NAFDAC SIWES TECHNICAL REPORT

CHAPTER ONE

1.0       INTRODUCTION

  PREAMBLE ABOUT STUDENT INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES)

The Students Industrial Work Experience Scheme (SIWES) is a skills development programme designed to expose and prepare students of universities and other tertiary institutions for the industrial work situation they are likely to meet after graduation.  It is also a planned and structured programme based on stated and specific career objectives which are geared towards developing the occupational competencies of students.

The Students Industrial Work Experience Scheme (SIWES) is the accepted training programme, which is part of the approved Minimum Academic Standard in the various degree programmes for all Nigerian Universities.  The scheme is aimed at bridging the existing gap between theory and practical of the knowledge acquired in the classroom by providing the needed exposure and experience in the industries, laboratory, and research institute. 

Prior to establishing the Scheme, industrialists and other employers of labour felt concerned that graduates of Nigerian universities were deficient in practical background studies preparatory for employment in industries and other organizations.  The employers thus concluded that the theoretical education being received in our higher institutions was not responsive to the needs of the employers of labour; hence the rationale for initiating and designing the scheme by the Industrial Training Funds (ITF), in 1973.

ITF, being a federal organization, was established by Decree 47 of 1971, and was charged with the responsibility of promoting and encouraging the acquisition of skills in industries and commerce with the view to generate a pool of indigenous trained manpower sufficient to meet the need of the company.

The need for Student Industrial Work Experience Scheme (SIWES) for students in higher institution of learning arose from the Federal Government’s directive that students should acquire practical knowledge of their respective disciplines in the real industrial environment in order to prepare them to meet challenges of life work situations and also give them opportunity of using sophisticated industrial equipment which are too expensive for universities to afford.

The scheme is a tripartite programme involving the students, the universities, and the employers of labour.  It is funded by the Federal Government of Nigeria and jointly coordinated by the Industrial Training Fund (ITF) and National Universities Commission (NUC), (Musa, 2009).

The aims and objectives of SIWES are summarised as follows:

1.      To provide students with an opportunity to apply their theoretical knowledge in real work situation, thereby bridging the gap between class work and actual practical work.

2.      To provide an avenue for students in Nigerian tertiary institutions to acquire industrial skills and experience in their course of study.

3.      To expose students to work methods and techniques involved in handling equipment and machineries that may not be available in their institutions.

4.      To prepare students for work ethics and the work situations they are likely to meet after graduation.

5.      To facilitate the transition from the university to the world of work, and enhance students’ contact for inter-job placement.

6.      To enlist and strengthen employers’ involvement in the entire educational process of preparing students for employment in industry.

       BRIEF HISTORY OF NATIONAL AGENCY FOR FOOD AND DRUG ADMINISTRATION AND CONTROL (NAFDAC)

The National Agency for Food and Drug Administration and Control (NAFDAC) was established by Decree No. 15 of 1993 as amended, as a parastatal of the Federal Ministry of Health, with the mandate to regulate and control quality standard for foods, drugs, cosmetics, medical devices, chemicals, detergents, and packaged water distributed in Nigeria, whether imported or locally manufactured. The organization was formed in 1993 under the country’s Health and Safety Law, to checkmate illicit and counterfeit products.

NAFDAC was established in response to the resolution of the World Health Assembly in 1988. This resolution stated that, in order to combat the threat that fake drugs posed to global health, countries should initiate a program for prevention and detection of counterfeit pharmaceutical products.

NAFDAC replaced an earlier body, the Directorate of Food and Drug Administration and Control of the Federal Ministry of Health, whose performance was limited by factors including legislations that were inadequate to discharge the production and distribution of fake drugs. Product registration was also almost non-existent.

It is undeniable that the production of adulterated and counterfeit drugs by unscrupulous individuals has been a major problem in Nigeria. In one 1989 incident, over 150 children died as a result of the presence of diethylene glycol in paracetamol syrup. The problem of fake drugs was so severe that neighbouring countries, such as Ghana and Sierra Leone, officially banned the sales of drugs, food products and beverages made in Nigeria. Such problems led to the establishment of NAFDAC, with the goal of eliminating counterfeit pharmaceutical products, food products and beverages that are not manufactured in Nigeria, and ensuring that available medication is safe and effective.

To achieve this mandate, the agency embarked on various activities. These include:

·         Inspecting regulated products at ports of entry and land borders.

·         Regulating and controlling the importation, exportation, manufacture, advertisement, distribution, sale, and use of drugs, cosmetics, medical devices, bottled water and chemicals.

·         Compiling and publishing relevant data resulting from the performance of the function of the agency or from other source.

·         Sponsoring such national and international conference as it may consider appropriate.

·         Liaising with relevant establishments within and outside Nigeria in pursuance of its functions.

·         Conducting appropriate tests and ensuring product compliance with standard specifications designated and approved by the council of effective control of quality of food, drugs, cosmetics, medical devices, water, and chemicals, along with their raw materials as well as their production processes in factories and other establishments.

·         Undertaking appropriate investigation into the production premises and raw materials for food, drugs, cosmetics, medical devices, bottled water and chemicals, and establishing relevant quality assurance system, including certification of the production sites and the regulated products.

·         Compiling standard specifications, regulations, and guidelines for the production, importation, exportation, sale and distribution of food, drugs, cosmetics, medical devices, bottled water and chemicals.

·         Undertaking inspection of imported food, drugs, cosmetics, medical devices, bottled water and chemicals, and establishing relevant quality assurance system, including certification of the production site and of the regulated product.

·         Undertaking the registration of food, drugs, medical devices, bottled water and chemicals.

·         Controlling the exportation and issue quality certification of foods, drugs, medical devices, bottled water and chemicals intended for export.

·         Establishing and maintaining relevant laboratories or other institutions in strategic areas of Nigeria as may be necessary for performance of its functions.

·         Pronouncing on the quality and safety of food, drugs, cosmetics, medical devices, bottled water and chemicals after appropriate analysis.

·         Undertaking measures to ensure that the use of narcotic drugs and psychotropic substances as well as other controlled substances, is not beyond standard limits.

·         Collaborating with National Drug Law Enforcement Agency (NDLEA) in measures to eradicate drug abuse in Nigeria.

·         Advising Federal, State and Local Governments, the private sectors, and other interested bodies regarding the quality, safety and regulatory provision of food, drugs, cosmetics, medical devices, bottled water and chemicals.

1.2.1    VISION STATEMENT OF NAFDAC

- Safeguarding the health of the nation.

1.2.2    MISSION STATEMENT OF NAFDAC

- To safeguard public health by ensuring that only the right quality drugs, food and other regulated products are manufactured, imported, exported, advertised, distributed, sold and used in Nigeria.

This is achieved through the regulation and control of activities that relates to NAFDAC regulated products.

1.2.3    DIRECTORATES OF NAFDAC

There are ten (9) directorates in the agency which play specific roles in achieving NAFDAC’s mandate. The Directorates are as follows:

1)      Administration and Human Resources Directorate: This directorate handles staff recruitment/appointment, promotion, transfer and posting in the agency. It is also involved in the documentation of new staff, prepares staff nominal roll and issues identity cards.

2)      Finance and Accounts Directorate: This directorate co-ordinates the general day-to-day financial functions of the agency, disburses funds of the agency as approved by the management, pays all staff salaries as at when due, prepares budget estimates for the agency etc.

3)      Directorate of Planning, Research and Statistics (PRS): This is a service directorate that is responsible for planning, researching, and collection of statistical data as well as co-ordinating and documenting the activities of all the other directorates for efficient achievement of the goals of the agency. It also co-ordinates pharmacy and medical internship, and industrial attachment training programme for the agency.

4)      Laboratory Services Directorate: This directorate is tasked with the analysis and pronouncement of the quality and safety of food, drugs, cosmetics, medical devices, chemicals, detergents, drinks, and bottled and packaged water. It also serves as reference laboratory for the other Government agencies.

5)      Establishment Inspection Directorate (EID): This directorate is responsible for good manufacturing practices, inspection of local establishments engaged in the manufacture, sales, storage, distribution, and use of food, drugs, medical devices, etc. It also investigates consumers’ complaints and alert notices.

6)      Ports Inspection Directorate (PID): This directorate is responsible for the regulatory activities concerning the movement of drugs, food, packaged water, cosmetics, etc. at all ports of entry and border posts, airports, and inlanders container terminals in the country.

7)      Enforcement Directorate: This directorate handles all matters concerning enforcement in all its ramifications which involve the prosecution of manufacturers and importers of fake products.

8)      Registration and Regulatory Directorate (R & R): This directorate undertakes the registration of drugs, food and other regulated products locally manufactured, imported, advertised, and sold in Nigeria. It also monitors national and international scientific development and initiatives that may affect public health, and develops appropriate measures to address it.

9)      Narcotics and Controlled Substances Directorate: This directorate controls the importation, manufacture, and sales of Narcotics and psychotropic substances with the main objective of ensuring that the drugs or other controlled substances like food items are available only for consumption purposes.

CHAPTER TWO

2.0       EQUIPMENT/WARES, FACILITIES, AND REAGENTS USED IN THE LABORATORY UNITS

DRUG UNIT/CHEMISTRY UNIT

Table 1: Table Showing Names of Equipments and their Manufacturers in drug lab:

S/N	NAME OF EQUIPMENT	MANUFACTURER
1	Analytical Balance	Sartorius
2	Ultrasonic Bath (Sonicator)	Grant
3	pH Meter	Accumet
4	Top Load Balance	Mettler Toledo
5	UV-Visible spectrophotometer	Perkin Elmer
6	Magnetic Stirrer and Hotplate	Jenway
7	Drying Cabinet	UNITEMP
8	Universal Dissolution Tester
9	Automatic Disintegration Tester
10	Polarimeter
11	Flask Shaker
12	Hardness Tester

APPARATUS:

Pipette fillers, Pipettes, Burettes, Beakers, Desiccators, Mortar and Pestle, Conical Flasks, Volumetric Flasks, Separating funnel, Spatula, Measuring Cylinders, Filter Funnels, Reagent Bottles, Evaporating dishes, Electric Oven, and Retort stand.

WATER UNIT

Table 2: Table Showing Names of Equipment and their Manufacturer in Water Laboratory. 

