1.1: Properties of Acrylamide 1.1.1: Physical Properties

INTRODUCTION
1.1: Properties of Acrylamide

1.1.1: Physical properties
Acrylamide or acrylic amide or 2-propenamide, a carcinogenic compound with a
chemical formula C3H5NO. Acrylamide was categorized in 2A group carcinogens by
researchers, who worked on cancer in International Agency. Acrylamide is α, β-
unsaturated colorless amide with low molecular weight of 71.09 gmol-1, boiling point:
136°C and a melting point: 84.5°C, which makes it extremely soluble in water and some
(polar) organic solvents including lower alcohols. Low volatility and hydrophilic nature
makes acrylamide difficult for analysis using classical analytical techniques,
particularly in complex matrixes [1].
1.1.2: Chemical properties

Solid acrylamide is fairly firm in surrounding conditions and has a fine mean life. Even
at melting point, no notable polymer formation is noticed. But, when temperature
slightly crosses above the melting point then, formation of liquid acrylamide starts
which polymerizes rapidly and exothermally.
Acrylamide (AA) is an unstable monomer holding an amide group (-CONH2) and a

double bond which has insufficient number of electrons. Acrylamide is highly reactive,
because of these two groups. Acrylamide has both acidic and basic nature. Double bond
is deactivated by carboxamide (electron withdrawing group) and the carbonyl (acidic)
group activation is not the best as the group in association. The structure of acrylamide
is seen in fig.1.1. So, acrylamide gives nucleophilic addition reactions. Ammonia,
amines, phosphines and bisulphites in alkaline conditions permits the reagents to react
with acrylamide making reactions reversible [2].
Figure 1.1: Chemical structure of acrylamide showing the functional groups of -

CONH2, C doubly bonded to CH2
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1.2: History of Acrylamide
The panic of AA started in 1997, when a herd of cows in southern Sweden begun
stumbling, crumbling and started to die, which exhibited that the cattle had been
drinking water carrying AA that was leaching out from a nearby mountain tunnel. Later
in 2002, the researchers of Stockholm University and Swedish Food Administration
(SNFA) reported that AA is potentially cancer-genic chemical and toxic which is
formed in many types of baked, processed and cooked foods at higher temperatures [3].
Acrylamide discovery in foods collected great attention of public and medical group.
Historically, acrylamide’s threat was only limited to water, but, the modern findings of
acrylamide formation in processed, fried and baked foods brought up the importance of
needing to understand the nature of the compound [4].
1.3: Mechanism of acrylamide formation

Asparagine, a nonessential amino acid, in union with various carbonyl compounds at
temperatures greater than 120°C, has been recognized as the precursor which associates
with sugar (fructose or glucose) for acrylamide formation in foods by the browning
procedure called maillard reaction to form acrylamide and other maillard products
(oxazoles, thiophenes etc.) as shown in figure 1.1 [5].
Potato products (French fries and chips), nuggets, coffee, biscuits, infant formulas,
roasted nuts, meat, chocolate products, crackers and corn flakes are among the food
items carrying the contents of acrylamide [6].
Figure 1.2: General mechanism for acrylamide formation through maillard

reaction
1.3.1: Two models for mechanism of acrylamide formation

Becalski and Yang in 2003 made 2 certain models concerning acrylamide formation.
The first model included a combination of 6 amino acids of aspartic acid, glutamine,
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asparagine, glutamic acid, lysine, and valine including glucose. The second model was
an easy kind which contained only asparagine and glucose [7].
1.3.1.1: Findings from the two models of acrylamide formation

The findings of first model yielded acrylamide 3300 ng only at high temperature of
175°C. The existence of glucose and asparagine at correct temperature has significance
in the development of acrylamide as it requires temperatures greater than 175 °C for 10
min. [8] .
Also, the acrylamide formation requires a significant kind and amount of precursors.
For acrylamide formation, it is necessary that both the di-carbonyl group (coming from
maillard reactions) or carbonyl group (coming from reducing sugar) are present [9].
1.3.2: Pathways of acrylamide formation

1.3.2.1: Route of asparagine through maillard reaction
The main route for acrylamide formation is asparagine pathway. Asparagine can be
altered to acrylamide via deamination and thermal decarboxylation reactions, but
reducing sugars should exist for reaction to move forward. A similar variation can be
increased through carbonyl group of compounds [10].
The first step involves a reaction between asparagine and reducing sugar to form a
Schiff base (intermediate I) via asparagine pathway. An intermediate II is formed
through decarboxylation of the carboxylate ion. Hydrogen gets separated from α-
hydroxy group (-OH) via the transportation of negatively charged α-carbon, as a result,
an intermediate III is formed. In the last step of degradation to acrylamide, the acidic
α-hydrogen is eliminated from six member ring by α-hydroxy anion which is seen in
figure 1.3 [11].
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Figure 1.3: A proposed mechanism which follows the maillard reaction, for
formation of acrylamide from asparagine and reducing sugar
1.3.2.1.1: Strecker reaction of asparagine

Other feasible routes include the Strecker aldehyde (intermediate) in asparagine
reaction. Mottram proposed that acrylamide formation in food throughout results from
reaction between reducing sugars and amino acids (maillard reaction). It is reported that
asparagine acts as a major amino acid in acrylamide production. Also, different color,
taste and aromas are also the products of maillard reaction. In Strecker degradation,
acrylamide is formed from intermediates reaction of amino acids with dicarbonyl
degradated products as seen in figure 1.4. So, amino acid is deaminated and de-
carboxylated for aldehyde formation, on reacting with carbonyl compound (sugar),
which then rearranges itself and reacts with degraded products to form acrylamide at
the end [12].
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Figure 1.4: Proposed pathways for the formation of acrylamide after Strecker
degradation of the amino acids asparagine and methionine in the presence of
dicarbonyl products from the maillard reaction. In asparagine, the side chain Z is
–CH2CONH2; in methionine, it is – CH2CH2SCH3
1.3.2.2: Different routes for acrylamide formation

Maillard reaction is a common word for the reaction between carbonyl compounds and
amines. Another compound produced from hexose degradation (Strecker degradation)
is hydroxymethylfurfural [13]. The routes of acrylamide formation from the maillard
reaction is shown in figure 1.5. The alternative pathways involve:
(1) 1-propenal or acrolein is an unsaturated aldehyde, cytotoxic compound. Acrylamide

is formed by the oxidation of acrolein to acrylic acid through an alternative pathway
under definite conditions in lipid-rich foods [14].
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Figure 1.5: Alternative formation routes of acrylamide via maillard reaction
system
Acrylamide could be formed from nitrogen-containing compounds and oils that are
present in foods. For the formation of acrylamide, the change in acrolein compound
takes place and acrylic acid was formed which reacted with ammonia and leaded to
breakage of compounds having nitrogen [15].
(2) Acrylamide is formed through thermal decomposition of carnosine, β-alanine and

aspartic acid with ammonia to convert acrolein to acrylic acid [16].
(3) Aminopropionamide acts an intermediate during acrylamide formation from

asparagine [17].
(4) Pyruvic acid is formed from dehydration of serine and desulfidation of cysteine
which is finally transformed to acrylamide [13].
(5) Acrylamide can also be formed through α-dicarbonyl- assisted Strecker dehydration
reaction of alcohol of asparagine (3-hydroxypropanamide) in one step, but reports have
suggested it’s limited significance [11].
(6) Acrylamide is formed by the breaking of amadori compounds (benzaldehyde and

styrene), decarboxylated amadori compound of asparagine and phenylalanine [18].
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(7) Higher levels of acrylamide are formed from sugar-asparagine adducts, (N-
glycosylasparagine), proposing an early maillard reaction as a source of acrylamide
[19].
1.4: Factors affecting acrylamide formation

Acrylamide formation is dependent on cooking processes, time, the quantity of
reducing sugars (asparagine), temperature, surface to volume ratio of food materials
and pH. These factors were taken into consideration through several effective measures
that include optimization of heat processing parameters, raw materials modification,
numerous compounds addition, proteins or amino acids blanching and pH modifiers.
Research showed that metal cations (mono-, di- and trivalent cations), efficiently
reduces acrylamide formation [20].
1.4.1: Effect of thawing conditions in correspondence to frying temperatures

The thawing (defrosting) state do not remarkably mark the production of acrylamide,
but microwave thawing method does because of low acrylamide and oil levels of French
fries and color qualities. Saturated fats produce low acrylamide while, unsaturated fats
produce high levels of acrylamide. For acrylamide formation the temperature needs to
be crossed at 140°C. But, as water boils at 100°C, acrylamide production cannot take
place in boiled foods [21].
1.4.2: Influence of frying process and cultivators

