Β-agonists In Urine Research Articles

Four Hapten Spacer Sites Modulating Class Specificity: Nondirectional Multianalyte Immunoassay for 31 β-Agonists and Analogues.

Immunoassay methods are important for monitoring β-agonists illegally used for reducing animal fat deposition in livestock. However, there is no simultaneous screening surveillance immunoassay for detecting various β-agonist chemicals that are possibly present in food. In this study, through the use of an R-(-)-salbutamol derivative as the immunizing hapten, an antibody recognizing 31 β-agonists and analogues was generated for the first time. Three-dimensional quantitative structure-activity relationship (3D QSAR) revealed that strong steric and hydrophobic fields around the hapten spacer near C-2, as well as a chirality at C-1', dominantly modulated the class specificity of the raised antibody. However, a hapten spacer linked at C-2' or C-1 would lead to a narrow specificity, and the spacer charge at C-6 could affect the raised antibody specificity spectrum. A class specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) was established with an ideal recovery ranging from 81.8 to 118.3% based on the obtained antibody. With a good agreement to the HPLC/MS method, the proposed ciELISA was confirmed to be reliable for the rapid surveillance screening assay of β-agonists in urine. This investigation will contribute to the rational design and control of the immunoassay specificity.

Pseudo template molecularly imprinted polymer for determination of 14 kind of β-agonists in animal urine by ultra-high-performance liquid chromatography-tandem mass spectrometry

A pseudo template molecularly imprinted polymer (MIP) for selective extraction of β-agonists was prepared using 4-hydroxyphenethyl alcohol as pseudo template molecule, methyl acrylic acid as functional monomer, and ethylene glycol dimethacrylate as cross-linker. Selectivity and adsorption capacity of prepared MIP were studied in detail. MIP selectively adsorbed 14 types of β-agonists. Advantages of the prepared polymer include non-template leakage in purification of target analytes and cheap cost. MIP was used to purify β-agonists in urine samples. The developed method was combined with ultra-performance liquid chromatography-tandem mass spectrometry. In optimum conditions, recoveries of the developed method ranged from 84.2% to 109.8% with low coefficients of variation at below 15%. Limits of detection and quantification for 14 target β-agonists in urine samples reached below 0.05ng/mL and 0.1ng/mL, respectively. The developed method based on MIP enrichment and purification were successfully applied to analysis of target analytes in 50 actual urine samples.

Magnetic graphene dispersive solid phase extraction-ultra performance liquid chromatography tandem mass spectrometry for determination of β-agonists in urine.

In this study, a magnetic graphene-based dispersive solid phase extraction method was first developed for extraction of β-agonists in urine. During the experiments, the absorbent amount, sample pH, extraction time, elution solution and elution time were optimized respectively. The optimized extraction method was finished within 10min, and showed high enrichment factors for 9 β-agonists (20-26 folds). Furthermore, this absorbent could be reused for at least 60 times. Then this extraction method was combined with ultra performance liquid chromatography triple quadrupole tandem mass spectrometry to determine the 9 drugs in urine. The limits of detection for the 9 drugs were in a range of 0.015-0.023ngmL-1, and the recoveries from the standards fortified blank urine were in a range of 60.2%-109.4%. Therefore, this method could be used as a simple, rapid, sensitive and accurate tool to determine trace level of β-agonists in urine.

A novel and sensitive screening method for β-agonists in porcine urine by using atmospheric solid analysis probe source coupled tandem mass spectrometry

A novel, sensitive and specific screening method for trace multi-residues of 13 β-agonists in porcine urine by combining atmospheric solid analysis probe (ASAP) source with tandem mass spectrometry (MS/MS) has been developed. Optimization experiments were carried out including the selection of solvent, probe temperature and monitoring ionic pairs. Using ASAP combined with rapid solid-phase extraction (SPE) procedures, not only the matrix interference of porcine urine was decreased, but also the sensitivity of the method was improved. Under the optimal conditions, the performance of the proposed method was studied. The linear correlation coefficients (R2) of calibration curves for 13 β-agonists were above 0.98 in the concentration range of 0.5 ng mL−1 to 300 ng mL−1. The limits of determination (LOD) for all target compounds were below 0.20 ng mL−1, recoveries of the developed method for different spiked concentrations varied from 58.5 to 85.7%. A single run for the analysis of 13 β-agonists in a urine sample was achieved within 1.0 min for ASAP-MS/MS detection. In addition to the fast analysis speed, MS/MS provided structural information for the confirmation of target analytes. Real porcine urine samples were investigated and the results were in accordance with the confirmatory determination results by using China’s agricultural industry standard method. The developed method can potentially be used for the screening of 13 β-agonists in porcine urine for the surveillance of livestock.

