Abstract: The acrylamide (AA) levels in marketing foods in gulf area are not investigated yet. An LC-MS/MS method for the determination of acrylamide in some selected food (local/imported) has been described. The samples were pre-dried, crushed/minced, degreased and mixed with [D3] acrylamide internal standard then acrylamide was water extracted at 60 °C in a ultrasonic bath. The aqueous solution was clean-up using a Carrez-Precipitation followed by centrifugation. The clean-up extract was then analyzed by LC-MS/MS. The method was applicable to detect acrylamide in different food types. The detection limit was as low as 30 μg kg-1. The AA level in different food groups were in order, mashed-roasted potato > fried pasta > soluble coffee > biscuits > potato chips > cocoa powder > crisp bread > fired rice > roasted Turkish coffee > cereal breakfast (corn) > butter cookies. The highest value of acrylamide (8974 μg kg-1) was detected in mashed-roasted potato, whereas the lowest value was detected in butter cookies (151 μg kg-1). The calculated average daily intake amounted to 34.03 μg AA/person/day which corresponds to 0.57 μg kg-1 body weight/day (body weight 60 kg). The outcome of this study has strongly recommended the necessity to conduct a large-scale survey in order to evaluate the levels of acrylamide in traditional foods. Thus, the true risk levels related to AA intake will be accurately estimated.
INTRODUCTION
In April 2002, both Swedish National Food Administration (SNFA, 2002) and University of Stockholm announced that certain cooked processed foods at relatively high temperatures contain significant levels of acrylamide.
Acrylamide (2-propenamide) is a highly water-soluble (2144 g L-1) compound with a low molecular weight (71.09). It is a reactive chemical, which is used as monomer in the synthesis of polyacrylamides used e.g., in purification of water, conditioning of soil and separation of proteins in analytical biochemistry and it is known as a component in tobacco smoke (Allan, 2002; Grives et al., 2002). Acrylamide has been classified as a group 2A carcinogen (probably carcinogenic to human) by the International Agency for Research on Cancer (1994). This might represent a potential threat to public health (FAO/WHO, 2002, 2005). Animal studies documented that high doses of acrylamide (> 203 mg kg-1) caused adverse developmental and reproductive effects in neonatal rodents (Friedman, 2003). For example, nerve degeneration, deficiency of intestinal enzymes, abnormal sperm and reduced fertility have been observed in treated rodents. On the other hand, in epidemiological studies for different types of cancer, it has not been possible to fined positive evidence for relationship between dietary acrylamide and cancer (Wirfalt et al., 2008). Neither in hospital-based control studies (Pelucchi et al., 2005) nor in epidemiological studies for large bowel, kidney and bladder cancer (Mucci et al., 2003) were observed. However, it is concluded that the statistical power of standard epidemiological studies is too low to detect an increased risk for cancer due to background exposure to acrylamide (Hagmar et al., 2005).
In view of this, the FAO/WHO consultation acknowledged the potential link between acrylamide and carbohydrate-rich food cooked excessively, for too long time or/and at very high temperature. The consultation considered that existing data is insufficient, especially outside Europe and North America to allow a quantitative estimate of health risks from dietary acrylamide and urged for more research on acrylamide in food in this area (FAO/WHO, 2002, 2005).
Arabian traditional carbohydrate-rich foods are an all-time favorite in Arab countries. These including a wide range of products, such as fried rice and pasta (Shaerria), Conaffa, Om Alli etc. Where such food exposed to high-temperature cooking e.g., roasting, backing, frying, grilling, which have the same manner of preparation with lots of western foods. Therefore, Arabian traditional carbohydrate-rich foods may represent high-risk levels of acrylamide under heating processes. To our best knowledge, there is a little information reported on the level of acrylamide in Arabian area.
The aim objectives of this study were to determine the levels of acrylamide in selected home-made and marketing foodstuffs known for its high concentration of acrylamide in Saudi market, to evaluate the effect of some traditional cooking processes on the level of acrylamide and further, risk assessment of dietary acrylamide.
