The mobile phase was acetonitrile (solvent A) and 0.03% acetic acid solution (solvent B). No solvent or 2 mL of water, ethanol, acetonitrile, ethanol/water 50/50 (v/v), and acetonitrile/water 50/50 (v/v) were tested. A good way to increase the amount of n‐3 fatty acids in animal feed is to use linseed (also known as flax or Linum usitatissimum) for its high α‐linolenic acid content (more than 50% of the fatty acids)3, 6, 7 or fish oil and microalgae for their eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content.8, 9 Unfortunately, at these high polyunsaturated fatty acids levels there is rapid oxidation to other less desirous productsd10-12; consequently, it is important to monitor this potential oxidation pathway in animal feed because it can lead to less polyunsaturated fatty acids in meat, milk, or eggs than expected. However, it was decided to quantify 4‐HNE using 4‐HNE‐D3 and 4‐HHE using MeMDA instead of 4‐HNE‐D3, according to preliminary results which were better when MeMDA was used as internal standard to quantify 4‐HHE. If you do not receive an email within 10 minutes, your email address may not be registered, The opinions expressed and arguments employed in this publication are the sole responsibility of the authors and do not necessarily reflect those of the OECD or of the governments of its Member countries. Learn more. Indeed, those three compounds were either not detected or detected with their chromatographic peak areas close to those of the background noise peaks in Corn Flakes®. The low, medium, and high levels of concentrations tested are detailed in Table 2. The best compromise between solubility and chromatographic peak areas was obtained for the 8 aldehydes with a DNPH concentration of 0.05 M in acetonitrile/acetic acid (9/1, v/v). The sample matrix is very acidic (20% HCL, ph 0). Most of the other published methods described in the literature were not validated and demonstrated to be fit‐for‐use for food or feed matrices. Unfortunately, no isotopic analogues of the 2,4‐nonadienal or 2,4‐decadienal were commercially available, therefore those two compounds were quantified using hexanal‐D12, the internal standard with the closest retention time. endstream endobj startxref As illustrated in Figure 2, the developed method was applied in ground Kellogg's® Corn Flakes® cereals and they were evaluated as ‘blank’ regarding their malondialdehyde, 4‐HHE and 2,4‐nonadienal contents. ChEBI. The formed aldehydes, including malondialdehyde (MDA), 4‐hydroxy‐2‐nonenal (4‐HNE), 4‐hydroxy‐2‐hexenal (4‐HHE), crotonaldehyde (CRT), benzaldehyde (BNZ), hexanal (HXL), 2,4‐nonadienal and 2,4‐decadienal can be relatively stable and some of them have been shown to be cytotoxic and genotoxic by reacting with proteins and nucleic acids.13-16 What is more, in 2011, in an advisory note from the Belgian Superior Health Council, MDA, crotonaldehyde, and 4‐HNE have been considered of major concern for human health.17 Until now, no maximum levels have been established for aldehydes in food or animal feed, mainly because of the lack of data in the literature about toxic concentrations, quantities contained in food or feed, and quantities of these oxidation products actually ingested. Other parameters were also assessed according to the recommendations of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use.22, 1,1,3,3‐Tetraethoxypropane (TEP), 2,4‐dinitrophenyl‐hydrazine (DNPH), crotonaldehyde, benzaldehyde, hexanal, 2,4‐nonadienal, 2,4‐decadienal, methylcrotonaldehyde, benzaldehyde‐13C, hexanal‐D12 and butylated hydroxytoluene (BHT) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Methylcrotonaldehyde (MeCRT), benzaldehyde‐13C, hexanal‐D12, 4‐HNE‐D3 and methylmalondialdehyde (MeMDA) were used as internal standards in the quantitative analysis. endstream endobj 931 0 obj <. The analysis of 2,4‐nonadienal and 2,4‐decadienal can also be performed but in a limited range of concentration and with a limited degree of accuracy. After checking that they were still suitable, the derivatization time and temperature were maintained at 60°C for 2 h, as previously optimized. Extractions of samples and injections of samples extracts were performed on three days. The validation was performed according to the Commission Decision 2002/657/EC and following the most commonly used approaches.24, 25 The Commission Decision 2002/657/EC promotes the use of Certified Reference Materials (CRM) to validate an analytical method. Separation and detection of aldehydes as dinitrophenylhydrazone derivatives were performed using a ThermoFinnigan Spectra System P4000 HPLC and a ThermoFinnigan LCQ Deca ion trap mass spectrometer, equipped with an electrospray source. Because the cost of the stable isotope analogue of 4‐HHE was prohibitively high, 4‐HNE‐D3 was bought instead and used for 4‐HHE and 4‐HNE quantification. Formaldehyde-2,4-DNPH may be used as an analytical standard for the determination of the analyte in air samples, food and feed products, and aquatic products by various chromatography techniques. At 2.4 mg/m . Target spiking levels, mean introduced concentrations (average of the real concentrations for all experiments), LOD, LOQ, repeatability, intermediate precision, recovery, linearity (R2), calibration curve equations, and uncertainty are summarized in Table 2. Dinitrophenylhydrazone derivatives were prepared by adding 750 μL DNPH solution (0.05 M in acetonitrile/acetic acid 9:1 (v/v)) to 750 μL of extract in an injection vial and reaction took place for 2h at 60°C. Please check your email for instructions on resetting your password. The information in this document is based on the present state of our knowledge and is applicable to the product with regard to appropriate safety precautions. LODs (the smallest quantity of the targeted substance that can be detected but not accurately quantified in the sample) are calculated within the e‐Noval software by either dividing the lower LOQ by 3.3 or by adding three times the residual standard deviation obtained from an ANOVA test to the y intercepts of the calibration curves. Subsequently, a validation protocol for