summary of phd thesis - usamv cluj1.2 liquid chromatographic method for the assessment of polycyclic...
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U�IVERSITY OF AGRICULTURAL SCIE�CES A�D
VETERI�ARY MEDICI�E CLUJ-�APOCA
DOCTORAL SCHOOL
FACULTATY OF VETERI�ARY MEDICI�E
Eng. A�CA �AGHIU
Summary of PhD THESIS
RESEARCH REGARDI�G THE I�CIDE�TS A�D HYGIE�IC
SIG�IFICA�CE O� POLYAROMATIC HYDROCARBO�S I� THE
FOOD CHAI�
SCIE�TIFIC COORDI�ATOR:
LASLO COR�EL, PhD Professor
CLUJ-�APOCA
2012
2
TABLE OF CO�TE�T
KEYWORDS .............................................................................................................................. 3
INTRODUCTION ...................................................................................................................... 3
CHAPTER 1: DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS IN
FOOD AND ENVIRONMENTAL FACTORS ......................................................................... 4
1.1 Samples taken in to analysis ....................................................................................... 4
1.2 Liquid chromatographic method for the assessment of polycyclic aromatic
hydrocarbon content in some foods and environmental factors ............................................ 4
CHAPTER 2: RESULTS OBTAINED FROM THE ASSESSMENT OF POLYCYCLIC
AROMATIC HYDROCARBONS IN FOOD AND ENVIRONMENTAL SAMPLES ............ 5
CHAPTER 3: THE HYGIENIC SIGNIFICANCE OF POLYCYCLIC AROMATIC
HYDROCARBONS ................................................................................................................... 6
3.1 Survey results ................................................................................................................... 7
3.2 Estimated daily intake of PAH`s from meat products ................................................... 10
CHAPTER 4: GENERAL CONCLUSIONS............................................................................ 11
REFERENCES ......................................................................................................................... 12
3
KEYWORDS: PAHs, HPLC-FLD, LC MS MS, extraction method, meat and meat
products, daily intake, smoking, bezno(a)pyrene.
I�TRODUCTIO�
Polycyclic aromatic hydrocarbons (PAHs) are a group of about 10,000 organic
compounds containing two or more fused aromatic rings that are formed and released during
incomplete combustion or pyrolysis of organic matter, during industrial processes and other
human activities.[1,2]
Compounds that are relevant considering their effect on human health and there
abundance in the environment are naphthalene, acenaphthylene, acenaphthene, fluorene,
phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene,
benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3,c,d]-pyrene,
dibenz[a,h]anthracene, benzo[g,h,i]perylene. [2, 3, 4, 5, 11]
The most studied PAH is benzo[a]pyrene, which is often used as a marker for PAH in
ambient air and food.[2]
Human’s major routes of exposure to PAH are from inhaled polluted air and food
products, especially the ones that suffer grilling, roasting and smoking processes. [2, 6, 7, 12]
Vegetables may also contain high values of PAHs due to the air pollution and the deposit of
these organic pollutants on the surface. [2, 6, 7]. PAHs are lipophilic compound and thus tend
to from complex bound with the fatty part of meat products. [1, 11]
Considering the complexity of the sample matrix and the low concentration in which
the analit is found, in order to have a sensitive, selective and stable method of analysis it is
essential have an easy, reliable and rugged method of extraction.
A number of studies have been done in order to determine PAHs level in different
food stuff. [1,2, 4, 12]. PAH’s are found in food due to environmental pollution or are formed
during the heat treatment operation that the food suffers in the fabrication process. [1,2,3,4]
Results indicate that the level of PHAs found depend on the temperature and time of heat
treatment that the food suffers, the type of wood used for obtaining smoke, fat content, etc.
[1,2,3,11]
4
CHAPTER 1: DETERMI�ATIO� OF POLYCYCLIC AROMATIC
HYDROCARBO�S I� FOOD A�D E�VIRO�ME�TAL FACTORS
1.1 Samples taken in to analysis
64 samples were analyzed, from which 10 were water samples, 7 soil samples and the
rest 47 where meat and meat products. The meat products were bought from the Cluj-Napoca
market and households from around the city. They had different fat content and different heat
treatment during their production. The fat content and temperature of the heat treatment are
very important facts that determine the level of PAH’s that accumulate in the meat products.
The samples were extracted using a liquid/liquid method.
