1. GIVE DETAIL PROTOCOLS FOR THE ANALYSIS OF
VITAMINS AND TOXICANTS IN FOOD
BY
OCHENI RABI OJOMA
14/7706/PGD
UNIVERSITY OF AGRICULTURE MAKURDI
DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY
ON
FOOD ANALYSIS AND INSTRUMENTATION TECHNIQUES
FSD 002
LECTURER
MR. A.I. SENGEV
DECEMBER, 4TH 2014

2. PROTOCOLS FOR THE ANALYSIS OF VITAMINS
ANALYSIS OF VITAMINS
Vitamins are a group of organic nutrients required in small quantities for a variety of
biochemical functions and which, generally, cannot be synthesized by the body and must
therefore be supplied in the diet.
The lipid-soluble vitamins are polar hydrophobic compounds that can only be absorbed
efficiently when there is normal fat absorption. They are transported in the blood, like any other
polar lipid, in lipoproteins or attached to specific binding proteins. They have diverse functions,
e.g., vitamin A, vision; vitamin D, calcium and phosphate metabolism; vitamin E, antioxidant;
vitamin K, blood clotting. As well as dietary inadequacy, conditions affecting the digestion and
absorption of the lipid-soluble vitamins - such as steatorrhea and disorders of the biliary system -
can all lead to deficiency syndromes, including: night blindness and xerophthalmia (vitamin A);
rickets in young children and osteomalacia in adults (vitamin D); neurologic disorders and
anemia of the newborn (vitamin E); and hemorrhage of the newborn (vitamin K). Toxicity can
result from excessive intake of vitamins A and D. Vitamin A and β-carotene (provitamin A), as
well as vitamin E, are antioxidants and have possible roles in atherosclerosis and cancer
prevention. (Fafana)
The water-soluble vitamins comprise the B complex and vitamin C and function as enzyme
cofactors. Folic acid acts as a carrier of one-carbon units. Deficiency of a single vitamin of the B
complex is rare, since poor diets are most often associated with multiple deficiency states.
Nevertheless, specific syndromes are characteristic of deficiencies of individual vitamins, eg,
beriberi (thiamin); cheilosis, glossitis, seborrhea (riboflavin); pellagra (niacin); peripheral
neuritis (pyridoxine); megaloblasticanemia, methylmalonicaciduria, and pernicious anemia
(vitamin B12); and megaloblasticanemia (folic acid). Vitamin C deficiency leads to scurvy.
This nutritional, biomedical and medical significance of vitamins makes its analysis very
imminent.

3. VITAMIN A (Vitamin A Concentrate (Oily Form) Synthetic)
Assay
To carry out the assay as rapidly as possible, avoiding exposure to actinic light and air, oxidizing
agents, oxidation catalysts (e.g. copper, iron), acids and prolonged heat; using freshly prepared
solutions. If partial crystallization has occurred, the material is homogenized at a temperature of
about 65 °C, but avoiding prolonged heating.
Examine by ultraviolet absorption spectrophotometry
Dissolve 25 mg to 100 mg, weighed with an accuracy of 0.1 percent, in 5 ml of pentane R* and
dilute with 2-propanol R1 to a presumed concentration of 10 IU/ml to 15 IU/ml. Verify that the
absorption maximum of the solution lies between 325nm and 327nm and measure the
absorbance at 300 nm, 326 nm, 350 nm and 370 nm.
Repeat the readings at each wavelength and take the mean values.
Calculate the ratio A_/A326 for each wavelength.
If the ratios do not exceed:
0.593 at 300 nm,
0.537 at 350 nm,
0.142 at 370 nm.
Calculate the content of vitamin A in International Units per gram from the expression:
(A326 x V x 1900) / (100 x m)
A326= absorbance at 326nm, m=mass of the preparation to be examined, in grams
1900= factor to convert the specific absorbance of esters of retinol into international
Units per gram

