A Novel Use of 3-Methyl-2-Benzothiazolinone Hydrazone Hydrochloride Monohydrate for Kinetic Spectrophotometric Determination of Captopril in Pharmaceutical Formulations

. A new, simple and sensitive kinetic spectrophotometric method has been proposed for the determination of captopril (CPT) in pharmaceutical formulations. The method is based on oxidation of 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH) by ferric chloride followed by its coupling with the drug to form green- yellow coloured product with absorbance maximum at 395nm. The concentration of CPT was calculated using the calibration equation for the rate data and fixed time methods. The linearity range was found to be 0.5– 22.5 µg mL -1 for each method. The correlation coefficients were 0.9994 and 0.9971 for rate data and fixed time methods respectively. The proposed methods were applied successfully for the determination of CPT in pharmaceutical formulations. Statistical comparison of the results shows that there is no significant difference between the proposed and official methods.

Kinetic spectrophotometric methods are becoming of great interest in pharmaceutical analysis [27]. Some specific advantages in the application of kinetic methods can be expected such as: selectivity due to the measurement of the evolution of the absorbance with the time of the reaction instead of the measure of a concrete absorbance value, sufficient accuracy and requires relatively simple and inexpensive instrumentation [28].
The present work describes the development of a simple and sensitive kinetic spectrophotometric method for the determination of CPT in pharmaceutical formulations. The method is based on the oxidative coupling reaction of 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH) with CPT in the presence of ferric chloride to form greenyellow coloured chromogen with absorption maximum at 395nm. Optimum conditions were established and the method was validated for linearity, sensitivity, accuracy and precision.

Apparatus
An Optizen UV2120 UV-VIS spectrophotometer with 1 cm quartz cells was used for all absorbance measurements under the following operating conditions: scan speed medium (400 nm/min), scan range 200 -1100 nm and slit width 1 nm. Spectra were automatically obtained by Optizen system software. pH measurements were made with ORION 250A (USA) with combined glass pH electrode.

Reagent and Chemicals
All chemicals were of analytical grade. Double distilled water was used throughout all experiments. Captopril (CPT), (C 9 H 15 NO 3 S), 217.28g.mol -1 was obtained from Kunshan chemical and pharmaceutical Co., Ltd, Its purity was found to be 99.5% according to the compendial method. 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH) was obtained from Fluka and ferric chloride was from Merck.

Formulations
Captomed tablets supplied by Medico Company (Homs, Syria), each tablet was labeled to contain 25 or 50 mg Captopril and Capoten tablets supplied by Unipharma (Damascus, Syria), each tablet was labeled to contain 25 or 50 mg Captopril.

Solutions
Standard stock solution of CPT (100 µg mL -1 ) was prepared by dissolving 10.05mg of CPT in 100mL of double distilled water. 1.0× 10 -2 M MBTH solution was prepared with double distilled water and 0.5% FeCl 3 solution was prepared with hydrochloric acid (0.1M) medium. Freshly prepared solutions were always used.

Rate data method
Aliquots 0.5-22.5 µg mL -1 of CPT stock solution (0.05-2.25 mL, 100 µg mL -1 ) were pipette into a series of 10 mL volumetric flasks. To each flask, 1.0 mL of 0.5% FeCl 3 solution was added, followed by 1.0 mL of 1×10 -2 M MBTH and then diluted to the volume with double distilled water at 25 º C. Flask contents were shaken and immediately transferred to the spectrophotometric cell and the increase in absorbance at 395 nm of each flask was recorded as a function of time over 4-16 min against reagent blank treated similarly. The rate data of the reaction (v) at different concentrations was obtained from the slope of the absorbance time curves. The calibration graphs were constructed by plotting the logarithm of the rate data of the reaction (log v) versus logarithm of molar concentration of CPT (log C).

Fixed time method
Aliquots 0.5-22.5 µg mL -1 of CPT stock solution (0.05-2.25 mL, 100 µg mL -1 ) were pipette into a series of 10 mL volumetric flasks. To each flask, 1.0 mL of 0.5% FeCl 3 solution was added, followed by 1.0 mL of 1×10 -2 M MBTH and then diluted to the volume with double distilled water at 25 º C. Flask contents were shaken and immediately transferred to the spectrophotometric cell. Absorbance of each sample solution was recorded at 395 nm against reagent blank treated similarly. The calibration curves were constructed by plotting the absorbance measured after a fixed time of (8 min) against the final concentration of the CPT.

