Electromembrane extraction of clenbuterol from swine urine for monitoring illegal use in livestock.

The illegal use of clenbuterol seriously endangers food safety and human health. Accurate monitoring of the illegal use of clenbuterol in livestock can efficiently prevent the clenbuterol residue pork products from entering the consumer market. Thus, in this study, a simple, rapid, and sensitive method for the determination of clenbuterol in swine urine was developed using electromembrane extraction combined with liquid chromatography-tandem mass spectrometry. It should be noted that the electromembrane extraction method presented many advantages of simple operation, fast mass transfer rate, good sample clean-up capability, and less organic solvent consumption. The effect of important factors on the extraction efficiency of clenbuterol was investigated. Under the optimal conditions, good linearity was achieved for clenbuterol over the range of 1-1000 ng/ml (linear correlation [R2 ] = 0.9996). The recoveries of clenbuterol in swine urine at three spiked levels ranged from 83.7% to 110.0% with relative standard deviation values lower than 9.7% (n = 4). The limits of detection and quantification for clenbuterol were 0.07 and 0.25 ng/ml, respectively. These results suggested that the proposed method has great potential for the extraction and determination of trace analyte in a complex sample matrix for monitoring illegal use in livestock.

An enzyme-free fluorometric nanoprobe for chloramphenicol based on signal amplification using graphene oxide sheets.

A sensitive and selective method for the determination of the antibiotic chloramphenicol (CAP) is described, which is based on double signal amplification and GO as an efficient fluorescence quencher. The nucleic acid probe is composed of three well-defined regions, viz. the signal probe I, the signal probe II, and the capture probe. The capture probe will bind to CAP specifically and the signal probes produce a significant fluorescence signal. One end of the signal probes is labeled with the fluorophore 6-carboxyfluorescein (FAM). The labeled probes can be adsorbed on graphene oxide (GO) via π-stacking interactions, upon which the green fluorescence of FAM (measured at excitation/emission wavelengths of 490/514 nm) is quenched. On addition of CAP, the aptamer/CAP complexes are formed, and this leads to the restoration of fluorescence due to the removal of the probes from GO. The double signal probes, together with GO as quencher, improve the fluorescence signal significantly and lower the detection limit. Under optimized conditions, the assay works in the 20- to 200-ppb CAP concentration range and has a 0.3-ppb detection limit. It is also successfully applied to the determination of CAP in spiked swine urine samples. The recoveries from spiked swine urine samples are between 97.73 and 108.56%, and the repeatability (expressed as the RSD) is between 4.66 and 8.90%. Graphical abstract The constructed DNA probes form a stable structure and bind to chloramphenicol specifically. One end of signal probes was labeled with the fluorophore 6-carboxyfluorescein (FAM). The detection sensitivity of chloramphenicol was significantly enhanced by using double signal amplification, which was superior to the traditional methods. The quantities of CAP can be achieved by fluorescence increment.

Immunochromatographic fluorometric determination of clenbuterol with enhanced sensitivity.

A method is described to enhance the sensitivity of an immunochromatographic assay for clenbuterol (CLE) by making use of dually-labeled gold nanoparticles (GNPs), background fluorescence blocking, and immunomagnetic separation. The GNPs were labeled with biotinylated antibody and streptavidin, respectively, and dually labeled GNPs were obtained via the biotin-streptavidin interaction to amplify the detection signal. The fluorescent signal was blocked by dually labeled GNPs and decreased as the dually labeled GNPs aggregation increases on nitrocellulose membrane, which derived from fluorescent polyvinylchloride card. However, fluorescence (measured at excitation/emission wavelengths of 518/580 nm) recovers when CLE reacts with dually labeled GNPs. Immunomagnetic separation was first applied for sample pretreatment. This can offset the matrix effect and improves the sensitivity and accuracy of the assay. Under the optimal conditions, the limits of detection of CLE visually were 0.25 µg·L-1. In addition, clenbuterol can be quantified in swine urine with a 0.03 µg·L-1 detection limit. This is 60-fold lower than current immunochromatography. Response is linear in the 0.06-0.59 µg·L-1 concentration range, and the recoveries from spiked swine urine range from 81 to 115%." Graphical abstract Schematic presentation of the strategies for improving sensitivity of immunochromatographic assay. It includes immunomagnetic separations, dually-labeled gold nanoparticles and background fluorescence blocking. The assay was applied to detect clenbuterol (CLE) in swine urine with an excellent performance.

