1. drug profile - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/32325/19/19_chapter...

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1. DRUG PROFILE

1.1 Perindopril

Perindopril [Figure 13.1], is an ACE inhibitor used in treatment of hypertension &

heart failure. It is converted into active metabolite Perindoprilat in the body [1]. It blocks

conversion of AT I to AT II & also inhibit degradation bradykinin which is a potent

vasodialator, thus decreases peripheral resistance, lowers B.P & produces mild natriuresis

[2]. Reduces B.P without increasing heartrate. Mostly used in patients having diabetes,

naphropathy,CHF left ventricular hypertrophy & post-myocardial infraction [3].

Figure 13.1: Molecular structure of Perindopril

Molecular formula : C19H32N2O5

Molecular weight : 368.46

Chemical name : (2S,3aS,7aS)-1-[(2S)-2-{[(2S)-1-ethoxy-1-oxopentan-yl]amino}

propanoyl]-octahydro-1H-indole-2-carboxylic acid.

Solubility : Perindopril is freely soluble in water and alcohol, sparingly

soluble in dichloromethane.

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1.1. Amlodipine

Amlodipine [Figure 13.2], is a dihydropyridine calcium channel blocker used in

management of hypertension & angina pectoris [1]. It has greater selectivity for vascular

smooth muscle [2]. It has longer t½. It has lesser 1st pass metabolism; hence

bioavailability is consistent [3-4].

Figure 13.2: Molecular structure of Amlodipine

Molecular formula : C20H25CLN2O5

Molecular weight : 408.88

Chemical name : Methyl ethyl 2-(2-aminoethoxymethyl)-4-(2-

chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-

dicarboxylate.

Solubility : Amlodipine is freely soluble in methyl alcohol,

sparingly soluble in dehydrated alcohol

Table 13.1: List of brand names of combined formulations of Perindopril and

Amlodipine [5-6]

S. No. Brand names Formulation Available strength

Address of manufacturer

1 COVERSYL-AM Tablet Perindopril Erbumine 8 mg

Amlodipine Besylate 4 mg

Serdia

Pharmaceuticals

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2. LITERATURE SURVEY

Several analytical methods have been reported for the determination of

Perindopril in pure drug, pharmaceutical dosage forms and in biological samples using

spectrophotometry [7], HPLC [8-9], HPTLC [10], LC-MS [11] and Gas Chromatography

[12] and also for the determination of Amlodipine in pure drug, pharmaceutical dosage

forms and in biological samples using spectrophotometry [13], HPLC [14-18], HPTLC

[19] and LC-MS [21-25]. Very few analytical methods using HPLC [26] and HPTLC

[27] have been reported for the simultaneous determination of Perindopril and

Amlodipine in combined dosage forms.

Sharma et al [7] developed a sensitive, selective and well validated

spectrophotometric method based on the formation of extractable colored complex of

drug with colouring agent Indigo Carmine. Two simple, precise and economical UV

methods have been developed for the estimation of Perindopril erbumine in bulk and

pharmaceutical formulations. Perindopril erbumine has the Zero order spectroscopic

method at 387.2 nm (Method A), Method B applied was Area under Curve (AUC) for

analysis of Perindopril erbumine in the wavelength range of 200-400 nm. A wavelength

maximum was found to be 387.2 nm. The detection and quantitation limits determined

were 0.13 and 0.27 mg/mL respectively. The result of analysis have been validated

statistically and also by recovery studies. From the percentage recovery and specificity

studies it was concluded that there was no interference of common additives during the

estimation. This proves the suitability of this method for the routine quality control

analysis of the Perindopril erbumine in formulation.

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Prameela Rani et al [8] developed a new high performance liquid

chromatographic (HPLC) method and validated for the determination of Perindopril

erbumine (PE) in pharmaceutical formulations. Optimum separation was achieved in 10

min using C18 column (250 mm × 4.6 mm, i.d., particle size 5 mm), and elution was

accomplished using a mobile phase (1mL/min). Detection was carried out using a UV

detector set at 215 nm. A linear relationship between mean peak area and concentration

of PE was observed in the range 4-20 µg/mL, with a detection limit of 2 µg /mL and a

quantization limit of 7.0 µg/mL. Intra-day and Inter-day precision, and accuracy of the

methods have been established according to the current ICH guidelines. The developed

method was successfully applied to the determination of PE in pharmaceutical

formulations. The results were statistically compared with those of the reference method

(UV method) by applying Student’s t-test and F-test. Accuracy, evaluated by means of

the recovery method, was in the range 99.00 - 100.5%, with precision (RSD) 0.865%. No

interference was observed from the coformulated substances. The proposed method was

successfully employed for the determination of PE in various pharmaceutical

preparations.

