Sodium 4-styrenesulfonate functionalized nanofibers mat as 96-well plate solid-phase extraction adsorbent for quantitative determination of multiple ß-agonists residues in pork samples.

In this work, sodium 4-styrenesulfonate functionalized polyacrylonitrile nanofibers mat (SS/PAN NFM) was firstly prepared and applied as 96-well plate solid-phase extraction adsorbent for quantitative determination of seven ß-agonists residues in pork samples. The functional modification endowed the SS/PAN NFM with superior adsorption performance for target ß-agonists. The adsorption process is spontaneous (ΔG < 0), the initial adsorption rate can reach 6.03-9.09 mg/g/min and the maximum adsorption capacity is calculated to be 48.3 mg/g at 298 K. Moreover, SS/PAN NFM can be reused for 12 times without degradation in adsorption capability. Combined with UPLC-MS/MS, the limits of detection can reach 0.006-0.24 µg/kg, the recoveries ranged from 87.2% to 111% and the relative standard deviations of intra-day and inter-day precisions were in the scope of 1.75%-11.6% and 5.08%-13.5%, respectively. The obtained results fully demonstrated the practicability of this method in preventing the hazard of ß-agonists residues.

Facile synthesis of magnetic sulfonated covalent organic framework composites for simultaneous dispersive solid-phase extraction and determination of ß-agonists and fluoroquinolones in food samples.

In this work, an efficient method for the determination of ß-agonists and fluoroquinolones was established, based on a mixed-mode sorbent of magnetic sulfonated covalent organic framework composites. By coupling with HPLC-MS/MS, the main factors that affect the extraction procedure were optimized. Under the optimal conditions, the proposed HPLC-MS/MS method was successfully utilized for the extraction of ß-agonists and fluoroquinolones in milk and pork meat samples. The method showed good linearities (R2 ≥ 0.9916), and low LOQs of 0.1-0.2 ng g-1 for ß-agonists and fluoroquinolones. The adsorption mechanism was investigated with the assistance of quantum chemistry calculation method, and it is worth noting that the sorbent relied mainly on the multiple adsorption mechanisms, including π-π stacking, hydrophobic, electrostatic attraction and hydrogen-bonding interactions. This work not only provides a simple method for the preparation of a mixed-mode sorbent, but also a routine analysis strategy for monitoring the illegal use of ß-agonists and fluoroquinolones.

The determination of ß-agonist residues in bovine tissues using liquid chromatography-tandem mass spectrometry.

We aimed to develop a rapid, simple and reproducible method based on LC-tandem mass spectrometry (LC-MS/MS) to analyze ß-agonist residues (clenbuterol, zilpaterol, ractopamine and isoxsuprine) in bovine tissues. The method was validated in accordance with the European Council Decision 2002/657/EC. The samples were homogenized, and then 10 mL of an acetate buffer was added to a 5-g sample. The sample was then centrifuged at 12,000 rpm and filtered. Sodium hydroxide (2 m) was added to adjust pH of the sample that was centrifuged again. The extract was filtered through a solid-phase extraction column. The residue was re-dissolved in 250 µL acetonitrile and then subjected to LC-MS/MS. The separation was done on a C18 column. The mobile phase consisted of 0.1% formic acid in deionized water and 0.1% formic acid in methanol. The mean recoveries of ß-agonists were in the range of 84.3%-119.1% with relative standard deviations (%RSDs) of 0.683%-4.05%. Decision limits and detection capabilities of the analytes ranged from 0.0960 to 4.9349 µg/kg and from 0.0983 to 5.0715, respectively. This method was used to detect four ß-agonists in 100 bovine muscle, 100 liver and 100 kidney tissues from a slaughterhouse. No residue was found above the maximum residue limit level.

Hydrophobic deep eutectic solvent-based dispersive liquid-liquid microextraction for the simultaneous enantiomeric analysis of five ß-agonists in the environmental samples.

