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.

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.

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.

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.

Quick and high-throughput quantification of ß-agonist residues in bovine liver, meat, milk, kidney, poultry, and egg using dispersive solid phase extraction.

A reliable liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (LC-Q-Orbitrap HRMS) method was developed for the simultaneous identification and quantification of 13 ß-agonist residues in bovine liver, meat, milk, kidney, poultry, and egg. Dispersive-solid phase extraction (d-SPE) using acetonitrile (ACN) was used to prepare the samples. The analyte in the extracts was separated on a reversed-phase Accucore aQ (50 mm × 2.1 mm, 2.6 µm) using a mobile phase of an aqueous solution containing 2 mM ammonium acetate and acetonitrile (ACN) 0.1 % formic acid. The method was validated in accordance with Commission Implementing Regulation (CIR) EU 2021/808 at six different concentrations ranging from 0.1 to 5 µg/kg. The mean recoveries ranged from 65 to 94 %, while repeatability and reproducibility values were all below 13 %. The linearity, as correlation coefficients (R2) ranged from 0.9955 to 0.9999. The decision limit (CCα) and detection capability (CCß) ranges were 0.11-0.13 µg/kg and 0.12-0.15 µg/kg, respectively. The limits of detection (LOD) and limits of quantification (LOQ) were in the range of 0.004-0.048 µg/kg and 0.010-0.075 µg/kg, respectively. Of the 180 samples that were collected from local markets in Egypt, 21.11 % had ß-agonist residues. The mean concentration (µg/kg) and detection frequency (%) of the most frequently found ß-agonist in the samples were as follows: terbutaline (2.63 µg/kg and 90 %), ractopamine (5.14 µg/kg and 23.3 %). The method's applicability was verified by successfully completing two rounds of proficiency testing (PT).

Application of ionic liquid as additive in determination of three ß-agonists by capillary electrophoresis with amperometric detection.

CE coupled with amperometric detection method was developed using ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate (EMImBF(4)) as additive for the simultaneous detection of clenbuterol (CLB), terbutaline (TER), and ractopamine (RAC) in feed. The effects of detection potential, concentration of EMImBF(4), pH, and concentration of the running buffer, separation voltage as well as injection time on the separation and detection of these three ß-agonists were investigated in detail. Under the optimum conditions: the detection potential at 1.05 V, 50 mmol/L Tris-HAc at pH 8.0 with 0.6% (v/v) EMImBF(4), electrokinetic injection 6 s at 16 kV and separation voltage at 16 kV, a baseline separation for these three analytes could be achieved within 11 min. Introduction of EMImBF(4) into the running buffer resulted in significant improvement in separation selectivity and enhancement in peak currents for those ß-agonists, especially for TER and RAC, which could not be separated in the running buffer without additive. The method exhibited wide linear range with LOD (S/N = 3) of 2, 1, and 2 nmol/L for CLB, TER, and RAC, respectively. The precision was determined in both intraday (n = 5) and interday (n = 3) assays, and the RSDs for both migration time and peak current were less than 6%. The proposed method was also applied to analyze ß-agonists in feed sample.

Matrix effects in the determination of ß-receptor agonists in animal-derived foodstuffs by ultra-performance liquid chromatography tandem mass spectrometry with immunoaffinity solid-phase extraction.

Matrix effects in determination of three ß-receptor agonists including salbutamol (SAL), clenbuterol, and terbutaline in animal-derived foodstuffs were studied by ultra-performance LC-MS/MS with cleanup of immunoaffinity SPE column (IAC). Some animal tissue samples including pig liver, swine muscle, and fish muscle were hydrolyzed by the mixed enzyme solution or HCl solution, and the cleanup efficiencies with SAL IAC, MCX SPE column, and C(18) -SCX tandem columns were examined and compared by using spiked experiments. The results showed that the matrix effects in the determination of SAL and terbutaline can be eliminated with SAL IAC cleanup, and the average recoveries of SAL were 77.4~81.5%, 79.0~80.3%, and 85.0~87.2% in pig liver, swine muscle, and fish muscle, respectively. The decision limit (ccα) and detection capability (ccß) for SAL in pig liver were 0.02 and 0.05 µg/kg, respectively.

