Development of a lateral flow immunoassay strip for screening of sulfamonomethoxine residues

A rapid lateral flow immunoassay screening method has been developed for the determination of sulfamonomethoxine (SMM) residues in swine urine. For this purpose, a specific monoclonal antibody (mAb), SMM4B9 for SMM, was generated and characterized. The mAb showed low cross-reactivity (not larger than 0.3%) to other sulfonamides and other potentially occurring analytes. Based on the competitive immunoassay principle, the strip was developed with the mAb SMM4B9 and applied to the screening of SMM residues. The test strip is made up of a sample pad, a gold-conjugate SMM4B9 reagent pad, a blotted test membrane containing a test line, a control line (a nitrocellulose membrane spotted with SMM-BSA and goat anti-mouse antibody, respectively), and an absorbent pad. The test could be accomplished within 8-10 min. It was shown that the sensitivity of the test strip was as low as 5 ng ml^-1 of SMM and the half of maximal inhibition concentration (IC₅₀) was calculated to be 10.78 ± 0.22 ng ml^-1 by relative optical density. In unaided visual assessment the detection limit of the strip was 15 ng ml^-1. For samples spiked at 20 and 30 ng ml^-1 the coefficient of variation (CV (%)) was between 2.3 and 7.1%. When the test strip was compared with high-performance liquid chromatography (HPLC) analysis for naturally contaminated swine urine samples, the difference in results was less than 6.1%. The data suggest that the method has advantages of high sensitivity, specificity, simplicity and speed of performance, as well as the characteristics of repeatability, reproducibility or accuracy and assurance. Therefore, the test strip is suitable to determine SMM residues in swine urine rapidly and reliably by quantitative, semi-quantitative or qualitative detection.     

Development of rapid immunoassays for the detection of ractopamine in swine urine

The monoclonal antibodies (mAbs) against ractopamine (Rac) were prepared and their properties identified by indirect competitive enzyme-linked immunoabsorbant assay (ELISA). The IC₅₀ of mAbs was 2.7 ng ml^?1 towards Rac or 9.3 ng ml^?1 towards Rac-glucuronides and no cross-reactivity (CR) towards other competitors except dobutamine (CR: 3.76%). Based on the mAbs, the Rac-kit (kit) and Rac-strip (strip) were developed to detect Rac residues in swine urine. The strip and kit assay could be performed within 5–10 min and 2 h, respectively, allowing the analysis of urine samples without the need for sample clean-up. The detection limits were 1 ng ml^?1 for kit and 3 ng ml^?1 with the unaided eye, and 0.2 ng ml^?1 with the Strip Reader for strip. The correlation coefficients (R ²) were 0.988 for kit in the range 0–128.0 ng ml^?1, and 0.987 for strip in the range 0–10.8 ng ml^?1. Comparing the gas chromatography-mass spectrometry (GC-MS) with the kit or strip in swine urine spiked with Rac standards, the differences ranged from 1.4% to 4.5% for kit and 1.0% to 4.7% for strip. However, the differences were greater than 54% for the kit and 55% for the strip test for the analysis of urine from swine treated with Rac. The results obtained from GC-MS using hydrolysed urine samples were generally in good agreement with those obtained from strip or kit using non-hydrolysed urine samples.     

Development of a Monoclonal Antibody-Based Immunochromatographic Assay Detecting Ractopamine Residues in Swine Urine

A lateral-flow immunochromatographic assay (LFIA) using monoclonal antibody was developed for the rapid detection of ractopamine residues in swine urine. The assay procedure could be accomplished within 5 min, and the results of this qualitative one-step assay were evaluated visually according to whether test lines appeared or not. When applied to the swine urines, the detection limit and the half maximal inhibitory concentration (IC₅₀) of the lateral-flow immunochromatographic test strip under an optical density scanner were calculated to be 0.1?±?0.013 and 0.71?±?0.056 ng/mL, respectively. The cut-off level was observed with the naked eye of 1 ng/mL for detection of ractopamine residue in swine urine. Parallel analysis of swine urine samples with ractopamine showed comparable results obtained from the LFIA and LC-MS/MS. Therefore, the described lateral-flow test strip can be used as a reliable, rapid and cost-effective on-site screening technique for the determination of ractopamine residues in swine urine.     

