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CHAPTER TWO: LITERATURE REVIEW

2.1: Poultry Production

Poultry production is one of the most important sectors in the agricultural setup of Uganda because it contributes to improve human nutrition and food security by being a leading source of high-quality protein in the form of eggs and meat (FAO, 2020). It acts as a key supplement to revenue from crops and other livestock enterprises, thus avoiding over-dependency on traditional commodities with inconsistent prices. It has a high potential to generate foreign exchange earnings through the export of poultry products to neighbouring countries (MAAIF, 2019).

Poultry is also highly prized in many social-cultural functions such as dowry and festivities. The poultry industry in Uganda is relatively new but the increasing investments in the livestock sector during the last two decades have led to a dramatic increase in the number of exotic chickens such as layers and broilers. However, the major problem is associated with further expansion of the poultry production due to the increasing market base which has led to usage of antibiotics for growth promotion and fighting diseases to meet with the demand and supply locally and internationally (Clarke. B, 2004; Gerber et al., 2007).

2.2: Antibiotics

2.2.1: Definition of Antibiotics

Antibiotics are chemical compounds that kill or inhibit the growth of microorganisms but cause little or no damage to the host,(Guardabassi, 2008).They are naturally produced by microorganisms such as fungi (e.g., penicillin) and bacteria (e.g., tetracycline) or can be semi-synthetically produced (e.g., amoxicillin) or totally synthetically produced (e.g., sulfonamides), (Guardabassi, 2008).

The term of antibiotic refers to a wide range of chemical substances derives from a natural, semisynthetic or synthetic way that affect antibacterial activity, by killing or inhibiting the growth of bacterial pathogens. (Mara C, 2001). Antibiotics have been used in poultry farming in large quantities since the 1940s. Since then, the antibiotics have been used on poultry in large quantities to enhance production in poultry. A wide range of antibiotics are used in poultry not only to treat diseases but also to maintain health, promote growth and enhance feed efficiency (Mara C, 2001).

They are widely used in the prevention and treatment of infectious diseases. Nowadays Antibiotics are widely prescribed for both therapeutic and prophylactic aims against microbial infections and as a growth promoting substances in animal and poultry farms. (Mara C, 2001). The incorrect use of these drugs in veterinary field can lead to the existence of residues in animal derived foods that affect human health (Mara C, 2001).

2.2.2: Classification of Antibiotics

According to Wang, 2012 Antibiotics can be classified in the following ways; Broad spectrum antibiotics where Broad-Spectrum Antibiotics are classes of antibiotics that work against an extensive and wide range of disease-causing bacteria and they target both gram positive and negative bacterial groups and they are often grouped by their abilities to act upon the different bacterial groups, examples include; Doxycycline, Minocycline, Aminoglycosides, Ampicillin, Amoxillin to mention (Wang, 2012). but a few while Narrow spectrum antibiotics are active against a selected group of bacterial types and they can act on either gram positive or negative but not both thus used for the specific infection which when causative organisms are known and examples include; Azithromycin, Clarithromycin, Erythromycin and Clindamycin.

2.2.3 Mode of action of Antibiotics

This involves the disruption of essential processes or structures in the bacterial cell and this is either through killing the bacterium or slowing down bacterial growth thus antibiotics can be bactericidal or bacteriostatic and this is achieved through Inhibition of cell wall synthesis, inhibition of protein synthesis, inhibition of nucleic acid synthesis, inhibition of the synthesis of essential metabolites and injuring the plasma membrane (Wang, 2012).

2.2.4: Common Antibiotics used in Poultry

A wide range of antibiotics are used in poultry not only to treat disease but also to maintain health, promote growth and enhance feed efficiency (Gaudin, 2004). Antibiotic usage has facilitated the efficient production of poultry, allowing the consumer to purchase, at a reasonable cost, high quality meat and eggs (Donoghue, 2003). Broiler chicken is often grown actively with antibiotics to attain maximum weight within a short period of time (Nonga, 2009).

