Bovine Urine Research Articles

English

The experiment was conducted at Department of Seed Science and Technology, Agricultural College and Research Institute, Madurai during 2014-2015, to find out the effect of organic seed priming with cow urine at different concentrations. The cluster bean seeds were soaked for 3 h with different concentrations viz., 2, 4, 6, 8 and 10% along with water and control (no treatment). The seed quality parameters viz., speed of germination, germination, root length, shoot length, vigour index I, vigour index II and dry matter production were evaluated. The best performance was observed in cow urine (2%) by recording highest seed quality parameters. The percentage increase over control was 10.52, 8.16, 9.8, 8.0, 16.34, 15.36 and 7.8 for speed of germination, germination percentage, root length, shoot length, vigour index I, vigour index II and dry matter production, respectively. Key words: Bovine urines, cluster bean, pre sowing seed treatment, vigour index-germination.

Pseudoendogenous presence of β-boldenone sulphate and glucuronide in untreated young bulls from the food chain

The administration of boldenone (bold) to bovines, either for growth promotion or therapeutic purposes, has been banned in the EU since 1981. It is, however, a pseudoendogenous hormone, thus its detection in bovine urine, in the form of α-boldenone conjugates, is considered fully compliant up to 2 ng ml−1. Greater attention has been placed on β-boldenone, the anabolic active epimer, whose conjugated form must be absent in urine. Recently, the identification of a biomarker representing unquestionable evidence of illicit treatment with bold or its precursor androstadienedione has been a major topic in the literature regarding the detection of residues in bovine urine, and β-boldenone sulphate is a candidate molecule. In this study, we used a method previously validated according to the European Commission Decision 2002/657/EC for the determination of sulphate and glucuronide conjugates of β-boldenone. We assessed the occurrence of these molecules in young bull urine, with the aim of understanding whether they could be of endogenous origin, and to check for a possible relationship with particular environmental and stress conditions. Urine samples from 56 young bulls were collected after transport stress, under non-stressful conditions and after transport and slaughter stress. Histopathological investigation of the hormone target organs, i.e. the bulbourethral and prostate glands, was also performed. The results indicate an inverse relationship between the presence and concentration of β-boldenone sulpho- and gluco-conjugates in urine, and stress conditions, expressed by the absence of detection at the slaughterhouse. No significant macroscopic and histologic lesions were detected. Our study indicates that β-boldenone sulphate could be a biomarker of treatment only at the slaughterhouse, while at the farm, in untreated animals (i.e. after a five-month period under the control of Official Veterinarians), sulphate and glucuronide metabolites were found with a frequency of 78% and 46%, respectively, showing the endogenous origin of boldenone.

Determination of veterinary antibiotics in bovine urine by liquid chromatography–tandem mass spectrometry

A follow-up of antibiotics (tetracyclines, fluoroquinolones, cephalosporins, penicillins and amphenicols) in the bovine urine is important for two reasons: to understand if they are still present in organism, and whether their occurrence in urine might be considered as an environmental risk. A validated HPLC–MS/MS method (Decision 2002/657/EC) for antibiotics determination in bovine urine was developed. CCα and CCβ were in the range of 0.58–0.83 and 0.55–1.1ngmL−1, respectively. Recoveries were 92–108%, with inter-day repeatability below 12%. Analysis of bovine urine revealed frequent presence of tetracyclines, which was related with animal’s age. The cause, most presumably, might be found in different therapeutic protocols applied for veal calves and young bulls enrolled in this study. Most abundant was oxytetracycline with highest level in veal calves (1718ngmL−1) vs. young bulls (2.8ngmL−1). Our results indicate the necessity of antibiotics monitoring in bovine urine before animals undergo further processing in the food industry.