Acrylamide formation increases with frying temperature but different results is seen
between cultivars. For example Red Pontiac extraordinarily increases acrylamide levels
170°C with increase in color quality. But, lower oil activity and higher moisture level
takes place due to rise in temperature. Acrylamide level in cultivar is because of
hydrolysis of sucrose during the frying stage [22].
1.4.3: Effect of pre-treatments and air frying

The degree of maillard reaction decreases as reducing sugars levels increase in raw
material and lowers for deep-oil-frying rather for air-frying irrespective of
pretreatments process. Air-frying decreases acrylamide levels by 90% corresponding
to conventional deep-oil-frying without applying pretreatment procedure. Although,
deep-oil fried products pretreated with solutions of citric acid, nicotinic acid, glycine at
1% NaCl lowered the acrylamide levels [23].
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1.4.4: Effect of natural extracts
When antioxidant extracts (cinnamon, green tea and oregano) are used before frying
then, the acrylamide levels decreases. So, pre-treatment with antioxidants before frying
yields valuable effects such as a reduction in acrylamide concentration, without any
notable changes in properties [24].
1.4.5: Effect of vacuum roasting

Vacuum-processed coffee with medium roast rate has approximately 50% less
acrylamide than classical roasting due to low pressure generation inside the oven
between the vacuum processes, as it used a stripping effect, avoiding acrylamide
buildup [25].
1.4.6: Effect of pH with inorganic salts

Acrylamide formation and removal are pH sensitive within range of 7 to 9. Metal
cations lowers pH due to aggressive migration of protons from ionizable functional
groups which contain oxygen, sulfur atoms or nitrogen atoms that part electrons with
hydrogen atoms. Higher pH allows rivalry between metal cations and protons and if the
pH is extremely high then, protons are eliminated before addition of metal ions (Na,
Ca, K salts). It is observed that metal cations allow the pH to increase. Lower pH
promotes formation of contaminants like hydroxymethylfurfural (HMF) and 3-
monochloropropane-1,2-diol (3-MCPD) [26].
1.4.7: Effect of sugars with storage time

Investigations reveal that acrylamide concentration is greatly affected by amount of
reducing sugars while, no correlation was seen with the level of asparagine. There is a
high potential of acrylamide formation and an increase in concentration of reducing
sugars, if storage temperature is lowered from 8-4°C. Surface-to-volume ratio is also
dependent on acrylamide formation [27].
Food products need to have low sugar level to evade browning of product which is
determined by the genotype and post- (mechanical stresses and storage conditions) and
pre-harvest factors (crop maturity, mineral nutrition, temperature during growth, and
irrigation) [28].
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1.4.8: Effect of water and starch
Studies revealed that besides time and temperature, the addition of water and the level
plays a significant role in acrylamide formation. Starch decreases acrylamide formation
while, minimum acrylamide formation is at 25-40% water level [29].
1.4.9: Effect of formulations, baking technology and enzyme modifiers

Baking technology and formulations strongly mark acrylamide levels in bakery
products. The fermentation time and addition of cysteine significantly lowers
acrylamide formation but, enzymes that contain bakery improvers shows no effect.
Prolonged heat treatment and reduced temperature including utilization of deck ovens
minimizes acrylamide [30].
1.5: Advantages of using acrylamide

High vinyl polymers (vinyl monomer) are manufactured from acrylamide. Many
acrylamide polymers are used as flocculants in board, metal industry, production of
coal and ores and in increasing the strength of paper. Polyacrylamides are widely
utilized for simplification and action of municipal and industrial wastes. Acrylamide
polymers disperse fog and soothe soil by breaking oil in water emulsions. Bacterial and
algal growth are also prevented in cooling towers and swimming pools by copolymers
of AA. Biochemists utilize acrylamide for polyacrylamide gel production in
electrophoresis. Other applications involve metal coating, photography, binders and as
an adhesive [31].
1.6: Harmful effects of acrylamide

Acrylamide forms polymers i.e. polyacrylamide which is a major concern due to the
occurrence of unreacted acrylamide monomer. Although, acrylamide is biodegradable
in non-sterile environments but, highly toxic to humans, while polyacrylamide remains
unaffected by microbial attack. Acrylamide is one of the major sources of oral cancer
because it majorly sweeps through skin [32].
1.7 Reduction of acrylamide

1.7.1: Reduction through asparginase
No such enzymes can reduce acrylamide but, bacterial l-asparaginase catalyzes the
breakage of l-asparagine to l-aspartic acid. Asparginase enzyme is active at high
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temperatures, which reflects the potential activity in the food processing industry. The
acrylamide is also reduced through blanching treatment also, reduces acrylamide [33].
1.7.2: Reduction through salts

Studies show that pretreatment with VOSO4 (mono-, di-, tri-valent metal actions) salt
reduces concentration of acrylamide formation in fried products. Investigations reveal
that when VO2+ binds asparagine then, a decrease in pH is seen which results in notable
reduction in formation of acrylamide [20].
1.8: Acrylamide and digestion inside human body

Acrylamide (AA) can be ingested, inhaled or absorbed through the skin and due to its
solubility, it is distributed throughout all body tissues, through passage in the blood and
is majorly found in the liver, brain, heart, kidneys, breast milk and placenta. In human
body, acrylamide is oxidized by cytochrome P450 and forms glycidamide (metabolite),
which has higher binding affinity for serum, DNA, hemoglobin and enzymes (in vivo)
to produce neurological damage and mutagen formation. Signs of acrylamide contact
constitutes weakness in muscles (skeletal) and ataxia [34].
1.8.1: Acrylamide and human health

In humans, when acrylamide gets in contact with skin it distributes itself to all tissues
and forms glycidamide, (reactive epoxide). It makes adducts with DNA and proteins
leading to genotoxicity and mutagenicity. The other products of maillard reaction tend
to increase or decrease acrylamide toxicity [35].
Acrylamide concentration significantly reduces after the gastrointestinal digestion. As

food swallowing causes a change in structure and composition by chewing, the change
in pH occurs along with, dilution and different enzymes present in mouth, stomach and
intestine act on to food [36].
It is important to identify what measures may be taken to decrease both the production
and effects of acrylamide in foods. Acrylamide causes cancer and neurotoxic effects
therefore its development in foods and the effect of this subjection on human health
needs to be particularly understood. It is also necessary that the method for determining
acrylamide in foods is not only applicable to a wide range of foods, but also adequately
strong for analyzing large numbers of samples [37].
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1.9: Acrylamide and neurotoxicity
When scientists first found about acrylamide, then numerous tests were performed on
mice to study about its neurotoxicity and the results revealed that mice developed
tumors and died so, it was made clear that acrylamide is neurotoxic in animals and
humans. Acrylamide induces dominant killings in male rodents because of
translocation (unusual rearrangement of chromosomes) carriers in post-meiotic germ
cells of male population. AA is similar to alkylating agents (ethylmethanesulfonate and
ethylene oxide) in inducing heritable translocations. The absence mutagenicity of AA
in bacteria indicates a different mechanism for AA’s germ cell mutagenicity [38].
Higher levels of AA causes neurotoxic effects in humans and lower levels from dietary
sources are not related with neurotoxic effects in humans. Exposures of acrylamide
from environment or from diet contributes remarkably to promotion and progression
neurodegenerative diseases in humans. Acrylamide binds to sulfhydryl groups on
cysteine residues of proteins which are responsible in membrane fusion process, causes
nerve endings degeneration and inhibition of axonal transport. As, nerve endings are
sites of type-2 alkene action, it is likely that interaction between exogenous and
endogenous sources of chemicals speed up the movement of neurodegenerative
diseases that is initiated by presynaptic damage and oxidative stress [39].
1.10: Reason for using food samples for determination of acrylamide

In work presented in this thesis, we have analyzed food samples for determination of
AA in it. In today’s society, everyone tends to eat from outside and people have hardly
any time to make any food at home, so, people rely on take-away. The people that do
make food at home have no idea what is in their food and what temperatures should
they cook at. They only know that more the food is brown the more it is crispier and
more is it cooked. Through my research work, I want to confirm the presence of
acrylamide formation that takes place in brown foods. As acrylamide is highly
carcinogenic and we should avoid more browning of foods and the ways which enhance
the formation of acrylamide.
1.11: Analytical techniques