MATERIALS AND METHODS
Chemicals
Acrylamide (99%) was from Sigma (MO, USA). [D3] acrylamide
internal standard ISTD (89%) was obtained from CIL (Andover, MA, USA).
Potassium hexacyanoferrat (3 H2O), Zinc sulphate (7H2O),
sodium chloride, sodium sulphate anhydrous and acetic acid were analytical
grade from Merck (Darmstadt, German). Acetic acid ethylester, methanol
(durability: 3 months), iso-hexan, acetonitril (min 99%) were HPLC grade
obtained from Merck (Darmstadt, German). Deionised water was purified
using a water purification system (Millipore, Mohlsheim, France).
Food Samples
Thirty-two samples of various foodstuffs (9 groups) were analyzed
in this study. The selected food groups were potato (mashed roasted and
chips), crisp bread, biscuit (Steak), cookies (Butter cookies), rice (fried),
pasta (Shaerria; like nodal), breakfast cereals, cacao and coffee (soluble
and roasted Turkish). Out of total samples, ten were collected from the
local stores. Half of it was locally produced while the other half was
imported. The last two samples; fried rice and pasta, were fried in the
Laboratory to simulate the cooking procedure of making rice in the Arabian
kitchen. A portion of soaked rice and dried fine pasta were deep-fried
in fresh vegetable oil at 170 ± 10 °C for 10 min until it became
substantial browning.
Acrylamide Analysis
In general, the given sample was pre-dried, crushed/minced, degreased
and mixed with deuterated internal standard [D3] then acrylamide
was water extracted at 60 °C in a ultrasonic bath. The aqueous solution
was clean-up using a Carrez-Precipitation followed by centrifugation.
The clean-up extract was then analysed by LC-MS/MS. In the case of complex
matrices, clean-up extract was directly re-extracted with ethylacetate
and acrylamide in organic phase was analysed by LC-MS/MS. The quantification
was done against the Internal Standard (Hoenicke, 2003).
Preparation of Sample
Damp groceries were minced in a meat grinder (Jupiter) and made homogeneous.
Dry groceries were crushed in a Retsch-mill and made homogeneous. Bread
and fine cakes and pastries have to be pre-dried carefully using Turbovap
500 concentrator. The spample material was mixed well and then filled
into a sample cup.
Processing of Sample
Depending on the groceries’ matrix a certain processing was chosen.
In the case of dry groceries containing starch (cornflakes, potato chips,
crisp-bread) and roasted coffee, two grams of the fine-grained or pulverized
sample was weighed in on a filter paper. The filter was directly set on
an aspiration apparatus to degrease the spample by arranging a slight
vacuum with altogether 80 mL iso-hexan. The residue wass then quantified
and transferred into a 150 mL beaker. After that it mixed with a 200 μL
ISTD-solution (c = 10 μg mL-1) and kept still for 30 min.
Then a portion of 20 mL of water was added and the spample suspension
was extracted at 60°C in an ultrasonic bath (Bandeline Sonderex Super
RK 510 H, Germany) for 30 min. Next 20 mL acetonitril and each of 500
μL Carrez I (150 g of potassium hexacyanoferrat/l) and Carrez II
(300 g of zinc sulphate/l) were adjoined, whereby the solution has to
be well-stirred after each adding up. Then, the aliquot was centrifuged
(Hettich EBA 85, Germany) at 4500 g for 10 min. The resulted supernatant
was membrane-filtered (Acrodisc 0.45 μm, Pall Gelman Lab., USA) in
an autosampler-vial.
In the case of samples containing high amount of fat (fats for frying), 40 mL iso-hexan were brought out on a 150 mL partition funnel. Two grams of spample were weighed in the partition funnel and finally dissolved. A portion of 20 mL water and 200 μL ISTD-solution (10 μg mL-1) were then added and sturdily shaken. After that 10 mL of the aqueous phase were transferred into a 150 mL beaker and then mixed with 10 mL acetonitril and each 500 μL Carrez I and Carrez II, whereby the solution has to be well-mixed after each adding up. Then centrifugation and filtration were followed as mentioned above.