A fluorometric clenbuterol immunoassay based on the use of organic/inorganic hybrid nanoflowers modified with gold nanoclusters and artificial antigen.

Organic/inorganic hybrid nanoflowers were synthesized from calcium phosphate and protein modified fluorescent gold nanoclusters and antigens. These nanoflowers are shown to be well suited labels for bioassay because they fulfill the functions of biological recognition and signal output. A fluorometric immunoassay was developed that was combined with immunomagnetic separation. In the detection system, the red fluorescence of the supernatant (measured at excitation/emission wavelengths of 360/640 nm) is found to be proportional to the clenbuterol (Clen) concentration after two immunomagnetic separations. The assay has a linear response in the 0.5 µg L-1 to 40 µg L-1 Clen concentration range, and 0.167 µg L-1 limit of detection. This makes it well suited for food safety monitoring. The average recoveries from spiked samples range from 92.7 to 109.1% (intra-assay) and 101.2 to 125.7% (inter-assay) with relative standard deviations of <11.6%. Spiked swine urine samples were analyzed by this method, and the results correlated well with data obtained by LC-MS/MS. Graphical abstract Fluorescent hybrid nanoflowers were fabricated with gold nanoclusters (BSA-AuNCs) and antigens. A fluorometric immunoassay based on the use of such nanoflowers and based on immunomagnetic separation was developed to detect clenbuterol residues in swine urine with satisfactory recoveries and acceptable accuracy.

Determination of Tranquilizers in Swine Urine by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

A rapid, reliable, and sensitive method was developed for the determination of ten tranquilizers in swine urine. Sample preparation was based on solid-phase extraction, which combined isolation of the compounds and sample cleanup in a single step. Separation was performed on a reversed phase C18 column by gradient elution with a chromatographic run time of seven minutes, consisting of 0.1% formic acid in water and acetonitrile as the mobile phase. Multiple reaction monitoring in positive mode was applied for data acquisition. Matrix-matched calibration was used for quantification and good linearity was obtained with coefficients of determination higher than 0.99. The average recoveries of fortified samples at concentrations between 0.05 and 10 µg/L ranged from 85% to 106% with interday relative standard deviations of less than 13% in all cases. The limits of detection and limits of quantification obtained for tranquilizers in the urine were in the ranges of 0.03⁻0.1 µg/L and 0.05⁻0.25 µg/L, respectively. The applicability of the proposed method was demonstrated by analyzing real samples; diazepam was detected at concentrations between 0.3 and 0.6 µg/L.

An Improved Label-Free Indirect Competitive SPR Immunosensor and Its Comparison with Conventional ELISA for Ractopamine Detection in Swine Urine.

Ractopamine (RCT) is banned for use in animals in many countries, and it is urgent to develop efficient methods for specific and sensitive RCT detection. A label-free indirect competitive surface plasmon resonance (SPR) immunosensor was first developed with a primary antibody herein and then improved by a secondary antibody for the detection of RCT residue in swine urine. Meanwhile, a pre-incubation process of RCT and the primary antibody was performed to further improve the sensitivity. With all the key parameters optimized, the improved immunosenor can attain a linear range of 0.3-32 ng/mL and a limit of detection (LOD) of 0.09 ng/mL for RCT detection with high specificity. Furthermore, the improved label-free SPR immunosenor was compared thoroughly with a conventional enzyme-linked immunosorbent assay (ELISA). The SPR immunosensor showed advantages over the ELISA in terms of LOD, reagent consumption, analysis time, experiment automation, and so on. The SPR immunosensor can be used as potential method for real-time monitoring and screening of RCT residue in swine urine or other samples. In addition, the design using antibody pairs for biosensor development can be further referred to for other small molecule detection.