Mohit et al [10] developed a new sensitive, simple, rapid and precise High

Performance Thin Layer Chromatographic (HPTLC) method for simultaneous

determination of both the drugs in Pharmaceutical dosage form. The method was based

on the separation of two drugs on plates precoated with silica gel 60F. The mobile phase

used was dichloromethane: methanol: glacial acetic acid in the ratio of 9.5:0.5:0.1 v/v/v.

Both the drugs showed considerable absorbance at 215 nm. Linearity was obtained in the

concentration range of 1-5 µg/band and 100-500 ng/band for Perindopril and Indapamide

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respectively. The method has been successfully applied to tablets and was validated

according to ICH Harmonized Tripartite guidelines.

Jignesh et al [26] developed an analytical method for quantitative determination

of Perindopril erbumine and Amlodipine besylate in a tablet formulation.

Chromatographic separation of the two drugs was achieved on an Eclipse XDB C-8 (150

mm x 4.6 mm), 5µm. The mobile phase constituted of buffer: acetonitrile (65:35 v/v) and

pH adjusted to 2.6 with dilute orthophosphoric acid was delivered at the flow rate 1.0

mL/min. Detection was performed at 210 nm. Separation was completed within 8 min.

Calibration curves were linear with correlation coefficient between 0.99 to 1.0 over a

concentration range of 8 to 60 mg/mL of Perindopril Erbumine and 10 to 75 mg/mL of

Amlodipine Besylate. The relative standard deviation (R.S.D) was found <2.0%.

Barole et al [27] developed a simple high-performance thin-layer

chromatographic (HPTLC) method for separation and quantitative analysis of

Amlodipine besylate and Perindopril erbumine in bulk and in pharmaceutical

formulations has been established and validated. After dissolving in methanol, sample

and standard solutions were applied to prewashed silica gel plates and developed with

dichloromethane: methanol: glacial acetic acid: (8.5:1.5:0.1ml v/v/v) as mobile phase.

Zones were scanned densitometrically at 238 nm. The Rf values for Amlodipine besylate

and Perindopril erbumine were 0.20±0.02 and 0.89±0.02 respectively. Calibration plots

were linear in the ranges 500–2500 ng/band for Amlodipine besylate and 6000–18000

ng/band for Perindopril erbumine; the correlation coefficients (r2) were 0.995 and 0.995

respectively. LOD and LOQ for Amlodipine besylate were 10.31 and 32.10 ng/band. For

Perindopril erbumine they were 179 and 544 ng/band, respectively. The suitability of this

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method for quantitative determination of these compounds was proved by validation in

accordance with the requirements of pharmaceutical regulatory standards. The method

was used for routine analysis of these drugs in bulk and in a formulation.

3. EXPERIMENTAL 3.1. Instrumentation

The author had attempted to develop and validate a liquid chromatographic

method for determination of Perindopril and Amlodipine using an isocratic Waters HPLC

system on a Xterra C18 column (100 mm x 4.6 mm, 5 µm). The instrument is equipped

with a 2695 binary pump with inbuilt degasser, 2487 Dual absorbance detector and

Rheodyne injector with 20 µL sample loop. A 20 µL Hamilton syringe was used for

injecting the samples. Data was analysed using Waters Empower 2 software. A double-

beam Elico SL-159 UV-Visible spectrophotometer was used for spectral studies.

Degassing of the mobile phase was done by using an ultrasonic bath sonicator. A

Shimadzu balance was used for weighing the materials.

3.2. Chemicals and Solvents

The reference samples of Perindopril (API) and Amlodipine (API) were obtained

from Sun Pharmaceutical Industries Ltd., Baroda, India. The branded formulation

(tablets) (COVERSYL-AM tablets containing 8 mg of Perindopril and 4 mg of

Amlodipine) were procured from the local market. HPLC grade acetonitrile and

analytical grade potassium dihydrogen phosphate was obtained from Qualigens Fine

Chemicals Ltd, Mumbai, India. Hydrochloric acid, sodium hydroxide, hydrogen peroxide

and orthophosphoric acid of analytical grade were obtained from Merck Chemicals Ltd,

Mumbai, India. Milli-Q water was used throughout the experiment dispensed through

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0.22 µ filter of the Milli-Q water purification system from Millipore, Merck KGaA,

Darmstadt, Germany.

3.3. The Phosphate buffer solution

Weigh about 7.0 grams of potassium dihydrogen phosphate and transfer to 1000

mL standard flask, add 400 mL of Milli-Q water mix and dilute to volume with Milli-Q

water, sonicate for five minutes and cool to room temperature, measure the pH of above

buffer solution and finally adjusted the pH to 3.0±0.05 with orthophosphoric acid

solution and filtered through 0.45 µ nylon filter.