In Uganda penicillin are among the most widely used group of antibiotics (Mitema, 2001). The uncontrolled and unlimited use of these antibiotics may however lead to the accumulation of undesirable residues in the animals treated and their products (Mitema, 2001).

Drug residues in the edible portions of the animal usually occur because the withdrawal period has not been observed (Mitema, 2001). Benzylpenicillin (penicillin G) is widely used in Kenya to treat specific infections and as a prophylactic (Lee, et al. 2001). It is administered as one or more of a variety of salts which are used to prolong the activity of the drug. These can be the soluble sodium or potassium salts or the longer acting procaine and benzathine salts (Lee, et al. 2001). Concern has been expressed over the possible presence of residues of this drug in foods of animal origin due to the occurrence of penicillin hypersensitivity in humans and development and transfer of antibiotic resistance between animals and man (Mitema, et al. 2001).

2.2.4.1: Tetracyclines

The tetracyclines were discovered in the 1940s, they are a family of antibiotics that inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the Ribosomal acceptor (A) site (Chopra, 2001).  Tetracycline consists of a common four-ring structure to which a variety of side chains are attached (Prescott, 2002). Chlortetracycline and oxytetracycline were the first members of the tetracycline group to be described (Michalova, 2004). Subsequently, a number of important semisynthetic tetracyclines were developed, e.g., doxycycline and minocycline (Michalova, 2004).

Tetracyclines are the most commonly prescribed antibiotics; they have played an important role in veterinary medicine because of their broad-spectrum activity and low cost, tetracyclines (TCs) including tetracycline (TC), Oxytetracycline (OTC), Chlortetracycline (CTC) and doxycycline (DC) are widely used in animals for both prevention, treatment and as feed additives to promote growth. (Mesgariabbasi, 2011).

The widespread utilization of TCs leads to an increasing resistance factor, so accurate monitoring by public health agencies is required (H. Matsumoto, 2000). Three different tetracycline resistance mechanisms have been described; active efflux of the antimicrobial, ribosomal protection and enzymatic inactivation of the drug. All these mechanisms are based on the acquisition of one or several tetracycline resistance determinants, which are widely distributed among bacterial genera. Additionally, mutations in the rRNA, multidrug transporter systems or permeability barriers may be involved in the resistance to several antimicrobials including tetracyclines (Michalova, 2004).

Figure 2.1: Chemical Structure of Tetracyclines

2.2.4.2 β-lactams

The β-lactam group is one of the most important families of antibiotics used in veterinary medicine and has been widely used for decades in animal husbandry (Konieczna, 2007). This group consists of penicillin and cephalosporins. The most common members of the penicillin used in veterinary practice are benzyl penicillin, amoxicillin, ampicillin and penicillin G (Kowalski, 2007). The extensive use of penicillin may cause the presence of their residues in food products of animal origin and may have side effects to consumers (Kowalski, & Konieczna, 2007). Moreover, penicillin residues in food products may be responsible of allergic reactions in humans and promote the occurrence of antimicrobials resistant bacteria (Kowalski, &Konieczna, 2007).

The cephalosporins are chemically related to the penicillin and both share the β-lactam ring structure (Woodward, 2009).  A number of cephalosporins, including cefalexin, cefuroxime, ceftiofur, cefquinome and cefotaxime are used in veterinary medicine in food animals. Due to increased emergence of cephalosporin resistant bacteria (especially E. coli and Salmonella) the FDA prohibited the usage of cephalosporins in food producing animals including poultry (Schmidt, 2012).

Figure 2.2: Chemical structure of β-lactams

2.2.4.3 Macrolides

Macrolides constitute a very important class of antibacterial compounds widely used in veterinary medicine to treat respiratory diseases (Stolker & Brinkman, 2005). These antibiotics are molecules with a central lactone ring bearing 12 or 16 atoms to which several amino and/or neutral sugars are bound (Stolker & Brinkman, 2005). The antibiotic action of macrolides is through the inhibition of protein synthesis by binding to the 50S ribosomal subunit of prokaryote organisms (Riviere, 2009).