Determination of acetylsalicylic acid and its major metabolites in bovine urine using ultra performance liquid chromatography

A new method based on ultra high performance liquid chromatography (UPLC) with photometric and fluorometric detection for the determination of acetylsalicylic acid and its main metabolites, namely gentisic, salicylic and salicyluric acids, in bovine urine samples is reported. Photometric detection was used for acetylsalicylic acid determination, whereas the native fluorescence of the metabolites was monitored using fluorometric detection. The separation was performed under isocratic conditions, using acetonitrile-phosphate solution (3.5mM, pH 3.5) (26:74, v/v) as the mobile phase. The retention times of the four compounds were lower than 2min, which are shorter than those achieved using conventional HPLC. Under the optimum separation conditions, the dynamic ranges and detection limits (ngmL(-1)) were: 0.2-2500, 0.09 for gentisic acid; 0.2-2500, 0.08 for salicylic acid and 2.5-15,000, 1.1 for salicyluric acid, using fluorescence detection, and 10-25,000, 2.2 for acetylsalicylic acid, using UV detection. Intra-day and inter-day precision data were assessed at two levels of concentration of each analyte using both detection systems. The selectivity of the method was checked by assaying different drugs of veterinary use showing that most of them did not interfere with the determination of the analytes. The method has been applied to the analysis of bovine urine samples, which only required a simple clean up step of the samples prior to injection in the UPLC system. A recovery study was performed, which provided values in the range of 80-100%. This fact proves the practical usefulness of this method as an ultrafast analytical tool for the therapeutic control of acetylsalicylic acid administration in bovine animals intended for food production.

Carbon and nitrogen leaching under high and low phosphate fertility pasture with increasing nitrogen inputs

Intensive pastoral land use is associated with increased use of phosphate (P) and nitrogen (N) to enhance food and fibre production, but the interaction of N and P, particularly on carbon (C) storage, is not well understood. Our objectives were to determine the quantity and forms of C and N leached and also the changes in soil stocks in association with progressively increasing urea additions in two similar soils with high and low phosphate (P) fertility. A pasture cut-and-carry lysimeter experiment was established in the Waikato region of New Zealand, using soils from sheep grazed farmlets with a P management history of either no P or high P additions. Treatments imposed were a continuation of no P and high P (31.5kgha−1y−1) inputs in combination with 0, 100, 200, 400kg urea–Nha−1y−1 in 50kg split dressings or a single spring application of 400kg Nha−1y−1 of bovine urine. The high P soil had greater dissolved organic carbon (DOC) leaching, and DOC leaching in both soils increased with increasing urea inputs. Soil C decreased in the high P soil with N inputs, although there was no correlation between the rate of N addition and C loss. Urea addition led to increased N leaching in both soils, but was reduced in the high P soil compared with the no P soil. Greater herbage production may have utilised more dissolved N in the high P than in the no P soils, which led to less N available for leaching in the high P soil. Urine additions also led to greater C and N leaching in both the no P and high P soils.

Effect-based detection of synthetic glucocorticoids in bovine urine

Challenges to testing for the illicit use of anabolic substances in meat-producing animals stem from the production of new synthetic compounds and the administration of low-dose cocktails to circumvent detection by the surveillance schemes of European Union member states. This work evaluated for the first time GR-CALUX, a highly sensitive reporter gene assay, as a screening tool for the detection of synthetic glucocorticoids in bovine urine. In order to verify the effect of natural corticosteroids on the method, the bioassay was tested first using blank urine samples collected at the farm and the slaughterhouse. Next, the dose–response curves were measured for the most commonly used synthetic glucocorticoids. The bioassay’s ability to detect them in spiked and incurred samples of bovine urine was then evaluated. Finally, its performance was compared against a commercially available ELISA kit ordinarily used in screening activities. GR-CALUX performance did not appear to be influenced by physiological levels of endogenous corticosteroids in the farm samples, whereas an increase in these hormones might invalidate the analysis in samples obtained at the slaughterhouse. Using pure compounds, GR-CALUX showed a high sensitivity toward the synthetic glucocorticosteroids tested in order of relative potencies: flumethasone ≫ dexamethasone > betamethasone > methylprednisolone > prednisolone. As expected, the bioassay failed to detect the prohormone prednisone. The results obtained from analysis of the spiked and incurred specimens reproduced those of the blank samples and the pure compounds. GR-CALUX is a promising screening tool for the detection of illicit treatments in meat-producing bovines. Its ability to detect the most commonly used synthetic glucocorticoids was comparable with the ELISA test. Importantly, it appeared to be less susceptible to matrix effects than ELISA.