Analysis of acrylamide is much difficult because the nature of the acrylamide formation
from Maillard reaction. If reducing sugars and asparagine are existing in concentrate
then, GC-MS procedure should be avoided. Additional drawback characteristic is
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absence of blank food mediums because of maillard reaction and its products that
include acrylamide [4].
Through literature study it has been found that acrylamide is carcinogenic in nature and
its detection in food has been found with various techniques such as liquid
chromatography, high performance liquid chromatography with an ion exclusion
column and UV detection, FTIR (Fourier Transform Infrared Spectroscopy), Gas
Chromatography coupled to Mass Spectrometry, portable vibrational spectrometers,
Gas chromatography coupled to an electron capture detector, flame photometric and
ionizing detector and nitrogen phosphorous detector [40].
1.11.1: Preferred method for acrylamide estimation

Upon literature survey and the availability of chemicals and instruments, High
Performance Liquid Chromatography (HPLC) was chosen as the best method for
estimating acrylamide in different foodstuffs (baked, processed and fried) because it
requires a lesser number of chemicals (acetone and n-hexane).
Reported work
In the present work, estimation of acrylamide in different sorts of foodstuffs was done.
Firstly, the samples were ground fine to powder then, defatted with n-hexane and
acetone was used to extract acrylamide from the samples. Finally, the sample was run
on HPLC for detection and estimation of acrylamide.
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Aims & objectives
The project has been designed by keeping in mind the following aims and objectives:
 To evaluate and analyze foods that contain acrylamide.

 To develop a HPLC method for routine estimation of acrylamide in different
food-stuffs.
 To study the factors which are involved in acrylamide formation.
 To increase awareness of common people about the cooking methods which
produce less AA.
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REVIEW OF LITERATURE
Saraji et al. (2019) investigated acrylamide in complex food matrices (bread, potato
chips and cookie) using green, eco-friendly technique of single drop micro-extraction
combined with gas chromatography-electron capture method. Experimental parameters
(limits of detection and quantitation, relative standard deviation and linearity) and
derivatization reaction yield were evaluated. Results revealed highest amount of
acrylamide in cookies and potato chips had lower amount while, the quantity of bread
was in between [41].
Zahra et al. (2018) used high-performance liquid chromatography for determining

acrylamide concentration mainly in branded and non-branded potato chips, cakes and
biscuits of Pakistan. Mean and the standard deviation showed differences but a notable
amount of acrylamide. Results showed that acrylamide was lower in the local vendor's
sample and higher in the branded one [42].
Haouet et al. (2016) proposed acrylamide determination in processed foods (potato

chips, salted bakery products, biscuits and wafers, sweet bakery products and
sandwiches) from vending machines using high-performance liquid chromatography.
Precision, accuracy, uncertainty, recovery and limits of quantification and detection
were examined. Results revealed that differences in acrylamide concentrations in the
same kinds of chips was due to the difference in the content of reducing sugar and
amino acid, while the lowest level was found in sandwiches [43].
Razia et al. (2016) investigated concentrations of acrylamide levels in processed cakes,

biscuits and potato chips using GC-MS. Carcinogenic acrylamide could be produced
due to the heat treatment of carbohydrate-rich foods at high levels. Results showed that
levels of acrylamide in chips were high and biscuits were in range, but in cakes it was
low [44].
Khoshnam et al. (2010) reported acrylamide analysis in a variety of chips HPLC via
acetone extraction. Recovery tests at different spike levels were calculated in different
days which comprised mean and relative standard deviation. Outcomes showed that all
potato chips samples had acrylamide in them and its value tend to decrease on different
days analysis which concluded that the suggested technique was applicable for routine
estimation of acrylamide because of the use of fewer chemicals [45].
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Yadav et al. (2018) detected acrylamide in processed foods (potato chips, biscuits,
cakes, nuts and cereals) by constructing an electrochemical paper-based biosensor using
hemoglobin nanoparticles (HbNPs). Nanoparticles were categorized using x-ray
diffraction (XRD), transmission electron microscopy (TEM), UV and atomic force
microscopy. Limit of detection, the percentage of recovery, coefficient of variation,
mean and standard deviation were evaluated including effects of storage stability on a
biosensor of paper electrode drop cast with HbNPs, pH, linear range and recovery.
Results were collected in concentrations of acrylamide in cyclic voltagramms with an
inverse linear curve due to sodium acetate buffer solution of acrylamide. The proposed
work showed that highest amount of acrylamide was present in chips the, biscuits,
cakes, cereal and finally least in nuts [46].
Norouzi et al. (2018) examined acrylamide in bread by ultrasonic assisted micro-

extraction technique. Statistical analysis (percentage recovery, relative standard
deviation, analysis of variance, limits of quantification and detection) quantitative and
qualitative analysis was performed. Acceptable results have shown that the proposed
technique was fast and a reliable sample-pretreatment method for determining traces of
acrylamide. Acrylamide found in all bread samples crossed the normal range and high
amount of acrylamide was found in all of them [47].
Laura et al. (2017) estimated acrylamide in dried fruits (peanuts, almonds, cashew nut,
chest nut etc.) and edible seeds by a dispersive solid phase extraction (QuEChERS:
quick, easy, cheap, effective, rugged and safe) using LC couples to MS detector. Effects
of two different QuEChERS pouches (NaCl and CH3COONa), water volume, solvent
injection and sample weight on acrylamide formation were studied. Validation was
done through statistical analysis (linearity, sensitivity, accuracy and repeatability).
Results described a higher quantity of acrylamide in peanuts and the proposed
technique required minimal solvent [48].
Wu et al. (2016) presented microchip electrophoresis (MCE) with laser-induced

fluorescence technique for analysis of acrylamide in potato chips and French fries at
very low concentrations. Optimization of derivatization, MCE separation and on-line
pre-concentration conditions was done experimentally and the effects of injection,
reversed-polarity and prolonged-FASS time were evaluated. Validation of this
technique was checked statistically (limit of detection and linearity). Results obtained
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were reliable, which showed that potato chips were having greater acrylamide in them
and this technique provided a shorter run time with little solvent and sample
consumption [49].
Daniali et al. (2016) examined oils and fats in heat treatment by liquid chromatographic
coupled to tandem mass spectrometry for observing the formation of acrylamide.
Oxidative values (peroxide, anisidine and iodine values) were also checked
experimentally and a direct correlation was observed with acrylamide formation.
Results showed that acrylamide formation was lesser in fats than in oil [50].
Altunay et al. (2016) worked on acrylamide determination in infant formulas, crackers

and potato and corn chips firstly by pre-concentration and estimated using flame atomic
absorption. Effects of pH, amount of ion-pairing reagent and metal ions, added
electrolyte and concentrations were studied. Results concluded that acrylamide was
highest in infant biscuits then, potato chips, crackers and corn chips had the least.
offered method was sensitive, simple, inexpensive, rapid and easy which gave good
precision and accuracy and has a vast working area [51].
Musa et al. (2015) compared two HPLC columns (Rezex ROA-organic acid and
Kinetex C18) for separating and evaluating acrylamide levels in fried and baked potato
based products, eggplant, gorrasa (baked), minnan (baked), taamia (fried) and Hilumur.
Linearity, limits of quantification and detection and the matrix effect authenticated the
method. Results revealed that the C18 column was best for routine analysis because it
run a large number of samples and determined acrylamide level in traces. Acrylamide
was greatest in fried eggplant, then comes fried potato, minnan, tamia, and least in
garrosa [1].
Zhao et al. (2015) used HPLC for determination of acrylamide by preparing carbon
nanotubes made up of chitosan in potato, flour and a variety of fried chips.
Characterization, optimization, accuracy including applicability of proposed method
was done experimentally and checked statistically. Results showed that acrylamide was
greater in potato chips and lesser in twisted crullers concluding that the suggested
method was novel one which detected traces of acrylamide in difficult food matrices
[52].
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Ayvaz et al. (2015) employed portable and handheld infrared spectrometer for
acrylamide screening and bench top system (GC-MS and LC-MS) for estimation in
potato chips (sweet, regular and seasoned). Models of partial least squares predicted
acrylamide levels in a good linear correlation with LC-MS and GC-MS and gave similar
results. Limits of quantification, detection and standard deviation were evaluated.
Results showed that higher acrylamide was found in sweet potato chips and lesser was
found in seasoned ones concluding that proposed method of portable systems required
less sample size and were cost effective [53].
Shamla et al. (2014) evaluated acrylamide concentrations in deep-fried snacks (potato

