Acetaldehyde (ethanal) is a genotoxic carcinogen, which may occur naturally or as an added flavour in foods. We have developed an efficient method to analyze the compound in a wide variety of food matrices. The analysis is conducted using headspace (HS) gas chromatography (GC) with flame ionization detector. Using a robot autosampler, the samples are digested in full automation with simulated gastric fluid (1 h at 37°C) under shaking, which frees acetaldehyde loosely bound to matrix compounds. Afterwards, an aliquot of the HS is injected into the GC system. Standard addition was applied for quantification to compensate for matrix effects. The precision of the method was sufficient (<3% coefficient of variation). The limit of detection was 0.01 mg/L and the limit of quantification was 0.04 mg/L. 140 authentic samples were analyzed. The acetaldehyde content in apples was
Acetaldehyde (ethanal) is carcinogenic in animal experiments [
In foods, acetaldehyde may occur either naturally or because of intentional addition as flavour compound [
The Netherlands Organization for Applied Scientific Research (TNO) database “volatile compounds in foods (VCF)” lists numerous studies on the occurrence of acetaldehyde but most of these were from the 1980s or earlier [
The most simplistic procedure for acetaldehyde analysis is the direct injection of a sample solution into a gas chromatograph with flame ionization detection (FID). Such a procedure can be used for analysis of alcoholic beverages without any further sample preparation and is also included in the EU reference methods for the analysis of spirits [
Prior to chromatographic measurement of acetaldehyde, derivatization using 2,4-dinitrophenylhydrazine was suggested, and the formed hydrazone can be measured using gas chromatography (GC) or high-performance liquid chromatography (HPLC) [
We have set focus on providing a sample preparation without losses as well as an improved acid digestion that simulates physiological conditions of the human stomach and therefore allows to estimate the exposure after oral consumption of foods.
Acetaldehyde (>99.5%) was purchased from Fluka. Sodium chloride was from Riedel-de-Haen, and pepsin from porcine gastric mucosa (800–2500 U/mg protein) was from Sigma-Aldrich. Hydrochloric acid (37%) and ethanol (>99.9%) were obtained from Merck. The simulated gastric fluid (SGF) was prepared according to USP 32 [
The sample types were selected according to risk oriented principles [
Liquid and semisolid foods were homogenized by shaking or stirring with a spoon. Dependent on consistency, the samples were weighed with help of a 20 mL disposable plastic syringe or using an Eppendorf pipette, with a tip that was cut off with scissors to facilitate the pipetting of semisolid samples. For quantification with standard addition, five aliquots (in the range of 1.2–2.0 g) of the sample were weighed with an accuracy of 10 mg into 20 mL headspace vials. After addition of 1.25 mL of SGF and the required acetaldehyde spiking, distilled water up to a total volume of 5 mL was added. The total time for sample preparation was 10–15 min per sample.
Solid foods were homogenized in a standard household mixer (Magic Maxx, ds-produkte GmbH, Gallin). For fruits and vegetables only the edible parts were used (e.g., bananas and oranges were peeled prior to homogenization). The homogenized samples were weighed similar to the liquid foods described above. Only completely dry or highly viscous samples were weighed using a spatula. The total time for sample preparation of the solid foods was 15–25 min per sample. The prepared headspace vials were stored at 5–8°C and generally analyzed on the same day, but never later than on the next day after preparation.
The HS-GC-FID system used for analysis was an Agilent model 6890N gas chromatograph in combination with a CTC Combi PAL autosampler. To simulate the physiological conditions inside the stomach, the samples were incubated for 60 min at 37°C under constant stirring in the oven of the autosampler. After that, 500
The limits of detection and quantification were determined according to German norm 32645 [
The precision (expressed as coefficient as variation) can be directly calculated for each sample from the calibration curve resulting from standard addition (5 aliquots measured per sample). Furthermore, we have measured one yoghurt sample several times with different amounts of sample weight (1.2, 1.4, and 1.6 g). The storage stability was evaluated by preparing three standard addition series of the same yoghurt sample and measuring them after 2, 9, and 16 days after preparation (the prepared headspace vials were stored at 5–8°C in the meantime).
