A survey of 75 commercially available, unpasteurised, spontaneously fermented vegetables was conducted, focusing on the culturable microbiota. Foodborne pathogens (Listeria monocytogenes, Salmonella sp.), indicator bacteria (generic E. coli, Enterobacteriaceae), and lactic acid bacteria (LAB) were analysed both at the day of purchase and at the end of shelf life. Selected samples were further examined for biogenic amines, and dominant culturable LAB were isolated and identified. Challenge tests evaluated the growth potential of L. monocytogenes during white cabbage fermentation. Results showed considerable variation in pH (3.1-4.3) and LAB counts (<1.0-8.8 log CFU/g), reflecting the spontaneous nature of the vegetable fermentations. No Salmonella sp. (detection in 25 g), L. monocytogenes (LOD 10 CFU/g), or E. coli (LOD 10 CFU/g) were detected, indicating no immediate food safety or hygiene concerns, although Enterobacteriaceae were present (LOD 10 CFU/g) in six samples, suggesting an inadequate (spontaneous) fermentation process to ensure die-off of enteric pathogens. LAB isolates were dominated by Pediococcus parvulus, Lactiplantibacillus plantarum, Levilactobacillus brevis, and Lentilactobacillus buchneri. High biogenic amine levels highlight an increased risk for adverse health effects related to spontaneous vegetable fermentations. Challenge tests confirmed that rapid acidification to pH <4.4, followed by a minimum 14-day holding period under optimal fermentation conditions, effectively limits pathogen outgrowth and/or survival. Good fermentation practices, together with a sensory evaluation are essential to ensure the microbiological safety, quality, and consistency of spontaneous vegetable fermentations.

Several analytical techniques, i.e. Near Infrared (NIR) and Mid-Infrared (MIR) spectroscopy, Hyper Spectral Imaging (HSI), Gas Chromatography coupled to Mass Spectrometry (GC-MS) and Proton-transfer Reaction Time-of-Flight Mass spectrometry (PTR-TOF-MS), combined with chemometrics, are examined to evaluate their potential for solving food authenticity questions on the case of rice. In total, 237 rice samples were analyzed in this study to examine origin and variety assessment and sourced from producing countries (Italy, Spain, Vietnam, Pakistan and Thailand). The Gaussian Process Latent Variable Model (GP-LVM) was applied as technique to obtain a meaningful compressed representation of the data in a two-dimensional space followed by a classification with a nearest neighbour algorithm. What concerns the origin assessment, GC-MS results score good across all countries, resulting in the most accurate method, with prediction rates ranging from 86% to 94%. Data fusion experiments of combination of GC-MS with NIR, or GC-MS with HSI resulted in prediction rates on origin assessment of more than 90%. Variety assessment was performed with these analytical techniques. Using single techniques, HSI achieved prediction values above 90% for all classes (96%–99%). For data fusion experiments of variety assessment combining GC-MS and NIR all prediction values were equal or higher than 92%; for GC-MS combined with HSI, prediction values were all 98%. The presented methodology can be successfully used for the authentication of rice.

Several analytical techniques, i.e. spectroscopic techniques as Near Infrared (NIR) and Mid-Infrared (MIR), Hyper Spectral Imaging (HSI), Gas Chromatography coupled to Mass Spectrometry (GC-MS) and Proton-transfer Re-action Time-of-Flight Mass spectrometry (PTR-TOF-MS), combined with chemometrics, are examined to evaluate their potential to solve different food authenticity questions on the case of oregano. In total, 102 oregano samples from one harvest season were analyzed for origin and variety assessment, 159 samples for adulteration -assessment and 72 samples for batch-to-batch control. The Gaussian Process Latent Variable Model (GP-LVM) was applied as technique to obtain a reduced two-dimensional space. A Random Forest Regression algorithm was used as regression model for the adulteration assessment. Prediction rates of more than 89% could be achieved for origin assessment. For variety assessment, prediction rates of more than 78% could be obtained. Batch-to -batch control could be successfully performed with NIR and PTR-TOF-MS. Detection of adulteration could be successfully performed from 10% on with HSI, NIR and PTR-TOF-MS.

