INTRODUCTION:

Perindopril is a non-sulfhydryl angiotensin-converting-enzyme inhibitor with antihypertensive activity. Perindopril upon hydrolysis is converted to its active form perindoprilat. This active metabolite is a potent, competitive inhibitor of ACE, the enzyme responsible for the conversion of angiotensin I to angiotensin II. Consequently, angiotensin II- mediated vasoconstriction and angiotensin II- stimulated aldosterone secretion from the adrenal cortex are inhibited and diuresis and natriuresis ensue.

Amlodipine, a dihydropyrimidine calcium channel blocker acts by inhibiting the movement of calcium ions across the cell membrane into vascular smooth muscles and myocytes. It is also used as an antihypertensive drug. These drugs are combined in a pharmaceutical dosage form for the treatment of mild to moderate hypertension and stable coronary artery disease^1-3.

A thorough review of literature reveals that several studies using analytical procedures such as HPLC, UV and UHPLC have been carried out for the determination of perindopril and amlodipine either alone or in combination^4-19. However no method has been reported for cleaning validation of perindopril and amlodipine in combination. Thus, it was thought worthwhile to develop a cleaning validation HPLC method for the simultaneous determination of perindopril and amlodipine as per ICH guidelines and use the developed method in performing assay of a marketed formulation.

MATERIALS AND METHODS:

Chemicals and Reagents:

Analytically pure amlodipine was obtained as a gift sample from Unichem Laboratories Ltd., Pilerne, Berdez, Goa while perindopril was obtained as a gift sample from Glenmark Pharmaceutical Ltd. Colvale, Bardez- Goa. All chemicals and reagents were of HPLC grade.

Equipment:

The liquid chromatographic procedures were carried out on AGILENT HPLC system equipped with WATERS SHERISORB CN (250mmx4.6mm, 5μm) column. OpenLAB software was used for analysis.

Cleaning Validation Method:

Preparation of standards:

Preparation of Standard stock solution of Amlodipine (100ppm):

About 5mg of amlodipine was weighed accurately and transferred into a 50ml volumetric flask, 30ml of diluent (50% methanol) was added and sonicated for 5 minutes and diluted to mark using diluent and mixed well.

Preparation of Standard stock solution of Perindopril (100ppm):

About 5mg of perindopril was weighed accurately and transferred into a 50ml of volumetric flask, 30ml of diluent was added and sonicated for 5 minutes and diluted up to the mark with diluent and mixed well.

Preparation of Mixed Standard solution (1 ppm):

One ml from each standard drug stock solution was taken and transferred to 100ml volumetric flask and the volume was made up with diluent.

Blank/ Diluent:

The blank/ diluent consisted of 50% methanol.

Chromatographic conditions:

Chromatography was performed on WATERS SHERISORB CN column (250mm×4.6mm, 5µm) with column oven temperature of 40^°C and run time of 7 min. The mobile phase used was phosphate buffer pH 6.5: methanol: acetonitrile (70:20:10, v/v/v) at a flow rate of 1.3ml/min. The injection volume was 10µL, and UV detector wavelength was 210nm.

System Suitability Testing:

The system suitability testing was performed by injecting 6 replicates of the mixed standard drug solution and chromatograms were obtained. The system suitability was established by calculating the theoretical plates, tailing factor, resolution and %RSD for the peak area.

Specificity:

Swabs used to evaluate the specificity for the proposed method were pretreated by transferring swab sticks in a clean test tube containing methanol and sonicated for 5 minutes and then drained. The whole procedure was repeated in water and then back in methanol. Two pretreated swabs were taken and put in two separate test tubes containing 10ml of diluent and sonicated for about 5 minutes with intermittent shaking. The resulting solution was injected in HPLC system as per test method to test swab interference.

Linearity:

To evaluate the linearity of the proposed method, a 10 ppm solution of amlodipine and perindopril was prepared by diluting 5ml of stock solution of each drug to 50ml using diluent. Dilutions over the concentration range 0.5-10μg/ml were prepared and the solutions were injected into the HPLC system. A calibration curve of Peak area v/s Concentration was plotted for both the drugs and the linear regression equations and correlation coefficients (r²) were calculated.