Resistance to macrolides is usually plasmid-mediated, but modification of ribosomes may occur through chromosomal mutation, resistance can occur either by decreasing entry into bacteria, synthesis of bacterial enzymes that hydrolyze the drug or modification of the target (ribosome) (Riviere, 2009).

Figure 2.3:Chemical structure of Macrolides

2.2.4.4 Aminoglycosides

Aminoglycosides are a large class of antibiotics that are characterized by two or more amino sugars linked by glycosidic bonds to an aminocyclitol component (Mingeot,1999). Aminoglycosides are broad-spectrum antibiotics and act primarily by impairing bacterial protein synthesis through binding to prokaryotic ribosomes (Mingeot-Leclercq, 1999). In veterinary medicine and animal husbandry, aminoglycosides are widely used in the treatment of bacterial infections, and have been added to feeds for prophylaxis and for growth promotion. Those most commonly used are gentamicin, neomycin, streptomycin and dihydrostreptomycin (Stead, 2000).

Figure 2.4: Chemical structure of Aminoglycosides

2.3: Administration of Antibiotics in Poultry

As in mammals, medications may be given to poultry by a variety of routes, but if individual birds may be treated (i.e., individual injection or by oral gavage), it is more effective to treat entire groups by mass application to the whole flock by drinking water (main way of administration) or feed (Ramatla et al., 2017).

2.3.1: Antibiotics Resistance

Due to the excessive and inappropriate use of antibiotics, there has been a gradual emergence of populations of antibiotics–resistant bacteria, which pose a global public health problem (Levy & Marshall, 2004) A resistant microbe is one which is not killed by an antimicrobial agent after a standard course of treatment (Levy & Marshall, 2004). Antibiotics used to combat infection forces bacteria to either adapt or die irrespective of the dosage or time span (Ahmed, 2012).  The surviving bacteria carry the drug resistance gene, which can then be transferred either within the species/genus or to other unrelated species (Ahmed, 2012).   Clinical resistance is a complex phenomenon and its manifestation is dependent on the type of bacterium, the site of infection, distribution of antimicrobials in the body, concentration of the antibiotics at the site of infection and the immune status of the patient (Ahmed, 2012).

2.4: Chicken Diseases Treated by Antibiotics

Under intensive farming methods, a broiler chicken lives a minimum of six weeks before slaughter. Free-range chickens are usually slaughtered at 8 weeks and organic at around 12 weeks (Kaneene, et al. 1997). To maintain this intensive production schedule, antibiotics are being excessively used in poultry farms for various purposes; such as prevention and control of disease (Miller, et al. 1997). Tetracycline (TC), Penicillin, lactams and Enrofloxacin are the most commonly used antibiotics in poultry especially broilers for prevention and treatment of various diseases (Miller, et al. 1997)   It is used for the treatment of air sacculitis (air-sac disease, chronic respiratory disease) caused by Mycoplasma gallisepticum and Escherichia coli; fowl cholera caused by Pasteurella multocida; infectious sinusitis caused by Mycoplasma gallisepticum; and infectious synovitis caused by Mycoplasma synoviae (Kaneene, et al. 1997). This intensive use of antibiotics poses a threat of chemical residues remaining in chicken products like meat and eggs (Kaneene, et al. 1997).   A wide range of antibiotics are used in poultry not only to treat diseases but also to maintain health, promote growth and enhance feed efficiency (Kaneene,& Miller, 1997)

2.5: Antibiotic Residues in Poultry Meat.

The term “residues” is used to describe all active principles and their metabolites, which persist in meats or other food products from animals that have been treated with the drug in question (Doyle, 2006).

