An Intravenous Pharmacokinetic Study of Cannabidiol Solutions in Piglets through the Application of a Validated Ultra-High-Pressure Liquid Chromatography Coupled to Tandem Mass Spectrometry Method for the Simultaneous Quantification of CBD and Its Carboxylated Metabolite in Plasma.

Cannabidiol (CBD) has multiple therapeutic benefits that need to be maximized by optimizing its bioavailability. Numerous formulations are therefore being developed and their pharmacokinetics need to be studied, requiring analytical methods and data from intravenous administration. As CBD is susceptible to hepatic metabolism, the requirement of any method is to quantify metabolites such as 7-COOH-CBD. We demonstrated that CBD and 7-COOH-CBD could be simultaneously and correctly quantified in piglet plasma by using an UHPLC-MS/MS technique. The validated method allowed for an accurate bioanalysis of an intravenously injected solution consisting of CBD-HPßCD complexes. The experimental pharmacokinetic profile of CBD showed multi-exponential decay characterized by a fast apparent distribution half-life (0.25 h) and an elimination half-life of two hours. The profile of 7-COOH-CBD was not linked with the first-pass metabolism, since 80% of the maximum metabolite concentration was reached at the first sampling time point, without any decrease during the period of study. A two-compartment model was optimal to describe the experimental CBD profile. This model allowed us to calculate macro-micro constants and volumes of distribution (Vss = 3260.35 ± 2286.66 mL) and clearance (1514.5 ± 261.16 mL·h-1), showing that CBD is rapidly distributed to peripheral tissues once injected and slowly released into the bloodstream.

Fully automated electrophoretically mediated microanalysis for CYP1A1 activity monitoring optimized by multivariate approach.

In this study, a fully automated incapillary system was developed to monitor the activity of CYP1A1 (Cytochrome P450, family 1, subfamily A, polypeptide 1) in physiological conditions. Ethoxycoumarin, the selected substrate, undergoes an inline bioreaction in the presence of CYP1A1 supersomes and Nicotinamide adenine dinucleotide phosphate reduced as cofactor, giving rise to hydroxycoumarin, the product that was assayed. The optimization of the experimental conditions was supported by the application of a design of experiment, providing a better understanding of electrophoretic mixing parameters that influence the metabolic reactions. The results obtained in optimal conditions were compared not only to those achieved after offline metabolization but also with liver microsomes. Finally, inhibition studies were conducted showing an important decrease of hydroxycoumarin formation using apigenin as CYP1A1 potent inhibitor. This study demonstrates the usefulness of our inline system for the fully automated in vitro metabolism studies and the screening of new CYP1A1 inhibitors.

Triphenylphosphonium salts of 1,2,4-benzothiadiazine 1,1-dioxides related to diazoxide targeting mitochondrial ATP-sensitive potassium channels.

The present work aims at identifying new ion channel modulators able to target mitochondrial ATP-sensitive potassium channels (mitoKATP channels). An innovative approach should consist in fixing a cationic and hydrophobic triphenylphosphonium fragment on the structure of known KATP channel openers. Such phosphonium salts are expected to cross the biological membranes and to accumulate into mitochondria. Previous works revealed that the presence of an (R)-1-hydroxy-2-propylamino chain at the 3-position of 4H-1,2,4-benzothiadiazine 1,1-dioxides KATP channel openers increased, in most cases, the selectivity towards the pancreatic-type (SUR1/Kir6.2) KATP channel. In order to target cardiac mitoKATP channels, we decided to introduce a triphenylphosphonium group through an ester link on the SUR1-selective (R)-7-chloro-3-(1-hydroxy-2-propyl)amino-4H-1,2,4-benzothiadiazine 1,1-dioxide. The new compounds were found to preserve an inhibitory activity on insulin secretion (SUR1-type KATP channel openers) while no clear demonstration of an impact on mitochondria from cardiomyocytes (measurement of oxygen consumption, respiratory parameters and ATP production on H9C2 cells) was observed. However, the most active (inhibition of insulin release) compound 17 was found to penetrate the cardiac cells and to reach mitochondria.

