Development and validation of a rapid LC-MS/MS method for the confirmatory analysis of the bound residues of eight nitrofuran drugs in meat using microwave reaction.

A rapid analytical method was developed and validated for the analysis of eight bound nitrofurans in animal tissue, shortening laboratory turnaround times from 4 to 2 days. The majority of methodologies for nitrofuran analysis focus on the detection of only four drugs (nitrofurantoin, furazolidone, furaltadone and nitrofurazone), and is time-consuming given the 16-h overnight derivatisation step and a double liquid-liquid extraction. In this study, the narrow scope of analysis was addressed by including further four important nitrofuran drugs (nifursol, nitrofuroxazide, nifuraldezone and nitrovin). Full chromatographic separation was achieved for the metabolites of all eight nitrofurans, using phenyl-hexyl column chemistry and a rigorous optimisation of the mobile phase additives and gradient profile. The conventional, lengthy sample preparation was substantially shortened by replacing the traditional overnight water bath derivatisation with a rapid 2-h microwave-assisted reaction, followed by a modified-QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction. This confirmatory method was fully validated in accordance with the new 2021/808/EC legislation, and was shown to perform satisfactorily when applied to incurred tissues. The decision limit (CCα) for the eight analytes ranged between 0.013 and 0.200 µg kg-1, showing abundant sensitivity given that the current RPA for nitrofurans is 0.5 µg kg-1. This innovative method can play a major role in the surveillance of the illegal use of nitrofuran drugs.

MRM3-based UHPLC-MS/MS method for quantitation of total florfenicol residue content in milk and withdrawal study profile of milk from treated cows.

Florfenicol is a broad spectrum antibacterial, licensed globally for treatment of animal and aquaculture diseases. In the EU, Canada and US it is not permitted for use in animals producing milk or eggs. There are no published methods for analysis of total florfenicol content in milk/milk products as these lack a hydrolysis step, failing to meet the marker residue definition. A method for determining total florfenicol content in milk that meets this definition is reported for the first time. Use of a UHPLC-MS/MS multiple reaction monitoring-cubed method improved the selective detection and quantitation of lower levels of florfenicol amine in milk compared to MRM only. Single laboratory validation data and withdrawal profile in bovine milk are presented. A withdrawal period of over 50 days is indicated in case of off-label use. Requirement for hydrolysis is demonstrated.

Verification of Bound Aminoguanidine as the Marker Residue for the Banned Antibiotic, Nitrovin, in Pig Tissues.

Nitrovin (NTV) belongs to a class of antibiotics called nitrofurans, which are classified as nonallowed pharmacologically active substances that do not have a maximum residue limit listed in EU legislation. The objectives of this study were to confirm aminoguanidine (AGN) as a suitable marker residue to monitor NTV abuse and to investigate its persistence in porcine tissues. In this work, pigs were fed with NTV-medicated feed (50 mg/kg), and tissues (kidney, muscle, and liver) and plasma were collected on different withdrawal days. All samples were analyzed for bound AGN, total AGN, and the parent drug NTV itself. The highest concentrations of AGN residues were found in the liver, while the lowest were in muscle. Parent NTV was only detected in the kidney at low levels on day 0 of withdrawal. The findings are in support of using AGN as the marker residue for monitoring the illegal use of NTV in animal-derived products.

Development of an optical biosensor based immunoassay to screen infant formula milk samples for adulteration with melamine.

The illegal adulteration of milk with melamine in 2008 in China led to adverse kidney and urinary tract effects in hundreds of thousands of children and the reported deaths of six. The milk had been deliberately adulterated to elevate the apparent protein content, and subsequently melamine was detected in many milk-related products which had been exported. This led to the banning of imports of milk and milk products from China intended for the nutritional use of children and to the implementation of analytical methods to test products containing milk products. An optical biosensor inhibition immunoassay has been developed as a rapid and robust method for the analysis of infant formula and infant liquid milk samples. A compound with a chemical structure similar to that of melamine was employed as a hapten to raise a polyclonal antibody and as the immobilized antigen on the surface of a biosensor chip. The sensitivity of the assay, given as an IC(50), was calculated to be 67.9 ng mL(-1) in buffer. The antibody did not cross-react with any of the byproducts of melamine manufacture; however, significant cross-reactivity was observed with the insecticide cyromazine of which melamine is a metabolite. When sample matrix was applied to the assay, a limit of detection of <0.5 µg mL(-1) was determined in both infant formula and infant liquid milk. The development of the immunoassay and validation data for the detection of melamine is presented together with the results obtained following the analysis of melamine-contaminated milk powder.