The term metabolite has not been defined it is generally accepted that it applies to any by-product of biotransformation of the initial active principle (Policy Guide, 2009).

Failure to observe the instructions for antibiotic use can lead to antibiotic residues entering chicken derived foods (Doyle, 2006 & Policy Guide, 2009).

Improper maintenance of treatment records or a failure to identify treated animals adequately may lead to their omission of these animals (Stobberingh, 2000).   This approach is problematic as these feed additives are usually used without prescription and for very long periods, in both large and small doses, which leads to drug residues entering chicken derived food (Bogaard & Stobberingh, 2000).

It is a common practice among poultry farmers to treat entire groups of birds despite there being only a few affected individuals (Doyle, 2006).  Such practices unintentionally and unnecessarily expose healthy individuals to antibiotics (Baker & Leyland, 1983). Additionally, many farmers use sub-therapeutic doses of antibiotics to prevent diseases and this of course will lead to antibiotic residues entering the human food chain (Doyle, 2006).  Moreover, antibiotics are prescribed inappropriately in cases of viral infection, which do not respond to such drugs. In most cases, however, only young growing animals and poultry are responsive to antibiotic-mediated health maintenance (Doyle, 2006).  And some cases there is overdose of these antibiotics administered to the chicken hence the excess doze ends up accumulating in the tissues longer the expected period as per the prescription. This leads to persistence of such residues in chicken products (Doyle, 2006).

2.5.1: Effects of Antibiotic Residues to Poultry

A number of possible adverse health effects of veterinary drug residues have been suggested (Stobberingh, 2000). These may include but not limited to the following; allergic or toxic reactions to residues, chronic toxic effects occurring with prolonged exposure to low levels of antimicrobials, development of antimicrobial-resistant bacteria in treated animals and these bacteria might then cause difficult-totreat human infections, disruption of normal human microbiota in the intestine (Bogaard & Stobberingh, 2000). The bacteria that usually live in the intestine act as a barrier to prevent incoming pathogenic bacteria from getting established and causing disease (Doyle, 2006). Antimicrobials might reduce total numbers of these bacteria or selectively kill some important species (Doyle, 2006).

Occurrence of antibiotic residues in animal-originated foodstuffs could pose consumers health risk (Heshmati, 2015).  Antibiotic residues in food are potential threat to direct toxicity in human and their low levels would result in death of intestinal flora, cause disease and the possible development of resistant strains which cause failure of antibiotic therapy in clinical situations (Heshmati, 2015).  However, the principal hazardous effect is likely to develop the resistance of bacteria following the ingestion of subtherapeutic doses of antimicrobials (Heshmati, 2015). The resistance could be transferred from non-pathogenic microorganisms to pathogenic ones, which would then no longer respond to normal drug treatment (Heshmati, 2015).

The illicit use of antibiotics could thus increase the risk of food-borne infection with antibiotic resistant pathogenic bacteria contaminating food taken by human (Heshmati, 2015). Other harmful effects related to antibiotic residues in food include immune pathological effects, autoimmunity, carcinogenicity (sulphamethazine, oxytetracycline, furazolidone), mutagenicity, nephropathy (gentamicin), hepato-toxicity, reproductive disorders, bone marrow toxicity (chloramphenicol), and allergy (penicillin) (Heshmati, 2015).

Toxic effects are not probable because residue contents in food are in very low concentrations (Heshmati, 2013). Nevertheless, some substances must receive particular attention owing to their harmfulness. Allergic reactions may also be produced in sensitive or sensitized individuals (Heshmati, 2013).

For protecting human from exposure of any veterinary residues, a withdrawal time has been determined (Heshmati, 2014).  The withdrawal time is defined as the time interval from administration of a drug to animal until slaughter to assure that drug residues in meat are below maximum residue limit (Heshmati, 2013).