chips, jack chips and plantain chips) using HPLC. Statistical analysis and recoveries
showed good extraction and efficient results for potato chips in which high amounts of
acrylamide were present. Results revealed reasonable levels of acrylamide in the
samples, potato chips being the highest and plantain chips the lowest, demonstrating a
danger of exposure [54].
Veni et al. (2014) performed acrylamide estimation using HPLC in potato chips.
Internal standard peak area, concentrations of acrylamide and reaction factors were
assessed from linear calibration curve of acrylamide. Mean, precision, limits of
quantification and detection and relative standard deviation were also investigated.
Results showed that this method was used only for estimating a higher amount of
acrylamide in potato chips [40].
Zhang et al. (2014) estimated acrylamide in starchy foodstuffs (cookies, chips, rice
crusts and buns) by HPLC, stationary phase is a reformed form of silica gel sorbent
(NCSi: tetraazacalix[2]arene[2]-triazine). Main influence factors of extraction such as
flow rate, composition and size of solvent and volume of sorbent were examined in the
step of pretreating of sample. Results showed high amount of acrylamide in potato chips
then cookies, rice crusts and buns had the least amount. In conclusion, that proposed
method enabled an efficient purification of acrylamide as the stationary phase sorbent
didn’t allow any interference (proteins, carbohydrates) to pass through [55].
Lim et al. (2014) proposed a simple, unique method for estimating acrylamide in
roasted coffee, French fry and potato chips by derivatizing with d-cysteine then,
quantitating with LC/MS/MS technique. Parameters such as acrylamide level, mean,
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precision, accuracy, standard deviation and limits of quantification and detection were
examined. Results showed that French fry was high in acrylamide and potato chips was
less while, coffee was in between the two. The applied method offered analysis of lesser
sample masses in a small run time [56].
Al-Janabi et al. (2013) quantified acrylamide level in different varieties of chips and
an Iraqi meal (Harissa). Acrylamide level, spiked recoveries, relative standard deviation
and limits of quantification and detection were assessed. Results showed that the
samples of potato chips showed a high level of acrylamide content which is considered
as a risky dosage for human health while Harissa sample exhibited less acrylamide
content. The given method used minimal amounts of reagents which avoided
production of toxic residues and could be applicable only for detection at higher levels
of acrylamide [57].
Choi et al. (2013) applied HPLC for estimating acrylamide in deep fried and baked
foods. Acrylamide contents do not show positive relation with browning of food.
Optimization of the chromatographic conditions, statistical examination including
effects of cooking processes were examined. Results concluded that fried food
contained higher acrylamide concentration and food additives could be used for the
reduction process while baked foods had lesser acrylamide [58].
Prashant et al. (2010) quantitated acrylamide concentration in processed chips and

puffs using normal phase HPLC. Acetonitrile was used as mobile phase in assessing
concentration of acrylamide. Results revealed that kurkure branded chips was highest
in acrylamide level and puffs was least. This technique required fewer chemicals in the
analysis [59].
Karasek et al. (2009) performed HPLC-MS/MS isotope dilution technique for

analyzing acrylamide in roasted chestnuts and as well as chestnut-based foods.
Influence of roasting time on acrylamide content was assessed experimentally. Results
exhibited greater acrylamide concentration in roasted chestnuts and lower in chestnut
products [60].
Kaya et al. (2009) determined contents of acrylamide using HPLC-MS in meshed

chips, coffee, meat and biscuit samples. Parameters such as selected ion monitoring of
forerunner ion of acrylamide was investigated and checked statistically. Results
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described that obtained values of acrylamide of all the samples were lesser as compared
to the given in literature [61].
Wang et al. (2008) developed a simple solid-phase extraction technique with

HPLC/UV for acrylamide estimation in deep-fried flour foods. Optimization of
chromatographic conditions, recovery tests, limits of quantitation and reproducibility
were investigated statistically and effect of cooking processes was examined
experimentally. Results showed that the proposed method resolved acrylamide from
interferences and concluded that samples which were browner showed more acrylamide
concentration [62].
Chen et al. (2008) surveyed 349 foods in China for estimating acrylamide by LC-
MS/MS system. Initial assessments of risk calculation and exposure of acrylamide from
foods in Chinese people were executed by merging the data found in current review.
The average exposure of acrylamide was estimated by body weight per day and results
concluded that high amounts of acrylamide was found in chips [63].
Geng et al. (2008) measured acrylamide concentrations in starchy Chinese foods using
LC-DAD. Calibration was done through recovery experiments (accuracy, precision and
relative standard deviation). Result analysis showed that the proposed method was
inexpensive and could easily be used for repetitive investigation of acrylamide levels
in starchy foods [64].
Zhang et al. (2007) used LC-MS/MS for examining acrylamide content in potato
crisps. Spiked recoveries, accuracy and relative standard deviation were calculated
from the detected levels of acrylamide. Results explained that the current quantitative
technique was useful for immediate acrylamide determination in many surveys [65].
Majcher et al. (2007) performed acrylamide analysis in fries, crackers and cookies
using GC-MS/MS. Measurement of sample color, effect of temperature and time and
correlation concerning acrylamide level and color were checked. Results revealed that
low fat foods have lesser acrylamide than in chips also, temperature showed a robust
influence on acrylamide level than time [66].
Zhang et al. (2007) performed GC coupled micro-electron capture detector and LC-
MS/MS technique for acrylamide estimation in potato chips. Limit of detection, limit
19
of quantitation, spiked recovery and relative standard deviation were inspected. Results
presented that concentration of acrylamide was greater in concentration in all samples
while the statistical analysis concluded that both techniques were appropriate for
acrylamide analysis and gave similar results [67].
Gökmen et al. (2006) developed a general preparatory technique for acrylamide

determination in thermally processed foods (potato chips, biscuits and coffee) using
LC-MS technique. The recoveries, relative standard deviations and limits of
quantitation were evaluated while valine was identified as a major co-extractive
(interfering peak) in the chromatogram. Results described that changing of the solvent
and further clean up by Oasis HLB cartridge decreased the concentration of interference
in chromatogram which gave satisfactory analytical results for a variety of food
complexes in acrylamide determination [68].
Şenyuva et al. (2006) determined acrylamide in potato and cereal foods using liquid
chromatography-mass spectrometry technique. The mean recovery and repeatability
were evaluated. Results showed valine as a major interfering co-extractive so, using a
solid phase extraction with a strong cation exchanger based sorbent and by
instrumentally adjusted delayed time, avoided the antagonistic influence of valine for
improving sensitivity, accuracy and precision of obtained chromatogram [69].
Amrein et al. (2006) determined levels of acrylamide in salted, smoked, caramelized,

raw and roasted almonds and almond based foods using GC-MS. Analysis of
acrylamide, free amino acids, color and moisture content was estimated. Results
demonstrated that roasted almonds was higher in acrylamide content while raw, cooked
and almonds enclosed no levels of acrylamide [70].
Paleologos et al. (2005) employed normal phase HPLC for estimation of acrylamide
and meth-acrylamide in chocolate cookies, chicken, coffee and potatoes. The influence
of acetonitrile on separation through chromatography was studied and analytical
characteristics such as relative standard deviation, limits of quantification and
detection, spiked recoveries were evaluated. Obtained chromatograms showed
acrylamide found was comparable with mean values of literature but, meth-acrylamide
was not spotted in the least of samples [71].
20
Murkovic et al. (2004) performed reverse phase HPLC for measuring acrylamide in a
variety of food types such as coffee, pizza, chips, cookie, bread etc. Statistical analysis,
recovery, reproducibility and linearity was studied. Results concluded that acrylamide
in foods was made through cooking of starchy products that traveled from top to bottom
[72].
Hoenicke et al. (2004) analyzed acrylamide from a wide variety of foodstuffs using
LC-MS/MS for larger number of samples and GC-MS/MS for difficult matrices. Mean,
the coefficient of variation, limit of quantification were evaluated through percentage
recovery. Results revealed that the measurement uncertainties of acrylamide through
recurrent analysis were due to inhomogeneity of sample matrices and stability of
compound (acrylamide) in food samples [73].
John et al. (2003) reported acrylamide levels in chips, coffee, bread and cereal using
(LC-MS/MS). Effects of sample size and solvent configuration on acrylamide levels
and column temperature were examined including accuracy, precision and recovery.
Results showed a correlation between acrylamide and oil while, acrylamide contents
were higher in chips which suggested cooking oil should be changed more often so that
acrylamide levels may be reduced [74].
Leung et al. (2003) analyzed acrylamide in different varieties of rice, rye, biscuit, and
wheat flour-based foods and deep-fried foods using LC-MS/MS. Acrylamide formation
started at the surface and then, breached in the matrix of food through diffusion of hot
oil. Results revealed that alterations in fat and moisture content (parameters) need to be
achieved during the cooking of foods for the lower level of acrylamide [75].
Takatsuki et al. (2003) determined acrylamide (AA) in cooked and processed foods
such as tea, coffee, cakes, bread crumbs, meat, fish etc. by LC-MS using column-
switching (four columns). Mean recoveries, standard deviation and limits were
evaluated through adding internal standard. Results revealed that foods containing
much water showed lower acrylamide content in relation with dry foods and greater
quantity in fries and lesser in cakes [76].
Rosén et al. (2002) reported acrylamide in cooked foods using LC-MS-MS. The
technique was authenticated statistically by mean, standard deviation and precision.
21
Fallouts concluded greater levels of acrylamide in mashed potatoes and the proposed
method gave limited qualitative information [77].
Mesias et al. (2018) performed estimation of acrylamide using liquid chromatography