Simultaneous determination of zearalenone, deoxynivalenol and their metabolites in bovine urine as biomarkers of exposure

A feeding trial with 30 dairy cows which were fed rations with three different concentrations of zearalenone (ZEA) and deoxynivalenol (DON) contaminated maize was carried out to examine the ZEA and DON concentration in urine. German Holstein cows (n=30) were divided into three groups (n=10 in each) which received diets with following toxin concentrations: CON (0.02 mg ZEA and 0.07 mg DON, per kg dry matter (DM)), FUS-50 (0.33 mg ZEA and 2.62 mg DON, per kg DM), FUS-100 (0.66 mg ZEA and 5.24 mg DON, per kg DM). For urine analysis, a reliable, cost-efficient and sensitive method for simultaneous determination of ZEA, DON and their metabolites was developed. The method comprises a solid phase extraction clean-up on Oasis HLB cartridges followed by LC-MS/MS measurement. ZEA, α-zearalenol, β-zearalenol, DON and de-epoxydeoxynivalenol (DOM) could be detected in the urine samples of the feeding trial. Thereby, DON was almost completely metabolised to DOM (83-98%) independent of the DON exposure. Moreover, conjugated toxins were the major urinary metabolites based on results of the analysis with β-glucuronidase treated and untreated samples. Furthermore, relationships between toxin intake and urinary toxin concentration could be established. In conclusion, increased urine toxin concentrations may hint on toxin exposure through the diets and thus the mycotoxins ZEA and DON and their detected metabolites could be used as biomarkers of exposure.

Direct glucose sensing in the physiological range through plasmonic nanoparticle formation.

Development of improved glucose detection has vast significance in both clinical and point of care settings. Herein, we present a novel, label-free, enzyme-free, colorimetric method of glucose detection that relies on the reduction of a gold salt precursor facilitated by physiological concentrations of glucose (1.25-50 mM). The concentration of glucose present during the reduction process results in nanoparticles of different size, which in turn change the color of solution. Through transmission electron microscopy (TEM), it was found that the nanoparticle size decreases as the glucose concentration increases. Kinetic characterization of nanoparticle formation shows rate constants change 5-8 orders of magnitude when comparing normal versus diabetic glucose concentrations. Assay versatility was also investigated through incorporation onto solid substrates as well as the addition of a filtering step, which produced relatively clear samples below the diabetic cut-off (10 mM glucose) and colored samples above. The colorimetric sensor was then found to also show similar color changes with glucose solutions containing biological interfering agents as well as samples with 20% serum. Last, the sensor was tested in solution containing 100% mouse serum and 100% bovine urine spiked with varying glucose concentrations, which resulted in smaller nanoparticle formation whose intensities were dependent on glucose concentration. The resulting color changes observed for this sensor in urine samples are directly compared with Benedict's reagent and are shown to be significantly more sensitive to lower concentrations of glucose in the diabetic relevant range.

MATHEMATICAL PROCESSING COMPARISON OF VOLTAMMETRIC DATA: APPLICATION WITH THE SIMULTANEOUS DETERMINATION OF FLUOROQUINOLONES

This paper describes a comparative study between the procedures of deconvolution and the second-order derivative of square wave voltammograms to achieve separation of the voltammetric peaks of levofloxacin (LEVO) and norfloxacin (NOR), for their simultaneous quantification in urine samples. The obtained results indicate that the use of second-derivative voltammograms coupled with carbon screen-printed electrodes is the most efficient approach for completely separating the voltammetric peaks of LEVO and NOR. In addition, this approach has produced detection limits lower than 1.0 µmol L-1 and a wide linear range for both drugs. The proposed method was successfully used to simultaneously determine LEVO and NOR in spiked human and bovine urine samples with recovery percentages close to 100% for all analyzed samples.