In vitro surfactant mitigation of gas bubble contact-induced endothelial cell death.

Interactions of gas embolism bubbles with endothelial cells, as can occur during decompression events or other forms of intravascular gas entry, are poorly characterized. Endothelial cells respond to microbubble contact via mechanotransduction responses that can lead to cell death or aberrant cellular function. Cultured bovine aortic endothelial cells were individually contacted with microbubbles. Cells were loaded with fluorescent dyes indicating calcium- and nitric oxide-signaling and cell viability. A surfactant, Pluronic F-127, and/or albumin were added to the culture media. Control experiments utilized calcium-free media as well as probe-poking in place of microbubble contact. We acquired fluorescence microscopy time-lapse images of cell responses to bubble and probe contact and determined contact effects on cell signaling and cell death. Calcium influx was essential for cell death to occur with bubble contact. Bubble contact stimulated extracellular calcium entry without altering nitric oxide levels unless cell death was provoked. Cell responses were independent of bubble contact duration lasting either one or 30 seconds. Microbubble contact provoked cell death over seven times more frequently than micropipette poking. Albumin and the surfactant each attenuated the calcium response to bubble contact and also reduced the lethality of microbubble contact by 67.4% and 76.0%, respectively, when used alone, and by 91.2% when used together. This suggests that surface interactions between the bubble or probe interface and plasma- and cell surface-borne macromolecules differentially modulate the mechanism of calcium trafficking such that microbubble contact more substantially induces cell death or aberrant cellular function. The surfactant findings provide a cytoprotective approach to mitigate this form of mechanical injury.

Screening method for the detection of residues of amphenicol antibiotics in bovine milk by optical biosensor.

An immunobiosensor assay was developed for multi-residue screening in bovine milk of the parent amphenicols, thiamphenicol and florfenicol, along with the metabolite florfenicol amine. A polyclonal antibody raised in a rabbit after immunisation with a florfenicol amine-protein conjugate was employed in the assay. Milk samples were subjected to acetonitrile extraction, reconstituted in buffer and diluted prior to biosensor analysis. Validation data obtained from the analysis of fortified samples has shown that the method has a detection capability of less than 0.25 µg kg-1 for florfenicol and less than 0.5 µg kg-1 for florfenicol amine and thiamphenicol. The cross-reactivity profile and validation data for the detection of these amphenicols is presented together with results obtained following the analysis of florfenicol incurred samples using the developed screening method along with a comparison of results obtained from the analysis of the same incurred samples using an MRM3 UPLC-MS/MS confirmatory method. Results are also presented obtained from the analysis of samples from both treated and non-treated animals which were co-housed and which show the potential for cross-contamination.

Feasibility of a clinical chemical analysis approach to predict misuse of growth promoting hormones in cattle.

A study was performed to determine if targeted metabolic profiling of cattle sera could be used to establish a predictive tool for identifying hormone misuse in cattle. Metabolites were assayed in heifers (n = 5) treated with nortestosterone decanoate (0.85 mg/kg body weight), untreated heifers (n = 5), steers (n = 5) treated with oestradiol benzoate (0.15 mg/kg body weight) and untreated steers (n = 5). Treatments were administered on days 0, 14, and 28 throughout a 42 day study period. Two support vector machines (SVMs) were trained, respectively, from heifer and steer data to identify hormone-treated animals. Performance of both SVM classifiers were evaluated by sensitivity and specificity of treatment prediction. The SVM trained on steer data achieved 97.33% sensitivity and 93.85% specificity while the one on heifer data achieved 94.67% sensitivity and 87.69% specificity. Solutions of SVM classifiers were further exploited to determine those days when classification accuracy of the SVM was most reliable. For heifers and steers, days 17-35 were determined to be the most selective. In summary, bioinformatics applied to targeted metabolic profiles generated from standard clinical chemistry analyses, has yielded an accurate, inexpensive, high-throughput test for predicting steroid abuse in cattle.