coupled to tandem mass spectrometry technique in french fries. Amount of reducing
sugars, moisture, color and polar (interfering) compounds were evaluated. Results
showed that acrylamide concentration could be reduced with an increasing degree of
automatization and control of the frying process [78].
Nguyen et al. (2017) investigated 5-hydroxymethylfurfural (HMF) and formation of

acrylamide in the course of baking biscuits. Effects of ratio of asparagine and reducing
sugars on acrylamide level and reaction rate constants were evaluated. Results
concluded that a lower concentration of amino acid and sugars (asparagine and fructose,
glucose) during biscuit baking gave lowered acrylamide and HMF formation [79].
Dutta et al. (2015) proposed a computer system for identification and liquid
chromatography-mass spectrometric for assessment of acrylamide in potato chips.
Upon image analysis of chips, a color map of acrylamide content was seen in the region
of interest. Results showed that this method was a novel influence in image handling
that gave accurate results and could be used commercially [80].
Geng et al. (2011) evaluated acrylamide content in starch-based foods by HPLC with
column derivatization. Acrylamide level, mean, precision, accuracy, standard
deviation, spiked recovery and limits of quantification and detection were evaluated.
Results showed that the proposed method was novel, inexpensive and a vigorous
substitute for conservative examination of acrylamide [81].
22
MATERIALS AND METHOD
3.1: History of presence of AA in samples and reasons of testing/analyzing these
samples
The samples used this study were chips, cakes, muffins, breads, fried items, biscuits,
noodles, milk powder, breakfast cereals, whole wheat chapatti, nuts, tea, coffee and
crackers. As acrylamide is formed due to the reaction of glucose sugar and asparagine
amino acid at temperatures greater than 120°C. So, most of the food contain sugars and
amino acids and upon consulting the literature, it was revealed that carbohydrate and
starch rich foods are able to go towards maillard reaction and form acrylamide. It was
also studied in literature that cooked foods at higher temperatures also have AA in them.
We analyzed such regularly used foodstuffs as samples for studying about acrylamide
formation and concentration. The starchy food stuffs included chips homemade and
packaged ones, the biscuits were both baked and processed ones, fried items included
nuggets, burger patties. While cakes, noodles, breakfast cereals, tea, coffee and crackers
were also used for their AA detection and estimation.
3.2: Samples
The samples used in the analysis involved 30 daily consumable foodstuffs (baked, fried
and processed products) that were purchased from the local stores of Lahore (Punjab;
Pakistan) and acrylamide was determined from them. The samples included the items
given below:
1. Bread 2. Kurkure chips 3. Sooper biscuit
23
4. Rusk 5. Pizza bread 6. Shawarma bread
7. Baked biscuits 8. Nuggets
9. Burger patties 10. French fries 11. Roasted nuts
12. Homemade chapatti 13. Wavy chips 14. Milo powder
24
15. Corn flakes 16. Bun 17. Tuc biscuit
18. Gala biscuit 19. Patties (brown part) 20. Plain tea cake
21. Oats bran 22. Cheetos 23. Lays masala
24. Zeera plus biscuit 25. Noodles 26. Muffin
25
27. Marie biscuit 28. Tea 29. Coffee
30. Crackers
Fig.3.1: Different food samples chosen for analysis of acrylamide
3.2.1: Classification of food items into cooked processed or baked

The food items are classified into processed on the basis of foods which are pruned,
peeled or packaged before eating which includes dry fried and oil fried, cooked foods
are classified on the basis of foods that are cooked with direct application of heat.
Baked foods are those which require heating in oven or any such apparatus which only
heats the food.
Table 3.1: List for the classification of samples into processed, cooked or baked
foods
SR# NAMES OF SAMPLES CLASSIFICATION OF FOODS
INTO PROCESSED, COOKED OR
BAKED
1. Bread (Dawn) Baked
2. Kurkure chips (Kurkure) Processed
3. Sooper biscuit (Peek Freans) Processed
4. Rusk (Shezan) Baked
26
5. Pizza bread (Local) Baked
6. Shawarma bread (Dawn) Baked
7. Baked biscuits (Gourmet) Baked
8. Nuggets (Sufi) Processed
9. Burger patties (Sufi) Processed
10. Fries (Local) Cooked
11. Roasted peanuts (kurkure) Processed
12. Homemade chapatti (Punjab Atta) Cooked
13. Wavy chips (Lays) Processed
14. Milo powder (Nestle) Processed
15. Corn flakes (Nestle) Processed
16. Bun (Dawn) Baked
17. Tuc biscuit (Lu) Processed
18. Gala biscuit (Lu) Processed
19. Baked patties (Gourmet) Baked
20. Plain tea cake (Gourmet) Baked
21. Oats bran (Quaker) Processed
22. Cheetos (Pepsi) Processed
23. Lays masala (Lays) Processed
24. Zeera plus biscuit (Lu) Processed
25. Noodles (Knorr) Processed
26. Muffin (Dawn) Baked
27. Marie biscuit (Peek Freans) Processed
28. Tea (Supreme) Processed
29. Coffee (Nescafe) Processed
30. Crackers (Local) Cooked
3.3: Preparation of samples

Firstly, all samples were made oil-free and finely ground to a powdered form. About 4
g of the sample was weighed using a weight balance and taken in a conical flask. 10 ml
n-hexane was added twice to de-fat the sample and shaken vigorously for 20 min. n-
hexane was decanted, leaving the sample to dry. 20 ml acetone and 0.1 ml distilled
water was added in the sample and shaken for 1 min. Acetone was filtered in a beaker
27
and evaporated till dry using a hotplate. Finally, 5 ml distilled water was added in the
dried beaker, filtered and collected in eppendorf tube for acrylamide analysis using an
analytical technique (HPLC) [45].
3.4: Preparation of working standard solution

0.01 g of acrylamide of analytical grade having purity >99% was obtained from Sigma
Aldrich laboratories and dissolved in 100 ml double distilled water to make 100 ppm
stock solution and dilution of 2 ppm was made from it. Further, 250 ppb dilution was
made from 2 ppm. The working standard solution of 250 ppb was taken for analysis.
3.5: Preparation of mobile phase

Mobile phase was made using methanol and distilled water for 100 ml (10:90), filtered
and sonicated for 20 min to avoid any bubbles or interferences during acrylamide
analysis [55].
3.6: Acrylamide analysis using High Performance Liquid Chromatography

(HPLC)
All the samples in eppendorf tubes were filtered again through 0.64 nylon filter paper
and injected in to the HPLC column. Flow rate was 0.1/min. at 40°C column
temperature, 202 nm wavelength and 10 min. The concentrations of acrylamide were
collected in form of graphs of peak area on y-axis and time on x-axis. The graphs that
are recorded in HPLC are known as chromatograms. So, the chromatograms obtained
from HPLC analysis were discussed detail in results section.
3.7: High Performance Liquid Chromatography (HPLC)

A type of column chromatography which thrusts the sample (analyte) in a moving phase
(solvent) having solid (stationary phase) with He or N2 as a carrier stream gas.
Compounds in a sample are classified and separated even if they are in trace levels
(parts per trillion) [82].
3.8: Principle of High Performance Liquid Chromatography (HPLC)

The principle of HPLC is material separation, which occurs on the basis of polarity and
molecular weight. When a mixture of compounds is introduced through the HPLC
column, it gets separate into its components before it exits from the column and travels
according to relative affinities. The component which has greater affinity towards the
stationary phase (adsorbent), travels slower [83].
28
Figure 3.2: Schematic diagram and parts of HPLC
3.9: Working of High Performance Liquid Chromatography (HPLC)