S/N	NAME OF EQUIPMENT	MANUFACTURER
1	pH meter	Accumet
2	Smart Spectrophotometer
3	Water Distillery	Distinction
4	LaMotte test kits	LaMotte

APPARATUS

Pipettes, Burettes, Beakers, Conical Flasks, Volumetric Flasks, Measuring

 Cylinders, Desiccators, Reagent Bottles, and Retort Stand

FOOD UNIT

Table 3: Table Showing Names of Equipment and their Manufacturer in Food Laboratory

S/N	NAME OF EQUIPMENT	MANUFACTURER
1	Heating mantle
2	Water bath	nickel electro
3	Electric Oven	Memmert
4	Centrifuge
5	Kjeldahl Equipment	Labconco
7	Drying Cabinet	UNITEMP
8	Moisture Analyzer	Sartorious
9	Analytical Balance	Mettler Toledo
11	Muffle furnace	AAF 1100

APPARATUS

Separating funnels, Pipettes, Burette, Beakers, Conical Flasks, Volumetric Flasks, Measuring Cylinders, Reagent Bottles, Spatula, Tripod Stand, and Retort Stand

MYCOTOXIN UNIT

Table 4: Table Showing Names of Equipment and their Manufacturers in Mycotoxin Unit

S/N	NAME OF EQUIPMENT	MANUFACTURER
1	Analytical Balance	Sartorius
2	Ultrasonic Bath (Sonicator)	Grant
3	Horizontal Orbital Shaker	VWR DS2
4	Vortex Mixer	Stuart
5	Drying Cabinet	UNITEMP
6	ELISA Reader	Stat Fax
7	Multi-Block Heater	Labline
8	Rotary Evaporator	Romerevap

APPARATUS

Micropipettes, Micropipette Tips, Beakers, Conical Flasks, Volumetric Flasks, Measuring Cylinders, Reagent Bottles, Spatula, Aflatoxin Test Kits, Melamine Test Kit, and Vials

 REAGENTS USED IN THE LABORATORY UNITS

The following reagents are commonly used in the laboratory units of NAFDAC:

a.       Acids : These include hydrochloric acid (HCl), perchlorics acid (HClO₄), glacial acetic acid (CH₃CO₂H), sulphuric acid (H₂SO₄), phosphoric acid (H₃PO₄), sodium dihydrogen phosphate (NaH₂PO₄), lead nitrate [Pb(NO₃)₂], orthophosphoric acid, silver nitrate (AgNO₃), boric acid (H₃BO₃), cupric acid, acetic acid (CH₃CO₂H), and ascorbic acid(C₆H₈O₆).

b.      Bases and Salts: These includes sodium hydroxide (NaOH), ammonium hydroxide (NH₄OH), sodium chloride (NaCl), sodium hydogen orthophosphate, potassium hydroxide (KOH), Meyers reagent, Fehling I and Fehling II, saturated sodium chloride, potassium iodide (KI), alcoholic potassium hydroxide (alc. KOH), and  ammonia (NH₃).

c.       Organic solvents: These include diethylether (C₂H₅OC₂H₅), ethanol(C₂H₅OH), chloroform(CHCl₃), petroleum ether (mixture of pentane and hexane), Hexane (C₆H₁₄), acetone(CH₃COCH₃), formaldehyde(CH₂O), acetonitrile (CH₃CN), methanol (CH₃OH), tetrahydrofuran, isopropanol (CH₃CHOHCH₃), dimethyl formamide, phenol, and alcohol.

d.      Indicators: These include phenolphthalein, screened methyl red, methylene blue, methyl red, methyl orange, phenanthroline, cresol purple, ortho-ludine, crystal violet, xylenol orange, mordant black II, solochrome black T (Eriochrome black T), starch indicator, bromocresol green, bromomethylene blue, and resorcinol.

e.       Buffers: These include sodium dihydrogen orthophosphate (NaH₂PO₄.2H₂O), phosphate buffer, ammonium chloride buffer, sodium hexane sulphonic acid buffer.

f.       Cleansing agents: Example is activated charcoal.

PREPARATION OF STOCK SOLUTIONS

Stock solutions are usually prepared for use in pharmaceutical chemistry and pharmaceutical control laboratories as follows:

Preparation of Buffer Solutions

·                     Standard Buffer Solutions of pH 4, 7 and 10

Aim: To prepare standard Buffers of pH 4, 7 and 10.

Materials: 100-ml volumetric flask, disodium hydrogen orthophosphate buffer of pH 6.9 (equivalent to buffer pH 7.0) and distilled water.

Procedure:  Buffer tablet of pH 4 was dissolved in a 100-ml volumetric flask with distilled water. It was shaken properly to dissolve, and was then made up to volume with more distilled water.

The same procedure was followed to prepare buffers 7 and 10.

·                     Ammonium Chloride (NH₄Cl) Buffer Solution of pH 10.8

Aim: To prepare ammonium chloride buffer solution of pH 10.8

Materials: Analytical balance, spatula, Ammonium chloride salt, 10M ammonia solution, 100-ml volumetric flask, and distilled water.

Procedure: 5.4g of ammonium chloride salt was weighed and dissolved in 20ml of distilled water. 35ml of 10M ammonia solution was added and diluted to the mark in a 100ml volumetric flask with distilled water.

·                     Sodium Hexane Sulphonic Acid Buffer

Aim: To prepare sodium hexane sulphonic acid buffer.

Materials: Analytical balance, spatula, measuring cylinder, 2000-ml volumetric flask, sonicator, sodium hexane sulphonic acid salt, glacial acetic acid, and distilled water.

Procedure: 1.8822g of Sodium Hexane sulphonic acid salt was weighed into a 2litre volumetric flask. 1500ml of distilled water was added and shaken to dissolve. 20ml of glacial acetic acid was added and made up to the mark with distilled water. The solution was sonicated in a sonicator to remove air bubbles.

Preparation of Salt Solutions

·                     24.818% Sodium ThiosulphatePentahydrate (Na₂S₂O₃.5H₂O) Solution

Aim: To prepare 24.818% sodium thiosulphatepentahydrate solution.

Materials: Analytical balance, spatula, Sodium Thiosulphate salt, 1000ml volumetric flask and Distilled water.

Procedure:  248.18g of the sodium thiosulphatepentahydrate salt was weighed and was transferred into a 1000ml volumetric flask, to which sufficient water was added. The flask was shaken properly to dissolve, and was made up to volume with more distilled water.

·                  0.05M Iodine solution

Aim: To prepare 0.05M iodine solution.

Materials: Analytical balance, spatula, iodine salt, and distilled water.

Procedure: 3.1725g of iodine was weighed into a 500ml volumetric flask and dissolved with adequate amount of distilled water. The solution was made up to the mark with distilled water

·                  0.1M Silver Nitrate (AgNO₃) Solution

Aim: To prepare 0.1M silver nitrate solution.

Materials: Analytical balance, spatula, silver nitrate salt, and distilled water

17.0g of AgNO₃ salt was weighed into a 1-litre volumetric flask and dissolved with adequate quantity of distilled water. The solution was made up to the 1-litre mark with distilled water.

Preparation of Acidic Solutions

·                        25% Hydrochloric Acid (HCl) Solution

Aim: To prepare 25% hydrochloric acid solution.

Materials: Measuring cylinder, 100-ml volumetric flask, concentrated hydrochloric acid, and distilled water.

Procedure: 25ml of concentrated Hydrochloric acid was measured under the fume hood into a 100-ml volumetric flask containing a considerable amount of distilled water, more distilled water was added to make up to the mark.

·                        1.0M Tetraoxosulphate (VI) Acid (H₂SO₄) Solution

Aim: To prepare 1.0M sulphuric acid solution.

Materials: Measuring cylinder, 500-ml volumetric flask, concentrated sulphuric acid (36.0%), and distilled water.

Procedure: 74ml of conc. H₂SO₄ was measured under the fume hood and was transferred into a 500ml volumetric flask containing some quantity of distilled water. The solution was then made up to 500ml with more distilled water.

Note: Acid was added to water and not water to acid.

The relation below was used to calculate the volume of concentrated H₂SO₄ to be measured in order to prepare 1.0M H₂SO₄ solution in a required amount:

Where:

M= Molar Mass of H₂SO₄ = 98g/mol

C= Concentration of H₂SO₄ to be prepared (Known) =1.0M

V= Volume of H₂SO₄ to be prepared (Known) =500ml

P= Percentage Purity or Assay (Usually found on the container’s label) =36.0%

D= Density of H₂SO₄ (Usually found on the container’s label) = 1.84g/cm³

V₁= 74ml

This is the method used to determine the volume of a stock solution required to prepare a given volume of known concentration of the solution.

·                     Ascorbic acid solution

Aim: To prepare ascorbic acid solution.

Materials: Analytical balance, spatula, measuring cylinder, 500-ml volumetric flask, ascorbic acid powder, and dimethyl formamide.

Procedure: 0.5g of Ascorbic Acid powder was weighed and dissolved in 5ml of water. It was diluted to 500ml with dimethylformamide and wrapped with foil paper.

Note: preparation should be done in the dark.

Preparation of Alkaline Solutions

·                           10M Ammonia solution

Given that the percentage purity and density of the stock ammonia solution are 25% and 0.903g/cm³, the required concentration was prepared using the dilution formula.

Aim: To prepare 10M ammonia solution.

Materials: Measuring cylinder, 100-ml volumetric flask, stock ammonia solution, and distilled water.

Procedure: 75ml of concentrated ammonia was measured under the fume cupboard using a measuring cylinder and was transferred into a 100ml volumetric flask. It was then diluted with distilled water to the 100ml mark.

·                           50% Sodium Hydroxide (NaOH)

Aim: To prepare 50% sodium hydroxide solution.

Materials: Analytical balance, spatula, 500-ml volumetric flask, beakers, sodium hydroxide salt, and distilled water.

Procedure: 250g of NaOH crystals were weighed and dissolved in a beaker with adequate amount of distilled water which was placed under the influence of running water. The dissolved NaOH was transferred into a 500ml volumetric flask and made up to the mark with distilled water.

Preparation of 15% Tween-Water Stock Solution

Aim: To prepare 15% tween-water solution

Materials: Measuring cylinder, 1000-ml volumetric flask, tween solution, and distilled water.

Procedure: 150ml of tween solution was measured and added to 850ml of deionized or distilled water in a 1-litre volumetric flask. The resulting solution was swirled to mix.

Preparation of 70% Methanol-Tween Solution

Aim: To prepare 70% methanol-tween solution

Materials: Measuring cylinder, 1000-ml volumetric flask, 15% tween-water stock solution, and 100% methanol.

Procedure: 300ml of 15% tween-water stock solution was measured and added to 700ml of 100% methanol in a 1000-ml volumetric flask. The solution was swirled to mix.

Preparation of 1% starch indicator.

Aim: To prepare 1% starch indicator

Materials: Analytical balance, 1000-ml volumetric flask, spatula, starch, and hot water.

Procedure: 10g of starch was weighed and dissolved in hot water and made up to volume with hot water in 1000ml volumetric flask.

Preparation of Fehling I Solution

Aim: To prepare Fehling I solution.

Materials: Analytical balance, spatula, copper (II) sulphate salt, 1000-ml volumetric flask, glass rod, and distilled water.

Procedure: 69.278g of copper (II) sulphate (CuSO₄) was weighed and was transferred into 1000-ml volumetric flask. Sufficient water was added, stirred and shaken properly to dissolve, after which it was made up to volume with more distilled water.

Preparation of Fehling II Solution

Aim: To prepare Fehling II solution.

Materials: Analytical balance, spatula, sodium hydroxide (NaOH) salt, sodium potassium tartarate salt, 1000-ml volumetric flask, glass rod, and distilled water.

Procedure: 100g of NaOH and 346g of sodium potassium tartarate salts were weighed and transferred into 1000-ml volumetric flask. Sufficient water was added, after which the mixture was stirred and shaken properly to dissolve. It was then made up to volume with more distilled water.

 CHAPTER THREE

            STORE

Store is a place where private organization or government properties are kept and protected against an unauthorized removal. Store with respect to NAFDAC is a place where chemicals and reagents, glassware, instruments stationeries are kept and protected against an unauthorized removal.