Hydroxylated analogues of ATP-sensitive potassium channel openers belonging to the group of 6- and/or 7-substituted 3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides: toward an improvement in sulfonylurea receptor 1 selectivity and metabolism stability.

Diversely substituted 3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides are known to be potent KATP channel openers, with several drugs being selective for the SUR1/Kir6.2 channel subtype. This work examined the biological activity, tissue selectivity, and in vitro metabolic stability of hydroxylated analogues of 3-isopropylaminobenzothiadiazine dioxides. Because of the presence of a chiral center, the R and S isomers were prepared separately and characterized. R isomers were systematically found to be more potent and more selective than S isomers on pancreatic tissue (compared to vascular smooth muscle tissue), leading to compounds with an improved sulfonylurea receptor 1 (SUR1) selectivity. An in vitro metabolic study revealed that 7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (1a) was rapidly biotransformed and led in part to a mixture of the corresponding (R)- and (S)-3-(1-hydroxy-2-propyl)amino-substituted derivatives. Radioisotopic experiments characterized one of the most potent and SUR1-selective enantiomers, (R)-7-chloro-3-(1-hydroxy-2-propyl)amino-4H-1,2,4-benzothiadiazine 1,1-dioxide 13a, as being a KATP channel opener. Moreover, 13a exhibited an enhanced metabolic stability. Such a compound can be considered as a new lead candidate displaying improved physicochemical (hydrosolubility) and pharmacological (tissue selectivity) properties as well as improved metabolic stability compared to its nonhydroxylated counterpart, 1a.

Development and validation of a ultra-high-performance liquid chromatography-UV method for the detection and quantification of erectile dysfunction drugs and some of their analogues found in counterfeit medicines.

Pharmaceutical counterfeiting is a permanently growing problem. Control laboratories are constantly analysing counterfeit medicines. In industrialised countries, one of the main counterfeited class of medicines are erectile dysfunction drugs. This paper describes the development and validation of a fast method to detect and quantify the three authorised phosphodiesterase type 5 inhibitors and five analogues. The method is based on the use of a sub-2 microns polar-embedded column with a gradient using acetonitrile as organic modifier and 10mM ammonium formate buffer (pH 3.5) as aqueous component of the mobile phase. The separation was achieved in less than 4.5 min. The method has also been compared to the registered HPLC method for the assay of Viagra(®) which was considered as the reference method. The method is also compatible with on-line coupling mass spectrometry and will significantly reduce analysis times and solvent consumption.

Impurity fingerprints for the identification of counterfeit medicines--a feasibility study.

Most of the counterfeit medicines are manufactured in non good manufacturing practices (GMP) conditions by uncontrolled or street laboratories. Their chemical composition and purity of raw materials may, therefore, change in the course of time. The public health problem of counterfeit drugs is mostly due to this qualitative and quantitative variability in their formulation and impurity profiles. In this study, impurity profiles were treated like fingerprints representing the quality of the samples. A total of 73 samples of counterfeit and imitations of Viagra(®) and 44 samples of counterfeit and imitations of Cialis(®) were analysed on a HPLC-UV system. A clear distinction has been obtained between genuine and illegal tablets by the mean of a discriminant partial least squares analysis of the log transformed chromatograms. Following exploratory analysis of the data, two classification algorithms were applied and compared. In our study, the k-nearest neighbour classifier offered the best performance in terms of correct classification rate obtained with cross-validation and during external validation. For Viagra(®), both cross-validation and external validation sets returned a 100% correct classification rate. For Cialis(®) 92.3% and 100% correct classification rates were obtained from cross-validation and external validation, respectively.

Optimization of micro-HPLC peak focusing for the detection and quantification of low hepcidin concentrations.

Micro-high-performance liquid chromatography is a miniaturized, economic and ecological chromatographic system allowing the use of reduced size chromatographic columns. Coupled with electrospray ionization tandem mass spectrometry, this technique can be used to detect and quantify low concentrations of peptides. In this study, hepcidin was used as the model compound and analysed using octadecylsilica stationary phase by means of a gradient elution mode at a flow rate of 4 µL/min. Several parameters were studied to optimize peak focusing. Using the methodology of experimental design, the mobile-phase gradient conditions and the sample composition were optimized in order to maximize the sensitivity and minimize retention time. Stability of the target peptide in solution was also demonstrated.

