Werabe University

College of Agriculture and Natural Resource

Department of Animal

science Research proposal

submission

DRUG QUALITY ASSESSMENT ON DIFFERENT VETERINERY

OXYTETRACYCLINEANTIBIOTICS make it specific

PREPARATIONS CIRCULATING IN AND AROUND SILTE ZONE,

CENTRAL ETHIOPIA

By

Dr. Numan Jemal Amdino (PI)

Dr. Redwan Anwar Mohammed (Co-In)

Dr. Ufaysa Gensa Tajudin Denur Hassen

(Co-In)
March, 2024
DECLARATION

We, the under signed WRU staffs, declared that this research proposal is our original work and it has
not been presented in our MSc/PhD thesis or for any other purpose at any university. We mean, we
solemnly declare that this work is not submitted to any other institution anywhere else for the award
of any academic degree, diploma or certificate.

Name of investigators Signature Date

1) Numan Jemal Amdino
Redwan Anwar Mohammed
2) Redwan Anwar Mohammed
Numan Jemal Amdino
3) Ufaysa GensaTajudin
Denur Hassen

This research proposal has been submitted for examination with our approval as reviewer, college
dean, research and publications director.

Approved by

( )

Reviewer Signature Date

Mulgeta Tesfaye ( )

Reviewer Signature Date

Serkalem Asefa ( )

Department Dean Signature Date

Hana Yasin ( )

College Dean Signature Date

Kedir Ebrahim (PhD, )

Research and Publication director Signature Date

Researchers involved in the study and the role they play

Numan Jemal (Dr.) – Principle investigator

Will design the study and verify the methodologies, will perform the laboratory analysis, played a
role in analysis of the data and revision final version of the manuscript

Numan Jemal (Dr.), Redwan Anwar (Dr.) and Ufaysa Gensa (Dr.) Tajudin Denur Hassen

; Co-investigators

Will perform the sample collection, write the draft manuscript

ii
TABLE OF CONTENTS

DECLARATION..................................................................................................................................................i
TABLE OF CONTENTS....................................................................................................................................iii
LISTS OF ABBERVATIONS.............................................................................................................................v
1. INTRODUCTION......................................................................................................................................1
1.1. Background of the Study........................................................................................................................1
1.2. Statement of the Problem.......................................................................................................................3
1.3. Significance of study...............................................................................................................................5
1.4. Objectives.................................................................................................................................................6
1.4.1. General objective...............................................................................................................................6
1.4.1. Specific objectives.............................................................................................................................6
2. LITERATURE REVIEW.........................................................................................................................7
2.1. Livestock Diseases....................................................................................Error! Bookmark not defined.
2.2. Veterinary pharmaceuticals....................................................................................................................7
2.3. Antibiotic use in domestic animals........................................................................................................8
2.4. Development of antimicrobial resistance in domestic animals...........................................................9
2.5. Counterfeited Vs. Substandard drug......................................................................................................9
2.6. High performance liquid chromatography (HPLC)..........................................................................10
2.6.1. High Pressure Pumps.......................................................................................................................11
2.6.2. Injector system.................................................................................................................................11
2.6.3. Column.............................................................................................................................................12
2.6.4. Detectors..........................................................................................................................................13
2.6.5. Location of spots..............................................................................................................................13
2.7. Pharmacopoeial and non-Pharmacopoeial tests................................................................................13
2.8. Properties of a primary standard........................................................................................................14
2.7. Oxytetracycline.....................................................................................................................................15
2.9.1 Mechanism of Action of Oxytetracycline.........................................................................................16
2.9.2 Determination of API of Oxytetracycline.........................................................................................16
2.10. Measurements / Uneven manufacturing quality of drug................................................................17

iii
3. METHODOLOGY; MATERIALS AND METHODS.............................................................................19
3.1. Description of the Study area..................................................................Error! Bookmark not defined.
3.2. Data collection, Study design, sample and sampling technique.......................................................21
3.3. Sample size and sampling.....................................................................................................................21
3.4. Facilities.................................................................................................................................................22
3.5. Chemicals and reagents........................................................................................................................23
3.6. Physicochemical Assessment of drug quality.....................................................................................23
3.6.1. Identification test.............................................................................................................................23
3.6.2. High performance liquid chromatography method for Assay of API..............................................23
3.6.3. Uniformity of weight test for tablets and filling to the volume for injectable preparations............26
3.6.4. Sterility Test.........................................................................................................................................27
3.7. Data management and analysis...........................................................................................................30
4. STUDY PERIOD – WORK PLAN............................................................................................................27
5. BUDGET DISTRIBUTION - COST BREAK DOWN.............................................................................31
6. REFERNCES...............................................................................................................................................34
Annex.................................................................................................................................................................39

iv
LISTS OF ABBERVATIONS

API Active pharmaceutical ingredient

APIQTC Animal Product and Input quality testing center

BP British pharmacopoeia

CSA Central statistical agency

DQA Drug Quality Analyst

GABA Gamma amino butyric acid

GDP Gross domestic product

HPLC High performance liquid chromatography

IGAD Inter-governmental aid development

IV Intravenous

Nm Nano meter

ODS Octadecyl-silica

OTC Oxytetracycline

RAPS Regulatory affairs professional society

RT Retention time

SD Standard deviation

SZPCASA Siltie Zone Plan Commission Annual Statistical Abstract

v
1. INTRODUCTION

1.1. Background of the Study

The livestock subsector has an enormous contribution to Ethiopia’s national economy and
livelihoods of many Ethiopians, and still promising to rally round the economic development of the
country. Livestock play vital roles in generating income to farmers, creating job opportunities,
ensuring food security, providing services, contributing to asset, social, cultural and environmental
values, and sustain livelihoods (Zeleke & Harris-Coble, 2021).

Production and Productivity of livestock sector in Ethiopia is not parallel with livestock population
size as it holds largest livestock population in Africa and the top ten in the world that is 70 million
cattle, 42 million sheep, 52 million goats, 8 million camels, and 56 million chickens (CSA, 2021).
This is mainly due to disease constraints, feed shortage and poor genetic potential of our indigenous
breeds. Among diseases, bacterial and parasitic diseases continue to be a major constraint on
profitable livestock production. They are usually characterized by lower outputs of animal products,
byproducts, manure and traction all contributing to production and productivity losses (FAO, 2004).

To tackle such challenges as measure for disease constraint we need to have drugs which are
effective, safe, fulfill quality standards set by official pharmacopeia and available for rural farmers
throughout the year. Unless the quality of the drugs are assured the presence and availability of the
drug in the market is not such important. Thus beside disease diagnosis and therapeutic intervention,
drug quality paramount is critical issue always to consider(WHO, 2018). In the four years since
launching its global surveillance and monitoring system, WHO says it has received more than 1500
reports of substandard or falsified medicines, with 42% of reports coming from sub-Saharan Africa,
and 21% each coming from the Americas and Europe (RAPS, 2017).

1
The safety and efficacy of drug products can be guaranteed when their quality is reliable and
reproducible from batch to batch. To ensure the requisite quality, drug manufacturers are required to
test their products during and after manufacturing and at various intervals during the shelf life of the
product (Kahsay & G/Egziabher A., 2010).

The quality of medicines is an integral part of access in light of ensuring that the pharmaceutical
products are fit for their intended use, comply with the requirement of the marketing authorization
and do not expose consumers to risks. To attain this objective there must be a system of quality
assurance, which incorporates aspects including product development, manufacture, distribution,
and storage. Many developing countries do not have an effective means of monitoring the quality of
generic drug products in the market. This results in widespread distribution of substandard and/or
counterfeit drug products. It was in view of this fact that the WHO issued guidelines for global
standard and requirements for the registration, assessment, marketing, authorization, and quality
control of generic pharmaceutical products (Pierre-Louis Lezotre MS, 2014)

Several brands of tetracycline imported and sold in veterinary shops, livestock markets and by drug
peddlers are routinely used by farmers without veterinary diagnosis or prescriptions (Fagbamila et
al., 2010). Most of these drug formulations have been reported ineffective against tetracycline
susceptible organisms (Okonko et al., 2009). (Cartwright & Baric, 2018) estimated that about 10%
of medicines sold in Africa are counterfeit of which antimicrobial agents being the most popular
target. Substandard products with lower-than-stated doses promote resistance, and those containing
no antimicrobial drug at all promote microbial dissemination (Okeke et al., 2007).

2
1.2. Statement of the Problem

Drug quality is currently receiving renewed international attention in response to increases in the
magnitude of counterfeit medications which are a threat to public health with many devastating
consequences for patients; increased mortality and morbidity and emergence of drug resistance. In
addition, physicians treating these patients lose their confidence in the medications use (Theodore et
al., 2007). Over the past decade, there has been an increase in public awareness of the existence of
counterfeit and substandard drugs which have been increasingly reported in developing countries
where drug regulations are ineffective (Cartwright & Baric, 2018).

Although practically all types of pharmaceutical products have been shown to be involved, the
existing data suggest that anti-infectious agents, particularly antibiotics and antiparasitic agents are
the most counterfeited products in developing countries (Theodore et al., 2007).

