Determination of perindopril erbumine by oxidative coupling reaction using 2, 6-dichloroquinone-4-chlorimide

Perindopril erbumine (Perindopril tert-butylamine salt) is a potent angiotensin-converting enzyme (ACE) inhibitor. It is used to treat the patients with hypertension and heart failure problems. A sensitive, inexpensive and precise analytical technique has been developed for the estimation of perindopril in bulk and formulations. The procedure involves the development of colour by forming an oxidative coupling reaction between drug (PPE) and reagent such as 2, 6-dichloroquinone-4-chlorimide (DCQC). The formed colored species were measured at lmax=520 nm. The developed method showed linearity within the concentration limits of 25-75 μg mL -1 . The linear correlation coefficient (r) and molar absorptivity were found to be 0.9999 and 3.285 x 10 3 mol -1 cm -1 . % Recovery ± SD values were in the range of 99.69 - 100.51 (+ 0.42 - ± 0.41) (n=3) which indicates the accuracy of the developed method. The interference of other excipients that are commonly present in formulations is found to be negligible. Precision and accuracy of the proposed method were confirmed by student t-test and F-tests at 95% confidence limits with (n-1) degrees of freedom. The validity parameters of proposed method were calculated by ICH guidelines.


Preparation of bulk drug and Gibb's reagent (DCQC) solution
Dissolved 100 mg of bulk drug perindopril erbumine in a minimum quantity of sodium hydroxide (0.1 molL -1 ) solution followed by dilution to 100 ml with distilled water to prepare the standard stock solution (mg/ml).
The released free erbumine was extracted with 10.0 ml chloroform. The aqueous solution free from erbumine was used as a stock solution. It is further diluted stepwise with distilled water to obtain working standard solutions of concentration (250 µg mL -1 ) 2, 6-dichloroquinone-4-chlorimide (DCQC) solution (Loba; 0.2%, 9.52 x 10 -3 mol L -1 ) was prepared by dissolving 20 mg of Gibb's reagent (DCQC) in 100 ml of isopropyl alcohol (IPA). AR grade of isopropyl alcohol ((IPA, Qualigens) solvent was used for the preparation of reagent and dilution of the test solution.

Instrumentation
Precise and accurate wavelength measurements were made using UV wavelength scanning double beam spectrophotometer UNICAM UV-500 (Thermo Electron Corporation, UK) and visible scanning spectrophotometer (SL-177, Elico India). Digital pH meter (Elico LI 120, India) was used for measuring the pH of the samples. All materials were weighed using Dhona 200D analytical balance (J S Enterprises, India) with an accuracy of ± 0.1 mg.

Procedure for formulations
Coversyl (Serdia Pharmaceuticals Pvt Ltd., India) Coversyl plus (Serdia Pharmaceutical Ind. Ltd., India), Perigard-DF (Glenmark Pharmaceuticals Ltd., India) and Aceon (Solvay Pharmaceuticals Inc.) containing perindopril erbumine were procured from the local market. Tablets equivalent to 2 mg, 4 mg and 8 mg per tablet respectively were selected for this study. Tablet powder equivalent to 100 mg was taken for extraction with chloroform (4 x 25.0 mL portions) and filtered. The filtrate was taken and extracted three times with sodium hydroxide (0.1 molL -1 ) using separating funnel. Stock solution (mg/mL) was prepared to dilute the aqueous alkali extract to 100 mL with deionized water. Working standard solution (250 µg mL -1 ) was made by diluting a portion of the above stock solution and analyzed as per the developed analytical method.

Calibration curve of perindopril erbumine by UV method
100 mg of bulk drug sample was dissolved in distilled water (100 mL) to prepare a stock solution (mg/mL). Working standard solution concentration (100 μg mL -1 ) was prepared from an aliquot portion (10.0 mL) of the above stock solution. The absorption spectrum was recorded on a spectrophotometer within the UV region against a reagent blank ( Figure.2). A portion of the working standard drug solution (1.0 -3.0 mL, conc.100 μg mL -1 ) was taken in a series of 10.0 mL calibrated tubes and diluted to 10.0 mL with double distilled water. The absorbance was measured at 204 nm against deionized water as blank. The concentration of the drug sample was calculated using its calibration curve ( Figure.3). The UV absorption method was chosen as a reference method.

Absorption spectrum of perindopril-DCQC system:
For the selection of analytical wavelength, the sample solution containing a fixed quantity of drug, DCQC solution and other furnished variables as outlined in the analytical procedure was scanned in the visible wavelength region 350 -800 nm against the reagent blank. The spectrum of the oxidative coupling product observed to have a maximum wavelength at 520 nm which was selected for the analysis. The spectrum of reagent blank against isopropanol solvent was also measured ( Figure 5).

