INTRODUCTION

Acrylamide (AA) is one of the dangerous compounds known in food chemistry. The problematic
nature is due to the presence of carboxylic and amide group in the compound which enables
nucleophilic addition reactions in alkali conditions[1]. Acrylamide causes cancer and tumors in
humans and neurodegenerative diseases. Acrylamide is placed in group 2A carcinogens by
International Agency of Chemical Research [2].

Acrylamide also known as plastic in local term. Many processed, cooked and baked foods contain
acrylamide and the main reason of formation of acrylamide is the browning procedure known as
Maillard reaction [3] between asparagine (amino acid) and glucose (sugar) at temperatures greater
than 120°C without the presence of any enzyme [4]. Mainly, the temperature regulates the
formation of acrylamide. In simple words, the browning of foods aids the formation but,
acrylamide formation starts at the top and makes its roots towards the bottom of the food [5]. Many
compounds other than acrylamide are also formed as a result of browning which is due to reaction
between amino acids and sugars other than asparagine and glucose [6]. These compounds are
detected from their flavor, taste, appearance and aroma so, that is why every food tastes and smells
different [7].

Many factors control acrylamide formation such as water [8], starch [9] and type of oil [10],
cultivars, pretreatments [11], natural extracts [12], pH [13], salt [14], formulations, baking time
and procedure [15] and precursors of Maillard reaction i.e. amino acid and sugar level [16]. Only
asparaginase enzyme reduces acrylamide formation, while no other enzyme is capable to reduce
acrylamide [17]. A slight change in any factor will result is increase, decrease or no formation of
acrylamide. Although AA is biodegradable in non-sterile environments but, highly toxic to
humans, while monomer of AA i.e. polyacrylamide remains unaffected by microbial attack [18].
There are many advantages of acrylamide such as its polymers are used as flocculants in paper,
textile industries and as a binder is photography [19].

When acrylamide gets in contact with human body, it oxidizes to glycidamide by cytochrome P450,
here the oxidized form has higher affinity for DNA, which binds to them and leads to mutagenicity
and genotoxicity and cause skeletal muscle weakness and ataxia [20]. Nerve endings degeneration
and inhibition of axonal transport is caused due to binding of AA to sulfhydryl groups on cysteine
residues that do membrane fission process. So, acrylamide causes cancers and neuro degenerative
diseases [21].

There are many methods to detect and estimate acrylamide i.e. Fourier Transform Infrared
spectroscopy, Gas Chromatography coupled to Mass Spectrometry, Liquid Chromatography with
tandem mass spectrometry [22], Adsorption Stripping Voltammetry [23], microchip
electrophoresis [24] but, these methods are time consuming and are expensive. But, High
performance liquid chromatography is simple, low cost and gives effective results for acrylamide
estimation using acetone. The concentration of acrylamide varies greatly in a food in such a way
that concentration is greater on the edges as they are crispier and brown. The acceptable limits of
average acrylamide intake is 0.3-0.8μg/kg body weight/day while, the level required to observe
neuropathy in humans is 0.5 mg/kg body weight/day. For neuropathy, the average amount is safe
in relation of food but, harmful enough to cause cancer in human beings.

It is impossible to completely remove acrylamide from our diet but, certain measures can definitely
be taken for its reduction such as avoiding more browning, and over cooking of food, cooking at
lower temperatures, usage of inorganic salts like NaCl and saturated fats, lower pH, blanching in
pretreatments, and using antioxidants (natural extracts). Overall, some serious steps should be
taken for controlling the acrylamide exposure because of its presence in our daily food items.

The current study involves the detection and estimation of acrylamide in food stuffs that are used
on daily basis by people i.e. cooked, processed and baked. In this matter, High Performance Liquid
Chromatography was employed because of the usage of lesser chemicals and lower cost.

MATERIALS AND METHODS

REAGENTS AND CHEMICALS

All the chemicals used in this study were purchased from Sigma Aldrich Laboratories.

COLLECTION OF SAMPLES
All 30 samples were purchased from local stores of Lahore, Punjab, Pakistan.

EXTRACTION OF ACRYLAMIDE
Acrylamide was extracted using acetone. All food sample were firstly dried, and grinded to a fine
powder using pastel and mortar. 5g of each sample was weighed in a flask and 10 ml of n-hexane
was added to de-fat the sample and shaken vigorously for 20 min. This step was done twice to
ensure the complete removal of fats from the sample. After this, hexane was decanted and sample
was dried using hotplate. 20 ml of acetone and 0.1 ml of distilled water was added in the sample
and shaken for 1 min. Acetone was filtered in a beaker and dried on hot plate. After the beaker was
completely dried, 5 ml of water was added, filtered and collected in an eppendorf tube for analysis
of acrylamide on HPLC [25].

PREPARATION OF STANDARD SOLUTION OF ACRLAMIDE

The standard of acrylamide was purchased from Sigma Aldrich laboratories. Stock solution was
prepared by dissolving 0.1g of AA in 100 ml to make 1000 ppm solution. From this stock solution,
2ppm was prepared by dilution. The running solution 250 ppb was prepared from 2 ppm solution
by diluting 12.5ml with 100 ml distilled water. The solution was kept away from sunlight and
stored for analysis.

ANALYSIS OF ACRYLAMIDE USING HPLC

Samples were analyzed on Breeze 2 HPLC system. C 18-AQ column with 250 mm was using used
Flow rate was 1 ml/min. with a temperature of 40°C at 202 nm with a retention time of 10 min.

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