To test for artefactual formation of acetaldehyde from ethanol during sample preparation or analysis, two standard addition series of an apple sample were prepared with and without addition of ethanol (250
Possible losses during sample preparation were tested as follows: (1) 50 mL of an acetaldehyde stock solution in a 100 mL measuring flask was left to stand open (i.e., without stopper on the flask) for 65 min in the 20°C water bath (normally, the flask are directly sealed after the pipetting of the standard, of course). (2) 100 mL of standard solution were filled into the mixer used for homogenization and mixed for 20 s similar to the samples.
During initial method development, it was noted that buttermilk did not contain any detectable acetaldehyde in the headspace if aqueous samples are analyzed. After addition of SGF, considerable amounts of acetaldehyde were found, however. To research the influence of SGF on the matrix, light microscopy was conducted (Axiostar plus, camera: AxioCam ICC1, Carl Zeiss GmbH, Oberkochen). Acetaldehyde was coloured using Schiff reagent (Merck).
Preliminary experiments had shown that the differences in matrix composition have massive influences on the recovery of acetaldehyde in the headspace. For this reason, external calibration with aqueous standards is not possible. Due to the diversity of matrices we wanted to analyze, it would also not have been possible to conduct calibration in matrix, with the additional problem of finding acetaldehyde-free matrices for spiking. It was also not possible to find a suitable internal standard with similar behaviour to acetaldehyde, and the use of mass spectrometry with the possibility to use isotopically labelled acetaldehyde was not possible for instrumental restrictions and cost reasons. For all these reasons, we decided to use standard addition according to the German norm 32633:1998 [
The basic calibration using external standards showed an acceptable linearity and precision in the working range (
Regression curve of the basic calibration including the determination of the detection limit in the lower range.
Examples of calibration in different matrices are shown in Figure
Standard addition curves of four selected samples compared to basic calibration.
Reduced response due to matrix increase demonstrated by a spiked apple sample.
With the exception of a single sample of roast coffee powder (
During the storage stability experiment, no significant difference in the results was seen between the yoghurt samples stored for 2, 9, or 16 days. The overall mean was 16.91 mg/kg (standard deviation 0.49 mg/kg, CV 2.90%). No artefactual formation of acetaldehyde was detected in the apple sample series with spiked ethanol (
Regarding the losses during sample preparation, the highest influence had the storage of the stock solution without stopper (4% loss of acetaldehyde during 65 min), while during homogenization only a minor loss of 2% occurred. The samples were stored in a fridge and the temperature in the samples during mixing was increased by a maximum of 7°C (in the case of a cheddar cheese). We assume that no massive losses of acetaldehyde occur (boiling point 20.1–20.8°C). The loss during sample preparation of solid matrices in the mixer is therefore deemed as acceptable, but unavoidable.
Our validation results show that the method has an acceptable performance for the use of analyzing food matrices. Due to the volatility of acetaldehyde, careful handling of the stock solutions is required to avoid losses.
The regression curves for different spiking levels of acetaldehyde in buttermilk are shown in Figure
Standard addition curves of a buttermilk sample measured with and without simulated gastric fluid (SGF).
Microscopic analysis of buttermilk samples after colouration with Schiff reagent.
The results from 140 samples are presented in Table
Results of acetaldehyde analyses in selected foods.