A review of methodology for the analysis of pyrethrin and pyrethroid residues in food of animal origin.

Pyrethrin and pyrethroid pesticides are commonly used in crop protection and animal health, to control pests. As a result, they can potentially transfer into food if good agricultural practice is not followed or even due to accidental contamination. The analysis of these compounds has been widely reported in crops and the environment. However, the analysis of pyrethrin and pyrethroids has not been reported frequently in foods of animal origin, particularly animal tissues. The focus of this review is to report on pyrethrin and pyrethroid analysis including key aspects such as chemistry, choice of target matrix, sample preparation, chemical analysis, legislation and method validation. This review shows that most methodologies for the analysis of these compounds are based on gas chromatography with the trend in recent years to move towards GC-MS or GC-MS/MS based platforms. This review shows that these compounds can also be satisfactorily analysed by LC-MS/MS, which can be advantageous because of shorter chromatographic run times. A wide range of sample preparation procedures have been applied in analytical methods and more complex protocols are required for GC applications, whereas more crudely prepared extracts can be analysed by LC-MS/MS. This review demonstrates that pyrethrin and pyrethroid residues should be included as analytes in multi-class analytical methods for pesticides and veterinary drug residues in animal derived foods.

Detection of Paralytic Shellfish Toxins in Mussels and Oysters Using the Qualitative Neogen Lateral-Flow Immunoassay: An Interlaboratory Study.

Paralytic shellfish toxins (PSTs) in bivalve molluscs represent a public health risk and are controlled via compliance with a regulatory limit of 0.8 mg saxitoxin (STX)⋅2HCl equivalents per kilogram of shellfish meat (eq/kg). Shellfish industries would benefit from the use of rapid immunological screening tests for PSTs to be used for regulation, but to date none have been fully validated. An interlaboratory study involving 16 laboratories was performed to determine the suitability of the Neogen test to detect PSTs in mussels and oysters. Participants performed the standard protocol recommended by the manufacturer and a modified protocol with a conversion step to improve detection of gonyautoxin 1&4. The statistical analysis showed that the protocols had good homogeneity across all laboratories, with satisfactory repeatability, laboratory, and reproducibility variation near the regulatory level. The mean probability of detection (POD) at 0.8 mg STX⋅2HCl eq/kg using the standard protocol in mussels and oysters was 0.966 and 0.997, respectively, and 0.968 and 0.966 using the modified protocol. The estimated LOD in mussels was 0.316 mg STX⋅2HCl eq/kg with the standard and 0.682 mg STX⋅2HCl eq/kg with the modified protocol, and 0.710 and 0.734 mg STX⋅2HCl eq/kg for oysters, respectively. The Neogen test may be acceptable for regulatory purposes for oysters in accordance with European Commission directives in which the standard protocol provides, at the regulatory level, a probability of a negative response of 0.033 on 95% of occasions. Its use for mussels is less consistent at the regulatory level due to the wide prediction interval around the POD.

Review of methodology for the determination of benzimidazole residues in biological matrices.

Benzimidazoles are anthelmintic agents widely used in the treatment of parasitic infections in a range of species and as fungicidal agents in the control of spoilage of crops during storage and transport. In this paper, the more important benzimidazoles are introduced and their pharmacological effects and physiochemical properties discussed. The metabolism of these drugs is described relating to the occurrence and persistence of residues in biological matrices, providing information for selection of suitable matrices and target residues for testing. Methods for determination of benzimidazoles are reviewed for a range of biological matrices. The importance of selecting suitable extraction and clean-up procedures is discussed, along with the difficulties encountered in adapting single residue methods to multi-residue methods. The importance of suitable detection systems for determination of benzimidazoles, namely, screening, HPLC, GC and confirmatory methods is described in detail. The future for benzimidazole residue analysis is discussed, focusing on selection of appropriate residues for screening methods and protocols for confirmation of benzimidazole residues.