Quick screening of priority β-agonists in urine using automated TurboFlow™-LC/Exactive mass spectrometry

This paper describes a method for the determination of priority β-agonists in urine based on a fully automated sample preparation procedure using an online TurboFlow™ chromatography clean-up step and determination with Orbitrap™ mass analyser technology. The principle of the method was the enrichment of the β-agonists after enzymatic hydrolysis overnight on a small column packed with a special stationary phase (TurboFlow™) while flushing away sample matrix and interfering compounds. Thereafter, the analytes were transferred onto an analytical column and detected by liquid chromatography/high-resolution mass spectrometry in full-scan mode at a resolution of R = 50,000 FWHM (full width at half maximum) and in higher energy collisional dissociation (HCD) scan mode at a resolving power of 10,000 FWHM. The optimisation of each step of the method, such as selection of the TurboFlow™ and analytical column as well as sample loading and elution parameters were performed using a standard solution containing salbutamol, clenbuterol and mabuterol at a concentration of 100 µg l−1. The developed automated sample preparation significantly improved the throughput and efficiency of the previously used screening method and it resulted in a considerable reduction in analysis time. Validation experiments including 24 β-agonists in urine gave decision limits (CCα) between 0.05 and 0.35 µg l−1. The repeatability of analyses for urine samples spiked at 0.5 µg l−1 was within the range of 5–26% and recoveries for all compounds were within 89–107%.

Development of a Fast and Simple Method for Determination of β-Agonists in Urine by Extraction on Empore Membranes and Detection by a Test Strip Immunoassay

A rapid screening method for β-agonists in cattle urine was developed that offers speed and protocol simplicity and that could be performed on the spot. The method consists of an extraction on Empore membranes with strong cation exchange properties and a test strip immunoassay. The Empore extraction provides a fast and simple cleanup of the urines and a concentration step with recoveries ranging from 50-80% for the β-agonists of the aniline type and around 10% for salbutamol and terbutaline. A test strip immunoassay is performed on the diluted extract. With the use of an antiperoxidase antibody, an internal negative control was included, to which all color reductions are compared. Although a portable colorimeter increased the sensitivity of the test, a visual interpretation was sufficient to give a correct yes/no answer. The whole test takes about 30 min and has a visual detection limit for clenbuterol of 1 ng/mL of urine. The test also shows good sensitivity for salbutamol, despite the low recovery with the Empore extraction.

Gas and liquid chromatography coupled to tandem mass spectrometry for the multiresidue analysis of β-agonists in biological matrices

β-Agonists are substances used in veterinary and human medicine for the treatment of pulmonary disorders. They have found a use as growth promoters to improve meat-to-fat ratios in cattle but they are not authorized for use in the European Union. Due to their presence in trace levels (usually less than 1 μg/kg), to the diversity of the illegally used compounds and to the complexity of the biological matrices analysed, the detection of these residues requires a very sensitive and specific method of determination. This work describes the strategy of analysis we developed for five β-agonists in urine and liver. The combination of improved solid- or liquid-phase extraction methods and LC or GC-MS-MS (in the multiple reaction monitoring mode) has shown to provide a system suitable for the control of these substances. The efficiency of extraction and the sensitivity and selectivity allow this multiresidue detection down to, and below, the UK regulatory level of 0.5 μg/kg. Moreover, the use of LC removes the need for the derivatisation step (cyclic methylboronate derivatives) which is required prior to GC-MS-MS analysis.

