Activation/Deactivation Free-Energy Profiles for the ß2-Adrenergic Receptor: Ligand Modes of Action.

We use enhanced-sampling simulations with an effective collective variable to study the activation of the ß2-adrenergic receptor in the presence of ligands with different efficacy. The free-energy profiles are computed for the ligand-free (apo) receptor and binary (apo-receptor + G-protein α-subunit and receptor + ligand) and ternary complexes. The results are not only compatible with available experiments but also allow unprecedented structural insight into the nature of GPCR conformations along the activation pathway and their role in the activation mechanism. In particular, the simulations reveal an unexpected mode of action of partial agonists such as salmeterol and salbutamol that arises already in the binary complex without the G-protein. Specific differences in the polar interactions with residues in TM5, which are required to stabilize an optimal TM6 conformation that facilitates G-protein binding and receptor activation, play a major role in differentiating them from full agonists.

Development of an HPLC-MS/MS Method for Chiral Separation and Quantitation of (R)- and (S)-Salbutamol and Their Sulfoconjugated Metabolites in Urine to Investigate Stereoselective Sulfonation.

The aim of this study was to develop and optimize a chiral HPLC-MS/MS method for quantitative analysis of (R)-/(S)-salbutamol and (R)-/(S)-salbutamol-4'-O-sulfate in human urine to allow for bioanalytical quantitation of the targeted analytes and investigations of stereoselectivity in the sulfonation pathway of human phase Ⅱ metabolism. For analytical method development, a systematic screening of columns and mobile phases to develop a separation via enantiomerically selective high performance liquid chromatography was performed. Electrospray ionization settings were optimized via multiple-step screening and a full factorial design-of-experiment. Both approaches were performed matrix-assisted and the predicted values were compared. The full factorial design was superior in terms of prediction power and knowledge generation. Performing a longitudinal excretion study in one healthy volunteer allowed for the calculation of excretion rates for all four targeted analytes. For this proof-of-concept, either racemic salbutamol or enantiopure levosalbutamol was administered perorally or via inhalation, respectively. A strong preference for sulfonation of (R)-salbutamol for inhalation and peroral application was found in in vivo experiments. In previous studies phenol sulfotransferase 1A3 was described to be mainly responsible for salbutamol sulfonation in humans. Thus, in vitro and in silico investigations of the stereoselectivity of sulfotransferase 1A3 complemented the study and confirmed these findings.

Temperature cycling-induced formation of crystalline coatings.

The surface of particles is the hotspot of interaction with their environment and is therefore a major target for particle engineering. Particles with tailored coatings are greatly desired for a range of different applications. Amorphous coatings applied via film coating or microencapsulation have frequently been described in the pharmaceutical context and usually result in homogeneous surfaces. In the present study we have been exploring the feasibility of coating core particles with crystalline substances, a matter that has rarely been investigated. The expansion of the range of possible coating materials to include small organic molecules enables completely new product properties to be achieved. We present an approach based on temperature cycles performed in a tubular crystallizer to result in engineered crystalline coatings on excipient core particles. By manipulating the process settings and by the choice of coating substance we are able to tailor surface roughness, topography as well as surface chemistry. Benefits of our approach are demonstrated by using resulting particles as carriers in dry-powder-inhaler formulations. Depending on the resulting surface chemistry and surface roughness, coated carrier particles show varying fitness for delivering the model API salbutamol sulphate to the lung.

Early potential evaluation of lead compounds from a DNA-encoded library by the determination of their thermodynamics through a chromatographic method based on immobilized ß2-adrenoceptor.

Early potential evaluation of lead compounds is critical to decrease downstream lead-optimization cycle times and clinical attrition rates for drug development. This increasingly necessitates the methodologies for accurately evaluating the potential compounds. This work immobilized ß2-adrenoceptor (ß2-AR) onto microspheres through Halo-tag mediated reaction. Characterizing the resulting microspheres by elemental and functional analysis, we utilized the immobilized receptor to determine the thermodynamics of terbutaline, tulobuterol, clorprenaline, salbutamol, and methoxyphenamine. The association constants correlated to their capacity factors on the column containing the immobilized ß2-AR, thus providing a possibility for early potential evaluation of lead compounds from complex matrices like a DNA-encoded library. By this model, the lead compound (XC267) was predicted to have an association constant higher than terbutaline, salbutamol, and methoxyphenamine, but lower than tulobuterol and clorprenaline. The binding interaction between XC267 and ß2-AR is a spontaneous endothermic process with an association constant of (6.62 ± 0.13) × 104 M-1 at 37 °C. The change of Gibbs free energy(ΔGθ), enthalpy change (ΔHθ), and entropy change (ΔSθ) was -28.49 kJ/mol, -10.58 kJ/mol, and 57.79 J/moL·K at 37 °C. By the semi-empirical rule of Ross, the driving force of the interaction between XC267 and ß2-AR was electrostatic interaction. Such binding force was also achieved by molecular docking. These results suggested that XC267 is a candidate to treat asthma by specific binding to ß2-AR. We reasoned that receptor chromatography is able to the early potential evaluation of lead compounds from complex matrices.