Modified dispersive liquid–liquid microextraction followed by high-performance liquid chromatography for the determination of clenbuterol in swine urine

A modified dispersive liquid–liquid microextraction (DLLME) technique combined with an HPLC-UV procedure was developed for the extraction and determination of clenbuterol in swine urine. The modification involved the selection of methyl tert-butyl ether (MTBE) as the dispersive solvent, which had a low solubility in aqueous samples, playing the part of dispersion with the help of violent shaking. MTBE improved the partition of clenbuterol into the extractant, and helped the formation of phase separation. Various factors affecting the extraction efficiency including selection of the organic extractant and the dispersive solvent, the volume of extractant and dispersive solvent, salt concentration, NaOH concentration and centrifugation time were evaluated and optimised. Under the optimal conditions, precision, linearity (correlation coefficient, r ^2?=?0.996 over the concentration range of 10–1000?ng?ml^?1), detection limit (2.4?ng?ml^?1) and enrichment factor of 55 were obtained. The modification to the DLLME made it suitable for analytes with pronounced solubility, especially when the compounds are highly polar and thus more difficult to extract effectively by DLLME. The procedure was suitable for the fast screening of clenbuterol residue in swine urine.     

Development of a polyclonal indirect ELISA with sub-ng g−1 sensitivity for the analysis of clenbuterol in milk,animal feed,and liver samples and a small survey of residues in retail animal products

An indirect competitive enzyme-linked immunosorbent assay (ELISA) with sub-ng g^?1 sensitivity for clenbuterol was developed. The antisera obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. All four antisera displayed high sensitivity with IC₅₀ and limit of detection (LOD) values lower than 0.9 and 0.03 ng ml^?1, respectively. The most sensitive ELISA was established with IC₅₀ and LOD values of 0.1–0.3 and 0.01–0.02 ng ml^?1, respectively, which are more than ten times lower than those reported in the literature. The cross-reactivity (CR) values of the four antisera with salbutamol, another frequently used β-agonist of similar molecular structure to clenbuterol, were estimated to be within 25–46%. No binding (CR < 0.01%) of nine other drugs which are frequently used in animal feeds was observed. The superior ELISA at optimal assay conditions was used for the analysis of five clenbuterol-fortified samples and the results were confirmed by high-performance liquid chromatography (HPLC). Acceptable recovery rates of 92.2–97.0% and intra-assay coefficients of variation of 1.3–5.3% were obtained. The proposed ELISA was highly correlated with HPLC (R ²= 0.9893, n = 5). Another sixteen samples were randomly collected from local markets and analysed by ELISA. The highest clenbuterol residues found in milk, feed, swine and chicken livers were 5.33, 64.04, 2.28 and 0.74 ng g^?1, respectively.     

Development and validation of an immunochromatographic assay for the rapid detection of quinoxaline-2-carboxylic acid,the major metabolite of carbadox in the edible tissues of pigs

A new method was developed for the determination of quinoxaline-2-carboxylic acid, the marker residue of carbadox, in the edible tissues of food-producing animals using a colloidal gold probe-based immunochromatographic assay. The highly specific polyclonal antibody (PcAb), which was very sensitive to N-butylquinoxaline-2-carboxylic acid (BQCA) with an IC₅₀ value of 2.38?ng?ml^?1, was selected for the development of an immunochromatographic assay (ICA). Only 5?min were required to perform this assay; it had a visual detection limit of 25?ng?g^?1 for quinoxaline-2-carboxylic acid. The results of the analysis of quinoxaline-2-carboxylic acid in animal tissues using the immunochromatographic assay showed good agreement with those obtained by HPLC. In conclusion, the method was rapid and accurate for screening residues of carbadox in the edible tissues of food-producing animals.     