CLASSIFICATION OF STORE

·         Consumable store

·         Non-consumable but expendable store

·         Non-consumable non-expendable

CONSUMABLE STORE

These are items that are used in the store or disappear from sight as is being used without valuable remnants. E.g. cement, drugs, chemical fuel, glassware and reagents.

NON-CONSUMABLE BUT EXPENDABLE STORE

These are items they required frequent replacement as being used. e.g. instruments, accessories, spare parts, tools, hardware e.t.c.

3.0   ANALYSES PERFORMED IN THE VARIOUS LABORATORY UNITS

     DRUG/CHEMISTRY UNIT

  INTRODUCTION

Drug unit of NAFDAC is responsible for carrying out various physical and chemical analyses on different drug samples that are brought to the agency. The major function of drug laboratory, therefore, is the physicochemical analysis of drugs for human and animal consumption to ensure that the product consistently complies with the stipulated International standard and these analysis are carried out through a systematic procedure such as: Registration and allocation of these drug samples, the use of adequate parameter for each sample and a potent assay to ascertain the percentage of active ingredient found in such drug samples.

The drugs could be either tablets, capsules, injectable or syrup and the standard operating procedures (SOP) used for the analysis of these drugs could be either the British Pharmacopeia (BP) or United State Pharmacopeia (USP).

A drug is a substance used to treat an illness, relieve a symptom, or modify a chemical process in the body for a specific purpose. These drugs are analyzed to know if they actually contain what the manufacturer claims and they are up to the amount claimed (i.e. the strength of the active pharmaceutical ingredient). The time taken for a drug active to be released (to be effective) in the body and the amount released are also taken into consideration.

A drug sample contains the following;

(I)                Excipient: An ingredient that is intentionally added to a drug for purposes other than the therapeutic or diagnostic effect at the intended dosage. It acts as a vehicle and as a binder for the active pharmaceutical ingredient. The true role of excipients will be to produce a stable, uniform product of high quality, and in some cases to influence the delivery of a drug to a desired location at the desired rate. Excipients have a major impact on the field of biopharmaceuticals, reason for which is necessary to ensure that they do not adversely affect the stability, dissolution rate, bioavailability, safety or efficacy of the active ingredient(s). Degradation of an excipient can negatively affect the drug’s physical or microbiological stability. Therefore, a good excipient must be inert so as not to react with the API and must be able to dissolve in physiological medium.

(II)       Active Pharmaceutical Ingredient (API): This is the active substance of a drug which produces the medical healing process. 

(III)     Coating: Most drugs are covered with a thin layer to enhance the taste by sugar coating. Other drugs are film coated or enteric coated to protect from light and delay release of API respectively. 

DOSAGE FORMS OF DRUGS

Drugs come in various dosage forms. The most commonly analysed dosage forms of drugs in the unit include tablets, capsules, syrups, suspensions, and injections.

 TABLETS

Tablets are solid dosage forms containing one or more active ingredients. They are obtained by single or multiple compressions and may be coated or uncoated. They are circular in shape and their surfaces are flat or convex and they should be sufficiently hard to withstand packaging, storage and transportation without breaking. Tablets may contain excipients such as binders, diluents, disintegration agents, substance capable of modifying the behavior of the dosage forms and the active ingredients in the gastrointestinal tracts, coloring matter etc. tablets can either be coated or uncoated.

i.       Coated Tablet: they are tablets covered with one or more layers of mixtures of substances such as natural or synthetic resins, polymers, sugars, waxes etc. the tablets are coated for a variety of reasons such as protection of the active ingredients from air, moisture, or light and masking of unpleasant taste and odors. Coated tablets are further groups into three main categories such as sugar-coated, film-coated and enteric-coated tablets.

ii.         Uncoated Tablet: The majority of uncoated tablets are made in such a way that the release of active ingredients is unmodified. A broken section, when examined under a lens, shows either a relatively uniform texture (single-layer tablets) or a stratified texture (multi-layer tablets). Uncoated tablets are also classified into three groups such as soluble tablets, effervescent tablets and tablets for use in the mouth.

CAPSULES: capsules are solid dosages forms with hard or soft shells. They are of various shapes and sizes, and contain a single dose of one or more active ingredients. The different categories of capsules that exist include hard, soft and modified release capsules. Capsules shells are made of gelatin or other substances which may be modified by the addition of substances such as glycerol and sorbitol.

Capsules shells and contents may contain excipients such as diluents, solvents, anti-microbial agents, sweeteners, colouring matter, flavouring substances etc. the contents should not cause deterioration of the shell.

 SYRUPS

A syrup is a liquid drug which may be sweet viscous or sweet non-viscous with a characteristic smell, used as medicine due to the drug actives present in it.

 SUSPENSION

This is a liquid with small pieces of drug that is originally in powder form prior to reconstitution with water. The drug is not completely dissolved in the solution (on reconstitution).

INJECTION

This is a non-viscous liquid drug that is not taken orally, but is administered into the body via a syringe. Injections are usually colorless, odorless, non-viscous, and non-turbid. They can be enclosed within

-          Ampoules (small bottles made of glass containing liquid drug that will be used for injection)

-          Transparent plastic containers or bottles

-          Amber-coloured bottles

-          Sachets (commonly referred to as drip injections)

-          An assembly consisting of injection water (either in plastic bottle or ampoule), white powder (contained usually in a labeled transparent bottle), and liquid drug (contained in an ampoule), all packed in a labeled hardboard package.

PHYSICAL EXAMINATION/ANALYSIS OF DRUG SAMPLES

This involves the visual examination of the samples, i.e. both the labeling and the packaging.

Every drug sample, as well as all samples to be analysed by NAFDAC, must have the following features:

·         Date of Receipt

·         Manufacturer’s name and address

·         Distributor’s name and address

·         Manufacturing date and expiry date

·         NAFDAC Registration number and Batch Number

Physical parameters of drugs are usually analysed based on the type of the sample via: tablets, capsules, powder, syrups, suspensions etc.

·         Drugs like tablets and capsules undergo the analyses below:

-       Description of physical appearance(colour, shape, contents and inscriptions)

-       Determination of weight uniformity from Average Weight and Individual weights

-       Disintegration test

-       Dissolution test

-       Hardness test

·         Injections, suspensions and syrups are analysed by

-       Description of physical appearance

-       Determination of weight per ml, pH, and filling volume

·         Powders (both oral suspensions and injection powders) are analysed by

-       Description of physical appearance

-       Determination of net weight and pH (of 10% solution of the sample, that is, 1g of sample in 10ml of distilled water). Injection powders and powders for oral suspensions are reconstituted before proceeding with the pharmacopeial assay.

UNIFORMITY OF WEIGHT TEST

This is the test carried out on tablets and capsules to determine the degree of uniformity of weight of the dosage units. Non-uniformity of weight indicates that there is disparity in the concentration of the API in the tablets or capsules. This should not be because each dosage unit is expected to have approximately the same amount of the API, hence the need for Uniformity 0f weight test.

Weight uniformity is ascertained by calculating the percentage deviation for each dosage unit and comparing to check whether, or not, the values are within the standard limits, as shown below for tablets and capsules, respectively.

DISINTEGRATION TEST

Complete disintegration is defined as that state in which any residue of the unit, except fragments of insoluble coating or capsule shell, remaining on the screen of the test apparatus or adhering to the lower surface of the disks, if used, is a soft mass having no palpably firm core. Reframe this definition, it is not straight forward.

Disintegration test is provided to determine whether tablets, capsules, granules or pills disintegrate within the prescribed time when placed in a liquid medium under standard experimental conditions. For the purposes of this test, disintegration does not imply complete solution of the unit or even of its active constituent.

This test is met if all of the dosage units have disintegrated completely. If 1 or 2 dosage units fail to disintegrate, the test is repeated on 12 additional dosage units. The test is met if not less than 16 of the total of 18 dosage units tested are disintegrated.

Aim: To determine the disintegration time lumefantrine tablets

Materials: Automatic Disintegration Machine (ADT) and 6 tablets of lumefantrine

Procedure: 1 dosage unit was placed in each of the six tubes of the basket rack. The ADT was switched on and the time was set for 15 minutes. The machine was maintained at 37 ± 2º C, using water as the immersion fluid. The basket rack was lifted from the fluid time after time to observe the dosage units. On complete disintegration, the disintegration time was noted and recorded.

Result:

The disintegration time for lumefantrine tablets was 14 minutes and 8 seconds.

Discussion:

The maximum disintegration time for plain tablets is 30 minutes. Since lumefantrine tablets are plain, it implies that the sample passed this test.

         HARDNESS TEST

The drug is not expected to be too hard (to ensure easy disintegration) or too soft (to withstand packaging and transportation). This measurement is achieved using the hardness tester machine which measures the force (N) required to break a certain drug. The length and diameter of drug is measured in millimeters, the values were inputted into the machine, the drug is placed within the jaws of the machine, and ok button was pressed to start. The accepted range is 40-300N.

WEIGHT PER VOLUME TEST

This is done for syrups, injection and suspension. It depends on the manufacturer’s declaration of volume of the medicine that will contain a certain mass of the active. For example, if the manufacturer claims that every 5ml contains 100mgof the active ingredient, then weight/5ml would be taken.

 NET WEIGHT TEST

This is done for drugs that are in powdered form, packaged in sachet or any container. This is achieved by taking the weight of the sample content and container, w_1, and then emptying the container and taking the weight of empty container, w₂. Difference between w₁ and w₂ is the net weight, w_3.

  CHEMICAL ANALYSIS OF DRUG SAMPLES

This involves the analysis of drug samples for quantification of the Active Pharmaceutical Ingredient (A.P.I) of the drug. Chemical analysis of these drug samples is basically the quantitative determination of the active pharmaceutical ingredient in sample. There are three (3) methods of chemical analysis, one of which is used depending on the pharmacopeial assay:

i.     Titrimetric method, which employs the use of bench reagents and titration.

ii.   Chromatographic method, which employs the use of a chromatograph like the HPLC (High Performance Liquid Chromatography).

iii. Spectrometric method, which employs the use of the Ultraviolet-Visible Spectrometer.

Quantitative determination of the drug’s API is mostly carried out spectrometrically. The use of the HPLC is mostly employed only for confirmatory purposes.

NOTE: Assay is the standard procedure for the determination of the amount of Active Pharmaceutical Ingredient (API) of the drug sample, whereas a pharmacopeia is a book published usually under the jurisdiction of the government and containing a list of drugs, their formulas, method of making medicinal preparations, requirements and tests for their strength and purity, and other related information. The two pharmacopeias that are most frequently consulted in the drug/chemistry unit are United States Pharmacopeia (U.S.P) and British Pharmacopeia (B.P).

ASSAY FOR DETERMINATION OF DIPHENHYDRAMINE HYDROCHLORIDE IN BENYLIN SYRUP

IUPAC Name: 2-(diphenylmethoxy)-N,N-dimethylethanamine

Molecular Formula: C₁₇H₂₁NOHCl

Diphenhydramine is a Histamine-1 Receptor Antagonist and its mechanism of action proceeds accordingly. It is a first-generation anti-histamine and ethanolamine with sedative and anti-allergic properties. Diphenhydramine competitively inhibits the Histamine-1 (H1) receptor, thereby alleviating the symptoms caused by endogenous histamine on bronchial, capillary and gastrointestinal smooth muscles. This prevents histamine-induced bronchoconstriction, vasodilation, increased capillary permeability, and GI smooth muscle spasms. Diphenhydramine is used as ingredient in common cold preparations. It has some undesired anti-muscarinic and sedative effects.