Rapid detection of sulfonamide antibiotics residues in swine urine by surface-enhanced Raman spectroscopy.

Surface enhanced Raman spectroscopy (SERS) combined with rapid pretreatment technique was used to determine sulfonamide antibiotics (sulfadiazine and sulfathiazole) residue in swine urine. Au nanoparticles (AuNPs) were synthesized as Raman enhance substrate and the extraction of swine urine was purified with primary secondary amine (PSA), octadecyl silane (C18) and graphitized carbon (GCB) to eliminate the interference of the matrix and different dosages of adsorbents (PSA, C18, GCB) were investigated. The results showed that the treatment with C18 of 150 mg, GCB of 200 mg and PSA of 200 mg were an excellent approach for rapidly detecting sulfonamide antibiotics residue in swine urine. Combined with density functional theory calculation (DFT), Raman characteristic peaks of 819, 1102, 1173, 1588 cm-1 and 825, 1127 cm-1 were selected for qualitative and quantitative assessment of sulfadiazine and sulfathiazole in swine urine, respectively. Based on raman characteristic peak of 819 cm-1, a good linear relationship between sulfadiazine concentration and Raman intensity was developed with R2 = 0.9912, and based on raman characteristic peak of 825 cm-1, a good linear relationship between sulfathiazole concentration and Raman intensity was developed with R2 = 0.9941. And recoveries for five unknown concentration samples predicted were 98.47 âˆ¼ 105.18% with relative standard deviation (RSD) of 1.53% ∼ 5.18%. This study demonstrated that SERS coupled with a quick, easy, cheap, effective, rugged, and safe (QuEChERS) method could be employed to rapidly examine the sulfonamide antibiotics residue in swine urine towards its quality and safety monitoring.

An ultrasensitive colloidal gold immunosensor to simultaneously detect 12 beta (2)-adrenergic agonists.

In this study, we first prepared a selective monoclonal antibody against 12 beta (2)-adrenergic agonists (Salbutamol, Clenbuterol, Brombuterol, Clenpenterol, Mabuterol, Carbuterol, Cimbuterol, Mapenterol, Pirbuterol, Terbutaline, Cimaterol, and Clenproperol). Then three haptens were designed and derived, among which, haptenS3 used the amino group of the salbutamol analog to derive a carboxyl group containing a spacer, which is unique to this study. The half-maximal inhibitory concentration (IC50) values were 0.35 ng/mL (Salbutamol), 0.42 ng/mL (Clenbuterol), 0.78 ng/mL (Brombuterol), 0.88 ng/mL (Clenpenterol), 1.34 ng/mL (Mabuterol), 1.38 ng/mL (Carbuterol), 1.71 ng/mL (Cimbuterol), 2.24 ng/mL (Mapenterol), 2.25 ng/mL (Pirbuterol), 2.27 ng/mL (Terbutaline), 3.49 ng/mL (Cimaterol), and 4.89 ng/mL (Clenproperol). We further developed a monoclonal antibody-based colloidal gold immunochromatographic test strip for screening and detecting 12 beta (2)-adrenergic agonists in swine urine and lamb samples. The immunochromatographic method developed in this study is a suitable tool for the on-site rapid detection and screening of beta (2)-adrenergic agonists in swine urine and lamb samples.

Influence of Selective Agents (EMJH-STAFF), Sample Filtration and pH on Leptospira interrogans Serovar Icterohaemorrhagiae Cultivation and Isolation from Swine Urine.