Reaction of ß-blockers and ß-agonist pharmaceuticals with aqueous chlorine. Investigation of kinetics and by-products by liquid chromatography quadrupole time-of-flight mass spectrometry.

The degradation of two ß-blockers (atenolol and propranolol) and one ß-receptor agonist (salbutamol) during water chlorination was investigated by liquid chromatography-mass spectrometry (LC-MS). An accurate-mass quadrupole time-of-flight system (QTOF) was used to follow the time course of the pharmaceuticals and also used in the identification of the by-products. The degradation kinetics of these drugs was investigated at different concentrations of chlorine, bromide and sample pH by means of a Box-Behnken experimental design. Depending on these factors, dissipation half-lives varied in the ranges 68-145 h for atenolol, 1.3-33 min for salbutamol and 42-8362 min for propranolol. Normally, an increase in chlorine dosage and pH resulted in faster degradation of these pharmaceuticals. Moreover, the presence of bromide in water samples also resulted in a faster transformation of atenolol at low chlorine doses. The use of an accurate-mass high-resolution LC-QTOF-MS system permitted the identification of a total of 14 by-products. The transformation pathway of ß-blockers/agonists consisted mainly of halogenations, hydroxylations and dealkylations. Also, many of these by-products are stable, depending on the chlorination operational parameters employed.

Covalent imprinted polymer for selective and rapid enrichment of ractopamine by a noncovalent approach.

A novel molecularly imprinted polymer (MIP) for the separation and concentration of ractopamine (RAC) was prepared by a covalent imprinting approach and the template was removed successfully by hydrolysis, so that four carboxylic acid groups were left in the cavities and could specifically rebind RAC through noncovalent interaction: hydrogen bonding. The conditions for synthesis of the MIP were optimized during the polymerization process, and a molar ratio of template-functional monomer complexes to cross-linker of 1:3 was confirmed. The adsorption capacity of the MIP was 4.1-fold that of the nonimprinted polymer, and the adsorption reaction reached equilibrium after 25 min at 50 mg L(-1) concentration. The results of the competitive adsorption test showed that the MIPs had specific recognition ability for the analyte RAC. In addition, the important factors affecting the efficiency of the method which was developed using the MIPs as a solid-phase sorbent for separation and determination of RAC combined with high-performance liquid chromatography with fluorescence detection were optimized. Under the optimum experimental conditions, the linear range of the calibration curve in the method was 0.05-5 µg L(-1) (R(2)=0.98) and the limit of detection (signal-to-noise ratio of 3) was 0.01 µg L(-1). The proposed method was applied to determination of RAC in spiked feedstuffs and urine samples, with recoveries ranging from 74.17 to 114.46% and relative standard deviation (n=3) below 4.55 in all cases.

Molecularly imprinted solid-phase extraction for the selective HPLC determination of ractopamine in pig urine.

A method was developed for the determination of ractopamine in pig urine using molecularly imprinted solid-phase extraction (MISPE) as the sample clean-up technique combined with high-performance liquid chromatography. The molecularly imprinted polymer (MIP) was synthesized in acetonitrile-triethylamine system using ractopamine (RAC) as the template and acrylamide as the monomer. The binding capacity of the polymer toward RAC was found to be about 2.57 mg of ractopamine/g of polymer. The optimal procedures for MISPE consisted of conditioning with 3 mL methanol, equilibrating with 3 mL of water, loading volume of <10 mL of aqueous sample (pH 7), washing with 3 mL water and 3 mL methanol, and eluting with 5 mL of 5% ammonia in methanol. In the four spiked samples with the levels of 0.01, 0.1, 1.0 and 5.0 µg/mL, the mean recoveries of analyte on the MIP were higher than 90% with relative standard deviation <10%, and significant differences between imprinted and non-imprinted materials were observed. The MIP selectivity was evaluated by checking 11 drugs with similar and different molecular structures to that of RAC. The characteristics of three-dimensional cavities and hydrogen bond interaction were regarded as the main factors that dominated the retention of RAC on the MISPE cartridge.