In this study, a simple and effective method was developed for the enantiomeric analysis of five ß-agonists (terbutaline, clorprenaline, tulobuterol, clenbuterol, and salbutamol) in water samples using deep eutectic solvent (DES) based dispersive liquid-liquid microextraction and chiral LC-MS. In such a framework, different kinds of hydrophobic DESs were tailored to examine their extraction ability for five ß-agonists from aqueous sample. After an initial screening, the primary factors affecting the extraction recovery of DES based dispersive liquid-liquid microextraction, such as hydrogen-bond acceptor/hydrogen-bond donor ratio, DES volume, type and volume of disperser solvent and so on, were investigated and optimized. Finally, the established method was validated and found to be linear, precise, and accurate. The method was successfully applied to analyze the five ß-agonists in water samples, which will help better understand the behavior of individual enantiomer and make accurate risk assessment on the ecosystem.

Analysis of 10 ß-agonists in pork meat using automated dispersive pipette extraction and LC-MS/MS.

An analytical procedure for the analysis of 10 ß-adrenergic agonists (cimaterol, terbutaline, salbutamol, isoxsuprine, ractopamine, cimbuterol, clenbuterol, brombuterol, mabuterol and mapenterol) in pork meat was developed and validated using LC-MS/MS. An automated dispersive pipette extraction (DPX) was employed on a Hamilton Microlab® NIMBUS96® platform to extract the analytes of interest prior to LC-MS/MS analysis. The extraction time was <20 min with a total LC-MS/MS run time of 9.6 min. The method was fully validated in accordance with the international guidelines (European Commission Decision 2002/657/EC and National Standards of People's Republic of China, GB/T 22286-2008) for limit of detection, limit of quantitation, carryover, extraction efficiency, matrix effects, linearity, and within and between-run precision. The proposed method can be successfully used in the routine determination of 10 ß-adrenergic agonists in pork and as a potential solution for compliance monitoring in regulatory laboratories.

Determination of ractopamine and salbutamol in pig hair by liquid chromatography tandem mass spectrometry.

A liquid chromatography tandem mass spectrometric method was developed for the determination of two ß-agonists (ractopamine and salbutamol) in pig hair samples. An isotope of ractopamine-d5 or salbutamol-d6 as an internal standard was used to carry out quantitative analysis. Concentrated sodium hydroxide was used to pretreat hair samples and then purified by the solid phase extraction (SPE) procedure. The extracted solution was evaporated and reconstituted for injection in the instrument with electrospray ionization (ESI) operating in a positive multiple-reaction-monitoring (MRM) mode. Ractopamine and salbutamol separation were performed on C18 analytical column under gradient condition. The internal standard calibration curve was linear in the range of concentration from 0.5 to 100 ng mL-1 (R2 > 0.995). Recoveries of this method estimated at three spiked concentrations of 100, 250 and 500 ng mL-1 in pig hair samples, were 79-82% for ractopamine and 77-96% for salbutamol. The corresponding inter-day and intra-day precisions expressed as relative standard deviation (RSD %) were 3.8-6.4% and 3.8-8.6%, respectively. The analytical time for one sample was 8 min. The detection limit of this method was 0.6 and 8.3 ng mL-1 for ractopamine and salbutamol, respectively. This developed method can be applied for monitoring the use of the ß-agonists salbutamol and ractopamine in swine feed incurred pig hair.

Detection of ß-agonists in pork tissue with novel electrospun nanofibers-based solid-phase extraction followed ultra-high performance liquid chromatography/tandem mass spectrometry.

A selective analytical method based on packed-fiber solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry (PFSPE-UPLC-MS/MS) has been developed for determination of six ß-agonists (clorprenaline, bambuterol, clenbuterol, brombuterol, mabuterol, and penbuterol) in pork tissue. Polystyrene-polymeric crown ether (PS-PCE) composite nanofibers were fabricated by electrospinning and utilized to prepare the homemade extraction columns. With optimal conditions, all analytes were separated very well and the blank pork did not disturb the determination, and the linearity is good in a range of 5.0µg/kg-25.0µg/kg. The recoveries were 79.3-110.1%. RSDs for intra-day were in the range of 1.5-10.5% and RSDs for inter-day were 4.7-11.8%. Above all, only 5mg of sorbent and 200µL of elution solvent were favorable to directly extract all analytes in a complex matrix. The method is simple and cost-effective, and has the potential to be applied to quantitatively analyze the concentrations of polar species in food samples containing complex matrix.

Preparation of hydrophilic interaction/ion-exchange mixed-mode chromatographic stationary phase with adjustable selectivity by controlling different ratios of the co-monomers.