Determination of Tetracyclines in Bovine and Human Urine using a Graphite-Polyurethane Composite Electrode

The performance of a bare graphite-polyurethane composite electrode (60 percent graphite, m/m) for the determination of tetracycline in human urine and oxytetracycline in human and bovine urine was evaluated. Using optimized differential pulse voltammetry, limits of detection as low as 2.6 micromoles per liter (tetracycline) and 1.7 micromoles per liter (oxytetracycline) were found in phosphate buffer at pH 2.5. The sample preparation included acidification and centrifugation to remove solid proteins followed by direct analysis. Recoveries between 96 and 100 percent were obtained for the determination of tetracycline and oxytetracycline in fortified urine.

Copper nanoparticles applied to the preconcentration and electrochemical determination of β-adrenergic agonist: An efficient tool for the control of meat production

A novel method for preconcentration and electrochemical detection of zinterol in bovine urine samples was developed. In order to improve the limit of detection, the surface of a screen-printed carbon electrode was modified with electrodeposited metal copper nanoparticles. The experimental electrodeposition optimization was performed using a central composite design (CCD), involving the variables: precursor concentration, potential and time applied. Copper nanoparticles were characterized by transmission electron microscopy, scanning electron microscopy, cyclic voltammetry, and energy dispersive X-ray spectroscopy. Mesoporous shuttle-like copper oxide nanoparticles were used for the preconcentration step to avoid interferences with many compounds present in the sample matrix. The optimal working conditions for the preconcentration approach were found by means of both two-level fractional factorial and CCD designs. The obtained enhancement factor for a sample volume of 30mL was 35 fold. The calibration curve showed linearity between 0.5 and 45ngmL−1 and the limit of detection was 0.16ngmL−1. The intra and inter assay coefficients of variability were below 4% and 5%; respectively.

Molecular detection of Leptospira spp. in the urine of cattle in northern Iran.

Leptospirosis is a zoonosis of worldwide distribution, caused by Leptospira interrogans and is considered as an emerging global public health problem. Transmission usually results from direct or indirect exposure to the urine or other body fluids of leptospiruric animals which may become a source of infection for human or other animals. Having a humid climate with plenty of annual rainfall, Guilan province is a suitable environment for maintaining Leptospira spp. Hence, early detection of Leptospira spp. in the host prompts control and protection, and the polymerase chain reaction (PCR) is a suitable method. The present report aimed to demonstrate the PCR analysis of bovine urine for detection of leptospiral DNA. A total of 98 urine samples were randomly collected from cattle bladder in Rasht abattoir of Iran and the presence of leptospiral DNA was assayed by PCR amplification of rrs (16S rRNA) gene and the results confirmed by nested PCR. Out of 98 urine samples in 42 samples leptospires DNA was identified with the frequency of 43%. The high presence of the organism in the urine of carriers is a serious threat to the dairy farms and to the public health which requires an effective control measure in the north provinces of Iran.

In situ N2O emissions are not mitigated by hippuric and benzoic acids under denitrifying conditions

