Acrylamide in Processed Foods

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are primarily two approaches for the analysis of acrylamide in food, based on gas ... According to the literature, neurotoxicity appears to be the only documented ...

Bulgarian Journal ofChemistry Volume 1 Issue 3 Article history: Received: 2 July 2012 Revised: 11 October 2012 Accepted: 11 October 2012 Available online: 12 October 2012

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Acrylamide in Processed Foods Valentina L. Christova-Bagdassarian*, Julieta A. Tishkova, Terry M. Vrabcheva National Center ofPublic Health and Analysis 15, Akad. Iv. Ev. Geshov 1431 Sofia, Bulgaria * Corresponding author. E-mail: [email protected]

The purpose of this paper is to briefly summarize the sources human expose to acrylamide, the reasons for its formation, toxicological problems associated with its occurrence, analytical methods for detection ofacrylamide, and ranges ofthe detected levels in various food products. Acrylamide can be industrially produced or formed in processed food by a Millard type of reaction between sugar molecules and the amino acid asparagine. The human exposure of acrylamide can come from both external sources and the diet. The content of acrylamide was significant in products regularly consumed in a range of quantities such as potato chips, French fries, bread, breakfast cereals, crackers, roasted coffee, and coffee substitutes. Accurate and sensitive analytical methods have been developed to obtain reliable quantitative data in various foodstuffs. A briefoverview ofthe analytical methods is described in this paper. There are primarily two approaches for the analysis of acrylamide in food, based on gas chromatography mass spectroscopy - GC-MS either with or without derivatization, and on liquid chromatography-tandem mass spectroscopy (HPLC-MS-MS). The various steps for sample preparation and clean-up are described. New methods have been developed recently for the determination of acrylamide, using modern techniques and providing an opportunity to analyze the amounts to 0.1 μg/kg. The analytical methods allow receiving a big set of data from many countries and assessing an exposure through diet. Studies of acrylamide help to make recommendations for lowering its levels in foods.

Dr. Valentina Bagdassarian is a head assistant professor at the department of“Food Contact Materials”, National Centre ofPublic Health and Analysis. She has greduated in “Technical Cybernetics in Chemical Industry” at the University of Chemical Technology and Metallurgy - Sofia and Applied mathematics in Sofia Technical University. She has obtained PhD in Moscow, Russia, in the field ofwater pollutions. She has over 50 articles in different areas - toxic gases and dust in the workplace air, analysis of pharmaceutical products, determination of chemical additives and contaminants in food. She shows a special interest in the field of pesticide analysis, risk assessment, plant protectionproducts registration. She is a member ofthe team that according to the requests ofthe Europian comission carried out the first Bulgarian national monitoring researches for acrylamide and for monochloropropanodiols in foods. She teaches courses and aftergraduate training for chemics and doctors.

Keywords: Acrylamide; analytical methods; foods; GC-MS; HPLC-MS

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Bulg. J. Chem. 1 ( 2012) 123-132

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1. Introduction

Acrylamide (CH2=CH–CONH2), an industrially produced α,β-unsaturated (conjugated) reactive molecule, is used worldwide to synthesize polyacrylamide. Polyacrylamide has found numerous applications as a soil conditioner, in wastewater treatment, in cosmetics, paper and textile industries, and in the laboratory as a solid support for the separation of proteins by electrophoresis. The effects of acrylamide on cells, tissues, animals, and humans have been extensively studied. The reports, that acrilamide is formed in foods during their processing by Millard reaction [1], are of interest for chemistry, biochemistry, and food safety. The human exposure of acrylamide can come from both external sources and the diet. A better understanding of its formation and distribution in food and its role in human health is needed. Therefore, an integrated review on the present data on the chemistry, analysis, metabolism, and toxicology of acrylamide is very useful. According to the literature, neurotoxicity appears to be the only documented effect of acrylamide in human epidemiological studies; reproductive toxicity, genotoxicity/clastogenicity, and carcinogenicity are potential human health risks on the basis of only animal studies. A better understanding of the pure acrylamide in general and its impact in a food matrix in particular can lead to the development of improved food processes to decrease the acrylamide content ofthe diet [2]. A brief overview of the analytical methods is described in this paper. 2. Physical and chemical properties, sources of acrylamide