Insights into the Impact of Nanostructural Properties on Powder Tribocharging: The Case of Milled Salbutamol Sulfate.

The impact of the crystallinity of organic solid materials on their tribocharging propensity is well reported. However, no unequivocal explanation about the potential underlying mechanism(s) could be found so far in the literature. This study reports the effect that different degrees of crystalline disorder has on the tribocharging propensity of a small molecular organic material, salbutamol sulfate (SS). Ball-milling was used to induce structural transformations in the crystalline structure of SS. Particles with different nanostructures were produced and analyzed for their solid-state, particle properties, and tribocharging. It was found that differences in the amorphous content among the processed particles and related moisture levels had an impact on powder tribocharging. A correlation between the latter and the nanostructural properties of the particles was also established. The presence of interfaces between nanodomains of different densities and shorter average lengths within the phases seems to lead to a mitigation of charge. This suggests that undetected, subtle nanostructural differences of materials can affect powder handling and processability by altering their tribocharging. The present findings demonstrate the nanostructural implications of powder triboelectrification, which can help toward the rational design of a wide variety of organic solids.

Membrane-Facilitated Receptor Access and Binding Mechanisms of Long-Acting ß2-Adrenergic Receptor Agonists.

The drugs salmeterol, formoterol, and salbutamol constitute the frontline treatment of asthma and other chronic pulmonary diseases. These drugs activate the ß2-adrenergic receptors (ß2-AR), a class A G protein-coupled receptor (GPCR), and differ significantly in their clinical onset and duration of actions. According to the microkinetic model, the long duration of action of salmeterol and formoterol compared with salbutamol were attributed, at least in part, to their high lipophilicity and increased local concentrations in the membrane near the receptor. However, the structural and molecular bases of how the lipophilic drugs reach the binding site of the receptor from the surrounding membrane remain unknown. Using a variety of classic and enhanced molecular dynamics simulation techniques, we investigated the membrane partitioning characteristics, binding, and unbinding mechanisms of the ligands. The obtained results offer remarkable insight into the functional role of membrane lipids in the ligand association process. Strikingly, salmeterol entered the binding site from the bilayer through transmembrane helices 1 and 7. The entry was preceded by membrane-facilitated rearrangement and presentation of its phenyl-alkoxy-alkyl tail as a passkey to an access route gated by F193, a residue known to be critical for salmeterol's affinity. Formoterol's access is through the aqueous path shared by other ß2-AR agents. We observed a novel secondary path for salbutamol that is distinct from its primary route. Our study offers a mechanistic description for the membrane-facilitated access and binding of ligands to a membrane protein and establishes a groundwork for recognizing membrane lipids as an integral component in the molecular recognition process. SIGNIFICANCE STATEMENT: The cell membrane's functional role behind the duration of action of long-acting ß2-adrenergic receptor (ß2-AR) agonists such as salmeterol has been a subject of debate for a long time. This study investigated the binding and unbinding mechanisms of the three commonly used ß2-AR agonists, salmeterol, formoterol, and salbutamol, using advanced simulation techniques. The obtained results offer unprecedented insights into the active role of membrane lipids in facilitating access and binding of the ligands, affecting the molecular recognition process and thus their pharmacology.

Superhydrophobic Surface for Enhancing the Bioavailability of Salbutamol Sulfate from Cross-Linked Microspheres: Formulation, Characterization, and in vivo Evaluation.