Food | Acetaldehyde content (mg/kg) | CV (%) |
---|---|---|
Ayran A (Turkish milk product) | 5.79 | 1.35 |
Ayran B (Turkish milk product) | 6.51 | 1.42 |
Ayran C (Turkish milk product) | 9.79 | 1.78 |
Buttermilk | 0.01 | 0.52 |
Crème fraiche | 1.78 | 1.21 |
Yoghurt with fruits (banana, granadilla, low-fat yoghurt) | 4.40 | 1.25 |
Yoghurt with fruits (apple, vanilla, low-fat yoghurt) | 3.35 | 0.54 |
Fruit-yoghurt (strawberry) | 2.77 | 1.03 |
Fruit-yoghurt (Raspberries, red-currant, low-fat yogurt) | 5.62 | 1.57 |
Yoghurt A 1 (low-fat yoghurt) | 17.42 | 0.24 |
Yoghurt A 2 (low-fat yoghurt) | 16.44 | 2.34 |
Yoghurt A 3 (low-fat yoghurt) | 16.89 | 1.46 |
Yoghurt B 1 | 6.05 | 1.61 |
Yoghurt B 2 | 6.15 | 1.81 |
Yoghurt B 3 | 6.36 | 1.42 |
Yoghurt C | 8.38 | 0.73 |
Yoghurt D | 12.35 | 0.10 |
Yoghurt E | 9.66 | 0.28 |
Yoghurt F 1 | 13.77 | 0.53 |
Yoghurt F 2 | 12.75 | 1.26 |
Yoghurt mild A (low-fat yoghurt) | 12.61 | 1.25 |
Yoghurt mild A | 8.48 | 1.55 |
Yoghurt mild B (low-fat yoghurt) | 7.27 | 1.07 |
Yoghurt mild D (low-fat yoghurt) | 9.43 | 1.32 |
Yoghurt mild A | 13.61 | 1.40 |
Yoghurt mild F (goat milk) | 2.40 | 0.76 |
Yoghurt mild G (sheep milk) | 11.54 | 0.76 |
Yoghurt mild H (sheep milk) | 11.07 | 0.19 |
Kefir mild A | 1.48 | 0.29 |
Kefir mild B | 0.01 | 0.18 |
Sour milk A | 1.19 | 1.52 |
Sour milk | 0.19 | 0.14 |
Sour cream A | 0.47 | 0.31 |
Sour cream B | 4.26 | 0.30 |
Sour cream C | 6.28 | 1.43 |
Cheddar cheese | 0.22 | 0.82 |
Fresh cheese A | 0.68 | 0.39 |
Fresh cheese B | 1.06 | 0.90 |
Gouda cheese | 0.16 | 0.22 |
Quark, fresh cheese (low fat) | 1.81 | 0.07 |
Quark, fresh cheese (20%) | 1.07 | 0.91 |
Quark C, fresh cheese (low fat) | 0.12 | 0.94 |
Quark D, fresh cheese (low fat) | 2.05 | 1.70 |
Pineapple | 0.63 | 1.09 |
Apple A (Elstar) | 1.81 | 0.73 |
Apple B (Pink Lady) | 0.32 | 0.06 |
Apple C (Jonagold) | 0.57 | 0.26 |
Apple D (Boskoop) | 0.40 | 0.29 |
Apple E (Tenroy Gala) | 0.52 | 0.89 |
Apple F.1 (Golden Delicious) | 2.39 | 1.06 |
Apple F.2 (Golden Delicious) | 2.35 | 1.01 |
Apple G (Granny Smith) | 0.76 | 1.77 |
Apricots | 1.57 | 1.07 |
Banana A.1 | 10.13 | 1.91 |
Banana A.2 | 16.36 | 2.36 |
Banana A.3 | 14.39 | 1.95 |
Banana A.4 | 18.27 | 2.33 |
Banana B | 2.21 | 1.13 |
Banana C | 14.78 | 2.33 |
Banana D | 1.88 | 0.43 |
Banana E | 7.52 | 1.07 |
Pear | 3.74 | 0.74 |
Strawberry | 1.29 | 1.57 |
Grapefruit | 3.23 | 0.30 |
Bilberries | 2.11 | 0.85 |
Kiwi fruit A | 0.73 | 1.30 |
Kiwi fruit B | 0.81 | 0.75 |
Mandarin | 0.78 | 0.95 |
Mango | 1.19 | 0.76 |
Orange A | 5.56 | 0.12 |
Orange B | 8.37 | 0.22 |