Deze studie had als doel de relatie te bepalen tussen de serumconcentraties van lidocaïne/monoethylglycinexylidide (MEGX) en hun effect op de verschillende lichamelijke stelsels bij het paard. Vijf gezonde paarden bij bewustzijn kregen een twee uur durende placebo-intraveneuze infusie gevolgd door een twee uur durende lidocaïne-infusie (bolus van 1,3 mg/kg in tien minuten gevolgd door een constante infusie van 0,05 mg/kg/min). De lidocaïne- en MEGX-serumconcentraties werden om de tien à vijftien minuten gemeten. Het evenwichtsverlies, de spiertrillingen, het ademhalings-, spijsverterings- en cardiovasculaire stelsel werden beoordeeld met behulp van verschillende niet-invasieve methoden. Tijdens de toediening van de lidocaïne-infusie was de gemiddelde (± standaarddeviatie) lidocaïne- en MEGXconcentratie respectievelijk 768,88 ± 93,32 ng/ml en 163,08 ± 108,98 ng/ml. De toediening van een lidocaïne- infusie beïnvloedde significant de aanwezigheid van trillen. Ook veroorzaakte ze een statistisch maar niet-klinisch significante afname van de systolische en diastolische bloeddruk, die beide correleerden met de lidocaïne- en MEGX-serumconcentratie. Verder verhoogde ze eveneens de duodenale contractiefrequentie, die correleerde met de serumlidocaïne-concentratie. In de voorliggende studie werden milde hypotensieve en prokinetische kortetermijneffecten van een lidocaïne-infusie waargenomen.

This study aimed to evaluate the relationship between the serum concentrations of lidocaine/ monoethylglycinexylidide (MEGX) and their effects on several systems in horses. Five healthy, conscious horses received a two-hour placebo intravenous infusion followed by a two-hour lidocaine infusion (bolus of 1.3 mg/kg over ten minutes followed by a continuous rate infusion of 0.05 mg/kg/min). Lidocaine and MEGX serum concentrations were sampled every ten to fifteen minutes during the experiment, and the presence of muscle fasciculations and loss of balance as well as the respiratory, digestive and cardiovascular systems of the five horses were evaluated by means of different non-invasive methods. During the lidocaine infusion, the mean (± SD) lidocaine and MEGX concentrations were respectively 768.88 ± 93.32ng/ml and 163.08 ± 108.98 ng/ml. The infusion of lidocaine significantly influenced the presence of fasciculations, caused a statistically but non-clinically significant decrease of systolic and diastolic blood pressures, which were both correlated with lidocaine and MEGX serum concentrations, and it increased the duodenal contractions frequency, which was correlated with the serum lidocaine concentration. In this study, mild hypotensive and prokinetic effects of short-term lidocaine infusion were observed.

The presence of mycotoxins in broiler feed can have deleterious effects on the wellbeing of the animals and their performance. Mycotoxin binders are feed additives that aim to adsorb mycotoxins in the intestinal tract and thereby prevent the oral absorption of the mycotoxin. The simultaneous administration of coccidiostats and/or antimicrobials with mycotoxin binders might lead to a reduced oral bioavailability of these veterinary medicinal products. This paper describes the influence of 3 mycotoxin binders (i.e., clay 1 containing montmorillonite, mica, and feldspars; clay 2 containing montmorillonite and quartz; and yeast 1 being a modified glucomannan fraction of inactivated yeast cells) and activated carbon on the oral bioavailability and pharmacokinetic parameters of the antimicrobials doxycycline and tylosin, and the coccidiostats diclazuril and salinomycin. A feeding study with 40 15 day-old broilers was performed evaluating the effects of long-term feeding 2 g mycotoxin binder/kg of feed. The birds were randomly divided into 5 groups of 8 birds each, i.e., a control group receiving no binder and 4 test groups receiving either clay 1, clay 2, yeast 1, or activated carbon mixed in the feed. After 15 d of feeding, both the control and each test group were administered doxycycline, tylosin, diclazuril, and salinomycin, consecutively, respecting a wash-out period of 2 to 3 d between each administration. The 4 medicinal products were dosed using a single bolus administration directly in the crop. After each bolus administration, blood was collected for plasma analysis and calculation of the main pharmacokinetic parameters and relative oral bioavailability (F = area under the plasma concentration-time curve (AUC(0-8) h) in the test groups/AUC(0-8) h in the control group)*100). No effects were observed of any of the mycotoxin binders on the relative oral bioavailability of the coccidiostats (i.e., F between 82 and 101% and 79 and 93% for diclazuril and salinomycin, respectively). Also, no significant effects could be noticed of any of the mycotoxin binders on the relative oral bioavailability of the antimicrobials doxycycline and tylosin (i.e., F between 67 and 83% and between 43 and 104%, respectively).

