Data generated during the validation of the method were processed with e‐Noval 3.0 software, from Arlenda (Liège, Belgium). The DNPH is not soluble in acetonitrile. Those six spiked samples were extracted simultaneously with the unknown samples, in order to build a calibration curve. Learn about our remote access options, Department of Food Sciences, Laboratory of Food Analysis, FARAH ‐ Veterinary Public Health, University of Liège, Quartier Vallèe 2, Avenue de Cureghem 10, Sart Tilman B43bis ‐ 4000, Liège, Belgium. The evaluated parameters were specificity/selectivity, recovery, precision, accuracy, uncertainty, limits of detection and quantification, using the concept of accuracy profiles. 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. During the three days of the validation, 20 ground cereal samples were spiked only with internal standards to be used as ‘blank’ samples and 60 ground cereal samples were spiked with internal standards and three different concentrations (low, medium, high; n=20 for each concentration) of aldehydes (Table 2), which correspond to the first, the fourth, and the sixth point of the calibration curve, respectively. Stock solution of malondialdehyde 1 mg/mL (MDA) was obtained by hydrolysis of 1,1,3,3‐Tetraethoxypropane (TEP) in trichloroacetic acid 5%., according to Regulation (EC) No 1907/2006 (REACH), IDENTIFICATION OF THE SUBSTANCE/MIXTURE AND OF THE COMPANY/UNDERTAKING, Relevant identified uses of the substance or mixture, 2.1 Classification of the substance or mixture, 6.1 Personal precautions, protective equipment and emergency procedures, 9.1 Information on basic physical and chemical properties, 11.1 Information on toxicological effects. These concentrations correspond to the first point of the matrix matched calibration curve. Number of times cited according to CrossRef: Nonenzymatic oxygenated metabolite of docosahexaenoic acid, 4(RS)‐4‐F4t‐neuroprostane, acts as a bioactive lipid molecule in neuronal cells. Water was of Chromanorm quality, while acetic acid 100% was of Normapur Quality and were both provided by VWR International (West Chester, PA, USA). The concentration range of the calibration curve was chosen to cover the range of concentrations observed previously in our laboratory for each aldehyde analyzed in oxidized animal feed containing high amounts of PUFAs. All of the standard solutions were kept for maximum 6 months at + 4°C. Their recovery rates ranged between 54 and 114% and coefficient of variation for the intermediate precision between 11 and 25% for these two compounds. A mixture of the six internal standards was prepared at a concentration of 25 (MeMDA, benzaldehyde‐13C and hexanal‐D12), 12 (MeCRT) and 0.8 ng/μL (4‐HNE‐D3) in water/ethanol 50/50 (v/v). 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 proposed extractions were tested to extract the dinitrophenylhydrazone derivatives of the eight aldehydes of interest and five internal standards and four times 1 mL hexane showed the best results. Suggestions for sample prep would be welcome. These parameters were determined during experiments conducted over three different days with ground Kellogg's® Corn Flakes® cereals as model matrix for animal feed and spiked at different levels of concentration. As quality control parameters, the correlation coefficients R2 associated with those curves had to be higher than 0.98, and only one point could deviate from the curve by more than 20% of the corresponding calculated value. Separation was achieved on an Atlantis T3 C18 column (3 µm, 2.1 x 150 mm), with an Atlantis guard column T3 C18 (3 µm, 2.1 x 10 mm), both from Waters Corporation (Milford, MA, USA). Where to find omega‐3 fatty acids and how feeding animals with diet enriched in omega‐3 fatty acids to increase nutritional value of derived products for human: what is actually useful? I know this method well. In order to solve this problem, the optimization of the concentration of DNPH, the amount and type of acid and the solvent to be used were investigated. Copyright © 2016 John Wiley & Sons, Ltd. Nowadays, many nutritional and health studies recommend a higher consumption of fat composed of polyunsaturated fatty acids (PUFA), mainly n‐3 polyunsaturated fatty acids.1, 2 Increasing the amount of n‐3 fatty acids in animal feed is a way to increase human intake of those compounds through the consumption of food from animal origin other than fatty fish, the major natural dietary source of long chain n‐3 fatty acids.3-5 Consequently, many products enriched with n‐3 fatty acids can be found in the market: meat, milk and dairy products, eggs, etc. 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). Validation was performed using the concept of accuracy profile.30, 31. A linear regression was used and no ‘fit weighting’ was applied. 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. The extraction was repeated a second time with 2.5 mL water/ethanol 50/50 (v/v) and supernatants were grouped and homogenized. Contact Technical Service for further support. The developed method was validated according to the criteria and procedure described in international standards. Derivatized standards were synthesized in our laboratory according to the derivation protocol explained in Section Sample preparation and infused directly in the mass spectrometer to optimize the MS tune parameters. Picograde hexane was from LGC Standard (Wesel, Germany). The analytical method presented in this paper is novel compared to what is commonly found in the literature since this method allows to quantify in the same run, MDA and 7 other aldehydes: 4‐HNE, 4‐HHE, CRT, BNZ, HXL, 2,4‐nonadienal and 2,4‐decadienal, in animal feed. 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).