4. Vitamin A Concentrate (Powder Form) Synthetic
Assay
Carry out the assay as rapidly as possible, avoiding exposure to actinic light and air, oxidizing
agents, oxidation catalysts (e.g. copper, iron), acids and prolonged heat.
Examine by Liquid Chromatography
Test solution (a). Introduce into a 50 ml volumetric flask, an amount of the preparation to be
examined, weighed with an accuracy of 0.1 percent and equivalent to about 120000 IU of
vitamin A.
Add 20 mg to 30 mg of bromelains R, 20 mg to 30 mg of butylhydroxytoluene R, 2.0 ml of
water R and 0.15 ml of 2-propanol R. Heat gently in a water-bath at 60°C to 65°C for 2 to 5 min.
Cool to below 30°C and add 20 ml of 0.1 M tetrabutylammonium hydroxide in 2-propanol.
Swirl gently for 5 min (an ultrasonic bath is recommended).
Dilute to 50.0 ml with 2-propanol R and homogenise carefully to avoid air-bubbles. Residue of
the matrix may cause more or less cloudiness of the solution.
Test solution (b). Introduce 20 mg to 30 mg of butylhydroxytoluene R into a 50 ml volumetric
flask, add 5 ml of 2-propanol R, 5.0 ml of test solution (a) and dilute to 50.0 ml with 2-propanol
R. Homogenise carefully to avoid air-bubbles. Filter before injection.
Reference solution (a).Introduce into a 50 ml volumetric flask about 120 mg of retinol acetate
CRS, weighed with an accuracy of 0.1 percent and dissolve immediately in 5 ml of pentane R.
Add 20 mg to 30 mg of butylhydroxytoluene R and 20 ml of 0.1 M tetrabutylammonium
hydroxide in 2-propanol. Swirl gently for 5 min (an ultrasonic bath is recommended) and dilute
to 50.0 ml with 2-propanol R.
Homogenise carefully to avoid air-bubbles. (Fafana)
Reference solution (b).Place 20 mg to 30 mg of butylhydroxytoluene R in a 50 ml volumetric
flask, add 5 ml of 2-propanol R, 5.0 ml of reference solution (a) and dilute to 50.0 ml with
2-propanol R, homogenize carefully to avoid air-bubbles. The chromatographic procedure may
be carried out using: - a stainless steel column 0.125 m long and 4 mm in internal diameter
packed with octadecylsilyl silica gel for chromatography R (5 μm), - as mobile phase at a flow

5. rate of 1 ml/min a mixture of 5 volumes of water R and 95 volumes of methanol R, - as detector
a spectrophotometer set at 325 nm, - a loop injector.
The assay is not valid unless:
- the chromatogram obtained with reference solution (b) shows a principal peak corresponding to
that of all-(E)-retinol, the retention time of all-(E)-retinol being about 3 min,
- there is no peak corresponding to unsaponified retinol acetate in the chromatogram obtained
with reference solution (b) at a retention time of about 6 min.
Inject a suitable volume of reference solution (b) in order to obtain an absorbance in the range of
0.5 to 1.0 at 325 nm and record the chromatogram using an attenuation so that the height of the
peak corresponding to vitamin A is not less than 50 percent of the full scale of the recorder.
Make a total of six injections. The relative standard deviation of the response for reference
solution (b) should not be greater than 1 percent. Inject the same volume of test solution (b) and
record the chromatogram in the same manner.
Calculate the content of vitamin A from the expression:
(A1 x C x m2)/A2 x m1
A1 area of the peak corresponding to all-(E)-retinol in the chromatogram obtained with test
solution (b),
A2 area of the peak corresponding to all-(E)-retinol in the chromatogram obtained with reference
solution (b),
C concentration of retinol acetate CRS in International Units per gram, determined by the
method below,
M 1 mass of the substance to be examined in test solution (a), in milligrams,
M 2 mass of retinol acetate CRS in reference solution (a), in milligrams.
Vitamin B1 (Thiamine Nitrate)
Dissolve 0.140 g in 5 ml of anhydrous formic acid R and add 50 ml of acetic anhydride R.
Titrate immediately with 0.1 M perchloricacid, determining the end-point potentiometrically and
carrying out the titration within 2 min. Carry out a blank titration.
1 ml of 0.1 M perchloric acid is equivalent to 16.37 mg of C12H17N5O4S