Development of mixed-mode chromatography (MMC) stationary phase with adjustable selectivity is beneficial to meet the needs of complex samples. In this work, surface-initiated atom transfer radical polymerization (SI-ATRP) using the mixture of two functional monomers was proposed as a new preparation strategy for MMC stationary phase with adjustable selectivity. The mixture of sodium 4-styrenesulfonate (NASS) and dimethylaminoethyl methacrylate (DMAEMA) underwent SI-ATRP to bond poly(NASS-co-DMAEMA) on the surface of silica to prepare hydrophilic interaction/ion-exchange mixed-mode stationary phase. Various analytes (neutral, acidic, basic analytes and strong polar nucleosides) were employed to investigate the retention behaviors. The influences of water content and pH of the mobile phase on the retention validated the mixed-mode retention mechanisms of HILIC and ion-exchange. The charge and polarity of stationary phase as well as the separation selectivity were conveniently manipulated by the ratio of NASS to DMAEMA monomer, and the use of DMAEMA in the mixture additionally endowed the column with the temperature-responsive characteristics. Moreover, the application of the developed column was demonstrated by the successful separation of nucleosides, ß-agonists and safflower injection. In a word, the proposed strategy can be potentially applied in the controllable preparation of MMC stationary phase with adjustable selectivity.

Antispasmodic and bronchorelaxant activities of Salsola imbricata are mediated through dual Ca+2 antagonistic and ß-adrenergic agonistic effects.

CONTEXT: Salsola imbricata Forssk. (Chenopodiaceae) has folkloric repute for the treatment of various gastrointestinal and respiratory ailments. OBJECTIVE: The present study investigates spasmolytic and bronchorelaxant effects of S. imbricata. MATERIALS AND METHODS: The crude aqueous-ethanol extract of the aerial parts of S. imbricata and its fractions, in cumulative concentrations (0.01-10 mg/mL), were tested on contractions of isolated rabbit jejunum and tracheal preparations. Furthermore, concentration response curves (CRCs) of Ca+2 and carbachol were constructed in the absence and presence of the extract. Standard organ bath methods were used. RESULTS: The crude extract relaxed spontaneous, K+ (80 mM) and carbachol (1 µM)-induced contractions in jejunum preparations with respective EC50 values of 0.40 (0.35-0.46), 0.69 (0.60-0.79) and 0.66 (0.57-0.75) mg/mL. It shifted Ca+2 CRCs rightward in nonparallel manner. In isolated tracheal preparations, the crude extract caused relaxation of K+ (80 mM) and carbachol (1 µM)-induced contractions with EC50 values of 0.86 (0.75-0.98) and 0.74 (0.66-0.84) mg/mL, respectively. It displaced carbachol CRCs rightward with suppression of maximal response. In both tissues, pretreatment with propranolol (1 µM) caused rightward shift in inhibitory CRCs of the extract against carbachol-induced contractions. The ethyl acetate fraction was found more potent in relaxing smooth muscle contractions than the parent extract and its aqueous fraction. DISCUSSION AND CONCLUSION: The results suggest that the spasmolytic and bronchorelaxant activities of S. imbricata are related to Ca+2 antagonistic and ß-adrenergic agonistic effects, thus justifying some of the traditional uses of the plant.

Prediction of retention in hydrophilic interaction liquid chromatography using solute molecular descriptors based on chemical structures.

