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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 β-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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A sensitive and enantioselective vancomycin chiral stationary phase high-performance liquid chromatography-tandem mass spectrometry method was developed for the determination of trantinterol enantiomers in human plasma. Baseline resolution was achieved using the vancomycin chiral stationary phase known as Chirobiotic V with polar ionic mobile phase consisting of acetonitrile-methanol (60:40, v/v) containing 0.01% ammonia and 0.02% acetic acid at a flow rate of 1.0 mL/min. Waters Oasis HLB C18 solid phase extraction cartridges were used in the sample preparation of trantinterol samples from plasma. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization. The calibration curve was linear in a concentration range from 0.0606 to 30.3 ng/mL in plasma, with the lower limit of quantification of 0.0606 ng/mL. The intra- and interday precision (relative standard deviation) values were within 9.7% and the accuracy (relative error) was from -6.6 to 7.2% at all quality control levels. The method was successfully applied to a study of stereoselective pharmacokinetics in human., (© 2015 Wiley Periodicals, Inc.)

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 β-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 β-agonist residues., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A simple and sensitive method based on the combination of solid-phase microextraction (SPME) and high-performance liquid chromatography with ultroviolet detection was developed for the simultaneous determination of clenbuterol, salbutamol and ractopamine in pig samples. Parameters of the SPME procedure affecting extraction efficiency, such as the type of fiber, extraction time, extraction temperature, ion strength, pH of sample and stirring rate, were optimized. The developed method was validated according to the International Conference on Harmonization guidelines. The calibration curves were linear over a range of 0.5-50 µg/L for clenbuterol and ractopamine, and 0.2-20 µg/L for salbutamol. The limits of detection were 0.1 µg/L for clenbuterol, 0.05 µg/L for salbutamol and 0.1 μg/L for ractopamine, respectively. The averages of intra- and inter-day accuracy ranged from 79.8 to 92.4%. The intra-day and inter-day precision were below 9.6% for the three analytes. This method exhibited the advantages of simplicity, rapidity and low solvent consumption, and was suitable for the monitoring of β2 -agonists residue in pig samples., (Copyright © 2013 John Wiley & Sons, Ltd.)

A high performance liquid chromatographic (HPLC) method using cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase (Lux Cellulose-1) for the separation of trantinterol hydrochloride enantiomers was developed. The method was based on normal phase model with n-heptane as the basal solvent of mobile phase. The influences of organic modifiers, mobile phase additives and column temperature on the retention and separation of the enantiomers were examined and discussed. It was demonstrated that the mobile phase additives had a dominant effect on the enantiomeric separation. No separation was observed with the addition of diethylamine only in the mobile phase, while the retention was increased and the enatiomeric separation was observed with the addition of trifluoroacetic acid. When both trifluoroacetic acid and diethylamine were added into the mobile phase, the enantioseparation was significantly improved with a resolution up to 4.0. Ethanol and isopropanol were investigated as the organic modifiers, and ethanol offered a better enatiomeric resolution for trantinterol hydrochloride. In the examined temperature range between 15 degrees C and 35 degrees C both separation factor and resolution were decreased with the increase of the column temperature. The optimized chiral HPLC method for the separation of trantinterol hydrochloride enantiomers involved a Lux Cellulose-1 column (250 mm x 4.6 mm, 5 microm), a mobile phase of n-heptane/ethanol/trifluoroacetic acid/diethylamine (88: 12: 0.3: 0.05, v/v/v/v) at a flow rate of 1.0 mL/min, a detection at 246 nm and a column temperature of 25 degrees C. The method is simple and rapid for the enantiomeric impurity determination of (-)-trantinterol hydrochloride bulk samples.

A simple and rapid high performance liquid chromatographic method has been developed for the direct resolution of clenbuterol enantiomers. The method involved the use of an amide type chiral stationary phase(CSP) made of (R)-1-naphthylglycine and 3,5-dinitrobenzoic acid known as the Chirex 3005 column. The effects of different contents of n-hexane, 1,2-dichloroethane and methanol in mobile phase on the chiral separation are discussed. The effects of column temperature and flow rate of mobile phase were also studied. The separation factor obtained was 1.32 and the resolution factor was 1.81 when using the optimized mobile phase composed of n-hexane-1,2-dichloroethane-methanol (54:38:8, volume ratio) at 17 degrees C and 1.0 mL/min. The mechanism of separation is also discussed.

More specific official methodology is needed to survey the illegal use of clenbuterol in animal production plus the synthesis of new compounds that currently elude routine analytical methods. The identification of a new adrenergic agonist, N1-(2-(4-amino-3,5-dichlorophenyl)-2-hydroxyethyl)-N1-isopropyl-propanamide (known as compound A) in animal feed has prompted studies to verify if the existing cleanup procedures developed for clenbuterol are really effective. This study considers the ion-exchange mechanism on cyanopropyl (CN), sulfonic cation exchange (SCX), mixed phase (MPH) (C8 + SCX), and nonendcapped C18 (C18NE) solid-phase extraction (SPE) columns. Results indicate that compound A (by contrast with clenbuterol) is not efficiently retained on the CN, SCX, and MPH SPE columns (recovery <10%). This finding thus leads to the development of a gas chromatography-tandem mass spectrometry procedure based on C18NE SPE that is able to purify both agonists from bovine livers spiked at 0.5, 1.0, and 2.0 ppb with a mean recovery of 93% for clenbuterol and 92% for compound A.

Clenbuterol molecularly imprinted polymers (MIPs) as chromatographic stationary phase for the solid-phase extraction (SPE) of the drug from biological samples have been prepared. Propylene columns filled with 500 mg of clenbuterol MIPs have been tested with respect to their loading capacity, memory effects, selectivity toward related drugs (mabuterol, clenproperol, clenisopenterol, ritodrine) and specificity toward interferences arising from heterogeneous matrices such as animal feeds, bovine urine and liver. Analytes were concentrated on Extrelut 20 columns and the residues resuspended in 70% acetonitrile. Application, washing and elution fractions were collected and analyzed by HPLC-diode array detection. Results indicate this MIP approach in SPE is extremely selective for clenbuterol, mabuterol, clenproperol and clenisopenterol (>95% found in the eluate), with a loading capacity of about 20 microg/100 mg of stationary phase. Ritodrine showed a recovery rate of 51%. The molecular recognition mechanism is so specific to allow clenbuterol detection and identification by conventional detectors at level of interest (ppb) also from complex matrices such as feeds, urine and liver.