INTRODUCTION: The aim of the work was to formulate salbutamol sulfate (SB) microspheres by using superhydrophobic surface (SHS) under different processing factors for improving its encapsulation efficiency, controling its release rate, and hence enhancing its bioavailability. METHODS: Cross-linked microspheres of chitosan (CN) and carrageenan (KN) were made on a SHS under a glutaraldehyde-saturated atmosphere. The formulations were designed and optimized based on 42 factorial design. Percentage encapsulation efficiency (%EE), particle size, swelling ratio, and in vitro release rate were characterized, and the in vivo performance of optimized formula was investigated in beagle dogs. RESULTS: The results showed that the prepared microspheres have a high %EE (97.11±0.78%) for F13. The swelling ratio was 4.2 at the end of the 8 hours for the optimized formula, and the in vitro release rate was controlled for 12 hours. In vivo study verified that there was a 1.61-fold enhancement in SB bioavailability from optimized formula (F13) compared to market tablet. CONCLUSION: The study suggested that microspheres prepared from CN/KN crosslinking on an SHS using glutaraldehyde atmosphere is a promising technique that can encapsulate and sustain the release of water-soluble drugs such as SB in addition to improving its in vivo pharmacokinetic profile.

Elucidating the Effect of Fine Lactose Ratio on the Rheological Properties and Aerodynamic Behavior of Dry Powder for Inhalation.

Dry powder inhaler (DPI) is recognized as the first choice for lung diseases' treatment. However, it lacks a universal way for DPI formulation development. Fine lactose is commonly added in DPIs to improve delivery performance; however, the fine ratio-dependent mechanism is unclear. Therefore, the objective of this study is to explore the influence of fine lactose ratio on DPI powder properties and aerodynamic behavior, and the fine lactose ratio-dependent mechanism involved during powder fluidization and lung deposition. Here salbutamol sulfate was used as a model drug, Lactohale® 206 as coarse carrier, and Lactohale® 300 as fine component; the mixtures were prepared at 1% drug content, with fine content up to 20%. It was shown that with the fine addition, flowability of the mixtures was improved, interaction among particles was increased, and the presence of fines could help to improve DPI's aerosolization performance. When the fines added were less than 3%, the "active site" hypothesis played a leading role. When the added fines were over 3% but less than 10%, fluidization enhancement mechanism was more important. After the added fines reaching 10%, aggregate mechanism started to dominate. However, FPF cannot be further increased once the fines reached 20%. Moreover, the correlations between FPF and dynamic powder parameters were verified in ternary mixtures, and cohesion had a greater impact on FPF than that of flowability. In conclusion, adding lactose fines is an effective way to improve lung deposition of DPI, with the concrete mechanism lactose fine ratio dependent.

Formulation, development, and in-vitro/ex-vivo evaluation of vaginal bioadhesive salbutamol sulfate tablets for preterm labor.

Preterm labor is the main cause of death and serious illness of both infants and pregnant women in Africa and worldwide. Parenteral and oral salbutamol sulfate as a B2 antagonist has been used for the treatment of preterm labor. The study aims are to formulate salbutamol sulfate non-invasive vaginal bioadhesive tablets to avoid the side effects of conventional formulations. Full factorial design 41 ×31 ×21 was used for the preparation of 24 vaginal bioadhesive tablet formulations. The independent factors were polymer type (Carbopol 934, HPMC 4000, HEC, and PEG 6000), polymer to drug ratio (1:1, 2:1, and 3:1), and diluent (lactose and mannitol). Vaginal bioadhesive tablets were evaluated for residence time and time required for release 50% of salbutamol sulfate T50% as dependent variables. The formulations were evaluated in terms of drug content, mass variation, hardness, friability, swelling index, residence time, and in-vitro drug release. Results revealed that polymer and diluent types are the most significant factors in both residence time and T50%. A strong positive correlation (0.91) between in-vitro and ex-vivo permeation was observed, which predict the best in-vivo performance of salbutamol vaginal bioadhesive tablet. Thus, salbutamol sulfate vaginal bioadhesive tablets could be a successful remedy for preterm labor.

Continuous Albuterol With Benzalkonium in Children Hospitalized With Severe Asthma.