Effects of oral exposure of pigs to deoxynivalenol (DON) sulfonate (DONS) as the non-toxic derivative of DON on tissue residues of DON and de-epoxy-DON and on DONS blood levels

The Fusarium toxin deoxynivalenol (DON) is of outstanding importance in pig nutrition because of its frequent occurrence in cereal grains at levels high enough to cause adverse effects such as a decrease in feed intake and impairment of the immune system. Thus, simple decontamination procedures would be useful. The present study aimed to examine the effects of wet preservation of triticale contaminated with DON and zearalenone (ZON) with sodium metabisulphite (SBS) on the treatment-related non-toxic derivative of DON (DON-sulfonate, DONS), and on ZON and its metabolites in blood and various physiological specimens of piglets. The uncontaminated control triticale (CON) and the DON-contaminated triticale (FUS) were included in the diets either untreated or SBS treated (CON-SBS, FUS-SBS) and fed to piglets for 28 days starting from weaning. The diet concentrations for DON were 0.156, 0.084, 2.312 and 0.275 mg kg^?1, for DONS were <0.05, <0.05, <0.05 and 1.841 mg kg^?1, and for ZON were <0.001, 0.006, 0.017, and 0.016 mg kg^?1 for each of CON, CON-SBS, FUS and FUS-SBS, respectively. DONS was present in the blood of piglets fed the FUS-SBS at a median concentration of 15.5 ng ml^?1 (3–67 ng ml^?1), while the median DON concentration amounted to 2 ng ml^?1 (0–5 ng ml^?1) at the same time. The median DON concentration in the blood of piglets fed the FUS diet reached a median concentration of 10.5 ng ml^?1 (5–17 ng ml^?1). Moreover, the relative differences between the DON concentrations in other physiological specimens (muscle, liver, kidney, bile and urine) in piglets fed the FUS-SBS and the FUS diet were comparable with the blood DON concentration differences. Although these differences can be taken as an indication for DONS stability after absorption and distribution further studies examining DONS in these other physiological specimens directly are necessary to substantiate this conclusion. Moreover, ZON and α-zearalenol could only be detected in bile and urine where their levels were not influenced by the SBS treatment.     

Quantitative determination of mycotoxins in urine by LC-MS/MS

Naturally occurring mycotoxins are responsible for a wide array of adverse health effects. The measurement of urinary mycotoxin levels is a useful means of assessing an individual's exposure, but the development of sensitive and accurate analytical methods for detecting mycotoxins and their metabolites in urine samples is challenging. Urinary mycotoxins are present in low pg ml^–1 concentrations, and the chromatographic identification of their metabolites can be obscured by other endogenous metabolites. We developed an analytical method focused on the selection of two appropriate multiple-reaction monitoring transition for unambiguous identification and quantification of carcinogenic aflatoxin M₁ (AFM₁), ochratoxin A (OTA) and fumonisin B₁, B₂ (FB₁, FB₂) in urine samples from a small volunteer group in a pilot study. AFM₁, OTA, FB₁ and FB₂ were concentrated selectively, interfering substances were removed using an immunoaffinity column (IAC), and mycotoxins were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in combination with a stable-isotope standard-dilution assay (SIDA). The method was sensitive enough to measure mycotoxins and their metabolites at pg ml^–1 levels in urine. The combination of LC-MS/MS and SIDA was critical to distinguishing pseudo-OTα interference from genuine OTα. Twelve urine samples contained OTA ranging from 0.013 to 0.093 ng ml^?1 (mean = 0.031 ng ml^?1). AFM₁ were detected in one sample at a 0.002 ng ml^?1 level, while FB₁ and FB₂ were undetectable in all 12 samples. None of the samples in this pilot study contained a detectable level of OTα, despite the presence of OTA, and this may suggest the need for further epidemiological investigation of OTA exposure in the Korean population.     