Ruminant urine patches deposited onto pasture are a significant source of greenhouse gas nitrous oxide (N2O) from livestock agriculture. Increasing food demand is predicted to lead to a rise in ruminant numbers globally, which, in turn will result in elevated levels of urine-derived N2O. Therefore mitigation strategies are urgently needed. Urine contains hippuric acid and together with one of its breakdown products, benzoic acid, has previously been linked to mitigating N2O emissions from urine patches in laboratory studies. However, the sole field study to date found no effect of hippuric and benzoic acid concentration on N2O emissions. Therefore the aim of this study was to investigate the in situ effect of these urine constituents on N2O emissions under conditions conducive to denitrification losses. Unadulterated bovine urine (0mM of hippuric acid, U) was applied, as well as urine amended with either benzoic acid (96mM, U+BA) or varying rates of hippuric acid (8 and 82mM, U+HA1, U+HA2). Soil inorganic nitrogen (N) and N2O fluxes were monitored over a 66day period. Urine application resulted in elevated N2O flux for 44days. The largest N2O fluxes accounting for between 13% (U) and 26% (U+HA1) of total loss were observed on the day of urine application. Between 0.9 and 1.3% of urine-N was lost as N2O. Cumulative N2O loss from the control was 0.3kgN2O–Nha−1 compared with 11, 9, 12, and 10kgN2O–Nha−1 for the U, U+HA1, U+HA2, and U+BA treatments, respectively. Incremental increases in urine HA or increase in BA concentrations had no effect on N2O emissions. Although simulation of dietary manipulation to reduce N2O emissions through altering individual urine constituents appears to have no effect, there may be other manipulations such as reducing N content or inclusion of synthetic inhibitory products that warrant further investigation.

Use of the nitrification inhibitor dicyandiamide (DCD) does not mitigate N2O emission from bovine urine patches under Oxisol in Northwest Brazil

Animal production systems are important sources of greenhouse gases (GHGs), especially methane (CH4) and nitrous oxide (N2O). GHG emissions from urine patches have been extensively studied in temperate climates, with few studies under tropical conditions. Here we examined the driving factors of N2O and CH4 emission from urine patches in the tropics, as well as the role of the nitrification inhibitor DCD (dicyandiamide) in mitigating emissions. We hypothesized that the high temperature and periodical rainfall can increase GHG emissions from urine patches through accelerating mineralization of urine-N. We measured CH4 and N2O emissions from beef cattle urine (360 kg N ha−1) in Rondonia state (Brazil, tropical climate), during two different seasons (winter and summer), with and without the application of DCD (10 kg ha−1). No effects of DCD on cumulative N2O emissions were detected in summer, but DCD retarded the main emission peak. During winter DCD increased N2O emissions from 10.8 to 39.2 mg N–N2O m−2 (p ≤ 0.05). Emission factors averaged 0.4 % for summer and 0.1 % for winter, which is significantly lower than the IPCC default value of 1 %. The climate, associated with soil (acidic pH, WFPS and low N content) and plant properties (biological nitrification inhibition) resulted in a low emission factor. We concluded that the IPCC default emission factor for tropical systems may be reduced, and that the application of DCD is not recommended in such systems.

Development and validation of a UPLC-MS/MS method to monitor cephapirin excretion in dairy cows following intramammary infusion.

Cephapirin, a cephalosporin antibiotic, is used by the majority of dairy farms in the US. Fecal and urinary excretion of cephapirin could introduce this compound into the environment when manure is land applied as fertilizer, and may cause development of bacterial resistance to antibiotics critical for human health. The environmental loading of cephapirin by the livestock industry remains un-assessed, largely due to a lack of appropriate analytical methods. Therefore, this study aimed to develop and validate a cephapirin quantification method to capture the temporal pattern of cephapirin excretion in dairy cows following intramammary infusion. The method includes an extraction with phosphate buffer and methanol, solid-phase extraction (SPE) clean-up, and quantification using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The LOQ values of the developed method were 4.02 µg kg−1 and 0.96 µg L−1 for feces and urine, respectively. This robust method recovered >60% and >80% cephapirin from spiked blank fecal and urine samples, respectively, with acceptable intra- and inter-day variation (<10%). Using this method, we detected trace amounts (µg kg−1) of cephapirin in dairy cow feces, and cephapirin in urine was detected at very high concentrations (133 to 480 µg L−1). Cephapirin was primarily excreted via urine and its urinary excretion was influenced by day (P = 0.03). Peak excretion (2.69 mg) was on day 1 following intramammary infusion and decreased sharply thereafter (0.19, 0.19, 0.08, and 0.17 mg on day 2, 3, 4, and 5, respectively) reflecting a quadratic pattern of excretion (Quadratic: P = 0.03). The described method for quantification of cephapirin in bovine feces and urine is sensitive, accurate, and robust and allowed to monitor the pattern of cephapirin excretion in dairy cows. This data will help develop manure segregation and treatment methods to minimize the risk of antibiotic loading to the environment from dairy farms.

