ß2-adrenergic receptor affinity chromatography with an interaction force analysis model: A method for analysis of active compounds targeting ß2-adrenergic receptor.

Asthma is one of the most prevalent diseases worldwide, and ß2-adrenergic receptor (ß2AR) agonists have been reported to be highly effective bronchodilators against this disease. In this study, we successfully constructed a novel CHO-ß2AR affinity chromatography (CHO-ß2AR/AC), which was evaluated by infrared spectroscopic and scanning electron microscope (SEM) analysis. In addition, CHO-ß2AR/AC model exhibited good selectivity and reliability with the relative standard deviation smaller than 5.6% after 30 days. Furthermore, an interaction force analysis model was developed based on CHO-ß2AR/AC. The results showed that the interaction force analysis model (Φ•E•pKa) exhibited a strong correlation with equilibrium dissociation constant (KD) (r2=0.9284, p=0.002) and a good correlation with logarithm of half-maximum effective concentration (pEC50) values (r2=0.7135, p=0.034). In addition, a pool of clinically approved drugs was screened by this CHO-ß2AR/AC model. Codeine wasfound to bind to and activate ß2AR with KD value of 4.10 × 10-7 M, leading to increased cyclic adenosine monophosphate (cAMP) production with EC50 of 6.49 × 10-7 M and reduction of intracellular Ca2+ concentration, which in turn relaxes bronchial contraction with EC50 of 2.62 × 10-6 M. Furthermore, the KD value and pEC50 of codeine were within the 95% prediction range of the interaction force analysis model. The results indicate that the CHO-ß2AR/AC with interaction force analysis model constructed in this study can be used to effectively and rapidly screen active compounds targeting ß2AR.

Sulfonate-bonded covalent organic polymer as mixed-mode sorbent for on-line solid-phase extraction of ß2-receptor agonists.

A novel sulfonate-bonded covalent organic polymer (COPTPBA-BPDA@SA) with mixed-mode interactions of hydrophobic and cation-exchange was synthesized and exploited as sorbent for on-line solid-phase extraction (on-line SPE) of ß2-receptor agonists. The successful synthesis of COPTPBA-BPDA@SA was confirmed by the characterization of SEM, XPS and FT-IR. Due to the excellent mixed-mode extraction ability towards the positively charged ß2-receptor agonists and the good anti-interference performance, COPTPBA-BPDA@SA was introduced into on-line SPE-HPLC system for selective extraction of ß2-receptor agonists in pork and pork liver. Via the optimization of the extraction condition, including formic acid percentage and ACN percentage in the sampling solution, the mixed-mode extraction mechanism of COPTPBA-BPDA@SA was investigated. The elution condition, such as the pH value, formic acid percentage and ACN percentage of the eluent was also optimized for the desired SPE performance. Under the optimized condition, COPTPBA-BPDA@SA revealed better purification performance than COPTPBA-BPDA without sulfonating. The LODs for ß2-receptor agonists were in the range of 0.08-0.22 µg/kg, and the recoveries in different samples at three spiked levels (0.4, 4.0, 8.0 µg/kg) were ranged from 83.2% to 98.5% with RSDs less than 5.2%, which indicated the satisfactory mixed-mode extraction ability of COPTPBA-BPDA@SA as well as the good applicability of the developed method.

Development of liquid chromatographic methods for enantioseparation and sensitive detection of ß-adrenolytics/ß2-agonists in human plasma using a single enantiomer reagent.

Enantioseparation of four commonly used ß-adrenolytics (bisoprolol, salbutamol, and carvedilol, marketed as racemic mixtures) has been achieved by both TLC and RPHPLC via an indirect approach. A new chiral reagent, (S)-naproxen benzotriazole ester, was synthesized and it was characterized by UV, IR, 1HNMR, elemental analysis and polarimetry. It was used to synthesize diastereomeric derivatives of the three ß-adrenolytics under microwave irradiation. TLC separation of diastereomeric derivatives was achieved which were then isolated by preparative approach; these were characterized and were used as standard reference for determining absolute configuration of diastereomeric derivatives and for establishing validated HPLC method for enantioseparation and sensitive detection of the three ß-adrenolytics in human plasma. Mobile phase in gradient mode containing methanol and aqueous triethylaminephosphate (TEAP) was successful for HPLC separation; conditions with respect to pH, flow rate, and buffer concentration were optimized. The method is capable to accurately quantitate ß-adrenolytics in human plasma with minimal sample clean-up and rapid separation by TLC and RPHPLC. The limit of detection values were 0.97 and 0.87ng/mL for diastereomeric derivatives of (S)- and (R)-bisoprolol, respectively, which are very low in comparison to literature reports.