Procedure for formulations
Ten tablets of each formulation were weighed and finely powdered. An accurately weighed amount of the powder equivalent to 10mg of CPT was dissolved in 20 mL methanol, mixed well for 10 min, and filtered through Whatman filter paper (No. 1). The methanol was evaporated to dryness. The remaining portion of solution was dissolved in a 100mL volumetric flask to the volume with double distilled water to achieve a concentration of 100 µg mL -1 approximately. The general procedures were then followed in the concentration ranges mentioned above.

Absorption spectra
The method involves the reaction of CPT with MBTH in the presence of an oxidant iron(III) chloride to give green-yellow colored products with maximum absorption at 395 nm (Fig. 2). Under reaction conditions, MBTH (I), on oxidation with Fe 3+ ions, loses two electrons and one proton forming an electrophilic intermediate (II), which is the active coupling species [29]. The electron density in these drugs studied is highest at the two and eight positions, which permits electrophilic substitution by reagents [30] (Scheme 1).

Optimization of reaction conditions
The optimum conditions for the development of methods were established by varying the parameters one at a time and keeping the others fixed and observing the effect produced on the absorbance of the colored oxidative coupling products.
The effect of various parameters such as volume of oxidant, volume of MBTH reagent, addition of buffer solutions and order of addition of reagents on color development and solvent for final dilution on the intensity were studied and the optimum conditions were established. A volume of 1.0 mL of 1×10 -2 M MBTH and 1.0 mL of 0.5% FeCl 3 were found to be optimal for maximum color development, since the absorbance was found to be maxima at the mentioned volumes (Fig. 3). Among the various oxidants NaIO 4 , K 2 Cr 2 O 7 , Ce(SO 4 ) 2 , FeCl 3 and KIO 4 were examined in combination with MBTH for oxidative coupling reaction. Ce (IV) and Fe (III) were responded for color development with MBTH. But MBTH-Fe (III) was found to be the best by virtue of absorbance values and stability considerations. Other water miscible solvents like methanol, ethanol and acetonitrile were found to provide no additional advantage, so distilled water is selected as a solvent for final dilution of the colored species. Addition of KCl-HCl, britton and acetate buffer solutions affected negativity on the formation of colored oxidative coupling product. Addition of drug, FeCl 3 and MBTH in that order gave maximum absorbance.

Quantitation methods
Because the intensity of the color increased at 395 nm with time (Fig. 4), this was used as the basis for a useful kinetic method for the determination of CPT. The rate data, rate constant, fixed absorbance and fixed time methods [31,32] were carried out and the most suitable analytical methods were chosen regarding the applicability, sensitivity, the values of the intercept and correlation coefficient (r).

Rate data method
The rate data of reaction would follow a pseudo order rate constant and obeyed the following rate equation: Where ν is the reaction rate, A is the absorbance, ∆A=A A , t is the measuring time, ∆t=t 2 -t 1 , k' is the pseudo order rate constant, C is the concentration of the CPT mol L -1 and n is the order of the reaction. A calibration curve was constructed by plotting the logarithm of the rate of reaction (log ν) versus logarithm of vitamin concentration (log C), which showed a linear relationship over the concentration range of 0.5-22.5 µg mL -1 (Fig. 5).

Rate constant method
The logarithm of the absorbance of reaction versus time for each concentration of CPT which studied over the concentration range of 10.0-22.5 µg mL -1 was calculated. Graphs of log absorbance versus time for CPT concentration in the range of 10.0-22.5 µg mL -1 (4.60×10 -5 -1.04×10 -4 M) were plotted and all appeared to be rectilinear. Pseudo order rate constant (k') corresponding to different CPT concentrations were calculated from the slopes multiplied by -2.303. Regression of C versus k' gave the following equation: k' = -0.6619C -0.0004156 (r=0.8566)

Fixed absorbance method
Reaction rate data were recorded for different CPT concentrations in the range 1-15 µg mL -1 (4.6×10 -6 -6.9×10 -5 M). A preselected value of the absorbance 0.2 was fixed and the time was measured in the seconds. The reciprocal of time (1/t) versus the initial concentration of CPT was plotted (Fig. 6) and the following equation of calibration graph was obtained.