The protocols are needed to be followed for HPLC. The sample is filtered through 0.64
µ nylon filter and mobile phase is filtered through vacuum and sonicated for 10 min.
only double distilled water is used for analysis because it is highly purified.
For this experiment, reversed phase is used because stationary phase is non polar and
hydrophobic (silica) and mobile phase (methanol and water) is polar. Firstly, mobile
phase is attached to a tube which pumps the eluent inside and is allowed to pass through
the instrument for washing purposes. After some time, the reading of HPLC stabilizes
and sample is injected into the instrument which passes through the column oven in
which stationary phase is present. An electronic signal of concentration of analyte is
collected from the detector in the form of peaks and finally reading is recorded.
The sample holding time with the stationary phase depends on the mobile phase and
the compound that is analyzed. As the sample passes the column, the analyte with lesser
interaction with stationary phase will pass out from the column faster. The results
obtained in the form of peaks are then analyzed for the presence of AA [84].
29
Figure 3.3: Instrumentation of High Performance Liquid Chromatography
3.10: Analysis of acrylamide values from chromatograms

In chromatography, the results are obtained in the form of chromatograms which
depicts the separation of components on a paper with its concentration. There are many
types of chromatogram obtained in chromatography such as ascending, descending,
circular chromatograms. But, the relative one is discussed here is chromatogram of
HPLC which separates the required component from a mixture of components and tells
us about the concentration of that compound. The value of acrylamide was calculated
from chromatograms (graphs) using values of standard concentration, area of sample
and area of standard. The formula was as follows:
Conc. of Acrylamide (ppb): Area of Sample × Conc. of Standard ---------------- (Eq.1)

Area of Standard
30
RESULTS
4.1: Presence or absence of acrylamide from chromatograms
All thirty samples were tested using HPLC for acrylamide levels and chromatograms
were obtained which showed only eight of the samples have the peak of acrylamide.
Following are chromatograms obtained from which a table for presence or absence of
acrylamide is drawn.
4.1.1: Chromatograms of standard and food samples

4.1.1.1: Chromatogram of standard of acrylamide
Acrylamide peak was observed at 4.467 s with a peak area of 834335 µV.
Figure 4.1: Chromatogram of standard of AA with its retention time, peak area,
percentage and height of peak
31
4.1.1.2: Chromatogram of bread
Acrylamide peak was observed at 4.517 s with a peak area of 3552 µV. Another peak
was also seen in the chromatogram, this was due to other interfering compounds which
were present inside the bread and showed solubility in the mobile phase.
Figure 4.2: Chromatogram of bread with its retention time, peak area, percentage
and height of peak
32
4.1.1.3 Chromatogram of kurkure chips
Acrylamide peak was observed at 4.517 s with a peak area of 2457 µV. Other peaks
were also obtained which was due to other compounds present which were also soluble
in mobile phase.
Figure 4.3: Chromatogram of kurkure chips with its retention time, peak area,
33
4.1.1.4: Chromatogram of sooper biscuit
No labelled peak of acrylamide was observed. Other peaks were also seen which
indicated other compounds soluble in mobile phase.
Figure 4.4: Chromatogram of sooper biscuit with its retention time, peak area,
34
4.1.1.5: Chromatogram of rusk
Acrylamide peak was observed at 4.517 s with a peak area of 5890 µV. Other peaks
were also seen which showed solubility of compounds other than acrylamide in mobile
phase.
Figure 4.5: Chromatogram of rusk with its retention time, peak area, percentage
and height of peak
35
4.1.1.6: Chromatogram of pizza bread
No peak of acrylamide was seen. But, a peak of other compound was seen which
indicated it’s solubility in mobile phase.
Figure 4.6: Chromatogram of pizza bread with its retention time, peak area,
36
4.1.1.7: Chromatogram of shawarma bread
No peak of acrylamide was seen. But, peaks of other compounds were seen which
showed solubility of compounds in mobile phase.
Figure 4.7: Chromatogram of sample of shawarma bread with its retention time,
peak area, percentage and height of peak
37
4.1.1.8: Chromatogram of baked biscuits
The peak of acrylamide was seen at 4.083 s with a peak area of 3020 µV. while, other
peaks showed solubility of other compounds in mobile phase.
Figure 4.8: Chromatogram of sample of baked biscuits with its retention time,
peak area, percentage and height of peak
38
4.1.1.9: Chromatogram of nuggets
Acrylamide peak was seen at 4.619 s with a peak area of 12719 µV. While other peak
in chromatogram showed it’s solubility in mobile phase.
Figure 4.9: Chromatogram of sample of nuggets with its retention time, peak area,
39
4.1.1.10: Chromatogram of burger patties
No peak of acrylamide was seen. While, other 2 peaks showed the compound’s
solubility in mobile phase.
Figure 4.10: Chromatogram of burger patties with its retention time, peak area,
40
4.1.1.11: Chromatogram of fries
No peak of acrylamide was seen. But the other peak indicated a compound which was
more soluble in mobile phase.
Figure 4.11: Chromatogram of fries with its retention time, peak area, percentage
and height of peak
41
4.1.1.12: Chromatogram of roasted almonds
Acrylamide peak was observed at 4.017 s with a peak area of 25637 µV. while, other
peaks indicated other compounds which were present in the sample and were soluble
in mobile phase and appeared on chromatogram.
Figure 4.12: Chromatogram of roasted almonds with its retention time, peak area,
42
4.1.1.13: Chromatogram of homemade chapatti
Acrylamide peak was observed at 4.685 s with a peak area of 3875 µV. While other
peaks showed solubility of compounds in mobile phase which were present in sample
and showed themselves in chromatogram.
Figure 4.13: Chromatogram of homemade chapatti with its retention time, peak
area, percentage and height of peak
43
4.1.1.14: Chromatogram of wavy chips
No peak of acrylamide was obtained. The peaks other than acrylamide showed the
presence of other compounds in sample and showed their solubility in mobile phase
and showed on chromatogram.
Figure 4.14: Chromatogram of wavy chips with its retention time, peak area,
44
4.1.1.15: Chromatogram of Milo powder
No acrylamide peak was seen. While, the peak showing in chromatogram was of the
compound present in sample and showed on the chromatogram because of the solubility
in mobile phase.
Figure 4.15: Chromatogram of Milo powder with its retention time, peak area,
45
4.1.1.16: Chromatogram of corn flakes (Koko crunch)
No peak of acrylamide was seen. While other 2 peaks showed presence of other
compounds in sample which showed on chromatogram due to solubility in mobile
phase.
Figure 4.16: Chromatogram of corn flakes with its retention time, peak area,
46
4.1.1.17: Chromatogram of bun
No peak of acrylamide was seen. While, the 2 peaks showed the presence of other
compounds in the sample and appeared on chromatogram due to solubility on mobile
phase.
Figure 4.17: Chromatogram of bun with its retention time, peak area, percentage
and height of peak
47
4.1.1.18: Chromatogram of Tuc biscuit
No peak of AA was seen. Other peaks indicated presence of other compounds in sample
which appeared due to solubility in mobile phase.
Figure 4.18: Chromatogram of Tuc biscuit with its retention time, peak area,
48
4.1.1.19: Chromatogram of gala biscuit
No peak of AA was seen. Other peaks were due to presence of compounds in sample
which were shown on chromatogram due to solubility in mobile phase.
Figure 4.19: Chromatogram of gala biscuit with its retention time, peak area,
49
4.1.1.20: Chromatogram of patties (brown part)
No peak of acrylamide was seen. But, the peak in chromatogram was due to presence
of other compound in sample which appeared because of solubility in mobile phase.
Figure 4.20: Chromatogram of patties with its retention time, peak area,
50
4.1.1.21: Chromatogram of plain tea cake
No peak of acrylamide was seen. While other peaks showed the presence of compounds
present in sample which were soluble in mobile phase and appeared on chromatogram.
Figure 4.21: Chromatogram of plain tea cake with its retention time, peak area,
51
4.1.1.22: Chromatogram of oats bran
No peak of acrylamide was observed. Other peaks indicated the presence of compounds
in sample which appeared on chromatogram due to solubility in mobile phase.
Figure 4.22: Chromatogram of oats bran with its retention time, peak area,
52
4.1.1.23: Chromatogram of Cheetos chips
Acrylamide peak was observed at 4.017 s with area of 30121 µV. Other peaks showed
the presence of compound present in sample and appeared on chromatogram because
of solubility in mobile phase.
Figure 4.23: Chromatogram of Cheetos chips with its retention time, peak area,
53
4.1.1.24: Chromatogram of lays masala
No acrylamide was detected but other compound’s peak was seen in chromatogram
which indicated the presence in sample and appeared due to solubility in mobile phase.
Figure 4.24: Chromatogram of lays masala with its retention time, peak area,
54
4.1.1.25: Chromatogram of Zeera plus biscuit
No acrylamide was detected but other compound’s peak was seen in chromatogram
Figure 4.25: Chromatogram of zeera plus biscuit with its retention time, peak
area, percentage and height of peak
55
4.1.1.26: Chromatogram of noodles
No acrylamide peak was obtained but other compound’s peaks were seen in
chromatogram which indicated the presence in sample and appeared due to solubility
in mobile phase.
Figure 4.26: Chromatogram of noodles with its retention time, peak area,
56
4.1.1.27: Chromatogram of muffin
No acrylamide was detected but other compound’s peaks were seen in chromatogram
Figure 4.27 Chromatogram of muffin with its retention time, peak area,
57
4.1.1.28: Chromatogram of marie biscuit
No acrylamide peak was seen but other compound’s peaks were seen in chromatogram
Figure 4.28: Chromatogram of marie biscuit with its retention time, peak area,
58
4.1.1.29: Chromatogram of tea
No acrylamide peak was seen but other compound’s peaks were seen in chromatogram
Figure 4.29: Chromatogram of tea with its retention time, peak area, percentage
and height of peak
59
4.1.1.30: Chromatogram of coffee
No acrylamide was detected but other compound’s peaks were seen in chromatogram
Figure 4.30: Chromatogram of coffee with its retention time, peak area,
60
4.1.1.31: Chromatogram of crackers
No acrylamide was observed but other compound’s peak was seen in chromatogram
Figure 4.31: Chromatogram of crackers with its retention time, peak area,
61
Table 4.1: List of samples for the presence or absence of acrylamide
SR# NAMES OF SAMPLES PRESENCE OR ABSENCE OF
ACRYLAMIDE (YES OR NO)
1. Bread Yes
2. Kurkure chips Yes
3. Sooper biscuit No
4. Rusk Yes
5. Pizza bread No
6. Shawarma bread No
7. Baked biscuits Yes
8. Nuggets Yes
9. Burger patties No
10. Fries No
11. Roasted peanuts Yes
12. Homemade chapatti Yes
13. Wavy chips No
14. Milo powder No
15. Corn flakes (Koko crunch) No
16. Bun No
17. Tuc biscuit No
18. Gala biscuit No
19. Baked patties No
20. Plain tea cake No
21. Oats bran No
22. Cheetos Yes
23. Lays masala No
24. Zeera plus biscuit No
25. Knorr noodles No
26. Muffin No
27. Marie biscuit No
28. Tea No
29. Coffee No
62
30. Crackers No
4.2: Analysis of acrylamide values