Protocol of the proposed method
Into a series of separating funnels (100.0 mL), a portion of working standard bulk drug solution concentration 250 μg mL -1 (10-5.0 mL), 1.0 mL of 9.52 x 10 -3 molL -1 DCQC was added and volume made up to 10.0 mL with isopropanol and kept it on a hot water bath for 20 min, then cooled to room temperature and the volume again made up to 10.0 mL with isopropanol. The absorbances of the colored species were measured at 520 nm against a reagent blank prepared simultaneously. The constancy of color species was found to be stable for 30 min. A calibration curve was drawn to calculate the amount of drug present (Figure 4). correlation coefficient, LOD, LOQ sensitivity, RSD, and percentage of error.

Linear relationship:
The developed analytical procedure showed the linear relationship within the beer's law range ((25 -75 µg mL -1 ). Beer's law plot (n = 6) was measured under optimum conditions and found consisting of linearity with a high correlation coefficient (r) value 0.9999. The standard calibration curve drawn at five concentration levels. Results are given in Table I

Limits of LOD and LOQ
Limit of detection (LOD) and Limit of quantification (LOQ) were calculated using the below-given expressions.
(LOD) = 3.3×S a / b (1) (LOQ) = 10×S a / b Where b is the slope of the calibrated curve and Sa is the standard deviation of the intercept.

Optimum conditions
The responses of several factors like the concentration of the DCQC, stability period of the complex formed, intensity of the colored species formed, were studied (Massart et al., 1988). The optimum conditions identified for the proposed method are 0.75-1.5 mL (0.0.71 -1.43×10 -3 molL -1 ) of the DCQC solution, laboratory temperature of (70 -80 0 C) and time required for colour development 15-30 minutes. In this method, 1.0 mL of (9.52 × 10 -3 mol L -1 ) DCQC, and 20 minutes essential for the highest color growth were found to be optimum conditions. The stability period of the oxidative coupling product was found as 30 min, afterward the absorbance was found to decrease which may be due to the decomposition of the oxidative coupling product.

Validations of analytical data
Following (ICH) guidelines (Tripartite, 2005) the developed method was validated for various optical and regression characteristics such as slope, intercept,

Sensitivity
The sensitivity of the developed method was measured in terms of molar absorptivity (εmax), limit of detection and the limit of quantification. Results of Molar absorptivity, LOD and LOQ are 3.285 x 10 3 L mol -1 cm -1 , 1.866 x 10 -1 μg mL -1 and 5.653 x 10 -1 μg mL -1 (Table I) Sandell's sensitivity Sandell's sensitivity (µg/cm 2 /0.001 Abs unit) is measured as the "smallest weight of a substance that can be detected in a column of the unit cross-section". Sandell's sensitivity is the concentration of the analyte (in µg mL -1 ) which gives an absorbance of 0.001 in a cell of path length 1 cm. Sandell's sensitivity value is found to be 1.344 x 10 -1 µg/cm 2

The selectivity of method:
Selectivity for the assay procedure was calculated by analyzing the standard drug sample solution in the presence of excipients (microcrystalline cellulose, magnesium stearate, lactose and titanium dioxide) that are commonly present in formulations. The results of the developed method indicated that no interference from the excipients present in formulations.

Precision
The precision of the analytical procedure expresses "the closeness of agreement between a series of measurements obtained from six determinations of sample solution under prescribed conditions". The intraday precision was calculated by measuring "absorbance of sample solution of particular concentration within the linearity range at regular intervals on the same day". The inter-day precision was calculated by measuring "absorbance of sample solution of same concentration at a fixed time in three consecutive days". The precision of the developed method expressed in terms of relative standard deviation (RSD) for the smallest concentration indicating good precision. Results are presented in Table I.

Accuracy
Accuracy of analytical procedure was calculated as "percentage of error between the measured mean concentrations and taken concentrations". The accuracy and precision were checked by comparing the result of the developed and UV reference method statistically through student t-and F-tests at theoretical values of 95% confidence limits with (n-1) degrees of freedom. It was observed that the values obtained for t-and F-tests for the proposed method are found to be lower than the tabulated values (Massart et al., 1988) of 2.57 and 5.05 respectively. % Recovery ± SD values were in the range of 99.69 -100.51 (+ 0.42 ± 0.41) (n=3) which indicates the accuracy of the developed method. Results of accuracy are given in Table II. Percentage of error (%) Confidence limit at 0.05 level 0.53 Confidence limit at 0.01 level 0.83 SCHEME 1 -Formation of oxidative coupling product between drug (PPE) and DCQC

Proposed scheme for the formation of coloured species
Earlier workers estimated the phenols or amines using 2, 6-dichloroquinone-4-chlorimide (DCQC) reagent (Rao et al., 1985). In the present investigation, the chemistry of color species is studied and found that the iminio group in perindopril couples directly with N-Cl in DCQC to get the colored product. The probable scheme for the formation of oxidative coupling product between drug and DCQC is presented in scheme 1.