Browsing by Subject "Acrylamid"

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Agronomic strategies to reduce potential precursors of acrylamide formation in cereals
(2020) Stockmann, Falko; Graeff-Hönninger, Simone
Food safety is of great importance as harmful substances formed during food processing can negatively affect human health. When the carcinogenic food ingredient acrylamide (AA) accidentally appeared in 2002, it was not expected that AA would take this much attention during the next years. Yet, after around 15 years of research, AA has finally been recognized as being harmful. In a first step, research focussed on food processing implications on AA formation. The impact of heat treatment, time of heating, baking agents, fermentation time, additives and enzymes were reported in several studies. Nevertheless, since 2011 food AA levels seem to stagnate or even increase in some years. Thus, the food industry did not show sufficient progress in reducing AA. Reducing sugars and amino acid free asparagine (Asn) are the main AA precursors. They can fluctuate in their content for instance in grain flour or potatoes shifting the focus of AA origin to the raw material. Thus, the production of raw material low in AA precursors seems important. However, lowering precursors of AA in the raw material necessitates suitable agronomic strategies to grow cereal species and cultivars low, especially in free Asn. Hence, the major goal of this thesis was to investigate the following questions concerning their impact on free Asn formation in cereals: 1. Which role does the management system plays, as organic vs. conventional farming systems highly differ in their cropping strategies? 2. What is the best nitrogen fertilization strategy when comparing organic vs. conventionally cropping systems? 3. Is there an impact of sulphur fertilization concerning sulphur amount and sulphur type? 4. Can expanding row distance and lowering seed density in low-input farming systems positively influence baking quality while keeping free Asn amounts low? 5. For organically grown cereals no level of free Asn was available. Thus, the question came up to which extent organically grown cereal species and cultivars including ancient grains like einkorn and emmer differ in free Asn. 6. Should free Asn be implemented in breeding programs if heritability is high? 7. Is there an impact of harvest timing on free Asn formation? Out of several field trials the following results were obtained: • The cropping system had a significant impact on grain yield, the level of free Asn and quality traits. Across all species, free Asn contents in flour were 26% lower under organic conditions compared to conventional farming. For wheat a maximum reduction of 50% in free Asn content was possible if organically produced. Spelt and rye were affected to a minor extend as only in single years organically grown cultivars showed up to 33% lower Asn contents. • Nitrogen (N) fertilization significantly influenced grain yield and baking quality in both cropping systems. In contrast, up to a certain amount of N free Asn was only affected to a minor extend. In particular, within the organic farming samples no significantly higher free Asn amounts were determined even if N fertilizer was raised or the N form was changed. A late N fertilization within the conventional cropping system increased crude protein content, while no clear effect was found on free Asn. Also, cultivars affected free Asn level significantly. Wheat cultivar Capo exhibited the lowest AA formation potential at a N supply of 180 kg N ha−1 while simultaneously reaching a crude protein content > 15% (conventional) and > 12% (organic). Thus, lowering free Asn by adjusting N treatments should not necessarily affect baking quality. In general, free Asn amounts in wheat varied widely both within cultivars and between cropping systems. Besides N, neither type nor amount of sulphur fertilization influenced free Asn significantly. • Extending row distance can increase quality traits protein and sedimentation value. Seed density was highly related to grain yield and test weight. Most importantly, free Asn was only minor affected by both treatments. Thus, larger row distances can be recommended to raise baking quality in organic farming systems without simultaneously affecting free Asn. Number of grains spike-1 seems to be related to free Asn (R2=0.72). This provides new insights on Asn synthesis during grain development and offers the opportunity to predict free Asn formation without expensive chemical analyzes. In contrast Asn and protein content did not show any relation while high protein contents in grain seem to lower AA amount in heated flour samples. • The impact of organically grown cereal species and cultivars in combination with marginal N supply on free Asn was clearly shown. A reduction potential of 85% was reached if rye was replaced by spelt. Surprisingly, the ancient species einkorn and emmer reached a very high free Asn content similar to rye. Heritability was high for wheat and spelt concerning locations, while regarding years, heritability was low for wheat but high for spelt and rye. For organically grown cereals, the relation between free Asn and AA formation was proven. Across species and years free Asn can serve as an indicator for AA formation (R2 of 0.69). • Harvest timing affects free Asn levels. In this context a delayed harvest can increase Asn significantly while shifting harvest 1-2 weeks earlier decreased Asn by up to 60% depending on cereal species and cropping system. After summarizing and stating the most promising steps in the frame of agronomic strategies to lower free Asn, a prediction tool for free Asn should be implemented that classifies the impact of agronomic strategies and leads to recommendations to farmers. Finally, the main riddle, that should be solved during the next studies is the question, why cereal species and cultivars differ in their Asn formation. This thesis gives some preliminary ideas but a much deeper insight is essential to establish long-term strategies to lower free Asn content.