This study compared serotonin concentrations in platelet poor plasma (PPP) from healthy horses and horses with surgical small intestinal (SI) colic, and evaluated their association with postoperative ileus, strangulation and non-survival. Plasma samples (with EDTA) from 33 horses with surgical SI colic were collected at several pre- and post-operative time points. Serotonin concentrations were determined using liquid-chromatography tandem mass spectrometry. Results were compared with those for 24 healthy control animals. The serotonin concentrations in PPP were significantly lower (P < 0.01) in pre- and post-operative samples from surgical SI colic horses compared to controls. However, no association with postoperative ileus or non-survival could be demonstrated at any time point. In this clinical study, plasma serotonin was not a suitable prognostic factor in horses with SI surgical colic.

For the first time, a sensitive and specific method was developed and fully validated for the quantification of the EU marker residue of tiamulin, 8-α-hydroxy-mutilin, in rabbit muscle and liver tissues using liquid chromatography combined with positive heated electrospray ionization triple quadrupole mass spectrometry. The mass spectrometer was operated in the selected reaction monitoring (SRM) mode with selection of the [M + H]+ ion in both quadrupoles 1 and 3, resulting in the SRM transition m/z 337.25 > 337.25 for quantification. Chromatography was performed using a Hypersil Gold C18 column using a gradient elution program with water and methanol as mobile phases. The sample preparation procedure for the analysis of 8-α-hydroxy-mutilin in liver and muscle samples consisted of three main steps: (1) extraction of the tissue matrix using 0.1 N hydrochloric acid/acetone (50/50, v/v), (2) hydrolysis of tiamulin and metabolites to 8-α-hydroxy-mutilin in alkaline medium at 45 °C, and (3) liquid–liquid extraction in acidic medium using ethyl acetate. This is the first method presenting fully validated results, encompassing a linearity of 50 to 2,000 μg/kg, within-run and between-run accuracy and precision, limit of quantification (50 μg/kg for both muscle and liver tissues), limit of detection (muscle, 11.9 μg/kg; liver, 20.6 μg/kg), extraction recovery (muscle, 66.2 %; liver, 75.5 %), signal suppression and enhancement (muscle, 51.7 %; liver, 43.3 %), carryover, applicability and practicability, and stability during storage and analysis. This novel method is therefore sensitive enough to be used for residue depletion studies of tiamulin in rabbits and for food safety monitoring with respect to MRL compliance of residues.

Sample preparation: 5 g milk + 12.5 μL of 10 μg/mL C6-CLO + 15 mL ACN/H2O (80/20, v/v) Vortex mixing – shaking – centrifugation SPE extraction (Oasis MAX) – elution 5% formic acid in MeOH Evaporation to dryness (50°C, N2) Resuspension: 125 μL ACN + 125 μL H2O HPLC conditions: Alliance type 2695 HPLC (Waters) Stationary phase: Zorbax® Eclipse Plus C18 column (Agilent) LC-MS/MS method EU Volume 8 VICH GL 49

⇒ 8-α-hydroxymutilin eluted at retention time (Tr) of 3.10 min (see Figure 1B) Sample preparation MS/MS conditions HPLC conditions Tiamulin is a diterpene antimicrobial agent with a pleuromutilin chemical structure, which is mainly active against Gram-positive micro-organisms and Mycoplasma. In rabbits, tiamulin is used for the treatment of Epizootic Rabbit Enteropathy. Maximum Residue Levels (MRLs) for rabbit tissues have been established by the European Union for the marker residue, i.e. the sum of metabolites that may be hydrolysed to 8-α-hydroxymutilin, at 100 μg kg-1 for muscle and 500 μg kg-1 for liver.1

A specific and sensitive LC-MS/MS method was developed for quantitative determination of closantel in bovine and ovine colostrum and tank milk. Sample preparation consisted of extracting milk samples with acetonitrile/acetone (80/20, v/v) followed by SPE clean-up with Oasis (R) mixed anion exchange columns. After elution with 5% formic acid in acetonitrile and evaporation to dryness, the residue was reconstituted in acetonitrile and water. HPLC separation was achieved on a Zorbax (R) Eclipse Plus C18 column and a gradient elution program with 1 mM ammonium acetate in water and acetonitrile. For closantel determination in bovine milk, the method was validated according to EU Volume 8 guidelines whereas for ovine milk both EU Volume 8 and VICH GL49 criteria were applied. The linear range of the method is between 10 and 2000 mu g/kg, the limit of quantification 10 mu g/kg and limit of detection is 0.63 and 0.32 mu g/kg for sheep colostrum and tank milk and 1.27 and 1.24 mu g/kg for cattle colostrum and tank milk, respectively. Both guidelines cover a similar set of parameters (linearity, accuracy, precision, limit of detection and limit of quantification), although the acceptance criteria might differ (accuracy and precision) or no specific acceptability ranges are specified in neither guidelines (LOD and LOQ). For some parameters, only one of the guidelines indicates acceptance criteria: EU Volume 8 for applicability, practicability and susceptibility and VICH GL 49 for linearity, specificity and analyte stability. (C) 2014 Elsevier B.V. All rights reserved.