Rapid analysis of β-agonists in urine by thermospray tandem mass spectrometry

A method is described for the analysis of β-agonists in urine of cattle. The method uses solid-phase extraction (SPE), followed by analysis of the resulting extract by flow injection thermospray tandem mass spectrometry (TSP-MS-MS). Sample preparation is performed using a mixed-bed SPE procedure using a sorbent having both hydrophobic and ionic properties. MS-MS analysis following thermospray ionization, is performed in single-reaction monitoring parent mode. In that way isotope dilution can be used for quantitation of clenbuterol. Data are presented on precision and accuracy for clenbuterol and related compounds. Furthermore, data acquisition was performed in full-scan neutral loss mode to indicate the suitability of flow injection analysis (FIA)-TSP-MS-MS for exploratory analysis. Detection of β-agonists in this mode is based on the presence of the N- tert.-butyl- β-ethanolamino moiety and, in that respect, detection of known as well as unknown compounds having this moiety will take place. This feature is exemplified by the analysis of samples containing several compounds.

Applicability of coupled-column liquid chromatography to the analysis of β-agonists in urine by direct sample injection I. Development of a single-residue reversed-phase liquid chromatography-UV method for clenbuterol and selection of chromatographic conditions suitable for multi-residue analysis

Optimisation procedures originally applied to coupled-column RPLC-UV for the residue analysis of polar pesticides were evaluated for the analysis of β-agonists in human and bovine urine using direct sample injection. Two approaches have been studied: (i) a multi-residue method (MRM) for the clean-up and separation of eight different β-agonists (isoprenaline, cimaterol, terbutaline, salbutamol, fenoterol, ractopamine, clenbuterol and mabuterol) and (ii) a single-residue method (SRM) focussed at the detection of clenbuterol residues in samples of urine. Both approaches provided efficient procedures to process urine samples automatically with coupled-column LC. Particular attention was paid to selecting analytical conditions suitable for thermospray MS detection, which is to be investigated in the near future. Though UV detection cannot offer enough selectivity for the simultaneous screening of a group of β-agonists, coupled-column RPLC-UV proved to be very powerful in SRM, allowing the detection of clenbuterol at the μg/1 level in filtered (0.45 μm) human and bovine urine after direct sample injection.

Multi-residue analysis for β-agonistic drugs in urine of meat-producing animals by gas chromatography—mass spectrometry

Two reliable gas chromatographic-mass spectrometric (GCMS) procedures for the unequivocal determination of β-agonists in urine of meat-producing animals are described. The β-agonistic drugs are extracted using disposable mixed solid-phase extraction columns. The urine sample (10 ml) is buffered (pH 6.0) and applied to a Clean Screen DAU cartridge. The analytes are eluted with ethyl acetate containing 3% (v/v) of concentrated (32%) ammonia solution. The extracted analytes are derivatized to either their trimethylsilyl (TMS) or cyclic 2-(dimethyl)silamorpholine (DMS) derivatives and analysed by GCMS under electron impact (EI) and positive-ion chemical ionization (PCI) conditions. Cyclic DMS derivatives proved to be useful for screening for or confirming the presence of clenbuterol analogues in the EI mode. If necessary, they could also be confirmed in the PCI mode using ammonia as reagent gas. With regard to TMS derivatives, their use provided a rapid and efficient method for screening for (EI) and confirming (PCI) the presence of at least thirteen β-agonists at the low ng ml −1 level.

Determination of β-agonists in urine by an enzyme immunoassay based on the use of an anti-salbutamol antiserum

An enzyme immunoassay (EIA) method for β-agonists has been developed using an antiserum raised in rabbits by immunization against 3- O-salbutamol succinate coupled to bovine serum albumin. Horse radish peroxidase coupled to the same salbutamol derivative was selected as tracer. The dose of salbutamol which caused 50% binding inhibition was 16.8 pg per well and the limit of detection of the EIA directly performed on diluted urine was 0.14 ng ml −1 in urine (when the variability of blank values in samples from untreated animals was taken into account). Owing to the large cross reactivities of the antibodies with a number of β-agonists, the present assay is promising for the simultaneous determination of salbutamol, clenbuterol, mabuterol, terbutaline, cimaterol and probably also other β-agonists with a tert-butyl or isopropyl group at the extremity of the aliphatic side chain of the molecule.