Quantitative structure-retention relationship (QSRR) models are developed to predict the retention times of analytes on five hydrophilic interaction liquid chromatography (HILIC) stationary phases (bare silica, amine, amide, diol and zwitterionic), with a view to selecting the most suitable stationary phase(s) for the separation of these analytes. The study was conducted using six ß-adrenergic agonists as target analytes. Molecular descriptors were calculated based only on chemical structures optimized using density functional theory. A genetic algorithm (GA) was then used to select the most relevant molecular descriptors and these were used to build a retention model for each stationary phase using partial least squares (PLS) regression. This model was then used to predict the retention of the test set of target analytes. This process created an optimized descriptor set which enhanced the reliability of the developed QSRR models. Finally, the QSRR models developed in the work were utilized to provide some insight into the separation mechanisms operating in the HILIC mode. Three performance criteria - mean absolute error (MAE), root mean square error of prediction scaled to retention time (RMSEP), and the number of selected descriptors, were used to evaluate the developed models when applied to an external test set of six ß-adrenergic agonists and showed highly predictive abilities. MAE values ranged from 13 to 25s on four of the stationary phases, with a somewhat higher error (50s) being observed for the zwitterionic phase. RMSEP values of 4.88-11.12% were recorded. Validation was performed through Y-randomization and chemical domain applicability, from which it was evident that the developed optimized GA-PLS models were robust. The high levels of accuracy, reliability and applicability of the models were to a large extent due to the optimization of the GA descriptor set and the presence of relevant structural and geometric molecular descriptors, together with descriptors based on important physicochemical properties, which establish a strong connection between retention time and meaningful chemical properties. The present strategy, while it is a pilot study, holds great promise for broader screening of HILIC stationary phases for desired separation, as well as for acquisition of information about molecular mechanisms of separation under chromatographic conditions.

Separation and elution order of the enantiomers of some ß-agonists using polysaccharide-based chiral columns and normal phase eluents by high-performance liquid chromatography.

In this study separation of enantiomers of 8 chiral ß-agonists were studied on 6 polysaccharide-based chiral columns in polar-organic and alcohol-hydrocarbon mobile phases. No separation of enantiomers was observed on any column with polar-organic mobile phase eluents such as pure methanol, ethanol or acetonitrile. Most of the chiral analytes were resolved into enantiomers when alcohol-hydrocarbon type mobile phases were used. The most successful column was Lux Cellulose-2 on which all 8 chiral analytes were baseline resolved into enantiomers at least with one mobile phase used. The reversal of enantiomer elution order was observed dependent on the chemistry of the chiral selector and the composition of the mobile phase.

Magnetic molecularly imprinted polymers for the determination of ß-agonist residues in milk by ultra high performance liquid chromatography with tandem mass spectrometry.

A simple, accurate, and highly sensitive analytical method was developed in this study for the determination of nine ß-agonists in milk. In this method, a new magnetic adsorbent of molecularly imprinted polymers/magnetic nanoparticles prepared by simple physical blending was adopted, which enabled magnetic solid-phase extraction. Thus, the resultant material can be separated from the solvent rapidly and conveniently by a magnet. Two kinds of molecularly imprinted polymer/magnetic nanoparticles materials were fabricated, and the characteristics of materials such as the ratio, pH, amount, desorption, and regeneration were investigated. The analytes were quantified by ultra high performance liquid chromatography coupled to an electrospray ionization tandem mass spectrometer operating in multiple reaction monitoring modes. The detection limit of the method was 0.003-0.3 µg/L, and the detection capability was 0.01-0.3 µg/L. The recoveries of these compounds were 65.7-114% at three spiked levels. Reproducibility represented by relative standard deviation was 11.2% or less. The method was successfully applied to the screening of real samples obtained from local markets and confirmation of the suspected target analytes.

Novel method for the rapid and specific extraction of multiple ß2 -agonist residues in food by tailor-made Monolith-MIPs extraction disks and detection by gas chromatography with mass spectrometry.

A quick and specific pretreatment method based on a series of extraction clean-up disks, consisting of molecularly imprinted polymer monoliths and C18 adsorbent, was developed for the specific enrichment of salbutamol and clenbuterol residues in food. The molecularly imprinted monolithic polymer disk was synthesized using salbutamol as a template through a one-step synthesis process. It can simultaneously and specifically recognize salbutamol and clenbuterol. The monolithic polymer disk and series of C18 disks were assembled with a syringe to form a set of tailor-made devices for the extraction of target molecules. In a single run, salbutamol and clenbuterol can be specifically extracted, cleaned, and eluted by methanol/acetic acid/H2 O. The target molecules, after a silylation derivatization reaction were detected by gas chromatography-mass spectrometry. The parameters including solvent desorption, sample pH, and the cycles of reloading were investigated and discussed. Under the optimized extraction and clean-up conditions, the limits of detection and quantitation were determined as 0.018-0.022 and 0.042-0.049 ng/g for salbutamol and clenbuterol, respectively. The assay described was convenient, rapid, and specific; thereby potentially efficient in the high-throughput analysis of ß2 -agonists residues in real food samples.