In the case of Cacoa powder, 2 g of the fine-grained spample were weighed then mixed with 50 mL water as well as with 200 μL ISTD-solution (c = 10 μg mL-1). After that it was extracted in an ultrasonic bath at 60 °C for 30 min. The extracted was transferred into a 250 mL centrifuge cup and mixed with 30 mL iso-hexan as well as with each 500 μL Carrez I and Carrez II. After each addition of a substance the solution was well-shaken. Then, it was centrifuged for 10 min at 4500 g. The aqueous phase was taken up and transferred into a 150 mL partition funnel where was mixed with sodium chloride until the saturation point then it was well-shaken. The aqueous phase was shake out two times with 50 mL ethylacetate. The united organic phases were given over a filter (filled with sodium sulphate) in a turbo-wap-container, narrowed down and then membrane-filtered in an autosampler-vial.
Identification and Quantification
LC-MS/MS analyses were performed using Sciex® API 2000 mass spectrometer
system coupled with an Agilent 1100 HPLC equipped with tempered well plate
autosampler, a pump and a pillar stove (Applied Biosystemes, Darmstadt,
Germany).
For HPLC analysis of the extracts, 40 μL were injected onto LiChro CART 250-4 LiChrospher®100CN (5 μm) preceded by LiChro CART 4-4 LiChrospher®100 RP-pre-pillar-cartridges (5 μm) (Merck, KGaA, Darmstadt, Germany).
The columns were eluted with a mobile phase composed of A: acetonitrile/acetic acid, 1% (50/50 v/v) and B: acetonitrile, run at 0-5 min 100% A, 6-9 min 100% B and 10-20 min 100% A. The flow rate was 0.7 μL min-1 and the MS/MS transitions (m/z) monitored for acrylamide were 72 and 55, those for [D3] acrylamide (the internal standard) were 75 and 58. The quantification and calibration was based on the 72 > 55 and 75 > 58 mass passages/ mass transitions. The calculation equation was as follow:
where, Area AA is a peak-area at 72 > 55 transitions, Area [D3]
AA is a peak-area at 75 > 58 transitions and EW is a specimen net weight
(g).
Additionally this analysis was integrated within the scope of official
accredited and validated analyses in the Laboratory of Food Analysis.
Analytical Quality Assurance
Analytical quality assurance measures were employed for acrylamide,
which involved inclusion in duplicate of 2 g of rolled oats spiked at
5 μg kg-1 (ppb) and reagent blank. The same series of
samples was also spiked with labeled acrylamide. Batches of samples were
deemed acceptable if spiked samples (with labeled acrylamide) indicated > 80% recovery rate.
Food Consumption Data and AA Intake
Food consumption data were based on a quantitative frequency questionnaire
answered by 100 female subjects aged 18-25 years who recorded their food consumption
during a week. All estimated intakes were adjusted for the individual’s self-reported
body weight (bw = 60 kg; WHO, 1983) and expressed as daily. AA daily intake
calculated according to the following model:
Eaters (yes or no), either 0 or 1 in proportion to percent eaters; Food amount, food consumption value (g) from survey data; AA level, acrylamide value from laboratory analysis (μg kg-1). Each data value equally likely on each iteration. Results are summed over foods and individuals.
RESULTS AND DISCUSSION
The calibration curve was computed using the area ratio of the acrylamide (72-55 m/z) peak to that of the D3-acrylamide (75-58 m/z). The correlation coefficient was R2 = 0.9991 (Fig. 1) and the detection limit was found to be 10 μ kg-1 while the quantification limit was set at 30 μ kg-1. The recovery was in the range of 80-100% with reproducibility is higher than 5%. A sample chromatogram is given in Fig. 2, which shows an extract of potato chips with AA level up to 2100 μg kg-1.
