INTRODUCTION:
The different bacterial and protozoa
infectious diseases remain a constant hazard to the human’s health. The use of antimicrobial
drugs plays a significant role in keeping infectious diseases under control.
The antiprotozoal drugs are extensively used in developing countries, in particular,
as well as, in the rest of the world.[1]
Metronidazole has bactericidal and antiprotozoal action. It is widely used in
the treatment of many anaerobic and certain protozoan and parasitic infections.[2]
Chemically, Metronidazole is (2-Methyl-5-nitroimidazole-1-ethanol),
molecular formula (C6H9N3O3) and
molecular weight 171.2g/mole. Physically is a white to pale-yellow crystalline
powder with a slight odor, bitter and saline taste, it darkens on exposure to
light, melting point 158–160oC, sparingly soluble in water but
soluble in dilute acid.[3,4]
Metronidazole is available in different pharmaceutical
formulations like a tablet, suspension, and suppository, topical and vaginal
cream and as an I.V. infusion. The tablet dosage form is the most widely
prescribed in the health care setting. There are several brand products of
Metronidazole tablets available in the pharmacies which are considered
pharmaceutically equivalent when they contain the same amount of active
ingredient in an identical dosage form and achieve the same compendia standard (i.e.
strength, purity and quality) but may differ in some characteristic such as
shape, packaging, additive material, and labeling requirement.[5]
The pharmaceutically equivalent drug products help the
healthcare providers in substituting the brand drug with the appropriate generic
equivalent in case of unavailability or cost unaffordability. This is
especially common with patients of a low-income in the developing countries
where the difference in cost between the brand and the generic medicines could
reach as high as ten folds. Hence, the World Health Organization agency (WHO)
has continuously supported the use of generic medicines. However, the
substitution is usually controversial and often met with doubt among the
patients and the physicians because of the fear of inefficient pharmacological
action or avoidable adverse effects.[6] Therefore, routine
laboratory testing of the drug products available in the market is crucial to
protect the public health; especially in the developing countries where the
less effective and substandard drugs have become a major challenge to the
health care services.[7] Therefore, it is necessary to perform such
monitoring on the approved medicines to adequately assess the quality,
therapeutic effectiveness and safety of medications for the larger public. This
quality control testing could be done during and after manufacturing as well as
at various intervals during the shelf life of a drug product. [8]
Many post-market monitoring studies were performed on
different drug products in different developing countries and were result in significant
specification. For examples, in Iraq, Musab et. al found that two out of four
products of carbamazepine tablet were bio-inequivalent to the brand drug
product.[9] In Nigeria, research work was done on Metronidazole tablets
by Adegbolagun et.al reported that some generic forms were chemically and
bio-pharmaceutically inequivalent to the brand Metronidazole product.[10]
Similarly, a study by Barone et.al on twenty-five generic product of piroxicam
capsule provides that more than 70% of the brands failed to meet the USP
requirements of pharmaceutical formulation parameters.[11]
Furthermore, Adegbolagun et. al assessed the drug content of ten generic
product of ciprofloxacin tablets, they found that three products contained
active ingredient lower than USP specifications of ciprofloxacin HCl content
(90–110%) while the rest confirm the USP specification.[12]
During the course of therapy for a certain disease,
the need to select one product among several generic products of the same
active ingredient is a field of interest to healthcare providers which requires
ascertaining the pharmaceutical and biopharmaceutical equivalency to the brand
drug formulation to ensure the therapeutic equivalency.[13] Therefore,
this study aimed to investigate and assess the pharmaceutical quality of five
different product of Metronidazole 500 mg tablets marketed in Mosul city by
using official and non-official standards test according to USP specifications,
in an attempt to emphasis that all the brands are pharmaceutically equivalent
or not. The assessment of tablets includes the evaluation of weight uniformity,
hardness, friability, disintegration time, in vitro dissolution study besides
determining the content of active pharmaceutical substance.
MATERIALS AND METHODS:
Materials:
1. Five different samples of, most commonly
available Metronidazole 500mg tablets in the Iraqi market, were purchased from
the retail pharmacy as shown in table (1).
2. Metronidazole standard powder was a
generous gift from AwaMedica® drug manufacture company, Erbil, Iraq.
3. Hydrochloric acid was supplied by the
Pharmaceutical Department, College of Pharmacy, University of Mosul.
Methods:
I-
Characterization of Metronidazole pure powder
1.
Determination of melting point
The
melting point was determined depending on the procedure stated by the USP. A
compact column of Metronidazole powder was prepared by inserting a small
quantity of the drug powder into a capillary tube, and then put it in Stuart
electrical apparatus.[14]
2.
Determination of λ max of Metronidazole
Metronidazole
solution of 25μg/ml in 0.1N HCl was prepared and scanned by spectrophotometer
from 200 – 400nm and then the λ max of
the drug was determined. [15]
3.
