17051885004g.laxmi Prasanna

QUALITYASSESSMENT, COMPARISION, DETECTION AND
DETERMINATION OF VARIOUS ADULTERANTS IN BRANDED AND

LOCAL MILK SAMPLES FROM DIFFERENT AREAS OF HYDERABAD
BY VARIOUS ANALYTICAL TECHNIQUES
THE DISSERTATION SUBMITTED TO OSMANIA UNIVERSITY IN

PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF
THE DEGREE
OF
MASTER OF PHARMACY
IN PHARMACEUTICAL ANALYSIS SUBMITTED BY
G. LAXMI PRASANNA B. Pharm
Redg. No. 170518885004
Under the Supervision of
Dr. P. SRIDEVI M.pharm, Ph.D
Associate Professor Head of the Department
Department of Pharmaceutical Analysis
Sri Venkateshwara College of Pharmacy Osmania University
Hyderabad
SRI VENKATESHWARA COLLEGE OF PHARMACY
OSMANIA UNIVERSITY HYDERABAD – 500 081
SEPTEMBER – 2020
DECLARATION
I, G. LAXMI PRASANNA, Student of M. Pharmacy (Pharmaceutical Analysis), bearing H.T.

No: 170518885004, Department of Pharmacy, Sri Venkateshwara College of Pharmacy
Hyderabad do here by declare that the work embodied in this dissertation entitled “QUALITY
ASSESSMENT, COMPARISION, DETECTION AND DETERMINATION OF
VARIOUS ADULTERANTS IN BRANDED AND LOCAL MILK SAMPLES FROM
DIFFERENT AREAS OF HYDERABAD BY VARIOUS ANALYTICAL
TECHNIQUES” is submitted to Osmania University for the partial fulfilment of the
requirements for the award of degree of Master of Pharmacy in Pharmaceutical Analysis under
Faculty of Pharmacy is the original research work carried out by me under the supervision of Dr.
P. SRIDEVI Head of the Department, Department of Pharmaceutical Analysis.
Further I hereby declare and inform that the contents presented in this thesis has not
been submitted by me for the award of any other degree or diploma of this or any other
University.
DATE:26/9/2020
G. LAXMI PRASANNA
PLACE:
Hyderabad Reg.No.170518885004
CERTIFICATE
This is to certify that the investigation described in this thesis entitled “QUALITY
VARIOUS ADULTERANTS IN BRANDED AND LOCAL MILK SAMPLES FROM
DIFFERENT AREAS OF HYDERABAD BY VARIOUS ANALYTICAL
TECHNIQUES”
submitted to Osmania University for the partial fulfilment of requirements for the award of
degree of Master of Pharmacy in Pharmaceutical Analysis under Faculty of Pharmacy
embodies the results and studies of a bonafide research work of G. LAXMI PRASANNA
under my supervision at Sri Venkateshwara college of Pharmacy and the contents of the
thesis do not form the basis for the award of any other degree or diploma to the candidate from
this or any other university elsewhere.
DATE:26/9/2020
Signature of Supervisor
Dr. SRIDEVDI PINGALI
M.Pharm, Ph.D
Department of Pharmaceutical Analysis
Sri Venkateshwara College of Pharmacy Hyderabad
CERTIFICATE
This is to certify that the investigation described in this thesis entitled “QUALITY
VARIOUS ADULTERANTS IN BRANDED AND LOCAL MILK SAMPLES
FROM DIFFERENT AREAS OF HYDERABAD BY VARIOUS ANALYTICAL
TECHNIQUES” submitted to Osmania University for the partial fulfilment of
requirements for the award of degree of Master of Pharmacy in Pharmaceutical
Analysis was carried out in Sri Venkateshwara college of Pharmacy and research
center, Hyderabad by Ms. G. LAXMI PRASANNA
DATE:26/9/2020
Dr. M. Bhagavan Raju

Professor and
Principal
Sri Venkateshwara College of Pharmacy Hyderabad

CONTENTS
Acknowledgement i
Abstract ii
List of tables iii
List of figures iv
List of abbreviations v
INTRODUCTION
1.1 Adulterant 1
1.2 Milk 2
1.3 Adverse effects by addition of adulterants in milk 6
1.4 Importance of milk 13
1.5 Definition of some common form of milk / milk products 14
1.6 Use of preservatives 16
1.7 High-performance liquid chromatography 17
AIM AND OBJECTIVE 32
PLAN OF WORK 33
LITERATURE REVIEW
4.1 Literature review 34
4.2 Conclusion from literature review 37
MATERIALS AND METHODS
5.1 Materials 38
5.2 Methods 39
5.2.1 Quantitative analysis of adulterants in milk 39
5.2.2 Qualitative estimation of oxytocin in milk 41
5.2.3 Qualitative analysis of diclofenac sodium in milk 42
5.2.4 Qualitative analysis of antibiotic residues in milk 42
5.2.5 Qualitative analysis of formaldehyde in milk 44
5.3 Analytical Method Validation of oxytocin, 45
diclofenac sodium, antibiotic residues and formaldehyde
RESULTS AND DISCUSSION
6.1 Quantitative analysis of adulterants in branded milk 46
6.2 Quantitative analysis of adulterants in local milk 47
6.3 Qualitative analysis of oxytocin in milk 48
6.4 Qualitative analysis of diclofenac sodium in milk 55
6.5 Qualitative analysis of antibiotic residues in milk 61
6.6 Qualitative analysis of formaldehyde in milk 71
6.7 Comparative studies 75
CONCLUSION 77
REFERENCES 78
ACKOWLEDGEMENT
The satisfaction and euphoria that accompany successful completion of any task would be
incomplete without mentioning of the people whose constant guidance and encouragement
made it possible. I take pleasure in presenting before you, my project which is a result of studied
blend of both research and knowledge.
I express my deep senses of privilege to my guide, Dr. P. SRIDEVI, HOD- Department of

Pharmaceutical Analysis of Sri Venkateshwara College of Pharmacy, for her invaluable
guidance, inspiring advice and constant encouragement with patience throughout the period of
my study and valuable suggestions, support, affections during my course.
I am very glad to take this opportunity to express my sincere thanks and gratitude to Dr. M.
BHAGAVAN RAJU, Principal, Sri Venkateshwara College of Pharmacy. I profusely
thank the Management of Sri Venkateshwara College of Pharmacy, Hyderabad for the
infrastructure and all other essential facilities and encouragement given to me during the study.
I would like to thank and express my sincere gratitude to Prof. Kavitha Waghray, Dean and
Head of The Department, Faculty of Pharmacy, Osmania University, V. Ramesh Kumar,
Chairperson, BOS in Pharmacy, Osmania University, Prof. Shyam Sunder, Principal Osmania
University Hyderabad, for accepting my work for the fulfilment of post-graduation degree.
I also take this opportunity in expressing my thanks to Mrs. K. Vinutha, Mrs. Anjali, Mr. P.
Nalla Kumar, Assistant Professor’s Sri Venkateshwara College of Pharmacy Research Centre,
Hyderabad for their encouragement at every step of this project, and providing best facilities to
carry out my work successfully.
My special thanks to Mrs. Mallika store in-charge and lab technician Laxmi and Non- Teaching
staff of Sri Venkateshwara College of Pharmacy who have helped directly and indirectly in the
successful completion of my dissertation
And I express my thanks to all, who contributed directly or indirectly in completing this work
and those who stood beside me during my work.
G. LAXMI PRASANNA
i
ABSTRACT
Adulteration is defined as an act of intentionally debasing the quality of food offered for sale either
by the admixture or substitution of inferior substances or by the removal of valuable ingredient.
Milk is an essential commodity of life as it is a source of calcium and other essential nutrients
required by the body. It is available in the market both locally and as a branded commodity. The
study was carried out keeping in view the recently emerging concern of adulteration of milk with
various illegal substances to increase its marketability. The aim of the study was to estimate
different types of adulterants in various marketed milk brands and local samples collect from
different areas of Hyderabad and to compare the quality of milk . Seven samples each of FSSAI
approved brands were collected and subjected to various standard tests. The milk samples were
estimated using different qualitative and quantitative tests and also Compared the level of
adulterants used in different branded milk samples by means of different chromatographic
techniques. The concentrations of oxytocin, Diclofenac, and Antibiotic residues of Vishaka
(1.9,0.092,0.09), Dodla (1.75,0.065,0.074), Heritage (1.8,0.075,0.087) were found to be the
closeness to the maximum residual limit whereas the samples like Sid farm milk , Amul and Vijaya
milk were found to be more safe and the concentration of adulterants in these brands are
comparatively less. on the other hand, the sample collected from local area is having the higher
concentrations of oxytocin, Diclofenac, and Antibiotic residues than MRL
Keywords: Adulteration; FSSAI, Quality of milk
ii
LIST OF TABLES
TABLE TITLE Page

No.
No.
1.1 Adverse effects of addition of adulterants in milk 5
1.2 Per captia of milk available in different areas 14

6.3 Chromatographic conditions of optimised method of 53

oxytocin
6.4 Results of Calibration curve of oxytocin 53
6.5 Results of method validation 54
6.6 Recovery studies of oxytocin 54
6.7 Chromatographic conditions of optimised method 59

diclofenac sodium
6.8 Results of Calibration curve of diclofenac sodium 60
6.9 Chromatographic conditions of optimised method of 65

antibiotic residue
6.10 Results of Calibration curve of antibiotic residue 66
6.11 Repeatability results of antibiotic residue 68
6.12 Data of accuracy 69
6.13 Results of limit of detection 70
6.14 Results of limit of quantification 71
6.15 System suitability of formaldehyde 71
iii
6.17 Data of precision 73
6.18 Ruggedness results of formaldehyde 73
6.19 Comparative studies of various milk samples 75
iv
LIST OF FIGURES
FIGURE FIGURE NAME Page

No.
No
1.1 milk 4
1.2 Adulterants of milk 5
1.3 Normal phase chromatography 19

1.4 Reverse phase chromatography 20
1.5 Schematic diagram of HPLC instrument 21
1.6 HPLC instrument 22
1.7 HPLC column 24
1.8 Schematic representation of size exclusion 26

chromatography
1.9 Pictorial representation of ion exchange 26

chromatography
6.1 Chromatographic conditions trail 1 48
6.5 chromatogram of optimised method 52
6.6 Calibration curve of oxytocin 53
6.7 Chromatogram of oxytocin 53
v
6.13 Calibration curve of diclofenac sodium 60
6.14 Chromatogram of diclofenac sodium 61
6.20 Calibration curve of antibiotic residue 66
6.21 Chromatogram of antibiotic residue 66
6.22 Chromatogram of blank 67
6.23 Chromatogram of working sample 67
6.25 calibration curve of formaldehyde 72
6.26 Chromatogram of blank sample 73
6.27 Chromatogram of standard formaldehyde 74
6.28 Chromatogram of sample 74
vi
LIST OF ABBREVIATIONS
ABBREVIATION DESCRIPTION
MRL Maximum residue limit
FSSAI Food safety and standards authority of India
RP-HPLC Reverse Phase High Performance Liquid

Chromatography
OT Oxytocin
DMAB Di methyl amino benzaldehyde
PPD Para phyenylene diamine
µg Micro gram
LOD Limit of detection
LOQ Limit of quantification
STD Standard deviation
µL Micro litres
mm Milli meter
cm Centimetre
EMEA European middle East Africa

NIH National institutes of Health
PBT Persistent bio cumulative and toxic

DDT Di-chloro di-phenyl tri-chloro ethane
EPA Environmental Protection Agency
FIFRA Federal Insecticide, Fungicide, Rodenticide Act
vii
FQPA Food Quality Protection Act
USDA United States Department of Agriculture
FDA Food and Drug Administration

PPP Plant Protection Product
EFSA The European Food Safety Authority
viii
CHAPTER 1 INTRODUCTION
INTRODUCTION
1.1 Adulteration:
Food Adulteration is an act of intentionally debasing the quality of food offered

for sale either by the admixture or substitution of inferior substances or by the removal of
some valuable ingredient, Food Adulteration takes into account not only the intentional
addition or substitution or abstraction of substances which adversely affect nature,
substances and quality of foods, but also their incidental contamination during the period
of growth, harvesting, storage, processing, transport and distribution.
“Adulterant” means any material which is or could be employed for making the food
unsafe or sub-standard or misbranded or containing extraneous matter;
Food is adulterated if its quality is lowered or affected by the addition of substances which
are injurious to health or by the removal of substances which are nutritious. It is defined
as the act of intentionally debasing the quality of food offered for sale either by the
admixture or substitution of inferior substances or by the removal of some valuable
ingredient.
Food is declared adulterated if:
• A substance is added which depreciates or injuriously affects it,

