[Determination of 12 prohibited veterinary drug residues in pig urine by ultra high performance liquid chromatography-tandem mass spectrometry].

A method was established for the simultaneous detection of 12 prohibited veterinary drugs, including ß2-receptor agonists, nitrofuran metabolites, nitroimidazoles, chlorpromazine, and chloramphenicol, in pig urine. The sample was pretreated by enzymolysis, acid hydrolysis/derivatization, and liquid-liquid extraction combined with solid-phase extraction. Detection was performed using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Ammonium acetate solution (0.2 mol/L, 4.5 mL) and ß-glucuronidase/aryl sulfatase (40 µL) were added to the sample, which was subsequently enzymolized at 37 ℃ for 2 h. Then, 1.5 mL of 1.0 mol/L hydrochloric acid solution and 100 µL of 0.1 mol/L o-nitrobenzaldehyde solution were added to the sample. The mixture was incubated at 37 ℃ for 16 h, and the analytes were extracted with 8 mL of ethyl acetate by liquid-liquid extraction. The lower aqueous phase obtained after extraction was extracted and purified using a mixed cation-exchange solid-phase extraction column. The extracts were combined, the extraction solution was blow-dried with nitrogen, and the residue was redissolved for determination. The samples were analyzed under multiple-reaction monitoring mode with both positive and negative electrospray ionization, and quantified using an isotope internal standard method. The correlation coefficients (r) of the 12 compounds were >0.99. The limits of detection (LODs) and quantification (LOQs) of chloramphenicol were 0.05 and 0.1 µg/L, respectively, and the LODs and LOQs of the other compounds were 0.25 and 0.5 µg/L, respectively. The mean recoveries and RSDs at 1, 2, and 10 times the LOQ were 83.6%-115.3% and 2.20%-12.34%, respectively. The proposed method has the advantages of high sensitivity, good stability, and accurate quantification; thus, it is suitable for the simultaneous determination of the 12 prohibited veterinary drug residues in pig urine.

Development of new matrix reference materials for quantitative urine analysis in drug tests.

Accurate quantitative analyses require standardized methods to control and improve the analytical process in the laboratory. The availability of urine reference materials (RMs) may offer a feasible option to improve the accuracy of urine analysis and to control matrix effects. This paper presents the complete process of the development of matrix RMs in urine, including sample preparation, homogeneity, and stability studies, as well as uncertainty assessment. A freeze-drying process was developed, and freeze-dried human and pig urine samples were prepared and verified to have comparable homogeneity to liquid samples and higher stability than liquid human, pig, and artificial urine samples at 4℃ or room temperature and under extreme conditions. A total of 21 authentic urine samples from August 2022 were measured with freeze-dried RMs and spiked urine samples, and the reliability of the quantification of the RMs was compared. The freeze-dried human urine matrix RM appeared to be an excellent tool for daily quality control, as it showed high stability and gave the most consistent results with spiked samples.

Demonstration of constant nitrogen and energy amounts in pig urine under acidic conditions at room temperature and determination of the minimum amount of hydrochloric acid required for nitrogen preservation in pig urine.

OBJECTIVE: The objectives were to demonstrate that the nitrogen and energy in pig urine supplemented with hydrochloric acid (HCl) are not volatilized and to determine the minimum amount of HCl required for nitrogen preservation from pig urine. METHODS: In Exp. 1, urine samples of 3.0 L each with 5 different nitrogen concentrations were divided into 2 groups: 1.5 L of urine added with i) 100 mL of distilled water or ii) 100 mL of 6 N HCl. The urine in open plastic containers was placed on a laboratory table at room temperature for 10 d. The weight, nitrogen concentration, and gross energy concentration of the urine samples were determined every 2 d. In Exp. 2, three urine samples with different nitrogen concentrations were added with different amounts of 6 N HCl to obtain varying pH values. All urine samples were placed on a laboratory table for 5 d followed by nitrogen analysis. RESULTS: Nitrogen amounts in urine supplemented with distilled water decreased linearly with time, whereas those supplemented with 6 N HCl remained constant. Based on the linear broken-line analysis, nitrogen was not volatilized at a pH below 5.12 (standard error = 0.71 and p<0.01). In Exp. 3, an equation for determining the amount of 6 N HCl to preserve nitrogen in pig urine was developed: additional 6 N HCl (mL) to 100 mL of urine = 3.83×nitrogen in urine (g/100 mL)+0.71 with R2 = 0.96 and p<0.01. If 62.7 g/d of nitrogen is excreted, at least 240 mL of 6 N HCl should be added to the urine collection container. CONCLUSION: Nitrogen in pig urine is not volatilized at a pH below 5.12 at room temperature and the amount of 6 N HCl required for nitrogen preservation may be up to 240 mL per day for a 110-kg pig depending on urinary nitrogen excretion.

Indirect competitive enzyme-linked immunosorbent assay based on a broad-spectrum monoclonal antibody for tropane alkaloids detection in pig urine, pork and cereal flours.