Detection of counterfeit Viagra® by Raman microspectroscopy imaging and multivariate analysis.

During the past years, pharmaceutical counterfeiting was mainly a problem of developing countries with weak enforcement and inspection programs. However, Europe and North America are more and more confronted with the counterfeiting problem. During this study, 26 counterfeits and imitations of Viagra® tablets and 8 genuine tablets of Viagra® were analysed by Raman microspectroscopy imaging. After unfolding the data, three maps are combined per sample and a first PCA is realised on these data. Then, the first principal components of each sample are assembled. The exploratory and classification analysis are performed on that matrix. PCA was applied as exploratory analysis tool on different spectral ranges to detect counterfeit medicines based on the full spectra (200-1800 cm⁻¹), the presence of lactose (830-880 cm⁻¹) and the spatial distribution of sildenafil (1200-1290 cm⁻¹) inside the tablet. After the exploratory analysis, three different classification algorithms were applied on the full spectra dataset: linear discriminant analysis, k-nearest neighbour and soft independent modelling of class analogy. PCA analysis of the 830-880 cm⁻¹ spectral region discriminated genuine samples while the multivariate analysis of the spectral region between 1200 cm⁻¹ and 1290 cm⁻¹ returns no satisfactory results. A good discrimination of genuine samples was obtained with multivariate analysis of the full spectra region (200-1800 cm⁻¹). Application of the k-NN and SIMCA algorithm returned 100% correct classification during both internal and external validation.

Determination of enantiomeric purity of S-amlodipine by chiral LC with emphasis on reversal of enantiomer elution order.

An LC method was developed and prevalidated for the enantiomeric purity determination of S-amlodipine in polar organic solvent chromatography using a chlorine-containing cellulose-based chiral stationary phase (CSP). The concentration of formic acid (FA) (0.01-0.2%) in the mobile phase containing acetonitrile as the main solvent was found to influence the elution order of amlodipine enantiomers as well as the enantioresolution. A reversal of the enantiomer elution order of amlodipine was only observed with chiral stationary phases with both electron-withdrawing (chloro) and electron-donating groups (methyl) on the phenyl moieties of the chiral selector, namely cellulose tris(3-chloro-4-methylphenylcarbamate) and cellulose tris(4-chloro-3-methylphenylcarbamate). The highest enantioresolution (Rs : 4.1) value was obtained at the lowest FA concentration (0.01%) using cellulose tris(4-chloro-3-methylphenylcarbamate) as the chiral selector and the enantiomeric impurity, R-amlodipine, eluted first under these conditions. Therefore, the mobile phase selected for the prevalidation of the method consisted of ACN/0.1% DEA/0.01% FA and the temperature was set at 25°C. The method was prevalidated by means of the strategy based on the total measurement error and the accuracy profile. The method was found to be selective and the limit of quantification was found to be about 0.05% for R-amlodipine, while the limit of detection was close to 0.02%.

Pre-study and in-study validation of a SPE-LC-MS-MS method for the determination of 5-S-cysteinyldopa, a melanoma biomarker, in human plasma.

The incidence of malignant melanoma has increased over the past decades, particularly in Caucasian population. This disease presents defavourable prognosis in terms of survey, especially when detection occurs at the metastatic phase. Reliable analytical methods for biomarker determination are thus an interesting tool in pathology detection and follow-up. In this context, a method using SPE-LC-ESI-MS-MS for the determination of 5-S-cysteinyldopa (5-SCD) in human plasma was optimized. The presence of matrix effect was investigated in details while 5-SCD stability was studied according to FDA requirements for the validation of bioanalytical methods. Pre-study and in-study validations of the entire method were then successfully performed by applying the approach based on total measurement error and accuracy profiles over a concentration ranges from 1.6 to 200 ng/ml. Good results with respect to accuracy, trueness and precision were obtained. The maximum risk of observing future measurements falling outside the acceptance limits during routine analysis was also estimated.

Evaluation of chlorine containing cellulose-based chiral stationary phases for the LC enantioseparation of basic pharmaceuticals using polar non-aqueous mobile phases.