Introduction: Serotonin (5-hydroxytryptamine) is an important signaling molecule in the body and a major regulator of gastrointestinal motility. It is released in increased quantities in several conditions, including intestinal ischemia-reperfusion and platelet activation. In surgical colic cases, changes in free plasma serotonin could hypothetically contribute to the development of equine postoperative ileus. Therefore plasma serotonin concentrations were compared in healthy horses and horses undergoing laparotomy for small intestinal colic. Materials and Methods: a total of 164 EDTA plasma samples were collected from 24 control horses and 34 colic horses. Samples from the colic horses were collected at admission, immediately after surgery and at least the first morning postoperatively. Platelet stabilizing agents aspirin (1 mM), clomipramine (1 µM) and phenelzine (10 µM) were added and samples were centrifuged to produce platelet poor plasma. Serotonin concentrations were determined with a validated liquid chromatography tandem mass spectrometry method (LC-MS/MS). Serotonin differences were analyzed using logistic regression. Results: Small intestinal surgical colic cases had significantly lower plasma serotonin concentrations than healthy horses (n = 55; p < 0.001; OR 0.96; 95% CI 0.94-0.98) at admission, with a trend towards lower concentrations postoperatively (n = 55; p = 0.08) and the next morning (n = 55; p = 0.08). Within the group of colic horses, there was no difference in plasma serotonin at admission between horses that developed ileus and horses with a normal intestinal recovery (n = 27; p = 0.91), nor between horses that survived to discharge and non-survivors (n = 31; p = 0.89). Conclusions: Plasma serotonin concentrations were lower in surgical small intestinal colic horses. There was no relation to development of ileus or postoperative survival. Variable results have been published concerning serotonin values in healthy and diseased horses. More studies using standardized sampling protocols and reliable measurement methods are needed.

Serotonin is regularly measured in equine platelet-poor plasma in research settings. However, reported reference values vary between studies, partially because plasma serotonin concentrations are very low and a reliable and affordable detection method is lacking. A simple, rapid, and sensitive method for serotonin determination in equine platelet-poor plasma using liquid chromatography--tandem mass spectrometry (LC-MS/MS) was developed and validated. Results of a commercially available enzyme-linked immunosorbent assay (ELISA) were compared to the LC-MS/MS results, in order to validate a test more suitable for use in a clinical situation. For LC-MS/MS, 500 µl of plasma was required, and deuterated serotonin was used as an internal standard. The sample preparation was based upon a simple liquid extraction into ethyl acetate. Chromatographic separation was performed with an acetic acid--acetonitrile mobile phase gradient elution. Linearity was demonstrated between 3 ng/ml and 100 ng/ml. A limit of quantification of 3 ng/ml was achieved, corresponding to a limit of detection of 0.10 ng/ml. Comparison of LC-MS/MS and ELISA with Passing-Bablok regression and Bland--Altman plotting showed a poor agreement between the 2 methods, with an increasing difference within the higher range of measurements. Caution is needed when extrapolating results from sources using different analytical techniques.

Two methods are presented for the determination of 'respectively' the plasma protein unbound and total concentration of acyclovir in horse plasma and body fluids: first, a liquid-liquid extraction was performed on plasma, combined with HPLC-fluorescence detection for the total plasma concentration; second a more sensitive method using high-performance liquid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC-HESI-MS/MS) was described for plasma and for body fluids analysis. To obtain the unbound concentration of acyclovir in plasma, a simple deproteinization step using a Microcon filter was performed. Ganciclovir was used as an internal standard. Analysis was carried out on an Inertsil 5 ODS-3 column for the HPLC-fluorescence method. For the LC-HESI-MS/MS method a PLRP-S column was used. The limit of quantification (LOQ) for the total concentration was set at 50 and 2 ng mL(-1) for the HPLC-fluorescence method and the LC-HESI-MS/MS method, respectively. The limit of quantification for the unbound concentration was set at 5 ng mL(-1) and at 2 ng mL(-1) for body fluids. The methods were successfully used to perform pharmacokinetic and clinical studies in horses after intravenous and oral dosage of acyclovir and its prodrug valacyclovir.