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.

Label-Free Impedimetric Immunosensors Modulated by Protein A/Bovine Serum Albumin Layer for Ultrasensitive Detection of Salbutamol.

The sensing properties of immunosensors are determined not only by the amount of immobilized antibodies but also by the number of effective antigen-binding sites of the immobilized antibody. Protein A (PA) exhibits a high degree of affinity with the Fc part of IgG antibody to feasibly produce oriented antibody immobilization. This work proposes a simple method to control the PA surface density on gold nanostructure (AuNS)-deposited screen-printed carbon electrodes (SPCEs) by mixing concentration-varied PA and bovine serum albumin (BSA), and to explore the effect of PA density on the affinity attachment of anti-salbutamol (SAL) antibodies by electrochemical impedance spectroscopy. A concentration of 100 µg/mL PA and 100 µg/mL BSA can obtain a saturated coverage on the 3-mercaptoproponic acid (MPA)/AuNS/SPCEs and exhibit a 50% PA density to adsorb the amount of anti-SAL, more than other concentration-varied PA/BSA-modified electrodes. Compared with the randomly immobilized anti-SAL/MPA/AuNS/SPCEs and the anti-SAL/PA(100 µg/mL):BSA(0 µg/mL)/MPA/AuNS/SPCE, the anti-SAL/PA(100 µg/mL): BSA(100 µg/mL)/MPA/AuNS/SPCE-based immunosensors have better sensing properties for SAL detection, with an extremely low detection limit of 0.2 fg/mL and high reproducibility (<2.5% relative standard deviation). The mixture of PA(100 µg/mL):BSA(100 µg/mL) for the modification of AuNS/SPCEs has great promise for forming an optimal protein layer for the oriented adsorption of IgG antibodies to construct ultrasensitive SAL immunosensors.

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%.

Investigation of deep eutectic solvents as additives to ß-CD for enantiomeric separations of Zopiclone, Salbutamol, and Amlodipine by CE.

The enantioseparation of chiral drugs via CE was first investigated using ß-CD as chiral additive and deep eutectic solvents (DESs) as auxiliary additive. The results showed that improved separation of tested chiral drugs was obtained in the presence of DESs and ß-CD compared to the single ß-CD separation system. With the optimized condition, resolutions of DESs applied ß-CD separation system for rac-Zopiclone, rac-Salbutamol, and rac-Amlodipine increased 3-4.2 times as single ß-CD separation system. The resolutions reached 4.74, 6.37, and 9.67, respectively. The results demonstrate that DESs are viable additives to CD system in CE for the separation of the chiral drugs.

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.

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.

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.

Source discrimination of drug residues in wastewater: The case of salbutamol.

Analytical methods used for pharmaceuticals and drugs of abuse in sewage play a fundamental role in wastewater-based epidemiology (WBE) studies. Here quantitative analysis of drug metabolites in raw wastewaters is used to determine consumption from general population. Its great advantage in public health studies is that it gives objective, real-time data about community use of chemicals, highlighting the relationship between environmental and human health. Within a WBE study on salbutamol use in a large population, we developed a procedure to distinguish human metabolic excretion from external source of contamination, possibly industrial, in wastewaters. Salbutamol is mainly excreted as the sulphate metabolite, which is rapidly hydrolyzed to the parent compound in the environment, so this is currently not detected. When a molecule is either excreted un-metabolized or its metabolites are unstable in the environment, studies can be completed by monitoring the parent compound. In this case it is mandatory to assess whether the drug in wastewater is present because of population use or because of a specific source of contamination, such as industrial manufacturing waste. Because commercial salbutamol mainly occurs as a racemic mixture and is stereoselective in the human metabolism, the enantiomeric relative fraction (EFrel) in wastewater samples should reflect excretion, being unbalanced towards one of two enantiomers, if the drug is of metabolic origin. The procedure described involves chiral analysis of the salbutamol enantiomers by liquid chromatography-tandem mass spectrometry (LC-MS-MS) and calculation of EFrel, to detect samples where external contamination occurs. Samples were collected daily between October and December 2013 from the Milano Nosedo wastewater treatment plant. Carbamazepine and atenolol were measured in the sewage collector, as "control" drugs. Salbutamol EFrel was highly consistent in all samples during this three-month period, but a limited number of samples had unexpectedly high concentrations where the EFrel was close to that observed of the un-metabolized, commercially available drug, supporting the idea of an external source of contamination, besides human metabolic excretion. Results showed that, when present, non-metabolic daily loads could be evaluated indicating an average of 4.12g/day of salbutamol extra load due to non-metabolic sources. The stereoselectivity in metabolism and enantiomeric ratio analysis appears to be a useful approach in WBE studies to identify different sources of drugs in the environment, when no metabolic products are present at useful analytical levels.