1.2 Liquid chromatographic method for the assessment of polycyclic aromatic
hydrocarbon content in some foods and environmental factors
2 methods of analysis were developed: one using LC MS MS 3200 Q TRAP from
Applied Biosystems and the other one using a HPLC with UV and FLD detectors 200 series
from Perkin Elmer (image 1). [9]
Image 1. HPLC FLD 200 series from Perkin Elmer
System Parameters:
• Flow Rate: 1,6mL/min
• Mobile Phase: A (H2O)
B (ACN)
5
• Column Temp: 25°C
• Injection Volume: 20 µL
• Column: ZORBAX Eclipse PAH 5µm, 4.6×150 mm column from Agilent
Technologies
• Wavelength: 254 nm for the UV detector, different wavelenghts appropriate for each
compound for the FLD detector
CHAPTER 2: RESULTS OBTAI�ED FROM THE ASSESSME�T OF
POLYCYCLIC AROMATIC HYDROCARBO�S I� FOOD A�D
E�VIRO�ME�TAL SAMPLES
Much lower concentrations of PAH`s were found in samples that were cold smoked
than in samples that were hat smoked. In table 1 are presented Average values obtained for
some meat product.[10]
Table 1
Average values obtained for some meat product
Crt
nr. Chemical compound
Summer
salami
(µg·kg-1
)
Hungarian
salami
(µg·kg-1
)
Pork
baloney
(µg•kg-1)
Pork
sausages
(µg·kg-1
)
Bacon
(µg•kg-1)
1 Naphthalene 13.0267 15.0674 8.0875 7.87480581 7,0953
2 Acenaphthene 21.4342 4.5204 7.4812 14.8989 0,0819
3 Fluorene 0.0011 ˂LQ* 0.0002 0.0003 ˂LQ
4 Phenanthrene 36.2035 9.9068 25.8693 35.5578 11,5655
5 Anthracene 1.4411 0.4533 0.4525 0.41444 0,4363
6 Fluoranthene 0.6266 0.8962 2.3732 2.03727 2,8927
7 Pyrene 7.7193 1.0861 2.8751 3.70149 2,0761
8 Benz[a]anthracene 4.2454 0.0349 2.4628 3.59074 1,5192
9 Chrysene 7.4973 0.1451 2.1781 3.81737 0,5178
10 Benzo[b]fluoranthene 45.6594 7.0258 74.0003 58.9585 88,6525
11 Benzo[k]fluoranthene 27.3575 0.4291 8.6940 16.3671 1,5415
12 Benzo[a]pyrene 2.8285 1.1381 0.7164 1.53717 0,2695
13 Dibenz[a,h]anthracene 3.3325 2.8721 3.9346 4.40805 3,5961
14 Benzo[ghi]perylene 8.4712 2.7365 2.8005 5.86746 0,2178
15 Indeno[1,2,3-
cd]pyrene 0.0073 ˂LQ 0.2380 0.13311 0,3112
PAHs total average value 179,8521 46.3116 213.6612 188.5553 103.0435
6
A lower concentration of PAH`s was found in summer salamis that were processed
using the traditional way (no products like liquid some and powder smoke was used).
Benzo(ghi)perylene had a very high concentration, 32,46 µg/kg, in smoked bacon and 15,49
µg/kg in smoked sausage produced in factories, this compound was not found the same
products that were traditionally smoked.[10]
In all samples benzo(a)pyrene was under the maximum level establish by the
regulation (max 5 µg/kg). Benzo(a)pyrene was found at values of 0,4-2 µg/kg. This
compound was found in commercial smoked samples with a concentration two times higher
than in traditional meat products. [10]
The average value of PAH`s found in meat products depending on the smoking type is
shown in image 2.
Imag. 2 Average total the PAH’s content depending on the smoking type
CHAPTER 3: THE HYGIE�IC SIG�IFICA�CE OF POLYCYCLIC
AROMATIC HYDROCARBO�S
A survey was conducted using a questioner. 108 adults form Cluj County were
selected using the non-random method, of unbound quotas, by gender and age. This applied
research was done in order to establish the customer’s behavior concerning meat products in
the Cluj County.
179,8521
149,5093667
103,0435
0
20
40
60
80
100
120
140
160
180
200
hot and cold hot very hot
µg/kg
PAHs total average value
7
3.1 Survey results
Survey participants were asked: “which is the meat product that they most often eat?”
