Investigations on the Transfer of Quinolizidine Alkaloids from Lupinus angustifolius into the Milk of Dairy Cows.

Lupin varieties with a low content of quinolizidine alkaloids (QAs) like blue sweet lupin (BSL) have long been used as a protein source for dairy cows. A health concern for humans may arise from the transfer of acute toxic QAs from feed into cow's milk. This study is the first to quantify the transfer of QAs from BSL into cow's milk with experimental and modeling methods. Four lactating dairy cows were subjected to two 7 day feeding periods with 1 and 2 kg/d BSL, respectively, each followed by a depuration period. BSL contained 1774 mg/kg dry matter total QAs. Individual milk samples were taken twice daily and QA contents in feed and milk determined with liquid chromatography-tandem mass spectrometry. Transfer of QAs into the milk was already seen with the administration of 1 kg/d BSL, with differences in transfer rates (TRs) between individual QAs. A toxicokinetic model was derived to quantify and predict QA feed-to-food transfer. For the four most prominent QAs, our model shows an α-half-life of around 0.27 d. TRs were obtained for six QAs and were between 0.13 (sparteine) and 3.74% (multiflorine). A toxicological assessment of milk containing QAs as measured in this study indicated a potential health concern.

Development and validation of two analytical methods for urea determination in compound feed, including pet food, and yeast using high-performance liquid chromatography coupled with fluorescence detection and tandem mass spectrometry.

Urea is authorised in the European Union (EU) as feed additive for ruminants. Because of its high molecular nitrogen content, it is a substance for potential protein adulteration in non-ruminant feed. The EU defines a spectro-colorimetric method as an official control method for the determination of urea in feed, whereas the Association of Official Analytical Chemists (AOAC) in the United States recommends an enzymatic method. Discrepancies between results obtained by these different approaches have been reported, especially at low concentrations. Therefore, we developed and validated two methods for urea determination in compound feed, including pet food, and yeast (Saccharomyces cerevisiae) over a wide concentration range using high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and fluorescence detection (HPLC-FLD) and compared performance with a commercial enzyme kit. Limit of detection (LOD) and limit of quantification (LOQ) were found to be 3 and 8 mg kg-1 for LC-MS/MS and 2 and 7 mg kg-1 for HPLC-FLD, respectively. For both methods, the variation coefficients ranged between 1.4% and 7.2% in ruminant feed used as reference material as well as spiked samples of complete feed for chicken, pet food for dogs and cats, as well as yeast. Recovery rates for spiked samples ranged from 86% to 105%. For real samples of complete feed for poultry, wet and dry pet food for cats and dogs and yeast amounts of urea between < LOD and 200 mg kg-1 relative to a feedingstuff with a moisture content of 12% were found. In comparison with the enzyme kit, the newly developed methods proved to be less time-consuming in sample preparation and more stable regarding matrix effects.

Interlaboratory validation of an LC-MS/MS method for the determination of melamine and cyanuric acid in animal feed.

Melamine and cyanuric acid have been mixed illegally into food and feed to increase the nitrogen content, which results in deceptively high protein contents. As a consequence, a maximum level for melamine of 2.5 mg kg-1 feed was established by the European Union under Directive 2002/32/EC. The Technical Committee (TC) 327 of the European Committee for Standardisation (CEN) commissioned the standardisation of a method for the analysis of melamine and cyanuric acid in animal feed. One main task in the standardisation process is the performance of a full international collaborative trial, which is described in this paper. After performing a pre-trial study, in the main study eight different feed samples with different concentration levels of melamine and/or cyanuric acid were distributed as double-blind samples to 13 participants. The minimum criterion of eight laboratories submitting results per sample is fulfilled for melamine but only partly for cyanuric acid. The evaluation showed for both analytes a Horwitz ratio (HorRat) well below 2, and meets the requirements stated in the appropriate international protocols. The results demonstrated that the method seems to be suitable for the analysis of melamine and cyanuric acid in animal feed.

Occurrence of fatty acid esters of 3-MCPD, 2-MCPD and glycidol in infant formula.