3.4. The mobile phase

A mixture of potassium dihydrogen phosphate buffer pH 3.0 and acetonitrile in

the ratio of 65:35 v/v was prepared and used as mobile phase.

3.5. The diluent

The potassium dihydrogen phosphate buffer pH 3.0 and acetonitrile mixture in the

ratio of 65:35 v/v was used as diluent.

3.6. Preparation of standard solution of the drug

About 40 mg of Perindopril and 20 mg of Amlodipine was accurately weighed

and transferred into a 100 mL clean dry volumetric flask containing 50 mL of diluent.

The solution was sonicated for 5 min and then volume was made up to the mark with a

further quantity of the diluent to get a concentration of 400 µg/mL for Perindopril and

200 µg/mL for Amlodipine (Stock solution). Further pipette 1 mL of the above stock

solution into a 10 mL volumetric flask and the volume was made up to the mark with the

diluents.

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3.7. Preparation of sample (tablet) solution

Twenty tablets were weighed and finely powdered. An accurately weighed

portion of powder sample equivalent to 40 mg of Perindopril and 20 mg of Amlodipine

was transferred to a 100 mL volumetric flask containing 50 mL of the diluent. The

contents of the flask were sonicated for about 10 min for complete solubility of the drug

and volume made up with further quantity of diluent. Then this mixture was filtered

through 0.45 µ membrane filter. Further pipette 1 mL of the above stock solution into a

10 mL volumetric flask and the volume was made up to the mark with the diluent.

4. METHOD DEVELOPMENT

For developing the method, a systematic study of the effect of various factors was

undertaken by varying one parameter at a time and keeping all other conditions constant.

Method development consists of selecting the appropriate wave length and choice of

stationary and mobile phases. The following studies were conducted for this purpose.

4.1. Detection wavelength

The spectra of diluted solutions of the Perindopril and Amlodipine in diluent were

recorded separately on UV spectrophotometer. The peaks of maximum absorbance

wavelengths were observed. The spectra of the both Perindopril and Amlodipine were

showed that a balanced wavelength was found to be 237 nm.

4.2. Choice of stationary phase

Preliminary development trials have performed with octadecyl and octyl columns

with different types, configurations and from different manufacturers. Finally the

expected separation and shapes of peak was succeeded in Xterra C18 column.

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4.3. Selection of the mobile phase

In order to get sharp peak and base line separation of the components, the author

has carried out a number of experiments by varying the composition of various solvents

and its flow rate. To effect ideal separation of the drug under isocratic conditions,

mixtures of solvents like water, methanol and acetonitrile with or without different

buffers in different combinations were tested as mobile phases on a C18 stationary phase.

A mixture of Phosphate buffer pH 3.0 and acetonitrile in the ratio of 65:35 v/v was

proved to be the most suitable of all the combinations since the chromatographic peaks

obtained were better defined and resolved and almost free from tailing.

4.4. Flow rate

Flow rates of the mobile phase were changed from 0.5-2.0 mL/min for optimum

separation. A minimum flow rate as well as minimum run time gives the maximum

saving on the usage of solvents. It was found from the experiments that 0.6 mL/min flow

rate was ideal for the successful elution of the analyte.

4.5. Run time

No interference in blank and placebo solutions for both drug peaks in the trail

injections with a runtime of 11.0 min.

4.6. Optimized chromatographic conditions

Chromatographic conditions as optimized above were shown in Table 13.2. These

optimized conditions were followed for the simultaneous determination of Perindopril

and Amlodipine in bulk samples and its combined tablet formulations. The

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chromatograms of standard and sample solutions of Perindopril and Amlodipine were

shown in Figure 13.3 and Figure 13.4. The chromatograms of stability studies of

Perindopril and Amlodipine were shown from in Figure 13.7 to Figure 13.10.

Table 13.2: Optimized chromatographic conditions for simultaneous estimation of

Perindopril and Amlodipine in combined tablet dosage form

Mobile phase : Potassium dihydrogen phosphate buffer pH 3.0:acetonitrile,

65:35 v/v

Pump mode : Isocratic

pH of Buffer : 3.0±0.05

Diluent : Potassium dihydrogen phosphate buffer pH 3.0:acetonitrile,

65:35 v/v

Column : Xterra C18 column, 100 mm x 4.6 mm, 5 µm

Column Temp : Ambient

Wavelength : 237 nm

Injection Volume : 20 µl

Flow rate : 0.6 mL/min

Run time : 11 min

Typical tR :-

Perindopril : 5.282±0.5 min

Amlodipine : 8.506±0.5 min

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Figure 13.3: Chromatogram of standard solution of Perindopril and Amlodipine

Figure 13.4: Chromatogram of sample solution of Perindopril and Amlodipine

5. VALIDATION OF THE PROPOSED METHOD

The proposed method was validated as per ICH [28-29] guidelines. The

parameters studied for validation were specificity, linearity, precision, accuracy,

robustness, system suitability, limit of detection, limit of quantification, and solution

stability.