Fixed time method
At preselected fixed time, the absorbance of green-yellow colored solution containing varying amounts of CPT was measured at 25 ºC and 395 nm. Calibration graphs were constructed by plotting the absorbance against the initial concentration of CPT at fixed time 4, 8, 12 and 16 min. The regression equations, correlation coefficients and linear ranges are given in Table 1. It is clear that, the slope increases with time and the most acceptable value of r was obtained for a fixed time of 8 min. Therefore, the fixed time of 8 min was utilized for the assay of CPT concentration.
As a result, the most acceptable values of the correlation coefficient and linear range were obtained for the rate data (t 1 =4; t 2 =8 min) and fixed time (8min) methods. Thus, they were used for the determination of CPT in pharmaceutical formulations.

Calibration graph
After optimizing the reaction conditions, the linearity range was found to be 0.5-22.5 µg mL -1 for each method. Analysis of the data gave the following regression equations shown in Table 2. Regression analysis indicates linear relationships with negligible intercepts. For fixed time (8min) method Sandell's sensitivity and molar absorptivity were calculated. Values of some analytical characteristics for the rate data (t 1 =4; t 2 =8 min), and fixed time (8 min) methods are shown in Table 2. Table 2. Analytical characteristics of the rate data and fixed time methods.

Parameters
Rate data (t 1 =4; t 2 =8 min) Comparison among the previous kinetic spectrophotometric methods for the determination of captopril are shown in Table 3. As it is seen in this table, some of the reported procedures are more sensitive but have drawbacks such as having a narrow linear range [11][12][13], involving heating [11], and some methods are not simple and required expensive or sophisticated instruments [10].

Stoichiometric relationship
The composition of colored oxidative coupling product was determined by Job's method of continuous variation and mole-ratio method [33], for fixed time (8 min) method. It is apparent from the data that a molar ratio of 1:1 and 1:2 CPT to MBTH (Fig. 7and 8 ).

Accuracy and precision
The accuracy and precision of the proposed methods were carried out by five determinations at three different concentrations. Percentage relative standard deviation (RSD %) as precision and percentage recovery as accuracy of the suggested methods were calculated and showed in Table 4. The values of relative standard deviations for different concentrations of CPT determined from the calibration curves. These results of accuracy and precision show that the proposed methods have good repeatability and reproducibility.
The proposed methods were found to be selective for the estimation of CPT in the presence of various tablet excipients. For this purpose, a powder blend using typical tablet excipients was prepared along with the drug and then analyzed. The recoveries were not affected by the excipients and the excipients blend did not show any absorption in the range of analysis.

Application to the pharmaceutical dosage forms
The rate data and fixed time methods of the proposed kinetic spectrophotometric method for determining CPT have been performed on commercial pharmaceutical tablets. The concentration of CPT was computed from its corresponding regression equations. The results of the proposed methods (rate data or fixed time) were statistically compared with those of the reported method [34], in respect to the accuracy and precision. The mean recovery values of the claimed amounts were between 99.39 and 101.64% ( Table 5).
The results of t-and F-tests indicated no significant differences between both the proposed and the reported methods at 95% confidence level. This indicated similar precision and accuracy in the analysis of CPT in tablets. F=3.61 a Five independent analyses. At 95% confidence level t-value is 2.776 and F-value is 6.26.

Conclusion
The developed kinetic spectrophotometric methods for the determination of CPT are simple, accurate and precise and hence can be used for the routine analysis of CPT in pharmaceutical formulations. The proposed methods are sensitive enough to enable determination of lower amounts of the drug, and compared with the previously reported methods in terms of accuracy and precision, which suggested non interference of formulations excipients in the estimation. These advantages encourage the application of the proposed methods in routine analysis of CPT in quality control laboratories as alternatives for the existing methods.