Construction of Calibration Curve of Metronidazole:
Calibration
curve of Metronidazole in 0.1N HCl was constructed by preparing a series of
solutions with different concentrations of Metronidazole in the range of 5-40μg/ml
from the stock solution containing 0.5mg/ml of Metronidazole (by dissolving 50mg
of drug powder in 100ml of 0.1N HCl). These solutions were then
analyzed spectrophotometrically at the wavelength of maximum absorbance using
Cary UV spectrophotometer. The measured absorbance of each sample was plotted
against the respective concentration.[15]
II-
Evaluation tests for the selected products:
1. Weight variation:
For
each brand twenty tablets were weighted individually by electrical balance (PMB
electrical balance, ADAM), then the average weight of the tablet was calculated
as well as the percentage deviation from the mean value were recorded.[16]
2.
Hardness:
The
crushing strength of a tablet was measured using Monsanto hardness tester. Five
tablets from each brand were randomly selected and the pressure at which each
tablet crushed was recorded in kg/cm2.[16]
3.
Friability test:
Ten
randomly selected tablets from each brand were previously weighed and placed in
the friabilator chamber. The friability apparatus was operated up to 100
revolutions, the difference in weight was calculated by weighing the tablet
after the test. The percentage of friability was calculated by using the following
formula:
W1
= the initial weight before the test
W2
= the final weight after the test
4.
Content uniformity test:
The
quantity of Metronidazole in each brand was determined according to the USP.
Ten tablets were weighed and crushed then weight equivalent to 500mg of
Metronidazole was measured and dissolved in 0.1N HCl, then diluted to the
required weight and the absorbance of the sample was measured by utilizing UV
spectrophotometer at 276nm and using 0.1N HCl as blank. Accordingly, the amount
of Metronidazole in each product was determined using the equation of the
calibration curve shown in figure 1.
5.
Disintegration test:
Six
tablets of each brand were selected and placed in a medium of 0.1N HCl 900 ml
at 37˚C in the cylindrical tubes of the basket into the disintegration
apparatus and the time at which no particle remained in the basket of the
system and passed out through the mesh at the bottom of the tube was recorded
as the disintegration time. Mean disintegration time was calculated for each of
the brands.
6.
In-vitro dissolution study:
The
dissolution studies were performed according to the USP paddle method, and the
release profile of Metronidazole from each branded tablet was performed using
900ml of 0.1N HCl at 37±0.5˚C and 75rpm. A volume of 5ml was withdrawn at
different time intervals from the dissolution medium and replaced immediately
with fresh 0.1N HCl medium. The samples were filtered and diluted (if needed)
to a suitable concentration with the same dissolution medium and assayed
spectrophotometrically at 276nm. The cumulative percentage of drug release was
calculated using the linear regression equation of the calibration curve.[17]
Statistical
Analysis:
The
results of different tests were analyzed by using Microsoft Excel 2010
calculating the mean ± Standard Deviation (SD).
RESULTS:
I-
Analytical evaluations of Metronidazole powder
Figure
1: The UV spectrum of Metronidazole dissolved in 0.1N HCl determined by using
Varian Cary UV spectrophotometer.
The
melting point for drug sample powder was found to be 156˚C, and the
UV/visible spectrophotometer scanning of Metronidazole solution gives the
spectrum shown in figure (1) with peak for λ max at 276nm.
While figure (2) shows the calibration curve by plotting the absorbance versus
concentration of different Metronidazole solutions gives a straight line with
high regression factor (R2 = 0.995) which mean that the chosen
concentration of metronidazole solutions obeys Beers law.
Figure
2: The spectrophotometry calibration curve of Metronidazole in 0.1N HCl
determined by using Varian Cary UV spectrophotometer (± SD).
B-
Quality control tests of selected product:
The
results of official and unofficial quality control tests that performed on
brand and four different generic products of Metronidazole are presented in
table no. (2) which summarized the result as an average value for tablet
hardness, weight uniformity, percent of friability, the average time of
disintegration test in addition to percent of Metronidazole content in each
sample products. Figure (3) shows the release profiles of different brands of
Metronidazole tablets by measuring the percent of drug released at a specific
time by applying the equation of Metronidazole calibration curve (each value
represent mean ± SD, n = 6).
Figure
3: Comparative dissolution profiles of the brand and the different generics of
the available Metronidazole
tablets in Iraq; (± SD).
DISCUSSION:
The
results of tests performed on Metronidazole powder like melting point and
spectrophotometer scan indicated the purity of the powder sample used in this
study, which is in agreement with the reported values stated in the USP-35.[14]
The generation of the calibration curve of the standard Metronidazole
solutions, as shown in figure (3), yielded a linear regression equation which
was used to determine the pure drug content of the tested tablets, and
measuring the percent of drug released during dissolution study.
The
hardness is a useful parameter to assess the ability of the individual tablet
to withstand the mechanical stress and keep structural integrity under different
conditions of processing, storage, transportation and handling before usage.