Acrylamide (or acrylic amide) is a chemical compound with the chemical formula C 3H5NO. Its IUPAC name is prop-2-enamide. It is a white odourless crystalline solid substance, soluble in water, ethanol, diethyl ether, and chloroform. Acrylamide is incompatible with acids, bases, oxidizing agents, iron, and iron salts. It decomposes to form ammonia аt ambient conditions. Thermal decomposition produces carbon monoxide, carbon dioxide, and nitrogen oxides [3]. Acrylamide is often used for the synthesis of polyacrylamides, commonly used as water-soluble thickeners. Acrylamide is known to cause cancer in animals and previous concerns have been focused on its use in occupational environments. Acrylamide can also 124 Bulg. J. Chem. 1 ( 2012) 123-132

be associated with cigarette smoking [4], occurs in many cooked starchy foods, and is of concern as a possible carcinogen [5]. 3. Absorption, distribution, metabolism and excretion

After oral administration, acrylamide is rapidly absorbed and widely distributed in all species that have been investigated (rats, mice, dogs, and miniature pigs). An autoradiography study in mice showed accumulation of acrylamide or its metabolites in the reproductive organs ofmales as well as rapid and extensive distribution to the developing fetuses in pregnant females. The compound was also excreted in the milk of lactating rats. The binding of acrylamide or its metabolites to RNA, DNA and proteins (such as haemoglobin) occurs in a range of tissues. Studies in rats have shown that direct conjugation of acrylamide and glutathione is a major route ofmetabolism. Evidence for glycidamide formation in humans was obtained from samples of haemoglobin taken from workers exposed to high levels of acrylamide [6]. Whereas both acrylamide and glycidamide form adducts with haemoglobin, only glycidamide forms DNA adducts in mice and rats. The levels of the glycidamideDNA adduct were similar in the different organs of rats and mice, showing that glycidamide is evenly distributed in the tissues like acrylamide [7]. The excretion of the parent compound and its metabolites is rapid and extensive, mostly via urine, with smaller amounts eliminated via faeces and exhaled CO 2. Acrylamide is toxic at high doses by oral route of administration. LD 50 values are in the range of107-203 mg/kg bw in rats. 4. Effects on human health

Acrylamide is a potential genetic and reproductive toxicant with mutagenic and carcinogenic properties [8]. In early 2002 a Swedish group reported that carbohydrate-rich foodstuffs processed or cooked at high temperatures contain relatively high levels of acrylamide [5,9]. These findings attracted considerable public and scientific attention worldwide as the study established that popular foodstuffs like potato chips, French fries, and bread, can contain substantial amount of this possibly carcinogenic substance. Since then, several government bodies and international organizations have initiated programs for investigation of acrylamide in foodstuffs. Meanwhile, many food processors have taken action to

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ofa Millard type ofreaction between sugar molecules and the amino acid, asparagine [1]. Figure 1. Formation ofacrylamide by the Maillard reaction [1].

