Assessment and
Comparison of Pharmaceutical Equivalence of
Amlodipine Besylate Tablets Available in Syria under Biowaiver Conditions
Maryana Salamah1, Youssef Alahmad1,2
1Department
of Pharmaceutical Chemistry and Drug Quality Control, Faculty of Pharmacy,
Al-Baath University, Homs, Syria.
2Department
of Pharmaceutical Chemistry and Drug Quality Control, Faculty of Pharmacy,
Arab University for Science and Technology, Hama,
Syria.
*Corresponding Author E-mail: maryana1734@gmail.com
ABSTRACT:
There are several generics of Amlodipine
besylate tablets locally manufactured by some Syrian pharmaceutical companies.
this study tries to evaluate the pharmaceutical and chemical equivalence of
amlodipine besylate tablets from 5 Syrian pharmaceuticals and comparison the
results with an innovator (Norvasc®). The pharmaceutical equivalence
and the quality for each generic were assessed through the evaluation of
standard tests such as uniformity of weight, friability, hardness,
disintegration, content uniformity, uniformity of dosage units and dissolution
rate. The assay was conducted by using HPLC (the analytical column was C 18,
150 x 4.6 mm I.D, 4μm particle size, the mobile phase was a mixture of 30%
acetonitrile and 70% 100 mM ammonium acetate pH 5 with flow rate of 1ml/min and
UV detection at 237 nm for amlodipine). All the generics complied with the
standard specifications for uniformity of weight (<±7.5%), friability (<1%),
content uniformity (85-115%), uniformity of dosage units (<15), hardness and
disintegration tests. The chromatographic system found to be suitable for
quantitative determination of amlodipine (the resolution was 13.587,
theoretical plates were 7080, tailing factor was 1.921, RSD% was 1.087). The
result of assay indicated that only (B, C, E) showed values out of the range
specified in the USP (90-110%) but all products released more than 75% of the
labeled quantity during 30 minutes. Only product D had similarity factor (f2)
>50. This confirms the need for continuous monitoring of the safety, quality
and efficacy of the marketed generic drugs with a view to bioequivalence and
agreement with pharmacopoeia standards.
KEYWORDS:
Amlodipine besylate, quality control, content uniformity, dissolution test,
pharmaceutical equivalence.
INTRODUCTION:
A generic drug is a medication created to
be the same as an existing approved brand-name drug in dosage form, safety,
strength, route of administration, quality, and performance characteristics[1].
In developing countries and especially during war and
crises period, it would be high importance to assess and control the quality of
medicines manufacturing in these countries[2,3]. Since the
pharmaceutical industry is not innovator, so a lot of companies compete to
produce generic products especially in tablets form.
Here comes the importance of pharmaceutical
equivalence and quality control of generic drugs to ensure its
interchangeability with brand-name drug[4].
The quality control parameters of tablets were
assessed through the evaluation of uniformity of weight, thickness test,
hardness test, friability test, disintegration test, dissolution test and
potency test according to the standard method[5].
The dissolution test used as a tool for assessment the
quality of oral pharmaceutical products[6]. It used in bioequivalence
studies according to the Biopharmaceutics Classification System (BCS) through
comparative in vitro dissolution profile similarity, which is called
“biowaiver” studies[7]. Biowaivers were established by WHO and FDA
for BCS Class I drugs (high solubility and high permeability)[8].
Amlodipine is a calcium channel blocker belonging to
the second generation of dihydropyridine [9]. It
inhibits calcium ion influx across cell membranes selectively, with a greater
effect on vascular smooth muscle cells than on cardiac muscle cells[10].
It used in the treatment of hypertension and chronic stable angina with a dose
of 5 mg according to WHO[11].
Amlodipine besylate is white to off-white, crystalline
powder with a molecular weight of 567.06. It is slightly soluble in water and
freely soluble in methanol. It has absolute bioavailability (60-65%), half-life
(35-50 h) and classified as BCS class I drug [12,13].
The aim of This study is to evaluate the
pharmaceutical equivalence of five generics of amlodipine besylate tablets
marketed in Syria and compare them with an innovator drug (Norvasc®).