Investigation of the role of environmental contamination in the occurrence of residues of the veterinary drug phenylbutazone in cattle.

Phenylbutazone is a non-steroidal anti-inflammatory drug licensed for use in horses to treat musculoskeletal disorders. It is not permitted in the European Union for use in animals destined for the food chain. Official statistics provided by the European Food Safety Authority (EFSA) show that 0.18% of bovines tested in the European Union between 2008 and 2014 for non-steroidal anti-inflammatory drugs were non-compliant, with phenylbutazone representing over 28% of these. Anecdotal evidence suggests animals that have not been treated with the drug may have produced non-compliant samples, possibly through some form of contamination. In this study, ultra-high-performance liquid chromatography coupled with mass-spectrometric detection was applied to bovine plasma samples to determine if detectable residues (CCα = 0.28 ng ml-1) may occur in untreated animals as a result of environmental contamination through normal farming practice. The study demonstrates that waste from animals treated with phenylbutazone, and spread on an area of pasture, can contaminate untreated bovines grazing the pasture many weeks later. It was determined that this contamination, which can persist over a significant period, may be due to the ingestion of as little as 30 µg phenylbutazone by a 500 kg bullock.

Use of Entertainment Elements in an Online Video Mini-Series to Train Pharmacy Preceptors.

Objective. To create an entertaining approach to training pharmacy preceptors. Design. A training program was developed to provide an innovative, entertaining, and flexible continuing education program for pharmacy preceptors. Three instructional design principles - providing an authentic context, offering a diversity of content, and engaging and maintaining attention - were foundational to this concept. The mini-series consisted of 12 online video episodes. Participants completed three reflective questions and one evaluation after watching each episode. Three months following completion of the training, a survey was distributed to analyze the long-term impact of the mini-series on precepting skills. Assessment. Two hundred two participants completed all 12 episodes. After completing the training series, the participants' confidence level in their knowledge pertaining to the objectives was significantly greater than before they started. Among the 32% of participants who responded to the three-month follow-up survey, the mean score for precepting confidence was 6.8 on a scale of 1 to 10 on which 1=no increase to 10=big increase. Also, 99% of participants indicated they would complete a similar training program and recommend to others. Conclusions. Feedback from the mini-series provides evidence of the effectiveness of its delivery format and use as a preceptor learning tool.

Screening method for the detection of residues of amphenicol antibiotics in bovine, ovine and porcine kidney by optical biosensor.

An immunobiosensor assay was developed for the multi-residue screening of the parent amphenicols, thiamphenicol and florfenicol, along with the metabolite florfenicol amine, in bovine, ovine and porcine kidney. A polyclonal antiserum raised in a rabbit after inoculation with a florfenicol amine-protein conjugate was employed in the assay. Sample homogenates were extracted directly into acetonitrile, reconstituted in buffer and diluted prior to biosensor analysis. Validation data obtained from the analysis of fortified samples has shown that the method has a detection capability (CCß) of less than 25µgkg-1 (1/2 MRL) for thiamphenicol in the kidney of all three species, less than 150µgkg-1(1/2 MRL) for florfenicol and florfenicol amine and less than 250µgkg-1 (1/2 MRL) for florfenicol and florfenicol amine in bovine/ovine and porcine kidney respectively. Intra-assay variation (n=10) was calculated at 4.5% and 2.6% at concentrations of 10µgkg-1 and 150µgkg-1respectively for bovine kidney while inter-assay variation (n=3) was determined to be 5.0% and 16.5% respectively at the same concentrations. The cross-reactivity profile and validation data for the detection of these amphenicols is presented together with the results obtained following the analysis of florfenicol incurred samples using the developed method.