Distribution and possible health effects of acrylamide is a concern among scientists, researchers, consumers and the public worldwide. As a result of collaboration between scientists and nutrition experts, existing data on the potential health effects ofacrylamide in food has been evaluated. Assessment of risk for people has been provided using the risk assessment approach [16], known as the margin of exposure (MOE). MOE is calculated by dividing the toxicity estimated from animal experiments by the estimated intake from food. The lower the MOE the greater the public health concern. The most sensitive carcinogenicity estimate from animal studies was 0.3 mg/kg bw/day. Human intake values of 0.001 mg and 0.004 mg acrylamide/kg bw represented intakes by the general population and high consumers, respectively. The MOE for the general population is calculated as 300, while the MOE for high consumers is 75. The assumed average acrylamide intake for the general population may be 1µg/kg bw/day, while high consumers may be 4 µg/kg bw/day. While there exists no generally accepted health based guidance value for acrylamide in foods, the WHO guidelines for drinking water quality is 0.5 µg/L; EU guideline is 0.1 µg/L; US regulations is based on treatment techniques, rather than a water quality standard for acrylamide [16]. Overall, the studies have confirmed that glycoconjugates of asparagine are the major source of acrylamide in foods under low moisture conditions and elevated temperatures in the presence of reducing sugars or a suitable carbonyl source. Moisture content and heat load (reaction time versus temperature) may be suitable parameters to minimize acrylamide formation. [17]. Acrylamide has been found in black olives [18], dried prunes [19] and dried pears [19]. Estimates for the proportion of acrylamide in adults’ diet coming from the consumption of coffee range from twenty to forty percent; prune juice has a high concentration of acrylamide though adults consume it in far smaller

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Bulg. J. Chem. 1 ( 2012) 123-132

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improve food-processing methods in order to reduce the level ofacrylamide occurring in their products. An EFSA statement in 2005 noted that there may be a potential health concern with the carcinogenic and genotoxic acrylamide. The statement endorsed the conclusions and recommendations of a previous risk assessment on acrylamide carried out by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). In this assessment JECFA concluded that acrylamide may indicate a human health concern and that efforts should be made to reduce exposure [10]. In 2009 Health Canada reported that they were assessing whether acrylamide which occurs naturally during the cooking of French fries, potato chips, and other processed foods is a hazard to human health and whether any regulatory actions need to be taken. They are currently collecting information on the properties and prevalence of acrylamide in order to make their assessment [11]. In March 2010 the European Chemical Agency added acrylamide to the list of substances of very high concern [12]. Following a recommendation from the European Commission in 2007, Member States were requested to perform a three-year monitoring of acrylamide levels and submit data to EFSA. In 2010 the Commission recommended that Member States should continue annual monitoring. In 2011 it was recommended that Member States carry out investigations in cases where the levels of acrylamide in food exceed the prescribed indicative values. The Commission will re-assess the situation by December 2012. [13]. In 2011 EFSA published its third report on acrylamide which compared data submitted in 2009 with previous data from 2007 and 2008 [10]. The report includes an exposure assessment to estimate the intake of acrylamide for different age groups as well as the major contributors to acrylamide exposure in the diets of consumers in Europe. Exposure estimations for the different age groups were comparable with those previously reported for European countries. Bulgaria took part in monitoring studies at 2008 and 2010 by sending national data for the contamination of acrylamide in 10 groups of products from the Bulgarian market [14,15]. Acrylamide levels in food are increased by prolonged heat treatment. Although the exact mechanism is not fully understood, the most common assumption is that acrylamide is formed as a product of the Maillard reaction. It is believed that the high levels of acrylamide are formed when potatoes and cereal products are fried, baked, toasted at high temperatures (> 120°C) because

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quantities [20]. Some of the major foods identified by JECFA as containing acrylamide [16] are: potato crisps (6-46% of samples), coffee (13-39%), French fries (16-30%), bread and toasts (10-30%), pastries and sweet biscuits (1020%), and other food items (< 10%). 5. Toxicological End Points