Development of an immunochromatographic assay for the β-adrenergic agonist feed additive zilpaterol

Zilpaterol is a β-adrenergic agonist feed additive approved in the United States to increase weight gain and improve feed efficiency of cattle. A zilpaterol immunochromatographic assay was developed as an economical and user-friendly rapid detection method for zilpaterol and validated using urine and tissue samples derived from animal studies. The assay sensitivity was 1.7–23.2 ng g^?1 or mL^?1 across a variety of feed and animal matrices and did not cross-react with clenbuterol or ractopamine. No sample pre-treatment of cattle and sheep urine was needed, but horse urine and feed required dilution; skeletal muscle required solvent extraction prior to testing. Of 32 incurred sheep urine samples tested, zilpaterol content was correctly identified in all but 2 samples. Horse urine containing >10 ng mL^?1 of incurred zilpaterol residue (n = 48) was correctly identified as zilpaterol positive. The assay correctly identified 0-day withdrawal sheep muscle samples as zilpaterol positive and the control and longer withdrawal day sheep muscle samples as negative. Zilpaterol was demonstrated to be stable in horse urine when stored at ?20°C for 7 years.     

A sensitive immunochromatographic assay using colloidal gold–antibody probe for rapid detection of fumonisin B1 in corn

A sensitive immunochromatographic assay (ICA) using a colloidal gold–antibody probe for the rapid detection of fumonisin B₁ (FB₁) in corn samples was developed. The colour density of the test line correlated with the concentration of FB₁ in the range 2–40 ng ml^–1 by the assay, and the detection limit for FB₁ was 2 ng ml^–1. The linear range for FB₁ was 50–1000 µg kg^–1, and the visual limit detection of the test was 1000 µg kg^–1 in corn samples. The ICA to detect FB₁ is sensitive, specific and rapid. Specific anti-FB₁ monoclonal antibody (mAb) and FB₁-ovalbumin (FB₁-OVA) conjugate antigen were prepared. FB₁ mAb, labelled with colloidal gold, was used as the probe on the immunochromatographic strip. FB₁-OVA and goat-anti-mouse IgG were coated onto a nitrocellulose (NC) membrane as test lines and control lines, respectively. FB₁ in samples will competitively combines the FB₁ mAb with the FB₁-OVA in an NC membrane and the results are directly observed by the colour of the detection and quality control lines. The concentrations of FB₁ mAb labelled with colloidal gold, detecting antigen and goat anti-mouse IgG, were optimised. The results indicate that the test strip is specific for FB₁, with no cross-reactivity to other toxins. The strip assay for FB₁ was simple, only needing one step without complicated assay performance and expensive equipment, and the total time for visual evaluation was less than 10 min. A survey of 24 corn samples from Hefei, China, was performed with the test strip and HPLC, and the detection results showed that the developed ICA and the HPLC were in excellent agreement. Hence, the developed ICA can be used as a method for rapid detection of FB₁ in corn samples.     

Rapid on‐site determination of melamine in raw milk by an immunochromatographic strip

A rapid and simple method was established based on gold nanoparticle‐labelled monoclonal antibody probes for the detection of melamine pollution in raw milk. The conditions for conjugation between the antibody and gold nanoparticles were optimised (pH 8.0, antibody concentration 5 μg mL^?1). It gives a single proportional to melamine concentration with a performance time of only 3 min. A practical calibration curve was established with a reader system with limit of detection calculated as 4.47 and 8.34 μg L^?1 with naked eyes. Three structural analogues, atrazine, desethyl‐desisopropyl‐atrazine and cyromazine, were used to test the specificity of the immunochromatographic strip, and small colour changes on the strip test line were found even at the 500 ng mL^?1 spiked level. Fifty raw milk samples were detected with this strip method, and the resulting data coincided well with results from gas chromatography tandem mass spectrometry. The above‐mentioned results showed that this test strip can be used for melamine screening in the daily monitoring of milk.     