Review of methodology for the determination of macrocyclic lactone residues in biological matrices.

The macrocyclic lactones (MLs) are probably the anti-parasitic agents most widely used in the treatment of food producing animals, poultry, aquaculture and crops. Ivermectin was the first macrocyclic lactone product to be licensed for use about 20 years ago. A number of alternative products such abamectin, doramectin, emamectin, eprinomectin, moxidectin, milbemycin and selamectin, have been marketed since. Because of the increase in the number of ML drugs, there has been a steady increase in the number of published analytical methods for determination of their residues. In this paper, the structure and properties of the different ML drugs available on the market are described. The occurrence and persistence of ML residues in food is discussed in relation to marker residues and current maximum residue limits (MRLs) as defined in the European Union (EU). Methodologies for determination of ML residues in biological matrices are described in terms of extraction and clean-up methods used for different matrices. Detection systems for determination of ML residues are discussed with a particular emphasis placed on new developments in screening technologies and liquid chromatography with fluorescence or mass spectrometry.

Instructional design framework for the sex and gender-specific health multimedia case-based learning modules.

The goal of the Sex and Gender Specific Health (SGSH) curriculum at the Texas Tech University Health Sciences Center (TTUHSC) is to advance the understanding of sex/gender differences, increase the awareness of gender-specific health issues, and improve the knowledge of sex and gender evidence-based medicine. The purpose of this paper is to explain the development and theoretical rationale for an important aspect of the curriculum: the SGSH Multimedia Case-Based Learning Modules (MCBLMs). The MCBLMs are designed to be used throughout the TTUHSC curriculum as a stand-alone or a supplementary instructional resource. The MCBLMs provide students with authentic learning opportunities that integrate the learning of SGSH with more traditional clinical knowledge and skills. The MCBLMs are specifically designed to enhance students' clinical reasoning and decision-making skills by portraying realistic clinical scenarios. In this way, students are able to practice effective SGSH as competent health-care professionals.

Evidence of non-extractable florfenicol residues: development and validation of a confirmatory method for total florfenicol content in kidney by UPLC-MS/MS.

The parent compound florfenicol (FF) is a broad-spectrum antibacterial compound licensed in the UK for use in cattle, pigs and the aquaculture industry. The analysis of porcine tissues in this study demonstrates that significant amounts of solvent non-extractable FF-related residues are present in incurred tissues (kidney and muscle) from treated animals. The results indicate that methods based on solvent extraction alone may carry a significant risk of reporting false-negative results. The use of a strong acid hydrolysis step prior to solvent extraction of tissue samples is necessary for an accurate estimate of the total tissue FF content. A robust and sensitive method for the determination of total FF residue content in kidney samples by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been developed and validated. This method covers the synthetic amphenicol drug FF and its metabolites, measured as the marker residue florfenicol amine (FFA) as per Commission Regulation (EU) No. 37/2010. Non-extractable and intermediate metabolites are converted to the hydrolysis product FFA, and then partitioned into ethyl acetate. Extracts are solvent exchanged prior to a dispersive solid-phase extraction step, then analysed using an alkaline reverse-phase gradient separation by UPLC-MS/MS. The method was validated around the maximum residue levels (MRLs) set out in Regulation (EU) No. 37/2010 for bovine kidney in accordance with Commission Decision No. 2002/657/EC. The following method performance characteristics were assessed during a single laboratory validation study: selectivity, specificity, sensitivity, linearity, matrix effects, accuracy and precision (decision limit (CCα) and detection capability (CCß) were determined).

Confirmation of synthetic glucocorticoids with liquid chromatography/mass spectrometry: organization and results of an international interlaboratory comparison test.