One in ten medicines in developing countries is substandard or fake . reference The findings come
from two new reports, one from WHO's global surveillance and monitoring system over the last four
years and another that pooled data from 100 literature reviews to examine the public health and
socioeconomic impact of substandard and falsified medicines(WHO, 2018). In the four years since
launching its global surveillance and monitoring system, WHO says it has received more than 1500
reports of substandard or falsified medicines, with 42% of reports coming from sub-Saharan Africa,
and 21% each coming from the Americas and Europe (RAPS, 2017); (Cartwright & Baric, 2018).

In 2003, WHO reported that fake drugs reported between 1999 and 2002 include analgesics and
antipyretics (6%), antimalarials (7%), anti-asthma and anti-allergy (8%), antibiotics (28%),
hormones and steroids (18%) and other therapeutic categories (33%). reference These problems
resulted in a weak therapeutic efficiency, selection of resistance strains and poor quality of
numerous drugs. Marketing of such drugs has been widely reported in Africa, Asia, and Latin
America. (Taylor et al., 2001). Lack of competent regulatory authorities and poor quality control
practices in some countries, have allowed the availability of poor quality drugs. These could be the
widespread counterfeiting of medicines, decomposition of the active ingredient in drug dosage form
due to high temperature and humidity of the storage condition, and inadequate quality assurance
systems during the manufacture of pharmaceutical products (Hebron et al., 2005).

3
Veterinary drug products those used in animals have also an effect on the health of human beings.
Obviously, this is due to pathogens which affects both human and animals share the same
characteristics. Thus, humans can obtain the pathogens those affects animals through a food chain or
other sources as a result pathogens develops resistance in animals also leads to development in
humans as well ((Palma et al., 2020); (Caneschi et al., 2023); (Salam et al., 2023); (Salam et al., 2023).

Drug resistance pathogens was due to the exposure of animals to the poor quality veterinary drugs,
those poor quality veterinary drug product arise from counterfeit drug, under or over concentration
of API during drug manufacturing , unsterile products and also circulation of unregulated veterinary
drug products in veterinary drug stores or retailers. Lack of simple analytical techniques and
adequate regulation in the evaluation of such products remain a challenge in developing countries
such as Ethiopia (Caneschi et al., 2023);(Palma et al., 2020).

In Ethiopia, Oxytetracycline injectable solution is one of the most drug mainly sold in the veterinary
pharmaceutical drug stores and used for treating many infectious diseases caused by bacteria
protozoa and ricktessials. reference But the response of pathogens to this drug was reduced at these
times. reference This was may be the reasons for the factors mention them mentioned above. There
are efforts of HPLC analysis on Albendazole, Ivermectin focusing centeral market Addis Ababa.
There was no sufficient study conducted in the country for in vitro quality assessment of
oxytetracycline by using HPLC and for sterility test primary emphasising the peripheral market
(Post Market Survey) like Siltie Zone. Therefore, this preliminary research study will be conducted
for sterility test and physico chemical evaluation of oxytetracycline circulating in Siltie Zone

4
1.3. Significance of study

Once the assessment is concluded, this study is expected to generate the following benefits:
 To provide base line data for further researchers and policy makers in the sub region
 To create awareness platform for local people about presence of substandard veterinary
drugs on the market based on research finding and to take precaution measure to mitigate the
problem
 To increase food resources of Ethiopia both in quantity and quality by implementing
effective monitoring and control methods of diseases and to improve herd fertility and
overall production of livestock under different production systems, taking into account
environmental, production as well as the tradition and culture of the people
 It will improve the practical knowledge of the researcher by recruiting out the factual
evidences regarding quality of veterinary drug status at the field level.
 Avoid financial loss due treatment failure that resulted from use of quality compromised drug

 It will serve as a spring board for further studies on animal health issues in relation to drug
quality
 It will provide basic information for stakeholders that aim to analyze the interaction of
livestock diseases, treatment cost and drug quality
 It will help the Zone to mitigate the problem so as to improve the situation and ensure better
livestock production and productivity in which cost of treatment is reduced using quality
veterinary drug
 The community, especially those participants of this study will be aware of veterinary
services in general and livestock drug handling, use and quality assessment in the particular.
 The investigators will trace back possible source and origin of veterinary drugs in the zone

5
1.4. Objectives

1.4.1. General objective

 To evaluate physicochemical quality and sterility test of OxytetracyclineOTC injectable

solution brands both formulated as short acting and long acting which circulating in Siltie
Zone. Should be inline with your title.

1.4.1. Specific objectives

 To assess the physical characteristics and aesthetic quality like packaging, leaflet, labelling
and registration status
 To identify and quantify assay of veterinary formulated products containing injectable
oxytetracycline by using HPLC method
 To evaluate whether the injectable oxytetracycline drug solution is sterile or not by using
standard microbiological procedures

 To carryout weight variation, content uniformity and filling to the volume of different
preparations circulating in the pharmaceutical markets and available for farmers use
 Evaluate the active pharmaceutical ingredient to check the presence of counterfeit or
substandard drugs destined for veterinary use based on laboratory evidence this is simply
analysis.

[1.5.] Research questions

 Wheather there is substandard or counterfeit Veterinary drugs circulating in and around

Siltie Zzone agro-ecology,
 If present the magnitude, specific geographical distribution, potential trade route or source

6
2. LITERATURE REVIEW

2.1. Livestock Diseases

Disease constraints are the major livestock production and productivity challenges of this nation.
One among solutions to tackle such challenges related to drug quality is making sure that all
available drugs for stock holders that take part in animal husbandry have drugs which are effective,
safe, fulfill quality standards set by official pharmacopeia and available for rural farmers throughout
the year. Unless the quality of the drug is assured the presence and availability of the drug in the
market is not such important. Thus beside disease diagnosis and therapeutic intervention, drug
quality paramount is critical issue always to consider (WHO, 2018).

2.2. Infectious Diseases

Infection is a complex mechanism caused by the entrance of living microbes (bacteria, fungi and
viruses) and other factors such as traumatism, physical and chemical agents which cause infection.
Large number of infectious diseases causes worldwide death, so early stage diagnosis may help to
reduce morbidity and mortality. Diagnosis of infectious disease involves the identification of the
root cause of infectious disorders. In spite of having many advanced techniques to identify and treat
infectious lesions, the world is facing a great threat of infectious diseases which is deleterious
(Saleha et al., 2018).

2.2. Veterinary pharmaceuticals

Veterinary pharmaceuticals are substances employed in the diagnosis, prevention, control and
treatment of animal diseases (Federal Office of Consumer Protection and Food Safety, 2016). From
those, Veterinary antibiotics are widely used as growth promoters and for therapies of infectious
diseases in livestock farming Worldwide, about 100,000 to 200,000 tons of antibiotics are used
annually (Jeong et al., 2010).
7
Just like food substances, pharmaceutical products can serve as media for growth and proliferation
of microorganisms. A drug may be microbiologically contaminated from the raw materials used in
its formulation, equipment from which it was made, from atmosphere of the production plant, from
the person operating the process, from the final container into which it was packed or during the
course of storage, transportation and even marketing. Some of the contaminants may be pathogenic,
whilst some others may grow even in the preservative (antibiotic) added. Antibiotics which are used
in treating bacterial diseases and in the preservation of pharmaceutical products have often been
found to be contaminated by microorganisms, because not all antibiotics are active against all
microorganisms, as such microbes can survive or even multiply in both syrup and capsules ( Arzai &
A.H., 2008).

2.3. Antibiotic use in domestic animals

Provision of complete animal health care necessitates the availability of safe, effective and
affordable drugs of the required quality in adequate amounts at all times. Veterinary medicinal
products (VMPs) are necessary to meet the challenges of providing adequate amounts of food for
the growing human population, as drugs prevent and cure diseases in food-producing animals.
Antibiotics are considered among the most commonly used classes of VMPs for several purposes,
including treatment and prevention of infections as well as growth promotion (Beyene et al., 2018).

In the livestock industry, antibiotics are used abundantly. There are three main uses of antibiotics in
livestock production. They can be used as a therapeutic, as a growth promoter and as a prophylactic.
Therefore antibiotics are necessary to society-used properly or not. The commercial importance of
antibiotics is not only for treating infections in humans but also for food production and keeping
animals and plants disease free (Kirkup et al., 2006).

Accordingly, Tetracyclines groups like, tetracycline, chlortetracycline, and oxytetracycline, which

exhibit broad-spectrum antimicrobial activity against a variety of animal diseases, are the most used
as animal growth promoters in livestock production. They are poorly metabolized in the digestive
tract of animals and most (50–80%) is excreted through faeces and urine as unmetabolized parental
compounds (Sarmah et al., 2006).
8
The growth promoting effects of antibiotics Like OTC were first discovered in the 1940s when
chickens fed by-products of tetracycline fermentation were found to grow faster than those that were
not fed those by-products. Since then, many antimicrobials have been found to improve average
daily weight gain and feed efficiency in livestock in a variety of applications (Gaskins et al., 2002).

2.4. Development of antimicrobial resistance in domestic animals

Resistance to antimicrobial drugs is becoming more serious throughout the world (Salam et al.,
2023). The principle behind the development of resistance is that bacteria in the guts of humans and
animals are subjected to different types, concentrations, and frequencies of antimicrobial agents.
Over time, selective pressure selects resistant bacteria that have specific fingerprints for resistance to
the antimicrobial agents that have been used (Troy et al., 2002).