• Cheaper or inferior substances are substituted wholly or in part.
• Any valuable or necessary constituent has been wholly or in part abstracted.
• It is an imitation.
• It is coloured or otherwise treated, to improve its appearance or if it contains any
added substance injurious to health.
• For whatever reasons its quality is below the Standard
Adulterated food is dangerous because it may be toxic and can affect health and it could
deprive nutrients essential for proper growth and development
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1.2 Milk:
Milk is the major source of strength for human beings as it contains a variety of nutrients
like casein, lactose, vitamins and minerals. As a large number of people consume milk
and milk products every day, there is a great need to monitor their quality and protect the
common man against the deleterious effects of adulterants.
Milk is a nutrient rich white liquid food; it is the primary source of nutrition. As an
agriculture product milk is extracted from farm animals, Adulteration of milk reduces the
quality of milk and even make it hazardous. Most commonly the milk is adulterated with
table sugars, starch, vegetable fats, urea, diclofenac sodium, antibiotic residues, oxytocin.
Milk is the major source of strength for human beings as it contains a variety of nutrients
like casein, lactose, vitamins and minerals. As a large number of people consume milk
and milk products every day, there is a great need to monitor their quality and protect the
common man against the deleterious effects of adulterants.
According to the literature, Moore et al.2012 reported that milk powder is the second most
likely food item being in the risk of adulteration after olive oil. Fischer, Schilter, Tritscher,
2011 2015
& Stadler, ; Singh & Gandhi, reported that Adulterants in milk mainly include
addition of vegetable protein, milk from different species, addition of whey and dilution
with water which are known as economically motivated adulteration.
Apart from the above less harmful adulterants, some of the major adulterants in milk
having severe adverse effects on human health are hormones, antibiotics, urea, formalin,
detergents, ammonium sulphate, boric acid, caustic soda, benzoic acid, salicylic acid,
hydrogen peroxide, sugars and melamine*.
Buffalo milk is a rare jewel in the cheese making world. It is much higher in fat than
cow’s milk, but also lower in cholesterol. What this means is that buffalo milk is much
richer, thicker, and creamier than cow’s milk. But there are way more cheeses out there
beyond those two. Buffalo milk is the most popular milk in Pakistan, India (which has 50
percent of the world’s buffalo), and Italy. Water Buffalo create on average about 12-18
lb of milk a day (compared with 50-60 lb for a Holstein cow), so their output of milk is
about the same as a goat but feeding requires about the same for a large cow.
Milk adulteration is a very common food fraud and is posing a big social problem in
today’s world. Apart from the ethical and economical issue, it also creates health hazards
Page 2
. Some of them are renal and skin disease, eye and heart problem and may also leads to
cancer. So, for preventing these, determination of milk adulteration is very important.
Most of the times, the adulteration is intentional to make greater profit, but sometimes it
may be due to the lack of proper detecting technology and confusion regarding
appropriate drug administration practices among the dairy farm workers. For example if
it is observed that after mastitis treatment of dairy animal there are traces of antibiotic
residues in milk. In the absence of proper guideline
about the lactation time and user friendly detector, often lactation is done in wrong time
leading to antimicrobial residues in milk (if lactation performed early) or wastage of milk
(if lactation starts late). Sometimes natural milk is adulterated with low value ingredient
like water, whey etc. and is known as ‘economic adulteration’. It is a very common
practice by the milk supplier to add water or ‘liquid-whey’ to milk to increase the volume
of milk. Diluted milk reduces its nutritional value, and contaminated water causes serious
health problems. Addition of water changes specific gravity of the milk and its natural
color gets destroyed. To Milk adulteration is a very common food fraud and is posing a
big social problem in today’s world. Apart from the ethical and economical issue, it also
creates health hazards . Some. So, for preventing these, determination of milk adulteration
is very important.
Milk adulterant and its detection
The measurement of milk quality is important for food safety, as well as in the production
process of the dairy industry. There are several sophisticated methods such as
chromatography, spectroscopy etc. are used to detect milk adulteration. Other analytical
techniques are freezing point osmometry, capillary electrophoresis, thermometric
sensors, mass spectrometry, least-squares support vector machine (LS-SVM) for
detection of adulterants (starch, whey, sucrose) in powdered milk. These conventional
methods of analysis of food products include expensive and sophisticated instruments.
Such instrumental assessment techniques are time consuming, tedious, expensive and
require elaborate sample preparation and in practice, expert human panels has to be
employed to judge the qualitative parameters in the food and beverage industry. This
method of assessment has some major drawbacks like fatigue, adverse mental state at
times, and individual variability of human experts. There are thousands of different
Page 3
biosensing techniques used for detection of milk adulteration which are very difficult to
categories.
A biosensor is an analytical tool that in intimate contact with a transducer converts a

biological signal into a measurable electrical signal. The biological components of the
biosensor are enzymes, whole cells, tissues, receptors, and antibodies. Many biosensors
are integrated with the electrical sensors to detect milk adulteration. Often lactose
concentration is used as a basic marker for the evaluation of milk quality and the detection
of abnormalities. It has been found that milk from cows suffering mastitis has low lactose
levels. Conzuelo et al. have reported an amperometric biosensors to detect the lactose
content of milk.
There has been extensive research in evaluating electronic noses for monitoring the
quality of milk. E-nose can monitor the aging of milk and can detect milk volatile
compounds. The two main components of an electronic nose are time consuming, tedious,
expensive and require elaborate sample preparation. And in practice, expert human panels
have to be employed to judge the qualitative parameters in the food and beverage industry.
This method of assessment has some major drawbacks like fatigue, adverse mental state
at times, individual variability of human experts.
There are thousands of different biosensing techniques used for detection of milk
adulteration which are very difficult to categories.
Fig:1.1 milk
Page 4
Fig:1.2 milk adulterants
Table:1.1Few adverse effects by the addition of adulterants in milk samples

include:
S.No. Name of the adulterant Adverse effect
1 Water Decreases the nutritive value of milk leads to

malnutrition in infants and children.
2 Detergents gastro – intestinal complications
3 Urea Renal toxicity as more urea content has to be

metabolized.
4 Hydrogen Peroxide Gastritis and inflammation of the intestine
5 Starch Fatal for diabetic patients
6 Carbonates and Disruption in hormone signaling that regulate

bicarbonates development and reproduction
7 Oxytocin Increases the risk of post-partum hemorrhage, early

puberty, memory loss etc
8 Antibiotic residues Carcinogenicity, reproductive disorders, bone marrow

toxicity etc
9 Formalin Carcinogenicity
Page 5
1.3 Adverse effects by addition of adulterants in milk

Formalin (Formaldehyde)
Formalin is a preservative and disinfectant that was primarily used to preserve dead
bodies and other biological samples. But, today this chemical is being used to increase
the lifespan of milk. Dairy farmers are using a few drops of formalin to save expenses of
refrigeration. But in this process, they are putting the milk consumers' health in jeopardy.
Some of the side effects of formalin include:
• Cancer
• Skin diseases
• Eye diseases
• Gastrointestinal diseases such as ulcers
• Kidney problems
Hydrogen Peroxide
Hydrogen Peroxide does a similar role to formalin. It is used to increase the shelf life of
milk and wards off bacterial growth that may turn milk unfit to consume. Even small
quantities of Hydrogen Peroxide consumption on a regular basis can lead to many health
hassles such as:
• Cardiac arrhythmia
• Gastrointestinal diseases
• Poor digestive system
Detergents
If you thought white milk with foam was the sign of its purity then you are wrong. This
effect can be attained by adding detergent to milk. Detergents give milk the whiteness
and thickness that would lure the end consumers. But it would cause many long term
health problems such as:
• Gastrointestinal diseases
• Kidney problems
Page 6
SUGAR
• Sugar can suppress your immune system.

• Sugar eaten during pregnancy and lactation can influence muscle force production
in offspring, which can affect an individual’s ability to exercise.
• Sugar can elevate glucose and insulin responses and return them to fasting levels
slower in oral contraceptive users.
• Sugar can increase reactive oxygen species (ROS), which can damage cells and
tissues.
• Sugar can cause hyperactivity, anxiety, inability to concentrate and crankiness in
children.
• Sugar can produce a significant rise in triglycerides.
• Sugar causes a decline in tissue elasticity and function – the more sugar you eat,
the more elasticity and function you lose.
• Sugar reduces high-density lipoproteins (HDL).
• Sugar can lead to ovarian cancer.
• Sugar can increase fasting levels of glucose.
• Sugar causes copper deficiency.
• Sugar can make tendons more brittle.
• Sugar can increase the levels of glucose in the blood much higher than complex
carbohydrates in a glucose tolerance test can.
• Sugar can cause two blood proteins – albumin and lipoproteins – to function less
effectively, which may reduce the body’s ability to handle fat and cholesterol.
• Sugar can contribute to Alzheimer’s disease.
• Sugar can cause platelet adhesiveness, which causes blood clots.
• Sugar can cause hormonal imbalance – some hormones become underactive and
others become overactive.
• Sugar can cause free radicals and oxidative stress.
• Sugar increases the concentration of bile acids in stool and bacterial enzymes in
the colon, which can modify bile to produce cancer-causing compounds and colon
cancer.
• Sugar combines with and destroys phosphatase, a digestive enzyme, which makes
digestion more difficult and can aggravate premenstrual syndrome (PMS).
Page 7
UREA
• Urea is generally added in the preparation of synthetic milk to raise the SNF value.
Potential symptoms are burning sensation in throat and chest; cough, dyspnea,
exercise-induced asthma (one case); redness, in eyes and skin, headache; nausea,
vomiting, lung damage-fibrosis, inflammation (HE11). Affected organs are
respiratory system, skin, eyes, it has also used medically as an abortifacient and a
skin moisturizer.
Salt consumption has been linked to:
• Exercise – induced asthma.

• Heartburn.
• Osteoporosis.
• Gastric cancer is associated with high level of sodium.
• Hypertension.
• Left ventricular hypertrophy (cardiac enlargement).
• Duodenal ulcer.
Starch: Potential symptoms are irritation of eyes, skin, mucous, membranes, rhinorrhea,
cough, chest pain, dermatitis.
Detergent: chemicals will cause health problem especially related to stomach and
kidneys.
So, it is important to consume milk from only the most reputed brands. Trusting on the
local milkmen is not a wise idea any more
Based on all the above facts, the present study is aimed to determine and compare various
adulterants in branded and local milk samples obtained from different places of
Hyderabad.
Apart from qualitative analysis of all the above adulterants, major adulterants like
oxytocin, diclofenac and antibiotic residues in milk samples can be determined
quantitatively by sensitive analytical methods as per FDA and FSSAI guidelines.
Page 8
Milk may be defined as the whole, fresh, clean lacteal secretion obtained by the complete
milking of one or more healthy milch animals, excluding that obtained within 15 days
before and 3 days after calving or such periods as may be necessary to render the milk
practically colostrums free and containing the minimum prescribed percentage of milk
fats and S-N-F. (Milk in technical aspect is defined as the whole, normal, clean and fresh
lacteal secretion obtained by milking a healthy animal 72 hours after calving.)
DICLOFENAC SODIUM
Diclofenac sodium (DFS) is a enteric coated tablets and is a benzene. Acetic acid
derivative. These tablets having the 75 mg (Light pink) for oral Administration. The
chemical name is (2-6 dichlorophynyl) amino benzene acetic acid monosodium salt. The
molecular weight is 318.14 and this molecular formula is C14H10Cl2NaO2 and it
has the following structural formula. In this case the diclofenac sodium (DFS) is
used to animals to prevent the some severe diseases.
COMMON SIDE EFFECTS OF DICLOFENAC SODIUM INCLUDES:
• Abdominal or stomach bloating, burning, cramping, or pain

• belching
• bloody or black, tarry stools
• cloudy urine
• constipation
• decrease in urine output or decrease in urine-concentrating ability
• diarrhea
• dizziness
• feeling of indigestion
• headache
Page 9
• increased bleeding time

