New Spectrophotometric Methods for Estimation of Perindopril Erbumine in Bulk and Pharmaceutical Formulations

Three rapid, simple, inexpensive, precise, and accu r te spectrophotometric procedures were developed for the determination of Perindopril erbu mine (PPE) using cobalt-thiocyanate , citric acid and 2,3dichloro-5,6-dicyano benzoquin one in bulk sample and in dosage forms. The procedures are based on the coordination comple x of the drug (electron donor) and the chromogenic reagents used. The colored products are ext acted into non aqueous solvents and measured spectrophotometrically at the optimum λmax for each complex. Regression analysis of Beer-Lambert plots showed good correlat ion in the concentration range of 30-70 μg mL 4-20 μg mL and 20-60 μg mL −1 for cobalt-thiocyanate , citric acid and 2,3dichloro-5,6-dicyano benzoquinone respectively. The apparent molar absorptivity, Sandell’s sensitivity, detection and quantification limits we re calculated. The developed methods were successfully applied for the determination of PPE in bulk and pharmaceutical formulations without any interference from common e xcipients and hence can be used for their routine analysis.


Preparation of reagents
All the chemicals and reagents used were of analytical grade and solutions were prepared using appropriate solvents.

For method M 2
Citric acid-acetic anhydride [CA/Ac 2 O solution (Fluka: 0.2% w/v, 3.26 x 10 -3 M)] [18]: Accurately 12 g of citric acid monohydrate was weighed and transferred to 250 mL beaker.The beaker was kept in ice water bath.To this beaker 5 mL of methanol and 20 mL of acetic anhydride were added.The mixture was stirred with a glass rod to complete the dissolution.The solution was transferred to 100 mL volumetric flask and final volume adjusted to mark with acetic anhydride.2.2.3.For method M 3 2,3-Dichloro-5,6-dicyano benzoquinone (DDQ) (8.8 x 10 -3 M): Prepared by dissolving 200 mg of DDQ in 100 mL of Acetonitrile.

For method M1
About 119.16 mg of PPE (equivalent to 100 mg of free base) was accurately weighed and transferred to 150 mL separating funnel.It was dissolved in 10 mL of water and 10 mL of 0.1 N sodium hydroxide was added to it drop wise to release the free base of the drug.The released free base was extracted 3 times with 20 mL portions of chloroform solvent and volume of the total chloroform extract was brought up to 100 mL with chloroform to get a standard stock solution of 1 mg/mL.

For method M2
The stock solution (1 mg/mL) of PPE was prepared by dissolving 100 mg of the drug in 100 mL water.This stock solution was further diluted to get the working standard solution of concentration 100 µg/mL.

For method M 3
The stock solution (1 mg/mL) of PPE was prepared by dissolving 100 mg of the drug in 100 mL of acetonitrile.This stock solution was used as such for this method.

Procedure for estimation
After systematic and detailed study of the various parameters involved in the optimization of the methods, the following procedures were recommended for the determination of PPE in bulk and pharmaceutical dosage forms.

Method M 1
Into a series of 125 mL separating funnels, PPE standard solution (1000 µg/mL) ranging from 0.3 to 0.7 mL was transferred and the volume in all separating funnels was adjusted to 2.0 mL with water.Then 5.0 mL of buffer (pH 2.0) and 5.0 mL of CTC were added.The total volume in each separating funnel was adjusted to 115.0 mL with distilled water.To each separating funnel 10 mL of nitrobenzene was added and contents were shaken for 5 min.The two phases were allowed to separate and the absorbance of the separated nitrobenzene layer was measured at 620 nm against a reagent blank.The amount of PPE was deduced from its Beer-Lambert's plot.

Method M 2
Aliquots of standard chloroformic PPE solutions containing (0.4 -2.0 mL of 100 µg/mL) were transferred to a series of 25 mL volumetric flasks and gently evaporated on a water bath to remove the chloroform.10 mL of CA/Ac 2 O reagent was added to each volumetric flask.The volumetric flasks were placed in a water bath and heated for 40 min.The contents were cooled to room temperature and the solution in each volumetric flask was made up to the mark with acetic anhydride and set aside for 15 min.The absorbance of colored solutions was measured at 580 nm against a reagent blank.The amount of PPE in sample was calculated from its calibration graph.