In this study, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a broad-spectrum monoclonal antibody for tropane alkaloids (TAs) was established for the rapid screening of atropine, scopolamine, homatropine, apoatropine, anisodamine, anisodine and L-hyoscyamine residues in pig urine, pork and cereal flour samples through a simple sample preparation procedure. The half inhibitory concentrations of atropine, homatropine, L-hyoscyamine, apoatropine, scopolamine, anisodamine and anisodine were 0.05, 0.07, 0.14, 0.14, 0.24, 5.30 and 10.15 ng mL-1, respectivelyThe detection and quantitative limits of this method for TAs in samples were 0.18-73.18 and 0.44-74.77 µg kg-1. The spiked recoveries ranged from 69.88% to 147.93%, and the coefficient of variations were less than 14%. Good correlation (R2 = 0.9929) between the results of the ic-ELISA and the high performance liquid chromatography-tandem mass spectrometry support the reliability of the developed ic-ELISA method.

A molecularly imprinted polymer based chemiluminescence array sensor for one-step determination of phenothiazines and benzodiazepines in pig urine.

The residues of phenothiazines and benzodiazepines in foods of animal origin are dangerous to consumers. For inspection of their abuses, this study for the first time reported on the use of a chemiluminescence array sensor for the simultaneous determination of four phenothiazines and five benzodiazepines in pig urine. Two molecularly imprinted polymers were coated in different wells of a conventional 96-well microtiter plate as the recognition reagents. After sample loading, the absorbed analytes were initiated directly by using an imidazole enhanced bis(2,4,6-trichlorophenyl)oxalate-hydrogen peroxide system to emit light. The assay process consisted of only one sample-loading step prior to data acquisition, so one test was finished within 10 min. The limits of detection for the nine drugs in the pig urine were in a range of 0.1 to 0.6 pg/mL, and the recoveries from the fortified blank urine samples were in a range of 80.3 to 95%. Furthermore, the sensor could be reused six times. Therefore, this sensor could be used as a simple, rapid, sensitive and reusable tool for routine screening for residues of phenothiazines and benzodiazepines in pig urine.

Influence of feedstock-to-inoculum ratio on performance and microbial community succession during solid-state thermophilic anaerobic co-digestion of pig urine and rice straw.

This study investigated the effect of the feedstock-to-inoculum (F/I) ratio on performance of the solid-state anaerobic co-digestion of pig urine and rice straw inoculated with a solid digestate, and clarified the microbial community succession. A 44-day biochemical methane potential test at F/I ratios of 0.5, 1, 2 and 3 at 55 °C and a 35-day large-scale batch test at F/I ratios of 0.5 and 3 at 55 °C were conducted to investigate the effects of F/I ratio on anaerobic digestibility and analyze microbial community succession, respectively. The highest cumulative methane yield was 353.7 m3/t VS in the large-scale batch test. Volatile fatty acids did not accumulate at any F/I ratios. The volatile solids reduction rate was highest at a F/I ratio of 0.5. Microbial community structures were similar between F/I ratios of 3 and 0.5, despite differences in digestion performance, suggesting that stable operation can be achieved at these ratios.

Natural incidence of zearalenone in Croatian pig feed, urine and meat in 2014.

The aim of this study was to determine the levels of zearalenone (ZEN) in different feed materials and feedstuffs for pigs, as well as in pig urine and pig meat following contaminated feed consumption. In total, 253 feed material and feedstuff samples were collected from Croatian pig farms. The results revealed the presence of ZEN in significant concentrations, the maximal being found in maize (5522 µg/kg), wheat (3366 µg/kg) and pig fattening feed (1949 µg/kg). In farms in which high feed contamination and pig hyperestrogenism were observed, samples of pig urine (n=30) and meat (n=30) were retrieved as well. The mean ZEN concentrations in pig urine and pig meat were 206±20.6 µg/L and 0.62±0.14 µg/kg, respectively. Despite high contamination of feedstuffs responsible for farmed pigs' intoxication, ZEN levels determined in pig meat were shown to be of little significance for human safety.

Pseudoendogenous origin of prednisolone in pigs from the food chain.

The debate about the origin of prednisolone in animal organisms has lasted for 5 years. Bovine species have been the most studied, but studies on humans and horses are also present in the literature. Even if prednisolone in pigs does not yet represent a problem for control agencies, interest has recently increased with regard to this species. To date, there has been just a single study in the literature about this topic, performed on 10 sows treated with prednisolone or a synthetic analogue of adrenocorticotropic hormone. We therefore initiated a study on 80 pigs, a number considered representative in relation to the expected frequency (prevalence) of prednisolone detection in urine collected at slaughter. Prednisolone was detected in urine both at the farm and at the slaughterhouse, with a concentration and frequency higher at slaughter. The presence of prednisolone was also studied in the adrenal glands, where the corticosteroids are produced in response to stress, and it was detected in 89% of the samples. These results, together with the similar behaviours of prednisolone and cortisol, i.e. a mutual rise in the two corticosteroids in urine collected at the slaughterhouse and the correlation between the concentrations of the two corticosteroids in the adrenal glands, seem to indicate an endogenous origin of prednisolone in pigs.