Pig Urine Samples Research Articles

Potentiometric Sensor Based on Molecularly Imprinted Polymer for Rapid Determination of Clenbuterol in Pig Urine

A polymeric membrane ion-selective electrode for the determination of clenbuterol is described.It is based on a molecularly imprinted polymer(MIP) as an ionophore for molecular recognition,which can be synthesized by precipitation polymerization using clenbuterol hydrochloride as a template molecule.Under the optimized conditions,the proposed membrane electrode exhibits Nernstian response to the protonated clenbuterol over the concentration range of 1.0×10-7-1.0×10-4 mol/L with a slope of 55.7 mV/decade,and the detection limit is 7.0×10-8 mol/L.The MIP-based sensor shows excellent selectivity,rapid response time and satisfactory long-term stability.The potentiometric sensor has been successfully applied to determine the clenbuterol in pig urine samples with recoveries between 98% and 107% and an analytical time of less than 3 min.

A Conductometric Sensor Based on Screen Printed Electrode Modified with Multi-walled Carbon Nanotubes and Molecularly Imprinted Membrane for Determination of Ractopamine in Pig Urine

First,multi-wall carbon nanotubes(MWCNs) were modified on the screen printed electrode(SPE),and then molecularly imprinted membrane(MIM) and non-imprinted membrane(NIM) were directly synthesized on the modified SPE by using the in-situ thermal polymerization techniques,respectively.A conductometric sensor for the detection of ractopamine was assembled by connecting the SPE modified with MWCNs-MIM and the other one coated with MWCNs-NIM to a conductometer through an electrode slot.Standard calibration curve was established and the determination of ractopamine in pig urine samples also was performed based on this sensor.Superficial characteristic of MIM and NIM was investigated using scanning electron microscope(SEM).The results showed that there were number of imprinted micro-pores on the surface of MIM.The sensor showed high selectivity and specificity towards the target ractopamine.The response of the sensor to concentration of ractopamine displayed a linear correlation over a range from 0.33 to 8.0 mg/L with a detection limit of 0.033 mg/L.The recoveries were 91%-98% based on pig urine samples for the determination of ractopamine on sites.

Simultaneous determination of four synthesized metabolites of mequindox in urine samples using ultrasound-assisted dispersive liquid–liquid microextraction combined with high-performance liquid chromatography

A novel pretreatment method termed ultrasound-assisted dispersive liquid–liquid microextraction (UADLLME) coupled with high-performance liquid chromatography-ultraviolet detector (HPLC-UV) was applied for the detection of four synthesized metabolites of mequindox in pig urine samples. A total volume of 200μL of methanol (dispersant) and 60μL of 1,1,2,2-tetrachloroethane (extract) were injected into 5.0mL of urine sample and then emulsified by ultrasound treatment for 4min to form a cloudy solution. The effect of several factors on the recovery of each metabolite was investigated by a fitting derivation method for the first time. Under optimum conditions, the method yields a linear calibration curve in the concentration range from 0.5 to 500μg/L and a limit of detection (LOD) of 0.16–0.28μg/L for target analytes. The recoveries ranged from 72.0% to 91.3% with a relative standard deviation (RSD) of less than 5.2%. The enrichment factors for the four compounds ranged from 75 to 95. Two pig urine samples were successfully analyzed using the proposed method.

Development of a Lateral Flow Colloidal Gold Immunoassay Strip for the Rapid Detection of Olaquindox Residues

A rapid immunochromatographic lateral flow test strip of competitive format has been developed for the specific determination of olaquindox (OLA) residues in pig urine and muscle tissues. The sensitivity of the test strip was found to be 1.58 ± 0.27 μg/kg and 1.70 ± 0.26 μg/kg of OLA in pig urine and muscle tissues, and the lower detection limit was 0.27 ± 0.08 μg/kg and 0.31 ± 0.07 μg/kg respectively. For negative pig urine and muscle samples spiked with 4, 12, and 36 μg/kg, the recovery range was 83.0-94.0% and 78.8-87.4% and the coefficient of variation scope [CV (%)] was 3.17-7.41% and 4.66-7.64% respectively. Parallel analysis of OLA samples from pig urine and muscle tissue showed comparable results from the test strip and HPLC. Each test requires 5-8 min, and the test strip can provide a useful screening method for quantitative, semiquantitative, or qualitative detection of OLA residues.

Towards the development of a portable sensor based on a molecularly imprinted membrane for the rapid determination of salbutamol in pig urine

A new type of conductometric probe based on a molecularly imprinted membrane (MIM) for the detection of salbutamol has been designed and fabricated. The probe consists of two parallel screen-printed electrodes (SPE). One of the SPEs was coated with a molecularly imprinted membrane using salbutamol as the template, and the other was modified with a non-molecularly imprinted membrane (N-MIM). Measurements of salbutamol were conducted after the conductometric probe had been connected to a commercial portable conductometer. Multi-sample or successive detections could be easily accomplished by replacing the one-off SPE coated with the salbutamol molecularly imprinted membrane with a new one. The conductometric response of the sensor to the concentration of salbutamol displayed a linear correlation over a range from 50 to 280 nM, with a detection limit of 13.5 nM. The recoveries reached 92.1–98.3% based on pig urine samples. In addition, the sensor based on this new type of probe demonstrated high sensitivity and selectivity for salbutamol.