• itching skin or rash
• loss of appetite
• nausea and vomiting
• pain in the chest below the breastbone
• pale skin
• severe stomach pain
• swelling
• troubled breathing with exertion
• unusual bleeding or bruising
• unusual tiredness or weakness
• vomiting of blood or material that looks like coffee grounds
• weight loss
OXYTOCIN
Oxytocin is a cyclic octapeptide hormone released by the posterior pituitary and showing
uterotonic and galactogenic activity. It is chemically L-Hemi-cystinyl-L-tyrosyl-L-
isoleucyl-L-glutaminylL-asparaginyl-L-hemi-cystinyl-L-propyl-L-leucyl glycinamide.
Its 20 membered ring consists of five amino acids- cysteine, tyrosine, isoleucine,
glutamine, and asparagines, further three amino acids, proline, leucine and glycinamide.
[Klaus Florey, Analytical profile of drug substances] Oxytocin is involved in the
contraction of uterus and milk ejection in receptive mammals. In the brain oxytocin is
classically viewed as primarily involved in the milk let down reflex and in the stimulation
of uterine smooth muscle during parturition. New inventions prove that when oxytocin is
injected into cows, there is a result over production of milk and traces of oxytocin can be
found in the same. When excess of oxytocin is found to be present in milk it may cause
headache, nausea, abdominal pain, drowsiness etc. Oxytocin Injections
These hormone injections are used to increase the milking capacity in cows and buffaloes.
Though the use of Oxytocin injections on cattle has been banned by the Indian
government yet its usage is still rampant across India. The injection causes hormone
release among the cattle. But the small quantity of oxytocin is passed into the milk and
can cause hormonal imbalance in people who are consuming the milk. Some of the side
effects caused by oxytocin include:
Page 10
• Early development of breasts in girls

• Gynecomastia (Male breast)
• Heart diseases (such as high or low blood pressure, cardiac arrhythmia, premature
ventricular contraction etc.)
• Kidney diseases
• Poor eyesight
• Loss of memory
• Slow heart rate
• Fast heart rate

• Premature ventricular complexes and other irregular heartbeats (arrhythmias)
• Permanent central nervous system (CNS) or brain damage, and death secondary to
suffocation
• Neonatal seizure
• Neonatal yellowing of skin or eyes (jaundice)
• Fetal death
• Low Apgar score (5 minute)
• Uteroplacental hypoperfusion and variable deceleration of fetal heart rate
• Inadequate fetal oxygen levels (hypoxia)
• Perinatal hepatic necrosis
• Fetal hypercapnia
• Severe decreases in maternal systolic and diastolic blood pressure, increases in heart
rate, systemic venous return and cardiac output, and arrhythmia.
ANTIBIOTIC RESIDUES
Since the early 1960s, there has been two-fold increase in per capita milk
consumption of developing countries. This increased demand of milk made it essential
to adopt extensive animal husbandry practices. Use of veterinary drugs for taking cure of
variety of ailments in farm animals is an integral component of such extensive
animal husbandry practices. Antimicrobial residues are currently most frequent
inhibitory substances found in milk, having
undesirable effects on milk quality, milk technological properties, dairy products
quality and human health problems. Food Safety and Standards Act, 2006 defines
veterinary drug residues as “the parent compounds or their metabolites or both in any
Page 11
edible portion of any animal product and include residues of associated impurities
of the veterinary drugs concerned” (FSSA, 2006). Presence of any drug or antibiotic
residue in milk and meat is considered as illegitimate and also lead to economic losses
to dairy industry.
Antibiotics used in dairy animals
In modern dairy farming system, antimicrobial drugs are used for both therapeutic and
prophylactic purposes. Pencillins, tetracyclines, sulphonamides and aminoglycosides
were most frequently used in lactating animals, which led to occurrence of their
residues in milk. Drugs are extensively used to promote the animal health, control
and treat the infection and to step up the production. Mastitis is the most prevalent and
economically important widespread disease in cattle and much of the veterinary
treatment of dairy cattle involves intramammary infusion of antibiotics to control
mastitis. The most likely cause of violative drug residues is the failure to observe
prescribed withdrawal times. The withdrawal time is the time required for the residue
of toxicological concern to reach safe concentration as defined by tolerance. However,
the extra label use of antibiotics (whenever a drug is used in a manner other that
which it is licensed for), mainly dosages deviating from recommendations of the drug
manufacturer fall under the main reason for occurrence of antibiotic residues in milk
after the end of the withholding period in India. The inappropriate use of veterinary
drugs and negligence regarding withholding periods of milk can lead to the presence of
residues of these compounds or their metabolites. Usage of antibiotics as preservatives
and as growth promoter has also been reported. Other major reasons for occurrence of
drug residues in milk are incorrect milking order of cows
and insufficient cleaning of milking cluster or milking installation. Few cases of
prolonged occurrences of residues in milk are related to veterinary error and
insufficient cleaning of milk contact surfaces after milking of treated cows. Education
on prudent use of antibiotics has been observed to be particularly lacking amongst
dispensers and prescribers of antibiotics.
Harmful consequences of antibiotic residues
Presence of antimicrobial residues in foods can cause health hazards in consumers such
as allergic reactions in sensitive persons. Antibacterial residues cause broad range of
health effects like allergy (penicillins), bone marrow aplasia (chloramphenicol),
ototoxicity etc. Major concern regarding dietary exposure of antimicrobial residues
is due to emergence of antibiotic resistant strains of pathogens, complicating the
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treatment for both human and animal diseases. Apart from health hazards, antimicrobial
residues in milk have also been linked to major technological problem in the dairy
industry. It is because, even residual quantities of antimicrobials in milk are
responsible for interference with starter culture activity which disrupts the manufacture
process of milk products. Antibiotic residues can also interfere with the methylene blue
reduction test, hence causing underestimation of the microbial load in milk. All of these
concerns may result in major threats to the society. Presence of veterinary drug residues
in food is thus a crucial food safety issue.
Common side effects of Antibiotic residues include:
• Transfer of antibiotic resistant bacteria to human
• Autoimmunity
• Carcinogenicity
• Hepatotoxicity
• Reproductive disorders
• Mutagenicity
• Nephropathy
• Bone marrow toxicity
• Allergy
• Teratogenicity
• Lung congention
1.4 Importance of milk:

• India ranks first in milk production, accounting for 18.5 per cent of world production,
achieving an annual output of 146.3 million tonnes during 2014-15 as compared to 137.69
million tonnes during 2013-14 recording a growth of 6.26 per cent.
• The Food and Agriculture Organization (FAO) has reported a 3.1 per cent increase in
world milk production from 765 million tonnes in 2013 to 789 million tonnes in 2014.
• The target for milk production in the country fixed by the Government for the year 2016-
17 was 163.7 million tonnes.
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• The per capita availability of milk in India has increased from 176 grams per day in
1990-91 to 322 grams per day by 2014-15. It is more than the world average of 294 grams
per day during 2013.
• The country’s estimated demand for milk is likely to be about 155 million tonnes by
2016-17 and around 200 million tonnes in 2021-22. To meet the growing demand, there
is a need to increase the annual incremental milk production from 4 million tonnes per
year in past 10 years to 7.8 million tonnes in the next 8 years (210 million by 2021-22),
(T Nanda Kumar, chairman, NDDB).
• India has about three times as many dairy animals as the USA, which produces around
75 million tons milk, over 80 per cent being kept in herds of 2 to 8 animals.
• Annual milk yield per dairy animal in India is about one tenth of that achieved in the
USA and about one fifth of the yield of a grass-fed New-Zealand dairy cow.
• A peculiar feature in our country is the wide variation between regions in respect of
consumption of milk.
Table1.2:percapita available in different years
Year Production million tonnes Per capita available (g/day)
1991-1992 55.7 178
1995-1996 66.2 197
2000-2001 80.6 220
2005-2006 97.1 241
2008-2009 108.5 258
2014-2015 146.3 322
*The per capita availability in different states varies widely.

• In 2001-02 India became the world leader in milk production.
• Recommended value of milk consumption is 250-450 g. (Minimum recommended by
NIN is 80 to 310 g/capita-day.
• Per-capita availability in India was only 114 g in 1975.
1.5 Definition of some common forms of milk/ milk products:
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Market milk Refers to fluid whole milk that is sold to individuals usually for direct
consumption. It excludes milk consumed on the farm and that used for the manufacture
of dairy products.
Pasteurized milk: It is the milk, which has been heated (processed) for at least 30
minutes at 63°C or 15 sec at 72°C (or any time temperature combination that is equally
efficient in approved and properly operated equipment). After pasteurization the milk is
cooled immediately to 5°C or below.
Sterilized milk :Milk which has been heated to a temperature of 100°C or above for such
lengths of time that it remains fit for consumption for at least 7 days at room temperature
(Usually the process condition is 108-111°C for 25-30 min).
Homogenized milk :It is the milk which has been treated in such a manner as to ensure
break up of fat globules to such an extent that after 48 hours of storage no visible cream
separation occurs in the milk and the fat percentage of the milk in the top 100 ml of milk
in a quart bottle or of proportionate volumes in containers of other sizes does not differ
by more than 10% of itself from the fat percentage of the remaining milk as determined
after thorough mixing (In sufficiently homogenized milk, the fat globules are subdivided
to 2 microns or less in diameter).
Soft-curd milk: It is the milk that forms a soft curd when coagulated with rennet or
pepsin under standard procedure (Curd tension <25 g).
Soft curd milk: is characterized by low casein content and low Ca content.
(Mother‟s milk is best for infants because it forms a soft curd when coagulated in the
stomach and is apparently more quickly digested than cow or buffalo milk)
Curd tension of human milk: 0 g, Boiled cow milk: 3 g, Homogenized milk: 14.5 g,
Pasteurized milk: 44.5 g, Raw milk: 55g) 4 Flavored milk Milk to which some flavors
have been added. When the term „milk‟ is used, the product should contain a fat per cent
at least equal to the minimum legal for the market milk. But when the fat level is lower
(1-2 per cent), the term drink is used.
Vitaminized milk: Milk in which one or more vitamins have been added.
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Irradiated milk: Milk in which Vitamin D content has been increased by exposure to U-
V rays.
Mineralized milk: Milk in which minerals have been added (Addition of vitamins and
minerals to milk is known as fortification and such milk is called fortified milk).
Frozen concentrated milk: The milk is partially concentrated and solidified by freezing
Fermented milk: Milk that has been made by employing selected microorganisms to
develop the characteristic flavor and/or body and texture.
(Fermentation has been defined as the metabolic process in which chemical changes are
brought about on an organic substratum, whether carbohydrates, proteins or fat, through
the action of enzymes liberated by specific microorganisms. In milk, the most important
fermentation is the lactic acid fermentation or souring of milk.)
a. Natural butter milk: It is a by-product of churning cream for butter making. Ripened
cream, which has undergone a clean, lactic fermentation, is usually preferred.
b. Cultured butter milk Obtained by inoculation and incubation of pasteurized skim

milk with lactic starter.
c. Acidophilus milk Produced by development in milk of a culture of Lactobacillus

acidophilus (It is claimed that acidophilus milk is therapeutic and has health promoting
properties).
1.6 Use of Preservatives:

1. Broad-spectrum activity: A critical necessity is the pernicious ability of the
preservative to act against all types of microorganisms in milk. The broader the spectrum
of the preservative, the better is its utility.
2. Efficient minimum inhibitory levels: The preservative should be effective at low

concentrations in milk to minimize sample dilution, costs, and expedite handling
procedures.
3. High water solubility: Because the average milk sample has about 87% water, it is
imperative that the preservative has the capacity to function against the microorganisms
in the aqueous phase; high water solubility also assures easy miscibility without excessive
agitation.
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4. Stability: The preservative must be stable under most storage conditions.
5. Presence of color: Unlike cosmetic preservatives, a desirable quality of a milk

preservative is that it imparts some color to the treated milk sample for identification and
safety concerns.
6. Compatibility: A good milk preservative should be just as effective in pooled milk

samples as in fresh samples from individual cows or other mammals and be suitable for
high fat as well as non fat milk samples.
7. Shelf-life activity: The milk preservative should be effective in milk for a period of six
months to one year (the expected time interval involved in litigations from the stage of
sampling till the stage of final chemical analysis through public health laboratory at
primary level and referral laboratory at the secondary level).
8. Toxicity and disposability: The preservative should be non-allergenic and exhibit no

demonstrable toxicity towards handlers or others coming in contact with it. Despite its
necessary biocidal property, it should not become an environmental hazard after disposal.
9. Economy: Cost should be minimal and the preservative should be readily available.
10. Dispensing ability: Dispensing in solid form, as in tablets, would be preferable to the
liquid form, because of the inherent difficulty in handling liquids and the greater accuracy
in dispensing solids
1.7 High Performance Liquid Chromatography (HPLC)