Principle:

The working principle of the uv-visible spectrometer is based on two laws: Beer’s law and Lambert’s law, which combine to give Beer-Lambert’s law. This law states that the intensity of monochromatic light transmitted through a solution decreases exponentially with increase in concentration of the compound (absorbing species) in the solution and increase in the path length of the cuvette or thickness of the solution. It is mathematically expressed as:

Where A is the measured absorbance, I_O is the intensity of the incident light at a given wavelength, I is the intensity of the transmitted light, L the path length of the cell, c the concentration of the absorbing species, E is a constant known as molar absorptivity or molar extinction coefficient, which is the fundamental property in a given solvent, at a particular temperature and pressure.

Every API has its absorption maximum, which is defined as the wavelength at which the molecules of the API will absorb the most or have the highest absorbance. Therefore, the concentration of the API can be ascertained given its absorption maximum. Quantitative determination of a drug’s active by spectrometric technique consists of sample preparation and operation of the uv-visible spectrometer to obtain the absorbance value, followed by computation of the active content by formula and, hence, the percentage active content of the drug.

Aim: To quantitatively determine diphenhydramine hydrochloride in benylin syrup.

Materials: UV-Visible spectrometer (with path length of 1cm), analytical balance, spatula, dropper, separating funnel, 100-ml beaker, 100-ml volumetric flask, 5-ml volumetric flask, measuring cylinder, distilled water, 20% NaOH solution, 0.1M NaOH solution, diethyl ether, and 0.1M HCl solution.

Procedure:

i.        Weight per 5ml

An empty 5ml volumetric flask was weighed. The syrup was transferred drop by drop into the volumetric flask up to the mark. The flask containing the syrup was then weighed.

ii.   Pharmacopeial Assay

15.0215g of the syrup was weighed using a dropper into a 100-ml beaker. The sample was then transferred into a separating funnel and the beaker was rinsed twice with 25ml of distilled water. 10ml of 20% NaOH solution was added and extraction was carried out with 20ml diethyl ether thrice. The combined ether extract was washed with 5ml of 0.1M NaOH solution twice. A second extraction was carried out with 20ml of 0.1M HCl solution four times. The combined ether extract was filtered into a 100-ml volumetric flask and was made up to volume with 0.1M HCl solution. The absorbance of the sample solution was taken at 254nm, using 0.1M HCl solution as blank.

Result:

Table 8: Table of Weights

Item	Weight in grams
Empty flask (W1)	7.3216
Flask and syrup (W2)	13.8244
Syrup (W3)	6.5028

Weight/5ml = W₂ - W₁ = 13.8244 – 7.3216

                    = 6.5028g

Weight taken = 15.0215g

Table 9: Table Showing the Absorbance at 254nm

Sample	Absorbance
Sample1	0.3817
Sample2	0.3913
Sample3	0.3973

Average absorbance =

                                 = 0.3901

Active Content =   dilution factor  1000

Dilution factor =

Dilution factor =

                        = 1 (This implies that there was no dilution)

Hence,

           Active Content = 1  1000

                   = 12.99mg

% Active Content =

                      =  100

                     = 96.2%

Discussion:

The concentration of diphenhydramine declared by the manufacturer was 13.5mg and the calculated concentration was 12.99mg and has a percentage concentration of 96.2%. The acceptable range set by NAFDAC is 90–120%. This means that the drug is within the range; hence it is considered satisfactory.

ASSAY FOR DETERMINATION OF AMOXYCILLIN TRIHYDRATE IN AQUAMOX CAPSULE

IUPAC Name: (2S,5R,6R)-6-{[(2R)-2-amino-2-(4-hydroxyphenyl)-acetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptanes-24-carboxylic acid

Molecular Formula: C₁₆H₂₅N₃O₈S

Methodology: UV-Visible Spectrometry

Procedure:

An equivalent of 250mg was weighed into 100ml flask. It was allowed to dissolves with 0.1M NaOH which was then make up to volume. It was filter and 2ml was measured from the resulting solution into 100ml flask and was made up to volume with distilled water. Absorbance was measured at 292nm (E₁=59).

Result:

Table 10: Table Showing the Weights of 5 Capsules

S/N	Weight of Content + Shell (g)	Weight of Shell (g)	Weight of Content (g)
1	0.6442	0.0268	0.6174
2	0.6682	0.0566	0.6116
3	0.6669	0.0560	0.6109
4	0.6776	0.0614	0.6162
5	0.6417	0.0555	0.5862

Average weight of the capsules   =0.60846g

Actual weight   Average weight

                         × 0.6085

             = 0.3043g

Weight Taken =  0.3058g

Table 11: Table showing the absorbance of amoxicillin trihydrate at 292nm

S/N	Absorbance
1	0.3531
2	0.3534
3	0.3530

Average Absorbance   = 0.3532

Active Content =   dilution factor  1000

  × 50 ×   × 1000

                    = 595.6097mg

% Active Content =

           

 × 100

 = 119.12%

Discussion:

The concentration of Amoxicillin trihydrate declared by the manufacturer was 500mg and the calculated concentration was 595.6097mg and has a percentage concentration of 119.12%. The acceptable range set by NAFDAC from the British pharmacopeia (B.P) and united state pharmacopeia (U.S.A) for Amoxicillin trihydrate is 90-120%. This means that the drug is within the range hence declared satisfactory and could be allowed to be sold.

ASSAY FOR LUMEFANTRINE DETERMINATION IN  ANTI-MALARIAL DRUG

IUPAC Name: 2-dibutylamino-1-{2,7-dichloro-9-[1-(4-chlorophenyl)-meth-(Z)-ylidene]-9H-fluoren-4-yl}-ethanol

Molecular Formula: C₃₀H₃₂C_l3NO

Methodology: UV- Visible Spectrometry

Lumefantrine is an anti-malarial agent that works only with artemether.

Procedure: An equivalent of 30mg was weighed accurately using analytical balance and was transferred  into 100ml volumetric flask, it was then make up to volume with chloroform(CHCl₃). 1ml was then measured from the resulting solution into 25ml flask; it was then made up to volume with the same solvent (chloroform). Absorbance was measured at 339nm (E₁=295)

Results:

Table 11: Table Showing the Weights of 5 Tablets

S/N	Weight (g)
1	0.6528
2	0.6779
3	0.6322
4	0.6639
5	0.7430

Average weight  = 0.6735g

Actual weight   Average weight

                          =   × 0.6735

              = 0.0420g

Weight taken    = 0.0424g

Table 12: Table Showing the Absorbance of Lumefantrine at 339nm

S/N	Absorbance
1	0.3472
2	0.3542
3	0.3436

Average Absorbance =  = 0.3483

Active Content =   dilution factor  1000

                  =   × 25 ×   × 1000

                    = 447.97mg

% Active Content =

                       =    × 100

= 93.3%  

Discussion:

The concentration of lumefantrine declared by the manufacturer was 480mg and the calculated concentration was 447.97mg and has a percentage concentration of 93.3%. The acceptable range set by NAFDAC from the British pharmacopeia (B.P) and United State Pharmacopeia (U.S.A) for lumefantrine is 90-120%. This means that the drug is within the range, hence declared satisfactory.

 ASSAY FOR ACETAMINOPHEN

Acetaminophen is an odourless drug that is slightly bitter in taste, which is in white crystalline powder form.

 Method: An equivalent of 37.5 was weighed into 50ml flask. 20ml of H₂O and 12.5ml of 0.1M NaOH was added and shaked for 15mins to dissolve and made up the volume with water. Five ml of the resulting solution was taken and diluted to 50ml. 5ml of 0.1M NaOH was taken and made up to volume with H₂O. Absorbance was measured at 257nm {E₁=715}.

Result:

Weight of tablet

S/N	Weight in grams	S/N	Weight in grams
1	0.5683	6	0.5745
2	0.5790	7	0.5901
3	0.5707	8	0.6006
4	0.5821	9	0.57739
5	0.57773	10	0.5812

Average weight of sample

Uniformity of weight

%deviation =  0.57976

                = 0.028988

Upper limit = average weight + %deviation

                = 0.57976 + 0.028988

                = 0.608748

Lower limit = average weight - %deviation

                = 0.57976 - 0.028988

                = 0.550722

Actual weight =  average weight

                   =  0.57976

                  = 0.043482

Weight taken = 0.0436g

Using a UV- Visible spectrophotometer, absorbance was taken and recorded as

Table showing the absorbance of acetaminophen at 257nm

Sample	Absorbance
Sample1	0.5352
Sample2	0.5297
Sample3	0.5404

Average absorbance =

                             = 0.5351

Concentration =   dilution factor  100

                    =  50  100

                   = 497.57

% concentration =

                      =  100

                     = 99.5%

Discussion:

Comparing the highest and the lowest weight of the tablet with the lower and higher limit of the drug, it can be observed that the weights fall within the range of the limit, hence it is satisfactory.

The concentration of acetaminophen as declared by the manufacturer is 500mg and the calculated concentration was 497.57 and has a percentage concentration of 99.5%. The range acceptable by NAFDAC is 95 – 105%. This means that the drug is within the range hence satisfactory and could be allowed to be sold.

 WATER UNIT

 INTRODUCTION

The parameters being determined in water unit are as follows:

1. Sensory parameters                                                                                                 

·         Appearance                                                                                                              

·         Odour

·         Taste                                                                                                                           

2. Physical Parameters

·         Net Volume                                                                                                                   

·         pH                                                                                                                                  

·         Total Dissolved Solids (TDS)

3. Chemical Parameters

·         Test For Free Dissolved Carbon Dioxide

·         Total Alkalinity Test

·         Total Hardness Test

·         Sulphate Test

·         Nitrite Test

·         Chloride Test

4. Metal Analysis

·         Potassium test

·         Sodium test

SENSORY PARAMETERS

The analyses carried out under this parameter are done with the aid of functional sensory organs such as the tongue, eyes and nose of the analyst. Portable water for consumption by convention is expected to be colourless, odorless and tasteless as such, any water sample without these characteristics is not considered fit for drinking.

TEST FOR WATER APPEARANCE

Test for water appearance provides an answer based upon the visual sighting of the water sample. It depends on the colour of the water sample. Pure and uncontaminated water is expected to be colourless. Any water sample that is coloured fails the appearance test and is, therefore, not accepted based on appearance. The eyes are used for this test. It requires an experienced analyst with good eyes; therefore it is usually carried out by the Head of the Unit.

TEST FOR WATER TASTE

Good suitable water for consumption is expected to be tasteless. Water taste arises as a result of the presence of mineral elements in the form of dissolved ions. This test helps to ascertain the level of water purification or good manufacturing and purification practices employed by the industry of factory. The tongue is used for this test. This is carried by an experienced analyst, usually the Head of the Unit.

TEST FOR WATER ODOUR

Pure water is expected to be odourless. Water odour arises from water pollution by microbial metabolic activities, poor sewage management, and/or poor industrial procedures. The nose is used for this test by well-trained personnel with good olfactory qualities; hence it is usually carried out by the Head of the Unit.

PHYSICAL PARAMETERS

Physical parameters are those that are determined without any use of chemical reagents. Therefore, no chemical reaction takes place in the physical analysis of water.