A novel aptasensor for electrochemical detection of ractopamine, clenbuterol, salbutamol, phenylethanolamine and procaterol.

ß-agonists are phenylethanolamines with different substituent groups on the aromatic ring and the terminal amino group which have the effect of nutrition redistribution and can accumulate in body tissues causing acute or chronic poisoning when consumed. Therefore, it is very important to establish a fast screening method for the detection of several kinds of ß-agonists in food safety control. In this study, the aptamer-agonists (AP-Ago) has screened out by Isothermal Titration Calorimetric method. AP-Ago was a single-strand DNA with 22 base-pairs. The dissociation constant (Kd) to phenylethanolamine (PHL) was 3.34 × 10(-5)mol L(-1). The AP-Ago based electrode was constructed by self-assembling on gold electrode. A label-free electrochemical aptasensor was then developed with AP-Ago-based gold electrode, which was sensitive to phenylethanolamine(PHL), clenbuterol (CLB), ractopamine (RAC), salbutamol (SAL) and procaterol (PRO). The detection limits were 0.04 ng/mL (RAC), 0.35 pg/mL (CLB), 1.0 pg/mL (PHL), 0.53 pg/mL (SAL) and 1.73 pg/mL(PRO), respectively, The detection time was 15 min. The reproductivity of the mentioned aptasensor is good with RSD of 2.09%. Comparing with ELISA and HPLC on ß-agonists detection in actual sample, this aptasensor is advantage of fewer steps and fast screen-detection of these five ß-agonists or their mixtures. This study suggests that the aptasensor can be developed to a rapid screening means with multi-ß-agonists (may be one or more) in sample.

Chiral resolution of salbutamol in plasma sample by a new chiral ligand-exchange chromatography method after its extraction with nano-sized imprinted polymer.

A new chromatographic procedure, based upon chiral ligand-exchange principal, was developed for the resolution of salbutamol enantiomers. The separation was carried out on a C18 column. (l)-Alanine and Cu(2+) were applied as chiral resolving agent and complexing ion, respectively. The kind of copper salt had definitive effect on the enantioseparation. Density functional theory (DFT) was used to substantiate the effect of various anions, accompanying Cu(2+), on the formation of ternary complexes, assumed to be created during separation process. The DFT results showed that the anion kind had huge effect on the stability difference between two corresponding diastereomeric complexes and their chemical structures. It was shown that the extent of participation of the chiral selector in the ternary diastereomeric complexes formation was managed by the anion kind, affecting thus the enantioseparation efficiency of the developed method. Water/methanol (70:30) mixture containing (l)-alanine-Cu(2+) (4:1) was found to be the best mobile phase for salbutamol enantioseparation. In order to analyze sulbutamol enantiomers in plasma samples, racemic salbutamol was first extracted from the samples via nano-sized salbutamol-imprinted polymer and then enantioseparated by the developed method.

Enhanced simultaneous detection of ractopamine and salbutamol--Via electrochemical-facial deposition of MnO2 nanoflowers onto 3D RGO/Ni foam templates.

In this paper, we report a facile method to successfully fabricate MnO2 nanoflowers loaded onto 3D RGO@nickel foam, showing enhanced biosensing activity due to the improved structural integration of different electrode materials components. When the as-prepared 3D hybrid electrodes were investigated as a binder-free biosensor, two well-defined and separate differential pulse voltammetric peaks for ractopamine (RAC) and salbutamol (SAL) were observed, indicating the simultaneous selective detection of both ß-agonists possible. The MnO2/RGO@NF sensor also demonstrated a linear relationship over a wide concentration range of 17 nM to 962 nM (R=0.9997) for RAC and 42 nM to 1463 nM (R=0.9996) for SAL, with the detection limits of 11.6 nM for RAC and 23.0 nM for SAL. In addition, the developed MnO2/RGO@NF sensor was further investigated to detect RAC and SAL in pork samples, showing satisfied comparable results in comparison with analytic results from HPLC.

Synthesis and application of boronic acid-functionalized magnetic adsorbent for sensitive analysis of salbutamol residues in pig tissues.