Russian botanist Tswett invented chromatography as a separation technique. He describes
in detail the separation of pigments, the coloured substances by filtration through column,
followed by developments with pure solvents.
High-performance liquid chromatography (HPLC) is the fastest growing analytical

technique for analysis of drugs. Its simplicity, high specificity and wide range of
sensitivity make it ideal for the analysis of many drugs in both dosage forms and
biological fluids.
According to IUPAC, chromatography is a physical method of separation in which

components will be separated or distributed between stationary and mobile phases. High
performance liquid Chromatography (HPLC) is the term used to describe liquid
Page 17
chromatography in which the liquid mobile phase is forced through the column at high
speed as a result, the analysis time is reduced by 1-2 orders of the magnitude relative to
classical column chromatography and the use of much smaller particles of the adsorbent
or support becomes possibly increasing the column efficiency substantially. The
importance of chromatography is increasing rapidly in pharmaceutical analysis for the
exact differentiation, selective identification and quantitative determination of
structurally closely related compounds. Another important field of application of
chromatographic methods is the purity testing of final products and the intermediates.
The reasons for the popularity of the method is its sensitivity, its ready adaptability to
accurate quantitative determinations, its suitability for separating non-volatile species or
thermally fragile ones and its wide spread applicability to substances that are of prime
interest to the industry. Sensitive detectors have transformed liquid column
chromatography into high speed, efficient, accurate and highly resolved method of
separation. The phenomenal growth in chromatography is largely due to the introduction
of the versatile technique called high-pressure liquid chromatography, which is
frequently called high-performance liquid chromatography. Both terms can be
abbreviated as HPLC
➢ High-pressure liquid-solid chromatography (HPLC) is rapidly becoming the

method of choice for separations and analysis in many areas. Most of the samples
that a r e dissolved can be separated on some type of HPLC column.
➢ HPLC is a form of liquid chromatography used to separate compounds that are
dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a
pump, an injector, a separation column, and a detector.
➢ Compounds are separated by injecting a sample mixture onto the column. The
different component in the mixture pass through the column at differentiates due to
differences in their partition behavior between the mobile phase and the stationary
phase. The mobile phase must be degassed to eliminate the formation of air bubbles.
The HPLC is the method of choice in the field of analytical chemistry, since this
method is specific, robust, linear, precise and accurate and the limit of detection is
low and also it offers the following advantages.
• Speed (many analysis can be accomplished in 20 min or less)

• Greater sensitivity (various detectors can be employed)
• Improved resolution (wide variety of stationary phases)
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• Reusable columns (expensive columns but can be used for many analysis)
• Ideal for the substances of low viscosity
• Easy sample recovery, handling and maintenance.
• Instrumentation leads itself to automation and quantification (less time and
less labor).
• Precise and reproducible
• Integrator itself does calculations.
• Only small sample required.
• Easily flexible to quantitative analysis.
• May be non destructive of sample
1.7.1 Modes of HPLC: [11]

1) Normal phase chromatography: The nature of stationary phase is polar and the
mobile phase is non-polar in this mode. In this technique, non-polar compounds
travel faster and are eluted first because of the lower affinity between the non-polar
compounds and stationary phase. The time for polar compounds to elute takes
longer time because of their higher affinity to the stationary phase, therefore
generally this method is not used in the pharmaceutical applications because most
of the drug molecules are polar in nature and hence take longer time to elute.
The stable functional groups which are used in a stationary phase with siloxane
connection are:
The stationary phase is usually silica and typical mobile phases are hexane,
methylene chloride, chloroform, diethyl ether, and mixtures of these.
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Fig 1.3: Schematic diagram of Normal Phase Chromatography
2) Reversed phase chromatography: Reversed phase mode is the most popular

mode for analytical and preparative separations of compounds of concern in biological
products, pharmaceutical formulations & API’s, chemical substances, food and
biomedical engineering. The stationary phase is non-polar hydrophobic packing with
octyl and octadecyl functional group bonded to silica gel and the mobile phase is a polar
solvent, often a partially or fully aqueous mobile phase. Polar substances prefer the
mobile phase and elute first. As the hydrophobic character of the solutes increases,
retention increases. Generally, the lower the polarity of the mobile phase, higher is the
eluent strength.
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Fig 1.4: Schematic diagram of Reverse Phase Chromatography
HPLC system
Fig 1.5 Schematic diagram of HPLC Instrument
The pump provides a steady high pressure without pulsation, and can be programmed to
vary the composition of the mobile phase during the course of the separation. Detectors
rely on a change in refractive index, UV-VIS absorption, or fluorescence after excitation
with a suitable wavelength to detect the separated compounds.
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Four types of liquid chromatography

1. Partition chromatography
2. Adsorption, or liquid-solid chromatography
3. Ion exchange chromatography
4. Size exclusion, or gel, chromatography
Composition of a liquid chromatography system

• solvent
• solvent delivery system (pump)
• injector
• sample
• column
• detectors (diode array)
• waste collector
• recorder (data collection)
Fig 1.6: HPLC Instrument
1.7.2 Instrumentation of a HPLC
As shown in the schematic diagram in (Figure 1.6), HPLC instrumentation includes a

pump, injector, column, detector and integrator or acquisition and display system. The
heart of the system is the column where separation occurs. Since the stationary phase may
be composed of micron-sized porous particles, a high-pressure pump is required to move
the mobile phase through the column. The chromatographic process begins by injecting
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the solute into the injector at the end of the column. Separation of components occurs as
the analytes and mobile phase are pumped through the column. Eventually, each
component elutes from the column as a peak on the data display. Detection of the eluting
components is important, and the method used for detection is dependent upon the
detector used. The response of the detector to each component is displayed on a chart
recorder or computer screen and is known as a chromatogram. To collect, store and
analyze the chromatographic data, integrators and other data-processing equipment are
frequently used.
Mobile Phase and Reservoir:
The type and composition of the mobile phase affects the separation of the components.
Different solvents are used for different types of HPLC. For normal-phase HPLC, the
solvent is usually nonpolar, and, in reverse-phase HPLC, the solvent is normally a mixture
of water and a polar organic solvent. The purity of solvents and inorganic salts used to
make the mobile phase is paramount. A general rule of thumb is to use the highest purity
of solvent that is available and practical depending on the particular application. The most
common solvent reservoirs are as simple as glass bottles with tubing connecting them to
the pump inlet.
Pumps:-
High-pressure pumps are needed to push the mobile phase through the packed stationary
phase. A steady pump pressure (usually about 1000–2000 psi) is needed to ensure
reproducibility and accuracy. Pumps are typically known to be robust, but adequate
maintenance must be performed to maintain that characteristic. Inability to build pressure,
high pressures or leakage could indicate that the pump is not functioning correctly. Proper
maintenance of the pump system will minimize down time.
Injectors:
The injector can be a single injection or an automated injection system. An injector for
an HPLC system should provide injection of the liquid sample within the range of 0.1-
100 mL of volume with high reproducibility and under high pressure (up to 4000 psi).
For liquid chromatography, liquid samples can be directly injected and solid samples need
Page 23
only to be diluted in the appropriate solvent. The function of the injector is to place the
sample into the high-pressure flow in as narrow volume as possible so that the sample
enters the column as a homogeneous, low-volume plug. To minimize spreading of the
injected volume during transport to the column, the shortest possible length of tubing
should be used from the injector to the column. When an injection is started, an air
actuator rotates the valve: solvent goes directly to the column; and the injector needle is
connected to the syringe. The air pressure lifts the needle and the vial is moved into
position beneath the needle. Then, the needle is lowered to the vial. The instruments are
different that can be operated manually or automatically. When operated manually a
single injection is administered, while if operated automatically up to 124 injections can
be performed simultaneously. By performing HPLC in an automatic setting it is more
time efficient and more than one sample can be tested at the same time. The sample is
drawn up into a sample loop by the syringe, metered by a stepper motor. The needle is
raised a second time to allow the vial to move away. Then, the needle is lowered a second
time and the air actuator reverses the valve, reconnecting the sample loop to the solvent
flow. The entire sample is flushed out of the injector, reaching the column as an undiluted
plug. Finally, the syringe stepper-motor moves the syringe plunger to the end of the
syringe sending the remaining solvent to the waste.
Columns:
Fig 1.7: HPLC column
The column is one of the most important components of the HPLC chromatograph
because the separation of the sample components is achieved when those components
pass through the column. The High performance liquid chromatography apparatus is
made out of stainless steel tubes with a diameter of 3 to 5mm and a length ranging from
10 to 30cm. Normally, columns are filled with silica gel because its particle shape, surface
properties, and pore structure help to get a good separation. Silica is wetted by nearly
every potential mobile phase, is inert to most compounds and has a high surface activity
which can be modified easily with water and other agents. Silica can be used to separate
Page 24
a wide variety of chemical compounds, and its chromatographic behaviour is generally

predictable and reproducible.
The column or stationary phase is the core of any chromatographic system. Columns are
commercially available in different lengths, bore sizes and packing materials. The use of
the correct combination of length and packing material in correlation with the appropriate
mobile phase can assist in the most effective separation of a sample compound. A variety
of column dimensions are available including preparative, normal- bore, micro- and mini-
bore and capillary columns. Different column dimensions can be used for different types
of separations and can utilize different packing materials and flow rates. The most widely
used packing materials for HPLC separations are silica-based. The most popular material
is octadecyl- silica (ODS-silica), which contains C18 coating, but materials with C1, C2,
C4, C6, C8 and C22 coatings are also available. Miscellaneous chemical moieties bound
to silica, as well as polymeric packing, are designed for purification of specific
compounds. Other types of column packing materials include zirconia, polymer-based
and monolithic columns. Theoretical plates relate chromatographic separation to the
theory of distillation and are a measure of column efficiency. The number of theoretical
plates (n) can be determined by the following equation.
where tR1 is the total retention time
w is the band width of the peak.
In general, LC columns are fairly durable with a long service life unless they are used in
some manner that is intrinsically destructive—for example, with highly acidic or basic
eluents or with continual injections of “dirty” biological or crude samples. Column
degradation is inevitable, but column life can be prolonged with proper maintenance.
Flushing a column with mobile phase of high elution strength following sample runs is
essential. When a column is not in use, it is capped to prevent it from drying out.
Particulate samples need to be filtered and when possible a guard column should be
utilized. Column regeneration could instil some life into a column, but preventive
maintenance is the key to preventing premature degradation.
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Usually, analytical columns are protected by a guard column which is an essence a

disposable (or sacrificial) top of the main analytical column. The guard column is the
final filter both mechanical and chemical. In addition to removing debris, it also can
adsorb undesirable sample components that otherwise might irreversibly bind and
possibly change the stationary phase of the analytical and preparative columns. Although
economy alone is not a persuasive argument for the use of guard columns, the need for a
long stable life of the analytical column to obtain reliable and reproducible results is
perhaps even more important. There are several column types, according to their function,
they can be classified as:
Size exclusion
In size exclusion the HPLC column is consisted of substances which have controlled pore
sizes and is able to be filtered in an ordinarily phase according to its molecular size. Small
molecules penetrate into the pores within the packing while larger molecules only
partially penetrate the pores. The large molecules elute before the smaller molecules.
Fig 1.8: Schematic representation of size exclusion chromatography
Ion exchange
In this column type the sample components are separated based upon attractive ionic
forces between molecules carrying charged groups of opposite charge to those charges
on the stationary phase. Separations are made between a polar mobile liquid, usually
Page 26
water containing salts or small amounts of alcohols, and a stationary phase containing
either acidic or basic fixed sites.
Fig 1.9: Pictorial representation of Ion exchange chromatography
Detectors:-
There are many different types of detectors that can be used for HPLC. The detector
is used to sense the presence of a compound passing through and to provide an electronic
signal to a data-acquisition device. The main types of detectors used in HPLC are
refractive index (RI), ultraviolet (UV-Vis) and fluorescence, but there are also diode
array, electrochemical and conductivity detectors. Each detector has its assets, limitations
and sample types for which it is most effective. Most applications in drug analysis use
detectors that respond to the absorption of ultraviolet radiation (or visible light) by the
solute as it passes through the flow-cell inside the detector. The recent development of
the so-called hyphenated techniques has improved the ability to separate and identify
multiple entities within a mixture. These techniques include liquid chromatography-mass
spectrometry (LC-MS), liquid chromatography- mass spectrometry-mass spectrometry
(LC-MSMS), liquid chromatography-infrared spectroscopy (LC-IR) and liquid
chromatography-nuclear magnetic resonance (LCNMR). These techniques usually
involve chromatographic separation followed by peak identification with a traditional
detector such as UV, combined with further identification of the compound with the MS,
IR or NMR.
Page 27
Types of Detectors
1) Absorbance (UV with Filters, UV with Monochromators)