TEST FOR NET VOLUME

Water net volume is the measure of packaged water samples in cm³ or ml. The volume declared by the manufacturer is compared with the actual volume measured in order to ascertain whether the actual volume agrees with the indicated volume. Filling or net volume is expected to be at least 95% so that the consumers are actually getting what they are paying for. Most samples are packaged and the volumes are usually given in centiliters (cl), where 1cl = 10ml.

Aim: To calculate the net volume of water sample.

Materials: Measuring cylinder and distilled water

Procedure: The water sample was poured into a measuring cylinder and the results were taken from the lower meniscus.

Result:

Declared volume = 50cl (500ml)

Measured volume = 48.0cl (480ml)

%Net volume =  100

                 =  100 = 96%

DISCUSSION:

From the minimum limit given above, it is evident that this water sample is satisfactory. This test gives the analyst opportunity to ascertain whether or not the declared volume of water by the manufacturer corresponds to the measured volume or it is within the range at least 95% and above. 

TEST FOR TOTAL DISSOLVED SOLIDS (TDS)

Total dissolved solid (TDS) is a measurement of all constituents dissolved in water. The principal inorganic anions dissolved in water include carbon, chloride, sulphates and nitrites. The principal cations are sodium, potassium, calcium and magnesium. For fresh water, salinity and TDS are equivalent. TDS provides a useful index to the suitability of a water supply for use.

Biological significance of TDS is that it serves as source of ions to the body and it also indicates the level of manufacturer’s quality.

Aim: To determine the concentration of total dissolved solids in water samples

Procedure:

TDS A

10ml of the water  sample  was transferred into a titration bottle, 3 drops of methyl orange indicator was then added and titrated using TDS reagent A (0.75% HCL). End point colouration was yellow. 

TDS B

10ml of the water sample  was transferred into a titration bottle and passed through an ion exchange resin to exchange the associated cations  (Na⁺, Ca²⁺ , Mg²⁺ etc for H⁺), 3 drops of methyl orange indicator was then added and titrated using TDS reagent B (1% NaOH). Endpoint colouration was reddish brown.

Result:

Amount of TDS in the sample =A+B.

Total dissolved solid (TDS) A = 1.8 ppm

Total dissolved solid (TDS) B = 0.8 ppm

Total dissolved solid in the sample = 1.8 + 0.8 = 2.6ppm

Table 13: Table Showing the Concentration of TDS in Water Samples

Sample	Conc. Of TDS(ppm)	Acceptable conc. Of TDS(ppm)	Comment
Sample 1	2.6		Satisfactory
Sample 2	15		Satisfactory

Discussion:

The samples all have concentrations of total dissolved solid TDS below the maximum range as shown in the  table above; it can therefore be declared satisfactorily and free for human consumption. 

TEST FOR pH

The measurement of pH plays an important role in quantifying and controlling acidity and alkalinity levels for industry and research. pH is a measure of the acidity or alkalinity of a substance. It is mathematically defined as the negative logarithm to base 10 of hydrogen ion concentration, as shown below:

                        pH = -log [H⁺]

It is sometimes referred to as the power of hydrogen ion in a solution.

By using a pH meter, the exact pH levels of water (or any other substance) can be most precisely determined. pH values generally range from 0 to 14, with a pH value of 7 being the neutral point, or the value of pure water. The pH values above the neutral point represent increasing alkalinity whereas those below the neutral point represent increasing acidity.

 Principle:

To measure pH, the meter receives a millivolt signal from a glass bulb electrode that is sensitive to hydrogen ions. Therefore the potential developed at the glass bulb is directly related to the pH of the substance. The glass bulb electrode is always paired with a reference electrode which completes the electrical measuring circuit and provides a stable reference point. These two electrodes can be separated, or they can be joined to create a combination electrode. The combination glass electrode makes a single connection to the pH meter which converts the electrode’s millivolt output to pH units, and displays the result.

The pH of water is a measure of the acid-base equilibrium of hydrogen ions in water, and it is controlled by the carbon dioxide-bicarbonate-carbonate (CO₂/HCO₃^2-/CO₃^2-) equilibrium system. Increase in carbon dioxide level beyond a certain limit causes a decrease in pH of water.

Aim: To determine the pH of water sample

Materials: pH meter, cotton wool, beakers, buffer 4.01, 7.00 & 10.00, and distilled water

Procedure:

-       To calibrate the pH meter by a three-point standardization:

The pH meter was turned on. The Mode knob was set to pH position and the %Slope knob 100%. The electrode and the ATC (Automatic Temperature Compensation) probe were immersed into the beaker containing buffer 4.01, with moderate stirring. The standardize knob was adjusted until the display indicated the pH of the buffer (4.01). The electrode and ATC probe were removed and cotton wool was used to carefully clean-up residual liquid. The pH meter was further standardized using the remaining two buffers, one after the other, by immersing the electrode and ATC probe into the buffer and adjusting the standardize knob.

-       To perform pH test on the sample:

The electrode and ATC probe were rinsed with distilled water and residual water was carefully cleaned up using cotton wool. These were then immersed into the beakers containing the samples, one after the other, in each case rinsing the electrode and ATC probe with distilled water before testing the next sample. The most stable pH reading was taken as the pH value for the sample in each case.

Result:

Table 14: Table Showing the pH values of the analysed samples at room temperature

Name of Sample	pH
Sample A	7.5
Sample B	6.8
Sample C	8.8

Discussion:

From the above result, Samples A and B passed the pH test while sample C failed because the accepted range of pH used by the agency, for consumable water is 6.5-8.5.

CHEMICAL PARAMETERS

 TEST FOR FREE DISSOLVED CARBONDIOXIDE (CO₂) IN WATER.

The amount of CO₂ in water depicts the number of micro organism present in the water sample or body, since CO₂ is a by-product of micro-organisms metabolic activities. They may serve as an indication of contamination by micro organisms. Prolonged exposure to moderate concentrations of carbon dioxide either in dissolved form or gaseous state is hazardous to health and can cause acidosis. An adverse effect of carbon dioxide on calcium – phosphorus metabolism is that it increases calcium deposit on soft tissues. Carbon dioxide is toxic to heart and causes diminished contractile force. It is also carcinogenic to the body (WHO water quality, 2004).

CO₂ in water behaves or acts as an acid as shown in the reaction below:

H₂O_(l)   +   CO_2(g)                                 H₂CO_3(aq)

            H₂CO_3(aq)                                             H⁺_(aq)   +   HCO₃^-_(aq)

            HCO₃^-_(aq)                                              H⁺_(aq)   +   CO₃^2-_(aq)

Free CO₂ in water was determined by titrating water sample with an alkaline solution (0.1% NaOH) using phenolphthalein indicator until a faint pink colour appear. The analysis was done immediately after the sample was open due to the ability of atmospheric CO₂ to dissolve in water, as it is denser than air which could lead to wrong results.

Aim: To determine the concentration of free dissolved CO₂ in water samples                 

Materials: Carbon dioxide LaMotte test kit and titration bottles with caps

Procedure: 20 ml of the water sample was transferred into a titration bottle. 2 drops of phenolphthalein as an indicator was then added and titrated against CO₂ Reagent A(0.1% NaOH). A pink colouration shows an end point has been reached.

 Result:

 Table 15: Table Showing the Results of the Concentration of Free CO₂ in Water Samples

Sample	Conc. Of CO2 (ppm)	Acceptable conc. Of CO2(ppm)	Comment
Sample 1	12	50	Satisfactory
Sample 2	10	50	Satisfactory

Discussion:

The samples all have concentrations of free dissolved CO₂ below the maximum range as shown in the table above: it can therefore be declared satisfactory and free for human consumption.

  TEST FOR NITRITE IN WATER.

Nitrite represents an intermediate stage in the nitrogen cycle; its presence is an indication of organic waste contamination which could be from sewage, industrial waste, manure etc. Nitrate may also leach into the ground water from artificial nitrogen fertilizer and find its way into public water and be converted to nitrite under anaerobic condition such as in the mouth when such water is taken. Nitrite causes damage to the body organs and when absorbed into the blood stream, combined with hemoglobin to form a blue pigment called methaenoglobin which reduces the ability of the blood to transport oxygen, and may result to a life threatening condition in babies called blue baby syndrome (methemoglobinemia).

Chemistry of qualitative test for nitrite

Aim: To qualitatively and quantitatively determine nitrite in water samples

Procedure

Qualitative Nitrite Test: 50ml of the water samples were measured into conical flasks, 1ml each of both Nitrite A and B were added, shaken and left to stand for ten minutes. The presence or absence of a pink coloration was noted which indicated the presence or absence of nitrite ions in the water samples.

Quantitative Nitrite Test: 10ml of sample water was measured in a cuvette and was scanned as blank. 5 ml water was removed from cuvette and made to mark with mixed acid reagent. Two (2) spoons of color developing reagent were then added. The mixture was left for 5 minutes before scanning in the spectrophotometer. The concentration was taken and recorded. 

Result:

Table 16: Table Showing Result for Qualitative Test of Nitrite

SAMPLE	Color formed	Inference	Comment
A	Colorless	Nitrite absent	Satisfactory
B	Pink color	Nitrite present	Unsatisfactory

Amount of Nitrite (PPM) = Nitrite conc. x 3.3

Amount of Nitrite (sample A) = 0.01 x 3.3

Amount of Nitrite (sample A) = 0.033ppm

Amount of Nitrite (sample B) = 0.09 x 3.3

Amount of Nitrite (sample B) = 0.297ppm

Discussion:

The maximum limit for nitrite is 0.2ppm. Thus, sample A has passed nitrite test while sample B has failed.

 TEST FOR CHLORIDE IN WATER.

Chloride is formed when chlorine gas dissolves in water. Chlorine is added to water during purification process because of its sterile ability to kill and destroy microorganisms. Chlorine is also a dietary mineral needed by the body for optimum health. Chlorine channels are important for setting cell resting membrane potential and maintaining proper cell volume. These channels conduct Cl^- as well as other anions such as NO₃^- , HCO₃^- etc.

Determination of chloride ion concentration therefore is by titration method. Silver ions react with chloride ions to form practically undissociated silver chloride. Excess ion together with potassium chromate (K₂CrO₄) as indicator form reddish-brown complex compound of silver chromate as shown in the reaction below:

Ag⁺ + Cl^-                                                            AgCl

2Ag⁺   + CrO₄^2-                                                   Ag₂CrO₄

Aim: To determine the concentration of chloride in water samples

Procedure:

15ml of sample was transferred into a titration bottle and 1drop of phenolphthalein was added, 3 drops of reagent A (5% potassium chromate) was then added into the titration bottle to give a yellow colour. It was then titrated using chloride reagent B (AgNO₃). Orange–brown colouration depicts its end point

 Result:

Table 17: Table Showing The Results of the Concentration of Chloride in Water Sample

Sample	Conc. Of Cl- in sample (ppm)	Acceptable Conc. Of Cl- in water (ppm)	Comment
Sample 1	101	200	Satisfactory
Sample 2	75	200	Satisfactory

 Discussion:

From the results obtained above, the concentrations of chloride in the water samples are within the range hence declared satisfactory and should be permitted for human consumption

TEST FOR SODIUM IN WATER                                                                                        

Sodium is a mineral element and an important element of the human body system. It controls fluid in the body and helps maintain the acid base level. About 40 % of the body’s sodium is contained in the bones, some are found within organs and cells and the remaining 55% is found in the blood plasma and other fluids outside cells. Sodium is important in proper nerve conduction, the passage of various nutrients into cells, and the maintenance of blood pressure.             