Regular use of antibiotics in humans or animals can lead to resistance among pathogens, and there is
growing concern that widespread antibiotic use has led to the emergence of some organisms
resistant to most or all antibiotics. The extent to which antibiotic use in livestock production
contributes to human health problems is a matter of controversy. The U.S. Food and Drug
Administration (FDA) has established that enough scientific evidence exists to support the agency’s
current restrictions on using medically important antibiotics for production purposes (USP, 2007).

2.5. Counterfeited Vs. Substandard drug

Counterfeiting is exact copy and can apply to both branded and generic products and may include
products with the correct ingredients, wrong ingredients, without active ingredients, with insufficient
quantity of active ingredient or with false or misleading packing; they may also contain different or
different quantities of impurities both harmless and toxic (Theodore et al., 2007). Whereas,
substandard drugs are genuine drug products which does not meet quality specifications set for
them. The term substandard is used to describe the quality status of genuine drugs produced by
legitimate manufacturer. Normally, manufacturers used specifications laid down by official
pharmacopoeias such as British pharmacopoeia (BP), United States pharmacopoeia (USP) and
European

9
pharmacopoeia (EP) for each drug that they produced, if a drug fails to meet the pharmacopoeias
specifications used for its formulations, the drug is classified as substandard (Karungamye, 2023).

2.6. High performance liquid chromatography (HPLC)

Since the origin of what is now called HPLC about 35 years ago, there has been a continuous
development of stationary phases for columns that provide improved separation with a wide variety
of compound type. Early HPLC separations were performed by liquid-liquid-partition
chromatography or by liquid-solid (adsorption) chromatography. While liquid-liquid
chromatography provided a wide range of potential separation selective activity, practical
limitations seriously restricted the usefulness of this approach. Maintaining a constant concentration
of the stationary liquid on the column support with a flowing proved mobile phase difficult and
cumbersome. This method required pre-saturating the mobile phase with the stationary phase and
maintaining close column temperature control. Such requirements also effectively eliminated the
possibility of conducting gradient elution separations that often were desired for separating
relatively non-polar samples using non-aqueous mobile phases. Also gradient elution separations
with this method were difficult and generally irreproducible because of problems in maintaining the
activity of the adsorbent with changing mobile phase polarity (Kirkland & J.J., 2004).

HPLC is a technique in analytical chemistry used to separate, identify and quantify each component
in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture
through a column filled with a solid adsorbent material. Each component in the sample interacts
slightly differently with the adsorbent material, causing different flow rates for the different
components and leading to the separation of the components as they flow out the column. High
performance liquid chromatography has been used for manufacturing (for example during the
production process of pharmaceutical and biological products), legal (e.g. detecting enhancement
performance drugs in urines), research (e.g. separating the components of complex biological
sample, or of similar synthetic chemicals from each other) and medical (e.g. detecting vitamin D
levels in blood serum) purposes (Gerber et al., 2004).

10
The separation technique based on a solid stationary phase and a liquid mobile phase and these are
achieved by partition, adsorption, or ion-exchange processes, depending upon the type of stationary
phase used. High performance liquid chromatography has distinct advantages over gas
chromatography for the analysis of organic compounds. Compounds to be analyzed are dissolved in
a suitable solvent, and most separations take place at room temperature. Thus, most drugs, being
non- volatile or thermally unstable compounds, can be chromatographed without decomposition or
the necessity of making volatile derivatives. Most pharmaceutical analyses are based on partition
chromatography and are completed within 30 minutes (USP-NF, 2023)

2.6.1. High Pressure Pumps

HPLC pumping systems deliver metered amounts of mobile phase from the solvent reservoirs to the
column through high-pressure tubing and fittings. Modern systems consist of one or more computer-
controlled metering pumps that can be programmed to vary the ratio of mobile phase components, as
is required for gradient chromatography, or to mix isocratic mobile phases (that is mobile phases
having a fixed ratio of solvents). However, the proportion of ingredients in premixed isocratic
mobile phases can be more accurately controlled than in those delivered by most pumping systems.
Operating pressures up to 5000 psi or higher, with delivery rates up to about 10 mL per minute are
typical. Pumps used for quantitative analysis should be constructed of materials inert to corrosive
mobile phase components and be capable of delivering the mobile phase at a constant rate with
minimal fluctuations over extended periods of time (USP-NF, 2023).

2.6.2. Injector system

Injectors after dissolution in mobile phase or other suitable solution, compounds to be

chromatographed are injected into the mobile phase, either manually by syringe or loop injectors, or
automatically by autosamplers. The latter consist of a carousel or rack to hold sample vials with tops
that have a pierceable septum or stopper and an injection device to transfer sample from the vials to
a loop from which it is loaded into the chromatograph. Some autosamplers can be programmed to
control sample volume, the number of injections and loop rinse cycles, the interval between
injections, and other operating variables (USP-NF, 2023).

11
The sample solution is introduced into the flowing mobile phase at or near the head of the column
using an injection system which can operate at high pressure. They contain fixed-loop and variable
volume devices which are operated manually or by an auto-sampler are used. Manual partial filling
of loops may lead to poorer injection volume precision (BP, 2005).

2.6.3. Column

Columns for most pharmaceutical analyses, separation are achieved by partition of compounds in
the test solution between the mobile and stationary phases. Systems consisting of polar stationary
phases and non-polar mobile phases are described as normal phase, while the opposite arrangement,
polar mobile phases and non-polar stationary phases, and are called reverse-phase chromatography.
Partition chromatography is almost always used for hydrocarbon-soluble compounds of molecular
weight less than 1000. The affinity of a compound for the stationary phase, and thus its retention
time on the column, is controlled by making the mobile phase more or less polar. Mobile phase
polarity can be varied by the addition of a second, and sometimes a third or even a fourth,
component (USP, 2000).

The columns are made of highly polished stainless steel usually having a column length of 10 to
30cm and an internal diameter of 4.5 to 5mm. Longer and larger pore size columns are available and
are used usually for commercial purposes. The most widely used stationary phase is silica
(SiO2.XH2O). The stationary phase consists of a network of siloxane linkages (Si-O-Si) in a rigid
three dimensional structure containing interconnecting pores. The pore size and the amount of
silanol groups are controlled in the manufacturing process. In a straight stationary phase column the
silanol groups are vital as they are involved in adsorption chromatography. Silica can be modified to
the reversed stationary phase. This is done by a controlled reaction of organochlorosilanes with the
silanol groups or the use of organoalkoxysilanes which modifies the surface of the silica. The
linkage of these hydrocarbons to the surface impacts a non-polarity to the surface and enhances
partitioning, thus the separation of lipophilic compounds. The most popular stationary phase
material used is the (ODS) Octadecyl-silica C18. Others includes octyl (C8), Phenyl (C6H5),
Cyanopropyl (CH2) 3-CN and aminopropyl (CH2) 3-NH2 groups. Pharmaceutical products contain
both lipophilic and polar groups. These groups are exploited during separation on columns (Okine et
al., 1999).

12
2.6.4. Detectors

Detectors many compendial HPLC methods require the use of Spectrophotometric detectors. Such a
detector consists of a flow through cell mounted at the end of the column. A beam of UV radiation
passes through the flow cell and into the detector. As compounds elute from the column, they pass
through the cell and absorb the radiation, resulting in measurable energy level changes. The detector
must have a broad linear dynamic range and compounds to be measured must be resolved from any
interfering substances. The linear dynamic range of a compound is the range over which the detector
signal response is directly proportional to the amount of the compound. For maximum flexibility in
quantitative work, this range should be about three orders of magnitude. HPLC systems are
calibrated by plotting peak responses in comparison with known concentrations of a reference
standard, using either an external or an internal standardization procedure. Five main types of
detectors are frequently used in HPLC. These are the electrochemical detectors, Fluorescent
detector, Refractive index detector, Mass spectrometers, Radioactivity detectors and the Ultra-Violet
visible detectors. Among these, the most widely used is the UV-Visible detectors (Elsa et al., 1996).

2.6.5. Location of spots

As most organic compounds are colorless, they must be made visible, preferably by a non-
destructive technique. Most compounds can be located by examining plate containing a
chromophore under a 254nm wavelength. In this process the absorbing compounds are seen as dark
spots. These spots can be ringed up with a pencil. Compounds that naturally fluoresce can be located
under UV lamp as colored spots. Another very important but destructive test is to spray ethanolic
sulphuric acid on the plate and gently warm in an oven. Organic material when treated in such
manner, char-up and are seen as dark spots (Ajibola & Francis, 1993).