1) IR Absorbance
2) Fluorescence
3) Refractive-Index
4) Evaporative Light Scattering Detector (ELSD)
5) Electrochemical
6) Mass-Spectrometric
7) Photo-Diode Array
Separation Techniques:
Isocratic versus Gradient Elution
Elution techniques are methods of pumping mobile phase through a column. In the
isocratic method, the composition of the mobile phase remains constant, whereas in the
gradient method the composition changes during the separation process. The isocratic
method is the simplest technique and should be the first choice when developing a
separation. Eluent gradients are usually generated by combining the pressurized flows
from two pumps and changing their individual flow rates with an electronic controller or
data system while maintaining the overall flow rate constant.
Derivatization
Derivatization of samples involves a chemical reaction that alters the molecular structure
of the analyte of interest to improve detection. In HPLC, derivatization of a drug is usually
unnecessary to achieve satisfactory chromatography. Derivatization is used to enhance
the sensitivity and selectivity of detection when available detectors are not satisfactory
for the underivatized compounds. Quantitative Analysis The quantification methods
incorporated in HPLC are borrowed mostly from gas chromatography methods. The basic
theory for quantitation involves the measurement of peak height or peak area. To
determine the concentration (conc.) of a compound, the peak area or height is plotted
versus the concentration of the substance. For peaks that are well resolved, both peak
height and area are proportional to the concentration. Three different calibration methods,
each with its own benefits and limitations, can be utilized in quantitative analysis: external
standard (std.), internal standard and the standard addition method.
Page 28
Internal Standard
Although each method is effective, the internal standard method tends to yield the most
accurate and precise results. In this method, an equal amount of an internal standard, a
component that is not present in the sample, is added to both the sample and standard
solutions. The internal standard selected should be chemically similar to, have similar
retention time and derivatize similarly to the analyte. Additionally, it is important to
ensure that the internal standard is stable and does not interfere with any of the sample
components. The internal standard should be added before any preparation of the sample
so that extraction efficiency can be evaluated. Quantification is achieved by using ratios
of peak height or area of the component to the internal standard.
Quantitation Method in HPLC:
Peak height or peak area measurements only provide a response in terms of detector
signal. This response must be related to the concentration or mass of the compound of
interest. To accomplish this, some type of calibration must be performed. Once the peak
height or peak area is measured, there are five principles evaluation methods for
quantifying sample.
A) Calibration by Standards: Calibration curves for each component are prepared

from pure standards, using identical injection volumes of operating conditions for
standards and samples.
The concentration of sample is read from its curve if the curve is linear
X= K x AREA
X= concentration of sample
K= proportional constant (slope of curve)
B) Internal Method: In this method a known quantity of the internal is injected

peak area ratio Vs concentration is ascertained. Then a quantity of the internal is
added to the raw sample prior to any sample pretreatment or separation operations. The
Page 29
material selected for the internal must be completely separated from the adjacent sample
components, should not interfere with sample components.
Peak area of sample component
Peak area ratio = Peak area of internal STD
Sample concentration = Peak area ratio x concentration of standard
C. Area Normalization: The technique used to examine the absolute purity of the
sample. The procedure is to total up the areas under all peaks then calculated the
percentage of the total area that is contributed by the compound of interest. For this
method the entire sample must be eluted, all components must be separated each peak
must be completely resolved .
E. Standard addition Method: The chromatogram· of the unknown is recorded, the

known amount of sample is added the chromatogram is repeated using same condition.
The increase in the peak area the original concentration will be computed by
interpretation.
1.7.4 The parameters that are affecting by the changes in chromatographic

conditions are:
➢ Column efficiency (N)
➢ Capacity factor (K' )
➢ Resolution factor (RS)
➢ Retention Factor (Rf)
➢ Retention time (Rt)
➢ Relative retention (Rr)
➢ Peak asymmetry factor (As)
1.7.5 SELECTIVITY OF HPLC-METHOD DEVELOPMENT
Page 30
Most of the drugs can be analyzed by HPLC-method because of several advantages like
rapidity, specificity, accuracy, precision, case of automation and eliminates tedious
extraction and isolation procedures. Some of advantages are:
1. Speed (analysis can be accomplished in 20 minutes or less)
2. Greater sensitivity (various detectors can be employed)
3. Improved resolution (wide variety of stationary phases)
4. Reliable columns (wide variety of stationary phases)
5. Ideal for substances of low volatility
6. Easy sample recovery, handling and maintenance
7. Easy programming of the numerous functions in each module.
8. Time programmable operation sequence, such as initiating operation of detector lamp

and pump to obtain a stable base line and equilibrated column before the workday begins.
9. Excellent reproducibility of retention times
10. An injection volume variable from 0.1 to 100 micro liters without any hardware
modification.
11. The flexibility of data analysis.
12. Suitable to avoid any interference from impurity.
13. Suitable for preparative liquid chromatography on a much large scale.
Page 31
CHAPTER 2 AIM AND OBJECTIVE
AIM:
Quality assessment, comparison, Detection and Determination of various adulterants in

branded and local milk samples from different areas of Hyderabad by various analytical
techniques.
Qualitative and quantitative estimation of various types of adulterants in different brands
of milk by using analytical techniques.
OBJECTIVE
• To identify different major and minor adulterants in the selected samples

qualitatively and quantitatively.
• To develop a simple, precise and accurate method for the estimation of adulterants
in milk by using UV SPECTROSCOPY and RP-HPLC
• To develop an optimised method for the estimation of major adulterants in milk

by using HPLC.
To compare the extent of adulteration in the selected samples
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CHAPTER 3 PLAN OF WORK
PLAN OF WORK
• To collect different types of milk brands.
• To perform qualitative tests for identification of adulterants like water, starch,

urea, detergent, vanaspathi, formalin, hydrogen peroxide, sugar, salt etc.
• To perform qualitative and quantitative analysis by using UV- spectroscopy and

RP-HPLC.
• Qualitative analysis for the detection of adulterants in the selected milk samples
by testing kit, lactometer and other testing procedures were performed.
• Analytical Method development using RP-HPLC
• Analytical Method validation studies
• Comparative studies
Page 33
CHAPTER 4 LITERATURE REVIEW
4.1 LITERATURE REVIEW:
SURESH KUMAR KRISHNAN (2010):
A simple, precise, accurate and validated reverse phase HPLC method has
been developed for the estimation of oxytocin in milk by the use of phenyl hexyl column
(250mm x 4. 6mm id, 5μm).The mobile phase used was a mixture of acetonitrile and 0.03
M phosphate buffer, pH 3.5 (21:79) at a flow rate of 1ml/min.The detection of oxytocin
was carried out at a wavelength of 197 nm. The retention time was found to be 4. 78 and
recovery was between 99 and 101 % which was performed by standard addition method.
CH.RAMAMOHAN ROA (2011) :
The numbers of antibiotics, pain killers are important role in the society particularly
animal and human life. In this case diclofenac sodium (DFS) is used to treat pain,
inflammatory disorders, and dysmenorrheal. Inflammatory disorder may include
musculoskeletal complaints to the animals and human to prevent the different pains.
Suppose after usage of this antibiotic the milk was contaminated with the residues of
Diclorofenac sodium. We examine 4 different she-buffalos for our research work. In our
research work collect the raw milk, boiled milk, and curd milk and some different food
stuff. And also analyzed for Diclofenac sodium (DFS) by using H.P.L.C at 258 nm with
UV detector. In this case satisfactory results were obtained
CH.RAMAMOHANA RAO, L.CYRIL ARUN KUMAR (2012):
New HPLC Methods, Method-CPC, Method –EFX are developed for analysis of
Chloramphenicol, Enrofloxacin in shrimp. Method-CPC developed with MeOH water,
Acetonitrile 15:50:35 v/v/v as mobile phase, C18 column as stationary phase, 246 nm
detector wave length, 7.2 PH of mobile phase. The calibration curve concentration range
is 0.2 µg/mL – 1.2 µg/mL. The L.O.Q and L.O.D concentrations are 0.05 µg/mL and 0.02
µg/mL respectively. The percentage of recovery is in between 98.9% to 99.6%. The
average recovery of Method-CPC is 99.27%.The Method –EFX developed for shrimp
tissue analysis for Eenrofloxacin. In this method mobile phase is Acetonitrile, Methanol,
water in the ratio of 17:13:70. Detector is 270 nm. 250 mm length C 18 column. PH of
mobile phase is 6.3. Run time 10 min. The recovery range of Method –EFX is 99.2% to
Page 34
99.75%. Calibration curve plotted in the range of 0.2-0.6 µg/mL. The L.O.D and L.O.Q
values of Method –EFX are 0.02 µg/mL and 0.04 µg/mL.
Manjari Mishra (2014):
Reported oxytocin in milk samples by precipitation with trichloroacetic acid and passed
through the solid phase extraction column. OT was eluted and evaporated to dryness
under a gentle stream of nitrogen. The residue was either dissolved in milli Q water or
buffer for analysis through HPLC or EIA. The intake assessment of OT through milk was
assessed through the Food Frequency Recall method employing a Food Frequency
Questionnaire. On the basis of milk consumption and the values of OT in milk, the actual
intake of OT was calculated. Results: In the present study, a total of 55 milk samples (39
milkman and 16 branded) were analyzed for occurrence of OT by EIA and UV-HPLC
from different locations of Lucknow, Uttar Pradesh (India). OT contamination in
milkman samples was found to be 21 pg/mL to 18.9 ng/mL with the mean value of 8.9
ng/mL . The average daily intake of OT in terms of mg/day/person was highest (2.3–2.4
mg/day/person) in 1–3-year age group. Conclusions: Since there is no prescribed level of
OT in milk and the intake of OT through this commodity is quite high there is need to
implement regulatory laws so that non-physiological OT exposure may not occur in
children which may have deleterious effects.
Luciana Carolina Bauer (2014):
The method proposed here for the simultaneous extraction of cholesterol and
cholesterol oxides, 7-ketocholesterol, and 25-hydroxycholesterol proved to be suitable,
and can be used with reliability for the determination of these compounds in milk. The
method could be valuable for the routine determination of cholesterol and cholesterol
oxides in milk.
Latifa Boultif (2014):

This study involved the optimization and validation of a High Performance Liquid
Chromatography (HPLC) method for the detection and quantification of oxytetracyclines
residues in cow’s milk as well as the adaptation of a technique for its extraction. Results
in these preliminary steps have already served to detect and quantify oxytetracycline
residues in a reduced number of samples. This allows us to initiate a meticulous study on
the presence and quantity of this antibiotic residues in milk produced in our region.
Page 35
AMRITA POONIA (2016):
Milk is a wholesome nutritious dairy product and is consumed by a majority of the

population world-wide for drinking as such, as well as via dairy products. However, the
practice of adulteration of milk invariably reduces its quality and may introduce
hazardous substances into the dairy supply chain jeopardising consumers’ health. Various
instances of adulteration of milk have been reported globally, wherein substances such as
extraneous water, foreign proteins, whey proteins, melamine and urea, vegetable or
animal fats, plus many minor constituents of milk fat have been added as potential
adulterants in milk and milk products. This review focusses on the different methods of
detection of these adulterants in milk using techniques such as DSC, RP-HPLC, LC-GC,
HPTLC.
Mahantesh Kurjogi (2019):
In the present study, antibiotic residues were detected in milk samples collected from the
dairy herds located in Karnataka, India, by microbiological assay. Subsequently, the
detected antibiotics were identified as azithromycin and tetracycline, by high-
performance liquid chromatography, further both the antibiotics detected in the cow milk
samples were found to be at high concentration (9708.7 and 5460 μg kg-1, respectively).
We then investigated the effects of temperature and pH on the stabilities of azithromycin
and tetracycline to determine the degradation rate constant k using first-order kinetic
equation. Results indicated that significant reduction in stability and antibacterial activity
of azithromycin solution when subjected to 70 and 100˚C for 24 h. While stability of
tetracycline was significantly reduced when subjected to 70 and 100˚C for 24 h. However
no significant reduction in antibacterial activity of tetracycline was observed at respective
temperatures when compared with that of control. In addition, the stabilities of
azithromycin and tetracycline were found to be decreased in acidic pH 4–5. The results
of the present study revealed the high risk of contamination of milk sample with
veterinary antibiotics and also demonstrated the effect of temperature and pH on stability
of antibiotics. Therefore the study suggests that the qualitative and quantitative screening
Page 36
of milk for the presence of antibiotics need to be strictly performed to ensure safe drinking
milk for consumer.
4.2 Conclusion from literaturereview:
• Very few analytical methods were reported for estimation of adulterants in milk
by using UV, HPLC.
• Till now no methods are reported for the comparission of different adulterants in
milk brands in Hyderabad.
• So keeping this point into consideration, in the present work an attempt has been
made to develop, validate, comparative studies for determination of adulterants in
milk brands.
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CHAPTER 5 MATERIALS AND METHODS
MATERIALS AND METHODS
5.1 Materials
S.NO. INSTRUMENTS MODELS
1 HPLC WATERS, software: Empower, 2695 separation