Although sodium is considered nontoxic it has been associated with high blood pressure. (US FDA)

Aim: To determine the concentration of sodium in water samples.

Procedure:

Sodium A

10ml of the water sample was transferred into a titration tube. Then 2-3 drops total alkalinity indicator (0.01%NaOH, Bromocresol green methyl red) was also added and it was titrated using reagent A (H₂SO₄). A brick-red colouration depicts its end point and the reading for sodium A is taken.

Sodium B

10ml of water sample was passed through a resin to de-ionize it and 2 drops of total alkalinity indicator was added and titrated using reagent B (0.1N NaOH). Expected end point colouration is blue-black.

Sodium C

Using a measuring cylinder 12.9mls of sample was transferred into a titration bottle and 5 drops of hard #5(1% Na₂SO₄, <1%NaOH, 4% sodium borate) was added and 1 tablet of hard #6(99% KCl). It was then titrated using reagent C (hard #7). A clear blue colouration depicts its end point.

 Result:

Maximum concentration of sodium in water sample  150 ppm

Total sodium in sample = (A+B) – C × 0.46

Sodium A = 50 ppm

Sodium B =70 ppm

Sodium C = 30 ppm

Total sodium in water = (50 + 70) – 30  0.46

                              = 90  0.46

                             = 41.4 ppm

Discussion:

The concentration of sodium present in the water samples is below the acceptable range set by NAFDAC. Hence it is declared satisfactory and should be permitted for human consumption.

TEST FOR TOTAL ALKALINITY IN WATER

Alkalinity is the degree to which water can neutralize acid. In other words, it is a measure of the buffering capacity of water.

Aim: To determine the concentration of total alkalinity in water samples

Procedure:

100ml of the water sample was transferred into the titration bottle, then 3 drops of phenolphtalein  indicator was added, followed by 3 drops of methyl orange. It was then titrated with 0.02N sulfuric acid.  When there is a colour from yellow to sunset yellow, it depicts the end point.

 Result:

 Table 18: Table Showing the Results of the Concentration of Total Alkalinity in Water Samples

Sample	Conc. Of total alkalinity (ppm)	Acceptable range (ppm)	Comment
Sample 1	49	100	Satisfactory
Sample 2	85	100	Satisfactory

Discussion:

Total alkalinity test determines the alkaline content of a water sample and the result gotten correlate with the pH of the sample. That is, once a sample has a total alkalinity of more than 100 ppm, and then such water samples are acidic when read on the pH meter. Therefore, sample 1& 2 have total alkalinity values less than the maximum hence the water sample is declared satisfactory. 

            FOOD UNIT

            INTRODUCTION

The Food Laboratory in NAFDAC is primarily concerned with the qualitative and quantitative test on all food samples brought in for analysis in order to certify the integrity of such product and to determine its fitness for consumption by the general public. The samples usually brought into the Food unit could be for the purpose of registration, renewal, routine check, pack extension or investigation. The particular analysis carried out on each sample depends on the classification or nature of the food sample and the ingredient(s) used in producing such food. Generally, most food samples undergo the proximate analysis which includes test for the moisture content, ash content, protein, fat, total carbohydrate which is further used to determine the total energy found in such sample(s).

Other analysis carried out in this unit includes test to check for adulteration, spoilage, additive or fortification in Milk and Milk products, oils and fats, fruits, flour, confectionaries, juices, cakes, icing sugars, custards, beers and wine products as well. The analyses are classified as either quantitative or qualitative.

3.3.2 CATEGORIES OF FOOD SAMPLES ANALYSED

Table 19: Table Showing Categories of Food Samples Analysed in Food Unit

S/N	Category of Food Sample
1	Milk and Milk Products
2	Meat and Meat Products
3	Fish and Fish Products
4	Fruit and Fruit Products
5	Cereals and Cereal Products
6	Tea, Coffee, and Cocoa Products
7	Fats and Oils
8	Baby and Infant Formula
9	Bread
10	Carbonated Drinks
11	Salt
12	Seasoning and Flavours
13	Sugar
14	Alcoholic Beverages
15	Melamine

            PARAMETERS DETERMINED ACCORDING TO FOOD TYPE

Parameters to be analyzed in each category of food are known based on the raw materials used and the manufacturer’s claim; hence parameters to be analyzed in each category differ. A common analysis done in almost all food categories is the proximate analysis.

Table 20: Table Showing Parameters Most Commonly Determined in Food Unit

S/N	FOOD PARAMETER	FOOD TYPE
1	Net Content	All packaged food types
2	pH	Yoghurt, fruit juice
3	Total Acidity	Yoghurt, fruit juice
4	Moisture Content	All food samples in solid form
5	Total Solids	All food samples in liquid form
6	Ash Content	Cereals, yoghurt, honey, fruit juice, tea
7	Fat Content	Yoghurt, cereals,
8	Protein	Cereals, yoghurt
9	Milk Solid Non-Fat (MSNF)	Yoghurt
10	Sucrose	Yoghurt, fruit juice, whisky/wine
11	Colour	Fruit juice
12	Fruit Juice Content	Fruit juice
13	Bromate	Bread, chinchin
14	Vitamin A	Cereals, oil
15	Alcoholic Strength	Whisky/Wine
16	Oil Adulteration	Oil
17	Esters	Whisky/Wine
18	Peroxide Value	Oil
19	Benzoic Acid	Yoghurt
20	Iodine Value	Salt

Other food parameters include saponification value, unsaponification value, acid value, acid insoluble, water extractible, caffeine content, matter volatile, mineral in oil, ascorbic acid, alcoholic content, melamine, fixed acidity, dextrose, monosodium glutamate, cocoa content, lycopene content,etc.

 PROXIMATE ANALYSIS

Proximate analysis is an analysis that is performed based on the six (6) classes of food. Therefore, in food unit, this analysis is carried out to determine:

-       Moisture Content

-       Fat Content

-       Ash Content

-       Protein Content

-       Total Carbohydrate

 MOISTURE CONTENT

 Principle:

Moisture content is the amount of moisture present in a food product. This test is based on LOD (Loss on drying) at an oven temperature of 105^oC. Besides water, other matter volatile at 105^oC will also be lost.

Aim: To determine the moisture content in food samples

Materials: Analytical balance, platinum dish, oven

Procedure: A dried cooled platinum dish was weighed (w₁) and  2g of the test sample was introduce   into the dish and weighed accurately (w₂). The dish was transferred and its content into an oven at 105^oC to dry for about 2 hours and the dish was removed and weighed (w₃).

 Result:

Weight of dish (w₁) =37.7775g

Weight of dish and sample (w₂) =40.7791g

Weight of dish and sample after drying in oven (w₃) =38.6997g

% moisture = (w₂ –w₃) / (w₂ –w₁) x 100 

               =    37.7775g  100

                  =  100

                  = 0.6927  100

                  =69.27%

 Discussion:

From the result obtained it could be seen that the food sample contains more moisture content  in it which may not be proper as it can provide a conducive environment for bacterial growth.

 ASH CONTENT

Principle:

The organic component of the food is burnt off in air; the residue is ash which consists of inorganic components in the form of their oxides.

Aim: To determine the ash content in food samples

Materials: Analytical balance, platinum dish, furnace

Procedure: A dry cool platinum dish was accurately weighed as (w₁) and about 2g of the food sample was spread evenly in the dish and weighed as (w₂). If the substance is moist, dry using water bath and char over hot plate in the fume cardboard until no more sooth is given off. Then it was transferred using a pair of tongs into a muffle furnace at 550^oC until fully ashed (colour changes to gray) and weigh as (w₃).

% Ash =  -  × 100

Result:

W₃ = 35.9516g

W₂ = 38.9980g

W₁ = 35.9503g

%Ash =  100

          =  100

 %Ash = 0.0426555%

Discussion:

The inorganic component of this food material is very low, as it represents less than 0.5% of the total food material which may imply low concentrations of minerals.

 NITROGEN AND CRUDE PROTEIN

Principle:

Kjeldahl nitrogen method is one of the most widely recognized methods employed for crude protein determination. This method involves three (3) stages as follows:

I. The Digestion Process

A general equation for the digestion process is shown below:

     Organic N + H₂SO₄              (NH₄)₂SO₄ + CO₂ + H₂O + Other by-productsfrom sample matrix

Essentially, digestion converts organic nitrogen (N) to ammonia (NH₃) and other organic matter to carbon dioxide (CO₂) and water (H₂O).Antibump is added to reduce bumping of the digestion mixture, as boiling concentrated H₂SO₄ solution results in explosion.

H₂SO₄ is an oxidising agent which digests the food by liberating organic nitrogen in the tri-negative state (N^3-). The liberated nitrogen is protonated to NH₃ , and then to ammonium ion (NH₄⁺⁾. The NH₄⁺ binds to the sulphate ion (SO₄^2-), thereby disallowing the loss of organic nitrogen as NH₃.

CuSO₄ salt serves as a catalyst, which functions by the lowering of activation energy of the reaction. A combination of titanium (IV) oxide, TiO₂, and CuSO₄ salts provides the most effective catalytic activity.

II. The Distillation Process

The 50% NaOH solution added makes the digest strongly alkaline, thereby liberating NH3 gas as in the equation below:

     (NH₄)₂SO₄(aq) + NaOH(aq)                      2NH_3(g) + Na₂SO_4(aq) + 2H₂O_(l)

The liberated NH₃ gas flows and condenses into the receiving flask via the condenser tip, which has been submerged in the acidic receiving solution.

Again NH₃ is trapped in acidic medium and, hence, stays in solution as a 1:1 ammonium borate complex, as shown in the equation below:

NH_3(g) + H₃BO_3(aq)                 NH₄⁺:H₂BO₃^-(aq) + Excess boric acid

The colour of the receiving solution changes from pink to blue as the NH3 collects.

N.B: The exact concentration of H₃BO₃ is not relevant because the titration directly measures the amount of NH₃ in the distillate by neutralising the complex.

III. The Titration Process

Titrating the solution with 0.05M H₂SO₄ solution exactly neutralises the ammonium-borate complex, with the consequent observation of a reverse colour change (blue to pink) as shown in the equation below:

2NH₄H₂BO_3(aq) + H₂SO_4(aq)                          (NH₄)₂SO_4(aq) + 2H₃BO_3(aq)--------------(4)

Aim: To determine the percentage of protein in food samples.

Materials:Kjeldahl apparatus, Top load balance, Digestion and Distillation set-ups, Retort stand with clamp, Burette, 250-ml Conical flask, Antibump, Copper (II) tetraoxosulphate (VI)(CuSO₄) salt, Concentrated tetraoxosulphate (VI) acid (H₂SO₄), 0.05M tetraoxosulphate (VI) acid, 2% Trioxoborate (III) acid (H₃BO₃), 50% Sodium hydroxide (NaOH) solution, Screened methyl red indicator, Distilled water

Procedure:

I. Digestion

1g of the sample was weighed and transferred into a digestion flask.A spatula-full of CuSO₄ salt was added, as well as 25ml of concentrated H₂SO₄ solution.A significant amount of antibump was added and the digestion flask was connected to a glass tube (with a condenser neck-off) whose joint was rubbed with vaseline.The whole digestion set-up was connected to the lower chamber of the Kjeldahl apparatus and the heat knob was turned on. (Sample was heated until a clear solution was obtained). 