The discovery of fatty acid esters of monochloropropanediol (MCPD) and glycidol generated during the refinement process in vegetable fats and oils caused concerns about possible adverse health effects. As these fats are components of infant formula, the current investigation of the MCPD and glycidyl ester contents in infant formula was necessary to update the data for risk assessment purposes. For the analysis of 3-MCPD, 2-MCPD and glycidyl esters in infant formula, an existing method for fats and oils had to be modified and validated. The fat fraction containing MCPD and glycidyl esters was extracted from infant formula by accelerated solvent extraction (ASE). The extracted fat was then analysed according to an established method for fats and oils. Glycidyl esters are converted to monobrompropanediol (3-MBPD) esters, MCPD and 3-MBPD esters hydrolysed subsequently and after derivatisation detected by GC-MS. Seven different products of infant formula, covering two types and five lots each, altogether 70 samples, were bought in retail markets and analysed. In all samples, 3-MCPD and glycidyl esters could be detected. Both 3-MCPD and glycidyl esters' concentration levels were found to be lower in comparison with earlier investigations described in the literature. The occurrence of 2-MCPD esters in infant formula was investigated for the first time and revealed concentrations about half of 3-MCPD ester concentrations.

Carryover of maduramicin from feed containing cross-contamination levels into eggs of laying hens.

Maduramicin is a coccidiostat authorized as feed additive in the European Union for chickens and turkeys for fattening but not for laying hens, considering the risk of residues in eggs. The unavoidable cross-contamination of non-target feed with coccidiostats is regulated by Commission Directive 2009/8/EC and resulting carry-over in food by Commission Regulation (EC) No. 124/2009. To verify the compliance of the maximum levels for maduramicin in feed (50 µg/kg) and eggs (2 µg/kg), the carry-over from feed into eggs was investigated. Diets containing 10, 30, and 50 µg of maduramicin/kg of feed were fed to laying hens. Feed, egg white, and yolk were analyzed by LC-MS/MS. Maduramicin residues were only detected in in egg yolk. Feeding the 10 µg/kg maduramicin diet resulted in maduramicin concentrations up to 2.5 µg/kg in whole eggs, already exceeding the maximum level. A carry-over rate of 8% maduramicin from feed into eggs was calculated.

Quantification of flavoring constituents in cinnamon: high variation of coumarin in cassia bark from the German retail market and in authentic samples from indonesia.

Coumarin is a flavoring which can cause hepatotoxicity in experimental animals and in a proportion of the human population. The tolerable daily intake (TDI) may be exceeded in consumers with high intake of cinnamon containing high levels of coumarin. The objective of this study was to determine these levels in cinnamon samples and to identify possible factors influencing them. A HPLC method to quantify coumarin and related constituents (cinnamaldehyde, cinnamic acid, cinnamyl alcohol, eugenol) in a single run was used. Results found in 47 cinnamon powder samples obtained from the German retail market confirmed high levels of coumarin in cassia cinnamon. A huge variation was observed in stick samples from two packages (range from below the limit of detection to about 10000 mg/kg). Cassia bark samples of five trees received directly from Indonesia were analyzed additionally. Interestingly, a high variation was observed in one of the trees, whereas no coumarin was detected in the samples of two other trees. In conclusion, coumarin levels in cassia cinnamon can vary widely even within a single tree.

Application of stable carbon isotope analysis to the detection of testosterone administration to cattle.

The use of anabolic substances is prohibited in food-producing animals throughout the European Union. No method is available to reliably detect the misuse of natural hormones in cattle. A method was developed to detect the abuse of testosterone in cattle fattening. Synthesized testosterone is rather depleted in the (13)C/(12)C ratio. Hence, the method is based on gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis of urine. To select testosterone metabolites and endogenous reference compounds (ERC), the concentration of urinary steroids of cattle was investigated. Dehydroepiandrosterone and androst-5ene-3beta,17alpha-diol were chosen as ERCs to show endogenous (13)C/(12)C ratios. Etiocholanolone and 5alpha-androstane-3beta,17alpha-diol were chosen as the most important testosterone metabolites. Other metabolites known from literature like epitestosterone were less promising. In principle, GC/C/IRMS is a nonspecific method because finally carbon dioxide is analyzed. Therefore, a dedicated cleanup procedure for the selected steroids was developed. By means of proposed confidence intervals in the isotopic composition of ERCs and metabolites, the administration of testosterone to cattle could be detected reliably. Differences of up to 11 per thousand on the delta-scale between ERC and testosterone metabolites were found after testosterone administration, whereas endogenous differences did not exceed 2 per thousand.