Trace level determination of beryllium in natural and flavored mineral waters after pre-concentration using activated carbon

The concentrations of beryllium (Be) in natural and flavored mineral water samples were determined by flame atomic absorption spectrophotometer (FAAS) after pre-concentration based on the complexation of Be⁺² with a mixture of acetylacetone (pentane-2,4-dione) plus morin (3,5,7,2′,4′-pentaoxyflavone) and adsorption on activated carbon. The adsorbed complex was eluted with 1.5?ml of 2.0?M HNO₃ and evaporated to dryness. After adding 1.5?ml of 2?M HNO₃ and centrifuging, Be in acid solution was determined by FAAS. To remove a number of metals present in water, EDTA was used as a chelating agent. Beryllium in mineral water samples was pre-concentrated by 500-fold, taking 750?ml as initial sample and 1.5?ml as the final volume. The relative standard deviations were sufficiently low for practical purposes and recoveries were up to 85%. Spiking experiments were performed in real samples to establish accuracy and recoveries. The limits of detection and quantification were 0.01 and 0.03?ng?ml^?1, respectively. Twenty samples were analyzed for their beryllium content using optimum parameters. The highest concentration of beryllium was found to be 0.94?±?0.15?ng?ml^?1 in a natural mineral water, while beryllium was not detected in five samples.     

Evaluation of three different microbial inhibition tests for the detection of sulphamethazine residues in the edible tissues of rabbit

The aim of the present study was to evaluate three microbial inhibition tests (MIT) based on inhibition of growth of the test organisms: (a) four plate test (FPT) containing Bacillus subtilis BGA, (b) screening test for antibiotic residues (STAR) containing Bacillus stearothermophilus var. calidolactis_ATCC 10149 and (c) the Premi®Test containing Bacillus stearothermophilus var. calidolactis. The tests were used to determine sulphamethazine (SMZ) residues in edible tissues of rabbit after oral administration up to day 15 of the withdrawal period (WP). A solvent extraction procedure was used to enhance the capability of the tests to detect SMZ residues at or below the maximum residue limit (MRL). Para-aminobenzoic acid (PABA) was employed to previously identify SMZ residues in the first stage of the residue screening. The presence of SMZ residues in the samples was confirmed and quantified by a validated HPLC method. The Premi®Test detected SMZ residues in the muscle and heart tissue up to day 9 of the WP, and in the liver, lungs and kidneys up to day 10 of the WP. The STAR detected SMZ residues in the edible organs of rabbits up to day 8 of the WP. The kidneys were positive up to day 5 of the WP, the liver until day 4 of the WP and the lungs until day 3 of the WP. No SMZ residues were detected in the muscle and heart. By using the solvent extraction procedure, SMZ residues were detected in the muscle extract up to day 10 of the WP and the muscle was positive until day 6 of the WP. No detection sensitivity was observed using the FPT. After solvent extraction, SMZ residues were detected in the muscle extract until day 8 of the WP and the muscle was positive until day 3 of the WP. No positive results were detected after the addition of PABA into/onto the agar medium. PABA at a concentration of 10 µg ml^?1 completely reversed the inhibitory activity of SMZ and enabled reliable identification of SMZ in the examined samples. Using HPLC, SMZ was detected in the muscle samples until day 10 of WP (0.02 mg kg^?1) and in the liver until day 12 of the WP (0.09 mg kg^?1). The results obtained by the HPLC method and the limit of detection (LOD) of screening tests for SMZ (FPT 0.4 µg ml^?1, STAR 0.2 µg ml^?1, Premi® Test 0.05 µg ml^?1) allowed us to state that the most suitable screening tests for the detection of SMZ residues in the edible tissues of rabbits at level corresponding to the MRL of 0.1 mg kg^?1, established for sulphonamides, are the Premi®Test and STAR in conjunction with the solvent-extraction procedure.     