MATERIALS AND METHODS:
Five different generic of amlodipine besylate 5 mg
tablets (A, B, C, D, E), brand-name of amlodipine besylate 5mg tablets (Norvasc®),
standard amlodipine besylate powder (Rakshit/India),
methanol (Honey well/Germany), hydrochloric acid 37% (Sigma/Germany),
acetonitrile (Sigma/Germany), ammonium acetate (Scharlau/Germany), phosphoric
acid (Sigma/Germany), Water for HPLC and distilled water.
Uniformity of Weight:
20 tablets were taken from each product. Each tablet
was weighed individually using analytical balance (Sartorius ENTRIS
64/Germany). The deviation and percentage deviation of each tablet from the
mean weight were calculated. Not more than two of the individual weights
deviated from the official standard (limit±7.5%) according to European Pharmacopeia[14].
Hardness test:
10 tablets from each product
were tested to determine its ability to resist pressure during manufacturing,
packing or transport using a hardness tester (Erweka TBH 125 TD/Germany). The
crushing force (KP) for each tablet was recorded and the average for the 10
tablets of each product was calculated[14].
Friability test:
A number of tablets were randomly taken from each
product its weight 6.5g. then placed inside the friability tester (Panomex
903/India). The tablets were then removed, dusted, and weighed together again.
The weight difference and the percentage friability were calculated for each
product (< 1% according to British Pharmacopoeia[15].
Disintegration Test:
The test was performed according to test A on 6 tablets - the
dimensions of the tested tablets are less than 18 mm- using basket-rack apparatus (Electrolab ED2AL/India). Each tablet was placed in each tube and disintegration
test was carried out in distilled water at 37 ± 2°C. The time when all the 6
tablets disintegrated and all the particles passed through wire mesh was
recorded as the ‘Disintegration Time’ of the tablets[14].
Uniformity of Dosage Units:
It was done using content uniformity according the
United States Pharmacopeia standard[16].
Calibration curve of amlodipine besylate in HCL 0.01 N
at 237 nm
A standard curve was created for amlodipine besylate
using pure drug powder to make 5 known concentrations (range between 0.005 and 0.03mg/ml). These standard curves were
established to verify accurate analysis of the drug.
Content Uniformity:
10 tablets were taken from each product. Each tablet
was crushed and dissolved separately in 50ml of methanol. They were then sonicated in ultrasonic
bath (Jeken/China) and filtered using 110mm filter paper, then took 10ml and the volume is completed
up to 100 ml with HCl 0.01N and sonicated in ultrasonic bath and filtered with syringe filter (0.45µm). The
absorbance of the filtrates was read in UV/visible spectrophotometer (Analytik Jena
SPECORD 50 PLUS/Germany) at 237 nm and the concentrations were determined.
Assay:
It was conducted on all products using High
Performance Liquid Chromatography HPLC (Agilent 1260 infinity 11/Germany). This
was done to determine the actual amount of amlodipine present in the 5mg tablet
formulation[17].
Chromatographic conditions for HPLC:
The analytical column was C 18, 150 x 4.6 mm I.D,
4μm particle size. Temperature of the column was maintained at 300C
and the data were analyzed by Agilent Technologies software. The mobile phase
was a mixture of 30% acetonitrile and 70% 100 mM ammonium acetate pH 5. The
mobile solvent was filtered under vacuum. The flow rate was 1ml/minute with
5μl injection while the detection wavelength was 237nm[17].
Standard preparation for HPLC:
Amlodipine besylate (1 mg/mL): Approximately 6.9mg of
standard amlodipine besylate powder was weighed on an analytical balance and
dissolved in 5mL of mobile phase. Then it was sonicated
in ultrasonic bath and filtered with syringe filter (0.45µm). The filtrate
was injected into the HPLC. Each concentration was evaluated from the calibration
plot of the standard [17].
System suitability:
For the system suitability test four parameters namely
relative standard deviation, tailing factor and theoretical plates and
resolution were studied. The standard solution of amlodipine besylate (1mg/ml)
was prepared and injected 5 times. Then the retention
time and Area Under Curve (AUC) were recorded. The percent relative standard deviation (RSD%) was
calculated for retention time[18].