Zearalenone, deoxynivalenol and aflatoxin B1 and their metabolites in pig urine as biomarkers for mycotoxin exposure

Methods to determine zearalenone (ZEA), deoxynivalenol (DON), aflatoxins (AF) and their metabolites in pig urine were developed as biomarkers for pig exposure to the mycotoxins in feed. Urine samples were incubated with β-glucuronidase to cleave conjugates, extracted and cleaned-up with solid phase and immunoaffinity columns, followed by HPLC with UV and fluorescence detection. Good recoveries (83-130%), low variation (2-10%), and low detection limits (0.3-9.9ng/ml) were obtained. The results of controlled AFB1 feeding trials found no difference in urine concentrations of AFB1 or AFM1 from pigs fed three different levels (127, 227, 327 µg/kg) of AFB1 in diets. The excretion of AFB1 and AFM1 in urine was on average 30% of the oral dose and the ratio AFB1 to AFM1 was around 23%. The analysis of 15 Vietnamese pig urine samples indicate a relatively high exposure of ZEA, DON and AF, which were found as toxin or metabolites in 47, 73, and 80% of the urine samples, respectively.

Integrated microfluidic immunoassay for the rapid determination of clenbuterol

An integrated microfluidic immunoassay system was established for high throughput analysis of clenbuterol. This system consisted of an integrated microchip and a linear confocal laser induced fluorescence (LIF) scanner. The microchip was composed of three layers: a fluidic channel layer, a PDMS membrane layer and a pneumatic control layer. The multi-layer chip was integrated with 36 pneumatic micro-valves and multiple micro-pumps to realize the flexible reagent delivery, facilitating the automatic assays with less consumption of samples and reduced analysis time. The homemade LIF scanner was able to simultaneously detect multi-channels and provide the potential capability of high throughput assays. The performance of the system was demonstrated by the determination of clenbuterol, one of the most widely used beta-agonists. Under the optimal conditions, the linear range and the limit of detection of clenbuterol were 0 approximately 5.0 ng mL(-1) and 0.088 ng mL(-1), respectively. The recovery rates determined with pig urine samples of 1.0 ng mL(-1) and 2.0 ng mL(-1) were 98.74% and 102.51% (n = 3), respectively. The total detection time was less than 30 min. The system had the potential application for rapid detection of multiple beta-agonists in clinical, pharmaceutical and chemical analyses.

Growth promotant abuse in meat-producing animals: Liquid chromatography-electrospray ionization tandem mass spectrometry for determination of β2-adrenergic agonist clenbuterol in pig urine

Clenbuterol, a β 2-adrenergic agonist, has been found to be applied illegally in meat-producing animals to modulate growth. For residue surveillance of clenbuterol in meat-producing animals and meat, it is essential to choose appropriate matrices as well as the high degree of sensitivity derived from spectrometric analysis of suspect tissues and body fluids. The aim of the study was to evaluate the suitability of urine as a matrix for clenbuterol residue determination throughout and after its subchronic repeated administration at a growth-promoting dose to food-producing pigs, using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) as a quantification and confirmation method. Mass spectral acquisition was done in multiple reaction monitoring (MRM) mode to provide a high degree of sensitivity, archieving a limit of detection and quantification at 0.11 and 0.30 μ g/kg, respectively. The mean recovery of the analyte fortified at 0.5 μ g/kg was 88.0% with acceptable inter- and intra-day relative standard deviations in a range of 7.2-9.3%. Clenbuterol was identified by two transitions (m/z 277>259 and m/z 277>203), and quantification was performed at the most intensive transition (m/z 277>203). Study results showed the mean clenbuterol concentrations in pig urine samples to oscillate widely during the 28-day treatment period. Maximal clenbuterol concentrations were achieved in urine on day 21 (96.40p ; 45.13 ng/mL) and then decreased to 45.78p ; 8.47 ng/mL on the last day of dosing period. After a 7-day withdrawal period, clenbuterol concentration in urine (0.45p ; 0.05 ng/mL) decreased below 0.5 ppb as currently accepted maximal residue level (MRL) for clenbuterol in liver as a regulated matrix for control clenbuterol abuse. Although the concentration of clenbuterol in pig urine several times exceeded MRL throughout the treatment period, suggesting acceptability of this matrix for determination of clenbuterol residues during fattening period, the significant depletion recorded during the first seven days of withdrawal showed the urine to be of limited value as a matrix in the control of clenbuterol abuse.

