J. Biochem. Biophys. Methods 69 (2006) 215 – 221 www.elsevier.com/locate/jbbm
Analysis of 3-aminopropionamide: A potential precursor of acrylamide Kristina Bagdonaite a,⁎, Gunilla Viklund b , Kerstin Skog b , Michael Murkovic a a
Institute for Food Chemistry and Technology, Graz University of Technology, Petersgasse 12/2, A-8010 Graz, Austria b Department of Food Technology, Engineering and Nutrition, Division of Applied Nutrition and Food Chemistry, Lund University, PO Box 124, SE-221 00 Lund, Sweden Received 13 October 2005; received in revised form 24 May 2006; accepted 26 May 2006
Abstract An analytical method for the analysis of 3-aminopropionamide (3-APA) based on derivatization with dansyl chloride and liquid chromatography/fluorescence detection was developed. We have analysed 3-APA formation in raw potatoes, grown and stored under different condition, green and roasted coffee beans and in freeze dried mixtures of asparagine with sucrose and glucose in molar ratio of 1:0.5, 1:1, and 1:1.5. In potatoes the 3-APA content varied depending on the potato variety. We detected 3-APA in potatoes up to 14 μg/g fresh weight. In the model experiment glucose had a stronger capacity to form 3-APA. The substance was formed at temperatures as low as 130 °C. However, in the model experiment with sucrose 3-APA was formed not below 150 °C. In heated mixtures with increasing molar ratio of sucrose at 170 °C we noticed a decrease of 3-APA and in the same mixtures at 150 °C we observed an increase of 3-APA. In coffee 3-APAwas not formed, neither in green nor in roasted beans. © 2006 Elsevier B.V. All rights reserved. Keywords: 3-Aminopropionamide; Dansyl chloride; Acrylamide; Coffee; Potato
1. Introduction Since acrylamide, a neurotoxin both to animals and humans and a probable carcinogen to humans substance [1], was recently detected in fried, roasted or baked food [2], several studies were carried out to investigate its formation pathway and ways of its reduction [3–5]. In recent studies, it was shown that for acrylamide formation, amino acids, especially asparagine, and ⁎ Corresponding author. Tel.: +43 316 873 6969; fax: +43 316 873 6971. E-mail address:
[email protected] (K. Bagdonaite). 0165-022X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbbm.2006.05.008
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reducing carbohydrates are needed [2,3]. Coffee attracts research as a significant source of dietary intake of acrylamide. It was reported that for northern countries (Norway, Sweden, and Denmark) coffee and potato products are the main foodstuffs for dietary acrylamide exposure [6,7]. 3-Aminopropionamide (3-APA) was recently suggested to be an intermediate in the formation of acrylamide in the Maillard reaction of asparagine with reducing carbohydrates [8]. 3-APA can also be formed in raw foodstuffs by enzymatic decarboxylation of asparagine. It is reported, that in the presence of 3-APA in food matrix acrylamide can be formed easily even under aqueous conditions during heating. In this pathway the Maillard reaction is not taking place; acrylamide can be formed even if there are no reducing sugars in the system. This can explain the fact, why acrylamide can be formed in raw materials containing low amounts of asparagine. In some studies for 3-APA detection LC-MS/MS [8], LC-MS techniques [9] were used. The purpose of our study was to develop an alternative method for analysis of 3-APA in foods. We decided to derivatize 3-APA with a fluorescence marker (dansyl chloride) [10,11] to increase the sensitivity and selectivity of the liquid chromatographic analysis. 2. Materials and methods 2.1. Chemicals and solvents Water was distilled twice and further purified using a water purification system (Simplicity, Millipore, Mohlsheim, France). Acetonitrile HPLC grade was purchased from Promochem (Wesel, Germany), 3-aminopropionamide hydrochloride 97% from ABCR (Karlsruhe, Germany). Other solvent and chemicals like HCl, dansyl chloride approx. 95% TLC were purchased from SigmaAldrich Chemie GmbH (Steinheim, Germany), and NaHCO3, L-asparagine anhydrous, D(+)glucose anhydrous, D(+)-sucrose, glycine, diethylether were from Fluka (Buchs, Switzerland). Potassium hexacyanoferrate(II) trihydrate and zinc acetate–dihydrate were purchased from Merck (Darmstadt, Germany). 2.2. Coffee For the analysis of 3-APA in green coffee beans three types of coffee were chosen: Indian Cherry AB Robusta (Coffea canephora, dry-processed), Liberia Robusta (Coffea canephora, dry-processed), Honduras s.h.g. Arabica (Coffea arabica, washed). For the roasting experiments Liberia Robusta (C. canephora, dry-processed), and Indian Plantation A Arabica (C. arabica, washed) were chosen. 2.3. Potatoes Potatoes of the variety Saturna grown by three different farmers under different conditions in Sweden were analysed in the experiments. After harvest (about 20 weeks) two of the samples have been stored at 8 °C, farmers 1 and 2, treated with a sprout inhibitor chloropropham, CIPC. The third sample from farmer 3 was stored at 4 °C. 2.4. Extraction of 3-APA from potatoes The potatoes (three tubers) were peeled and cut into small pieces, 150 g of them were taken to a beaker and 150 ml of purified water were added. The sample was homogenized in a laboratory