The discrimination ability of three cellulose-based chiral stationary phases (CSPs) was evaluated towards the enantiomers of basic drugs, using ACN as the main solvent in polar organic mobile phases. The study was focused on CSPs containing cellulose tris(3-chloro-4-methylphenylcarbamate) (3-Cl-4-MePC), cellulose tris(4-chloro-3-methylphenylcarbamate) (4-Cl-3-MePC) or cellulose tris(3,5-dichlorophenylcarbamate) (3,5-diClPC) as the chiral selector. The behaviour of these CSPs was studied systematically in order to investigate the influence of the presence and position of the chlorine substituents on the phenylcarbamate moieties on the retention and resolution of the enantiomers. The evaluation was made with three different generic mobile phases, namely ACN/0.1%DEA/0.1% TFA (DEA, diethylamine), ACN/0.1%DEA/0.2% FA and ACN/0.1%DEA/0.2%AcA, deduced from the previous study. The nature of the acidic additive and of the chiral selector was found to be particularly important for the retention and enantioresolution of these basic compounds. High-resolution values could be obtained for most studied enantiomers with these CSPs, clearly demonstrating the interest of using them in combination with polar organic mobile phases. However, significant differences in enantioresolution between the CSPs have been observed for many compounds, indicating that these phases seem to be quite complementary.

Generic systems for the enantioseparation of basic drugs in NACE using single-isomer anionic CDs.

The enantioseparation of 10 basic drugs was evaluated in NACE systems using heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)-ß-CD (HMAS-ß-CD). For this purpose, a D-optimal design with 21 experimental points was applied. Four antifungal agents (econazole, isoconazole, miconazole, sulconazole), three local anesthetics (bupivacaine, mepivacaine and prilocaine), two sympathomimetics (salbutamol and terbutaline) and one ß-blocker (carvedilol) were selected as basic model analytes. The influence on the enantiomeric resolution of anionic CD and BGE anion concentrations as well as the BGE anion nature was investigated. For all studied analytes, the enantiomeric resolution was shown to be significantly influenced by the CD concentration. Based on the observed results, a generic NACE system was recommended, namely 20mM HMAS-ß-CD and 10mM ammonium camphor SO(3)(-) in methanol acidified with 0.75 M formic acid. Moreover, this NACE system was compared to previous conditions with heptakis(2,3-di-O-methyl-6-O-sulfo)-ß-CD (HDMS-ß-CD) or heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-CD (HDAS-ß-CD). Finally, two generic systems using either HDAS-ß-CD or HMAS-ß-CD were proposed and evaluated for the enantioseparation of ketamine and norketamine after incubation of ketamine in phenobarbital-induced male rat liver microsomes systems.

Development and validation of a nonaqueous capillary electrophoretic method for the enantiomeric purity determination of a synthetic intermediate of new 3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans using a single-isomer anionic cyclodextrin derivative and an ionic liquid.

The enantiomeric purity determination of a synthetic intermediate of new 3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans, i.e. 4-amino-2,2-dimethyl-6-ethoxycarbonylamino-3,4-dihydro-2H-1-benzopyran, was successfully carried out using an anionic cyclodextrin (CD) derivative combined with a chiral ionic liquid (IL). In order to obtain high resolution and efficiency values, the addition of a chiral IL, i.e. ethylcholine bis(trifluoromethylsulfonyl)imide (EtChol NTf(2)), to the background electrolyte containing heptakis(2,3-di-O-methyl-6-O-sulfo)-ß-CD (HDMS-ß-CD) was found to be essential. A simultaneous increase in separation selectivity and enantioresolution seems to indicate a synergistic effect of HDMS-ß-CD and EtChol NTf(2). The best enantioseparation of the key intermediate was achieved using a methanolic solution of 0.75M formic acid, 10mM ammonium formate, 1.5mM HDMS-ß-CD and 5mM EtChol NTf(2). Levamisole was selected as internal standard. The optimized conditions allowed the determination of 0.1% of each enantiomer in the presence of its stereoisomer using the method of standard additions. The NACE method was then fully validated with respect to selectivity, response function, trueness, precision, accuracy, linearity and limits of detection and quantification.

