Athira v c

ANALYSIS OF CHEMICAL
RESIDUES IN MILK
SUBMITTED BY
ATHIRA VC
19-MSVM-03

CONTENTS
 INTRODUCTION
Analysis of ;
 ANTIBIOTICS
 PESTICIDES
 HEAVY METALS
 DETERGENTS

INTRODUCTION
 Chemical contaminants in milk and dairy products
may known to be contributory factors in
several diseases such as cancer, heart disease,
Alzheimer's and Parkinson disease.
 They get accumulated in milk through feeds,
veterinary drugs, milk utensils, etc.
 Major chemicals which contaminate milk are;
antibiotics, heavy metals, pesticides and detergents.

ANTIBIOTIC RESIDUES
 The most frequently and commonly used
antimicrobials associated with milk are
antibiotics.
 Different groups of antibiotics which are
available to treat infected dairy cattle; the most
common groups include:-
1. β-lactams (e.g. penicillin)
2. Sulphonamides (e.g. sulfamethazine)
3. Aminoglyclosides (e.g. streptomycin)
4. Tetracyclines (e.g. tetracycline)

ANTIBIOTIC TOLERENCE LIMIT
(mg/kg)
AMPICILLIN 0.01
STREPTOMYCIN 0.02
TETRACYCLIN 0.1
SULFAMIDINE 0.025

ANALYSIS OF ANTIBIOTIC RESIDUES
 Various analytical methods used to determine
antibiotic residues in milk are:
1. Chromatographic methods
2. Microbiological methods
3. Immunochemical tests

Chromatographic Methods
 Most reliable technique for quantitative detection
of antibiotic residues.
1. Gas Chromatography (GC)
 Principle: The sample solution injected into the
instrument enters a gas stream which supports the
sample into a separation tube known as the
column.
 The milk sample is dissolved in a suitable solvent
before the analysis.
 A detection system detects the amount of antibiotic
residue in the sample.

2. Thin Layer Chromatography (TLC)
 Principle: Different compounds will have different
solubilities and adsorption to the stationary and
mobile phase between which they are to be
partitioned.
 A suitable solvent system is used during the
analysis.
 Protein precipitation is done before analysis at the
time of sample preparation.
 Supernatent of the sample after a centrifugation
process is taken for the analysis.

3. High Pressure Liquid Chromatography
(HPLC)
 Principle: HPLC works based on the distribution of
the sample between a mobile phase and a stationary
phase. Depending on the chemical structure of the
sample the molecule are retarded while passing
though the phase.
 Reagents used depends on the antibiotic which is to
be analyzed.
 Extraction of sample is done before analysis.
 A detector system detects the quantity of antibiotic
residue.

Microbiological Methods
 Microbiologically a screening test is done in the
antibiotic residue analysis of milk.
 Reconstitute skim milk is used as the control
during the test.
 1:25 aqueous solution of 2,3,5-triphenyl
tetrazolium chloride is used as the indicator
reagent.
 Comparison in intensity of red colour with
control gives the result.
 A lighter shade of sample than control indicates
presence of antibiotic substances.

IMMUNOCHEMICAL TESTS
 They are capable of detecting low levels of
antibiotic residues in milk.
 These tests are rapid , sensitive, cost effective and
require little sample clean-up for analysis.
 Enzyme linked immunosorbent assay (ELISA) is
mainly performed in the case of antibiotic residue
analysis of milk.

KIT TESTS
 There are readymade receptor assay kits available for
analysis of antibiotic residue in milk.
 They are rapid assays which provide results within a
short time.
 Presence of antibiotic residue can be observed by
comparing the colour change with color bands
provided in the kit.

 Betastar kit
 A rapid detection assay for
antibiotics in cow, goat and
sheep milk.
 A self-contained kit for 25
tests, containing 25 individual
vials of lyophilized receptor, 1
container with 25 dipsticks, 1
syringe and 25 tips.
 The test employs binding of
reagents linked to gold
particles.

Interpretations:-
 No red color- invalid test
 First band intensity more than reference band-
negative test
 First band intensity lower than reference band-
positive test
 Absence of first band- highly positive

Trisensor kit
 Requires two element, a reagent
containing labeled receptor and
a dipstick with two membrane
captures lines that turns from
green to red.
 Limit of detection is around
25ppb.
 Provide results in 6 minutes.
Interpretation:-
 No color lines- Invalid result
 Test line prominent than
control line- Negative test
(<25pbb)

 Test line is less visible than control line- Positive
(>25pbb)
 Absence of test line- highly positive

SNAP TEST
 Introduced by IDEXX laboratories an American
company.
 Commonly used device to determine antibiotic
residues in milk.
 Results in 6 minutes.
 Raw milk can be tested directly from a tanker or
refrigerator.