2.7. Pharmacopoeial and non-Pharmacopoeial tests

Standard of uniformity of weight is applied to tablets or bolus and capsules which are supplied in
unit dose forms and uniformity of volume (filling to volume) to single dose pro-injections because
they are subject to more variation than comparable preparations supplied in multi-dose forms. This
test is performed to maintain the uniformity of weight of each tablet which should be in the
prescribed range, as stated in the pharmacopoeia when twenty tablets are selected at random and
13
a uniformity test

14
performed, not more than two tablets should deviate from the average weight by a greater
percentage as illustrated below and not even one should deviate by twice that value. The mean and
standard deviation (SD) were determined (Thahera et al., 2012).
Table 1: Specification of weight variation.
Pharmaceutical form Average weight (mass) Percentage deviation
Tablets (uncoated and film-coated 80 mg or less 10%
More than 80 mg and less than 250 mg 7.5%
Capsules, Granules (uncoated, single Less than 300 mg 10%
dose) and
300 mg or more 7.5%
Powders (single dose)
More than 40 mg 10%
Powders for Parenteral Use (single-dose)
Suppositories and Pessaries All weights (masses) 5%

Source: (USP, 2007)

2.8. Properties of a primary standard

A primary standard should as far as possible meet the following requirements like it should be
readily available in highly purified form, should not be wetable in air at ordinary and fairly high
temperature, have high equivalent weigh (this will reduce the effects of small weighing errors), be
readily soluble in the chosen solvent under the analytical conditions and it should give no interfering
products during titration ((Ajibola & Francis, 1993)). Active ingredient content (Active
Pharmaceutical Ingredient, API): The content of the active substance is of significance to the
analyst. The presence of the active ingredient and also in the right amount determines the efficacy of
the drug. Determination of active ingredient content of any drug formulation is also crucial in
eliminating substandard and counterfeit drugs (BP, 2005).

15
Overview of effects of sub-therapeutic drug dose as typical property of substandard drugs

Under dose (<95%) Drug with acceptable API % content Over dose (>105%)

Sub therapeutic dose Required therapeutic effect Undesirable effect

Therapeutic goal is achieved

Treatment Failure

Toxicities

Frequent use

Predispose for Drug Resistance

Figure 5.The relationship between Percentage (%) of API content of the unit drug and respective
effects (Ozawa et al., 2022)

2.7. Oxytetracycline

Oxytetracycline is first discovered as result of work of researcher in American trade organization

from streptomyces fungal species. It has been extracted from fungus streptomyces rimosus for first
time by Alexander F in 1950 (Aranda, 2006). It possesses antibacterial activity by binding to the
30S ribosomal subunit of a susceptible organism. Following ribosomal binding it interferes with the
binding of aminoacyl-tRNA to the messenger RNA molecule/ribosome complex; this disrupts the
bacterial protein synthesis and for this reason oxytetracycline drug is classified under bacteriostatic
group, which stop the growth of bacteria (Chopra et al., 2001).

16
Oxytetracycline (OTC) is one of the most commonly used antibiotics in animal production. It
belongs to the tetracycline group of antibiotics. OTC is a compound produced in broth by
fermentation from Streptomyces rimosus. It has broad-spectrum of antimicrobial activity. In
particular, it is active against Gram-positive and Gram-negative bacteria, Rickettsia, Coxiella,
Chlamydia, Mycoplasma, and some protozoa. This antibiotic is widely used in the treatment of
respiratory (pharingitis, pneumonia, bronchitis), urinary, and gastrointestinal infections, because of
its activity and good penetration into the tissues. Oxytetracycline is generally well tolerated when
administered orally or intramuscularly as an aqueous solution (Dowling & Russell, 2000); (Korchi
et al., 2000).

Oxytetracycline consider as broad-spectrum antibiotics that effect growth of both gram positive and
negative bacteria in animal and human being and act against other microbial infection like
Rickettsia, Mycoplasma, Chlamydia. It used against trachoma, urinary and skin infection as well as
in treatment of chronic respiratory diseases like pneumonia, Hemophilus influenza while in high
doses, it effect growth of protozoal disease in animals like thileria. There is formulation of
Oxytetracycline as injectable solution at concentration (10, 20, and 40 %) for veterinary use (USP,
2015).

OTC is hygroscopic in aqueous solutions and only slightly soluble in water with a neutral pH and
highly soluble in lipid. It forms salts with acids and bases, the salt most commonly used being
hydrochloride and it also readily binds to divalent and trivalent cations such as magnesium and
calcium (Giguère et al., 2006)

2.9.1 Mechanism of Action of Oxytetracycline

Oxytetracycline functions by binding to the 30S subunit of the ribosome, thereby preventing the
translocation of amino acids from the transfer-RNA to the messenger-RNA and subsequently
prevents the elongation of peptide chains. By way of this mechanism of action, oxytetracycline is
bacteriostatic

2.9.2 Determination of API of Oxytetracycline

17
Different analytical methods are reported for the determination of API and identification of
oxytetracycline in different dosage or preparation form which includes, parentral , powdered and
Iontment for quality evaluation such as HPLC with UV detection (Owusuwaa, 2016), bioassay and

18
fluorometry for assay methods. Among high-performance liquid chromatographic (HPLC) methods
reported for the analysis of oxytetracycline, a large number concern reversed-phase bonded packing
materials involving complicated aqueous-organic eluents, with pH in the range 1–2.5, where a
number of bonded packing materials are unstable so that column life can be short. Furthermore,
most published methods failed to introduce an internal standard which is most useful in quantitation.

The HPLC analysis method of oxytetracycline described in the present study is characterized by
sufficient accuracy, precision and reproducibility, as well as sensitivity and selectivity. Codeine
proved to be a very suitable internal standard for this purpose. At the working pH conditions, no
epimerization products at C-4 were noticed in the chromatograms of commercial pharmaceuticals
(Papadoyannis et al., 1999).

2.10. Measurements / Uneven manufacturing quality of drug

Accurate definitions of poor quality medicines are essential. The World Health Organization (WHO)
uses the umbrella term, ‘Substandard/Spurious/Falsely labelled/Falsified/Counterfeit medical
products (SSFFC). ‘Substandard medicines are officially defined as“authorized medical products
that fail to meet either their quality standards or specification,or both”and may result through poor
manufacturing, shipping or storage conditions, or when the drug is sold beyond the expiration date.
Falsified medicines are defined as“medical products that deliberately/fraudulently misrepresent their
identity, composition or source (Karungamye, 2023). Poor quality medicines directly harm patients
by denying them access to potentially life-saving active pharmaceutical ingredients (API), or
exposing them to toxins. If poor quality medicines contain less than the intended API, pathogens can
become exposed to drug concentrations in the ‘ mutation selection window’ —high enough to exert
a selection pressure but too low to kill all of the pathogens (Ozawa et al., 2022)

Quality control is a part of good manufacturing practices sometimes neglected in developing

countries. The WHO compendium on pharmaceutical manufacture describes the importance of
having quality-control staff who are separate from production staff, working in an independent
department. Quality-control staff should verify that everything that is a part of the factory's
product, including
19
packaging, starting materials, intermediates, and finished products, meets requirements. The market
for active ingredients has been especially volatile in recent years because of increasing costs of raw
materials and growing environmental regulation in India and China. And, because the cost of API is
by far the largest fraction of overall cost, a small reduction in active ingredient can vastly increase
the profit margin. The water filtration system is a high risk for microbial growth in any
pharmaceutical plant and should be monitored vigilantly. Microbial contamination is more of a
threat in countries with poor water quality; much equipment cannot run on erratic power supplies
(Ashbolt, 2004)

Storage conditions of medicines is very important. Improper storage of products could lead to
breakdown of active ingredients in a formulation. For Oxytetracycline, reported degradation
products include 4-epioxytetracycline, α- and β-apooxytetracycline. Research has shown that
Oxytetracycline can be broken down by moisture, light, different PH (Zhao-jun et al., 2019)

Table 1. Determinants or Aspects of Medicine Quality

Characteristics Justification
Identity The correct active ingredient is present
Purity The medicine is not contaminated with potentially harmful substances.
Potency The correct amount of active ingredient is present, usually between 95
and 110 percent of the labeled amount.
Uniformity Consistency of, shape, and size of the dosage form do not vary.
Stability The activity of the medicine is ensured for the period of time stated on
the product label, that is, until the expiration date.
Pharmacopoeial standard A medicine is of good quality if its characteristics meet the standards
described in a widely accepted pharmacopoeia such as the British
Pharmacopoeia (BP), European Pharmacopoeia, International
Pharmacopoeia (IP), or United States Pharmacopeia (USP).
Source: (WHO, 2018).
Active Pharmaceutical Ingredient (API): The content of the active substance is of significance to
the analyst. The presence of the active ingredient and also in the right amount determines the
efficacy of the drug. Determination of active ingredient content of any drug formulation is also
crucial in eliminating substandard and counterfeit drugs (BP, 2005).
20
3. METHODOLOGY; MATERIALS AND METHODS

3.1. Description of the Study area

This study will be carried out in selected Weredas of Siltie Zone (nine administrative districts).
According to (SZPCASA, 2022), Siltie zonal administration is among one of the 7 zones and 3
special Wereds that forms central Ethiopia Region covering an area of 2700.04 Square Kilometers.
Zonal capital town is Werabe located on the main road from Addis Ababa to Hossana just 172kms
apart from Addis Ababa. Zonal Administration contains 10 Weredas and 5 town administrations
namely, Dalocha, Silti, Misrak Silti, Lanfuro, Mito, Sankura, West Azernet Berbere, East Azernet
Berbere, Hulbarag, Wereabe Town Administration, Tora Town Administration, Kibet Town
Administration, Dalocha Town Administration, and Alemgebeya Town Administration.

