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
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
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,
percentage and height of peak

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,
percentage and height of peak

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

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,
percentage and height of peak

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
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

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,
percentage and height of peak
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,
percentage and height of peak

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

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,
percentage and height of peak

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
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,
percentage and height of peak
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,
percentage and height of peak
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,
percentage and height of peak

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

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,
percentage and height of peak

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,
percentage and height of peak

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, percentage
and height of peak
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,
percentage and height of peak
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,
percentage and height of peak

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,
percentage and height of peak

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,
percentage and height of peak

4.1.1.25: Chromatogram of Zeera plus biscuit

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 4.25: Chromatogram of zeera plus biscuit with its retention time, peak area,
percentage and height of peak

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,
percentage and height of peak
4.1.1.27: Chromatogram of muffin
No acrylamide was detected but other compound’s peaks were seen in chromatogram
which indicated the presence in sample and appeared due to solubility in mobile phase.

Figure 4.27 Chromatogram of muffin with its retention time, peak area, percentage
and height of peak
4.1.1.28: Chromatogram of marie biscuit
No acrylamide peak was seen but other compound’s peaks were seen in chromatogram
which indicated the presence in sample and appeared due to solubility in mobile phase.
Figure 4.28: Chromatogram of marie biscuit with its retention time, peak area,
percentage and height of peak

4.1.1.29: Chromatogram of tea

No acrylamide peak was seen but other compound’s peaks were seen in chromatogram
which indicated the presence in sample and appeared due to solubility in mobile phase.
Figure 4.29: Chromatogram of tea with its retention time, peak area, percentage and
height of peak

4.1.1.30: Chromatogram of coffee

No acrylamide was detected but other compound’s peaks were seen in chromatogram
which indicated the presence in sample and appeared due to solubility in mobile phase.
Figure 4.30: Chromatogram of coffee with its retention time, peak area, percentage
and height of peak

4.1.1.31: Chromatogram of crackers

No acrylamide was observed but other compound’s peak was seen in chromatogram
which indicated the presence in sample and appeared due to solubility in mobile phase.
Figure 4.31: Chromatogram of crackers with its retention time, peak area,
percentage and height of peak

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
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

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 NAME NAME

CONC. CONC. CONC.
1. Nuggets Baked Homemade
3.811 0.904 1.161
Biscuits Chapatti
2. Kurkure Bread 1.064
0.736
Chips
3. Cheetos Rusk
9.025 1.764
Chips
4. Roasted
7.681
Peanuts

Processed ˃ Baked ˃ Cooked

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
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.

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:

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].

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.