Comparison and combination of spectroscopic techniques for the detection of counterfeit medicines.

During this study, Fourier transform infrared spectroscopy (FT-IR), near infrared spectroscopy (NIR) and Raman spectroscopy were applied to 55 samples of counterfeit and imitations of Viagra and 39 samples of counterfeit and imitations of Cialis. The aim of the study was to investigate which of these techniques and associations of them were the best for discriminating genuine from counterfeit and imitation samples. Only the regions between 1800-400 cm(-1) and 7000-4000 cm(-1) were used for FT-IR and NIR spectroscopy respectively. Partial least square analysis has been used to allow the detection of counterfeit and imitation tablets. It is shown that for the Viagra samples, the best results were provided by a combination of FT-IR and NIR spectroscopy. On the other hand, the best results for the Cialis samples were provided by the combination of NIR and Raman spectroscopy (1400-1190 cm(-1)). These techniques not only permitted a clear discrimination between genuine and counterfeit or imitation samples but also the distinction of clusters among illegal samples. This might be interesting for forensic investigations by authorities.

Optimization of the LC enantioseparation of chiral pharmaceuticals using cellulose tris(4-chloro-3-methylphenylcarbamate) as chiral selector and polar non-aqueous mobile phases.

The resolving power of a new commercial polysaccharide-based chiral stationary phase, Sepapak-4, with cellulose tris(4-chloro-3-methylphenylcarbamate) coated on silica microparticles as chiral selector, was evaluated toward the enantioseparation of ten basic drugs with widely different structures and hydrophobic properties, using ACN as the main component of the mobile phase. A multivariate approach (experimental design) was used to screen the factors (temperature, n-hexane content, acidic and basic additives) likely to influence enantioresolution. Then, the optimization was performed using a face-centered central composite design. Complete enantioseparation could be obtained for almost all tested chiral compounds, demonstrating the high chiral discrimination ability of this chiral stationary phase using polar organic mobile phases made up of ACN and containing an acidic additive (TFA or formic acid), 0.1% diethylamine and n-hexane. These results clearly illustrate the key role of the nature of the acidic additive in the mobile phase.

Association of two single-isomer anionic CD in NACE for the chiral and achiral separation of fenbendazole, its sulphoxide and sulphone metabolites: application to their determination after in vitro metabolism.

A NACE method was developed for the separation of fenbendazole (FBZ), a prochiral drug giving rise to chiral (oxfendazole or OFZ) and nonchiral (FBZ sulphone or FBZSO(2)) metabolites. First, the effect of the nature and the concentration of CD as well as that of the acidic BGE on the enantiomeric separation of OFZ were studied. OFZ enantiomers were completely resolved using a BGE made up of 10 mM ammonium formate and 0.5 M TFA in methanol containing 10 mM heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-CD and 10 mM heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-CD. Moreover, the NACE method was found to be particularly well suited to the simultaneous determination of FBZ, OFZ enantiomers, and FBZSO(2). Thiabendazole was selected as an internal standard. The CD-NACE potential was then evaluated for in vitro metabolism studies using FBZ as a model case. The OFZ enantiomers and FBZSO(2) could be detected after incubation of FBZ in the phenobarbital-induced male rat liver microsomes systems.

New fluorinated 1,2,4-benzothiadiazine 1,1-dioxides: discovery of an orally active cognitive enhancer acting through potentiation of the 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid receptors.

In the search of a potent cognitive enhancer, a series of 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides have been synthesized and evaluated as positive allosteric modulators of the AMPA receptors. In the present work, we focused our efforts on the insertion of mono- or polyfluoro-substituted alkyl chains at the 4-position of the thiadiazine ring in an attempt to enhance the pharmacokinetic behavior of previously described compounds. Among all the described compounds, 7-chloro-4-(2-fluoroethyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide, 12b, was shown to exert a strong activity on AMPA receptors in vitro and a marked cognitive-enhancing effect in vivo after oral administration to Wistar rats. Considering its in vivo activity, the metabolic degradation of 12b was studied and compared to that of its nonfluorinated analogue 9b. Taken together, results of this study clearly validated the positive impact of the fluorine atom on the alkyl chain at the 4-position of benzothiadiazine dioxides on activity and metabolic stability.