module, UV detector
2 UV/VIS spectroscopy LABINDIA UV 3000+, UV WIN SOFTWARE
3 pH meter Adwa-AD 1020
4 Weighing machine Sartorius-CPA, Afcoset ER-200A
5 Potentiometer Eleco Digital Potentio Meter Model-118
6 Conductometer Digisun electronics DI-909
7 Water bath Sisco instrument
8 sonicator Soltec, spincotech instrument
9 Centrifuge Remi
CHEMICALS
S.NO CHEMICALS MANUFRACTURER
1 Iodine Merck
2 Soya bean powder S D fine cheme limited
3 Sliver nitrate FINER chemical LTD
4 Potassium di chromate reagent S D fine cheme limited
5 Para phenylene diamine FINER chemical LTD
6 Phosphate buffer S D fine cheme limited
7 Resorcinol FINER chemical LTD
8 Hydro chloric acid S D fine cheme limited
9 Sulphuric acid Fischer scientific
Page 38
10 Ammonia
11 Methanol S D fine cheme limited
12 Ethanol Fischer scientific
13 Ethyl ether Fischer scientific
14 Petroleum ether Fischer scientific
15 Diclofenac sodium FINER chemical LTD
16 Antibiotics FINER chemical LTD
17 Oxytocin
5.2 METHODS
5.2.1 minor constituents by Quantitative analysis of adulterants in milk
WATER: The presence of water in milk detected by putting a drop of milk on a polished
slanting surface the drop of pure milk flows slowly leaving a white trial behind it whereas, milk
adulterated with water will flow immediately without leaving a mark.
STARCH: To 5ml of milk sample boiled, cooled added a few drops of tincture of iodine or
iodine solution formation of blue color indicates the presence of starch.
UREA: Take tea spoon of milk into test tube add half tea spoon of soybean or arhar powder
mixup contents by shaking the test tubes after 5 minutes dip red litmus paper in it remove the
paper after half a min change in color from red to blue indicates the presence of urea.
DETERGENT: Shake 5-10 ml of sample with equal amount of water lather indicates the
presence of detergent.
GLUCOSE/INVERT SUGAR: Take a strip of diacertic strip and dip in milk for 30 sec to one
min the strip changes the color then it shows the sample of milk contains glucose if there is no
change in the color of the strip then glucose is absent.
Page 39
VANASPATHI: Take 3ml of milk in test tube add 10 drops of HCl and mix 1 tea spoon full of
sugar after 5 min examine the mixture the red coloration indicates the presence of vanaspati
FORMALIN: Take 3ml of milk in test tube add 10 drops of HCl and mix 1 tea spoon full of
sugar after 5 min examine the mixture the red coloration indicates the presence of vanaspati.
Formalin enhances the life of milk so it is added for preservative purpose
Use of formalin shown variable results regarding its ability to preserve samples. Various
reasons, such as use of substandard formalin, storage period, storage temperature, nature of the
sample preserved, have been proposed to explain this variability. The preservative has also
been shown to affect various physico-chemical properties as well as the fat, protein, lactose
and total solids content during storage. This has created a problem and brought industry in
direct conflict with the regulatory agencies. Many research workers have studied the
compositional changes in milk samples as affected by formalin preservation
SALT: Take 5ml of silver nitrate reagent in a test tube add 2-3 drops of potassium dichromate
reagent add 1 ml of milk in above test tube and mix thoroughly. If the contents of the test tube
turns yellow, then milk contains salt. If it turns to chocolate or reddish brown color then sample
free from salt.
Addition of salt in milk is mainly resorted to with the aim of the increasing the corrected
lactometer readings.
HYDROGEN PEROXIDE: To 5ml milk sample add 3 drops of paraphenylenediamine and

shake well change in color of the milk to blue conforms that the milk adulterated with Hydrogen
peroxide
SUGAR: Take 3 ml of milk in a test tube and add 2 ml of HCl heat the test tube after adding
50mg of resorcinol. The red coloration indicates the use of sugar in milk
BORIC ACID: Take 3 ml of milk in test tube add 20 drops of HCl shake dip a yellow paper
strip remove the same after 1 min a change in color from yellow to red followed by a change
from red to green by adding a 1 drop of ammonia solution indicates boric acid is present.
Page 40
To prepare yellow paper strips of filter paper in an aqueous solution of the turmeric, and dry it
up.
REMOVAL OF FAT: The lactometer reading will go above 26.
TOTAL SOLIDS IN MILK: To determine the total solids content of milk, 5g of milk sample
was placed in pre- dried and tarred duplicate crucibles, which were labeled with a pencil. Then
after evaporating the milk sample to dryness on steam bath, the sample was kept at 102⁰ C in a
hot air oven (model 101- 1A Tianjin Taisite Inst. Co. Ltd) for 3 hours. The dried samples are
taken out from the oven, cooled to room temperature and then weighed (Richardson, 1985).
Total solids (TS percent) = (Weight of dried sample) ×100
sample weight
TITARABLE ACIDITY IN MILK: The Titratable acidity (TA) of both yoghurt and milk are
determined using the method described by (Marth 1978). Sample (9 g) was weighed in 100 ml
wide mouth flask. 20 ml of fresh distilled water (in case of yoghurt only) was added and then
titrated against 0.1 N NaOH after adding 3-5 drops of 1 percent phenolphthalein solution until
persistent (30 sec.) faint pink color was observed. The Titratable acidity was expressed as
percentage of lactic acid using the following formula Lacticacid(percent) = ml N alkali ×0.009
ml of sampleused × 100
MAJOR CONSTITUENTS BY QUALITATIVE ANALYSIS
5.2.2 QUALITATIVE ESTIMATION OF OXYTOCIN BY USING RP-HPLC METHOD
Preparation of mobile phase: Acetonitrile:0.03M phosphate buffer, pH3.5(Dil. Ortho

phosphoric acid)
Preparation of Standard solution: It is prepared in 10 ml volumetric flask by dissolving

accurately weighed quantities of oxytocin (10 mg) in HPLC grade water followed by dilution
up to the mark with HPLC grade water (1000 μg / ml ).
Preparation of milk Sample: concentration of milk was prepared by taking 1 ml of sample

solution with 1 ml of ice cold solution of acetone. This solution was mixed and centrifuged at
3500 rpm in the centrifugal apparatus. After centrifugation process, the acetone layer was taken
Page 41
and mixed with 1 ml of petroleum ether. This solution was mixed and kept for 5 minutes. After
5 minutes, the ether layer was discarded and the lower layer was evaporated to dryness, to this
0.2 ml buffer solution was added and filtered. The filtered solution was injected.
5.2.3 QUALITATIVE ESTIMATION OF DICLOFENAC SODIUM BY USING RP-

HPLC METHOD
Preparation of mobile phase: ACN: Phosphate buffer pH 7.0 (50:50v/v)
Preparation of standard solution: Weighed accurately 100mg of diclofenac sodium in 100ml

of volumetric flask and add 25ml of mobile phase.
Preparation of milk sample: Take 4 test tubes each test tube having 4 ml of methanol and 1
ml of milk sample and mixed well with the help of shaker system. After shaking well to filtrate
the each sample with Whitman filter paper then the samples were ready for analyzing with
HPLC Peak. Now from the filtrate milk sample to take 20µL Sample was injected into the
H.P.L.C . Then observed H.P.L.C Report. Basing on the calculation the drug value by using
statistical formula. The sample (liquid 20 % v/v, solid 20 %w/v) preparation was performed by
shaking with a mixture of Water-Methanol (30%:70% v/v) followed by ultra-filtration.
Prepared samples are injected in to H.P.L.C to estimate quantity of Diclofinac sodium.
5.2.4 QUALITATIVE ESTIMATION OF ANTIBIOTIC RESIDUES BY USING RP-

HPLC
Preparation of buffer (pH 4.0): Mobile phase were prepared by dissolving 5.04 gm Disodium
hydrogen phosphate and 3.01 gm potassium dihydrogen phosphate in sufficient water to
produce 1000 ml adjust the pH with glacial acetic acid.
Preparation of mobile phase : Prepared a required volume of degassed mixture of buffer and
acetonitrile in the ratio of 55:45 v/v.
Preparation of diluents: Use mobile phase as diluents.
Preparation of standard solution:
• Weighed and transferred accurately 100 mg of ciprofloxacin working standard into a 100
ml clean, dry volumetric flask. (1000µg/ml).
Page 42
• Diluted 1mL standard stock solution to a 100 mL volumetric flask with mobile phase as
diluents and mixed well. (10 µg/ml).
• Further diluted 2,4,6,8,10 ml standard stock solution with mobile phase as diluents and
mixed well. Results in 0.2, 0.4, 0.6, 0.8, 1 µg/ml concentrations respectively.
Preparation of sample solution:
Liquid phase extraction:
• Weigh 4gm of sample in a clean beaker. The homogenization was done with the addition of
10ml phosphate buffer pH 6.5.
• After proper mixing, protein contents of these samples were precipitated with the adddition
of 2ml trichloroacetic acid (30%).
• Then these samples were taken into properly cleaned and sterilized centrifuge tubes for
centrifugation. The centrifugation was performed at 7000rpm for 15 min.
• Then filtration of the supernatant was performed with the help of whatsmann filter paper and
funnel, then these filtrates was extracted with an equal volume of diethyl ether to perform
defatting.
• Then by using cleaned and sterilized separating funnel, these mixtures separated from each
other and upper oily layer was discarded but only bottom layer was collected.
• This extraction of supernatant was repeated twice with diethyl ether, and then these extracts
were evaporated until dryness.
• The dried extract was reconstituted with 2ml mobile phase as diluents.
• Then filtered with 0.45µ membrane filter.
5.2.5 QUALITATIVE ANALYSIS OF FORMALDEHYDE CONTENT IN MILK
Preparation of mobile phase: Acetonitrile and water at a ratio of 45:55.
Page 43
Preparation of standard solutions: A solution of standard of known concentrations of

formaldehyde (8 ppm) were prepared.
Preparation of sample solution: Accurately measured 40 ml of marketed product was taken

into a centrifuge tube with 4 ml of trichloroacetic acid, to precipitate out the fats and proteins
present in milk and milk products. Then it was centrifuged for 10 min at 3500 rpm. The
supernatant was separated and then filtered using a Whatman 41 filter paper to obtain the final
sample and injected to HPLC.
Chromatographic conditions: The separation was achieved using a 5 µm particle sized

octadecylsilyl (ODS) column (250 mm × 4.6 mm i.d.) (Phenomenex, USA). The mobile phase
consisted of acetonitrile and water at a ratio of 45:55. The mobile phase flow rate was 2.0
ml/min and the injection volume was 20 µl. The analyses were performed at 345 nm for
formaldehyde
5.3 ANALYTICAL METHOD VALIDATION OF OXYTOCIN, DICLOFENAC

SODIUM , ANTIBIOTIC RESIDUES AND FORMALDEHYDE.
Method validation study was performed based on the current pharmaceutical regulatory
guidelines i.e., ICH Q2 (R1). A number of parameters such as precision, accuracy, specificity,
linearity, ruggedness were investigated for this purpose.
System suitability: For the evaluation of system suitability, the repeatability, retention time
and tailing factor of six replicates of working standard of formaldehyde (8 ppm) were used and
percentage relative standard deviation (%RSD) values were calculated in each case.
Linearity: The linearity was checked by analyzing different concentrations of formaldehyde

(2 – 16 ppm). Calibration curves were made using MS Excel 2007 for each standard
component. The regression line was calculated as Y = mX + c, whereX was the concentration
of standard and Y was the response (peak area expressed as AU).
Limits of detection (LOD) and limits of quantitation: (LOQ): LOD and LOQ were
calculated according to ICH Q2 (R1) recommendations in accordance with the 3.3s/m and
10s/m criteria respectively, where ‘s’ is the standard deviation of the peak area and ‘m’ is the
slope of the calibration curve determined from linearity investigation.
Page 44
Accuracy (recovery test): Recovery test was done by injecting a sample of known
concentration of standard solutions. Then percent recoveries (mean ± %RSD of six replicates)
were calculated.
Precision: Repeatability (intra-day precision) and intermediate precision (inter-day precision)

of the methods were determined by using the solution of standard formaldehyde (8 ppm) and
the solutions were analyzed in six replicates on the same day (intra-day precision) and daily
for six times over a period of three days (inter-day precision).
Ruggedness: Ruggedness of the method was determined by analyzing six assay sample
solutions of standard formaldehyde (8 ppm) by two analysts in the same laboratory to check
the reproducibility of the result. The percentage recovery and %RSD were calculated in both
cases.
Page 45
CHAPTER 6 RESULTS AND DISCUSSION
RESULTS AND DISCUSSION
6.1 QUANTITATIVE ANALYSIS OF ADULTERANTS IN MILK