Within the framework of a European Union (EU) research project entitled "Food Safety Screening: Synthetic Glucocorticoids (QLK1-1999-00122)," an international interlaboratory ring test was organized to compare and evaluate different liquid chromatography/mass spectrometry (LC/MS) confirmatory methods that are applied in European monitoring programs for detecting the use of synthetic glucocorticoids. Liver and urine samples of bovines treated with synthetic glucocorticoids were collected and sent to the participants of the study for analysis. Participants received 3 liver and 3 urine samples and were free to use either their own LC/MS method or an LC/MS-based method developed during the EU research project. The residue concentrations in the samples were calculated as the mean of the concentrations reported by each laboratory. The mean dexamethasone concentration of liver sample L1 was calculated as 2.27 microg/kg [relative standard deviation (RSD) 43%, n = 9], which exceeds the maximum residue level (MRL) of 2 microg/kg. Three of the 9 laboratories (33%) reported concentration levels less than 2 microg/kg, resulting in obviously false compliant results. The overall mean concentration of flumethasone in liver sample L2 was calculated as 3.27 microg/kg (RSD 33%, n = 8). Applying a comparable limit for flumethasone of 2 microg/kg, 8 of the 9 laboratories would have obtained a correct noncompliant result. As for the blank liver sample, 1 participant found a false noncompliant result. The urine sample U1 contained prednisolone residues at a mean concentration of 1.58 microg/kg (RSD 43%, n = 9). Four out of 9 results were less than a theoretical minimum required performance level (MRPL) of 2 microg/kg. The calculated concentration of dexamethasone in urine sample U3 was 5.21 microg/kg (RSD 62%, n = 9). One of the 9 results was lower than 2 microg/kg. Urine sample U2 was correctly reported as blank by all participants.

The production and characterisation of dinitrocarbanilide antibodies raised using antigen mimics.

Polyclonal antibodies were produced to detect the coccidiostat nicarbazin. Due to structural constraints of the active component of nicarbazin, dinitrocarbanilide (DNC), three different compounds that shared a common substructure with DNC were used as antigen mimics. The compounds (N-succinyl-L-alanyl-L-alanyl-L-alanine 4-nitroanilide (SAN), L-glutamic acid gamma-(p-nitroanilide) (GAN) and p-nitrosuccinanilic acid (NSA)) were conjugated to a carrier protein and used in the immunisation of rabbits. Five different polyclonal sera were produced and consequently characterised. The antibodies exhibited an IC(50) range of 2.3-7.6 ng/ml using a competitive ELISA procedure. Serum from one rabbit, R555, exhibited an IC(50) of 2.9 ng/ml for DNC and cross-reactivity studies showed that this serum was specific for DNC and did not cross-react with other coccidiostats such as halofuginone, toltrazuril or ronidazole.

Determination of ivermectin in bovine liver by optical immunobiosensor.

A rapid and sensitive biosensor immunoassay was developed for residues of the antiparasitic agent ivermectin in bovine liver. A detection limit of 19.1 ng g(-1) was achieved. The sensor employed was a Biacore optical instrument based on surface plasmon resonance. 5-O-succinoylivermectin-apo-transferrin conjugate was used to produce monoclonal antibody while a second derivative, ivermectin-oxime, was immobilised onto the surface of a sensor chip. A range of assay parameters (flow rate, injection time, temperature) and extraction techniques were investigated. In the final assay procedure, ivermectin was extracted with acetonitrile followed by C(8) SPE clean-up. Matrix effect was minimised by increasing the flow rate to 25 microl min(-1) and reducing the sample injection time to 2 min. The average value for liver samples spiked at 100 ng g(-1) (the MRL for the drug) and 50 ng g(-1) concentrations were 93.7 and 43.2 ng g(-1), respectively.

Examination of the effect of increasing the number of radiation beams on a radiation treatment plan.

Within the confines of least-squares operations, it is possible to quantify the effect of the addition of treatment fields or beamlets to a treatment plan. Using linear algebra and eigenvalue perturbation theory, the effect of the increase in number of treatments is shown to be equivalent to adding a perturbation operator. The effect of adding additional fields will be negligible if the perturbation operator is small. The correspondence of this approach to an earlier work in beam-orientation optimization is also demonstrated. Results are presented for prostate, spinal and head and neck cases, and the connection to beam-orientation optimization is examined.