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homogenizer (Büchi Mixer B-400, Flawil, Switzerland). 5 ml of Carrez I K4[Fe(CN)6]·3H2O 150 g/l and 5 ml of Carez II Zn(CH3COO)2·2H2O 230 g/l solutions were added in order to precipitate proteins. The homogenate was centrifuged at 3500×g for 30 min (Hermle Z320, Wehingen, Germany). Thereafter the supernatant was centrifuged for a second time at 15 000×g for 10 min. 6 ml of the extract were transferred into 15-ml centrifuge tubes and 6 ml of 0.25 M NaHCO3 were added in order to adjust the pH to 8. 2.5. Coffee roasting 10.0 g of green beans were roasted in glass dishes in an oven at 150 to 240 °C for 5 to 15 min. Before roasting, the glass dishes were preheated for 10 min. After roasting the samples were immediately put on ice for 15 min and were ground in an analytical mill. The analysis was carried out directly after milling. 2.6. Coffee sample preparation for the derivatization with dansyl chloride Coffee beans were ground with an analytical mill (green coffee beans additionally milled with a ball mill in order to get a fine powder). 100 to 500 mg of the powder were balanced in 14-ml centrifuge tube and mixed with 7 ml of 0.1 M HCl. After 10 min of ultrasonic treatment and centrifugation at 3500×g for 30 min 5 ml of the supernatant were transferred to a 10-ml volumetric flask. The pH was adjusted to ∼8 with 0.25 M NaHCO3 and filled up to 10 ml with water and filtered. 2.7. Preparation of asparagine mixtures with sucrose and glucose Asparagine and sugars in molar ratio of 1:0.5, 1:1 or 1:1.5 were dissolved in 20 ml of purified water in a round flask. Samples were frozen (− 20 °C) overnight and freeze-dried to get a fine dry powder. Freeze-dried asparagine, sucrose or glucose mixtures were heated in 4-ml vials at 130, 150 and 170 °C for 7 min. After heating the samples were cooled for 40 s in the air (20 °C) and for an additional 15 min on ice. The heated samples were dissolved in 3 ml of 0.25 M NaHCO3 (pH ∼ 8). After the aliquots had been sonicated for 10 min (Transsonic T460, Singen, Germany, 35 kHz), 500 μl of the solution were centrifuged for 5 min, diluted if necessary 10 times and then derivatized with dansyl chloride. 2.8. Derivatization with dansyl chloride Our samples, in order to be able to detect them by using high performance liquid chromatography with fluorescence detection, were derivatized with dansyl chloride (5-[dimethylamino]naphthalene1-sulfonylchloride). The reaction scheme is shown in Fig. 1. The derivatization was done according to the method previously described by Moret et al. [12] with small modifications. 100 μl of the dansyl chloride solution (5 mg/ml in acetone) were added to 100 μl of sample in a test tube. The mixture was thoroughly mixed and left in the dark overnight. In order to eliminate excessive dansyl chloride, 20 μl of a glycine solution (100 mg/ml) were added after the reaction and left for another 15 min at ambient temperature. The sample was then extracted twice with 1 ml of diethyl ether. The combined extracts were dried under a stream of nitrogen (purity 5.0) and the residue was re-dissolved in 700 μl of acetonitrile.
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Fig. 1. Reaction scheme of 3-aminopropionamide into sulphonamide.
2.9. HPLC–FLD operating conditions For HPLC analysis 10 μl of the sample were injected into a HP 1100 liquid chromatograph (Hewlett Packard, Waldbron, Germany) with a fluorescence detector, equipped with cooling autosampler set at 6 °C, quaternary pump, degasser. The HPLC column MERCK Lichrosphere 60 Select B 120 × 4 mm ID, 5 μm particle size with a precolumn was used. Analysis was performed at ambient temperature. As a mobile phase 10% of acetonitrile and 90% water was used. The gradient elution programme was held to 30% of acetonitrile at 2 min, increased to 70% at 12 min and held until the end of the run (20 min) with a flow rate of 1 ml/min. Post time was set to 6 min. Dansyl chloride derivatives were detected with a fluorescence detector, set at λEx = 320 nm and λEm = 500 nm. Quantification was done by external calibration. Standard solutions of 3-APA in 0.25 M NaHCO3 (200 to 1000 ng/ml) were used. The limit of detection (LOD) was determined as 17 ng/ml software supported using Validata (version 1.0). In this program the calculation of the LOD is based on the standard deviation of the residues of the linear calibration. The limit of quantification (LOQ) was determined as 30 ng/ml, RSD of 3.2%. The typical chromatogram of the standard 3-APA (1000 ng/ml) and the sample (asparagine and sucrose mixture heated to 150 °C for 7 min) is given in Fig. 2. 3. Results and discussion It was recently reported, that 3-APA is a main precursor in acrylamide formation [4,9]. As a transient intermediate it is formed during thermal degradation of asparagine with carbohydrates, mainly glucose and fructose. It can also be formed in raw foods, where enzymatic reactions take place (under aqueous conditions, warm temperatures). Potato products, such as French fries, chips and crisps, are foodstuffs, wherein the highest amounts of acrylamide were detected [3,13,14]. Acrylamide is not formed when the food is heated to temperatures below 120 °C [15,16]. In the pathway of the formation of acrylamide Stadler et al. showed, that in presence of free asparagine and α-dicarbonyls 3-APA can be formed during the Maillard reaction [4]. However, 3-APA can also be formed enzymatically from asparagines by a simple decarboxylation. In raw potatoes, stored at temperatures above 0 °C 3-APA was detected as well [8]. In our experiments the results for potatoes from different farmers did not differ significantly (Table 1). According to Granvogl et al. [8] the amount of 3-APA should be higher in the potatoes stored at warmer temperatures. The data presented here do not support this conclusion. For this study potatoes were stored at rather low temperatures (8 and 4 °C). Due to similar storage conditions the amount of 3-APA in potatoes differ not a lot. In addition, the content of 3-APA can
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Fig. 2. Typical chromatogram of a 3-APA standard solution (1000 ng/ml) and a sample (asparagine mixture with sucrose heated to 150 °C for 7 min).