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5.1. Specificity

A study conducted to establish specificity of the proposed method involved

injecting blank and placebo using the chromatographic conditions defined for the

proposed method. It was found that there is no interference due to excipients in the tablet

formulation and also found good correlation between the retention times of standard and

sample. The specificity results are shown in Table 13.3.

Table 13.3: Specificity study

Name of solution Retention time (min)

Blank No peaks

Perindopril 5.28

Amlodipine 8.51

5.2. Linearity

Linearity was performed by preparing mixed standard solutions of Perindopril and

Amlodipine at different concentration levels including working concentration mentioned

in experimental condition i.e., 20.0 to 100.0 µg/mL for Perindopril and 10.0 to 50.0

µg/mL for Amlodipine respectively. Twenty micro litres of each concentration was

injected in duplicate into the HPLC system. The response was read at 237 nm and the

corresponding chromatograms were recorded. From these chromatograms, the mean peak

areas were calculated and linearity plots of concentration over the mean peak areas were

constructed individually. The regressions of the plots were computed by least square

regression method. Linearity results were presented in Table 13.4 & 13.5 and linearity

plots are shown in Figure 13.5 & 13.6.

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Table 13.4: Linearity study of Perindopril

Table 13.5: Linearity study of Amlodipine

Level Concentration of Perindopril (µg/mL) Mean peak area

Level-1 20 420293

Level-2 40 793838

Level-3 60 1181098

Level-4 80 1522084

Level-5 100 1919281

Slope 18631

Intercept 49452

Correlation Coefficient 0.999

Level Concentration of Amlodipine (µg/mL) Mean peak area

Level-1 10 821061

Level-2 20 1528220

Level-3 30 2194775

Level-4 40 2874126

Level-5 50 3613261

Slope 69303

Intercept 12719

Correlation Coefficient 0.999

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Figure 13.5: Linearity plot of Perindopril

Figure 13.6: Linearity plot of Amlodipine

5.3. Precision

Precision is the degree of repeatability of an analytical method under normal

operational conditions. Precision of the method was performed as system precision,

method precision and intermediate precision.

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5.3.1. System precision

To study the system precision, five replicate mixed standard solutions of

Perindopril and Amlodipine were injected. The percent relative standard deviation (%

RSD) was calculated and it was found to be 0.28 for Perindopril and 0.28 for Amlodipine

respectively, which are well within the acceptable criteria of not more than 2.0. Results of

system precision studies are shown in Table 13.6.

Table 13.6: System precision

Injection number Area of

Perindopril

Area of

Amlodipine Acceptance criteria

1 790956 1515057

The %RSD of peak

areas of Perindopril

and Amlodipine

should not be more

than 2.0

2 785055 1506206

3 788935 1516059

4 788753 1512946

5 789934 1516721

Mean 788727 1513398

SD 2234 4266

%RSD 0.28 0.28

5.3.2. Method precision

The method precision study was carried out on five preparations from the same

tablet samples of Perindopril and Amlodipine and percent amount of both were

calculated. The %RSD of the assay result of five preparations in method precision study

was found to be 0.83 and 0.76 for Perindopril and Amlodipine respectively, which are

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well within the acceptance criteria of not more than 2.0. The results obtained for assay of

Perindopril and Amlodipine are presented in Table 13.7.

Table 13.7: Method precision

Sample number % Assay

Perindopril Amlodipine

1 100.6 99.0

2 100.4 100.5

3 99.6 99.5

4 101.1 100.3

5 99.0 98.8

Mean 100.14 99.62

SD 0.835 0.759

%RSD 0.83 0.76

5.3.3. Intermediate precision

The intermediate precision study was carried out by different analysts, different

columns, different reagents using different HPLC systems from the same tablet of

Perindopril and Amlodipine and the percent amount of Perindopril and Amlodipine was

calculated. The %RSD of the assay result of six preparations in intermediate precision

study was 0.27 and 0.25 for Perindopril and Amlodipine respectively, which are well

within the acceptance criteria of not more than 2.0. The results of intermediate precision

study are reported in Table 13.8.