Rapid Determination of Clenbuterol in Pork by Direct Immersion Solid-Phase Microextraction Coupled with Gas Chromatography-Mass Spectrometry.

Direct immersion solid-phase microextraction (DI-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was developed for rapid analysis of clenbuterol in pork for the first time. In this work, a low-cost homemade 44 µm polydimethylsiloxane (PDMS) SPME fiber was employed to extract clenbuterol in pork. After extraction, derivatization was performed by suspending the fiber in the headspace of the 2 mL sample vial saturated with a vapor of 100 µL hexamethyldisilazane. Lastly, the fiber was directly introduced to GC-MS for analysis. All parameters that influenced absorption (extraction time), derivatization (derivatization reagent, time and temperature) and desorption (desorption time) were optimized. Under optimized conditions, the method offered a wide linear range (10-1000 ng g(-1)) and a low detection limit (3.6 ng g(-1)). Finally, the method was successfully applied in the analysis of pork from the market, and recoveries of the method for spiked pork were 97.4-105.7%. Compared with the traditional solvent extraction method, the proposed method was much cheaper and fast.

Current status and recent advantages in derivatization procedures in human doping control.

Derivatization is one of the most important steps during sample preparation in doping control analysis. Its main purpose is the enhancement of chromatographic separation and mass spectrometric detection of analytes in the full range of laboratory doping control activities. Its application is shown to broaden the detectable range of compounds, even in LC-MS analysis, where derivatization is not a prerequisite. The impact of derivatization initiates from the stage of the metabolic studies of doping agents up to the discovery of doping markers, by inclusion of the screening and confirmation procedures of prohibited substances in athlete's urine samples. Derivatization renders an unlimited number of opportunities to advanced analyte detection.

Development of a Efficient and Sensitive Dispersive Liquid-Liquid Microextraction Technique for Extraction and Preconcentration of 10 ß2-Agonists in Animal Urine.

Dispersive liquid-liquid microextraction (DLLME) coupled with ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) was developed for the extraction and determination of 10 ß2-agonists in animal urine. Some experimental parameters, such as the type and volume of the extraction solvent, the concentration of the dispersant, the salt concentration, the pH value of the sample solution, the extraction time and the speed of centrifugation, were investigated and optimized. Under the optimized conditions, a good enrichment factors (4.8 to 32.3) were obtained for the extraction. The enrichment factor show that the concentration rate of DLLME is significantly higher than other pretreatment methods, and the detection sensitivity has been greatly improved. The calibration curves were linear, the correlation coefficient ranged from 0.9928 to 0.9999 for the concentration range of 0.05 to 50 ng mL(-1) and 0.1 to 50 ng mL(-1), and the relative standard deviations (RSDs, n = 15, intra and inter-day precision) at a concentration of 5 ng mL(-1) were in the range of 1.8 to 14.6%. The limits of detection (LODs) for the 10 ß2-agonists, based on a signal-to-noise ratio (S/N) of 3, were in the range of 0.01 to 0.03 ng mL(-1). The proposed method was used to identify ß2-agonists in three types of animal urine (swine, cattle, sheep), and the relative recoveries from each matrix were in the range of 89.2 to 106.8%, 90.0 to 109.8% and 89.2 to 107.2%, respectively.

Identification of nuclear factor-κB inhibitors and ß2 adrenergic receptor agonists in Chinese medicinal preparation Fuzilizhong pills using UPLC with quadrupole time-of-flight MS.