UPLC-ESI-MS/MS for determining trans- and cis-vitamin K1 in infant formulas: method and applications

A simple and sensitive method of determining vitamin K₁ isomers (cis-and trans-forms) was developed for routine monitoring of supplemented infant formulas. The proposed method involves the applications of the lipase hydrolysis and the liquid-to-liquid extraction. After the hydrolysis and extraction procedures, the vitamin K₁-enriched extract was directly injected into the ultra-performance liquid chromatography–tandem mass spectrometry system (UPLC-MS/MS). The components were detected using electrospray ionization (ESI) in positive-ion and quantified by multiple-reaction monitoring (MRM) mode. trans-vitamin K₁ was separated from the biologically inactive cis-isomer through a C₃₀ column (4.6?×?150?mm, 3?μm), and vitamin K₁-d₇ was used as an internal standard. Calibration curves were linear over the range of 9.3–464.75?ng?ml^?1 for trans-vitamin K₁ (r ²?>?0.999) and 1.71–85.25?ng?ml^?1 for cis-vitamin K₁ (r ²?>?0.999). The limit of detection for trans- and cis-vitamin K₁ was 0.011?μg 100?g^?1 and 0.01?μg 100?g^?1. The limit of quantification was 0.037?μg 100?g^?1 and 0.031?μg 100?g^?1, respectively. The intra- and inter-batch variations (RSD%) were less than 5?%. The proposed method was applied to determine vitamin K₁ isomers in milk-based, soy-based and rice-based infant formulas, and the cis-vitamin K₁ isomer contributes to 7.05–17.21?% of the total vitamin K₁ in certain infant formulas.     

Deoxynivalenol exposure assessment in a cohort of pregnant women from Bradford,UK

Deoxynivalenol (DON) is a ubiquitous contaminant of cereal crops in temperate regions of the world. It causes growth faltering and immune suppression in animals. Limited information is available on DON exposure in UK subpopulations. The objective of this study was to provide DON exposure assessment in a subset of pregnant women scheduled for an elective caesarean in a large multi-ethnic mother/infant birth cohort from Bradford, UK. Women aged 16–44 years (n?=?85) provided a urine sample for DON analysis in the last trimester of pregnancy, and concurrently completed a food-frequency questionnaire (FFQ). The urinary DON biomarker was detected in all measured samples (geometric mean (GM)?=?10.3?ng?DON?mg^?1 creatinine, range?=?0.5?116.7?ng?mg^?1). Levels were higher in women classified as South Asian in origin (GM: 15.2?ng?mg^?1; 95% CI?=?10.7?21.5?ng?mg^?1) compared with non-South Asians (GM?=?8.6?ng?mg^?1; 95% CI?=?6.6?11.8?ng?mg^?1), p?=?0.02). Estimated DON intake from FFQ data and typical levels of DON contamination of food suggest that this was mainly due to higher levels of exposure from bread, particularly daily intake of DON from chapattis in South Asians (estimated mean?=?2.4?µg?day^?1; 95% CI?=?1.2, 3.7?µg?day^?1) compared with non-South Asians (estimated mean?=?0.2?µg?day^?1; 95% CI?=?0?0.4?µg?day^?1), p?<?0.001. This is the first biomarker demonstration of DON exposure in pregnant women, and several urinary DON levels were the highest ever recorded in any study. A larger survey within this birth cohort is warranted to investigate any potential risk to mothers and their babies, from DON exposure during pregnancy.     

Simultaneous determination of five quinoxaline-1,4-dioxides in animal feeds using an immunochromatographic strip

An immunochromatographic (ICG) strip was developed for the simultaneous quantitative determination of five quinoxaline-1,4-dioxides in animal feed. For this purpose, polyclonal antibodies (PcAb) with group-specific quinoxaline-1,4-dioxides were conjugated to colloidal gold particles as the detection reagent for ICG strips to test for quinoxaline-1,4-dioxides. This method achieved semi-quantitative detection of quinoxaline-1,4-dioxides within 5–10 min. The visual lower detection limits of the strip for quinocetone, cyadox, carbadox, mequindox and olaquindox were 10, 15, 15, 20 and 20 ng ml^?1, respectively. Using an ICG strip reader, the 50% inhibitions (IC₅₀ values) were calculated to be 9.1, 13.5, 16.6, 20.2 and 21.3 ng ml^?1 for quinocetone, cyadox, carbadox, mequindox and olaquindox, respectively. When used to analyse samples of animal feed, acceptable recovery rates of 77.5–99.5% and coefficients of variation (CVs) of 4.3–10.7% were obtained. Levels measured with the ICG strip for 10 spiked samples were confirmed by HPLC with a high correlation coefficient of 0.9965 (n = 10). In conclusion, the method was rapid and accurate for simultaneous determination of five quinoxaline-1,4-dioxides antibiotics in animal feed.     