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.

Chiral separations of some beta-adrenergic agonists and antagonists on AmyCoat column by HPLC.

Sixteen beta-adrenergic antagonists namely acebutalol, alprenolol, atenolol, bisoprolol, bopindolol, bufurolol, carazolol, celiprolol, indenolol, metaprolol, nebivolol, oxprenolol, practolol, propranolol, tertalol, and timolol, and two beta-adrenergic agonists namely cimeterol and clenbuterol were resolved on AmyCoat (150 x 46 mm, 3 microm size of silica particle) by using (85:15:0.1, v/v/v), (90:10:0.1, v/v/v), and (95:05:0.1, v/v/v) combinations of n-heptane, ethanol, and diethylamine solvents, respectively. The flow rates used were 0.5, 1.0, 2.0, and 3.0 ml/min with detection at 225 nm. The values of capacity, separation, and resolution factors ranged from 0.38 to 19.70, 1.08-2.33, and 1.0 and 4.50, respectively. The maximum and minimum resolutions were achieved for celiprolol and bufurolol, respectively. The chiral recognition mechanisms were also discussed. The values of validation parameters were calculated.

Investigation of ractopamine-imprinted polymer for dispersive solid-phase extraction of trace beta-agonists in pig tissues.

Ractopamine, as an alternative beta-agonist to clenbuterol, is more and more used as leanness-enhancing agent in the swine industry. This work presents a new molecularly imprinted polymer (MIP) using ractopamine as template for dispersive solid-phase extraction of trace ractopamine and the structural related beta-agonists in animal tissues. The binding properties and selectivity of MIP were investigated. High selectivity in polar environment was found, since the extraction capacity of ractopamine with the MIP was 4.5-fold as much as that with the non-imprinted polymer in acetonitrile. Cross-selectivity investigation indicates that the MIP preferentially binds the template and then the structural analogues according to their molecular similarity. Thermodynamic and kinetic investigation was performed to interpret the specific adsorption and molecular recognition of the MIP for ractopamine. Standard free energy, standard enthalpy, and standard entropy were determined. Related information suggested that adsorption of ractopamine onto MIP was an exothermic, spontaneous process. The MIP can be applied as dispersive solid-phase extraction material for enrichment of ractopamine, isoxsuprine, fenoterol and clenbuterol in complex samples before HPLC analysis. The method revealed detection limits of 0.20-0.90 microg/L, recoveries of 83.8-115.2 and 85.2-110.2% for the spiked pig muscle and pig liver, respectively, with the RSD from 2.5 to 8.8%.

Improved simultaneous enantioseparation of beta-agonists in CE using beta-CD and ionic liquids.

In this study, approaches to improve chiral resolutions in simultaneous enantioseparation of beta-agonists by CE via a CD inclusion complexation modified with ionic liquids (ILs) are described. Different types of ILs, including tetraalkylammonium-based ILs, alkylimidazolium-based ILs and alkylpyridinium-based ILs, were examined and compared for controlling the EOF in order to improve resolutions of beta-agonists enantiomers. In this regard, tetraalkylammonium-based ILs were more effective because they could be used at much higher concentrations than other types of ILs. N-octylpyridinium hexafluorophosphate gave poor resolutions of beta-agonists enantiomers. In addition, when different ILs were mixed to use, they would present particular properties of their own. Moreover, the presence of ILs was essential in the chiral separations of (+/-) salbutamol, (+/-) cimaterol and (+/-) formoterol, which were reportedly not enantioseparated by using the buffer electrolytes containing only beta-CD as a chiral selector.