Table:6.1 quantitative analysis of branded milk sample
S.NO ADULTERANT M-VI MAM M-D M-SF M-VJ M-JS M-HG
1 Water ✓ ✓ ✓ ✓ ✓ ✓ ✓
2 Starch - - - - - - -
3 Urea - - - - - -
4 Detregent - - - - - - -
5 Glucose/invert sugar - - - - - - -
6 Vanaspati - - - - - - -
7 Formalin ✓ ✓ ✓ - ✓ ✓ ✓
8 Salt - - - - - - -
9 Hydrogen peroxide - - - - - - -
10 Sugar - - - - - - -
11 Boric acid - - - - - - -
12 Titerable acidity 35% 28.5% 40% 50% 50% 50% 50%
13 Chloride content 5.2% 16% 6.66% 14.2% 2% 3.3% 16%
By this quantitative analysis we came to know that the branded milk sample contains
adulterants such as (water and formalin)
Page 46
6.2 QUANTITATIVE ANALYSIS OF ADULTERANTS IN LOCAL SAMPLE
S.NO ADULTERANT Sample Sample Sample Sample Sample
1 2 3 4 5
1 Water + + + + +
2 Starch - - - - -
3 Urea - - - - -
4 Detergent + - + + -
5 Glucose/invert sugar - + + + +
6 Vanaspati - - - - -
7 Formalin - - - - -
8 Salt - - - - -
9 Hydrogen peroxide - - - - -
10 Sugar - - - - -
11 Boric acid - - - - -
12 Titerable acidity 50% 32.5% 41% 40% 42%
13 Chloride content 2.2% 1.6% 6.66% 1.2% 2%
Table:6.2 quantitative analysis of local milk sample
By this quantitative analysis we came to know that the local milk sample contains
adulterants such as (water, glucose, detergent).
Page 47
6.3 QUALITATIVE ANALYSIS OF ADULTERANTS IN MILK SAMPLES
TRIALS:
TRIAL-1: oxytocin
Mobile phase:Methanol : water (70:30)
Figure 6.1 chromatogram of trail 1
Observation: Splitting of peak is observed so another trial is performed
Discussion: So, the mobile phase composition has been changed for next trails.
Page 48
TRIAL-2: oxytocin
Mobile phase: Methanol: water (80:20)
Figure 6.2chromatogram of trail 2
Observation: The peak shape was not clear so another trial is performed
Discussion: The column has been changed for next trails.
Page 49
TRIAL-3: oxytocin
Mobile phase: Methanol : Acetonitrile (50:50)
Page 50
TRIAL-4: oxytocin
Mobile phase: phosphate buffer: Acetonitrile (70:30)
Page 51
6.3.1 OPTIMIZED METHOD FOR OXYTOCIN
Mobile Phase: acetonitrile:phosphate buffer (21:79)
Column:Dikma, C18, 250X4.6mm, 5µm
Flow Rate: 1.0ml/Min
Column Temperature: 25˚C
Sample Temperature: 25˚C
Volume: 10µL
Run time: 10min
Detector: UV detector
Figure 6.5 chromatogram of optimised method
S.No NAME RETENTION AREA % RESOLUTION TP

TIME HEIGHT
1 Oxytocin 4.7333 648.44 73.37 1.07 1498.5
Table:6.3 optimised parameters of axytocin
Page 52
6.3.2 CALIBRATION CURVE OF OXYTOCIN
S.NO CONCENTRATION AUC

1 0.5 1105
2 1 2045
3 1.5 3650
4 2 3845
5 2.5 4423
6 3 5897
Table:6.4 calibration of oxytocin
7000
CALIBRATION CURVE OF OXYTOCIN
y = 1956.5x
6000 R² = 0.9913
5000
4000
3000
2000
1000
0
0 0.5 1 1.5 2 2.5 3 3.5
Fig:6.6 calibration curve of oxytocin
Figure 6.7 Chromatogram of oxytocin
Page 53
METHOD VALIDATION STUDIES
S.No Parameters Results
1 Theoretical plates (N) 3521
2 Resolution 6.7
3 Linearity range (μ g/ml) 0.5 -2.5
4 Percentage recovery %(Accuracy) 99.8
5 LOD (μg/ml) 3
6 LOQ (μg/ml) 9
7 Tailing factor 1.184
8 Capacity factor 0.86
Table:6.5 Method validation
Recovery Studies
Name of the Percentage Recovery %RSD
drug
100%level 100% level
Oxytocin
99.8 0.002
*Mean of 6 observation
Table: 6.6 recovery studies of oxytocin
Page 54
6.4 QUALITATIVE ANALYSIS OF DICLOFENAC SODIUM IN MILK
TRAIL 1: Diclofenac sodium trail-1
Mobile phase: Methanol : acetonitrile(50:50)
Observation: Appropriate peak is observed so another trial is performed
Page 55
TRAIL 2: Diclofenac sodium
Mobile phase: Methanol: acetonitrile (70:30)
Discussion: So, the mobile phase has been changed for next trails.
Page 56
Mobile phase: Acetonitrile :water (70:30)
Page 57
Mobile phase:Methanol :water (80:20)
Page 58
6.4.1 OPTIMIZATION OF DICLOFENAC SODIUM
Mobile phase 70:30 (methanol : water )
Column C18, 250×4.6mm
pH 5.0
Wave length 258nm
Flow rate 1 ml/min
Run time 6 Min
Retention time 9.18
Figure 6.12 chromatogram of optimised method of diclofenac sodium
S.No NAME RETENTION AREA % RESOLUTION TP

TIME HEIGHT
1 Diclofenac 9.10 4790 80.29 1.25 1594.3
Table:6.7optimization studies of diclofenac sodium
Page 59
6.4.2 Calibration curve of diclofenac sodium by HPLC
The chromatogram of pure drug at concentration 10µg/ml is shown in the fig . The
standard calibration curve and table for Diclofenac sodium is shown in Figure
2nd & Table 1 respectively. A linear correlation was found between area under the curve
and concentration in the ranges 6 to 16µg/ml for diclofenac sodium. The retention time
was observed as 9.15min. The r2 of the calibration curve was found to be 0.998.
CALIBRATION CURVE OF DICLOFENAC SODIUM

500000
450000 y = 27 918x
R² = 0.9996
400000
350000
300000
250000
200000
150000
100000
50000
0
0 2 4 6 8 10 12 14 16 18
S.No. Concentration (µg/mL) AUC 220 nm
1 0 0
2 6 170493
3 8 233270
4 10 285990
5 12 338344
6 14 384983
7 16 438985
R2 0.998
Slope 27403
Intercept 6207
Table:6.8 calibration of diclofenac sodium
Page 60
Figure:6.14 chromatogram of diclofenac sodium
6.5 QUALITATIVE ANALYSIS OF ANTIBIOTIC RESIDUES
Trail-1: Antibiotic residue
Mobile phase: MeOH: water
Figure:6.15 Chromatogram of trail-1
Observation: Tailing of peak is observed so another trial is performed
Page 61
Mobile phase: Methanol: acetonitrile (50:50)
Observation: Tailing of peak is observed so another trial is performed
Page 62
Mobile phase: buffer : acetonitrile (30:70)
Page 63
TRAIL 4: Antibiotic residue
Mobile phase: phosphate buffer: acetonitrile (30:70)
Observation: The analytical peak was not good because of broad peak.
Discussion: so the mobile phase composition has been changed for next trail.
Page 64
6.5.1 Optimization of Antibiotic residue
Column Kromosil C18 column (150mm X 4.6m)5µ
Mobile phase Phosphate buffer 4.0:Acetonitrile (55:45 v/v)
Flow rate 1.0ml per min
Wavelength 258nm
Injection volumn 20µL
Run time 10min
Table:6.9 optimized method of antibiotic residue
Figure:6.19 chromatogram of optimizes method of antibiotic residue
Observation: The analytical peak was good. The plate count also, tailing factor, and the
resolution was found to be satisfactory. The condition is taken as optimized method.
Page 65
6.5.2 CALIBRATION CURVE OF CIPROFLOXACIN
Calibration curve: The linearity of method was evaluated by using standards over a
calibration range of 2 - 400µg/ml for fluoroquinolones in milk sample.
Acceptance criteria: > or = 0.999 r2 (coefficient of correlation)
Calibration curve of ciprofloxacin

35000
R² = 0.9987
30000
25000
20000
15000
10000
5000
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Figure:6.20 calibration curve of antibiotic residue
S.NO. Concentration AUC
1 0.2 5763
2 0.4 11200
3 0.6 15738
4 0.8 20644
5 1 25887
6 1.2 29889
Table:6.10 calibration of antibiotic residue
Figure:6.21 Chromatogram of ciprofloxacin
Page 66
6.5.3 Method validation studies of ciprofloxacin
Specificity
A solution of 20µl of blank, standard and sample was injected into chromatographic
system separately and chromatogram were recorded.
Figure:6.22 Chromatogram of blank solution
Chromatogram of working standard solution of ciprofloxacin
Figure:6.23 Chromatogram of working sample
Specificity formula = sample halfwidth/standard halfwidth x 100
=19.5/18 x 100
=108.3
Discussion: There were no interfering peaks in blank, standard and sample at retention
Page 67
times these analytes in this method. So this method was specific.

REPEATABILITY
Conc Given Conc obtained Conc Given (µg/mL) Conc obtained

(µg/ml)
0.5 0.35 1 0.8
0.5 0.37 1 0.86
0.5 0.39 1 0.9
0.5 0.4 1 0.87
0.5 0.38 1 0.83
0.5 0.36 1 0.92
AVG 0.375 AVG 0.863333
STD 0.018708 STD 0.044121
RSTD 4.988877 RSTD 5.110546
Conc given (µg/ml) Conc obtained

1.5 1.2
1.5 1.29
1.5 1.35
1.5 1.37
1.5 1.42
1.5 1.32
AVG 1.325
STD 0.075565
RSTD 5.702984
Table:6.11 repeatability
Average= 0.854
Standard deviation=2.69/5 =0.538
Page 68
RSD=Std/avg x 100
=62
ACCURACY:
CONC. CONC. OBTAINED
AREA
GIVEN(µg/ L) %RECOVER Y
0.5 1721.3 0.35 70

0.5 1743.8 0.37 74
0.5 1769.3 0.39 78
0.5 1786.8 0.4 80
0.5 1789.9 0.38 76
0.5 1793.9 0.36 72
1 2419.1 0.8 80
1 2426.1 0.86 86
1 2438.1 0.9 90
1 2443.1 0.87 87
1 2461.1 0.83 83
1 2464.1 0.92 92
1.5 3987.6 1.2 80
1.5 3932.4 1.29 86
1.5 3928.9 1.35 90
1.5 3943.9 1.37 91
1.5 3939 1.42 94
1.5 3941.9 1.32 88
table :6.12 accuracy
Page 69
figure: 6.24Chromatogram of accuracy
Acceptance criteria: The percentage recovery was found to be within the limit (70-
94%)
Discussion: The results obtained for recovery at 0.5%, 1%, 1.5% are within the limits.
Hence method is accurate
LIMIT OF DETECTION
Conc given Area Conc obtained

0.2 3732 0.35
0.4 6732.5 0.37
0.6 10616.3 0.39
0.8 13545.9 0.4
1 16284.9 0.38
Table:6.13 limit of detection

Avg=0.378, Std deviation=2638.781
LOD=3*STD/M
=0.318
Observation: The LOD of ciprofloxacin was found to be 0.318.
LIMIT OF QUANTIFICATION:
Page 70
CON GVN AREA CON OBT

0.2 3732 0.35
0.4 6732.5 0.37
0.6 10616.3 0.39
0.8 13545.9 0.4
1 16284.9 0.38
Table:6.14 limit of quantification
Avg= 0.378
Std= 2638.781
LOQ=10 x std/m
=1.062
Observation: The LOQ of ciprofloxacin was found to be 1.062.
6.6 QUALIITATIVE ANALYSIS OF FORMALDEHYDE IN MILK
Validation of the method System suitability.
The results (Mean ± %RSD of six replicates of the standards) of the parameters
indicated good performance of the chromato-graphic system.
PEAK AREA RETENTION TIME TAILING FACTOR

(Mean ± %RSD) (Mean ± %RSD) (Mean ± %RSD)
216226.2 ± 0.80 15.273 ± 0.98 1.117 ± 0.58
Table:6.15 system suitability
Linearity: The linearity was checked by analyzing 2, 4, 8, 12 and 16 ppm formaldehyde

solutions. The linear regression equation for the calibration curve (figure 1) was found
Y = 27951X - 5528 (X=concentration and Y= peak area) with the correlation coefficient
(R2 ) of 0.999.
Page 71
Figure:6.25 calibration curve of formaldehyde
Limit of detection (LOD) and limit of quantitation. (LOQ). The LOD and LOQ of
formaldehyde were found to be 0.4 and 1.15 ppm, respectively.
Accuracy (recovery test). The overall recoveries of the standards are summarized
in table 6.16. The method showed good recovery.
Results of accuracy determination.
Amount used (ppm) eq. to Amount recovered % Recovery