Accuracy:

Accuracy of the method was ascertained by carrying out recovery studies. For recovery at 100% level, 1ml of amlodipine and perindopril 10ppm solution were dispersed over 4’’ x 4’’ stainless steel plates and dried using a hand held dryer. After drying, the plate was swabbed and the swabbed samples were collected in a test tube containing 10ml of diluent, mixed and sonicated for about 5 min with intermittent shaking to result in approximately 1ppm solution. The procedure was performed in triplicate to result in 3 samples which were then injected as per test method.

Precision:

The evaluation of precision was done by injecting six replicates of amlodipine and perindopril mixed drug standard solution at Limit of Quantitation (LOQ) level. The peak areas were recorded and the % relative standard deviation for perindopril and amlodipine were calculated.

Limit of detection and limit of quantitation:

The limit of detection and limit of quantitation of perindopril and amlodipine was determined based on “Signal to Noise ratio” method. A 0.5ppm solution of the two drugs was injected and the concentration which gave a Signal to Noise ratio of about 3 for limit of detection and about 10 for limit of quantitation was derived. The solutions were prepared, injected and response was recorded.

Robustness:

Robustness of the method was demonstrated by bringing about deliberate changes in the buffer pH of mobile phase, flow rate and column temperature and calculating its impact on system suitability parameters.

Assay Method:

Preparation of drug standard stock solutions:

Preparation of stock solution of perindopril (150 ppm):

About 15mg of perindopril was weighed accurately and transferred into a dry 200ml volumetric flask. To this 140ml of diluent was added and sonicated for about 10 min. Volume was made up to mark with diluent.

Preparation of stock solution of amlodipine (250 ppm):

About 25mg of amlodipine was weighed accurately and transferred into a dry 200ml volumetric flask. To this 140ml of diluent was added and sonicated for about 10 min. Volume was made up to mark with diluent.

Preparation of mixed standard solution of perindopril and amlodipine:

A mixed standard solution of the drugs was prepared by taking 5ml of respective stock solutions into a 100ml volumetric flask. Volume was made up to mark with diluent and mixed thoroughly. This resulted in a concentration of 6ppm and 10ppm of perindopril and amlodipine respectively.

Preparation of test sample:

Five tablets, containing 20mg of perindopril and 25mg of amlodipine were weighed accurately and average weight of the tablets was recorded. The tablets were dropped into 200ml volumetric flask employing the tablet drop technique for assay. To this 140ml diluent was added and sonicated for about 10 min. Volume was made up to mark with diluent. The solution was centrifuged for 10 min at 5000rpm. A volume of 5ml of supernatant was transferred to a 100ml volumetric flask and diluted to mark using diluent and mixed thoroughly to get final concentration of 5ppm perindopril and 6.25 ppm amlodipine. The solution was injected and chromatogram recorded. The above procedure was performed in triplicate and the % assay of the formulation was calculated.

Blank:

Diluent was used as blank.

System Suitability Testing:

The mixed standard solution was injected five times and peak areas recorded. The System Suitability was established by calculating the resolution, % RSD, tailing factor and the theoretical plates.

RESULTS AND DISCUSSION:

Preliminary experiments were carried out to achieve optimized chromatographic conditions for simultaneous determination of amlodipine besylate and perindopril erbumine. Mobile phase consisting of phosphate buffer (pH 6.5): methanol: acetonitrile (70:20:10, v/v/v) at a flow rate of 1.3ml/min was optimised for the estimation of drugs at detector wavelength of 210nm. The drugs gave sharp peaks with WATERS SHERISORB CN (250mm×4.6mm, 5µm) column at column oven temperature of 40°C. An injection volume of 10µl was used and runtime was set at 7 min.

The results of system suitability experiments are depicted in table 1. A representative chromatogram (figure 1) shows the retention time of perindopril and amlodipine as 3.260 and 4.153 min respectively. The results for system suitability parameters indicate that the values were within the acceptable criteria and hence the system was suitable for the intended analysis.

Figure 1: Representative chromatogram for mixed standard of perindopril and amlodipine

Table 1: Summary of System Suitability Parameters

System Suitability parameters	Observed values		Acceptance criteria
System Suitability parameters	Perindopril	Amlodipine	Acceptance criteria
% RSD (n=6)	4.1	1.14	NMT 10.0%
Average Theoretical plates	6903	8089	NLT 2000
Average Tailing factor	1.085	1.165	NMT 2.0
Resolution	5.20		NLT 1.5

To check specificity (swab interference), prepared swab solutions were injected into the HPLC system. A representative chromatogram for specificity (swab interference) is shown in figure 2. As seen, the chromatogram showed no interference at the retention time of the drugs. Hence, there was no swab interference, the method was specific and the swabs were suitable for use.