vary not only between the cultivars [8], but also, according to our experiments, between the growing conditions. However, the detected amount of 3-APA was more than ten times higher than the values reported by Granvogl et al. [8]. This could be due to the significant longer storage time (about 20 weeks) in our experiment in comparison to 5 and 12 days. In our studies 3-APA was not detected in raw coffee material. To our knowledge, a similar enzyme that decarboxylates asparagine to 3-APA is not reported for coffee. In roasted coffee we could not detect 3-APA either. Since 3-APA is formed in course of the Maillard reaction we expected this substance in roasted coffee as well. However, using different conditions for roasting (150–240 °C, 5 to 15 min) 3-APA could not be detected in any sample. This could be because the roasting conditions were not perfectly chosen or other reaction pathways predominate or the ratio of sucrose to asparagine occurring in coffee does not give 3-APA anyway. Some experiments on the formation of 3-APA in model systems were provided at low moisture conditions [4,9] as well as aqueous conditions [9]. In our experiment, when anhydrous mixtures of asparagine and sugars (sucrose and glucose) were heated at 130, 150 and 170 °C, glucose showed to have a stronger capacity to form 3-aminopropionamide (Fig. 3). In asparagine mixtures with glucose 3-APA was detected already at 130 °C. In the mixture with sucrose 3-APA was detected only at higher temperatures (150 °C). This could be explained by the fact that sucrose is not as strongly reacting as glucose in the Maillard reaction. As we know, for the formation of 3-APA reducing sugars such as glucose or fructose are needed [9]. Due to the feature, that sucrose, as a nonreducing sugar, cannot react with asparagine directly (first it needs to be heated to the temperature >150 °C to decompose to reactive carbonyl compounds) [17], in our studies 3-APA was not detected in the mixtures of asparagine and Table 1 Content of 3-APA in raw potatoes with standard deviation and relative standard deviation (n = 3) Potato sample
3-APA amount (μg/g)
RSD (%)
Farmer 1 Farmer 2 Farmer 3
9.5 ± 0.02 14 ± 0.1 11 ± 0.1
0.2 1.0 0.8
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Fig. 3. Formation of 3-aminopropionamide in mixtures of asparagine and sucrose (molar ratio 1:0.5, 1:1 and 1:1.5) as well as asparagine and glucose (molar ratio 1:0.5, 1:1 and 1:1.5) heated to 130, 150 and 170 °C.
sucrose, heated at temperatures below 150 °C. Furthermore, in heated mixtures with increasing molar ratio of sucrose at 170 °C we noticed a decrease of 3-APA. But in the same mixtures heated to 150 °C we observed an increase of 3-APA. We observed a high 3-APA formation in the asparagine mixtures with glucose heated to 150 °C. The molar ratio of carbohydrates to amino acid in the samples did not have much influence for 3-APA formation. 4. Conclusions Knowing the way of formation of acrylamide and its precursors can lead to developing the ways of its reduction and decreasing the toxicity. In our model experiments the highest amount of 3-APA was detected at 150 °C independent on the molar ratio between asparagine and glucose. The fact that we could not detect 3-APA in coffee (neither green nor roasted) can lead to a discussion about other possible pathways of acrylamide formation in coffee. 3-APA is formed in potatoes due to favoured enzymatic reactions and it seems that the amount of this acrylamide precursor depends on storage conditions and time, as well as the growing conditions. Acknowledgements This study was financed by Commission of the European Communities, specific RTD programme “Food Quality and Safety”, FOOD-CT-2003-506820, “Heat-generated food toxicants – Identification, characterisation and risk minimisation”. It does not necessarily reflect its views and in no way anticipates the Commission's future policy in this area. References [1] IARC. Some industrial chemicals. Lyon, France: International Agency for Research of Cancer; 1994. [2] Friedman M. Chemistry, biochemistry, and safety of acrylamide. A review. J Agric Food Chem 2003;51:4504–26.
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