Veterinary drug residue contents in animal-originated food depend on various factors such as drug dosage, type and age of animal, feeding, disease status, poor manage-ment, extra-label drug use, withdrawal time, and route of administration (Codex Alimentarius Commission, 2001). Human health problems resulting from intake of sub-chronic exposure levels of Oxytetracycline include gastrointestinal disturbances (Baker and Leyland, 1983) .Teratogenic risk to the fetus, allergic reactions (Schenk and Collery, 1998) and development of resistant pathogens for human and animals (Bogaard and Stobberingh, 2000) as well as Carcinogenicity caused by Oxytetracycline (Donoghue, 2003).

2.5.2: Maximum Residue Limits

Maximum residue limits mean the maximum concentration of residue resulting from the use of a veterinary medicinal product, which may be legally permitted or recognized as acceptable in or on a food, allocated to individual food commodities (Myllyniemi, 2004). It is based on the type and amount of residue considered to be without any toxicological hazard for human health as expressed by the Allowed Daily Intake (ADI), or on the basis of a temporary ADI that utilizes an additional safety factor (Myllyniemi, 2004). The Codex Alimentarius Commission (CAC) is a commission jointly sponsored by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO). It is a collection of international food standards, guidelines, and codes of practice that protect the health of consumers and ensure fair practices in food trade.

The Codex Alimentarius covers food safety matters (residues, hygiene, additives, contaminants, and quality matters (product descriptions, quality classes, labelling, and certification). Codex established the Codex Committee on Residues of Veterinary Drugs in Food in 1986. Codex has defined 590 MRLs for at least fifty-nine veterinary drugs. Most countries use Codex MRLs as a basis for establishing their national regulations for veterinary drug use, but still other organizations make their own MRLs to be used in their countries (Table 2.3) shows the MRLs of some antimicrobial residues as stated by Codex Alimentarius Commission (Hsu, 2008).

Table 2.1: MRLs of Antibiotics (Codex Alimentarius, 2018 a)

2.5.3: Withdrawal Period (WP)

The withdrawal period is defined as the interval between the times of the last administration of a drug and the time when the animal can be safely slaughtered for food, milk or eggs can be safely consumed (Codex Alimentarius, 2018) The withdrawal period provides a high degree of assurance to both producers and consumers that concentration of residues in foods derived from treated animals will not exceed the MRLs (Codex,2018). Each antimicrobial has a WP which depends on drug type, drug concentration, route of administration, animal kind and the animal product as demonstrated in (Table2.4).  All antibiotics are labelled with the appropriate WP, whether it is hours, days or weeks.

Table 2.2: Withdrawal periods of antibiotics used in poultry production (Codex Alimentarius, 2018 a)

2.6: Prohibition of Some Antibiotics

The extensive use of antibiotics as feed additives for long time may contribute to the development of resistant bacteria to drugs that are used to overcome infections. These microbes pose a potential risk for humans if they are transferred to people (Castanon, 2007). Many European countries banned using antibiotics as food additives (Castanon, 2007).  Sweden prohibited in 1986 the use of additives belonging to the groups of antibiotics in feeding stuffs. Avoparcin was banned in Denmark (1995) and Germany (1996) (Castanon, 2007). spiramycin was prohibited in Finland (1998) because this product was used in human medicine, and virginiamycin was prohibited in Denmark (1998).  Also zinc bacitracin was banned because its use in human medicine as treatment skin infections (Castanon, 2007).

Chloramphenicol, a broad-spectrum antimicrobial, was previously widely used in veterinary and human medicine (Stolker, 2005). Reports of aplastic anaemia in humans arising from its use led to its ban in the USA and European Union (EU) in 1994. Thiamphenicol and florfenicol were permitted as substitutes (Stolker, 2005). Nitrofurans, particularly furazolidone, furaltadone, nitrofurantoin and nitrofurazone for livestock production was completely prohibited in the EU in 1995 due to concerns about the carcinogenicity of the drug residues and their potential harmful effects on human health (Vass, 2008).