Simultaneous determination of major B-trichothecenes and the de-epoxy-metabolite of deoxynivalenol in pig urine and maize using high-performance liquid chromatography–mass spectrometry

A selective analytical method based on high-performance liquid chromatography (HPLC), combined with atmospheric pressure chemical ionisation (APCI −) mass spectrometry (MS), has been developed for simultaneous determination of B-trichothecenes and the major metabolites of deoxynivalenol. The method allows simultaneous analysis of nivalenol (NIV), deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-AcDON), 3-acetyldeoxynivalenol (3-AcDON), fusarenon X (Fus-X) and de-epoxydeoxynivalenol (DOM-1). The method is based on one-step sample clean-up using a multifunctional MycoSep column. A linear gradient mobile phase system, consisting of water:acetonitrile:methanol (H 2O:ACN:MeOH) at a flow-rate of 1 ml/min, and a Polar-RP C18 column, were utilised to obtain the best resolution of all tested compounds along with column and equilibrating within 30 min. Dexamethasone (Dex) was used as internal standard. The developed method shows good repeatability for inter- and intra-day precisions as well as good linearity of calibration curves ( r 2 ranged from 0.9936 to 0.9998). Average recoveries for tested compounds in both matrices have been determined ranging from 63.7 to 102.3% and limit of quantification (LOQ) ranged from 25 to 150 ng/g. The utility and practical impact of the method is demonstrated using contaminated pig urine and maize samples.

Determination of zearalenone and its metabolites in urine and tissue samples of cow and pig by LC-MS/MS

For the sensitive and selective determination of zeranol, taleranol, α-zearalenol, β-zearalenol and zearalenone in animal urine and tissue a LC-MS/MS method has been developed. Sample preparation included extraction of meat samples and enzymatic digest of urine samples followed by solid-phase extraction with RP-18 columns for sample clean-up. Mass spectrometric determination was carried out with an atmospheric pressure chemical ionisation interface (APCI) in the multi-reaction monitoring mode (MRM). Using the negative ion mode detection limits between 0.1 and 0.5 ppb and determination limits between 0.5 and 1 ppb could be achieved. With zearalanone as internal standard, a linear range between 0.5 (1.0) and 100 ppb in urine samples (cow; pig) and between 1 and 100 ppb in meat samples (cow, calf, pig) could be established. Depending on the biological matrix and analyte standard deviations were below 8.2 %, with recovery rates between 86 and 102 % in spiked samples. The applicability of the method was demonstrated via several contaminated cow and pig urine samples.

Determination of zeranol, taleranol, zearalenone, α- and β-zearalenol in urine and tissue by high-performance liquid chromatography-tandem mass spectrometry

An LC-MS/MS method has been developed for the sensitive determination of the anabolic compound zeranol, its epimer taleranol and the mycotoxins zearalenone, α-zearalenol and β-zearalenol in animal urine and meat samples. Sample preparation included extraction of meat samples and enzymatic digest of urine samples followed by solid phase extraction with RP-18 columns for sample clean-up. Mass spectrometric determination was carried out with an atmospheric pressure chemical ionisation interface (APCI) in the multi-reaction monitoring mode (MRM). Using the negative ion mode, a limit of detection between 0.1 and 0.5 ppb and a limit of determination between 0.5 and 1 ppb could be achieved. With zearalanone (ZAN) as internal standard, a linear range between 0.5 (1.0) and 100 ppb in urine samples (cow, pig) and between 1 and 100 ppb in meat samples (cow, calf, pig) could be established. Depending on the biological matrix and analyte, standard deviations were below 8.5%, with average recovery rates around 86% to 102% in spiked samples. The usefulness of the method developed was demonstrated via several contaminated cow and pig urine samples.

Detection ofLegionella DNA in human and guinea pig urine samples by the polymerase chain reaction

A detection system for Legionella DNA in urine samples based on the polymerase chain reaction (PCR) was developed and tested on infected guinea pigs and patients suffering from pneumonia. Results were compared with standard methods for diagnosis of Legionnaires' disease. A primer system was selected which amplifies a 108 bp DNA fragment of the 5S rRNA gene. The sensitivity of the PCR system was one femtogram of extracted Legionella DNA. Three methods were tested for pretreatment of urine samples. Of these, the Geneclean II kit (Bio 101, USA) gave the best results for artificially contaminated urine samples as well as those from infected guinea pigs or patients. Thirty-seven urine samples from 15 guinea pigs intraperitoneally infected with either Legionella pneumophila serogroup 1, 3 and 6 or Legionella micdadei, 26 urine samples of 21 patients suffering from pneumonia, and 30 control samples of patients with urinary tract infection (UTI) were tested. Legionella DNA was detected in 29 of the guinea pig urine samples; whereas, urinary antigen detection using EIA was positive in only 20 of the samples. PCR was also positive in the samples of 11 patients with pneumonia, 9 of which were confirmed by other microbiological methods, such as culture, direct fluorescent antibody test, urinary antigen detection and antibody testing. However, of the 30 control samples from patients with UTI, three samples yielded positive results. The results demonstrate that Legionella DNA is excreted in the urine of infected individuals and that the PCR shows a higher degree of sensitivity than EIA to the detection of soluble Legionella antigen in urine.(ABSTRACT TRUNCATED AT 250 WORDS)