The results obtained from the analysis showed acrylamide values in parts per billion.
Concentrations of AA were calculated from equation 1 as follows:
Conc. of Acrylamide (ppb): Area of Sample × Conc. of Standard ---------------- (Eq.1)

Area of Standard
Among the 8 food samples detected with AA, the highest amount of acrylamide was
found in the sample of cheetos chips (9.025 ppb) while, the lowest was seen in kurkure
chips (0.736 ppb) and all the samples showed AA values beyond the acceptable range.
Table 4.2: Concentrations of acrylamide in samples

SR # NAMES OF SAMPLES CONC. OF ACRYLAMIDE
1. Bread 1.064 ppb
2. Kurkure chips 0.736 ppb
3. Rusk 1.764 ppb
4. Baked biscuits 0.904 ppb
5. Nuggets 3.811 ppb
6. Roasted peanuts 7.681 ppb
7. Homemade chapatti 1.161 ppb
8. Cheetos chips 9.025 ppb
63
4.3: Comparison of acrylamide values in processed, baked and cooked foods
From the obtained results, the acrylamide levels observed in foods can be classified in
terms of acrylamide values obtained from processed (dry fried and oil fried), cooked
and baked foods. This can be seen in table 4.3 which showed a comparison of the
obtained values in different foods. The mean values were calculated of each group
(cooked, processed and baked) and placed in decreasing order of acrylamide amount.
Table 4.3: Comparison of acrylamide levels in processed, baked and cooked foods
SR AA IN PROCESSED AA IN BAKED AA IN COOKED
# FOODS (ppb) FOODS (ppb) FOODS (ppb)
NAME CONC. NAME CONC. NAME CONC.
1. Nuggets 3.811 Baked 0.904 Homemade 1.161