Due to emergence of fluoroquinolone-resistant bacteria especially Campylobacter and Salmonella, the Food and Drug Administration (FDA) in 1977 banned the use of fluoroquinolones in treating poultry but the use of sarafloxacin and enrofloxacin in poultry was permitted, but an increase in fluoroquinolone-resistant Campylobacter spp in poultry was linked to increased incidence of infection with resistant Campylobacter spp. in humans.

Finally, FDA in 2005 prohibited the usage of enrofloxacin in poultry and sarafloxacin were withdrawn by the producer, thus usage of any members of fluoroquinolones in poultry species is illegal by FDA (Davis, 2009).

2.7: Cooking effect on Antibiotic Residues

To determine the effect of cooking process on antibiotic residues (ABR), a study investigated the effect of cooking and cold storage on ampicillin, chloramphenicol, oxytetracycline, streptomycin and sulphadimidine residues in meat, the study showed that active ABR might be detected in animal tissue after roasting, grilling and prolonged cold storage (O’brien and Conaghan, 1985). They concluded that it would be unwise to rely on cooking or cold storage to minimize or destroy such residues (O’brien & Conaghan, 1985). The only way to ensure no residues would appear to be the strict observance of the WP for each drug administered to domestic animals (O’brien, & Conaghan, 1985).

In another study, researchers investigated the effects of various ordinary cooking procedures (boiling, roasting and microwaving) on tetracyclines (TC) residues in chicken meat (Abou-Raya, 2013).  The obtained data revealed that the reduction of TC residues in cooked samples was related to cooking procedures, cooking time and TC agents. The losses of TC residues increased with prolonged cooking time (Abou-Raya, 2013).  Doxycycline was the most heat stable of TCs, less than 50% of the initial residue concentration was decreased in boiling and microwaving for 40 and 80 minutes respectively (Abou-Raya, 2013).

In contrary, a different study concluded that oxytetracycline was the most heat labile. The time required to destroy more than 90% of the initial level of oxytetracycline (OTC) in breast meat was 15, 40 and 60 minutes for microwaving, boiling and roasting, respectively, OTC residues in breast meat were not detected after microwaving for 20 minutes (Al-Ghamdi, 2000). Generally, sufficient cooking temperature and time can have a significant effect on the losses of TC residues and provide an additional margin of safety for consumers (Al-Ghamdi, 2000). To determine the effect of different cooking processes (microwaving, roasting, boiling, grilling and frying) on enrofloxacin residues in chicken muscle, investigators used liquid chromatography mass spectrometry (LC-MS) method to evaluate stability of enrofloxacin in natural incurred chicken samples after cooking (Al-Ghamdi, 2000). They conducted the study on different parts of chicken (Muscles, thigh muscles and liver).

The study showed that enrofloxacin remained stable in boiling water for three hours (Al-Ghamdi, 2000). On the other hand, the amount of residue increased in the case of roasting and grilling.

Also, it was noticed that when there was a reduction in residues percentage, the lost amount of analyte was found in water or exudates. These results rendered the investigators to inferred that cooking procedures did not affect the levels of quinolones (Lolo, 2006). In another study also evaluated the effects of different cooking processes on enrofloxacin residues in chicken muscle, liver and gizzard of broilers tissue from broiler chickens, results showed that enrofloxacin residues were reduced after different cooking processes. In cooked meat and gizzard, the most reduced levels of the residue were due to the boiling method. A high residue levels remained stable after microwave cooking/ heating (Lolo, 2006).

It was concluded that cooking processes cannot destroy the total amounts of this drug but it can only decrease their amounts and most of the residues in boiling process are excreted from tissue to cooking fluid during the boiling process (Javadi, & Khatibi, 2011). Thus, exposure to residues can be reduced by discarding any juice that come from the edible tissues as they are cooked. Among the various agents affecting antimicrobial residues after the cooking process, it was found that cooking time and temperature can play major roles (Javadi, &Khatibi, 2011).