Papaya | 0.83 | 0.83 |
Grapes (red) | 0.91 | 1.78 |
Lemon | 3.92 | 1.93 |
Apple puree | 0.41 | 1.91 |
Fruit preparation with apples and bananas | 0.37 | 1.16 |
Fruit preparation with bananas and yoghurt | 1.41 | 0.32 |
Fruit preparation with pears | 1.17 | 0.31 |
Mandarins (canned) | 3.13 | 0.02 |
Banana chips (roasted) | 0.98 | 1.11 |
Cucumber | 1.56 | 0.46 |
Carrot | 1.91 | 1.81 |
Garlic | 5.60 | 1.22 |
Cabbage turnip | 2.88 | 1.10 |
Capsicum (yellow) | 0.17 | 2.48 |
Capsicum (red) | 0.10 | 1.19 |
Beetroot | 0.15 | 0.47 |
Tomato | 0.05 | 1.81 |
Onion | 1.06 | 0.03 |
Pickled gherkin | 2.61 | 1.25 |
Sweet corn (canned) | 1.29 | 0.45 |
Sauerkraut (canned) | 2.37 | 1.42 |
Asparagus (canned) | 0.40 | 1.74 |
Carrots (canned) | 1.60 | 1.17 |
Peas (canned) | 4.49 | 2.61 |
Fresh beans (canned) A | 1.01 | 0.75 |
Fresh beans (canned) B | 1.01 | 1.50 |
Lentils (canned) | 0.10 | 0.57 |
Strawberry jam | 0.26 | 0.31 |
Plum puree | 0.97 | 1.84 |
Honey | 1.01 | 0.60 |
Wheat and rye bread | 1.50 | 1.63 |
Rye whole-meal bread with pumpkinseed | 2.68 | 0.62 |
Vinegar | 2.61 | 1.35 |
Mustard | 0.15 | 0.65 |
Lemon flavour for baking | 26.32 | 1.27 |
Orange flavour | 1416 | 0.83 |
Pineapple juice (direct juice) | 0.01 | 0.85 |
Apple juice (direct juice) | 5.72 | 0.60 |
Banana nectar A | 0.26 | 0.95 |
Banana nectar B | 0.45 | 1.25 |
Peach nectar | 0.52 | 1.51 |
Orange juice (from concentrate) | 1.83 | 0.92 |
Orange juice (direct juice) | 5.89 | 0.94 |
Smoothie strawberry banana | 3.06 | 1.58 |
Grape juice (direct juice) | 0.97 | 1.85 |
Ice tea (peach flavour) | 4.32 | 1.05 |
Energy drink A | 1.08 | 1.43 |
Energy drink B | 0.06 | 0.82 |
Energy drink C | 0.36 | 0.27 |
Soft drink (with fermented cranberry) | 3.49 | 0.92 |
Soft drink (with fermented quince) | 0.32 | 0.75 |
Soft drink (with fermented herbs) | 0.33 | 1.16 |
Cola | 0.28 | 0.66 |
Apple soft drink | 7.54 | 1.07 |
Cherry soft drink | 0.93 | 1.68 |
Orange soft drink A | 16.30 | 2.28 |
Orange soft drink B | 14.01 | 0.25 |
Wild beery soft drink | 2.39 | 2.18 |
Carrot juice (fermented) | 1.14 | 0.67 |
Carrot juice (direct juice) | 2.49 | 0.82 |
Tomato juice (from concentrate) | 0.15 | 1.37 |
Instant coffee A (powder) | 35.51 | 0.44 |
Instant coffee A (2 g per 180 mL) | 0.26 | 0.35 |
Instant coffee B (powder) | 31.20 | 1.70 |
Coffee, roasted A (powder) | 40.14 | 3.28 |
Coffee, roasted A (powder) | 1.15 | 1.19 |
Coffee, roasted B (powder) | 36.26 | 2.49 |
Earl Grey tea (leaves) | 9.84 | 0.99 |
Green tea (leaves) | 1.35 | 0.34 |
GC/FID chromatogram of a yoghurt sample (8.4 mg/kg acetaldehyde).