Determination of a large set of ß-adrenergic agonists in animal matrices based on ion mobility and mass separations.

While the coupling of traveling wave ion mobility spectrometry (TWIMS) and mass spectrometry is mainly reported for structural purposes, we studied its potential in enhancing compounds analysis such as growth promoters used in livestock animals at trace concentrations. ß-Adrenergic agonists have been selected as model compounds since they exhibit a range of close physicochemical properties leading to analytical issues using classical approaches. In this paper, the potential of Synapt G2-S (Q-TWIM-TOF MS) has been investigated for sensitive and specific detection of a range of these synthetic phenethanolamines in various complex biological matrices (retina, meat, and urine) from bovine considered as relevant in the context of detecting ß-adrenergic agonists use in animals. In particular, the specificity of the additional information provided by the TWIMS (i.e., collision cross section) together with the interest of the extra dimension of separation is discussed.

Higenamine 4'-O-ß-d-glucoside in the lotus plumule induces glucose uptake of L6 cells through ß2-adrenergic receptor.

Hypoglycemic effect is an efficient means to modulate elevated blood glucose levels in patients with diabetes. We found that the extract of lotus plumule (the germ of Nelumbo nucifera Gaertn. seed) showed potent glucose uptake enhancement activity against L6 myotubes, which results in a hypoglycemic effect. This activity was further investigated, and an active constituent was identified as a single bioactive compound, higenamine 4'-O-ß-d-glucoside. Mechanistic studies employing phosphatidylinositol 3-kinase (PI3K) inhibitor, AMP-activated protein kinase (AMPK) inhibitor, or adrenergic receptor antagonist showed that the compound induced its activity through ß2-adrenergic receptor. Patients with type II diabetes mellitus frequently develop insulin resistance. Owing to the differences between the mechanism of action of insulin and of the isolated compound, the compound or lotus plumule itself may have the possibility of modulating blood glucose levels in insulin-resistant patients effectively.

Development and validation of a sensitive method for simultaneous determination of eight ß2-agonists in pork by ultrasonic-assisted extraction and liquid chromatography/tandem mass spectrometry.

A simple and sensitive ultrasonic-assisted extraction coupled with liquid chromatography-tandem quadrupole mass spectrometry was developed for the simultaneous determination of eight ß2-agonists (i.e., phenylethanolamine, fenoterol, formoterol, clenbuterol, ractopamine, salbutamol, terbutaline and tulobuterol) in pork. The recovery rate of ultrasonic-assisted extraction was compared with that of enzymolysis. Results showed that ultrasonic-assisted extraction is a satisfactory method for the treatment of pork samples, yielding ß2-agonist recovery rates of 82.0-114.0%. In contrast, most of the recovery rates obtained by enzymolysis were <80%. Linearities obtained ranged from 0.1 to 16.0 µg kg(-1) for terbutaline, formoterol, tulobuterol and phenylethanolamine, 0.2 to 16.0 µg kg(-1) for salbutamol, fenoterol and ractopamine, and 0.2 to 40.0 µg kg(-1) for clenbuterol. The correlation coefficients were >0.9945 for all analytes. The lower limits of quantification were between 0.086 and 0.20 µg kg(-1). The applicability of the proposed method for detecting and quantifying ß2-agonists was demonstrated in the analysis of 50 pork samples.

Simple semi-automated portable capillary electrophoresis instrument with contactless conductivity detection for the determination of ß-agonists in pharmaceutical and pig-feed samples.

An inexpensive, robust and easy to use portable capillary electrophoresis instrument with miniaturized high-voltage capacitively coupled contactless conductivity detection was developed. The system utilizes pneumatic operation to manipulate the solutions for all flushing steps. The different operations, i.e. capillary flushing, interface rinsing, and electrophoretic separation, are easily activated by turning an electronic switch. To allow the analysis of samples with limited available volume, and to render the construction less complicated compared to a computer-controlled counterpart, sample injection is carried out hydrodynamically directly from the sample vial into the capillary by manual syphoning. The system is a well performing solution where the financial means for the highly expensive commercial instruments are not available and where the in-house construction of a sophisticated automated instrument is not possible due to limited mechanical and electronic workshop facilities and software programming expertise. For demonstration, the system was employed successfully for the determination of some ß-agonists, namely salbutamol, metoprolol and ractopamine down to 0.7ppm in pharmaceutical and pig-feed sample matrices in Vietnam.