Astronomically, it is roughly in between 7043'-8010' N latitude and 37086' -38053' E longitude. It is

bordered with Hadiya Zone in the South, East Gurage Zone in the north, Gurage Zone in the west,
Mareko special wereda in the north east, Oromia Region in the east and Halaba Zone in south-east.
The population of the zone is about 1,240,036 where 48.90% male and 51.10% female. Concerning
the settlement, 78.07% and 21.93% of the population lives in rural and urban areas, respectively.
(SZPCASA, 2022)

The zone has three different agro-climatic conditions; high land (Dega and Wurch) and Weyna-
Dega. The average temperature ranges from 12-26oC and the average annual rainfall ranges from
780- 1818mm. Above 80% of the population engaged in agriculture. According to Zonal sector and
Weredas reports of 2022, there are 36 urban and 200 rural kebeles in the zone. (SZPCASA, 2022).

According to the (CSA, 2021) Livestock population of the zone are Cattle (547,666), Sheep
(331,455), Goats (227,592), Horses (33,160), Mules (2,040), donkey (126,539), Poultry (805,968)
and Beehives
(27,869).

21
Figure 6: Map of study area, Siltie zone, South Ethiopia (SZPCASA, 2022).

22
3.2. Scope of the study

The study will target different veterinary pharmacy shops, clinics and smallholder or commercial
farms, abattoirs and laboratories located in the zone, but practical visits on veterinary infrastructures
and the way they provide the service will take place. The investigation is all about identification of
the all status of veterinary drug handling, use, quality and sterility assessment and then to
recommend possible ways of intervention.

Sample collection procedure Study Design and Population

The sample will be collected/purchased randomly from different veterinary pharmacy shops, clinics
and farms, then brought to the laboratory of veterinary drug, animal product and feed quality
assessment center which is located in Akaki Kaliti sub-city under Animal feed and veterinary drugs
administration and control authority. During sampling, safety protocol is considered to avoid factors
that reduces the quality of the drugs for example, light since OTC is light sensitive despite that
storing at room temperature is advisable. In this study and to run one sample two vials will be used
for each brands during physicochemical evaluation and only one vials will be used in the case of
sterility test.

3.3. Sampling Method and Sample size determination

According to the report released by WHO's Global Surveillance and Monitoring System over the last
four years and another that pooled data from 100 literature reviews to examine the public health and
socioeconomic impact of substandard and falsified medicines suggested that 1 in 10 Medicines in
developing countries is substandard (RAPS, 2017). Thus the rate of prevalence of substandard
veterinary pharmaceutical product is expected to be 10 % and the confidence interval taken chosen
as 95% and precision 5%. Thus, the sample size is calculated according to (Thrusfield,
2007)Thrusfield (2007). By substituting these values in the formula, the sample size become 138.
N =1.962 (pexp) (1-pexp)

23
 d2
Where: N= required sample size, Pexp=Expected prevalence, d=Absolute precision (usually 0.05)

But here the number of sample size required depends on availability of different brands of
Oxytetracycline on market during studies, hence sample size adjustment will b employed as follow.

i.e. N = No*n/ No +n (Sample size adjusted =population size * sample size required/ population size +
sample size required) Where N = sample size adjusted, No = population size or max sample available
and n = required sample size (Thrusfield, 2007).

N (sample size adjusted) = Population size * sample size required

Population size + sample size required

Total Sample size required was 138/ districts; excluding reform town considering livestock population
and assuming together with districts with corresponding town as one.

i.e. 138/11 = 12 for highland districts and 13 for mid land districts
13/x, 12/x, where proportional factor for Farms, Clinics and Vet Pharmacy

3.4. Facilities

 Analytical Balance  Spoon and spatula

 Analytical Balance  Ultrasonic Sonicator
 Aluminum foil  Magnetic Stirrer
 Piston and mortar  Labeling paper
 Volumetric flasks  Shaker
 Gradating cylinders  Vortex mixer
 Beaker ,100ml  Syringe filter
 Duran bottle 1000ml  Syringe
 Erlenmeyer flask 250 ml  Funnels
 Pipette 5ml,10ml, 1ml  Filtration units
 Whatman Filter paper  Column

24
 HPLC  UV spectrophotometry
 Sample vials

25
3.5. Chemicals and reagents

High performance liquid chromatography graded solvents like methanol (Sisco research
laboratories Pvt. Ltd India), orthophospharic acid, Acetonitrile (Mfg. date 09/2015, Batch No.
2189056), hydrochloric acid 0.1N, oxytetracycline, reference material 200mg (USP-CRM USA,
potency of 100%, lot number :), Triethalmine and HPLC graded double distilled water will be
used in the analysis process.

3.6. Physicochemical Assessment of drug quality

3.7.1. Identification test

One among the approaches of identification test indicated in USP (2015) is retention time (RT)
that is to compare the retention time (RT) of the OTC sample with that of the RT of the standard.
(USP-38-NF, 2015).

3.7.2. High performance liquid chromatography method for Assay of API

Cleaning agent: to avoid the effect of carry over; remnants of the previous processed sample
(1:1 of Acetonitrile and water) that can clean both organic and inorganic particles respectively

Mobile phase preparation: 4.075ml of orthophosphoric acid will be added into 1000ml of
distilled water (produced in the lab using TKA Gen Pure UV–TOC/UF + TKA Pacific UP 20,
Thermo Fisher Scientific) and then after mixed well; the PH of the solution will be adjusted to
2.7 with triethylamine. And then 650ml will taken from this solution and mixed with 1000ml
HPLC grade Methanol and 250ml HPLC grade Acetonitrile and sonicated for 30min and this
will be passed through suitable filter of 0.5µm or fine pore size and used as mobile phase.
Therefore the volume ratio is, 0.4% Orthophosphoric acid in distilled water:, acetonitrile,:
methanol (65 : 25: 10).

26
Diluent preparation: 37% of 0.85ml (0.1N HCL) will mixed in 1000ml of distilled water and
then sonicated for 15min.

Mobile phase preparation: a filtered and degassed mixture of acetonitrile, methanol, and water
at ratio of (106:55:39) will be prepared (USP-38-NF, 2015).
Reagents USP standard Least Ratio Adjustment ratio Manufacturer/AVICO*
Acetonitrile/CH3CN 25
Methanol/CH3OH 10
Water/H2O -
0.4% Orthophosphoric 65
Acid
Acetic acid - - -

Table 2. Mobile Phase preparation i.e. for 1000 ml (250 ml CH3CN, 100 ml CH3OH and 650
ml .4% Orthophosphoric acid) * manufacturer working Certificate will be used.

Mobile phase calculation = number of injection * Running time * flow rate

As case example we need 409 ml of mobile phase for OTC injectable for single batch running

Standard preparation: 1mg/ml of USP oxytetracycline reference standard will be prepared by

taking two RS, as ref1 and ref2. 0.025g of OTC reference standard (RS) will be accurately
weighed and transfer to a 25ml volumetric flask and dissolve in a diluting solution and mix on a
vortex mixer to dissolve well. Then 10ml of this stock solution will be transferred in to another
100ml volumetric flask, and further dilute with distilled water to volume, and mix, and then
transfer to HPLC vials by using syringe filter.

Sample preparation: First, 2 vials of OTC will be added into one beaker and mix well. Then
Equivalent of 1mg/ml concentration of RS will be calculated depending on present of sample
API like 10%, 20%, then amount of sample taken will be diluted to half of volumetric flask and
sonicated for few minutes and after cooling to at room temperature the solution will be filled
with diluent to the volume and mixed well. Then 10ml from the stock solution will be taken and
added

27
in to 100ml of volumetric flask and diluted to the volume by distilled water and mixed well
again. Afterward, the assay preparation take in to a syringe, and pass through 0.45µm syringe
filter and fill in to ¾ of 1.5 ml HPLC vial, and ready for assay determination (USP, 2015).

Chromatographic system: The liquid chromatography system will be equipped with UV-Vis
spectrophotometer as a detector and we will use a 245 nm detection wave length and a 4.6mm by
25cm column that contains 5 particle size. According to the official pharmacopoeial method,
the flow rate is about 1.5 ml/min. The total chromatography run time will be adjusted to 13
minutes. The tailing factor should not be more than 2; the column efficiency should not be less
than 1000 theoretical plate; the relative standard deviation for replicate injection should not be
more than 2.0% (USP-38-NF, 2015).

Chromatographic system:

Set parameters USP standard Manufacturer/AVICO

Column 5 µm, 4.6 mm *25 cm 5 µm, 4.6 mm *25 cm/ C18
Detection UV 245-nm UV 245-nm
Flow rate 1.5 ml/min 1.2 ml/min
Column Temperature not indicated 35˚C
Injection Volume 20 µL 20 µL
Run Time not indicated 15 min

Table 3. Chromatographic condition for OTC API determination (USP, 2015), AVICO –
manufacturer Standard working Certificate will be used.

Procedure: Separately injecte equal volumes (about 10 µL) of the standard preparation and
assay preparation into the chromatograph, will record the chromatograms, and the responses for
the major peaks will be measured

Calculation: the % of label claim of OTC in the portion of injection will be taken by the formula
will be calculated as follow

28
 (Cs/Cu)(ru/ rs) *100

Where Cs - is the concentration, in mg per ml, of USP OTC RS in the Standard

Cu -is the concentration, in mg per ml, of in the assay preparation

ru -is the total peak response of obtained from assay preparations

rs -is the total peak response of obtained from standard preparation

Simply as ratio of peak area of sample to peak area of standard multiplied by potency of standard

Percentage Content (%) = Peak of area of Sample * 100

Peak area of standard
Percentage Content (%) =

Milligram Content (mg) =

According to official compendia of (USP-38-NF, 2015), OTC for injection contains an amount
of OTC Hydrochloride equivalent to not less than 90% and not more than 120% of labeled
amount of oxytetracycline ( C22H24N2O9).