Biscuits Chapatti
2. Kurkure 0.736 Bread 1.064
Chips
3. Cheetos 9.025 Rusk 1.764
Chips
4. Roasted 7.681
Peanuts
Processed ˃ Baked ˃ Cooked
64
DISCUSSION
Acrylamide is a carcinogen, formed in foods by the reaction of reducing sugar (glucose)
and amino acid (asparagine) at temperature greater than 140-160°C through a non-
enzymatic procedure known as maillard reaction. Acrylamide is highly reactive due to
the presence of an amide group (-CONH2) and a double bond. So, it gives nucleophilic
addition reactions. Many factors such as frying temperature and process, modifiers, pre-
treatments, pH, addition of salts and sugars, conc. of water and starch and shelf life of
foods effect greatly on acrylamide formation. Although monomers of acrylamide
(vinyl) are widely used in metal industry but, toxic for humans because it remains
unaffected by microbial attack and is one of the major sources of oral cancer. When
acrylamide gets in contact with body it sweeps through the skin and distributes itself
throughout the whole body via blood and reaches to different sites of cells, tissues,
organs, lymph nodes etc. Acrylamide oxidizes itself into an epoxide which is highly
reactive (glycidamide) that forms adducts with DNA and alters the structure which
leads to mutagenicity and genotoxicity. 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 should be taken in consideration that not every brown thing contains acrylamide but,
maillard reaction is taking place in every brown food. The flavor, aroma, taste,
appearance and many other products are due to maillard reaction. This can be made
clear from examples of maillard reaction other than acrylamide such as: meaty roasted
type of food is due to thiophenes, green nutty sweet is due to oxazoles, meaty burnt
caramel like appearance is because of furans, sweet caramel burnt food is due to
furanones, cracker like cereal is acylpyridines, bitter burnt astringent is alkylpyridines,
cereal like nutty is due to pyrroles, cooked roasted or toasted food is pyrazines,
melanoidions is the end product of maillard reaction which is a brown polymeric
substance that gives different types of colors to the cooked food.
There were thirty samples tested for AA in this study and only eight of them had
acrylamide in them. Different techniques including GC-MS, LC-MS, microchip
electrophoresis and HPLC has confirmed the presence of AA in homemade potato chips
(French fries) [49] and processed chips [59]. Different potato chips samples were used
in our study including homemade chips from fresh potatoes and fresh oil and processed
65
ones. Razia S. along with her coworkers, measured acrylamide amount using HPLC
and concluded that potato chips showed the highest amounts of AA, biscuits showed
AA level in range while cakes showed lower conc. of AA [44]. Takatsuki S. and his
team cited that by using LC-MS, acrylamide levels were high in corn chips than corn
based snacks [76]. In our results it was found that Cheeetos chips (corn chips) had the
highest amount of AA (9.025 ppb) while, kurkure chips had the lowest amount of AA
(0.736 ppb) and no peak of AA was detected in lays masala and homemade potato chips
(French Fries).
Nygen H. T. and his associates investigated acrylamide levels using HPLC in course of
baking biscuits and concluded that lower conc. of amino acid and sugar, lowered AA
formation [79] and Vaclavik L. and his companions also studied acrylamide levels
using high throughput mass spectrometric analysis and concluded that acrylamide
concentrations were positively correlated with baking time and temperature and
observed highest acrylamide levels in biscuits baked at 200°C for 15 min [85]. Our
results showed that bakery baked biscuits had AA (0.904 ppb) while packed biscuits
(Sooper, Tuc, Gala, Zeera plus and Marie biscuits) had no AA peak.
Acrylamide formation and higher levels were seen in bread by a researcher Claus A.
and his coworkers by using LC-MS/MS. Their results demonstrated that acrylamide
would form in bread and bread products but, lower levels would be observed when
reduced temperature, prolong heat treatment and deck oven are utilized [86]. We tested
5 bread samples (bun, shawarma bread, pizza bread, rusk, dawn bread) and it was found
2 out of 5 samples showed acrylamide peak. The highest amount of acrylamide was
seen rusk (1.764 ppb) and lowest in bread (1.064 ppb) while, no peak was seen for bun,
shawarma bread and pizza bread.
Jagerstad M. and his team talked about acrylamide’s genotoxicity in cooked foods.
They said that cooking conditions and dietary habits causes different types of DNA
damage: nucleotide alterations and chromosomal aberrations by forming carcinogenic-
DNA adducts [87]. In our results, homemade whole grain chapatti had AA (1.161 ppb).
Altunay N. and his classmates studied acrylamide levels in different crackers using
flame atomic absorption and found that sesame and spices crackers had the highest
amount of AA while pure crackers had the lowest level of AA [51]. Our results found
no acrylamide in crackers sample.
66
Acrylamide concentration was measured in roasted peanuts and other dried fruits by
Laura and her associates by using LC-MS and a significant concentration was measured
in dried prunes and peanuts which agreed with literature value [48]. Our results showed
acrylamide peak of roasted peanuts (7.681 ppb).
Tateo F. and his team reported acrylamide levels in fast food items by GC-MS and said
that chicken nuggets had the highest amount of acrylamide and burger patties had
slightly lower AA but, reasonable enough to cause toxicity [88]. In our results, AA peak
was seen in nuggets (3.811 ppb) while no peak detecting AA was observed in baked
and processed patties.
In literature, acrylamide levels was measured through different techniques using HPLC
in cakes and muffins which were obtained from vending machines by Haouet N. and
his coworkers. They concluded that sweet bakery products (cakes and muffins) showed
reasonable AA levels than sandwich categories [43]. Our work revealed that plain tea
cake and muffin had no AA in them.
Yoshida M. and his colleagues measured acrylamide levels in different types of tea
using GC-MS and concluded that acrylamide level in green tea was not as high as in
roasted tea. And roasting time, temperature and tea leaves effected the conc. of
acrylamide [89]. But, in our study the tea sample didn’t show any peak of acrylamide.
Senyuva H. Z. and his classmates studied acrylamide levels in breakfast cereals using
LC-MS and found lower but a reasonable amount of AA [69]. In our work, corn flakes
and oats bran showed no peak of acrylamide.
Takatsuki S. and his team also measured acrylamide levels in processed foods (roasted
coffee beans, cocoa powder and instant noodles) using LC/MS and found that coffee
beans had the highest amount of acrylamide and instant noodles had the lowest while
cocoa powder was in between the two values of AA [76]. Our results showed no
acrylamide peak for the three samples (coffee, noodles and Milo powder).
The twenty two samples didn’t have acrylamide but the samples did show some peaks
other than acrylamide which concluded that some other interfering compounds were
present in them. The decreasing order of the determined acrylamide values from eight
samples are shown below:
67
Cheetos ˃ Roasted peanuts ˃ Nuggets ˃ Rusk ˃ Homemade Chapatti ˃ Bread ˃
Baked biscuits ˃ Kurkure chips
OR
9.025 ˃ 7.681 ˃ 3.811 ˃ 1.764 ˃ 1.161 ˃ 1.064 ˃ 0.904 ˃ 0.736
From the measured values, the foods were grouped accordingly and arranged in
decreasing order of processed, cooked and baked items for finalizing which group
contains higher amount of acrylamide.
Processed foods ˃ Baked foods ˃ Cooked foods
The analyzed quantities of acrylamide were beyond the limits and were greater enough
to cause different types of cancers in human body. Wen acrylamide gets in contact
through skin and spreads to tissues and then penetrates towards organs and causes
cancer by producing mutations in DNA makeup by binding on to DNA and protein.
When acrylamide gets in contact with nerve endings, an inhibition of axonal transport
occurs and cause neurodegenerative diseases. Acrylamide cannot be avoided
completely but some measures can definitely be taken to prevent more damage [39].
68
Conclusion
Acrylamide is a toxic carcinogen formed at higher temperatures in foods. All food
samples were tested for acrylamide presence and concentration and eight samples were
found to have acrylamide. Acrylamide was the highest in Cheetos corn chips and lowest
in Kurkure chips. The bread sample showed shocking acrylamide concentrations as, we
daily eat bread in breakfast. So, more toasting of the bread should be avoided and same
is the case for homemade whole wheat chapatti. Rusk, nuggets, baked biscuits, and
roasted peanuts showed levels of acrylamide beyond normal range. It is clear from the
results that these eight food items should be consumed in lesser quantity and cooking
or heating way should be improved for minimum acrylamide formation.
After calculating the mean values of processed, baked and cooked foods, we can say
that in case of processed foods, the acrylamide content was higher than cooked or baked
while, cooked foods was having the least amount of acrylamide so, the order for
acrylamide level will go as:
Processed foods ˃ Baked foods ˃ Cooked foods
While food processing, cooking and baking, the amounts of sugar, water and inorganic
salts, effect of pH and type of oil should be kept in mind. As, it leads to the formation
of a dangerous chemical compound i.e. acrylamide.
69
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1.1: Properties of Acrylamide 1.1.1: Physical Properties

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INTRODUCTION

1.1: Properties of Acrylamide

1.1.2: Chemical properties

Acrylamide (AA) is an unstable monomer holding an amide group (-CONH2) and a

Figure 1.1: Chemical structure of acrylamide showing the functional groups of -

1.3: Mechanism of acrylamide formation

Figure 1.2: General mechanism for acrylamide formation through maillard

1.3.1: Two models for mechanism of acrylamide formation

1.3.1.1: Findings from the two models of acrylamide formation

1.3.2: Pathways of acrylamide formation

1.3.2.1.1: Strecker reaction of asparagine

1.3.2.2: Different routes for acrylamide formation

(1) 1-propenal or acrolein is an unsaturated aldehyde, cytotoxic compound. Acrylamide

(2) Acrylamide is formed through thermal decomposition of carnosine, β-alanine and

(3) Aminopropionamide acts an intermediate during acrylamide formation from

(6) Acrylamide is formed by the breaking of amadori compounds (benzaldehyde and

1.4: Factors affecting acrylamide formation

1.4.1: Effect of thawing conditions in correspondence to frying temperatures

1.4.2: Influence of frying process and cultivators

1.4.3: Effect of pre-treatments and air frying

1.4.5: Effect of vacuum roasting

1.4.6: Effect of pH with inorganic salts

1.4.7: Effect of sugars with storage time

1.4.9: Effect of formulations, baking technology and enzyme modifiers

1.5: Advantages of using acrylamide

1.6: Harmful effects of acrylamide

1.7 Reduction of acrylamide

1.7.2: Reduction through salts

1.8: Acrylamide and digestion inside human body

1.8.1: Acrylamide and human health

Acrylamide concentration significantly reduces after the gastrointestinal digestion. As

1.10: Reason for using food samples for determination of acrylamide

1.11: Analytical techniques

1.11.1: Preferred method for acrylamide estimation

 To evaluate and analyze foods that contain acrylamide.

Zahra et al. (2018) used high-performance liquid chromatography for determining

Haouet et al. (2016) proposed acrylamide determination in processed foods (potato

Razia et al. (2016) investigated concentrations of acrylamide levels in processed cakes,

Norouzi et al. (2018) examined acrylamide in bread by ultrasonic assisted micro-

Wu et al. (2016) presented microchip electrophoresis (MCE) with laser-induced

Altunay et al. (2016) worked on acrylamide determination in infant formulas, crackers

Shamla et al. (2014) evaluated acrylamide concentrations in deep-fried snacks (potato

Prashant et al. (2010) quantitated acrylamide concentration in processed chips and

Karasek et al. (2009) performed HPLC-MS/MS isotope dilution technique for

Kaya et al. (2009) determined contents of acrylamide using HPLC-MS in meshed

Wang et al. (2008) developed a simple solid-phase extraction technique with

Gökmen et al. (2006) developed a general preparatory technique for acrylamide

Amrein et al. (2006) determined levels of acrylamide in salted, smoked, caramelized,

Mesias et al. (2018) performed estimation of acrylamide using liquid chromatography

Nguyen et al. (2017) investigated 5-hydroxymethylfurfural (HMF) and formation of

1. Bread 2. Kurkure chips 3. Sooper biscuit

7. Baked biscuits 8. Nuggets

9. Burger patties 10. French fries 11. Roasted nuts

12. Homemade chapatti 13. Wavy chips 14. Milo powder

21. Oats bran 22. Cheetos 23. Lays masala

24. Zeera plus biscuit 25. Noodles 26. Muffin

3.2.1: Classification of food items into cooked processed or baked

3.3: Preparation of samples

3.4: Preparation of working standard solution

3.5: Preparation of mobile phase

3.6: Acrylamide analysis using High Performance Liquid Chromatography

3.7: High Performance Liquid Chromatography (HPLC)