Method development and validation: solid Phase extraction-ultra performance liquid chromatography-tandem mass spectrometry quantification of pirlimycin in bovine feces and urine.

Pirlimycin, a lincosamide antibiotic, is one of the most commonly used antibiotics for the treatment of mastitis in dairy cows. Assessment of pirlimycin loadingto the environment via fecal and urinary excretion is critical to develop efficient management strategies to reduce environmental pollution by the livestock industry. Therefore, the aim of this study was to develop and validate an analytical method to identify and quantify pirlimycin in bovine feces and urine. Samples were extracted with methanol- phosphate buffer and cleaned up by SPE before analysis for pirlimycin using UPLC-MS/MS. This method was sensitive (LOQ 1.47 ng/g wet feces, 0.90 ng/mL urine), accurate (recovery, 80-108%), and precise (repeatability, 2.3-13%; reproducibility, 2.3-14%) for both bovine feces and urine. With the application of this method to samples collected in the first 10 h and then every 24 h for 120 h following intramammary dosing (50 mg/cow; n = 3 cows), pirlimycin was detected at 40.5-287 ng/g and 46.1-254 ng/mL in feces and urine, respectively. This robust, sensitive, and accurate method can be used to assess the fate and environmental impact of antibiotics used on farms.

Simultaneous determination of seven β2-agonists in human and bovine urine by isotope dilution liquid chromatography–tandem mass spectrometry using compound-specific minimally 13C-labelled analogues

Seven β2-agonist (clenproperol, clenbuterol, salbutamol, bronbuterol, ractopamine, clenpenterol and clencyclohexerol) were determined simultaneously in human and bovine urine by isotope dilution LC–ESI-MS/MS in a triple quadrupole instrument. The method is based on the application of multiple linear regression in combination with compound-specific minimally 13C-labelled analogues. Additionally, the increase of the bandpass of the first quadrupole during the selected reaction monitoring (SRM) measurement procedure allowed the simultaneous quantification of the seven compounds at sub ngg−1 levels in a single chromatogram without resorting to a methodological calibration graph. Recovery values at concentration levels between 5.0 and 0.05ngg−1 ranged from 95 to 110% in fortified bovine urine and from 91 to 108% in human urine, with relative standard deviations lower than 5% except for salbutamol and ractopamine. The proposed methodology was validated by analyzing the certified reference material BCR-503 (lyophilized bovine urine) certified for clenbuterol and salbutamol. The limits of detection (LOD) for a sample volume of 10mL of both human and bovine urine was found to be lower than 0.012ngg−1 for all compounds, except to salbutamol in bovine urine which was of 0.029ngg−1. The use of compound-specific isotopically labelled analogues minimally labelled in 13C minimized the occurrence of isotope effects and corrected for matrix effects during ESI ionization and can be efficiently applied for the quantification of ultra-trace concentrations of β2-agonists in human and bovine urine.