Ultrasensitive and Specific Detection of Salbutamol in Swine Feed,Meat, and Urine Samples by a Competitive Immunochromatographic Test Integrated with Surface-Enhanced Raman Scattering

Salbutamol (SAL), a kind of β-agonist which can enhance the lean meat-to-fat ratio, has been inhibited as an additive used in animal feeds for livestock production in many countries due to its harmful effect to the consumers. In this study, an ultrasensitive and specific competitive immunochromatographic test (ICT) integrated with surface-enhanced Raman scattering (SERS) for the detection of SAL was described. The immunoprobe was prepared by immobilizing polyclonal antibody against SAL on the surface of Au@Ag nanoparticles in which the Raman reporter (4-mercaptobenzoic acid, MBA) had been sandwiched. After ICT procedures, the specific SERS signals generated from MBA on the test line of the ICT strip were measured for the quantitative determination of SAL. The assay was completed in 15 min. The IC₅₀ and the limit of detection (LOD) values of the assay for SAL were 0.028 ng mL^?1 and 3.0 pg mL^?1, respectively. There was no cross-reactivity (CR) of the assay with other three β-agonists (clenbuterol, phenylethanolamine A, and ractopamine), showing high specificity of the assay. Spiking experiments indicated that the average recoveries (n?=?3) of SAL from swine feed, meat, and urine samples were in ranges of 98.4–105.2 % with the relative standard deviations (RSDs) of 1.7–7.8 %. The results demonstrated that the proposed ICT was a feasible method for ultrasensitive and specific detection SAL in swine feed, meat and urine samples, and could be extended for the detection of other target analytes.     

Unravelling a vicious circle: animal feed marketed in Costa Rica contains irregular concentrations of tetracyclines and abundant oxytetracycline-resistant Gram-positive bacteria

Diverse tetracyclines are used to prevent and control bacterial infections in livestock and farmed fish. These drugs are administered through the diet, but farmers seldom check whether feed contains antibiotic-resistant bacteria that may colonise their crops or transfer their resistance traits to species of veterinary relevance. To examine whether antibiotic dosage defines the abundance of antibiotic-resistant bacteria in animal feed, we determined the concentration of parental compounds and epimers of oxytetracycline (OTC), doxycycline, tetracycline and chlortetracycline, as well as the abundance and resistance level of OTC-resistant bacteria in samples of fish (n = 21), poultry (n = 21), swine (n = 21), and shrimp feed (n = 21) marketed in Costa Rica. Fish feed contained the highest amounts of tetracyclines (119–8365 mg kg^?1) and the largest proportion of bacteria resistant to 10 μg ml^?1 (1.8–92.4%) or 100 μg ml^?1 of OTC (12.5–63.8%). Poultry (78–438 mg kg^?1) and swine (41–1076 mg kg^?1) feed had intermediate concentrations of tetracyclines and OTC-resistant bacteria (0.2–66% and 0.3–49%, respectively), whereas shrimp feed showed the lowest amounts of tetracyclines (21.5–50.3 mg kg^?1), no OTC and no culturable OTC-resistant bacteria. In line with these results, the MIC₅₀ of OTC for 150 isolates from fish and poultry feed was > 256 µg ml^?1, while that of 150 bacteria isolated from swine feed was 192 µg ml^?1. Phenotypic tests, fatty acid profiles and proteotypic analyses by matrix-assisted laser desorption/ionisation-time of flight mass-spectroscopy revealed that most OTC-resistant isolates were Gram-positive bacteria of low G+C% content from the genera Staphylococcus and Bacillus. Clear correlations between OTC dosage and feed colonisation with OTC-resistant bacteria were seen in medicated feed for fish (r = 0.179–0.651). Nonetheless, some unmedicated feed for fish, swine and poultry contained large populations of OTC-resistant bacteria, suggesting that raw materials and manufacturing processes may also influence carriage of OTC-resistant bacteria in animal feed.     