Leptospira spp. cause the zoonotic disease leptospirosis, which occurs in numerous mammalians worldwide. Isolation is still important for serotyping and genotyping of Leptospira, which in turn is essential for epidemiological surveillance of leptospirosis and the development of diagnostic tests and vaccines. However, isolation of Leptospira from clinical specimens is inherently insensitive. This study was conducted to examine the influence of selective agents, sample filtration, sample pH and the use of phosphate buffered saline (PBS) buffer for sample storage to improve the success of cultivation and isolation of Leptospira interrogans serovar Icterohaemorrhagiae from swine urine. EMJH (Ellinghausen McCullough, Johnson and Harris) medium including the selective agents sulfamethoxazole, trimethoprim, amphotericin, fosfomycin and 5-fluorouracil (STAFF) increased the success of Leptospira isolation from spiked swine urine samples. Sample filtration yielded only negative results. Isolation in EMJH-STAFF was successful from swine urine with a density as low as 104 Leptospira/mL, and urine with pH ≤ 7 impaired the cultivation rate. Cultivation and isolation were not improved by the addition of PBS to spiked urine samples prior to storage for 24 h at 4 °C. The results of the study demonstrate that cultivation and isolation of leptospires from swine urine can be improved by enhanced methods.

Selective extraction and detection of ß-agonists in swine urine for monitoring illegal use in livestock breeding.

The illegal use of ß-agonists often endangers animal-derived food safety. In this study, a selective detection method for ß-agonists in swine urine was established via the combination of polymeric ionic liquid-molecularly imprinted graphene oxide-miniaturized pipette tip solid-phase extraction and high-performance liquid chromatography. It is worth noting that this method relied mainly on the designed adsorbent, which presented a rich adsorption mechanism, fast mass transfer rate, and high selectivity, and was successfully utilized in the selective extraction of ß-agonists from swine urine samples. The proposed method has low LOD (0.20-0.56 ng/mL), high recovery (94.9-107.9%), and high reusability (4 times, 91.9-108.8%), which indicates its high potential as a selective, sensitive, accurate, and nonfatal method for monitoring the illegal use of ß-agonists in the livestock breeding stage.

Sensitive and Matrix-Tolerant Lateral Flow Immunoassay Based on Fluorescent Magnetic Nanobeads for the Detection of Clenbuterol in Swine Urine.

The lack of sensitivity and poor matrix tolerance are the main bottlenecks of the lateral flow immunoassay (LFIA). Here, a sensitive and matrix-tolerant method that integrated immunomagnetic separation and fluorescent lateral flow immunoassay (IMS-FLFIA) based on fluorescent magnetic nanobeads was developed to detect the clenbuterol (CLE) residue in swine urine. The limit of detection (LOD) of IMS-FLFIA is 4 times lower than that of traditional colloidal gold LFIA. This method, which exhibits similar LOD and linearity range in both phosphate-buffered saline and urine swine, is highly correlated with liquid chromatography-tandem mass spectrometry for the detection of real swine urine samples. The result indicated that IMS-FLFIA has a universal resistance to the swine urine matrix. The merits of this assay, high sensitivity, matrix tolerance, accuracy, and specificity, ensure a promising future in detection of veterinary drug residues.

Development of molecular imprinted column-on line-two dimensional liquid chromatography for selective determination of clenbuterol residues in biological samples.

A novel method coupling molecular imprinted monolithic column with two-dimensional liquid chromatography was developed and validated for the analysis of clenbuterol in pork liver and swine urine samples. The polymers were characterized by using Fourier transform infrared spectroscopy, nitrogen adsorption desorption analyses, frontal analysis and the adsorption of selectivity. The results indicated that the imprinted columns were well prepared and possessed high selectivity adsorption capacity. Subsequently, the MIMC-2D-LC (molecular imprinted monolithic column-two dimensional liquid chromatography) method was developed for the selective analysis of clenbuterol in practical samples. The accuracy ranged from 94.3% to 99.7% and from 93.7% to 99.6% for liver and urine, respectively. The relative standard deviation (RSD) of repeatability was lower than 8.6% for both analyses. The limit of detections was 16ng·mL(-1) for liver and 25ng·mL(-1) for urine, respectively. Compared with the reported methods, the disturbance of endogenous impurity could be avoided by the 2D-LC method.