Ammonia volatilisation is not the dominant factor in determining the soil nitrate isotopic composition of pasture systems

• NH 3 volatilisation in urine patches is not directly reflected in δ 15 N of residual soil N. • NO 3 − isotopic enrichment depends on mineralisation, nitrification, and denitrification. • NO 3 − in grazed pastures can range from −10‰ to +15‰ (δ 15 N) and −5‰ to +5‰ (δ 18 O). Nitrate dual isotopes (δ 15 N NO 3 − and δ 18 O NO 3 − ) are increasingly used to assess the sources and sinks of nitrogen (N) pollution in freshwater systems. However, the application of this methodology to pasture agroecosystems is currently limited by the lack of information on how, or even if, the primary N inputs to the systems (livestock urine and urea fertiliser) are expressed in the isotopic signature of exported NO 3 − . To remedy this gap, direct measurements of fractionation during ammonia volatilisation were linked with changes in the concentration and isotopic composition of the residual soil inorganic N pool (NO 3 − , nitrite, and ammonium) following the addition of differing levels of bovine urine and urea fertiliser. Ammonia volatilisation, with a δ 15 N enrichment factor of +35 ± 5‰, removed from 5 to 40% of N inputs from the different treatments, which should have enriched the residual inorganic N pool to 25‰ and 3‰, respectively. However, this fractionation did not propagate into the soil NO 3 − pool due to a combination of urine-induced mineralisation (up to 120 μg N g soil −1 day −1 in the high urine treatment) and on-going nitrification. Consequently, NO 3 − measured within the treatments was not as enriched in 15 N as the values typically ascribed to excreta-N sources. Up-scaling these results, the whole-pasture NO 3 − isotopic composition primarily reflected time since fertilisation, regardless of urine inputs. These findings necessitate expanding the range of δ 15 N NO 3 − values ascribed to livestock sources to encompass values as low as −10‰, highlighting the need to account for post-deposition soil N cycling in order to accurately define NO 3 − isotopic source ranges.

Simultaneous determination of resorcylic acid lactones, β and α trenbolone and stilbenes in bovine urine by UHPLC/MS/MS.

Treatment of cattle with α-zearalanol (zeranol, α-ZAL), a resorcylic acid lactone (RAL), is illegal in European Union countries. Zearalenone, a common contaminant of cattle feed, is also a RAL and there is evidence that it, or its metabolites, can be converted in vivo to α-ZAL (or to β-zearalanol, β-ZAL). To determine whether an animal has been treated with α-ZAL it is necessary to quantify separately all the RALs. This work presents the simultaneous determination in urine of RALs, β-trenbolone (β-TB) and its metabolite α-trenbolone (α-TB) and the stilbenes diethylstilbestrol (DES), dienestrol (DEN) and hexestrol (HEX) using Ultra High Performance Liquid Chromatography/Mass Spectrometry (UHPLC/MS/MS). Several chromatographic UHPLC columns were tested in order to achieve chromatographic separation of the analytes and the results are shown. Baseline separation of all compounds was not possible, so that careful consideration of the MRM transitions was necessary. The separation chosen for the validation work used a 100mm×2.1mm×1.7μm Phenyl column eluting with a gradient of acetonitrile/methanol/water. The method validation according to EU Decision 657/2002 included linearity, within laboratory reproducibility and trueness, decision limit (CCα) and detection capability (CCβ). For all compounds the method was linear in the range 2-12μg/l (1 and 6μg/l for DES) with determination coefficients greater than 0.97 and linear residuals below 20%. Within laboratory reproducibility was lower than 25% and trueness less than 11% for all compounds and concentration levels. CCα ranged from 0.6μg/l (DES) to 1.6 (α-TB) and CCβ was 0.8μg/l (α-zearalenol) to 1.9μg/l (α-TB).

Electroanalytical Determination of Danofloxacin in Biological Samples Using Square Wave Voltammetry

The voltammetric behavior of danofloxacin (DFX) has been studied, in aqueous solution, on a glassy carbon electrode using square wave voltammetry (SWV) as electroanalytical technique. After optimization of the experimental conditions, DFX was analyzed in spiked biologic samples using a Britton-Robinson buffer with pH = 5.0 as the supporting electrolyte. Oxidation occurs at 0.98 V vs. Ag/AgCl in a two-electron process controlled by adsorption of the electrogenerated products on the electrode surface. A acceptable recovery was obtained for assay of spiked biologic samples, with value of 98.7% for the swine urine and 95.3 % for the bovine urine.