II. Distillation

After complete digestion, 200ml of distilled water was added, as well as 85ml of 50% NaOHsolution , to the digest. The measuring cylinder used to measure the NaOH solution, was rinsed with 50ml distilled water and the content transferred to the digestion flask.Antibump was added and the distillation set-up was connected to the upper chamber of the apparatus.50ml of 2% H₃BO₃ was measured and transferred into a receiving flask.3 drops of screened methyl red indicator were added.The receiving flask was placed at the middle chamber of the apparatus, and the delivery tube was immersed into the pinkish solution in the receiving flask.The heat knob of the upper chamber was turned on for distillation to begin, and about 200ml of the resulting bluish solution was collected for titration.

III. Titration

After complete distillation, the bluish receiving solution was then titrated with 0.05M H₂SO₄ solution until a permanent pink colour was observed, which indicated the end point.

Result:

Titre Value, TV = 6.5ml

% N = TV x 0.0014    x 100

                  W

W is the weight of sample taken.

                                                                            = 0.91%

\% Nitrogen = 0.91%

                       

Percentage protein, %P, is calculated by multiplying the %N by the Jones factor, F, corresponding to the protein source, as shown below:

% Protein = %N ´ F

Where F = 5.70 for wheat flour

\ % Protein = 0.91% ´ 5.70 = 10.44%

\% Protein = 5.2%                           

 Discussion:

Since percentage protein of the sample was found to be 5.2%, it implies that the sample passed because the value is above the minimum limit for the food type, which is 5%.

 FAT CONTENT 

Principle:

Heating with concentrated HCl dissolves the fat and other materials the fat is then extracted with suitable solvents (diethyl ether). This method is called Werner schmid method.

Aim: To determine the fat content in food sample

Materials: Analytical balance, water-bath, oven, separating funnel, measuring cylinder, boiling tube, conical flask, concentrated HCl solution, ethanol, diethyl ether

Procedure: 2g of the sample was weighed into a boiling tube. 10ml of conc. HCl was added and put in a boiling water bath until solid particles dissolve and until mixture becomes brown. It was then taken off and cooled, then transferred into a separating funnel. 10ml of ethanol and 30ml of diethyl ether were added and shaken to dissolve;   it was then allowed to stand for some minutes so as to separate.

A clean dried conical flask (w₁) was weighed and the ether layer was transferred into the flask. The extraction was repeated twice with 25ml of diethyl ether and the extract was evaporated in a water bath. The fat was dried at 105⁰C in an oven, cooled and weighed (w₂).

 Result:

Weight of sample (w) = 2g

Weight of conical flask (w₁) =36.4310g

Weight of sample and conical flask (w₂) =36.6757g

% Fat = (w₂-w₁) / (w) x 100 

         =  100

         = 0.12235  100

         = 8.157%

TOTAL CARBOHYDRATE:

% Total Carbohydrate = 100 – (%moisture content +%ash +%protein + %fat)

                     = 100 – (69.27 + 0.042655 + 10.44 + 8.157)

                     = 100 – 87.9097

                     = 12.09%

ENERGY (kcal)

Energy (Kcal) = (total carbohydrate×4) + (protein×4) + (fat×9)

                     = (12.09×4) + (10.44×4) + (8.157×9)

                      =48.36 + 41.76 +73.413                   

                      =163.533Kcal 

Discussion:

From the percentage of all nutrients measured in the food sample, it is obvious that the food has its primary nutrients as carbohydrate and proteins while fat is the secondary nutrient.

To determine whether this food sample is satisfactory for human consumption, it must first be compared with the manufacturers claim and if it falls within the range, then it has passed or else taken out of market.

OTHER PARAMETERS ANALYSED IN DIFFERENT FOOD SAMPLES.

 BENZOIC ACID TEST:

Benzoic acid (C₆H₅COOH) is the simplest of the aromatic carboxylic acids, a family of organic compounds containing the carboxyl (-COOH) group. It occurs in the form of white crystal needles or thin plates. Many naturally occurring plants contain benzoic acid, including most types of berries and the natural product called gum benzoin, a plant common to the islands of java, Sumatra and Borneo. Benzoic acid and its sodium and potassium salts may pose moderate health hazards to those who work directly with them. They may cause skin, nose and eye problems if inhaled or deposited on the body. NAFDAC requirement of benzoic acid in food products is ≤ 300ppm

Aim: To determine the benzoic acid content of a sample

Materials: 250ml conical flask, 100ml measuring cylinder, separating funnel, beakers, retort stand, saturated NaCl, 10% NaOH, 25% HCl, blue litmus paper, red litmus paper, absolute ethanol, chloroform (CHCl₃).

Procedure: 

10g of NaOH was accurately weighed and  dissolved in 100ml volumetric flask to obtain 10%NaOHand the NaCl was dissolved in distilled water to obtain saturated NaCl. 50ml of the sample was measured using a 50ml measuring cylinder and was transferred into 250ml volumetric flask. 50ml of the saturated salts solution was added and neutralized it with 10% NaOH. Red litmus paper was used to confirm if the alkaline solution has really been obtained and was made up to the mark on the 250ml volumetric flask with remaining saturated NaCl and was allowed to stand for 2 hrs. It was then filtered and 100ml of the filtrate was taken into a separating funnel and 1ml of 25% HCl was added and 5ml excess HCl to neutralized mixture. It was then confirm with blue litmus paper which changes to red to confirm whether neutralization reaction is complete. It was then extracted with 40, 30, 30,20ml of chloroform (CHCl₃) and the lower layer was collected in a conical flask and evaporated to dryness using a hot water bath. 30ml of ethanol was used to dissolve the residue in the conical flask dehydrated. It was then titrate against with 0.05M NaOH, using phenolphthalein as indicator. A brown colour formation was the end point depicted.

Result:

After the analysis, the following result was obtained

Titre value (T.V) = 1.2ml

                                                            = 146.4ppm

Discussion:

From the result obtained above, the sample is said to have passed the analysis because NAFDAC requirement of benzoic acid in food products is £ 300ppm.

SUGAR DETERMINATION IN DIFFERENT FOOD SAMPLE

Principle:

Sugar in yoghurt is lactose sugar which is broken down by the bacteria culture to glucose and galactose, this breakdown gives a sour result. Sugar (sucrose) is added to it to sweeten it.

Lane and Eynon’s method is the method employed for sugar determination.

Aim: To determined %content of both reducing sugar and invert (total) sugar in different food samples

Materials: Conical flask, activated charcoal, filter paper, funnel, water bath, burette, measuring cylinder, retort stand, clamp, conical flask, heating mantle, pair of tongs, methylene blue indicator, Fehling I & II solution, concentrated HCl solution, 50% NaOH solution

Procedure:

25ml of the sample was measured using a measuring cylinder and was transferred into a 250ml volumetric flask and activated charcoal (which serves as a clarifying agent) was also added. It was then made up to volume with distilled water. The mixture was then filtered using filter paper and a funnel and the clear filtrate was collected in a conical flask.

i.        Reducing sugar

From the filtrate above, 50ml was transferred into a burette. 10ml of a mixture in equal ratio of Fehling I & II was prepared and 15ml of the clear filtrate sample contained in the burette was run into the mixture of Fehling I & II contained in a conical flask and was placed in a heating mantle to boil. After boiling the colour changed brick red, 3-5 drops methylene blue indicator was added, the colour remains unchanged. Failure of colour to change to blue shows high concentration of sugar which requires dilution of stock. But if it changes to blue on addition of methylene blue indicator, allow boiling and further titrating with mixture until brick red coloration is obtained. For this sample, dilution was carried out by diluting 50ml of the remaining filtrate in 100ml volumetric flask, it was then made up to volume with distilled water (50%v/v)

Result:

After the analysis, the following result was obtained

Titre value (T.V) = 27.2ml (1 dilution)

Where;

                    R.S = Reducing sugar =?

DF = Dilution factor =

EQV = Equivalent = 51.4 (according to Codex Alimenterious Commission)

T.V = titre value = 27.2ml

            =3.78%

ii.         Invert sugar (Total sugar)

From the filtrate obtained earlier, 50ml of the clear filtrate was transferred into a 100ml volumetric flask. 10ml of conc. HCl was also added. It was then allowed to stand for 24hours (1 day). After 24hrs, 50% NaOH and 3 drops of phenolphthalein indicator were added into the filtrate to neutralize it and give a pink colour, it was then made up to mark with distilled water.  50ml of the filtrates was transferred into a burette. 10ml of a mixture in equal ratio of Fehling I & II was prepared and 15ml of the clear filtrate sample contained in the burette was run into the mixture of Fehling I & II contained in a conical flask and was placed in a heating mantle to boil. After boiling the colour changed brick red, 3-5 drops methylene blue indicator was added, the colour remains unchanged. Failure of colour to change to blue shows high concentration of sugar which requires dilution of stock. But if it changes blue on addition of methylene blue indicator, allow boiling and further titrating with mixture until brick red coloration is obtained. For this sample dilution was carried out by diluting  50ml of the remaining filtrates in 100ml volumetric flask, it was then make up to volume with distilled water (50%v/v)

Result:

After the analysis, the following result was obtained

Total (invert) sugar

Titre value (T.V) = 25.2ml (1 dilution)

Where; T.S = Total sugar (invert)

DF = Dilution factor = EQV = Equivalent = 51.2(according to Codex Alimenterious commission (CAC)

T.V = Titre value = 25.2ml

                                   

      = 8.13%

%Sucrose

          % Sucrose = (%invert sugar - %reducing Sugar) ´ 0.95

                           = 8.13 - 3.78) ´ 0.95

                           = 4.13%

 TEST FOR BROMATE IN BREAD

Principle:

Bromate in bread is determined by redoximetry, which involves the oxidation-reduction reaction taking place between the bromate ion (BrO₃^-) and the iodide ion (I^-). The bromate in bread is reduced in acidic medium by potassium iodide to give bromide ion (Br^-) and iodine. In the presence of starch, a blue-black complex is formed by reaction between the iodine formed and starch, thereby indicating the presence of bromate. The reaction is shown below:

        BrO₃^-_(aq)   +   6I^-_(aq)    +    6H⁺_(aq)                                       Br^-_(aq)    +    3I_2(s)    +    3H₂O_(l)

Aim: To check for the presence of potassium bromate (KBrO₃) in bread.

Materials: Crucible, 1% starch indicator, 10% KI solution, 1:7 HCl solution

Procedure: The inner portion of the bread sample was taken and put into a crucible, and then 1% of starch indicator was added to mash the sample using a pasture pipette; and equal volume of 10% KI and 1:7HCL was also added to the bread sample.

Result:

Table 21: Table showing the presence or absence of potassium bromate (KBrO₃)   in bread

Sample	Status	Colour if present	Comment
Sample1	Present	Blue black	Unsatisfactory
Sample2	Absent	No colour change	Satisfactory

 Discussion:

Under standard conditions KBrO₃ is a white crystalline powder freely soluble in water. In a dilute aqueous solution, in high concentration, KBrO₃ strongly irritates the gastric mucous membrane, leading to nausea and sometimes vomiting.