Salbutamol (SAL) is the most widely used ß2 -agonist drug for asthma and chronic obstructive pulmonary patients, but it is also often abused as feed additive. In recent years, the abuse of SAL has led to a large number of food safety incidents. Therefore, the monitoring of SAL residues in animal products is very important. A highly selective boronate affinity magnetic adsorbent was synthesized and developed for detection of trace levels of SAL residues in pig tissue samples. The obtained Fe3O4@SiO2@FPBA(4-formylphenylboronic acid) magnetic adsorbent showed good adsorption ability to catechol and SAL, and then it was successfully applied as special magnetic solid-phase phase extraction adsorbent coupled with high-performance liquid chromatography (HPLC) for simultaneous isolation and determination of cis-diol compounds. The binding capacity of catechol and SAL reached 96 and 50 µmol/g, respectively. The method was successfully established for the detection of trace levels of SAL in pig tissue samples. The linear range extended from 0.32 to 800 µg/kg (R(2) = 0.9994). The limit of detection of SAL was 0.19 µg/kg. The recoveries were satisfactory (89.5-108.0%) at three spiked levels with RSD between 2.1 and 11.3%. These results indicated that the method has potential for enrichment and detection of trace levels of SAL residual in animal food products.

Highly selective and sensitive detection of ß-agonists using a surface plasmon resonance sensor based on an alkanethiol monolayer functionalized on a Au surface.

Immunosensor surfaces for surface plasmon resonance (SPR) have been constructed using a functionalized succinimidyl propanethiol monolayer as a linker to immobilize ß-agonist protein conjugates on a Au surface. Because ß-agonist is a small molecule, an indirect competitive inhibition immunoassay was used for detection. The lowest detection limits for ractopamine and salbutamol were 10 ppt (10 pg mL(-1)) and 5 ppt (5 pg mL(-1)), respectively. The fabricated immunosensor surface can be used again for detection after regeneration in 0.1 M sodium hydroxide. It was found that the same sensor surface could be reused for performing over 100 rapid immunoreactions. Moreover, one immunosensing-regeneration cycle requires only 600 s. The fabricated immunosensor surfaces were characterized using SPR and scanning tunneling microscopy observation. In the kinetic study of the indirect competitive immunosensing inhibition, the affinity constant (K1) of salbutamol antibody was smaller than the K1 of ractopamine antibody. Compared to a previous study of clenbuterol detection, it was concluded that the high K1 was coupled with low sensitivity. In the selectivity study, both immunosensor surfaces provided >90% of confidence level for the specific detection of ß-agonist compounds. The fabrication of highly selective and sensitive sensor surfaces for detecting ß-agonist compounds was confirmed.

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.

Combined solid-phase microextraction and high-performance liquid chromatography with ultroviolet detection for simultaneous analysis of clenbuterol, salbutamol and ractopamine in pig samples.

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 ß2 -agonists residue in pig samples.

Salbutamol extraction from urine and its stability in different solutions: identification of methylation products and validation of a quantitative analytical method.

Salbutamol is commonly used in asthma treatment, being considered a short-effect bronchodilator. This drug poses special interest in certain fields of chemical analysis, such as food, clinical and doping analyses, in which it needs to be analyzed with quantitative precision and accuracy. Salbutamol, however, is known to degrade under certain conditions and this is critical if quantitative results must be generated. The present work aimed to investigate salbutamol extraction from urine samples, to determine whether salbutamol is unstable in other solvents as well as in urine samples, to elucidate the structures of the possible degradation products and to validate an analytical method using the extraction procedure evaluated. Stability investigations were performed in urine at different pH values, in methanol and acetone at different temperatures. Semi-preparative liquid chromatography was performed for the isolation of degradation products, and gas chromatography coupled to mass spectrometry as well as nuclear magnetic resonance were used for identification. Three unreported methylation products were detected in methanolic solutions and had their structures elucidated. Urine samples showed a reduction in salbutamol concentration of up to 25.8% after 5 weeks. These results show that special care must be taken regarding salbutamol quantitative analyses, since degradation either in standard solutions or in urine could lead to incorrect values.

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.

Screening, recognition and quantitation of salbutamol residues in ham sausages by molecularly imprinted solid phase extraction coupled with high-performance liquid chromatography-ultraviolet detection.

A highly selective molecularly imprinted solid phase extraction (MISPE) coupled with liquid chromatography-ultraviolet detection was developed for the determination of salbutamol (SAL) in ham sausages. New molecularly imprinted polymers (MIPs) were synthesized with phenylephrine as dummy template and it revealed good affinity to SAL in methanol-acetonitrile system. Adsorption capacity of the MIPs was evaluated by dynamic adsorption experiments. The MIPs were used as SPE sorbent for the selective clean-up and pre-concentration of SAL in ham sausage samples. The results showed that the matrix compounds presented in ham sausage samples could be removed effectively and the recoveries of SAL at three spiked levels were ranged from 82.6 to 100.5% with the relative standard deviation (RSD) of less than 3.6%. This method is simple and sensitive, and is therefore an alternative tool to the existing methods for analyzing residual SAL in biological samples.

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.

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.