INTRODUCTION: Fuzilizhong Pills (FZLZ), a modified form of a famous traditional Chinese medicine (TCM) Lizhong Wan in Treatise on Febrile Diseases and consisting of Panax ginseng C.A.Mey. (Ren Shen), Aconitum carmichaelii Debx. (Fu Zi, Zhi), Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. or Glycyrrhiza glabra L. (Gan Cao), Atractylodes macrocephala Koidz. (Bai Zhu) and Zingiber offcinale Rosc. (Gan Jiang), show strong clinical therapeutic effects for dyspnea and pulmonary oedema. However, the bioactive compounds are still unclear. In this study, FZLZ was analysed using a rapid detection method to identify its anti-inflammatory and spasmolytic constituents. OBJECTIVE: To develop a simple screening method to detect the anti-inflammatory and spasmolytic constituents of FZLZ. METHODS: Ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry combined with dual-bioactive (NF-κB and ß2 -adrenergic receptor) luciferase reporter assay systems was employed. RESULTS: Two ß2 -adrenergic receptor agonists (salsolinol and higeramine) and three terpenoidal analogues of NF-κB inhibitors such as ginsenosides derivatives, triperpenoids derivatives and diester-diterpenoid aconitum alkaloid derivatives were characterised. Mesaconitine, flaconitine, ginsenosides Rb2, Rf, Rg2, F1 and Ro were considered to be new NF-κB inhibitors. Furthermore, IL-8 detection by enzyme linked immunosorbent assay confirmed the anti-inflammatory effects of the potential NF-κB inhibitors. CONCLUSION: Compared with conventional fingerprints, activity-integrated fingerprints that contain both chemical and bioactive details offer a more comprehensive understanding of the chemical composition of plant materials. This strategy clearly demonstrated that dual bioactivity-integrated fingerprinting is a powerful tool for the improved screening and identification of potential dual-target lead compounds in complex herbal medicines.

Sports doping: emerging designer and therapeutic ß2-agonists.

Beta2-adrenergic agonists, or ß2-agonists, are considered essential bronchodilator drugs in the treatment of bronchial asthma, both as symptom-relievers and, in combination with inhaled corticosteroids, as disease-controllers. The use of ß2-agonists is prohibited in sports by the World Anti-Doping Agency (WADA) due to claimed anabolic effects, and also, is prohibited as growth promoters in cattle fattening in the European Union. This paper reviews the last seven-year (2006-2012) literature concerning the development of novel ß2-agonists molecules either by modifying the molecule of known ß2-agonists or by introducing moieties producing indole-, adamantyl- or phenyl urea derivatives. New emerging ß2-agonists molecules for future therapeutic use are also presented, intending to emphasize their potential use for doping purposes or as growth promoters in the near future.

Simultaneous determination of 10 ß2-agonists in swine urine using liquid chromatography-tandem mass spectrometry and multi-walled carbon nanotubes as a reversed dispersive solid phase extraction sorbent.

A simple and inexpensive pretreatment procedure was developed for 10 ß2-agonists (clenbuterol, ractopamine, salbutamol, bambuterol, penbuterol, tulobuterol, clorprenaline, mabuterol, cimaterol and terbutaline) in swine urine using dispersive solid phase extraction (dSPE) with multi-walled carbon nanotubes (MWCNTs). The sample was analysed after purification by ultra high performance liquid chromatography-positive electrospray ionisation tandem mass spectrometry (UHPLC-ESI-MS/MS). The pH value of the swine urine, extraction time, type and amount of MWCNTs and type of eluent were optimised to increase the sample throughput and sensitivity. The samples were quantified using clenbuterol-D9, ractopamine-D6 and salbutamol-D3 as internal standards. The recoveries of the target compounds from swine urine samples at pH 10.0 were most efficient when using 20 mg of MWCNTs with a 30-50 nm outer diameter and a length of 10-30 µm, while a mixture of water/methanol (90:10, v/v) with 0.5% formic acid was shown to be the most suitable solvent for desorbing the compounds from the MWCNTs. The proposed method was validated according to the European Commission Decision 2002/657/EC, which determines linearity, specificity, decision limit (CCα), detection capability (CCß), recovery, precision and stability.

Bioactivity-based liquid chromatography-coupled electrospray ionization tandem ion trap/time of flight mass spectrometry for ß2AR agonist identification in alkaloidal extract of Alstonia scholaris.