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Table 13.8: Intermediate precision study of Perindopril

Injection number Area of

Perindopril

Area of

Amlodipine Acceptance criteria

1 798173 1533531

The %RSD of peak

areas of Perindopril

and Amlodipine

should not be more

than 2.0

2 800725 1537321

3 802996 1543169

4 802947 1542100

5 803014 1540454

Mean 801571 1539315

SD 2137 3916

%RSD 0.27 0.25

5.4. Accuracy

The accuracy of the method was determined by standard addition method. A

known amount of standard drug was added to the fixed amount of pre-analyzed tablet

solution. Percent recovery was calculated by comparing the area before and after the

addition of the standard drug. The standard addition method was performed at three

concentration levels of 50%, 100% and 150%. The solutions were analyzed in triplicate

at each level as per the proposed method. The percent recovery and % RSD at each level

was calculated and results are presented in Table 13.9 & 13.10. Satisfactory recoveries

ranging from 99.65 to 101.40 for Perindopril and 98.30 to 100.81 for Amlodipine

respectively were obtained by the proposed method. This indicates that the proposed

method was accurate.

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Table 13.9: Recovery study for Perindopril

%Concentration(at specification

Level)

Mean peak area

Amount of Perindopril

spiked (mg)

Amount of Perindopril recovered

(mg)

%Recovery Mean

Recovery

50% 419284 19.92 20.20 101.40%

100.41% 100% 791215 39.90 39.98 100.20%

150% 1176715 60.12 59.91 99.65%

Table 13.10: Recovery study for Amlodipine

%Concentration(at specification

Level)

Mean peak area

Amount of Amlodipine

spiked (mg)

Amount of Amlodipine recovered

(mg)

%Recovery Mean

Recovery

50% 817072 10.0 10.04 100.81%

99.6% 100% 1520125 20.09 20.06 99.59%

150% 2185048 30.59 30.40 98.3%

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5.5. Robustness

The robustness study was performed by slight modification in flow rate of the

mobile phase, pH of the buffer and composition of the mobile phase. Mixed samples of

both Perindopril and Amlodipine containing 20 µg/mL of Amlodipine and 40 µg/mL of

Perindopril concentration were analyzed under these changed experimental conditions. It

was observed that there were no marked changes in chromatograms, which demonstrated

that the developed method was robust in nature. The results of robustness study are

shown in Table 13.11 & 13.12.

Table 13.11: Robustness study for Perindopril

Condition Mean area % assay % difference

Unaltered 784003 100.2 -

Flow rate at 0.5 mL/min


776248

789625

100.9

99.2

0.7

1.0

Mobile phase:

• Buffer(70):Acetonitrile(30)


778621

788541

100.6

99.4

0.4

0.8

Table 13.12: Robustness study for Amlodipine

Condition Mean area % assay % difference

Unaltered 1505860 99.9 -



1485689

1513254

101.3

99.5

1.4

0.4

Mobile phase:



1496147

1536112

100.6

98.1

0.6

1.8

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5.6. System suitability

System suitability was studied under each validation parameters by injecting six

replicates of the standard solution. The system suitability parameters are given in Table

13.13 & 13.14.

Table 13.13: System suitability for Perindopril

Parameter Tailing factor Theoretical plates

Specificity study 1.52 2270

Linearity study 1.42 2115

Precision study 1.31 2089

Robustness study



Mobile phase:



1.15

1.21

1.08

1.18

2152

2284

2063

2197

Table 13.14: System suitability for Amlodipine

Parameter Tailing factor Theoretical plates

Specificity study 1.47 5234

Linearity study 1.42 5019

Precision study 1.35 4082

Robustness study



Mobile phase:



1.21

1.26

1.36

1.17

4301

5024

4052

5052

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5.7. Limit of detection and Limit of quantification

Limit of detection (LOD) is defined as the lowest concentration of analyte that

gives a detectable response. Limit of quantification (LOQ) is defined as the lowest

concentration that can be quantified reliably with a specified level of accuracy and

precision. For this study six replicates of the analyte at lowest concentration were

measured and quantified. The LOD and LOQ of Perindopril and Amlodipine are given in

Table 13.15 & 13.16.

Table 13.15: LOD and LOQ of Perindopril

Parameter Measured value (µg/mL)

Limit of detection 0.17

Limit of quantification 0.59

Table 13.16: LOD and LOQ of Amlodipine

Parameter Measured value (µg/mL)

Limit of detection 0.03

Limit of quantification 0.10

5.8. Solution stability

To determine the stability of Perindopril and Amlodipine in solution, the standard

and sample solution were observed under room temperature. Any change in the retention

time, peak shape and variation in response was compared to the pattern of chromatogram

of freshly prepared solution. The solution stability results are shown in the Table 13.17 &

13.18.