According to official compendia of (USP-38-NF, 2015) OTC tablet should contains 90 – 110%
of the labeled amount of OTC while 95 – 105% of the labeled amount of OTC for injectable
preparations, but range is higher by manufacturers

3.7.3. Uniformity of weight test for tablets and filling to the volume for injectable preparations

The uniformity of mass between tablets of each brand will be evaluated against Pharmacopoeial
specification limit. For this purpose, twenty (20) tablets from a particular brand will be released
from the aluminum blister pack and weighed individually and collectively. The differences
between the masses of the individual tablets and the mean from the 20 tablets will be compared
each other. The percentage deviations will be also calculated. This method will be repeated for
the other brands and filling to the volume for injectable preparations by evaluations 10 vial from
each brand (USP-38-NF, 2015)
The term quality should be operationally defined.
You should bring methods how to carryout weight variation, content uniformity and filling to the
29
volume of drugs

30
[4.] STUDY PERIOD
[5.]
[6.] The entire work will be completed by the end of June, 2025, provided that all the required materials
and budgets allocated for the study is fulfilled as per the schedule.
[7.]
N Activities Ja Fe M A M J J A F M

1 Literature        
search

2 Proposal   
develop
ment

3 Sample      
collectio
n

4 Sample      
processin
g at
VDAF –
QAC
5 Data entry 

and
analysis

6 Paper write 

up

7 Submission √ 

of paper

Paper 

8 presentat
ion

31
 Take this out of method part
Sterility Test

Facilities used for sterility test; Autoclave, incubator, conical flask, balance, hot plate, test tubes,
aluminum foil, biosafety cabinet, forceps, measuring cylinders, media dispenser, spoon, test tube
rack,

Chemicals and reagents; Dehydrated thioglycollate media and Soyabean casein digest media,
Distilled water, Alcohol 70%.

Facilities used for Physicochemical quality test; High performance liquid chromatography
equipped with a UV-vis detector (SPD-20A/20AV, Shimadzu Corporation, Japan) used for
determination of API content in the study sample and also for identification. Fume hood, PH
meter, magnetic stirrer, Aluminum foil, analytical balance, different types of Volumetric flasks
(50ml, 100ml, 250 and 500ml); graduating cylinders, beakers, Duran bottles, pipette of different

whatman filter paper (90mm diameter), HPLC vials of 1.5 ml capacity and column (250mm X
4.6mm column packed with ODS silica gel).

According to USP 2015, Sterility test will be applied to substances or preparations which, according
to the Pharmacopoeia, are required to be sterile. However, a satisfactory result only indicates that
no contaminating microorganism will be found in the sample examined in the conditions of the
test.

Precautions against microbial contamination: The test for sterility will be carried out under aseptic
conditions. In order to achieve such conditions, the test environment has to be adapted to the way
in which the sterility test will be performed. The precautions take to avoid contamination are
such that they do not affect any microorganisms which are to be reveal in the test. The working
conditions in which the tests are perform are monitor regularly by appropriate sampling of the
working area and by carrying out appropriate controls

32
Culture media and incubation temperatures: The following culture media will be found to be
suitable for the test for sterility. Fluid thioglycollate medium is primarily intend for the culture of
anaerobic bacteria; however, it will also detect aerobic bacteria. Soya-bean casein digest medium
is suitable for the culture of both fungi and aerobic bacteria. Fluid thioglycollate medium is to be
incubated at 30–35 °C. Soya-bean casein digest medium is to be incubated at 20–25 °C.

Direct inoculation Method. Transfer the quantity of the preparation to be examined directly into
the culture medium so that the volume of the product is not more than 10% of the volume of the
medium, unless otherwise prescribed.

If the product to be examined has antimicrobial activity, carry out the test after neutralizing this
with a suitable neutralizing substance or by dilution in a sufficient quantity of culture medium.
Media used for dilution will be Soya-bean casein digest medium. Incubate the inoculated media
for not less than 14 days. If the material being tested renders the medium turbid so that the
presence or absence of microbial growth cannot be readily determined by visual examination, 14
days after the beginning of incubation transfer portions (each not less than 1 ml) of the medium
to fresh vessels of the same medium and then incubate the original and transfer vessels for not
less than 4 days.

If no evidence of microbial growth is found, the product to be examined complies with the test
for sterility. If evidence of microbial growth is found the product to be examined does not
comply with the test for sterility, unless it can be clearly demonstrated that the test will be invalid
for causes unrelated to the product to be examined. Therefore the principle behind sterility test
by direct inoculation method is if microorganisms are placed in a medium which needs nutritive
materials and water and kept at favourable temperature, the organism will grow and their growth
can be indicated by turbidity in the original media (USP, 2015).

33
3.8. Data management and analysis

The data will be checked, coded and entered in to Microsoft excel work sheet and will be
analyzed using descriptive statistics like percentage, mean (average of content) and RSD to
compare API of standard with sample ( assay) of different classes, brands, batches and
Preparations of OTC.

34
4.[8.] STUDY PERIOD

The entire work will be completed by the end of June, 2025, provided that all the required
materials and budgets allocated for the study is fulfilled as per the schedule.

No Activities Jan Feb Mar Apr May Jun Jul Aug Fe Mar
b

1 Literature search        

2 Proposal   
development

3 Sample collection      

4 Sample processing      
at VDAF – QAC

5 Data entry and 

analysis

6 Paper write up 

7 Submission of √ 
paper

Paper presentation 

35
 5.[9.] BUDGET BREAK DOWN

1: Cost for transport, food, bed and parking during sample collection from study area and processing
in Addis Ababa using high tech HPLC at APIQTC
Travels Unit of Cost/unit Food & Total cost Reason of transport
travel travel Bed or parking (ETB)
(ETB) cost/travel
Addis Ababa to 5 trip 500 ≥22,500 27,500 Sample processing
worabe & vice versa Using high tech HPLC
Worabe to Silti and 5 trip 50 500 3,000 Sample collection
vice versa
Worabe to Alicho and 5 trip 100 3000 4,000 Sample collection
vice versa
Worabe to Sankura 5 trip 100 3000 4,000 Sample collection
and vice versa
Worabe to Merab 5 trip 200 3000 5,000 Sample collection
Azernte and vice versa
Worabe to Misrak 5 trip 150 2500 4,000 Sample collection
Azernte and vice
versa
Worabe to Lanforo 5 trip 100 3000 4,000 Sample collection
and vice versa
Worabe to Meto and 5 trip 70 700 1,400 Sample collection
vice versa
Worabe to Dalocha 5 trip 50 500 1,000 Sample collection
and vice versa
Within Worabe town 5 trip 100 500 1,000 Sample collection

Subtotal = 54,900 ETB

2: Cost of material used during sampling and analysis
Video-camera Number 1 Smart android Smart android Take necessary
phone phone images of
substandard drugs
Smart android phone Pcs 1 18000 18000 To check registration
or mini tablet status by
international
numbering system
and VDFAQAC

36
 Subtotal = 0 20,000 ETB

3: Data entering, analysis and stationery cost

Pen Dozen 2 600
Pencil Dozen 3 240
Marker with different Pcs 10 300 Labeling the
Provided
color sample
by University
Ruler Pcs 5 150
A4-size paper Pack 7 800 Provided Displaying the
by University HPLC Result
Note-Book 1 1 500
Flash disc 16 GB 3 500 Storage of
Chromatographic
figures
Photocopy
Print 1000
CD-RW Number 5 200 Provided by For taking data
APIQTC as from server for
support traceability **
Internet service 5 hr/day 5 day/week 30 cents/min 19,800
expense =20 =18
day/month ETB/hr=90ET
=220 day/ B/ day(5hr)
11 month
Binding and PCS 20 50 1000
laminating
Subtotal = 20,800 ETB
4: Costs of reagents and chemicals that used for API measurement
5 and 10 CC Pack 30 150 4500 For sample transfer to
Disposable syringe vial that can load to
HPLC
Glove and mouth mask Pack 10 each 180 1800 PPE – personal
protective equipment
Acetonitrile/CH3CN Bottle
Methanol/CH3OH Bottle
Acetic acid Bottle
Standard Bottle
Subtotal = 0 ETB Subtotal = 25,090 ETB
4: Costs of reagents and chemicals that used for API measurement

37
Acetonitrile/CH3CN Bottle Provided by APIQTC as support

Fee given
for

38
 APIQTC
as support
Data per-diem 30 383*2 22,980
Entering cost
Data per-diem 30 383*2 22,980
Analyzing cost
Fee for Duty 3*6060 724480 130,320
DQA 86,400
Fee for Duty 2*60 383200 45,9602
sample 4,000
collectors
Subtotal = 222,240156,360
Total 256,350
10% contingency 25,635
Grand Total 281,985
TOTAL BUDGET = 54, ,900 + 20,000 + 25,090 + 156,360 = >281,985 ETB

Table 2: Summary of Budget break down

S.N Category of Expense Sub Total

01 Cost for transport, food, bed and parking during sample collection from study 54,900
area and processing in Addis Ababa using high tech HPLC at APIQTC
02 Cost of material used during sampling and analysis 20,000
03 Data entering, analysis and stationery cost 25,090
04 Costs of reagents and chemicals that used for API measurement
05 Allowance / per-diem cost 156,360
Total 256,350
10% contingency 25,635
Grand Total 281,985

39
6.[10.] ETHICAL CONSIDERATION

Informed consent will be obtained from all participants, and their privacy and confidentiality will
be ensured throughout the study.