Figure 2: A representative chromatogram of swab interference

Linearity was determined at five levels over the concentration range of 0.5μg/ml to 10μg/ml. The solutions for linearity study were injected into the HPLC system and the results of linearity study are shown in table 2. The calibration curves for both the drugs are shown in figure 3 and figure 4. The correlation coefficient (r²) values were > 0.999 for both the drugs. The linearity range was thus established as 0.5-10µg/ml for both the drugs.

Figure 3: Calibration curve for perindopril

Figure 4: Calibration curve for amlodipine

Table 2: Summary of linearity data for perindopril and amlodipine

Parameters	Perindopril	Amlodipine
Linearity range	0.5 µg/ml-10 µg/ml	0.5 µg/ml-10 µg/ml
Correlation Coefficient (r²)	0.9994	0.9996
Linear Regression Equation	85866x - 4988.8	215877x – 3030.4

Accuracy studies were carried out by performing % recovery at 100% concentration. The mean % recovery at 100% as shown in table 3 was found to be within the acceptable range. Hence the method was found to be accurate.

Precision studies were carried out at LOQ level. The mixed standard solution was injected 6 times. The results as depicted in table 3 gave % RSD values less than 10.0%. Hence the method was found to be precise at the LOQ level.

Table 3: Results for accuracy and precision study for perindopril and amlodipine

API	Mean % recovery at 100% (n=3)	% RSD at LOQ (n=6)
Perindopril	102.20	9.00
Amlodipine	84.20	2.94
Acceptance criteria	70.0%-110.0%	NMT 10.0%

LOD value was found to be 0.2µg/ml for perindopril and amlodipine and the LOQ value was found to be 0.5 µg/ml for perindopril and amlodipine. Hence the method was found to be sensitive.

For robustness studies the mixed drug standard solution was injected in six replicates by employing change in pH, column oven temperature and flow rate. It was observed that there were no significant changes in system suitability parameters with any of the above tested method parameters, which demonstrated that the method was robust.

Assay Method:

System Suitability Testing:

The system suitability for assay was established by calculating % RSD, theoretical plates, tailing factor and resolution of five replicates of mixed standard solution. The results are depicted in table 4. As seen in table 4 the system suitability parameters complied with the acceptance criteria. Hence the system was suitable for the analysis of perindopril and amlodipine drug combination.

Table 4: Summary of system suitability parameters

System Suitability Parameters	Observed values		Acceptance criteria
System Suitability Parameters	Perindopril	Amlodipine	Acceptance criteria
% RSD (n=5)	1.5	1.7	NMT 2.0%
Average Theoretical plates	3700	3721	NLT 2000
Average Tailing factor	1.15	1.48	NMT 2.0
Resolution	2.36		NLT 1.5

Assay of formulation:

The method developed for cleaning validation was applied for analysing marketed tablet formulation. The results of % assay in triplicate, as seen in table 5 were within the acceptable limits of 90% to 110% for both perindopril and amlodipine.

Table 5: Assay results of perindopril and amlodipine

Sample	Retention time (min)	Mean % assay	% RSD n=3
Perindopril	3.06	104.10	0.50
Amlodipine	3.58	100.10	1
Acceptable limits		90% to 110%	NMT 2%

CONCLUSION:

A simple, accurate, precise and economical RP-HPLC method for cleaning validation and assay has been developed for the estimation of perindopril and amlodipine in combined tablet dosage form. The method was validated as per ICH guidelines for specificity, linearity, LOD, LOQ, accuracy, precision and robustness. The method was applied to the analysis of marketed combination. The results obtained lead to the conclusion that the target of developing a common chromatographic method for both cleaning validation and assay has been achieved. This efficient method can be used to estimate perindopril and amlodipine as independent components as well as in combination.

ACKNOWLEDGEMENT:

The authors are thankful to Unichem Laboratories Ltd., Pilerne, Berdez, Goa and Glenmark Pharmaceutical Ltd Colvale, Bardez-Goa for gift samples of amlodipine and perindopril respectively.

CERTIFICATE OF CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 24.11.2019 Modified on 07.02.2020

Research J. Pharm. and Tech. 2020; 13(12):5919-5923.

DOI: 10.5958/0974-360X.2020.01033.1