Validation and application of a liquid chromatography-tandem mass spectrometry based method for the assessment of the co-occurrence of mycotoxins in maize silages from dairy farms in NW Spain.

The first objective of this study was the validation of an efficient multi-analyte method for the simultaneous detection and quantification of mycotoxins in maize silage, by reverse-phase liquid chromatography coupled with electrospray ionisation triple quadrupole mass spectrometry (LC-HESI-MS/MS). A simple liquid/solid extraction was performed either with clean-up on Mycospin 400 columns or without any clean-up. Almost all the target mycotoxins showed highly-suppressed signals in the presence of a matrix, emphasising the need to quantitate mycotoxins by means of matrix-matched calibrations. An alternative validation method based on ISO 11843 and on a single factor balanced design was implemented. The achieved average recoveries from spiked samples at three levels ranged from 60% to 122% with relative standard deviations (rsd) below 11%. Limits of Detection (LODs) and Limits of Quantification (LOQs) were between 0.02-17.1 µg kg-1 and 0.06-57 µg kg-1. The calculated repeatability and within-lab reproducibility ranged from 5.2 to 23.2% and from 7.2 to 23.9%, respectively. Finally, the decision limit and detection capacity, CCα and CCß, were calculated for all mycotoxins having regulated/recommended contents in feed. The validated method was applied to 148 samples collected over two years in 19 dairy farms from Galicia (NW Spain). Of the analysed samples, 62% contained at least one mycotoxin. Zearalenone (ZEA), deoxynivalenol (DON), fumonisins B1 and B2, roquefortine C, α-zearalenol, ß-zearalenol, enniatins B and B1, andrastin A, marcfortine A, verruculogen and mycophenolic acid were quantified, the highest average detection frequency being for enniatin B (51%). DON, mycophenolic acid and ZEA plus metabolites (α-zearalenol, ß-zearalenol) were the most abundant mycotoxins.

Occurrence and stability of masked fumonisins in corn silage samples.

Corn plants contaminated with Fusarium verticilloides were harvested at two dates and ensiled in laboratory silos. The stability of Fumonisins B1 and B2 (FB1, FB2) was studied and the effects of inoculant use (Lactobacillus buchneri) and of the ensiling time were assessed. The occurrence of masked fumonisins was also investigated. After the extraction of the free forms (FB1, FB2), the residue was subjected to an alkaline hydrolysis resulting in the release of derivates subsequently detected by LC-HESI-MS/MS. The ensiling time has shown to be a significant factor for the evolution of free FB1 and FB2 at the 2nd harvest date. Moreover, the use of inoculant had a significant effect on the stability of hidden and total FB1 at the two harvest dates. Samples previously ensiled and exposed to aerobic conditions for 7days have shown higher fumonisin levels than those not exposed to air. Our results showed that 65% and 39% of the total FB1 and FB2 contents in silage were due to hidden fumonisins.

Effects of calf diet, antioxidants, packaging type and storage time on beef steak storage.

The effect of basal dietary supplemented with vegetable oils plus vitamin E (sunflower, soybean, linseed and a basal diet control), type of packaging (MAP or vacuum), addition of natural antioxidant (grape seed, rosemary) and storage time (0, 7, 14 and 21 days) on lipid oxidation, color stability, vitamin E content, and total aerobic bacterial counts in steaks of Longissimus thoracis was studied. The triple interaction diet × time × packaging affected oxidative stability, redness and yellowness of the meat. TBARS values did not increase with time in vacuum-packaged samples for all dietary treatments. However, samples from MAP and control showed the highest TBARS values after 21 days of storage (0.72 mg MDA/kg of meat, P<0.05). Both exogenous antioxidant extracts and MAP maintained low total aerobic counts in steaks until the 21st day. Calves should be fed a diet supplemented with L-VE, stored in MAP and treated with grape seed extract to extend the shelf life of their meat.

Effect of weaning status on lipids of Galician Blond veal: total fatty acids and 18:1 cis and trans isomers.

The effect of weaning at different ages (NW=not weaned, W5=5.5 months and W2=2 months) on fatty acids (FA) of the Longissimus thoracis (LT) muscle was studied in 36 Galician Blond (GB) calves. Total FA (TFA) were determined by gas chromatography (GC) and 18:1 isomers by a combination of reversed-phase high performance liquid chromatography (HPLC) and GC. NW group showed higher (P<0.001) values of n-3 polyunsaturated FA (PUFA), conjugated linoleic acid (CLA) and 18:1 trans-11 compared to LT from W5 and W2 calves. W2 calves showed the highest levels of n-6 PUFA (P<0.01), 18:1 trans-10 and 18:1 trans-6/7/8 (P<0.001). Generally, W5 calves had intermediate values for TFA and 18:1 isomers. As the suckling period was longer, GB milk and veal FA profiles became more similar, it seems that muscle FA were partially transmitted by the milk FA intake due to the persistence of the reticular grove closing reflex.