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Table 13.17: Solution stability of Perindopril

Standard solution Sample solution

Time

(hours) Response % variation

Time


Initial 784125 - Initial 784056 -

12 773968 1.3 12 772953 1.4

24 770812 1.7 24 770658 1.7

Table 13.18: Solution stability of Amlodipine

Standard solution Sample solution

Time


Time


Initial 1515840 - Initial 1501945 -

12 1509654 0.4 12 1498363 0.2

24 1497886 1.2 24 1476247 1.7

5.9. Stability studies

In order to demonstrate the stability of both standard and sample solutions during

analysis, both solutions were analyzed over a period of 24 hours at room temperature.

The results show that for both solutions, the retention time and peak area of Perindopril

and Amlodipine remained almost similar (%RSD less than 2.0) and no significant

degradation within the indicated period, thus indicated that both solutions were stable for

at least 24 hours, which was sufficient to complete the whole analytical process. Further

forced degradation studies were conducted indicating the stability of proposed method.

The results of the degradation studies are shown in the Table 13.19.

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Table 13.19: Forced degradation study results for Perindopril and Amlodipine

Stress

Conditions

Degradation

Time (Hrs)

Perindopril Amlodipine

% Assay % Degradation % Assay % Degradation

Control -- 99.5 -- 99.8 --

Acid 1 87.2 -12.3 85.9 -13.9

Base 1 82.8 -16.7 81.7 -18.1

Peroxide 1 83.1 -16.4 85.3 -14.5

Thermal 48 82.5 -17 81.2 -18.6

Control sample




contents of the flask were sonicated for about 10 min for complete solubility of the drug

and volume made up with further quantity of diluent. Then this mixture was filtered

through 0.45 µ membrane filter. 5.0 mL of this filtrate was further diluted to 50 mL with

mobile phase.

Acid degradation sample




contents of the flask were sonicated for about 10 min for complete solubility of the drug.

Then 5 mL of 5N acid (Hydrochloric acid) were added, refluxed for 60 minutes at 60°C,

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then cooled to room temperature, neutralized with 5N base (Sodium hydroxide) and

diluted to volume with diluent. Filtered about 25 mL of the above sample solution

through 0.45 µ membrane filter. Pipetted 5 mL of the above filtered sample solution into

a 50 mL volumetric flask and diluted to volume with diluent. Typical chromatogram of

acid degradation for Perindopril and Amlodipine is shown in Fig. 13.7.

Figure 13.7: Chromatogram of acid degradation showing Perindopril and

Amlodipine

Base degradation sample





Then 5 mL of 5N base (Sodium hydroxide) were added, refluxed for 60 minutes at 60°C,

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then cooled to room temperature, neutralized with 5N acid (Hydrochloric acid) and

diluted to volume with diluent. Filtered about 25 mL of the above sample solution

through 0.45 µ membrane filter. Pipetted 5 mL of the above filtered sample solution into

a 50 mL volumetric flask and diluted to volume with diluent. Typical chromatogram of

base degradation for Perindopril and Amlodipine is shown in Fig. 13.8.

Figure 13.8: Chromatogram of base degradation showing Perindopril and

Amlodipine

Peroxide degradation sample





Then 2 mL of 30% hydrogen peroxide were added, refluxed for 60 minutes at 60°C, then

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cooled to room temperature and diluted to volume with diluent. Filtered about 25 mL of

the above sample solution through 0.45 µ membrane filter. Pipetted 5 mL of the above

filtered sample solution into a 50 mL volumetric flask and diluted to volume with diluent.

Typical chromatogram of peroxide degradation for Perindopril and Amlodipine is shown

in Fig. 13.9.

Figure 13.9: Chromatogram of oxidative degradation showing Perindopril and

Amlodipine

Thermal degradation sample

Twenty tablets were weighed and finely powdered. The powder is exposed to heat

at 105°C for about 2 days. An accurately weighed portion of powder sample equivalent to

40 mg of Perindopril and 20 mg of Amlodipine was transferred to a 100 mL volumetric

flask containing 50 mL of the diluent. The contents of the flask were sonicated for about

10 min for complete solubility of the drug. Filtered about 25 mL of the above sample

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solution through 0.45 µ membrane filter. Pipetted 5 mL of the above filtered sample

solution into a 50 mL volumetric flask and diluted to volume with diluent. Typical

chromatogram of thermal degradation for Perindopril and Amlodipine is shown in Fig.

13.10.

