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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
432
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
442
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
Flow rate at 0.7 mL/min
776248
789625
100.9
99.2
0.7
1.0
Mobile phase:
• Buffer(70):Acetonitrile(30)
• Buffer(60):Acetonitrile(40)
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 -
Flow rate at 0.5 mL/min
Flow rate at 0.7 mL/min
1485689
1513254
101.3
99.5
1.4
0.4
Mobile phase:
• Buffer(70):Acetonitrile(30)
• Buffer(60):Acetonitrile(40)
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
Flow rate at 0.5 mL/min
Flow rate at 0.7 mL/min
Mobile phase:
• Buffer(70):Acetonitrile(30)
• Buffer(60):Acetonitrile(40)
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
Flow rate at 0.6 mL/min
Flow rate at 1.0 mL/min
Mobile phase:
• Buffer(55):Acetonitrile(45)
• Buffer(45):Acetonitrile(55)
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
(hours) Response % variation
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
(hours) Response % variation
Time
(hours) Response % variation
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
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. 5.0 mL of this filtrate was further diluted to 50 mL with
mobile phase.
Acid degradation sample
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.
Then 5 mL of 5N acid (Hydrochloric acid) were added, refluxed for 60 minutes at 60°C,
450
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
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.
Then 5 mL of 5N base (Sodium hydroxide) were added, refluxed for 60 minutes at 60°C,
451
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
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.
Then 2 mL of 30% hydrogen peroxide were added, refluxed for 60 minutes at 60°C, then
452
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
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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.
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