6. Identification
First identification: A, C.
Second identification: B, C.
A. Infrared absorption spectrophotometry
Comparison: Ph. Eur. Reference spectrum of thiamine nitrate.
B. Dissolve about 20 mg in 10 ml of water R, add 1 ml of dilute acetic acid R and 1.6 ml of 1 M
sodium hydroxide, heat on a water-bath for 30 min and allow to cool. Add 5 ml of dilute sodium
hydroxide solution R, 10 ml of potassium ferricyanide solution R and 10 ml of butanol R and
shake vigorously for 2 min. The upper alcoholic layer shows an intense light-blue fluorescence,
especially in ultraviolet light at 365 nm. Repeat the test using 0.9 ml of 1 M sodium hydroxide
and 0.2 g of sodium sulphite R instead of 1.6 ml of 1 M sodium hydroxide.
Practically no fluorescence is produced.
C. About 5 mg gives the reaction of nitrates
VITAMIN B2 (RIBOFLAVINE)
Assay
Carry out the assay protected from light. Dissolve 0.100 g in 150 ml of water R, add 2 ml of
glacial acetic acid R and dilute to 1000.0 ml with water R. To 10.0 ml add 3.5 ml of a 14 g/l
solution of sodium acetate R and dilute to 50.0 ml with water R. Measure the absorbance at the
maximum at 444 nm.
Calculate the content of C17H20N4O6 taking the specific absorbance to be 328.
Identification
A. Dissolve 50.0 mg in phosphate buffer solution pH 7.0 R and dilute to 100.0 ml with the same
buffer solution. Dilute 2.0 ml of the solution to 100.0 ml with phosphate buffer solution pH 7.0.
Examined between 230 nm and 350 nm, the solution shows an absorption maximum at 266 nm.
The specific absorbance at the maximum is 580 to 640.(Fafana)

7. VITAMIN B6 (PYRIDOXINE HYDROCHLORIDE)
Assay
In order to avoid overheating in the reaction medium, mix thoroughly throughout and stop the
titration immediately after the end-point has been reached. Dissolve 0.150 g in 5 ml of anhydrous
formic acid R. Add 50 ml of acetic anhydride R. Titrate with 0.1 M perchloricacid, determining
the end-point potentiometrically. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is
equivalent to 20.56 mg of C8H12ClNO3.
Identification
First identification: B, D.
Second identification: A, C, D.
A. Dilute 1.0 ml of solution S to 50.0 ml with 0.1 M hydrochloric acid (solution A). Dilute 1.0
ml of solution A to 100.0 ml with 0.1 M hydrochloric acid. Examined between 250 nm and 350
nm, the solution shows an absorption maximum at 288 nm to 296 nm. The specific absorbance at
the maximum is 425 to 445. Dilute 1.0 ml of solution A to 100.0 ml with a mixture of equal
volumes of 0.025 M potassium dihydrogen phosphate solution and 0.025 M disodium hydrogen
phosphate solution. Examined between 220 nm and 350 nm, the solution shows 2 absorption
maxima, at 248 nm to 256 nm and at 320 nm to 327 nm.The specific absorbances at the maxima
are 175 to 195 and 345 to 365, respectively.
B. Examine by infrared absorption spectrophotometry, comparing with the spectrum obtained
with pyridoxine hydrochloride CRS.
C. Examine the chromatograms obtained in the test for related substances. The principal spot in
the chromatogram obtained with test solution (b) is similar in position, colour and size to the
principal spot in the chromatogram obtained with reference solution (a).
D. Solution S gives reaction (a) of chlorides.
Solution S
Dissolve 2.50 g in carbon dioxide free water R and dilute to 50.0 ml with the same solvent.

8. Test solution (a). Dissolve 1.0 g of the substance to be examined in water R and dilute to 10 ml
with the same solvent.
Test solution (b). Dilute 1 ml of test solution (a) to 10 ml with water R. Pyridoxine
hydrochloride
VITAMIN C (ASCORBIC ACID)
Assay
Dissolve 0.150 g in a mixture of 10 ml of dilute sulphuric acid R and 80 ml of carbon dioxide-
free water R. Add 1 ml of starch solution R.Titrate with 0.05 M iodine until a persistent violet-
blue colour is obtained. 1 ml of 0.05 M iodine is equivalent to 8.81 mg of C6H8O6.
Identification
A. Dissolve 0.10 g in water R and dilute immediately to 100.0 ml with the same solvent.
To 10 ml of 0.1 M hydrochloric acid, add 1.0 ml of the solution and dilute to 100.0 ml with water
R. Measure the absorbance at the maximum at 243 nm immediately after dissolution. The
specific absorbance at the maximum is 545 to 585.
B. Examine by infrared absorption spectrophotometry, comparing with the spectrum obtained
with ascorbic acid CRS. Examine the substance prepared as discs containing 1 mg.
C. Solution S. Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same
solvent.
The pH (2.2.3) of solution S is 2.1 to 2.6.
D. To 1 ml of solution S add 0.2 ml of dilute nitric acid R and 0.2 ml of silver nitrate solution
R2. A grey precipitate is formed.
Note
1 ml of 0.05 M iodine is equivalent to 9.91 mg of C6H7NaO6 (Sodium ascorbate).
1 ml of 0.05 M iodine is equivalent to 8.81 mg of (C6H7O6)2Ca (Calcium ascorbate).
VITAMIN D3 (CHOLECALCIFEROL powdered form)
Assay
Avoid exposure to air and actinic light.