Trace analysis of acrylamide by high-performance thin-layer chromatography coupled to mass spectrometry
(2011) Alpmann, Alexander; Schwack, Wolfgang
Planar-chromatography (High-Performance Thin-Layer Chromatography, HPTLC) is a rapid and cost-effective offline separation method. Through advances in the automatization of each step the system reproducibility, from application and development to detection, has been improved. This makes planar-chromatography a highly reliable technique. HPTLC shows a couple of features that make it unique. There is great flexibility concerning application, development and detection that distinguishes HPTLC from other techniques. Especially the parallel development of up to 36 tracks per plate, the possibility of pre-chromatographic derivatization on the stationary phase, application volumes from nL up to mL, two-dimensional development, automated single or multiple development, and the multiple detection with different methods (UV, fluorescence, bioluminescence, etc.) have to be emphasized. A further advantage over column- (LC) and gas-chromatography (GC) is the single use of the stationary phase. This leads to a high tolerance towards sample matrix and allows for reducing sample preparation. Because of these aspects, planar-chromatography is an interesting tool for each analyst. However, in the last years hyphenation with mass spectrometry (MS) did not make great advancements in comparison to HPLC and GC: thus, planar-chromatography became less attractive. Therefore an existing universal hyphenation (ChromeXtract by Dr. Luftmann), that was based upon a plunger for elution, was improved (publication 1). The original version of the plunger did not allow any elution from glass backed plates, since they broke easily under the pressure applied during clamping. It was difficult to adjust the pressure depending on the experience of the operator. Furthermore, solvent leakage was possible because of insufficient sealing of the cup-point. For a reproducible contact pressure that was independent from the experience of the operator, a commercial torque wrench was used for clamping of the plates. This guaranteed reproducible contact pressure. The installation of a small plastic buffer into the plunger ensured a slight kind of attenuation. This decreased the frequency of leakage from over 50 % to below 5 %. An important criterion of applicability of this hyphenation is the repeatability of the extractions and thus the measurements. Thus, zones of xanthylethylcarbamat (XEC) and dansylpropanamid (DPA) were extracted after chromatographic development. Their specific masses were detected in positive ESI-mode. The relative standard deviation of the signal in single-ion-monitoring (SIM) mode was 18.6 % for XEC and 8.7 % for DPA. Linearity was given in the range of 10 to 200 ng/zone with a very good correlation coefficient (r > 0.9919). The limit of quantification at an S/N-ratio of 10 was calculated by means of the blank signal and amounted 52 and 160 pg/zone for XEC and DPA, respectively. Additionally, the influence of the elution solvent on the extraction of the HPTLC-plate and signal intensity was demonstrated with tests using different solvents. The second publication addressed the application of planar-chromatography hyphenated with MS by means of the modified ChromeXtractor on the determination of acrylamide in drinking water. The strict limit within the EU of 0.1 mug/L until then was only controlled through costly methods that were almost exclusively based on GC-MS or LC-MS/MS after applying intensive clean-up procedures. Thus it was aimed to develop a low priced and rapid alternative method for routine analysis based on HPTLC. Therefore a pre-chromatographic in-situ derivatization of acrylamide with a fluorescence marker was used. The product was detected densitometrically after chromatographic separation. During development of the method, the mass of the reaction product was determined for analysis of the derivatization step. With the aid of the modified ChromeXtract the product could be directly extracted from the plate and transferred to MS. The exact mass proved that instead of the originally used fluorescence marker dansylhydrazine the dimethylaminonaphthaline(Dan)-sulfinic acid reacts with acrylamide. Consequently, dansulfinic acid was synthesized and used for derivatization. To take advantage of the high tolerance of planar-chromatography towards various sample matrices, an approach was searched in order to skip sample preparation. However the necessity to use excess of reagent led to high background fluorescence. This allowed only a limit of detection of 20 mug/L. Thus, sample preparation and analyt enrichment was necessary to obtain a method able to control the maximum concentration. In accordance with DIN 38413-6 concerning determination of acrylamide in drinking water, activated carbon was used for analyte enrichment by means of solid phase extraction (SPE). An internal standard (dimethylacrylamide) was added prior sample preparation. The final extract was analysed as described. In spiked samples of drinking water, a 1000-fold lower limit of detection of 0.02 mug/L and a very good mean reproducibility across the whole system was shown, which suffices to control the maximum amount. A comparative study with measurements by LC-MS/MS revealed satisfactory correlation. Thus, for the first time a planar-chromatographic method for the determination of acrylamide at ultra-trace levels were presented. The third publication addresses the application of the developed method on a very complex food matrix like coffee. Several publications reported problems during determination of acrylamide in coffee. Therefore the extremely high tolerance of planar-chromatography towards sample matrix effects was used, allowing for a shortened sample preparation. The idea of a rapid method was followed by the extraction of commercial coffee samples by means of accelerated solvent extraction (ASE). This allowed for higher throughput during sample preparation. To remove a part of the co extracted matrix, the whole ASE-extract was cleaned by SPE with activated carbon and evaporated to a defined volume. This represented a simplification of common multistage extraction methods and clean-up steps, that aim for complete removal of co extracted matrix prior injection into LC- or GC-systems. In accordance with determination of acrylamide in drinking water, the extract was derivatized in-situ with the fluorescence marker Dansulfinic acid and detected densitometrically after chromatographic separation. The concentration of acrylamide was quantified by means of parallel preparation of three standard additions. Systematic errors and the influence of the sample were corrected by the calibration within the matrix. The linearity of the calibration (between r = 0.9825 and 0.9995) were acceptable. Good values were reached for the limit of quantification (48 mug/kg) and repeatability (rsd 3 %). After method development the acrylamide concentration of commercial coffee samples was determined, showing results being consistent with literature findings. Thus the applicability of the newly developed method to complex food samples was demonstrated. In summary, the present work shows the applicability of planar-chromatography hyphenated with mass spectrometry for sensitive determination of acrylamide. It was possible to quantify the analyte at ultra-trace levels using less instrumental effort and time than usual. Quantification in complex sample matrices was feasible in spite of a simplified sample preparation. These applications prove the relevance of planar-chromatography to solve current analytical problems.