Their responses (Table 2) were quite varied; pork sausages and chicken meat have the highest
share (image 3). [8]
Table 2
The most consumed meat products
Meat products �umber of respondents
turkey ham 2
pig fat 2
Sibiu salami 3
pork sausage 5
chicken ham 6
chicken thighs 6
chicken wings 6
baloney 6
hot dogs 8
Prague ham 9
salami 10
loin 10
summer salami 11
grilled chicken breast 12
specialties 12
Total number of respondents 108
turkey ham
2%
pig fat
2%Sibiu salami
3% pork sausage
5%
chicken ham
5%
chicken thighs
6%
chicken wings
6%
baloney
6%
hot dogs
7%Prague ham
8%
salami
9%
loin
9%
summer salami
10%
grilled chicken
breast
11%
specialties
11%
Image 3 The share of meat products consumption
Criteria that determine the purchase of meat products are presented in Table
Experience, appearance and price have the largest share (image
small part in purchase of meat products, which shows a lack of confide
in advertising campaigns. It is an important aspect given that economic investments made
advertising.[8]
Criteria for buying meat products
Criteria
Price
Manufacturer
Product organoleptic
Advertising
Recommendation of a person
Experience
Something else
Total number of respondents
Image 4 Criteria that determine the purchase of meat products
Purchase places of meat products are presented in Table
largest share fallowed by producer’s stores. (Image
Advertising
4%
Recommendati
on of a person
4%
Experience
33%
Something else
Criteria that determine the purchase of meat products are presented in Table
Experience, appearance and price have the largest share (image 4). Advertising plays a very
small part in purchase of meat products, which shows a lack of confidence that buyers have
in advertising campaigns. It is an important aspect given that economic investments made
Criteria for buying meat products
�umber of respondents
15
17
Product organoleptic characteristics 30
4
Recommendation of a person 5
36
1
Total number of respondents 108
Criteria that determine the purchase of meat products
Purchase places of meat products are presented in Table 4. Supermarkets have the
largest share fallowed by producer’s stores. (Image 5)
Price
14%
Manufacturer
16%
Product
organoleptic
characteristics
28%Advertising
Something else
1%
8
Criteria that determine the purchase of meat products are presented in Table 3.
). Advertising plays a very
nce that buyers have
in advertising campaigns. It is an important aspect given that economic investments made in
Table 3
. Supermarkets have the
Purchase places of meat products
Purchase place
Supermarket
Producer’s stores
Meat store
Market place
Local store
Somewhere else
Total number of respondents
Image
To assess the consumption levels of meat products, respondents were asked to
estimate their weekly consumption of meat products
Type of meat products that is most consumed varies with age; young people prefer
chicken products while the older prefer pork products.
In general people with the age of 25
meat products.[8]
Producer’s
stores
22%
Meat store
Purchase places of meat products
Purchase place �umber of respondents
65
Producer’s stores 24
11
0
5
Somewhere else 3
Total number of respondents 108
Image 5 Purchase places of meat products
To assess the consumption levels of meat products, respondents were asked to
estimate their weekly consumption of meat products (image 6).
products that is most consumed varies with age; young people prefer
chicken products while the older prefer pork products.
In general people with the age of 25-35 have the most diversified consumption of
Supermarket
60%
Meat store
10%Local store
5%
Somewhere
else
3%
9
Table 4
To assess the consumption levels of meat products, respondents were asked to
products that is most consumed varies with age; young people prefer
35 have the most diversified consumption of
Somewhere
else
10
122,79
162,86 169,05158,81
70,99
90,36 98,29 75
0
20
40
60
80
100
120
140
160
180
under 25
years old
25-34 years
old
35-44 years
old
45-55 years
old
Qu
an
tity
co
nsu
me
d (
g/
da
y)
Men
Women
Image 6 Meat consumption evolution
With regard to consumption of dishes like barbecue, sausage and mici, 60 (56%)
respondents said that they don’t frequently consume these products and 48 (44%) sad that
they commonly consumed such products. The biggest consumers of these products are men
with ages between 25-44 years. Consumers of such products prefer to prepare them at home
and only a small part of them purchase these products already made in cafeterias or
supermarkets. A total of 69 (64%) respondents answered that they also consume other
smoked products such as smoked fish or smoked cheeses. Regarding the effects of smoked
products have on human health a large number (48%) of respondents said they have no effect
on human health. Those who responded that smoked products affect human (38%) had ages
between 25-34 years.
Negative effects that respondents believe that the smoked products have are: effect on
bile, gallbladder, liver, colon and a small number (9 respondents) has sad these products have
carcinogenic effect. 31 of the respondents where males and 77 women, with ages between 20-
55, with different education and with a monthly income of 600-5000 lei.
3.2 Estimated daily intake of PAH`s from meat products
In order to estimate the daily intake of PAH`s from meat products he results obtained
from the 47 meat and meat samples were correlated with the results obtained using the
survey.
Estimated daily intake of PAH`s from meat products is presented in table 5.
11
Tabel 5
Estimated daily intake of PAH's due to meat products consumtion(µg/day)
Total PAH
(µg/day)
Total PAH
(µg/week)
Total PAH
(µg/month)
Total PAH
(µg/year)
Women
Under 25 years 77,8436 544,9051 2335,307 28412,91
25-34 years 173,3110 1213,177 5199,329 63258,5
35-44years 107,5584 752,909 3226,753 39258,82
45-55 years 82,1210 574,8468 2463,629 29974,16
Men
Under 25 years 135,1012 945,7082 4053,035 49311,93
25-34 years 175,6531 1229,572 5269,593 64113,38
35-44years 184,4148 1290,904 5532,444 67311,41
45-55 years 173,3010 1213,107 5199,029 63254,85
CHAPTER 4: GE�ERAL CO�CLUSIO�S
• The recovery obtained with the liquid/liquid extraction are very good. It is an easy,
reliable and rugged method, optimal for the extraction of meat and meat products.