9. Test solution. Introduce into a Saponification flask a quantity of the preparation to be examined,
weighed with an accuracy of 0.1 percent, equivalent to about 100000 IU. Add 5 ml of water R,
20 ml of ethanol R, 1 ml of sodium ascorbate solution R and 3 ml of a freshly prepared 50 per
cent m/m solution of potassium hydroxide R. Heat under a reflux condenser on a water-bath for
30 min. Cool rapidly under running water. Transfer the liquid to a separating funnel with the aid
of two quantities, each of 15 ml, of water R, one quantity of 10 ml of alcohol R and two
quantities, each of 50 ml, of pentane R. Shake vigorously for 30 s. Allow to stand until the two
layers are clear. Transfer the lower aqueous alcoholic layer to a second separating funnel and
shake with a mixture of 10 ml of alcohol R and 50 ml of pentane R. After separation, transfer the
aqueous-alcoholic layer to a third separating funnel and the pentane layer to the first separating
funnel, washing the second separating funnel with two quantities, each of 10 ml, of pentane R
and adding the washings to the first separating funnel. Shake the aqueous-alcoholic layer with 50
ml of pentane R and add the pentane layer to the first funnel.
Wash the pentane layer with two quantities, each of 50 ml, of a freshly prepared 30 g/l solution
of potassium hydroxide R in alcohol (10 per cent V/V) R, shaking vigorously, then wash with
successive quantities, each of 50 ml, of water R until the washings are neutral to
phenolphthalein. Transfer the washed pentane extract to a ground-glass-stoppered flask.
Evaporate the contents of the flask to dryness under reduced pressure by swirling in a water-bath
at 40 °C. Cool under running water and restore atmospheric pressure with nitrogen R.
Dissolve the residue immediately in 5.0 ml of toluene R and add 20.0 ml of the mobile phase to
obtain a solution containing about 4000 IU/ml.
Reference solution (a).Dissolve 10.0 mg of cholecalciferol CRS without heating in 10.0 ml of
toluene R and dilute to 100.0 ml with the mobile phase.
Reference solution (b).Dilute 1.0 g of cholecalciferol for performance test CRS to 5.0 ml with
the mobile phase. Heat in a water-bath at 90 °C under a reflux condenser for 45 min and cool.
Reference solution (c).Dissolve 0.10 g of cholecalciferol CRS without heating in toluene R and
dilute to 100.0 ml with the same solvent.

10. VITAMIN E (ALPHA – TOCOPHEROL)
Assay
Examine by gas chromatography
Internal standard solution.
Dissolve 1.0 g of squalane R in cyclohexane R and dilute to 100.0 ml with the same solvent.
Test solution (a). Dissolve 0.100 g of the substance to be examined in 10.0 ml of the internal
standard solution.
Test solution (b). Dissolve 0.100 g of the substance to be examined in 10.0 ml of cyclohexane
R.
Reference solution (a).Dissolve 0.100 g of -tocopheryl acetate CRS in 10.0 ml of the internal
standard solution.
Reference solution (b).Dissolve 10 mg of the substance to be examined and 10 mg of α-
tocopherol R in cyclohexane R and dilute to 100.0 ml with the same solvent.
Reference solution (c).Dissolve 10 mg of all-rac-α-tocopheryl acetate for peak identification
CRS in cyclohexane R and dilute to 1 ml with the same solvent.
Column:
• Material: fused silica,
• Size: l = 30 m, Ø = 0.25 mm,
• Stationary phase: poly(dimethyl) siloxane R (film thickness 0.25 μm).
• Carrier gas: helium for chromatography R.
• Flow rate: 1 ml/min.
• Split ratio: 1:100.
Temperature:
• column: 280 °C,
• injection port and detector: 290 °C.
Detection: flame ionisation.
Injection: 1 μl of test solution and