2.7: Methods Used in Analysis of Antibiotic Residues

Antibiotic residue analysis is mainly performed by chromatography separation method which is the separation of a mixture by passing it in solution or suspension or as a vapor (as in gas chromatography) through a medium in which the components move at different rates (Coskun, 2016). It is used to separate proteins, nucleic acids, or small molecules in complex mixtures thus an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis (Coskun, 2016). Proteins can be purified based on characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase (Coskun, 2016). Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography. Column chromatography is one of the most common methods of protein purification (Coskun, 2016).

Various chromatography methods have been developed so as to achieve a satisfactory separation within a suitable time interval and some of these include column chromatography, thin-layer chromatography (TLC), paper chromatography, gas chromatography, ion exchange chromatography, gel permeation chromatography, high-pressure liquid chromatography, liquid chromatography (LC), liquid chromatography-tandem mass spectrometer (LC-MS/MS), Enzyme linked immunosorbent assay (ELISA), and affinity chromatography (Coskun, 2016).

2.7.1: Thin Layer Chromatography (TLC)

This is also a solid-liquid ad- sorption chromatography and in this method stationary phase is a solid adsorbent substance coated on glass plates. As adsorbent material all solid substances used in column chromatography (alumina, silica gel, cellulose) can be utilized. The mobile phase travels upward through the stationary phase and the solvent travels up the thin plate soaked with the solvent by means of capillary action driving the mixture priorly dropped on the lower parts of the plate with a pipette upwards with different flow rates thus the separation of analytes is achieved. This upward travelling rate depends on the polarity of the material, solid phase, and of the solvent (Coskun, 2016).

2.7.2: Liquid Chromatography-Mass Spectrometry (LC-MS)

This technique allows structural, and functional analysis, and purification of many molecules within a short time and yields perfect results in the separation, and identification of amino acids, carbohydrates, lipids, nucleic acids, proteins, and other biologically active molecules with the Essential components being solvent depot, high- pressure pump, commercially prepared column, detector, and recorder and computerized system for controlling the duration of separation material is accrued (Ramatla et al., 2017). The mobile phase passes through columns under 10–400 atmospheric pressure, and with a high (0.1–5 cm/sec) flow rate, this technique, use of small particles, and the application of high pressure on the rate of solvent flow increases separation power, of LC rendering analysis completion within a short time (Coskun, 2016). This method was utilized for this research because it the most commonly used technique coupled with its affordability and accessibility within the country (Faleye et al., 2018).

2.7.3: LC-MS/MS Parameters

A triple-quadrupole B.08.00 (B8023.0) mass spectrometer, also known as QQQ, is a tandem DA method in which the first and third quadrupoles act as mass filters and the second causes fragmentation of the analyte through interaction with a collision gas it is a radiofrequency only quadrupole, and can be used in either SIM or scan mode. For structural mass spec, a common sequence is product ion scan, precursor ion scan, and neutral loss scan, followed by selected reaction monitoring (SRM) or multiple reaction monitoring (MRM). Increased selectivity, improved S/N, lower limits of quantitation, wider linear range, and improved accuracy are some of the benefits of this technique, which encompasses triple-quad LC-MS, GC-MS, GC-MSD, and ICP-MS.

2.7.4: High Performance Liquid Chromatography (HPLC)

This technique is similar to LC however, the difference is that in LC the solvents travel by force of gravity while in HPLC, high-pressurized pump to drive the solvent (Coskun, 2016).

2.7.5: Enzyme Linked Immunosorbent Assay (ELISA)

This technique involves coupling antibody or antigen to assay enzymes which combines the specificity of antibody and sensitivity of assay enzymes to primarily detect antigens through assay antibody or antibodies through assay antigens. This makes it costly and not readily available compared to HPLC (Tajik et al., 2010)