• The amount of solvents used is low. The Florosil column retains all the fat that is let
after the extraction, assuring an impurity free sample.
• No matrix effect was observed for the compounds of interest in the analyzed samples,
they have the same retention time as the standard solution.
• Lower quantification limits were obtained using HPLC FLD than LC MS MS.
• Bezo[a]pyren, which is the only PAh’s that has maximum level legislation, was found
in all samples below the maxim limit, 5 µg/kg. Other PAHs that have mutagenic and
carcinogenic effect on mice, like Benzo[b]fluoranthene, were found in very high
values more than 20 time the value of Benzo[a]pyrene.
• Different values of PAHs were found in meat products, made by the same
manufacturer but sold in different shops proving that the level of PAH’s found in the
samples is also due both to the heat treatment and an incorrect storage and transport of
the products.
• The most consumed meat products are sausages followed by chicken products.
12
• The most important criteria in purchasing a meat preparation are experience,
appearance of the product and manufacturer. Price is only the third which shows that
respondents attach importance to food quality.
• The fact that previous experience firstly demonstrated that respondents are not willing
to try new products.
• Only 4% of respondents said that advertising is a factor that determines the purchase
of meat, which shows a low trust in advertising campaigns.
• Respondents under the age of 25 have the heights consumption on chicken meat
products.
• Heights diversity in meat products consumption is in the population with ages
between 25-34 years.
• Respondents between 35-44 years old are the most carful regarding what they eat. At
this age group is also the height meat consumption. These can be also explained by
the fact that they have the biggest income.
• From the 17 priority PAHs benzo(b)fluoranthene has the highest daily intake of 1,62-
5,37 µg/day in women and 2,62-6,17 µg/day in men, values that are much higher then
daily intake of benzo(a)pyrene.
• Respondents with ages between 25-34 years have the highest daily intake of
benzo(a)pyrene.
REFERE�CES
1. Agency for Toxic Substances and Disease Registry (ATSDR), 2009, Case Studies in
Environmental Medicine, Toxicity of Polycyclic Aromatic Hydrocarbons (PAHs).
2. Chen, B.H., and Lin, Y.S., 1997, Formation of Polycyclic Aromatic Hydrocarbons
durinprocessing of duck meat, J. Agric. Food Chem., 45, pp. 1394-1403.
3. EC (European Communities), 2001c, 2001, Ambient Air Pollution by Polycyclic
Aromatic Hydrocarbons (PAH) - Position Paper Annexes, (prepared by the Working
70 Group On Polycyclic Aromatic Hydrocarbons).
4. Farhadian, A., Jinap, S., Faridah A,, Sakar, Z. I., 2010, Determination of polycyclic
aromatic hydrocarbons in grilled meat, Food Control, Volume 21, Issue 5, pp. 606-
610.
13
5. Fetzer, J. C., 2000, The Chemistry and Analysis of the Large Polycyclic Aromatic
Hydrocarbons, Polycyclic Aromatic Compounds (New York: Wiley) 27 (2), p.143.
6. Food Standards Agency, 2005, PAHs in dietary supplements, Food Survey
Information Sheet 86/05.
7. Food Standards Agency, 2006, Polycyclic aromatic hydrocarbons (PAHs) in baby
foods and infant formulae, Food Survey Information Sheet 09/06.
8. �aghiu, Anca, M.O. Naghiu, C. Laslo, 2012, Applied research to assess the
consumption of meat products in cluj county, Agricultura, agricultural practice and
science journal, Vol. 79, nr. 1-2.
9. �aghiu, Anca, D. Simedru, C. Laslo, A. Mihaltan, 2012, An easy and reliable
method for PAH extraction from food samples, Agricultura, agricultural practice and
science journal, Vol. 79, nr. 1-2.
10. �aghiu, Anca, R. Laslo, D. Simedru, C. Laslo, 2012, The present of PAH’s in
meat products found on the Cluj-Napoca market, Agricultura, agricultural practice
and science journal, Vol. 79, nr. 1-2.
11. Wretling, S., A. Eriksson, G.A. Eskhult, B. Larsson, 2010, Polycyclic aromatic
hydrocarbons (PAHs) in Swedish smoked meat and fish, Journal of Food Composition
and Analysis, Volume 23, Issue 3, pp. 264-272.
12. US EPA, 1984, Review and evaluation of the evidence for cancer associated with air
pollution. EPA-450/5-83-006R, U.S. Environmental Protection Agency, Arlington.