Figure 13.10: Chromatogram of thermal degradation showing Perindopril and

Amlodipine

6. RESULTS AND DISCUSSION

The present study was aimed at developing a simple, sensitive, precise and

accurate HPLC method for the simultaneous estimation of Perindopril and Amlodipine

from bulk samples and their tablet dosage forms. A non-polar C18 analytical

chromatographic column was chosen as the stationary phase for the separation and

simultaneous determination of Perindopril and Amlodipine. Mixtures of commonly used

solvents like water, methanol and acetonitrile with or without buffers in different

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combinations were tested as mobile phases. The choice of the optimum composition is

based on the chromatographic response factor, a good peak shape with minimum tailing.

A mixture of buffer and acetonitrile in the ratio of 65:35 v/v was proved to be the most

suitable of all the combinations since the chromatographic peak obtained was well

defined, better resolved and almost free from tailing. The retention times of the

Perindopril and Amlodipine were found to be 5.28 and 8.51 min respectively.

The linearity was found satisfactory for the drugs in the range 20.0-100.0 µg/mL

for Perindopril and 10.0-50.0 µg/mL for Amlodipine (Table 13.4 & 13.5). The regression

equation of the linearity curve between concentrations of Perindopril and Amlodipine

over its peak areas were found to be Y=18631X+49452 (where Y is the peak area and X

is the concentration of Perindopril in µg/mL) and Y=69303X+12719 (where Y is the

peak area and X is the concentration of Amlodipine in µg/mL) respectively. Precision of

the method was studied by repeated injection of tablet solution and results showed lower

%RSD values (Table 13.6-13.8). This reveals that the method is quite precise. The

percent recoveries of the drug solutions were studied at three different concentration

levels. The percent individual recovery and the %RSD at each level were within the

acceptable limits (Table 13.9 & 13.10). This indicates that the method is accurate. The

absence of additional peaks in the chromatogram indicates non-interference of the

commonly used excipients in the tablets and hence the method is specific.

The deliberate changes in the method have not much affected the peak tailing,

theoretical plates and the percent assay. This indicates that the present method is robust

(Table 13.11 & 13.12). The system suitability studies were carried out to check various

parameters such as theoretical plates and tailing factor (Table 13.13 & 13.14). The lowest

455

values of LOD and LOQ as obtained by the proposed method indicate that the method is

sensitive (Table 13.15 & 13.16). The solution stability studies indicate that both the drugs

were stable up to 24 hours (Table 13.17 & 13.18). The forced degradation studies

indicate that both the drugs were stable in stability studies (Table 13.19).

7. CONCLUSION

The proposed stability-indicating RP-HPLC method was simple, specific,

sensitive, accurate and precise and can be used for simultaneous analysis of Perindopril

and Amlodipine in bulk samples and its tablet dosage forms.

8. REFERENCES 1. Martindale, The Complete Drug Reference, 33th Edn, pharmaceutical press, p. 985 &

838, (2002).

2. The Merck Index, 13th Edn., Merck Research Laboratories, Merck & Co., White

House Station, NJ, USA, p. 1286 & 86, (2001).

3. Wilson and Gisvolds, Text book of organic medicinal and pharmaceutical chemistry,

11th Edn, Lippincott-Williams & Wilkins, Philadelphia. U.S.A, p. 649 & 631, (2004).

4. H.L.Sharma & K.K.Sharma, Principles of pharmacology, 2nd Edn, Paras’s medical

publishers, Delhi, p. 254 & 270, (2012).

5. CIMS (Current Index of Medical Specialities), UBM Medica India Pvt. Limited,

Bangalore, p. 88, Jan-Apr, (2012).

6. IDR Drug Triple i Compendium (Indian Drug Review), UBM Medica India Pvt.

Limited, Bangalore, p. 179, 5, (2012).

7. Sharma. S and Sharma. M. C. UV-spectroscopic method for the Perindopril erbumine

in pharmaceutical formulations using Indigo carmine. Research American-Eurasian

Journal of Scientific Research, 6(4), 210-216 (2011).

456

8. Prameela Rani. A and Bala Sekaran. C. A validated RP-HPLC method for the

determination of Perindopril erbumine in pharmaceutical formulations. International

Journal of Pharm Tech Research, 1(3), 575-578 (2009).

9. Medenica. M, Ivanovic. D, Maskovic. M, Jancic. B and Malenovic A. Evaluation of

impurity level of Perindopril tert-butylamine in tablets. Journal of Pharmaceutical

and Biomedical Analysis, 44, 1087-1094 (2007).

10. Mohit. G.D, Kailesh. B.G and Mrinilani. C.D. Development and validation of

stability indicating HPTLC method for the determination of Perindopril erbumine.

International Research Journal of Pharmacy, 1(1), 428-435 (2010).