Sample preparation for HPLC:
20 tablets were weighed individually from each generic
and the average weight was calculated. The tablets were crushed with a mortar. Equivalence sample of average
weight was taken and
transferred into a 5ml sample bottle. This was dissolved with 5ml of mobile
phase and sonicated in ultrasonic bath and filtered with syringe filter
(0.45um). The filtrate was injected into the HPLC. Each concentration was
evaluated from the calibration plot of the standard[17].
Dissolution Test:
It was used to determine in vitro release of Amlodipine
besylate tablets using USP dissolution apparatus II (Panomex/India)[16].
Each vessel was cleaned with distilled water, rinsed with HCL 0.01N and filled
with 500ml of HCL 0.01N at 37±0.5 C̊. Six tablets from each
product were evaluated. Each was placed in a dissolution vessel and the system
was powered. Samples (5mls) were obtained at 5,10, 15, 20, 25, 30 minutes. Each
5ml sample was filtered with a syringe filter (0.45μm). Finally, the absorbance’s
were taken at 237 nm. Analysis was performed by UV/visible spectrophotometer.
Analysis of dissolution data:
A model independent approach is recommended by US FDA
guidance for dissolution data equivalence involving use of similarity factor (f2).
The similarity factor is a measurement of the similarity in the dissolution (%)
of two curves.
where n is the number of dissolution sample times and
Rt and Tt are the individual or mean percent dissolved at each time point for
the reference and test products respectively.
According to the FDA guidance, f2 values from 50-100 %
ensure similarity of two dissolution profiles. The dissolution profiles may be
established as comparable without additional mathematical estimation when drug
dissolution is more than 75% within 30 minutes[19,20].
RESULT AND DISCUSSION:
It is important to perform quality control testing of
the dosage forms to assess the relationship between physicochemical
characteristics and in vitro release of the drug. This could explain
drug’s behavior in vivo.
Uniformity of Weight:
Table 1 shows some of the physical characteristics of the samples
studied. All the individual weights deviated from the official standard less
than ±7.5%, so all the tested tablets passed the test for weight uniformity.
Hardness and friability tests:
Table 1 shows that the mean tablet hardness for the
tested products ranged from 6.061 to 16.09 KP and all passed the test. The
result of tablet friability test shows that virtually all tested products had
impressive friability values ranging from 0.0% to 0.4% w/w (Table 1). According
to BP no batch should have a friability value greater than 1.0%w/w; therefore,
all the tested products passed the test.
Disintegration test:
Table 1 shows that all samples met the requirements of
disintegration test according to BP, within 15 mins for uncoated tablets though
the values varied widely among the samples.
Table.1. Physical characteristics of tablets amlodipine
besylate tablets
|
Product
|
Uniformity of Weight (mg)
|
Hardness ± SD (KP)
|
Friability (%)
|
Disintegration time
|
|
Average weight ± SD
|
Deviation range (%)
|
|
n=20
|
n=10
|
*
|
n=6
|
|
Norvasc®
|
202.5 ± 0.002
|
2.074, -3.604
|
16.09 ± 6.687
|
0%
|
38 sec
|
|
A
|
199.5 ± 0.001
|
0.776, - 0.526
|
8.341 ± 1.171
|
0%
|
4 min.15 sec
|
|
B
|
99.2 ± 0.001
|
1.349, -1.067
|
7.044 ± 0.306
|
0%
|
13 sec
|
|
C
|
183.2 ± 0.001
|
0.684, -0.897
|
6.453 ± 0.354
|
0%
|
52 sec
|
|
D
|
200.7 ± 0.002
|
1.711, -2.672
|
12.362 ± 1.191
|
0%
|
11 sec
|
|
E
|
204.0 ± 0.002
|
1.185, -2.097
|
6.061 ± 0.512
|
0.4%
|
10 sec
|
(* n= number of tablets its weight 6.5 gr)
Uniformity of Dosage Units:
A linear relationship between the absorbance and the
concentration of amlodipine besylate in HCL 0.01N is observed. The regression
equation is Y= 0.4252X and the correlation coefficients (r) of the linear
regression of the calibration curves is 0.9992.
Content uniformity:
The results obtained showed that all tested tablets
gave individual content within the limits of 85 to 115% of the average content
according to USP specification (see table 2). All tested products showed acceptance
value less than 15, therefore, all the tested products showed uniformity of
dosage unites according to USP.