Although chromatographic fingerprinting combined with chemometrics, is a rational method for the quality control of traditional Chinese medicine (TCM), chemometrics cannot fully explore the relationship between chemical information and the efficacy of the potential activity. In the present work, a cell-based ß2 adrenergic receptor (ß2AR) agonist functional evaluation model coupled with high-performance liquid chromatography was developed to screen the potential ß2AR agonist components in the alkaloidal extract of Alstonia scholaris leaves. Using a liquid chromatography with ion trap time-of-flight mass spectrometry (LCMS-IT-TOF) system, the potential bioactive compounds in the prescription were identified and deduced based on the mass spectrometric fragmentation patterns, tandem mass spectrometry (MS/MS) data, and relevant literature. Several new ß2AR agonists of indole alkaloids were successfully found, and their activities were confirmed through an in vivo relaxant test on guinea pig tracheal muscles. The developed method is rapid and reliable compared with conventional fingerprinting and showed high sensitivity and resolution for the identification of ß2AR agonists in TCM prescriptions. This strategy clearly demonstrates that bioactivity-integrated fingerprinting is a powerful tool not only in screening and identifying potential lead compounds and in determining the therapeutic material basis of Chinese herbal prescriptions, but also in supplying suitable chemical markers for their quality control.

Determination of ß-blockers and ß-agonists using gas chromatography and gas chromatography-mass spectrometry--a comparative study of the derivatization step.

There is a growing demand for the rapid screening of multiple ß-blockers and ß-agonists in a single analytical run in clinical toxicology, antidoping control, forensic and environmental science. Although GC-MS is very often used to determine pharmaceuticals from these groups of drugs, the literature data on the derivatization and MS analysis of mixtures of these compounds is limited. This paper compares and evaluates derivatization procedures for the determination of six ß-blockers (acebutolol, atenolol, metoprolol, nadolol, propranolol, pindolol) and two ß-agonists (salbutamol, terbutaline) using GC techniques. Nineteen different derivatizing reagents (nine of them used for the first time with almost all the drugs) were employed in order to obtain a single derivative for each target compound with the greatest effectiveness of this reaction. Trimethylsilylation, tert-butyldimethylsilylation, acylation (e.g. trifluoroacetylation), combined trimethylsilylation and acylation, and the formation of cyclized silyl derivatives were carried out and the mass spectra (EI, 70 eV) recorded. The influence of the reaction time and temperature on these procedures was investigated. Additionally, the effects of the type of solvent and the amount of added trimethylchlorosilane (TMCS) on the silylation of the target compounds using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) were tested. Among of the five mentioned above derivatization procedures applied - trimethylsilylation was found to be the most effective for derivatizing the analytes. The best results were obtained with a 1:1 (v/v) mixture of 99% BSTFA+1% TMCS and ethyl acetate at 60 °C for 30 min. The MS data for different types of ß-blocker and ß-agonist derivatives is presented. The information in this paper is valuable for scientists working on the determination of ß-blockers and ß-agonists in biological and environmental matrices.

Effect of chromatographic conditions on liquid chromatographic chiral separation of terbutaline and salbutamol on Chirobiotic V column.

In this study, a method for enantioseparation of terbutaline and salbutamol was established using Chirobiotic V column as a stationary phase. Polar ionic mode applying mobile phase containing ammonium nitrate in 100% ethanol, pH 5.1 was found to give the best separation. The salt concentration in the mobile phase and pH value were found to be the most important chromatographic factors affecting separation. Separation of enantiomers of these two basic analytes was complete in less than 10 min without applying ammonium trifluoroacetate (ATFA) or triethylamine (TEA) salts.

Chiral separation of the ß2-sympathomimetic fenoterol by HPLC and capillary zone electrophoresis for pharmacokinetic studies.

The development of methods for the separation of the enantiomers of fenoterol by chiral HPLC and capillary zone electrophoresis (CZE) is described. For the HPLC separation precolumn fluorescence derivatization with naphthyl isocyanate was applied. The resulting urea derivatives were resolved on a cellulose tris(3,5-dimethylphenylcarbamate)-coated silica gel column employing a column switching procedure. Detection was carried out fluorimetrically with a detection limit in the low ng/mL range. The method was adapted to the determination of fenoterol enantiomers in rat heart perfusates using liquid-liquid extraction. As an alternative a CE method was used for the direct separation of fenoterol enantiomers comparing different cyclodextrin derivatives as chiral selectors.