40
7.[11.] BIBLOGRAPHY

Ajibola, & Francis. (1993). Experimental Pharmaceutical Chemistry. Shaneson TI Ltd.

Arzai, & A.H. (2008). Evaluation of microbiological purity of some brands of tetracycline sold
in kano state, Nigeria. Bayero Journal of Pure and Applied Sciences,, 1(1), p.104 – 107.
Ashbolt, N. J. (2004). Microbial contamination of drinking water and disease outcomes in
developing regions. Toxicology 198(1), pP 229–238. https://doi.org/doi:
10.1016/j.tox.2004.01.030
Beyene, T., Gurmu, F., Feyisa, A., Henok., H., Tariku, J., Ayana, D., Tadesse, F., H/mariam, E.,
Desisa, F., Stegeman, & J.A. (2018). Veterinary Medicinal Product Usage among food
animal producers and its health implications in central Ethiopia. BMC veterinary
research, p.14:409.
BP. (2005). British Pharmacopoeia (BP) CD soft ware, system simulation limited, monograph
on Albendazole. Appendix XVII G. Friability of Uncoated Tablets. .
Caneschi, A., B., A., B, A., & Z, A. (2023). . The Use of Antibiotics and Antimicrobial
Resistance in Veterinary Medicine, a Complex Phenomenon: A Narrative
Review. Antibiotics Basel(12 (3)), 487.
https://doi.org/10.3390/antibiotics12030487

Cartwright, R., & Baric, A. (2018). The rise of counterfeit pharmaceuticals in Africa (06), pP 1-
16. https://enact-africa.s3.amazonaws.com/site/uploads/2018-11-12-counterfeit-
medicines-policy-brief.pdf
Chopra, I., Roberts, M., & 2001. (2001). Tetracycline antibiotics: mode of action, applications,
molecular biology, and epidemiology of bacterial resistance. Microbiology and
Molecular Biology Reviews, 65(2), p.232-260.
CSA, C. S. A. (2021). Original data from https://www.statsethiopia.gov.et/our-survey-reports/.
Retrieved from https://public.knoema.com/pbwfnlf/livestock-statistics-of-ethiopia.
Dowling, P. M., & Russell, A. M. (2000). Pharmacokinetics of a longacting oxytetracycline-
polyethylene glycol formulation in horses

41
J.vet. Pharmacol. Therap, 23, pP 107-110.
Fagbamila, I., Kabir, J., P, A., Omeiza, G., Ankeli, P., Ngulukun, S., Muhammad, M., Umoh, J.,
& A. (2010). Antimicrobial screening of commercial eggs and determination of
tetracycline residue using two microbiological methods. Int. J. Poultry Sci, 9 (10):

, pP 959-962.
FAO. (2004). Guidelines of resistance management and integrated parasite control in
ruminants Animal Production and Health Division, Food and Agriculture Organization
of the United Nations Rome, .
Gaskins, R., H., Collier, C., C., Anderson, & B., D. (2002). Antibiotics as growth promotants:
mode of action. Animal Biotechnology, 13, p.29–42.
Gerber, F., Krummen, M., Potgeter, H., Roth, A., Siffrin, C., Spoendlin, & C. (2004). Practical
aspects of fast reversed-phase high-performance liquid chromatography using 3μm
particle packed columns and monolithic columns in pharmaceutical development and
production working under current good manufacturing practice. Journal of
Chromatography A, 1036(2), p.127–133.
https://doi.org/doi:10.1016/j.chroma.2004.02.056
Giguère, S., Prescott, J.F., Baggot, D., J., Walker, R.D., Dowling, & P.M. (2006).
Antimicrobial Therapy in Veterinary Medicine (4th edition. ed.). Blackwell Publishing.
Hebron, Y., Tetteyy, J.A., Pournamdari, M, Watson, & D.G. (2005). The chemical and
pharmaceutical equivalence of sulphadoxine/ pyrimethamine tablets sold on the
Tanzanian market, p.575-581. J. Clin. Pharm. Ther, 30, pP 575-581.
Jeong, J., Song, W., Cooper, W.J., Jung, J., Greaves, & J. (2010). Degradation of tetracycline
antibiotics: Mechanisms and kinetic studies for advanced oxidation/reduction processes.
Chemosphere, 78, p. 533-540.
Kahsay, G., & G/Egziabher A. (2010). Quality Assessment of the Commonly Prescribed
Antimicrobial Drug, Ciprofloxacin Tablet, Marketed in Tigray, Ethiopia. 2 (1), p. 93-107.
https://doi.org/10.4314/mejs.v2i1.49656
Karungamye, P. (2023). Counterfeit and substandard drugs in Tanzania: A review.
Forensic Science International Reports, 7.
https://doi.org/10.1016/j.fsir.2022.100302
Kirkland, & J.J. (2004). Development of some stationary phase for reversed-phase PHLC.

42
Journal of Chromatography A,, 1060(1-2), p.9-21.

43
Kirkup, B., C., & Jr. (2006). Bacteriocins as oral and gastrointestinal antibiotics, theoretical
considerations, applied research, and practical applications. Current Med. Chem, 13(27),
p. 3335-3350.
Korchi, E., G.I., Prats, C., Arboix, M., & B., P. (2000). Disposition of oxytetracycline in
pigs after i.m. administration of two long-acting formulations. Journal of Veterinary
Pharmacology Therapy. 24, pP. 247-250.
Okeke, I.N., Aboderin, O.A., Byarugaba, D.K., OJO, K.K., & J.A., O. (2007). Growing Problem
of Multidrug-Resistant Enteric Pathogens in Africa. Emerging Infectious Diseases,
13(11): , 1640-1646.
Okine, N.N.A., Ayim, J.S.K, & Kwakye S.K. (1999). Analysis of Albendazole by
HPLC method, West African Journal of Pharmacopeia, . West African Journal of
Pharmacopeia, 13(1), p. 41-44. .
Okonko, I.O, Soleye, F.A., Amusan, T.A., Ogun, A.A., Ogunnusi, T.A., & Ejembi. (2009).
Incidence of Multi-Drug Resistance (MDR) Organisms in Abeokuta,
Southwestern, Nigeria. Global J. Pharmacol., 3(2), pp 69-80

Owusuwaa. (2016). Quality assessment of veterinary pharmaceuticals: A case of oxtetracycline

and praziquantel. Msc Thesis, kwame nkrumah university of science and Technology,
College of health science kumasi, Ghana.
Ozawa, S., Chen, H.-H., Lee, Y.-F. A., Higgins, C. R., & Yemeke, T. T. (2022). Characterizing
Medicine Quality by Active Pharmaceutical Ingredient Levels: A Systematic Review
and Meta-Analysis across Low- and Middle-Income Countries. Am J Trop Med Hyg.,
106(6), pP 1778–1790. https://doi.org/doi: 10.4269/ajtmh.21-1123
Palma, E., Tilocca, B., & Paola Roncada, M. d. i. (2020). Antimicrobial Resistance in Veterinary
Medicine: An Overview. Int J Mol Sci., 21(6). https://doi.org/doi: 10.3390/ijms21061914
Papadoyannis, I. N., V.F., S., & L.A., K. (1999). A rapid high-performance liquid
chromatographic (HPLC) assay for the determination of oxytetracycline in commercial
pharmaceuticals. Journal of Pharmaceutical and Biomedical Analysis, 23(2000), p. 275–
280.
Pierre-Louis Lezotre MS, P. (2014). International Cooperation, Convergence and Harmonization
of Pharmaceutical Regulations

44
A Global Perspective. Elsevier,science direct pP 7-170. https://doi.org/doi: 10.1016/B978-0-12-
800053-3.00002-1
RAPS. (2017). WHO 1 in 10 medicine in developing countries is substandard or fake
http:/www.raps.org/Regulatory focus/News
Salam, M. A., Al-Amin, M. Y., Salam, M. T., Pawar, J. S., Akhter, N., Rabaan, A. A., &
Alqumber, M. A. A. (2023). Antimicrobial Resistance: A Growing Serious Threat for
Global Public Health. 11(13), pP.1946. https://doi.org/doi: 10.3390/healthcare11131946.
Saleha, T., Syed, F., Askari, & R.Ummar, A. (2018). Tetracycline: Classification, Structure
Activity Relationship and Mechanism of Action as a Theranostic Agent for Infectious
Lesions-A Mini Review. Biomedical Journal of Scientific & Technical Research, 7(2).
Sarmah, A.K., Meyer, M.T., Boxall, & A.B.A. (2006). global perspective on the use, sales,
exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the
environment. Chemosphere, 65, p.725-759.
SZPCASA. (2022). Annual Statistical Abstract 2022/23; socio-economic data of siltie zone
Thahera, P.D., Latha, A.K., Shailaja, T., Nyamathulla, S., Uhumwangho, & M.U. (2012).
Formulation and evaluation of Norfloxacin gastro retentive drug delivery systems using
natural polymers. International Current Pharmaceutical Journal, , 1(7), p.155-164.
Theodore, I.K., P.I., Rafailidis, & M.E. (2007). Counterfeit or substandard antimicrobial drugs: a
review of the scientific evidence. Journal of Antimicrobial Chemotherapy, 60, p.214 –
236. DOI: 10.1093/jac/dkm109
Thrusfield. (2007). Veterinary Epidemiology. (3rd ed ed.). Blackwell Science Ltd, a Blackwell
publishing company. http://librodigital.sangregorio.edu.ec/librosusgp/28347.pdf
Troy, S., M., Rose, J. B., M., T., Jenkins, Farrah, S. R., & 2002., J. L. (2002). Microbial source
tracking: current methodology and future directions. . Appl. Environ. Microbiol., 68,
5796–5803.
USP-38-NF. (2015). United state pharmacopeia (USP). National formulary USP
official Monograph. twin book parkway.
USP-NF. (2023). The United States Pharmacopeial Convention