(80% - 120%) respectively (ppm)
6.4 6.22 97.14±0.49

7.2 6.98 96.94±0.51
8 7.45 93.59±0.39
8.8 8.46 96.17±0.53
9.6 9.08 94.55±0.5
Table:6.16 accuracy data
Precision: Repeatability (intra-day precision) and intermediate precision(inter-day
precision) of the method were determined by using the solution of standard
formaldehyde (8 ppm) and the solutions were analyzed in six replicates on the same
day.
Page 72
Table : 6.17. Precision of the method.
Spike level (%) Intra-day (Mean ± %RSD) Inter-day (Mean ± %RSD)
100 216226±0.80 215636±0.55
Table:6.18. Ruggedness study of the method.
Amount found % Recovery ± % Amount found % Recovery ± %

(ppm) RSD (ppm) RSD
7049 93.59 ±0.39 7.51 94.26 ±0.51
Figure:6.26 Chromatogram of blank sample
Page 73
Figure:6.27 Chromatogram of standard formaldehyde
Figure:6.28 Chromatogram of sample
Page 74
6.7 Comparative study:
S.NO. Milk brands Oxytocin Concentration MRL

µg/mL µg/day/person
1 Visakha 1.9 2.3-2.4
2 Amul 1.1 2.3-2.4
3 Dodla 1.8 2.3-2.4
4 Sid farm milk 0.9 2.3-2.4
5 Vijaya 1.2 2.3-2.4
6 Jersy 1.6 2.3-2.4
7 Heritage 1.7 2.3-2.4
8 Local milk sample 2.3 2.3-2.4
Table:6.19 comparative studies of oxytocin
S.NO. Milk brands Diclofenac sodium MRL

Concentration (µg/ml) (µg/mL)
1 Visakha 0.092 0.1
2 Amul 0.041 0.1
3 Dodla 0.065 0.1
4 Sid farm milk 0.012 0.1
5 Vijaya 0.034 0.1
6 Jersy 0.056 0.1
7 Heritage 0.075 0.1
8 Local milk sample 0.084 0.1
Table:6.20 comparative studies of diclofenac sodium
Page 75
S.NO. Milk brands Ciprofloxacin MRL

Concentration (ppm/kg) (ppm/kg)
1 Visakha 0.09 0.1
2 Amul 0.051 0.1
3 Dodla 0.074 0.1
4 Sid farm milk 0.025 0.1
5 Vijaya 0.045 0.1
6 Jersy 0.053 0.1
7 Heritage 0.087 0.1
8 Local milk sample 0.093 0.1
Table:6.21 comparative studies of ciprofloxacin
Page 76
CHAPTER 7 CONCLUSION
CONCLUSION
At the end of the project we hope to establish various easy detecting qualitative and
quantitative techniques for the identification of adulteration in milk samples and to
estimate the level of adulteration in each sample. Estimation of major adulterants like
antibiotic residues, hormones and diclofenac sodium by RP-HPLC method in different
branded milk sample, also Compared the level of adulterants used in different branded
milk samples by means of different chromatographic techniques. The concentrations of
oxytocin, Diclofenac, and Antibiotic residues of Vishaka, Dodla, Heritage were found to
be the closeness to the maximum residual limit whereas the samples like Sid farm milk,
Amul and Vijaya milk were found to be more safe and the concentration of adulterants in
these brands are comparatively less. on the other hand, the sample collected from local
area is having the higher concentrations of oxytocin, Diclofenac, and Antibiotic residues
than MRL.
Page 77
CHAPTER 8 REFERENCES
REFERENCES
1. MENG HH, CHEN DD, QI KZ. Simultaneous Determination of Ampicillin and

Amoxicillin Residues in Dairy Milk by High Performance Capillary
Chromatography [J]. Progress in Veterinary Medicine. 2008;1.
2. Kavitha MP, Kumar KS. RP-HPLC method development and validation for the
estimation of oxytocin in milk. Int J Chem Tech Res. 2010;2(2):1340-3.
3. Rani R, Medhe S, Raj KR, Srivastava M. Standardization of HPTLC method for
the estimation of oxytocin in edibles. Journal of food science and technology.
2013 Dec 1;50(6):1222-7.
4. Beard Jr EL. The american society of health system pharmacists. JONA'S

healthcare law, ethics and regulation. 2001 Sep 1;3(3):78-9.
5. Ra C. QUALITATIVE AND QUANTITATIVE AN MILK AND DAIRY

PRODUCTS.
6. Mazumdar K, Dutta NK, Dastidar SG, Motohashi N, Shirataki Y (2006).

"Diclofenac in the management of E. coliurinary tract infections". In Vivo20(5):
613–619.PMID17091768.
7. Subramanian S, Ross Nw Mackinnon SL. Comparison of antimicrobial activity in

the epidermal mucus extracts of fish. Comp BiochemPhysiol B, 150: 85-92. 10.
8. Mat Jais am, Matori MF, Kittakoop P, Sowanborirux K. Fatty acid composition
in mucus and roe of Haruan, Channastriatus, for wound healing. Gen Pharmacol
30: 561-563. 11.
9. Larson .K, Hermann. W, Moller. P, Sanchez. D., Preparative High performance

Liquid Chromatography of peptides on a new reverse phase packing material,
Kromasil, C18, Ferring Pharmaceuticals, Sweden. Journal of chromatography
October 1998, 450(1), 71-80.
10. Sandra Pereira FUKUDA., Salvador Massaguer ROIG., Louis Fransisco

PRATA., Correlation between acidic ninhydrin and HPLC detection methods to
evaluate addition of whey in milk. Veterinary University, Brazil, EDP sciences
2004.
Page 78
CHAPTER 8 REFERENCES
11. Azad T, Ahmed S. Common milk adulteration and their detection techniques.
International Journal of Food Contamination. 2016 Dec 1;3(1):22.
12. Urbanke W, Luf W, Brandl E. Use of HPLC for control of the adulteration of milk
and milk products of different species. Zeitschrift fuer Lebensmittel Untersuchung
und Forschung. 1992.
13. Poonia A, Jha A, Sharma R, Singh HB, Rai AK, Sharma N. Detection of
adulteration in milk: A review. International journal of dairy technology. 2017
Feb;70(1):23-42.
14. Swathi JK, Kauser N. A study on adulteration of milk and milk products from
local vendors. International Journal of Biomedical and Advance Research.
2015;6(09):678-81.
15. Zachar P, Šoltés M, Kasarda R, Novotný J, Novikmecová M, Marcinčáková D.
Analytical methods for the species identification of milk and milk products.
Mljekarstvo/Dairy. 2011 Jul 1;61(3).
16. Harding F, editor. Milk quality. New York: Blackie Academic & Professional;
1995 Dec 31.
17. Rao PS, Sharma R, Rajput YS. Direct estimation of sialic acid in milk and milk
products by fluorimetry and its application in detection of sweet whey adulteration
in milk. Journal of dairy research. 2012 Nov;79(4):495-501.
18. Anand, S.P. and Sati, N. Artificial preservatives and their harmful effects: looking
toward nature for safer alternatives. Int. J. Pharm. Sci. Res. 4, 2005, 2496-2501
19. Chanda, T., Debnath, G.K., Hossain, M.E., Islam, M.A., Begum, M.K.
Adulteration of raw milk in the rural areas of Barisal district of Bangladesh.
Bangladesh J. Anim. Sci. 2012, 41, 112-115.
20. Korpan, Y.I., Gonchar, M.V., Sibirny, A.A., Martelet, C., El’skaya, A.V., Gibson,
T.D., and Soldatkin. Development of highly selective and stable potentiometric
sensors for formaldehyde determination. Biosens. Bioelectron 2000.
Page 79
S.NO ROLL NO. NAME OF CONFERENCES PUBLICATION
THE ATTENDED DETAILS
CANDIDATE
1 170518885004 G. Laxmi Participated and presented Published a paper
Prasanna a poster on synthesis of entitled “simultaneous
different dipeptides from 4- estimation of
hydroxy isoleucine and Anti-Reteroviral drugs
evaluation of their anti- by using various
diabetic activity analytical techniques”.
INTERNATIONAL IJCPS 2019, Aug, vol

CONFERENCE ON (2)
DRUG DISCOVERY
Participated and presented Published a paper
a poster on simultaneous entitled “comparative
estimation of anti- studies on seasonal
Reteroviral drugs by using variations of essential
various analytical oil composition of
techniques Ocimum species”
DIGITALISATION: 2020
CHALLENGES AND IJRAR,VOL.7,ISSUE1
OPPORTUNITIES IN
TRANSFORMING THE
PHARMA AND
HEALTHCARE
Participated in three day Published a paper
workshop on “HANDS ON entitled “A review on
TRANING ON COVID 19 and
ANALYTICAL challenges of pharma
INSTRUMENTATION” profession during
IN pandemic conditions”
JNTU-H
2020 IJCRT VOL8,
ISSUE 9
Participated in the webinar
ROLES OF NANO
TECHNOLOGY IN ANTI-
VIRAL AND ANTI-
BACTERIALS DRUGS
ORGANISED BY BVRIT
Participated and presents a
poster on Alzheimers
disease
INNOVATION IN
PHARMACEUTICAL
RESEARCH

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QUALITYASSESSMENT, COMPARISION, DETECTION AND

DETERMINATION OF VARIOUS ADULTERANTS IN BRANDED AND

THE DISSERTATION SUBMITTED TO OSMANIA UNIVERSITY IN

SRI VENKATESHWARA COLLEGE OF PHARMACY

OSMANIA UNIVERSITY HYDERABAD – 500 081

I, G. LAXMI PRASANNA, Student of M. Pharmacy (Pharmaceutical Analysis), bearing H.T.

Dr. M. Bhagavan Raju

Sri Venkateshwara College of Pharmacy Hyderabad

1.3 Adverse effects by addition of adulterants in milk 6

1.4 Importance of milk 13

1.5 Definition of some common form of milk / milk products 14

1.6 Use of preservatives 16

1.7 High-performance liquid chromatography 17

AIM AND OBJECTIVE 32

4.1 Literature review 34

4.2 Conclusion from literature review 37

MATERIALS AND METHODS

5.2.2 Qualitative estimation of oxytocin in milk 41

5.2.3 Qualitative analysis of diclofenac sodium in milk 42

5.2.4 Qualitative analysis of antibiotic residues in milk 42

5.2.5 Qualitative analysis of formaldehyde in milk 44

5.3 Analytical Method Validation of oxytocin, 45

diclofenac sodium, antibiotic residues and formaldehyde

RESULTS AND DISCUSSION

6.1 Quantitative analysis of adulterants in branded milk 46

6.2 Quantitative analysis of adulterants in local milk 47

6.3 Qualitative analysis of oxytocin in milk 48

6.4 Qualitative analysis of diclofenac sodium in milk 55

6.5 Qualitative analysis of antibiotic residues in milk 61

6.6 Qualitative analysis of formaldehyde in milk 71

6.7 Comparative studies 75

I express my deep senses of privilege to my guide, Dr. P. SRIDEVI, HOD- Department of

Keywords: Adulteration; FSSAI, Quality of milk

TABLE TITLE Page

1.1 Adverse effects of addition of adulterants in milk 5

1.2 Per captia of milk available in different areas 14

6.2 Quantitative analysis of adulterants in branded milk 47

6.3 Chromatographic conditions of optimised method of 53

6.4 Results of Calibration curve of oxytocin 53

6.5 Results of method validation 54

6.6 Recovery studies of oxytocin 54

6.7 Chromatographic conditions of optimised method 59

6.8 Results of Calibration curve of diclofenac sodium 60

6.9 Chromatographic conditions of optimised method of 65

6.10 Results of Calibration curve of antibiotic residue 66

6.11 Repeatability results of antibiotic residue 68

6.12 Data of accuracy 69

6.13 Results of limit of detection 70

6.14 Results of limit of quantification 71

6.15 System suitability of formaldehyde 71

6.17 Data of precision 73

6.18 Ruggedness results of formaldehyde 73

6.19 Comparative studies of various milk samples 75

6.20 Comparative studies of various milk samples 75

6.21 Comparative studies of various milk samples 76

FIGURE FIGURE NAME Page

1.2 Adulterants of milk 5

1.3 Normal phase chromatography 19

1.6 HPLC instrument 22

1.7 HPLC column 24

1.8 Schematic representation of size exclusion 26

1.9 Pictorial representation of ion exchange 26

6.1 Chromatographic conditions trail 1 48