Evaluation of matrix solid-phase dispersion extraction for 11 ß-agonists in swine feed by liquid chromatography with electrospray ionization tandem mass spectrometry.

A sensitive liquid chromatography with tandem mass spectrometry method was developed for the determination of 11 ß-agonists (clenbuterol, salbutamol, ractopamine, terbutaline, fenoterol, cimaterol, isoxsuprine, mabuterol, mapenterol, clenproperol, and tulobuterol) in swine feed. This rapid, simple, and effective extraction method was based on matrix solid-phase dispersion. The limit of quantification of clenbuterol, cimaterol, mabuterol, salbutamol, terbutaline, mapenterol, clenproperol, and tulobuterol was 1 µg/kg and that of ractopamine, fenoterol, and isoxsuprine was 2 µg/kg. The recoveries of ß-agonists spiked in swine feeds at a concentration range of 1-8 µg/kg were >83.1% with relative standard deviations <9.3%. This rapid and reliable method can be used to efficiently separate, characterize, and quantify the residues of 11 ß-agonists in swine feeds with advantages of simple pretreatment and environmental friendliness.

Rapid analysis of three ß-agonist residues in food of animal origin by automated on-line solid-phase extraction coupled to liquid chromatography and tandem mass spectrometry.

An automated online solid-phase extraction with liquid chromatography and tandem mass spectrometry method was developed and validated for the detection of clenbuterol, salbutamol, and ractopamine in food of animal origin. The samples from the food matrix were pretreated with an online solid-phase extraction cartridge by Oasis MCX for <5 min after acid hydrolysis for 30 min. The peak focusing mode was used to elute the target compounds directly onto a C18 column. Chromatographic separation was achieved under gradient conditions using a mobile phase composed of acetonitrile/0.1% formic acid in aqueous solution. Each analyte was detected in two multiple reaction monitoring transitions via an electrospray ionization source in a positive mode. The relative standard deviations ranged from 2.6 to 10.5%, and recovery was between 76.7 and 107.2% at all quality control levels. The limits of quantification of three ß-agonists were in the range of 0.024-0.29 µg/kg in pork, sausage, and milk powder, respectively. This newly developed method offers high sensitivity and minimum sample pretreatment for the high-throughput analysis of ß-agonist residues.

Development of an immunoaffinity chromatography column for selective extraction of a new agonist phenylethylamine A from feed, meat and liver samples.

Phenylethanolamine A (PA) is a new emerged ß-adrenergic agonist that has been illegally used as an animal feed additive for growth promotion in China. In this study, an immunoaffinity chromatography (IAC) column for selective extraction of PA from swine feed, meat and liver samples was developed. The IAC column was constructed by covalently coupling specific polyclonal antibody (Ab) against PA to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. The extraction conditions including loading, washing and eluting solutions were carefully optimized. Under optimal conditions, the IAC column was characterized in terms of maximum capacity, selectivity, extraction recovery and stability. The maximum capacity of the ICA for PA extraction was found to be 239.4ng. For selectivity testing, 100ng of other three ß-adrenergic agonists (clenbuterol, ractopamine and salbutamol) was separately loaded onto the column, and it was observed that the tested compounds could not be captured on the column, e.g. the column could only selectively recognize PA. The recovery of the IAC for PA extraction was found within 96.47-101.98% when 10, 50 and 100ng PA were separately loaded onto IAC column. The IAC column was also applied to real sample extraction. Swine feed, meat and liver samples were collected and spiked with PA in range of 1.0-20ngg(-1). The spiked and unspiked samples were extracted by IAC column and measured by high performance liquid chromatography (HPLC). It was found that there was no detectable PA in the blank samples, and the extraction recoveries of the IAC for PA from the spiked samples were within 89.48-104.89%. The stability of the column was also tested. It was showed that after 35 times repeated usage, 60% of the maximum capacity was still remained. The proposed IAC was proven to be a feasible extraction method for PA from different matrices with the properties of high maximum capacity, selectivity, extraction efficiency and stability.