BACKGROUND AND OBJECTIVES: The albuterol dropper bottle used to prepare solutions for continuous nebulization contains the preservative benzalkonium chloride (BAC). BAC, by itself, has been shown to cause bronchospasm. We hypothesized that BAC would decrease the therapeutic efficacy of albuterol in patients with acute asthma exacerbations. METHODS: We performed a retrospective cohort study comparing the clinical outcomes of patients <18 years of age receiving continuous nebulized albuterol with and without BAC. For the primary end point (duration of continuous albuterol nebulization), we compared the 2 groups with Kaplan-Meier estimate of survival curves, conducted a log-rank test of difference, and adjusted for baseline characteristics using multivariable Cox regression. A P value <.05 was considered significant. RESULTS: A total of 477 patients were included in the analysis (236 exposed to BAC and 241 controls). The duration of continuous nebulization was significantly longer in the BAC group than in the control group (median of 9 vs 6 hours; 15.7% required continuous nebulization compared to 5.8% of controls at 24 hours). The control group was 79% more likely to stop continuous nebulization at any particular point in time (hazard ratio 1.79; 95% confidence interval: 1.45 to 2.22; P < .001) and 43% more likely to stop additional respiratory support (hazard ratio 1.43; 95% confidence interval: 1.16 to 1.75; P < .001). CONCLUSIONS: BAC is a functional albuterol antagonist associated with a longer duration of continuous albuterol nebulization treatment and additional respiratory support, suggesting that preservative-free albuterol formulations are safer for use in continuous nebulization.

Preparation of a thiols ß-cyclodextrin/gold nanoparticles-coated open tubular column for capillary electrochromatography enantioseparations.

Inspired by the distinct chemical and physical properties of nanoparticles, here a novel open-tubular capillary electrochromatography column was prepared by electrostatic assembly of poly(diallydimethylammonium chloride) onto the inner surface of a fused-silica capillary, followed by self-adsorption of negatively charged SH-ß-cyclodextrin/gold nanoparticles. The formation of the SH-ß-cyclodextrin/gold nanoparticles coated capillary was confirmed and characterized by scanning electron microscopy and energy dispersive spectrometry. The results of scanning electron microscopy and energy dispersive spectrometry studies indicated that SH-ß-cyclodextrin/gold nanoparticles were successfully coated on the inner wall of the capillary column. The performance of the SH-ß-cyclodextrin/gold nanoparticles coated capillary was validated by the analysis of six pairs of chiral drugs, namely zopiclone, carvedilol, salbutamol, terbutaline sulfate, phenoxybenzamine hydrochloride, and ibuprofen. Satisfactory enantioseparation results were achieved, confirming the use of gold nanoparticles as the support could enhance the phase ratio of the open-tubular capillary column. Additionally, the stability and reproducibility of the SH-ß-cyclodextrin/gold nanoparticles coated capillary column were also investigated. Then, this proposed method was well validated with good linearity (≥0.999), recovery (90.0-93.5%) and repeatability, and was successfully used for enantioseparation of ibuprofen in spiked plasma samples, which indicated the new column's potential usage in biological analysis.

Using Polar Ion-Pairs to Control Drug Delivery to the Airways of the Lungs.

The rapid absorptive clearance of drugs delivered to the airways of the lungs means that many inhaled medicines have a short duration of action. The aim of this study was to investigate whether forming polar ion-pairs can modify drug absorption to slow down clearance from the airways. Salbutamol was used as a model drug and was formulated as ion-pairs in an aqueous solution with three negatively charged hydrophilic counterions: sulfate (molecular weight (MW) 142), gluconate (MW 218), and phytate (MW 736) (association constants of 1.57, 2.27, and 4.15, respectively) and one negatively charged hydrophobic counterion, octanoate (MW 166) (association constant, 2.56). All of the counterions were well tolerated by Calu-3 human bronchial epithelial cells when screened for toxicity in vitro using conditions that in silico simulations suggested maintain >80% drug-counterion association. The transport of salbutamol ion-pairs with higher polar surface area (PSA), i.e., the sulfate (PSA 52%), gluconate (PSA 50%), and phytate (PSA 79%) ion-pairs, was significantly lower compared to that of the drug alone (PSA 30%, p < 0.05). In contrast, the octanoate ion-pair (PSA 23%) did not significantly alter the salbutamol transport. The transport data for the gluconate ion-pair suggested that the pulmonary absorption half-life of the ion-paired drug would be double that of salbutamol base, and this illustrates the promise of increasing drug polarity using noncovalent complexation as an approach to control drug delivery to the airways of the lungs.

Investigation of the Interaction Mechanism between Salbutamol and Human Serum Albumin by Multispectroscopic and Molecular Docking.