Method M 3
To a series of 10 mL volumetric flasks, aliquots of standard PPE solutions (free base) containing 200.0 to 600.0 µg were transferred and 1.0 mL of DDQ was added to each volumetric flask.The final volume was made up to the mark with acetonitrile.The absorbance of the solutions was measured at 404 nm against its reagent blank.The amount of PPE was computed from its Beer-Lambert's plot.

For method M 1
Twenty tablets (Aceon) were taken in a mortar and crushed to a fine powder.From this tablet powder equivalent to 50 mg of PPE was taken and dissolved in 40mL of water.The solution was filtered and diluted to 50 mL with the same solvent.It was further diluted appropriately with the same solvent and amount of PPE was estimated by following the same procedure as described in bulk samples for method M 1 .

For method M 2
Tablet powder equivalent to 50 mg of PPE was accurately weighed and transferred to 150 mL separating funnel.Then it was dissolved in 10 mL of water and 10 mL of 0.1 N sodium hydroxide was added to it drop wise to precipitate the free base of the drug.The released free base was extracted 2 times with 20 mL portions of chloroform solvent and volume of the total chloroform extract was brought up to 50 mL with chloroform to get a standard stock solution of 1 mg/mL This stock solution was used in determination of PPE in formulations by following the procedure just as described in bulk samples for method M 2 .

Method M 3
Powder equivalent 50 mg of PPE was taken and sample was extracted into 50 mL acetonitrile.After performing appropriate dilutions, the amount of PPE in the sample was determined by following the procedure as described in bulk sample for method M 3 .

Method M 1
CTC has been proved to be a valuable chromogenic reagent for the detection and determination of pharmaceutical compounds containing tertiary amino compounds and phenothiazines.The colored species formed is the coordination complex of the drug (electron donor) and the central metal atom of CTC, which is extracted into non-aqueous solvents (usually nitrobenzene) from aqueous solution.The green colored complex formed between the reagent and PPE can be attributed to presence of cyclic tertiary nitrogen.

Method M 2
When basic tertiary amines are heated with a solution of citric acid (or cis-aconitic anhydride) in acetic anhydride, a red-violet colored molecular internal salt is formed.The trans-configuration of aconitic acid initially forms through dehydration besides cis-aconitic anhydride and subsequently yields α; γ-anhydride develops violet color in the presence of tertiary amine which allows the colorimetric determination of class of compounds.
Formation of an internal salt of aconitic anhydride with tertiary nitrogen in PPE is responsible for red-violet color formation.The probable sequence of reactions based on analogy is presented in scheme 1 (PPE).

Method M 3
Compounds with cyclic tertiary nitrogen of aliphatic nature (like PPE) function as electron donor and participates in charge transfer interaction with substituted quinones (DDQ).The colored species formation appears to be due to formation of radical anion.The formation of a radical anion in such molecular interactions has been established by electron spin resonance measurements.Based on analogy, reaction is given in scheme 3.
In order to ascertain the optimum wavelength of maximum absorption (λ max ), the spectra were scanned in the wavelength region of 400-800 nm against a corresponding reagent blank.The reagent blank absorption spectrum of each method was recorded against solvent employed in each method.
Beer's law limits, molar absorptivity, sandell's sensitivity and optimum photometric range for PPE in each method developed with mentioned reagents were calculated.Least square regression analysis was carried out for getting the slope, intercept and correlation coefficient values.These are recorded in Table1.Interference studies were conducted to see the influence of excipients with the proposed methods.The accuracy of the methods was evaluated by estimating the amount of PPE in previously analyzed samples to which known amounts of PPE was spiked.The accuracy of the methods was also confirmed by comparison of the results obtained by proposed and reference methods .The results of accuracy were given in Table 2. Some of the commercially available formulations were procured from the local market and analyzed by the developed methods and the results comply with the labeled claim.

Conclusion
The proposed method is simple, rapid and accurate and can be opted in routine analysis of drug formulation.The accuracy and reproducibility of the method was statistically validated by recovery studies.The proposed method was found to be superior to some of the reported spectrophotometric methods with respect to simplicity, selectivity, linear dynamic range of applicability and cost effectiveness.

Scheme 3 Table 1 .
Optical characteristics, regression data and precision of the proposed methods for PPE.
* Average of six determinations ; ** Average of three determinations.

Table 2 .
[20,21]and recovery of PPE in dosage forms[20,21].Average±standard deviation of six determinations, the t and F-values refer to comparison of the proposed method with reference method.Theoretical values at 95 % confidence limits t = 2.571 and F = 5.05.** Average of five determinations. *