12601 Twinbrook Parkway

Rockville, . https://www.uspnf.com/official-text/proposal-statuscommentary/uspnf-2023

45
USP. (2000). The United States Pharmacopoeia (USP) 24 Revisions, the National
Formulary, United States Pharamacopeial Convention.
USP. (2007). United state pharmacopeia (USP) Pharmacists’ Pharmacopeia 2dn edt the unite
state pharmacopeial convention. 12601 Twinbrook Parkway, Rockville, MD 20852.
USP. (2015). United state pharmacopeia (USP). National formulary USP official Monograph,
USP 38 NF 33 1, Pp.221-290.the United States pharmacopoeial convention. 12601 twin
book parkway, Rockville, md 20852. .
WHO. (2018). Quality Assurance of pharmaceuticals.
Zeleke, M., & Harris-Coble, L. (2021). Ethiopia’s Livestock Systems: Overview and Areas
of Inquiry. Gainesville, FL,.
Zhao-jun, L., Wei-ning, Q., Yao, F., Yuan-wang, L., Shehata, E., & Jian, L. (2019).
Degradation mechanisms of oxytetracycline in the environment. Journal of Integrative
Agriculture, 18(9), pP 1953-1960. https://doi.org/DOI:10.1016/S2095-3119(18)62121-5

Annex

Annex: HPLC with computer system that display activities and results used in the research

46
Figure 7: HPLC series with computer system that display activities and results will be used
in the research

47
 CV of the PI

PERSONAL INFORMATION Dr. Numan Jemal Amdino

University of Gondar
Place of Birth: Ethiopia
Sex: Male | Date of birth: 13/06/1993 | Nationality :
Ethiopia
Marital Status: Single
+251928762642

[email protected]

WORK EXPERIENCE
8/10/2018 – 08/09/2019: As Instructor as Lecturer and researcher of Veterinary Science
College of Agriculture and Natural Resource in the department of Veterinary
Science at Assosa University, Assosa, Ethiopia
PO BOX:18; Web site: http://www.asu.edu.et/en
Teaching: Veterinary Physiology. Veterinary Anatomy, Veterinary
Pharmacology, Clinical Pathology and Ethnology and Animal Welfare and
clinical Practice courses
09/09/2019 - Present As Instructor as Lecturer and researcher of Animal Science
College of Agriculture and Natural Resource in the department of Animal
Science at Werabe University, Worabe, Ethiopia
PO BOX: 46; Web site: http://www..wru.edu.et/en
Main Responsibilities
 Teaching : Animal Behaviour and Welfare, Animal Health
and Disease Control
 Senior seminar coordinator
 Conducting research on veterinary drug quality
assessment circulating in and around siltie zone, South
Ethiopia
 Serving Community on Clinical service

EDUCATION

48
01/ DOCTOR IN VETERINARY MEDICINE (DVM)
09/ University of Gondar, Gondar, Ethiopia
20 PO BOX: 46; Web site http://www.uog.edu.et
:

49
12-
28/ Seminar: Organic Livestock production: challenges and opportunities
06/ DVM thesis: Drug Quality Assessment on Ivermectin Preparations Circulating in
20
18 Addis Ababa Pharmaceutical Market

08/10/2018-08/12/21019 Induction training for Teacher Educators from Assosa Univeristy

Principal Subject:
 Reflective Teacher Educator
 Managing Learning in class
 Action Research in Organization
TRAINING/Certificate/Diploma
July 28, 2018 Infectious disease and Clinical pharmacy in the Ethiopian Public
Health institute – by the Ohio state university guests.

Oct, 2018 Induction training – on instructional planning, harmonized curriculum

and modular approach

July 23-July 27 2018 -International trade and Public Health training

Sep,09/ 2020 Agricultural Biotechnology, Hungary

MOTHER TONGUE(S) Amharic

OTHER LANGUAGES UNDERSTANDING SPEAKING WRITING
SPOKEN
SPOKEN
LISTENING READING PRODUCTIO
INTERACTION
N

ENGLISH
EXCELLENT EXCELLENT EXLEENT EXCELLENT Excellent
ARABIC
EXCELLENT EXCELLENT EXCELLENT GOOD Good

COMMUNICATION SKILLS Very good communication skills gained while working my thesis and externship, in
Addis Ababa & Gondar University, and also through Training in various institution
in
Addis Abba

50
ORGANI  Research and Community service committee chairperson in the department of Veterinary
ZATIONA
L/MANA Science (From Feb 08/2019- Sep 8/2019)-,Assosa University Good Leadership and
GERIAL communication -by organizing training & doing department tasks,
SKILLS
 Very good knowledge and skill of veterinary pharmacology, Veterinary Physiology.
Veterinary Anatomy, Clinical Pathology and Ethnology and Animal Welfare and clinical
Practice courses
 Very good skill in data management & data analysis with R software, SPSS & STATA
 Extremely motivated to do scientific work
 Very good skill at running drug quality assessment computer programs such as LC
solution gained through my stay in veterinary drug and animal feed quality assessment
center, Addis Ababa. Advanced browsing skill

Recognition and award

1. Certificate of recognition for excellent Academic staffs performance evaluation in the Assosa
University during 1st semester of 2011 E.C

2. Certificate of appreciation for delivery of tutorial program for female students during 1 st semester of
2011 E.C from Asossa University, Ethiopia

3. One Among top scorer students in the DVM graduation class of 2010 E.C at University of Gondar
with excellent cGPA 3.87 with rank of VGD

4. Academic Excellency award in 2008 E.C from University of Gondar from UoG Academic
V/President (Certificate + Necklace made from silver 18 g)

5. Academic Excellency award in 2007 E.C from University of Gondar from UoG President (Certificate
+ UoG logo)

Publications
1. Product quality evaluation of marketed veterinary Ivermectin formulations in Addis Abeba
Volume 10, Issue 4, December 2023, Pages 156-163. 10.48307/IAHSJ.2023.409755.1036

REFERENCES

51
 1. Dr. Elias Kebede (DVM, MSc, Assistant Professor and Department head): College of Veterinary
Medicine and Animal Sciences - at University of Gondar, Ethiopia.
Email:[email protected]/[email protected], Phone: +251912882236.
2. Professor Mersha Chanie (Professor of Veterinary Pathology): College of Veterinary Medicine and
Animal Sciences- at University of Gondar, Ethiopia. Email: [email protected] – now v/p of
research and community service in the university of Gondar

Yours faithfully,

Numan Jemal (DVM)

52
53
54
47
48
49
50
51
 CV of the Co-In 1

Name Redwan Anwar Mohammed

Date of Birth & Date June, 1993
Sex Male
Nationality Ethiopian
Address
Email [email protected] / [email protected]
Phone +251926042372 / +251904839986
Academic Qualification DVM from Hawassa University (2009-2014)
MSc in Veterinary Epidemiology from Hawassa University (2021-
2023)
MSc Thesis Qualification Grade ‘Verygood’
Skills Familiar with Some Softwares (installing and working with)
Example; SPSS, STATA and R for MS Excel, MS Word, and MS
PowerPoint
Volunteer Service I am ambitious to serve the community in the coming years
Language Speaking Reading Writing Listening
English Very good Excellent Excellent Very good
Amharic Excellent Excellent Excellent Excellent
Siltigna Excellent Very good Very good Excellent

Hobbies and Interests

 Reading books, Playing with my friends, Browsing internet, Public service
and volunteer works, and Visiting different places

52
Publication
Journal, year, vol.,
No Title vs author/s
page no.
1 Prevalence and Associated Risk Factors of Tick Infestation Int. J. Adv. Res.
On Cattle In and Around Jimma Town, South Western Biol. Sci., 2020,
Oromia, Ethiopia 7(6): 20-33.
Hussen Aliye , Redwan Anwar
2 Major Constraints of Veterinary Service Delivery in Global Veterinaria,
Ethiopia: A Review. 2022, 24 (3): 147-
Redwan Anwar Mohammed 152.
3 Ethno-Veterinary Use of Medicinal Plants in the Selected Journal of Animal
Districts of Siltie Zone, Southern Ethiopia. Health, 2023,
Ufaysa Gensa, Dureti Ensarmo and Redwan Anwar 3(2):1-16.

53
54