Salbutamol (SBAL), a kind of short-acting beta 2-adrenergic agonist, has been mainly used to treat bronchial asthma and other allergic airway diseases clinically. In this study, the interaction mechanism between salbutamol and human serum albumin was researched by the multispectral method and molecular docking. The fluorescence intensity of HSA could be regularly enhanced with the increase of SBAL concentration. Both the results of the multispectral method and molecular docking showed that SBAL could bind HSA with van der Waals force and hydrogen bonds. The binding mechanism was further analysed by UV-Vis and synchronous fluorescence spectra. The contents of the secondary structure of free HSA and SBAL-HSA complex were evaluated using CD spectra.

Flow microdialysis sampling-chemiluminescent detection coupled with molecular docking for the investigation of binding behavior between salbutamol and DNA.

The investigation of the binding behavior between drug and DNA provides basic information for understanding pharmacological and toxicologic mechanisms of many drugs. Herein, a facile chemiluminescent (CL) method for investigating the binding behavior between salbutamol and calf thymus DNA (ct-DNA) was established by utilizing flow microdialysis sampling technique. In a reaction equilibrium solution of salbutamol and ct-DNA, free salbutamol was extracted by a microdialysis probe, and then injected into a flow-injection CL detection system to quantitate its concentration. The binding constants of salbutamol acquired by Klotz analysis and Scatchard analysis were 2.97 × 104 M-1and 2.99 × 104 M-1, respectively. Salbutamol showed one sort of binding site on ct-DNA. Meanwhile, the three-dimensional spatial structure of the binding mode was investigated by molecular docking. The results indicate that the binding mode of salbutamol to ct-DNA was groove binding. The hydrogen bonds were primary driving force for the direct recognition of salbutamol by ct-DNA. This proof-of-principle method paves a pathway to investigate the binding behavior between small-molecular drug and DNA, and provides a theoretical guidance for designing DNA-targeting drugs.

Impact of drug particle shape on permeability and cellular uptake in the lung.

The generation of inhalable sized particles (1-5 µm) usually involves a particle-processing step; most commonly milling but spray drying has shown to be a suitable alternative. Besides particle size, processing may affect other particle properties, like shape and solid-state. For example, spray drying of salbutamol sulphate leads to spherical shaped predominantly amorphous particles whereas jet milling frequently maintains the irregular shape and the crystallinity of the raw material. The aim of the present study was to investigate whether particle properties, especially shape, change the biological action of the inhaled particles as well. Therefore, highly water soluble salbutamol sulphate and poorly water soluble budesonide were compared regarding dissolution, permeation and preferential uptake by epithelial cells compared to macrophages after jet milling and spray drying. For both drugs the spray dried, predominantly amorphous, particles resulted in lower respirable fractions, but higher permeability and cell uptake rates compared to the needle shaped, predominantly crystalline particles. The distinct particle properties did not affect the dissolution behaviour of salbutamol sulphate. In turn for drugs with lower solubility (budesonide), spray dried particles dissolved slower compared to jet milled particles. Preferential uptake by macrophages was higher for spray dried particles, suggesting that processing may improve targeted delivery. The comparison between murine cell lines and human monocyte derived macrophages primary cells showed similar trends in rate and preference of particle uptake.

Effect of Roughness on the Dispersion of Dry Powders for Inhalation: a Dynamic Visualization Perspective.

Dry powder inhalers have attracted more interest over the years in every aspect related to them. Interestingly, when focusing on the effects of particle morphology of the active or carrier (excipient), it is generally regarded particle size and shape to influence drug availability of aerosolized particles. However, to date, few studies have examined the effect of texture, i.e., roughness, on this relationship. The main objective of the present work is to gain a closer understanding of the influence of carrier morphology on the aerosolization performance of dry powder inhaler formulations. Image analysis and microscopy were used to visualize the aerosolization process. It is considered that the scale of morphological features on the surface of the carrier particles is responsible for the dispersion of the powder formulation, separation of the drug/carrier, and entrainment from a dry powder inhaler. Thus, for this study, the carrier particles of different surface roughness were mixed with micronized salbutamol sulphate. Aerosolization in vitro testing was used to evaluate the performance. The results indicate a connection between the qualitative surface roughness of coarse carriers and aerosolization performance during powder dispersibility. This investigation demonstrated that indeed, powder dispersion, a dynamic process, is influenced by the scale of the carrier morphology.

Uptake and Effects of the Beta-Adrenergic Agonist Salbutamol in Fish: Supporting Evidence for the Fish Plasma Model.