Sample 1 developed a blue black colour which signifies the presence of KBrO₃ which is not safe for human consumption but sample 2 remained colorless which signifies the absence of potassium bromate (KBrO₃), hence can be consumed.

COLOUR DETERMINATION IN ORANGE DRINKS

Aim: To confirm the presence of color additive in orange juice.

Reagents: Amyl alcohol (propanol) and concentrated HCl.

Materials: Separating funnel, conical flask, retort stand, beaker and UV-Spectrophotometer.

Principle:

Organic sources of colors such as quinine yellow and sunset yellow are soluble in organic solvents such as amyl alcohol and hexane. At such, they are extracted with such solvents under acidic condition provided by the conc. HCl.

Procedure:

20ml of the sample was taken into a separating funnel; unto it 20ml of amyl alcohol was added. Then 10ml of concentrated HCl was added, the separating funnel was closed and was properly shaken. The amyl alcohol layer was carefully extracted and the absorbance taken using UV-Spectrophotometer at the wavelength of 300nm- 700nm and amyl alcohol as blank.

Result:

There was a peak observed on the graph at 450nm.

Discussion of Result:

This shows the presence of color additive in the sample probably tetrazine yellow, because yellow-tetrazine shows high absorbance at 450 wavelength. Therefore the sample is possibly adulterated. Thus, it has failed color analysis.

DETERMINATION OF TECHNICAL INVERT SUGAR IN HONEY

Natural honey must not contain technical invert sugar. Feihe’s test detects the presence of 5-hydroxy methyl furfuraldehyde which is present in invert sugar prepared by non-enzymatic method.

Method: Fiehe’s test (1890)

Aim: To confirm the presence of added sucrose in Honey.

Materials: Separating funnel, crucible, measuring cylinder, weighing balance and beaker, resorcinol solution, diethyl ether.

Procedure: 10ml of the Honey was measured into a crucible. 5ml of diethyl ether was added and stirred thoroughly. 2ml of the ether extract was evaporated over the water bath. 1ml of 1% resorcinol solution in HCl was added to the residue to observe any color change present.

Observation: A cherry red colour was observed. This therefore, confirmed the presence of added technical invert sugar in the Honey sample.

Discussion of Result:

The honey sample has failed this parameter because it is not expected of honey to contain any sucrose, but only its natural fructose.

MYCOTOXIN UNIT

INTRODUCTION:

Mycotoxin laboratory is primarily concerned with the analysis of food products for mycotoxins. It also carries out the analysis of non- nutritive sweeteners, vitamins and melamine in milk and milk products. Mycotoxin is a toxic secondary metabolite produced by organisms of the fungus kingdom commonly known as mould. Mycotoxins are major contaminant of food crops in farmlands which usually cause spoilage which decreases yield in time of harvest. Mycotoxin has always been a threat to health as far back as 18^th – 19^th centuries when it manifested as “Ergotism”, in two forms which are dangerous, affecting the blood supply to extremities and convulsive which affects the central nervous system. Responsible for this 18^th and 19^th century damage to humanity are Ergot alkaloids, these compounds produced as toxic mixture of alkaloids in the sclerotia of species of claviceps which are pathogens of various grass species. Below are various mycotoxins and their precursors. Recently, aflatoxin B₁ have been threat to humanity in that it is a potent carcinogen and has been directly related to adverse health effects such as liver cancer in many animal species. Aflatoxins are largely associated with commodities such as cotton, peanuts, spices, maize, groundnut, barley wheat etc.

 STEPS FOR MYCOTOXIN ANALYSIS

Sampling: This includes grinding/homogenizing using warring blender and weighing of the sample using top load balance.

Extraction: Removal/separation of toxin from matrix into extraction solvent. This can be achieved using ultraturrax or warring blender. Mycotoxin does not dissolve readily in water but does in organic solvents such as chloroform, methanol, and acetonitrile and dichloromethane or a mixture of these solvents and water.

Clean-Up: Removal of other materials or interferences (co-extractives) that was extracted along with the toxins example is colour. This can be achieved using mycosep (multifunctional) clean-up columns or immuno-affinity clean-up columns (when using HPLC).

Evaporation: Since mycotoxins occur at nanogram levels, there is need to reduce the amount of solvent to be able to detect the toxin therefore concentrating the volume of clean-up extract to a smaller one. This can be achieved using rotary evaporator or heating block with the aid of nitrogen stream.

 AFLATOXINS

Aflatoxins are toxic and carcinogenic. They are secondary metabolites of the fungi Aspergillusflavusand Aspergillusparasiticus. There are four classes of aflatoxins: B_1, B_2, G_1, G₂, which are named according to their respective fluorescent properties. Aflatoxin   B₁ is the most frequently encountered of the group and the most toxic. Aflatoxins can be found mainly in cereals, corn, peanuts, cottonseeds and nuts.

Aflatoxins can cause liver disease in animals and may cause decreased production (milk, eggs, animal weight, etc. Aflatoxin B₁ is human carcinogenic and may contribute to human liver cancer. Consequently, NAFDAC has set action limit on food products likely to contain aflatoxin:

For ‘ready to eat’ food products = 4ppb

For food products for further processing is 10ppb.

AFLATOXIN ANALYSIS USING ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

Principle:

The assay is a direct competitive enzyme linked Immunosorbent assay (ELISA). Aflatoxins are extracted using 70% methanol. The extracted sample and enzyme conjugated aflatoxin are mixed and added to the antibody coated micro wells. Aflatoxin in the sample and control standard are allowed to compete with enzyme conjugated aflatoxin for the antibody binding sites. After washing, an enzyme substrate is added and a blue color develops. The intensity of the color is inversely proportional to the concentration of aflatoxin in the sample. A stop solution is then added which changes the color to yellow. The micro wells strips are measured optically using the ELISA reader with an absorbance filter of 450nm.

Aim: To determine the quantitative level of total aflatoxin (B₁, B₂, G₁ and G₂)

Procedure:

A representative sample was obtained and grinded in a blender so that 75% will pass through a 20 mesh screen, then 5g of the sample was measured and 25ml of 70/30 (v/v) methanol/water extraction solution was added to sample. Extraction is always done in the ratio of 1:5 (w/w) of sample extraction solution respectively. The sample was then vigorously shaked using orbital shaker at 250 rpm for 3mins.The sample was then allowed to settle, and then filter the top layer of extract and filtrate was then collected.

200µl of conjugate was pipetted using a micro litre pipette and dispensed into the mixing wells, 100µl of standard and sample was pipetted and added to mixing wells. Each well was mixed carefully by pipetting up and down 3 times and immediately 100µl of contents was transferred from each dilution well into a corresponding antibody coated micro well. It was then incubated at room temperature for 15mins. The content of the antibody coated micro well strips was then emptied into waste container and washed by filling with distilled water and then dumping the water from the micro well strips using a tissue. Repeat this 4 times. 100µl of the substrate was pipetted into each micro well stripand Incubated at room temperature for 5mins after which a blue colour is developed. 100µl of stop solution was measured and dispensed into each micro well strip. On adding stop solution colour changes from blue to yellow, micro well strip was then read using ELISA reader at the wavelength of maximum absorption of 450nm

Result:

Absorbance was 0.7060

Concentration was 13.3ppb

 Table 20: Table showing the absorbance and Concentration of the standards

Standard	Absorbance	Concentration
C1	2.334	0.0
C2	2.001	1.0
C3	1.828	2.0
C4	1.313	4.0
C5	0.827	10.0
C6	0.554	20.0

Discussion:

From the results obtained, the concentration of Total aflatoxin in kilishi was 13.3ppb and the acceptable range as set by NAFDAC for kilishi which is ready to eat food is 4µg/kg.

That is, the concentration of aflatoxin in kilishi exceeds the acceptable range, hence it is not advisable for consumption, and therefore the product is unsatisfactory and cannot be in the market for sale.

Mycotoxin has been termed as contaminants and carcinogenic especially to food grown on fields, both before and after harvest. Hence during storage of harvested food, it should be done appropriately to reduce any chances of Mycotoxin contamination. Consumers ofkilishishould also take heed, as they are prone to aflatoxin contamination which could have adverse health effect either now or in the long run.

MELAMINE ANALYSIS USING ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

                                   

Melamine is an organic base with the chemical formula of C₃N₆N₆ and the IUPAC name of 1, 3, 5triazine-2, 4, 6-triamine. Melamine became a topic of much discussion in early 2007 when veterinary scientists determined it to be the cause of hundreds of pet deaths because of pet food contamination. Prior to this report, melamine has been regarded as non- toxic or minimally toxic. Some manufacturers intentionally add melamine to their products as a means of improving nutritional value. It is a poison that can cause damage to the kidney (e.g. kidney stones). Some milk suppliers add melamine to artificially inflate the protein level. It is the high nitrogen- 66% that gives it the analytical characteristics of protein molecules. Only proper processing of these dairy products ensured reduction of melamine to minimal level allowed for consumption.

Aim: To determine the concentration of melamine in powdered milk sample

Principle:

The melamine assay is a direct competitive enzyme-linked immunosorbent assay (ELISA). It is extracted from a sample by vortex or sonication. The extracted sample and enzyme - conjugated melamine are pipetted into the antibody-coated micro well. Melamine from the samples and control standards are allowed to compete with enzyme-conjugated melamine for the antibody binding sites during the first incubation period. The micro wells are then washed with laboratory grade water. After the washing step, a substrate is added to the wells and blue colour develops. The intensity of the colour is inversely proportional to the concentration of melamine in the sample or standard. A stop solution is then added which changes the colour from blue to yellow. The micro wells strip are measured optically using the ELISA reader with an absorbance filter of 450nm.

Procedure:

1g of the sample was accurately weighed and was transferred into the centrifuge tube, and then 5ml of the distilled water was added into the samples in the centrifuge tube. The sample was centrifuge for 20min at 3000rpm using the centrifuge machine. The middle layer of extract and filtrate was then collected using 300ul micropipette into a new centrifuge tube. 150ul of diluents was pipetted and dispensed into the antibody coated wells. 50ul of the standard and conjugate was pipetted and was added into the antibody coated wells. It was then incubated for 30min at room temperature. The content of the micro well strips was then emptied into waste container and washed by filling with distilled water and then dumping the water from the micro wells using a tissue, it was repeated four more times. 100ul of the substrates was pipetted into each micro wells strips and incubated at room temperature for another 30min after which a blue colour developed, 100ul of the stop solution was measured and dispensed into each micro wells strips. On adding stop solution the colour changes from blue to yellow. The micro well strips were then read using ELISA reader with the absorbance filter of 450nm.  

Results:

Absorbance= 0.359

Concentration=4.4ppb

Table 21: table showing the Absorbance and Concentration of the Standard (ppb)

STANDARD	ABSORBANCE	CONCENTRATION(ppb)
C1	0.396	0.0
C2	0.295	20.0
C3	0.162	100.0
C4	0.070	500.0

                                 

From results obtained, the concentration of melamine in the powdered milk sample was 0.11ppm and the acceptable range set by NAFDAC for melamine consumption should not exceed 2.5ppm

That is, the concentration of melamine in the powdered milk sample is within the range hence it is declared satisfactory and should be allowed for human consumption.