11. Deepak. S.J, Subbiah. G, Mallika. S, Umesh. C.P and Pranav. S. First LC-MS/MS

electrospray ionisation validated method for the quantification of Perindopril and its

metabolite Perindoprilet in human plasma and its application to bioequivalence study.

Journal of Chromatography B, 837, 92-100 (2006).

12. Lin. S.J, Wu. H.L, Chen. S.H and Wen. Y.H. Derivitization-Gas chromatographic

determination of Perindopril. Analytical Letters, 29, 1751-1762 (1996).

13. Khopade. S.A and Jain. N.K. Difference spectrophotometric determination of

Amlodipine besylate. Indian Drugs, 37, 351-353 (2000).

14. Shimooka. K, Sawada. Y and Tatematsu. H. Analysis of Amlodipine in serum by a

sensitive high performance liquid chromatographic method with amperometric

detection. Journal of Pharmaceutical and Biomedeical Analysis, 7, 1267–1272

(1989).

457

15. Yeung. P.K.F, Mosher. S.J and Pollak. P.T. Liquid chromatography assay for

Amlodipine: chemical stability and pharmacokinetics in rabbits. Journal of

Pharmaceutical and Biomedical Analysis, 9, 565–571 (1991).

16. Josefsson. M and Norlander. B. Coupled-column chromatography on a Chiral-AGP

phase for determination of Amlodipine enantiomers in human plasma: an HPLC

assay with electrochemical detection. Journal of Pharmaceutical and Biomedical

Analysis, 15, 267–277 (1996).

17. Tatar. S and Atmaca. S. Determination of Amlodipine in human plasma by high-

performance liquid chromatography with fluorescence detection. Journal of

Chromatography B, 758, 305–310 (2001).

18. Avadhanulu. A.B, Srinivas. J.S and Anjaneyulu. Y. Reversed phase HPLC

determination of Amlodipine in drugs and its pharmaceutical dosage forms. Indian

Drugs, 33, 36-40 (1996).

19. Ilango. K, Kumar. P.B and Prasad. V.R.V. Simple and rapid HPTLC determination of

Amlodipine in pharmaceutical dosage forms. Indian Journal of Pharmaceutical

Science, 59, 336 (1997).

20. Bresford. A.P, Marcrac. P.V and Stopher. D.A. Analysis of Amlodipine in human

plasma by gas chromatography. Journal of Chromatography B, 420, 178 (1987).

21. Yasuda. T, Tanaka. M and Iba. K. Quantitative determination of Amlodipine in serum

by liquid chromatography with atmospheric pressure chemical ionization tandem

mass spectrometry. Journal of Mass Spectrometry, 31, 879 (1996).

22. Carvalho. M, Oliveira. C.H, Mendes. G.D, Sucupira. M, Moraes. M.E and De Nucci.

G. Amlodipine bioequivalence study: quantification by liquid chromatography

458

coupled to tandem mass spectrometry. Biopharmaceutical Drug Disposition, 22, 383–

390 (2001).

23. Streel. B, Laine. C, Zimmer. C, Sibenaler. R and Ceccato. A. Enantiomeric

determination of Amlodipine in human plasma by liquid chromatography coupled to

tandem mass spectrometry. Journal of Biochemical Biophysical Methods, 54, 357–

368 (2002).

24. Zhong. D, Chen. X, Gu. J, Li. X and Guo J. Applications of liquid chromatography–

tandem mass spectrometry in drug and biomedical analyses. Clinical Chimica Acta,

313, 147–150 (2001).

25. Feng. Y, Zhang. L, Shen. Z, Pan. F and Zhang. Z. Analysis of Amlodipine in human

plasma by liquid chromatography–mass spectrometry. Journal of Chromatographic

Science, 40, 49–53 (2002).

26. Jignesh. P, Ajay. P, Patel. M.B, Nimesh. P and Rashmika. P. Analytical method

development and validation of Amlodipine besylate and Perindopril erbumine in

combined dosage form by RP-HPLC. International Journal of Pharm Tech Research,

3(2), 801-808 (2011).

27. Barole. T.C, Gandhi. S.P, Ladke. A. V and Damle. M C. Validated HPTLC method

for the determination of Amlodipine besylate and Perindopril in bulk and its

pharmaceutical dosage form. The Pharma Review, September-October (2011).

28. ICH Harmonised Tripartite Guideline. Validation of Analytical Procedures: Text and

Methodology, Q2(R1), USA, 1-13 (2005).

29. ICH Harmonised Tripartite Guideline. Stability Testing of New Drug Substances and

Products, Q1A(R2), USA, 1-18 (2003).

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