PROCEDURE
 Add milk to the tube by using
pipette up to indication line
and swirl.
 Pour onto the well.
 Snap when the sample reaches
the activation window.
 Keep for 6 minutes.
Interpretations:-
 Test result darker than
control- Negative
 Test result lighter than
control- Positive

PESTICIDE RESIDUES
 Pesticide residues are accumulated in milk from
feeds provided to the cattle.
 Dichlorodiphenyltrichloroethane (DDT), aldrin,
endosulfan, Parathion,N-methylcarbamate are the
major pesticides could be seen in milk.
 A maximum residue level (MRL) is the highest level
of a pesticide residue that is legally tolerated in milk.

ANALYSIS OF PESTICIDE RESIDUES
Gas-Liquid Chromatography (GLC)
 Mainly used for the detection of organophosphates
and carbamate residues.
 Principle: GLC runs on the principle partition. In
GLC the components of vaporize samples are
fractioned due to partition between a gaseous mobile
phase and a liquid stationary held in column.
 Extraction of sample is done before analysis.
 Sample quantity should not be less than 0.5kg.
 An electron capture (EC) detector system is used for
the detection.

Multiresidue Method
 Performed for the detection of chlorinated
pesticides like aldrin, endosulfan, etc..
 Principle: Thoroughly mixed test portion is extracted
with acetonotrile. Fat is extracted from milk and
partitioned between petroleum ether and acetonitrile.
 Purification of sample is done by chromatographic
methods.
 Amount of residue is measured by using gas
chromatographic method.

HEAVY METALS
 Listed as priority pollutants by the United States
Environment Protection Agency (UEPA).
 Pd, Cd, As, Hg
 Heavy metals accumulated in the milk through paper,
paints ,etc.. that are consumed by animals during
grazing.
 Analysis of heavy metals can be done by various
chromatographic and spectrometric methods.

METAL MAXIMUM PERMITTED LEVEL
IN MILK (ppb)
LEAD 0.02
ARGON 0.1
CADMIUM 0.1
MERCURY 0.25

Detection of Lead in milk
 It is done by spectrometer.
 Standard solution, intermediate solution and working
solution are required for the analysis.
 Sample preparation is done by drying the suspected
milk sample.
 1N nitric acid is used in the sample preparation for
the dissolving of lead.
Calculation: Pb(ppm)= (sample reading-blank
reading).final dilution weight of sample

DETERGENTS
 Detergents get accumulated in milk during cleansing
of milk utensils and also used as an adulterant.
 Both acid and alkali detergent accumulation
contaminates milk.

ANALYSIS OF DETERGENT RESIDUES
 Both sample milk and pour milk treated with
methylene blue and chloroform solutions.
 Ethanol is used for the protein precipitation.
 Difference in color of methylene blue in the
chloroform layer of suspect sample with pure milk
gives result.
 Appearance of more intense blue color in sample
indicates the presence of detergents.

Test For Anionic Detergents
 This method can detect presence of 0.15% level of
laboratory grade in milk.
 A sample preparation is done by warming the
suspected milk sample.
 A immediate analysis is done when temperature of
sample reaches at room temperature.
 1ml of methylene blue dye and 2ml chloroform used
as reagents.
 Difference in intensity of blue colour in lower and
upper layer gives the result.

Interpretations:-
 Relatively more intense color in the lower layer
-presence of detergent in milk.
 Relatively more intense color in the upper layer
- absence of detergent in milk.

REFERENCE
 “CHALLENGES TO CONTEMPORARY- DAIRY ANALYTICAL
TECHNIQUES”-The Royal Society of Chemistry Burlington House,
London W1V 0BN
 “Quality Assessment of Milk & Milk Products”- D.K. Thompkinson
 fssai
 “Guidance document food safety management systems food industry guide
to implement gmp/ghp requirements”. Based on Part II &III of Schedule 4
of Food Safety & Standards (Licensing & Registration of Food
Businesses) Regulation, 2011

Athira v c

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