11. Reference solution.
Run time: twice the retention time of all-rac-α-tocopheryl acetate.
Relative retention with reference to all-rac-α-tocopheryl acetate (retention time = about 15 min):
squalane = about 0.4; impurity A = about 0.7; impurity B = about 0.8; impurity C = about 0.9;
System suitability:
• resolution: minimum 3.5 between the peaks due to impurity C and all-rac- -tocopheryl acetate
in the chromatogram obtained with reference solution
VITAMIN K (MANADIONE)
Principle
Menadione content is determined by U.V. spectrophotometry at 250 nm by comparison with
Menadione R.S. solution.
Reagents and Equipment
Menadione RS.
2-Methyl-1,4-naphthoquinone dried over silica gel for at least 4 hours.
Dichloro methane.
Methanol.
Analytical balance.
Normal laboratory glassware.
UV/Vis Spectrophotometer.
Assay
Preparation of test sample solution
Transfer about 80 mg of Menadione (dried over silica gel for at least 4 hours), accurately
weighed into a 250 ml volumetric flask; dissolve and dilute to volume with dichloro methane.
Transfer 2.0 ml of this solution into a 100 ml volumetric flask, bring to volume with methyl
alcohol.
Preparation of reference standard solution
Transfer about 80 mg of Menadione RS, accurately weighed, into a 250 ml volumetric flask;
dissolve and dilute to volume with dichloro methane.

12. Transfer 2.0 ml of this solution into a 100 ml volumetric flask, bring to volume with methyl
alcohol. The concentration of Menadione RS in the standard preparation is about 6.4 μg per ml.
Determine the absorbance of standard solution and test solution in 1 cm cells at 250 nm. Use a
solution of 1 ml of dichloro methane in 50 ml of methanol as blank.
Expression of Results
The content of Menadione is expressed in percent as follows:
Ts = (ASxWStdxTStd)/ AStdx WS
where:
TstdContent of Menadionepercent in standard
TS Content of Menadionepercent in sample
AStdStandard Solution Absorbance
ASSample Solution Absorbance
WStdStandard weight (in mg)
WS Sample weight (in mg)
ANALYSIS OF TOXICANTS IN FOOD
Food safety assessment depends upon the determination of toxic materials in foods. It is
important to develop accurate analytical methods to interpret the data correctly. Almost by
definition, toxicants are present in very low levels because substances with any significant level
of any toxicant are rejected as foods. This is illustrated by the fact that a distaste for a particular
food is developed after it is associated with an episode of illness. A decision-tree protocol for
testing the safety of food components has been proposed by the Scientific Committee of the
Food Safety Council in the United States. (Food Safety Authority,2009)
The qualitative and quantitative analyses of toxicants in foods are the principal tasks of food
toxicology. When toxicity is discovered in a food, the analyst’s first job is to identify the toxic
material(s) in the food. The analysis of toxicants requires both an assay for detecting the poison
and a method for separating it from the rest of the chemicals in the food. In order to allow legally
binding conclusions about toxicant levels in foods, the U.S. government monitors a set of
approved methods which have a certain set of criteria about quality. For example, unless
circumstances warrant, the recovery by the method must be at least 80%. Additionally, certain
physical processes are required when preparing the testing samples.(Food Safety
Authority,2009)
ANALYSIS OF TOXIC HEAVY METALS (ARSENIC, MERCURY, LEAD, CADMIUM
AND TIN) IN FOOD
Heavy metals are described as those metals which, in their standard state, have a
specific gravity (density) of more than about 5 g/cm 3 (Gian C.C., Zaheer D., Christian D.F.

13. et al, 2009). Heavy metal pollution is a result of increasing industrialization throughout
the world, which has penetrated into all sectors of the food industry. Because of that,
the World Health Organization (WHO) classifies heavy metals as one of the risks people
are exposed to through food.
Commission Regulation 333/2007 lays down the sampling methods and the methods of
analysis. The Regulation also lays down requirements for laboratories carrying out
analyses, core requirements being accreditation, competence in the specific analyses
and on-going participation in inter-laboratory studies for the determination of the metals
in food matrices.
There are usually two major steps to trace element determination, sample digestion and detection
method. There are four main methods of sample digestion commonly reported by laboratories:
(1) dry ashing of the sample in a conventional oven;
(2) microwave digestion of the sample in a strong acid;
(3) acid digestion of the sample by heating in a pressure vessel;
(4) dissolving the sample directly into acid. Some laboratories also extract the metals in 2-
methylhexan-2-one [isobutyl methyl ketone (IBMK)]. The detection methods most frequently
are atomic absorption spectrometry (AAS) and inductively coupled plasma (ICP) techniques.
Atomic absorption spectrometry includes flame AAS (CVAAS) for mercury determination and
hydride generation (HG), commonly used for arsenic determination. Inductively coupled plasma
techniques usually use mass spectrometry (ICP-MS) and ICP-optical emission spectrometry
(ICP-OES). Other techniques which are still used by some laboratories include anodic stripping
voltammetry, colorimetry, spectrophotometry, ion chromatography, polarography and titration.
However, these last few techniques for metal determination have largely been replaced by the
methods of AAS and ICP.
Collection of Samples .
Inclusion and Exclusion Criteria Inclusion criteria for the milk samples are: (a) formula
samples(b) should meet the criteria of Codex Stan 72-1981; (c) must be originally contained and
distributed in tin foil; and (d) must not be expired. with.
Preparation of Milk Samples for AAS 5 grams from each sample was placed in different
crucibles and heated in a maple furnace at 700°C for 3 hours to vaporize all other constituents