In this study, food samples from nine food groups (11 sub-groups) were analyzed for their AA contents. It is important to note that present results cannot provide guidance in a consumer’s choice between food products and brands; however, it could be used as a general guide to assess the AA levels in a selected segment of the Saudi food supply.
| Fig. 1: | Caliberation curve of Acrylamide |
| Fig. 2: | LC/MS-MS chromatogram of potato chips containing
2100 μg kg-1 acrylamide and 2000 μg kg-1
[D3]-labelled acrylamide |
| Table 1: | Acrylamide (AA) levels in selected food and drink groups
in Saudi market compared with published survey data |
NI: No Information; 1Friedman (2003), 2USFDA
(2008), 3Svensson et al. (2003), 4Leung
et al. (2003), 5Hoenicke et al. (2004),
6Murkovic (2004), 7Şenyuva and Gõkmen
(2005), 8Claus et al. (2008) and 9Zhang
et al. (2007) |
|
The levels of AA varied considerably between single foodstuffs within food groups (Table 1). Regarding the mean AA levels, food and drink groups were in the order mashed roasted potato (8974 μg kg-1) > fried pasta; Shaerria (1600 μg kg-1) > soluble coffee (816 μg kg-1) > biscuit (810 μg kg-1) > potato chips (620 μg kg-1) > cocoa (526 μg kg-1) > crispbread (439 μg kg-1) > fried rice (430 μg kg-1) > roasted Turkish coffee (282 μg kg-1) > breakfast cereal (215 μg kg-1) > butter cookies (151 μg kg-1). It is postulated that higher AA concentrations up to above 1000 μg kg-1 were determined in the two food groups roasted potato and Shaerria, where the lowest concentration was observed in the cookies butter group. Moreover, soluble coffee contained a higher amount of AA compared with roasted Turkish one. Arabian traditional carbohydrate-rich foods may represent consumer concern since, deep frying of rice and pasta raised AA level considerably as compared to raw materials (< 30 μg kg-1).
It is postulated that the present results are in comparable with published data however, it showed a wide range of AA for the same group of food (Table 1). This high variability among the survey data is mainly resulted from the variable heating production processes (°C/time) and variable composition of raw materials. The coffee had the highest contribution to our AA dietary intake, especially the soluble type compared to Turkish roasted coffee meanwhile; steak biscuits had the same high contribution to AA dietary intake (Fig. 3).
| Fig. 3: | Dietary intake of Acrylamide from each analyzed food groups
|
The way of cooking could represent a health risk factor such as the case of fried pasta and rice further, present study confirmed the input of potato chips towards AA intake (Fig. 3).
The average estimated daily intake amounted to 34.03 μg AA/person/day which amounted to 40 μg/p/d in the case of adding coffee to the daily diet. This is corresponding to 0.57 μg AA/kg body weight/day (body weight of 70 kg).
The FAO/WHO assumes that the range of dietary intake of AA has a range of 0.3-0.8 μg kg-1 body weight/day in the developing countries.
It is concluded from the present study that the levels of acrylamide in different food groups are great varied which could be reflectd the differences in food composition (e.g., nature of carbohydrate; free amino acid content) and heat treatment (e.g., tempature, time, type of heat convection). Hence, it is strongly recommended that large-scale research studies regarding the levels of acrylamide in traditional and markting Gulf foods should be conducted to give a validating evaluation about acrylamide dietary intake. Further, look for innovating a technological processes able to reduce it. Meanwhile, for nutritional and preventive consideration it is suggested that risk groups (children and adolescents) should eat a balanced and varied diet. This includes high amount of vegetables and fruit with moderate consumption of cereals, fried rich-carbohydrate foods and coffee. Hence, AA presumably would be present in acceptable levels in nutritionally balanced diet however; levels in individual foods should be as low as reasonably achievable (FAO/WHO, 2002).
ACKNOWLEDGMENTS
We are most grateful to Applied Biosystem, Germany to help us in carrying over the determination of acrylamide.