A New Extraction Method for the Determination of Oxytocin in Milk by Enzyme Immune Assay or High-Performance Liquid Chromatography: Validation by Liquid Chromatography–Mass Spectrometry

There has been a controversy regarding the use of exogenous oxytocin (OT) in milking cattle which may have toxicological consequences during nonphysiological exposure. In the present study, a new sensitive extraction method for OT was developed followed by enzyme immune assay (EIA) or high-performance liquid chromatography (HPLC) analysis. The extraction of OT in milk involves two steps: (1) TCA precipitation of milk proteins and (2) solid-phase extraction (SPE) cleanup process. Without these steps, analysis of OT in milk was not possible. Utilizing EIA as a quantitative tool the limit of detection (LOD) and limit of quantitation (LOQ) were found to be 7.74 and 10.3 pg?ml^?1, precision in terms of intra- and interday coefficient of variation was below 13 % (%RSD, N?=?8), while percent recoveries were between 85 and 92 %. Utilizing UV-HPLC, the LOD, LOQ, precision, and recovery values were found to be 4.1 ng?ml^?1, 9.8 ng?ml^?1, 2–10 %, and 84–91 %, respectively. OT was found to be stable against adverse temperature (up to 100 °C) and pH (2 to 10) and simulated gastric fluid digestibility assay. Four milk samples collected from the market were analyzed, which showed that TCA precipitation and SPE steps are mandatory and the results were validated by LC-MS showing mass ion peak at 1 kD.     

Inactivation of yeast and filamentous fungi by the lactoperoxidase-hydrogen peroxide-thiocyanate-system

The antifungal activity of the lactoperoxidase (LPO) system with glucose oxidase (GOD) as source of hydrogen peroxide was determined in salt solution and in apple juice. The test organisms Rhodutorula rubra and Saccharomyces cerevisiae were cultivated aerobically in apple juice, Mucor rouxii was grown on wort agar adjusted to pH 4.5. Aspergillus niger and Byssochlamys fulva were kept on malt extract agar. Spores of the filamentous fungi were harvested by suspension in salt solution supplemented with Tween 80® and checked microscopically. The antifungal activity of the combined enzyme system was tested with initial counts of approx. 10⁵ cfu · ml^?1 (yeast cells or spores) suspended in salt solution supplemented with 25 mg · l^?1 thiocyanate and 20 g · l^?1 glucose or in apple juice supplemented with the same amount of thiocyanate. The tests were performed with 25 ml of the medium in 100 ml Erlenmeyer flasks shaken at 28 °C under aerobic conditions. Inactivation was achieved for all test organisms in both media. The yeast strains were found to be least stable while B. fulva was most resistant. A combination of 5 U · ml^?1 LPO with 0.5 to 1 U · ml^?1 GOD was sufficient for complete inactivation of this mold in salt solution within 2 h. The enzyme system also showed antifungal activity in apple juice at acid pH (3.2), although its effectiveness was reduced. In this medium, B. fulva was inactivated by 20 U · ml^?1 LPD and 1 U · ml^?1 GOD within 4 h. R. rubra and S. cerevisiae were unable to survive in apple juice at 5 U · ml^?1 LPO combined with 1 U · ml^?1 GOD. For inhibition by GOD alone, higher amounts of this enzyme were needed and even then only M. rouxii and R. rubra have been affected within the concentration range tested (maximum 3 U · ml^?1).