New haptens and antibodies for ractopamine.

In this work, three unreported immunizing haptens of ractopamine (RAC) were synthesized and used to produce highly sensitive and specific polyclonal antibody. The spacer arms of haptens for coupling to protein carrier were located on different position of RAC with different length. High affinity polyclonal antibodies were obtained and characterized in terms of titer and sensitivity by using enzyme-linked immunosorbent assay (ELISA). The best antibody employed in a heterologous competitive ELISA exhibited an IC50 value as low as 0.12ngmL(-1) and could not recognize other 10 ß-agonists including clenbuterol and salbutamol. The heterologous competitive ELISA was preliminary applied to swine urine and the results showed the new antibody was sufficiently sensitive and specific, and potentially used for the detection of RAC at trace level in real samples.

Metabolomic analysis of swine urine treated with ß2-agonists by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

The illegal use of ß2-agonists in livestock production was previously detected by efficient methods based on mass spectrometry to control the residues of these drugs. Nevertheless, such methods still remain a challenging task for authorities who monitor these residues because the use of "cocktails" composed of mixtures of low amounts of several substances as well as the synthesis of new compounds of unknown structure prevent efficient prevention of illegal use of growth-promoting agents. Here, we outlined a metabolomics-based strategy for detecting the use of "cocktails" composed of mixtures of low amounts of three ß2-agonists via urine profiling. Urine profiles of controls and swine treated with mixture of low amounts of three substances (clenbuterol, salbutamol, and ractopamine) were analyzed with ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The metabolic differences between controls and ß2-agonists-treated groups were compared using multivariate data analysis. Fourteen metabolites were identified related with the ß2-agonists treatment, while two co-biomarkers, 2-indolecarboxylic acid and fluorometholone acetate, either in single or "cocktails" of low-dose mixture of clenbuterol, salbutamol, and ractopamine, could be considered as diagnostic markers for the detection of illegal use of ß2-agonists. The results of depletion study demonstrated that it is practical to use the markers for monitoring of ß2-agonists.

Preparation of stir cake sorptive extraction based on poly(4-vinylbenzoic acid-divinylbenzene) monolith and its application in sensitive determination of ß-agonists in milk and swine urine samples.

In this study, a new stir cake sorptive extraction (SCSE) based on poly(4-vinylbenzoic acid-divinylbenzene) (VBADB) monolith was prepared. The effect of preparation conditions of monolith on extraction efficiencies was investigated in detail. Several characteristic techniques, such as elemental analysis, infrared spectroscopy, mercury intrusion porosimetry and scanning electron microscopy were used to characterize the monolithic material. The combination of SCSE-VBADB with high-performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS) detection was developed for sensitive determination of ultra-trace ß-agonists in milk and swine urine samples. In order to obtain the optimal extraction conditions of SCSE-VBADB for ß-agonists, several extractive parameters, including pH values and ionic strength in sample matrix, extraction and desorption time were optimized. Under the optimum conditions, the limits of detection (S/N=3) for the target analytes were 0.007-0.030 µg/L in milk and 0.002-0.011 µg/L in swine urine, respectively. Excellent method reproducibility was achieved in terms of intraday and interday precisions, indicated by the RSDs of both <10.0%, respectively. Finally, the proposed method was successfully used to detect ß-agonists in different milk and swine urines samples. Acceptable recoveries ranged from 50.3% to 113% and 50.1% to 92.2% for milk and swine urine samples, respectively; and the RSDs for reproducibility were less than 8.0% for target analytes in all real samples.