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

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.

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

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

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

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)

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.

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.

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.

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

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.

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.

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

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,

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

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