Coupling of liquid chromatography/tandem mass spectrometry and liquid chromatography/solid-phase extraction/NMR techniques for the structural identification of metabolites following in vitro biotransformation of SUR1-selective ATP-sensitive potassium channel openers.

SUR1-selective ATP-sensitive potassium channel openers (PCOs) have been shown to be of clinical value for the treatment of several metabolic disorders, including type I and type II diabetes, obesity, and hyperinsulinemia. Taking into account these promising therapeutic benefits, different series of 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides structurally related to diazoxide were developed. In view of the lead optimization process of the series, knowledge of absorption, distribution, metabolism, excretion, and toxicity parameters, and more particularly the metabolic fate of these compounds, is a fundamental requirement. For such a purpose, two selected promising compounds [7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ 73) and 7-chloro-3-(3-pentylamino)-4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ 157)] were incubated in the presence of phenobarbital-induced rat liver microsomes to produce expected mammal in vivo phase I metabolites. The resulting major metabolites were then analyzed by both mass spectrometry (MS) and NMR to completely elucidate their chemical structures. The two compounds were also further incubated in the presence of nontreated rats and human microsomes to compare the metabolic profiles. In the present study, the combined use of an exact mass liquid chromatography (LC)/tandem MS platform and an LC/solid-phase extraction/NMR system allowed the clarification of some unresolved structural assessments in the accurate chemical structure elucidation process of the selected PCO drugs. These results greatly help the optimization of the lead compounds.

Effect of the nature of the single-isomer anionic CD and the BGE composition on the enantiomeric separation of beta-blockers in NACE.

The separation of ten beta-blockers has been investigated in NACE systems using heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-CD (HDMS-beta-CD) and heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-CD (HDAS-beta-CD). The influence on enantioresolution, mobility difference and selectivity of the nature of both anionic CD and BGE anion as well as their concentrations were studied by means of a multivariate approach. A D-optimal design with 25 experimental points was applied. For all studied analytes, the enantiomeric resolution was shown to be significantly influenced by both CD nature and concentration. Except for two compounds, HDAS-beta-CD was found to give higher enantioresolution values than HDMS-beta-CD. The best enantioseparation for all compounds was achieved in the presence of a high chiral selector concentration and for most of them at a low BGE anion concentration. For each investigated compound, operating conditions leading to the best enantiomeric resolution were deduced. A generic NACE system was then recommended, namely 10 mM ammonium acetate and 40 mM HDAS-beta-CD in methanol acidified with 0.75 M formic acid. This generic system was able to completely resolve the enantiomers of all beta-blockers, with a R(s) value of at least 4. Finally, the optimal conditions obtained modelling resolution, mobility difference and selectivity were compared.

Enantioresolution of basic pharmaceuticals using cellulose tris(4-chloro-3-methylphenylcarbamate) as chiral stationary phase and polar organic mobile phases.

A polysaccharide-based chiral stationary phase (Sepapak-4), with cellulose tris(4-chloro-3-methylphenylcarbamate) as chiral selector, has been investigated in liquid chromatography (LC). Its enantioresolution power was evaluated towards 13 basic amino-drugs with widely different structures and polarities, using polar organic mobile phases. After preliminary experiments, acetonitrile was selected as the main mobile phase component, to which a low concentration of diethylamine (0.1%) was systematically added in order to obtain efficient and symmetrical peaks. Different organic solvents were first added in small proportions (5-10%) to acetonitrile to modulate analyte retention. Polar organic modifiers were found to decrease retention and enantioresolution while hexane had the opposite effect, indicating normal-phase behaviour under these conditions. The addition of an organic acid (formic, acetic or trifluoroacetic acid) was found to strongly influence the retention of the basic amino drugs in these nonaqueous systems. The nature and proportion of the acidic additive in the mobile phase had also deep impact on enantioresolution. Therefore, the studied compounds could be subdivided in three groups in respect to the acidic additive used. All analytes could be enantioseparated in relatively short analysis times (10-20 min) using these LC conditions.