In milk products, a correlation between acetaldehyde and fat content was not detectable. Goat milk products had lower acetaldehyde contents than cow milk products. This can be explained by its higher glycin concentration, which acts as inhibitor of threonine-aldolase, which may produce acetaldehyde from threonine [
In fruits, the highest acetaldehyde contents were found in bananas and in citrus fruits. Some apple varieties (Granny Smith, Elstar) showed higher contents than the other varieties, but the number of samples analyzed does not allow any conclusions on influence of variety. There could also be an influence of other factors not controlled, for example, environment during storage, climate, country of origin, and so forth. It would be interesting, however, to further investigate if certain varieties of apples are especially susceptible for acetaldehyde content. While all fruits were generally analyzed in fresh state, we made an experiment with bananas and followed the acetaldehyde content during ripening (Figure
Changes in acetaldehyde content of bananas during ripening.
The fruit juices had in general less acetaldehyde than the corresponding fresh fruits. Causative could be on the one hand losses of the volatile compound during pressing or concentration of the juice, as well as a dilution effect in products with less than 100% fruit content. The content in direct juices was for the same reason higher than in juices from concentrate. This is consistent with previous observations [
Strikingly high acetaldehyde contents were detected in lemonades or soft drinks that only contain low amounts of fruit juice (apple drink 7.5 mg/kg, orange soft drink 15 mg/kg). In view of the results found naturally in the fruits, these contents can only be explained if acetaldehyde has been added as flavour compound, which is consistent with the labelling of the products (“flavour” was given in the ingredients list).
Compared to the literature, our survey results were generally consistent compared to the previous data. The exception are the results of Lund et al. [
The Joint FAO/WHO Expert Committee on Food Additives (JECFA) [
From the acetaldehyde content found in our survey for each food group and the estimated intake of each group for a selected population, the acetaldehyde exposure can be estimated. Regarding the exposure to acetaldehyde on a population basis, the food intake assessed during the German National Nutrition Survey II [
Estimation of total acetaldehyde exposure (in
Acetaldehyde content in the food | Consumption (men) | Consumption (women) | ||||||
Average | Fifth percentile | Median | 95th percentile | Average | Fifth percentile | Median | 95th percentile | |
Average | 42 | 1 | 20 | 161 | 44 | 2 | 27 | 147 |
Fifth percentile | 6 | 1 | 4 | 17 | 6 | 1 | 5 | 16 |
25th percentile | 12 | 1 | 8 | 38 | 14 | 1 | 10 | 39 |
Median | 24 | 1 | 14 | 83 | 28 | 2 | 19 | 86 |
90th percentile | 105 | 1 | 44 | 431 | 106 | 4 | 61 | 372 |
95th percentile | 119 | 1 | 49 | 487 | 120 | 5 | 70 | 418 |
99th percentile | 137 | 2 | 60 | 547 | 142 | 6 | 87 | 483 |
Acetaldehyde exposure estimation due to the food groups analyzed in this study.
This exposure estimated in our study is considerably lower than the previous assumptions (e.g., from JECFA or FEMA), which were derived from very old occurrence data or industrial production amounts for the use as food flavour additive. This shows the need for further research on acetaldehyde in foods, as the exposure situation appears to be far from well characterized.
Nevertheless, the margin of exposure (MOE) calculated according to our previous studies [
We think that this preliminary risk assessment justifies further studies into acetaldehyde exposure from food, and risk managers should also consider the possibility to reduce the exposure by disallowing the practice of acetaldehyde addition as a flavour compound.
Iris Woock is thanked for preparing the microscopic photographs.