Toxicological reference values for acrylamide are defined in the studies of exposure to pure substance. Exposure to large doses of acrylamide can cause damage to the male reproductive glands. Direct exposure to pure acrylamide by inhalation, skin absorption, or eye contact irritates the exposed mucous membranes, as the nose, and can also cause sweating, urinary incontinence, nausea, myalgia, speech disorders, numbness, paresthesia, and weakened legs and hands. In addition, the acrylamide monomer is a potent neurotoxin, causing the disassembly or rearrangement of intermediate filaments [21,22]. Ingested acrylamide is metabolised to a chemically reactive epoxide, glycidamide. In June 2002, the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) issued a report about the health implications of acrylamide in food. After study, the Consultation concluded that the "no observed adverse effect level" (NOAEL) for acrylamide neuropathy is 0.5 mg/kg bw/day and the NOAEL for fertility changes is four times higher than for peripheral neuropathy. The estimated average chronic human dietary intake is in the order of 1 μg/kg bw/day. This provides a margin between exposure and the NOAEL of500 [23,24]. The WHO and FAO established that the safe limit of 0.5 mg/kg bw/day pertains only to neuropathy. There has not been an established safe dietary limit of acrylamide as it pertains to causing cancer, since there is limited relative data. Hence, a woman weighing 132 pounds (60 kg) could safely consume 30 mg of acrylamide daily without neuropathy; a man weighing 180 pounds (82 kg), about 41 mg; a child weighing 40 pounds (18 kg), 9 mg [24,25,26]. According to DiNovi and Howard, 2004 [20], in a single day, а child can eat 13 kg (29 lb) of French fried potatoes, а woman can drink 86 kg (~86 L, or 23 US gal) of prune juice, and а man can eat 29 kg (64 lb) of ovenbaked potatoes, and each of them will have ingested less than 50% ofthe NOAEL ofacrylamide.

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One study reanalysed a population-based Swedish case-control study encompassing cases with cancer of the large bowel, bladder, and kidney, and 538 healthy controls [27]. Researchers assessed the impact of dietary acrylamide by linking extensive food frequency data with acrylamide levels in certain food items recorded by the Swedish National Food Administration. Unconditional logistic regression was used to estimate odds ratios, adjusting for potential confounders. They found consistently a lack of an excess risk, or any convincing trend, of cancer of the bowel, bladder, or kidney in high consumers of 14 different food items with a high (range 300–1200 µg/kg) or moderate (range 30–299 µg/kg) acrylamide content. Also, Unexpectedly, an inverse trend was found for large bowel cancer (P for trend 0.01) with a 40% reduced risk in the highest compared to lowest quartile. It was found that dietary exposure to acrylamide in amounts typically ingested by Swedish adults in certain foods has no measurable impact on risk of three major types of cancer. However, that relation of risk to the acrylamide content of all foods could not be studied. [27]. 6. Analytical methods

Because the content of acrylamide was significant in products regularly consumed in a range ofquantities such as potato chips, French fries, bread, breakfast cereals, crackers, roasted coffee, and coffee substitutes, accurate and sensitive analytical methods are needed to obtain reliable quantitative data in various foodstuffs. Methods published prior to 2002 did not suffice the analysis of acrylamide in processed foods at low levels because of their complexity ofmatrices, insufficiency ofselectivity or design which was intended for other objects (water, soil, textile materials et etc.) [28-30]. Numerous methods were developed in the past years to determine the acrylamide monomer, especially in water, biological fluids, and foods (sugar, field crops, mushrooms). Most of the methods used in the past were TLC and GC [3134]. However, the modern analytical methods have to comply with European criteria for efficiency and to generate reliable data that can be used for intake assessment. There are primarily two approaches for analysis of acrylamide in food, based either on gas chromatography mass spectroscopy – (GC-MS) with or without derivatization [35-39] or on the tandem liquid chromatography – mass spectroscopy (HPLC-MS-MS) [9,40].

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6.1. GC-MS

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The GC-MS techniques are either based on bromination of the analyte or on direct analysis without derivatization. A direct GC-MS analysis can be accompanied by several difficulties which, include a rather nonselective MS spectrum (due to the low molecular weight) and a potential formation of acrylamide in the injection port from the coextracted precursors (asparagine and reducing sugars) if present in the final extract. Also, the high solubility of acrylamide in water in comparison to organic solvents complicates sample preparation for GC [41]. The latter approach is less laborious and in both reported cases employs liquid–liquid extraction of the analyte. In the method reported by Biedermann et al. [42], the determinative step is either positive ion chemical ionization in the selected ion monitoring (SIM) mode or electron impact ionization achieving a level of detection (LOD) of around 50 and

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