C. Solans, J.J. Aramayona, L.J. Fraile, M.A. Bregante and J.R. Castillo, Talanta, 1998, 47, 1245-1254.

4. Y. Shah, S. Khanna, V.S. Dighe and K.C. Jindal, Indian Drugs, 1990, 27, 363-364.

5. P.M. Lacroix, N. Beaulieu, T.D. Cyr and E.G. Lovering, J. Pharm. Sci., 1989 78, 243-246.

6. R.T. Sane, S.M. Purandare, V.G. Nayak, M.D. Joshi, V.R. Bhate and V.R. Nerurkar, Indian Drugs, 1989, 26, 623-625.

7. P.V. Devarajan and V.V. Dhavse, J. Chromatogr. Biomed. Appl., 1998, 706, 362-366.

8. Y. Kuwahara and H. Nishi, Yakugaku Zasshi, 1998, 118, 456-463.

9. C. Coors, H.G. Schulz and F. Stache, J. Chromatogr., 1995, 717, 235-243.

10. H. Soini, M.L. Riekkola and M.V. Novotny, J. Chromatogr., 1994, 680, 623-634.

11. Z.Z. Leng, X.Y. Hu, X.M. Jiang and R.J. Yu, Fenxi Huaxue, 1995, 23, 332-335.

12. Z. Leng, X. Hu, P. Feng and W. Bai, Fenxi Yiqi, 1993, 3, 15-18.

13. J. Wang, P.A.M. Farias and J.S. Mahmoud, Analyst (London), 1986, 111, 837-839.

14. L. Cunningham and H. Freiser, Anal. Chim. Acta, 1984, 157, 157-162.

15. N. Rahman and S.N.H. Azmi, J. Microchem., 2000, 65, 39-43.

16. R. Bindu and A.K. Chandrasekharan, Indian Drugs, 1994, 31, 168-169.

17. B.V. Kamath, K. Shivram and A.C. Shah, J. Pharm. Biomed. Anal., 1993, 11, 407-409.

18. H.D. Revanasiddappa and P.G. Ramappa, Indian Drugs, 1995, 32, 534-536.

19. A.S. Amin, R. El Sheikh, F. Zahran and A.A. Gouda, Spectrochimica Acta, 2007, 67, 1088-1093.

20. R. El Sheikh, F. Zahran and A.A. Gouda, Spectrochimica Acta, 2007, 66, 1279-1287.

21. Y.M. Issa, A.F. Shoukry and R.M. El Nashar, J. Pharma. Biomed. Anal., 2001, 3, 379-386.

22. Validation of Analytical Procedure Methodology ICH Harmonized Tripartite Guidelines, 1996, 1.

23. Green J M, A practical guide to analytical method validation, Analytical Chemistry, 1996, 305A.

24. British Pharmacopoeia, HerMajasty’s, Stationary Office, London, 2005.

25. S. Dowdy and S. Weardern," Statistics for Research", N.Y. Wiley, 1983.

26. J.H. Yoe and A.L. Jones, Ind. Eng. Chem. Anal., Ed., 1944, 16, 14.

27. P. Job, Camp. Rend. (Paris), 1925, 180, 928; Ann. Chim. (Paris), 1928, 9, 133; Ann. Chim. (Paris), 1936, 6, 97.

28. W.C. Vosburgh and G.P. Cooper, J. Am. Chem. Soc., 1941, 63, 437.

29. D.M. Schnur, Y.H. Yuh, and D.R. Dalton, J. Org. Chem., 1989, 54, 3779.

30. B.L. Podlogar and D.J. Raber, 1989, 54, 5032.

31. N.L. Allinger, J. Comput. Chem., 1993, 14, 755.

Received on 12.05.2011 Modified on 05.06.2011

Asian J. Research Chem. 4(9): Sept, 2011; Page 1489-1502