the developed method was set‐up, based on various performance parameters. Data generated during the validation of the method were processed with e‐Noval 3.0 software, from Arlenda (Liège, Belgium). n) Water solubility completely soluble o) Partition coefficient: noctanol/ water log Pow: -0,54 at 25 °C p) Auto-ignition temperature 524,0 °C q) Decomposition temperature no data available r) Viscosity no data available s) Explosive properties Not explosive t) Oxidizing properties The substance or mixture is not classified as oxidizing. Calibration points were injected before each series of unknown samples and the extract spiked at a concentration corresponding to the central point of the calibration curve was injected one more time after all the samples. For the 4‐HHE, 2,4‐decadienal and 2,4‐nonadienal, the lower 95% tolerance interval is higher than the acceptance limit of 50% for the “high” level of concentration (66.3, 139.2, and 108.5%, respectively). In addition, during each of the three days of the validation, six ground Kellogg's® Corn Flakes® cereal samples were spiked with increasing concentrations of aldehydes to be used as calibration curves, leading to a total of 98 ground cereal samples. 3. and at 5% breakthrough, the Waters sampler had a capacity at <10% RH of 55 µg, and at >85% RH a capacity of 77 µg. Stock solutions of the other aldehydes were prepared by dissolving the compound in water/ethanol 50/50 (v/v). The solid central line represents the bias and the dots correspond to the relative back–calculated concentrations, plotted with respect to their targeted concentrations (expressed in percentage of variation from the targeted concentration). Correspondence to: Caroline Douny, Department of Food Sciences, Laboratory of Food Analysis, FARAH ‐ Veterinary Public Health, University of Liège, Quartier Vallée 2, Avenue de Cureghem 10, Sart Tilman B43bis ‐ 4000 Liège, Belgium. These concentrations correspond to the first point of the matrix matched calibration curve. 965 0 obj <>/Filter/FlateDecode/ID[]/Index[930 46]/Info 929 0 R/Length 152/Prev 387843/Root 931 0 R/Size 976/Type/XRef/W[1 3 1]>>stream A mixture of the 8 aldehydes of interest at a concentration of 8 (4‐HHE and 4‐HNE), 25 (hexanal) and 20 ng/μL (for the 5 other aldehydes) was prepared in water/ethanol 50/50 (v/v). Prior to extraction, the addition of a post‐derivatization solvent to increase the aqueous phase was investigated. 2005-03-26. Accuracy refers to the closeness of agreement between the test result and the accepted reference value.32 The accuracy takes into account the total error, related to the test results. The parameters relevant to evaluate the performances of the developed method were selectivity/specificity, linearity, recovery, precision (repeatability and intermediate precision), accuracy, limit of detection (LOD), and limit of quantification (LOQ). 2,4-dinitrophenylhydrazine is a C-nitro compound that is phenylhydrazine substituted at the 2- and 4-positions by nitro groups. Samples were extracted, derivatized and analyzed in three independent series. Picograde hexane was from LGC Standard (Wesel, Germany). The analytical procedure is considered as valid within the working range if the limits of the accuracy profile (dashed lines) are narrower than the acceptance limits (external dotted lines). The capacity of DNPH-coated silica gel samplers was found to vary with relative humidity (RH) in addition to concentration of formaldehyde. Working off-campus? What else can I use to solublize the DNPH that wont interfere with the CAA reaction? Create . I know this method well. I . 930 0 obj <> endobj Accuracy profile of the 8 aldehydes, computed with e‐Noval 3.0 (Arlenda). Eight aldehydes that can be formed after PUFA oxidation were chosen to evaluate the lipid oxidation from linseed in animal feed (MDA, 4‐HNE, 4‐HHE, CRT, BNZ, HXL, 2,4‐nonadienal and 2,4‐decadienal (Figure 1)) and a liquid chromatography coupled to tandem mass spectrometry detection (LC‐MS/MS) method was developed to measure their concentration as dinitrophenylhydrazone derivatives in animal feed. Log in or register to post comments; Tue, 04/07/2009 - 15:50 #2. For validation purposes, it was decided to use ground Kellogg's® Corn Flakes® cereals to mimic animal feed in calibration curves and for the validation of the method, knowing that this cereal contains 0.9% fat only and is thus poorly sensitive to oxidation. After checking that they were still suitable, the derivatization time and temperature were maintained at 60°C for 2 h, as previously optimized.26. To avoid the quick clogging of the chromatographic and spectrometric systems, an extraction of the dinitrophenylhydrazone derivatives obtained was considered. The above information is believed to be correct but does not purport to be all inclusive and shall be used only as a guide. By continuing to browse this site, you agree to its use of cookies as described in our,, I have read and accept the Wiley Online Library Terms and Conditions of Use, The acute and long‐term effects of dietary fatty acids on vascular function in health and disease, Omega‐6 fatty acids and risks for cardiovascular disease: a science advisory from the American Heart Association subcommittee of the Council on nutrition, physical activity, and metabolism; council on cardiovascular nursing; and council on epidemiology and prevention, Effect of dietary fatty acids on incorporation of long chain polyunsaturated fatty acids and conjugated linoleic acid in lamb, beef and pork meat: a review, Effects of fatty acids on meat quality: a review, Long‐term feeding of conjugated linoleic acid and fish oil to laying hens: effects on hepatic histopathology, egg quality, and lipid components, Effect of a linseed diet on lipid composition, lipid peroxidation and consumer evaluation of French fresh and cooked pork meats, Effect of dietary lipid on the content of conjugated linoleic acid (CLA) in beef muscle, Impact of different omega‐3 polyunsaturated fatty acid (n‐3 PUFA) sources (flaxseed, Isochrysis galbana, fish oil and DHA Gold) on n‐3 LC‐PUFA enrichment (efficiency) in the egg yolk.