The fish plasma model (FPM) predicts the fish blood plasma concentration of a pharmaceutical from the water concentration to which the fish is exposed and compares it with the human therapeutic plasma concentration (Hther PC) with the postulate that no adverse toxic effects occur below the Hther PC. The present study provides several lines of evidence supporting the FPM for the beta-adrenergic agonist salbutamol, a small cationic molecule at ambient pH. Salbutamol exhibited very low acute toxicity to early and adult life stages of fish. Biomass reduction in fish early life stages was the most sensitive apical endpoint, with no-observed-effect concentrations (NOECs) in the low mg/L range after continuous exposure for up to 120 d. Given that predicted and measured environmental concentrations are at least 1000-fold lower, the risk of salbutamol in freshwater is deemed very low. Increase in heart beat rate and decrease in total triglyceride content in fish also occurred at the low mg/L range and resembled effects known from humans. This finding supports the FPM assumption of conserved targets in fish with similar functionality. Plasma concentrations measured in adult and juvenile fish exposed to water concentrations at approximately the NOECs exceeded Hther PC and even approached plasma concentrations toxic to humans. This result confirms for salbutamol the FPM hypothesis that no adverse (i.e., population-relevant) toxic effects occur in fish below the Hther PC. Environ Toxicol Chem 2019;38:2509-2519. © 2019 SETAC.

Delivered Lung Dose and Aerodynamic Particle Size Distribution of Salbutamol Pressurized Metered Dose Inhaler After Living Under Patients' Realistic Retention Environments.

Background: The impact of inhalers' postdispensing, real-life temperature and relative humidity (RH) environments on their delivered dose (DD) and aerodynamic particle size distribution (APSD) is usually overlooked. This work evaluated the salbutamol DD and APSD of Ventolin® Evohaler® (V) inhalers already been used and stored by respiratory patients. Methods: Adult patients, prescribed V for ≥3 months before study enrollment, were dispensed both new V to use and portable, handheld electronic temperature and RH data loggers to keep close to the given V before returning them both after 2-3 weeks. Patients' enrollment took place during summer (VS) and winter (VW) seasons. The returned V was then in vitro evaluated using the Next Generation Impactor, and compared with control V (VC) counterparts stored under 21°C and 46% RH. Results: The VS survived in fluctuating habitats of 21.2°C-40.4°C and 16.2%-63.2% RH, which significantly (p < 0.05) decreased the salbutamol DD from 80.4 to 70.5 µg compared with VC. This 12.3% DD reduction was accompanied with a decrease in the fine particle dose from 26.2 to 20.4 µg (p < 0.05), and an increase in the mass median aerodynamic diameter from 2.3 to 2.5 µm (p < 0.05). The VW and VC had equivalent DD and APSD. Conclusion: Patients using V are expected to receive smaller lung doses during the hot summer season compared with intentionally well-kept VC. To have equivalent lung deposition, V users should be advised to retain their inhalers around 20°C with minimal daily environmental fluctuations during summer times.

In situ immobilization of sulfated-ß-cyclodextrin as stationary phase for capillary electrochromatography enantioseparation.

In this work, a novel sulfated-ß-cyclodextrin (S-ß-CD) coated stationary phase was prepared for open-tubular capillary electrochromatography (OT-CEC). The capillary was developed by attaching polydopamine/sulfated-ß-cyclodextrin (PDA/S-ß-CD) onto the gold nanoparticles (AuNPs) coated capillary which was pretreated with polydopamine. The results of scanning electron microscopy (SEM) and energy dispersive X-ray analysis spectroscopy (EDS) indicated that polydopamine/sulfated-ß-cyclodextrin was successfully fixed on the gold nanoparticles coated capillary. To evaluate the performance of the prepared open tubular (OT) column, the enantioseparation was carried out by using ten chiral drugs as model analytes. Under the optimal conditions, salbutamol, terbutaline, trantinterol, tulobuterol, clorprenaline, pheniramine, chlorpheniramine, brompheniramine, isoprenaline and tolterodine were baseline separated with the resolution (Rs) values of 3.25, 1.76, 2.51, 1.89, 3.17, 2.17, 1.99, 1.72, 2.01 and 3.20, respectively. Repeatability of the column was studied, with the relative standard deviations for run-to-run, day-to-day and column-to-column lower than 5.7%.