14. and leave the heavy metals as a pure ash. The ash was cooled to room temperature before being
dissolved in a 5 ml solution of nitric acid (1:6) to compound with the heavy metals, if present.
The solution was subsequently heated and evaporated to half its volume using a hot plate. The
resulting solution was then poured into a volumetric flask, or Erlenmeyer flask, and topped up to
25 ml with distilled water.
Preparation of Standard of Selected Heavy Metals The selected heavy metals were Arsenic,
Lead, and Mercury. For each of the selected metals, three standards were set for the calibration
of the AAS. These are as follows: 1.0000 ppm, 1.5000 ppm, and 2.0000 ppm. The calibration
curve of well prepared standards and an accurate Atomic Absorption Spectrophotometer should
present as a linear curve.
Atomic Absorption Spectrophotometric Analysis Analysis of the heavy metal contents sample
was done with the use of the Atomic Absorption Spectrophotometer (AAS) following the 7000B
Method of EPA (Environmental Protection Agency) for flame absorption spectrophotometry.
The AAS not only detects the presence of heavy metals, but, if present, it is also designed to
provide the concentrations in parts per million (ppm). Three trials were run on each sample in
every replicate of the heavy metal and the averages of the concentrations were then taken and
compared to standard stated by the Food and Agriculture Organization/World Health
Organization Joint Expert Committee on Food Additives (JECFA).
Atomic Absorption Spectrophotometry relies heavily on the Beer-Lambert Law. The electrons of
the atoms in the atomizer can be promoted to higher orbitals for a short amount of time by
absorbing a set quantity of energy i.e. light of a given wavelength. This amount of energy or
wavelength is specific to a particular electron transition in a particular element, and in general,

15. each wavelength corresponds to only one element. This gives the technique its elemental
selectivity. As the quantity of energy put into the flame is known, and the quantity remaining at
the other side (at the detector) can be measured, it is possible, for Beer-Lambert law, to calculate
how many of these transitions took place, and thus get a signal that is proportional to the
concentration of the element being measured. (Gian C.C., Zaheer D., Christian D.F. et al, 2009)
Data Assessment
Data was gathered from all trials and organized in tables. The concentrations, in parts per
million (ppm), of samples that yielded positive results for the presence of heavy metals were
converted into mg/kg body weight, then compared to the Provisional Tolerable Weekly Intake
limit of these heavy metals as set by the Food and Agriculture Organization/World Health
Organization Joint Expert Committee on Food Additives (JECFA). The results were simply
assessed as values greater
than or within the tolerable weekly limit of toxic heavy metals. No statistical analysis was
employed.
RESULTS
Calibration curves for each heavy metal were set to ensure the accuracy of the Atomic
Absorption Spectrophotometer and to establish that results of the determination proper were true
and reliable. Standards with the concentration of 1.000 ppm, 1.500 ppm, and 2.000 ppm,
respectively, were set for the calibration of the Atomic Absorption Spectrophotometer. The
calibration curve of well prepared standards and an accurate AAS should present as a linear
curve. The data on the calibration for Arsenic, Lead and Mercury are seen were tabulated in
ppm

16. REFERENCES
Fefana EU food additives and premixtures association method of analysis of vitamins,
provitamins&a chemically well-defined substance having a similar biological effect Pp. 1-51
Food Safety Authority of Ireland. (2009).Mercury, lead, arsenic, cadmium and tin in food
Toxicology factsheet series issue no.1
Gian, C.C., Zaheer, D., Christian, D.F.et al (2009). Analysis of toxic heavy metals(arsenic, lead
and mercury) in selected infant formula milk commercially available in Philippines by ASSE-
International Scientific Research Journal ISSN: 2094-1749 Vol: 1