This physical property influenced by many factors like formulation ingredients,
processing parameters, and storage conditions.[18] The tablet
hardness was measured as kg/cm2 for the tested product of
Metronidazole tablets, the result showed that the highest value was 13.7kg/cm2
for MT-E, and the lowest value was 7.7kg/cm2 for MT-B. As per
British and United State Pharmacopeia, a force of about 4-6 kg/cm2
is the minimum requirement for satisfactory tablet.[19,14] Thus, the
tablets of all the products were sufficient for proper hardness. It is
noteworthy to mention that the high values of hardness for all selected product
may be due to the film coat applied on the surface which adds to the mechanical
strength, this notification is in agreement with another study applied on the
coated tablet.[20]
The
friability testing is a method which is also performed to determine the
physical strength of compressed tablet upon exposure to mechanical shock and
attrition. According to the USP specification, if the friability is not more
than 1% means that the tablet formulation is acceptable.[14] As
shown in table (2), the values of friability for all selected brands were
relatively low and this fact may be related to the presence of coat around
tablet which indicates that all the brands are mechanically stable and met the
pharmacopoeial requirements which are in agreement with another study applied
on Metronidazole tablets.[21]
The
weight uniformity test is a critical factor in pharmaceutical manufacturing as
it serves as an indicator to Good Manufacturing Practice (GMP) to ensure the
reproducibility of the drug product from batch to batch and to confirm the drug
content in each tablet with the label strength.[22] The average weight of each tested tablet product was
calculated and the deviation from the average value was compared to it. The
lowest and highest weights were 660.4mg and 967.6mg for brand MT-B and MT-E,
respectively. However, the deviation from the average weight value for each
brand was not more than 1.65%, therefore, all the four brand and generic products
tested in this study fulfilled the pharmacopeial limitations for uniformity of
weight.[14]
Thus
all the commercial Metronidazole products possessed acceptable uniformity of
weight as the deviation of the tested tablets from average weight for each
product is not more or less than 5% which complies with the pharmaceutical
specification. The average content of analyzed Metronidazole tablets by the UV
spectrophotometric method was found to be in the range of 95.8% – 101.6% (table
2). The highest amount of Metronidazole was presented with brand MT-A which
included 101.6% of the labelled amount. Whereas the lowest amount of
Metronidazole content was found in brand MT-B which calculated as 95.8% of the
labelled amount. Consequently, all types of branded products had drug content
within the acceptable range of pharmacopeial specifications that are ranging
from 475 mg – 525mg for 500mg Metronidazole tablets.
Regarding
the dissolution study, in-vitro or pharmaceutical availability by dissolution
investigation provides useful and reliable information concerning in-vivo
bioavailability of drug content. These tests measure the rate and extent of drug
substance released from a dosage form by recording the percent of drug released
at a specific time.[23] The dissolution limitation for the
film-coated immediate-release tablet should not be less than 85% of the
labelled amount in 60 min.[14]
Figure
(3) shows the release profiles of different tested Metronidazole brand tablets
and the generic one after 10 min. of dissolution study. Brand MT-B showed the
highest drug release rate whereas brand MT-C had the slowest release rate
within the same time intervals. After 30 min. brand MT-D had maximum drug
release almost 100% of drug content while brand MT-C showed minimum drug
release at the same time 84.6%.
Comparison
of the dissolution data revealed that all the tested brands met the USP
specification for the dissolution limitation, and possessed satisfactory
dissolution profiles. Similarity factor (f2) has been calculated
using the following equation:
For
comparing the release data of branded tablets to the generic product release
behavior as it considered the reference for it. According to the FDA guidance,[24] the dissolution
profiles are similar if f2 values are between 50 and 100.[25]
The f2 values of the different brands were calculated and presented
in the table (3). It showed that brand MT-C and MT-E are within the
above-mentioned requirements, and can be considered pharmaceutically
equivalent. Therefore, the physicians can prescribe them interchangeably in
case of unavailability or for reducing the cost of the treatment.
Table
3: Comparison of dissolution profiles of four brand versus generic
Metronidazole tablets.
|
Brand comparison generic
|
Similarity factor (f2)
|
|
MT-B vs. MT-A
|
48.02
|
|
MT-C vs. MT-A
|
67.24
|
|
MT-D vs. MT-A
|
55.87
|
|
MT-E vs. MT-A
|
73.35
|
CONCLUSION:
The
present research work was done to evaluate the pharmaceutical properties and
in-vitro bioavailability of four different generic products in addition to the
brand of Metronidazole tablet marketed in Iraq. Various official and unofficial
quality-control tests were applied in an attempt to assess whether these four
brand products are pharmaceutically equivalent or not regarding the generic
product. The results indicated that nearly all the tested brands of
Metronidazole tablets were satisfactory as they successfully passed all the
pharmacopoeial requirements for oral tablets. Based on the dissolution studies
and the calculated similarity factor values, it was found that brand MT-C and
MT-E more closely equivalent to the innovator product by exhibiting highly
similar release behaviours. Therefore, the tested products can be recommended
for the healthcare providers to be substituted with the innovator product when
it is difficult to obtain it.
ACKNOWELGDEMENT:
The authors acknowledge the
scientific support provided by the University of Mosul and the College of
Pharmacy. Also, we are grateful for the Awa Medica Pharmaceutical Company for
providing us with the pure metronidazole powder.
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