Table.2. Uniformity of Dosage Units of amlodipine
besylate tablets
|
Product
|
Average content % ± SD n= 10
|
Percent of content range (%)
|
RSD (%)
|
Acceptance Value
|
|
Norvasc®
|
101.00 ± 0.016
|
99.83 - 103.97
|
0.016
|
0.039
|
|
A
|
109.65 ± 0.915
|
107.52 - 110.86
|
0.834
|
10.346
|
|
B
|
104.21 ± 0.644
|
103.15 - 105.29
|
0.617
|
4.255
|
|
C
|
111.12 ± 0.598
|
110.41 - 112.46
|
0.538
|
11.055
|
|
D
|
110.13 ± 0.551
|
109.38 - 110.81
|
0.499
|
9.951
|
|
E
|
112.00 ± 0.5601
|
110.93 - 112.77
|
0.501
|
11.845
|
Assay:
The system suitability
tests are parameters that confirm the validity of chromatographic system. Table
3 shows that RSD% was less than 2% (the retention time was acceptable 9.342
min), the peak was nature and symmetry, tailing factor was less than 2% and the
resolution was satisfactory. The result indicated that the chromatographic
system was adequate for the intended analysis.
Table 3: System suitability data
|
Parameter
|
Amlodipine
|
|
Theoretical plates/meter
|
7080
|
|
Asymmetry factor
|
1.692
|
|
Tailing factor
|
1.921
|
|
RSD%
|
1.087
|
|
Resolution
|
13.587
|
The results obtained from the assessment of the
percentage content of active ingredient of the labeled amount showed that three
products (Norvasc®, A, D) passed the test with values within USP specification,
while products (B, C, E) gave values outside the
monograph specifications (90-110%). (see figure 3).
Dissolution Test:
Assessment of biowaiver criteria confirmed that
amlodipine besylate tablets are very rapidly dissolving having dissolution
amount greater than 75% at 30 min and within the range specified in the USP
(90-110%). Dissolution profiles of the tested products were comparable to that
of the reference product using similarity factor (f2), the similarity factor
was found to be 28.05 and 92.03. table 4 shows that only product D has
similarity factor >50, therefor, dissolution profile for product D is
similar with Norvasc® dissolution profile.
Table.4. Dissolution data and dissolution profile
comparison using f2
|
Product
|
Q%
|
|
5 min
|
10 min
|
15 min
|
20 min
|
25 min
|
30 min
|
|
Norvasc®
|
101.86
|
103.29
|
104.35
|
104.97
|
105.40
|
105.83
|
|
A
|
47.25
|
65.96
|
93.17
|
101.80
|
103.70
|
105.72
|
|
f2
|
28.05
|
|
B
|
71.32
|
83.37
|
86.43
|
88.23
|
88.94
|
91.49
|
|
f2
|
34.60
|
|
C
|
81.02
|
85.23
|
90.67
|
94.40
|
95.26
|
99.45
|
|
f2
|
42.36
|
|
D
|
99.29
|
102.53
|
104.56
|
105.24
|
105.77
|
107.29
|
|
f2
|
92.03
|
|
E
|
81.29
|
86.24
|
86.78
|
87.12
|
87.85
|
89.13
|
|
f2
|
37.40
|
CONCLUSION:
This study showed that all tested products complied
the biowaiver condition of dissolution for BCS I drugs. The physicochemical properties
of amlodipine besylate tablets were comparable. there was a wide variation in
the results of the hardness test between the studied products. All products
passed the tests for friability (< 1%) and uniformity of weight
(deviation less than ±7.5%). Three tested products gave values within the
monograph specifications (90-110%), while products (B, C, E) failed the test
with values outside of proxy USP specification. In this study, as expected for
highly soluble compound, amlodipine, it was observed that for all products, at
least 75% release in 30 min. This confirms the need for continuous monitoring
of the safety, quality and efficacy of the marketed generic drugs with a view
to bioequivalence and agreement with pharmacopoeia standards. This study
further highlights the concerns over the quality of drug products marketed in a
developing country.
ACKNOWLEDGEMENT:
We would like to thank Human Pharma for pharmaceutical industry for allowing
access to their laboratory for the evaluation of the samples’ physicochemical properties
and provide
materials for this research.
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