Enantiomeric separation of amlodipine and its two chiral impurities by nano-liquid chromatography and capillary electrochromatography using a chiral stationary phase based on cellulose tris(4-chloro-3-methylphenylcarbamate).

In this work, a novel polysaccharide-based chiral stationary phase, cellulose tris(4-chloro-3-methylphenylcarbamate), also called Sepapak 4 has been evaluated for the chiral separation of amlodipine (AML) and its two impurities. AML is a powerful vasodilatator drug used for the treatment of hypertension. Capillary columns of 100 µm id packed with the chiral stationary phase were used for both nano-LC and CEC experiments. The optimization of the mobile phase composed of ACN/water, (90:10, v/v) containing 15 mM ammonium borate pH 10.0 in nano-LC allowed the chiral separation of AML and the two impurities, but not in a single run. With the purpose to obtain the separation of the three pairs of enantiomers simultaneously, CEC analyses were performed in the same conditions achieving better enantioresolution and higher separation efficiencies for each compound. To fully resolve the mixture of six enantiomers, parameters such as buffer pH and concentration sample injection have been then investigated. A mixture of ACN/water (90:10, v/v) containing 5 mM ammonium borate buffer pH 9.0 enabled the complete separation of the three couples of enantiomers in less than 30 min. The optimized CEC method was therefore validated and applied to the analysis of pharmaceutical formulation declared to contain only AML racemate.

Preparation and application of teicoplanin functionalized polymeric monolith for enantioseparation of chiral drugs.

A novel chiral stationary phase (CSP), based on a monolithic organic polymer chemically modified with teicoplanin, was fabricated within a 100 µm I.D. fused silica capillary. The teicoplanin was firstly derivatized with 2-isocyanatoethyl methacrylate (ICNEML) and then thermally co-polymerized with the crosslinker ethylene dimethacrylate (EDMA) in presence of porogens (methanol and dimethylsulfoxide). The optimal experimental conditions (e.g., concentration and ratio of the reagents), considering enantioresolution and permeability, were systematically investigated. The prepared monolith was evaluated using scanning electron microscopy, and the column exhibited quite good morphology. In order to further evaluate the enantioresolving power of the poly(ICNEML-teicoplanin-co-EDMA) monolith, a series of basic and acidic chiral compounds were analyzed using an isocratic mode of polar organic solvents (methanol and acetonitrile) or the same solvents in combination with water (reversed-phase) by nano-liquid chromatography. Five mandelic acids and six derivatized amino acids were enantioresolved under reversed-phase mode (Rs = 1.22-3.47 and α = 1.43-6.33). This monolithic teicoplanin-CSP was also effective in the enantioseparations of 17 amino alcohol drugs employing polar-organic phase mode (MeOH/ACN/TEA/HOAc (80/20/0.03/0.055, v/v/v/v)). Ten of them were baseline enantioresolved (alprenolol, betaxolol, clenbuterol, isoproterenol, metoprolol, pindolol, propranolol, salbutamol, sotalol, tertatolol) (Rs = 1.55-2.48 and α = 1.21-1.55), while the others were partially enantioseparated (Rs = 1.14-1.48).

Enantioseparations with cellulose tris(3-chloro-4-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography.

Cellulose tris(3-chloro-4-methylphenylcarbamate) was coated onto native and aminopropylsilanized silica in order to prepare chiral stationary phases (CSPs) for enantioseparations using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). The effect of the chiral selector loading onto silica, mobile phase composition and pH, as well as separation variables on separation of enantiomers was studied. It was found that CSPs based on cellulose tris(3-chloro-4-methylphenylcarbamate) can be used for preparation of very stable capillary columns useful for enantioseparations in nano-LC and CEC in combination with polar organic mobile phases.

Enantioseparation of the antidepressant reboxetine.

The enantioseparation of reboxetine by HPLC was investigated using chiral stationary phases (CSPs) containing cellulose Tris(3,5-dimethylphenyl)carbamate on silica gel (Chiralcel OD column) as the chiral selector. Reversed phase HPLC was the technique of choice for the analytical enantioseparation of reboxetine, while the chiral semipreparative separation was obtained with the same CSP, but in normal phase conditions. The effects of the mobile phase pH and composition on analytical retention, enantioselectivity and resolution were investigated. The best performance was obtained using a mobile phase composed of 0.5M sodium perchlorate at pH 6 and acetonitrile in the 60/40 (v/v) ratio. The semipreparative separation has allowed obtaining pure enantiomers, but required the preparation of reboxetine free base. Different n-hexane/alcohol mixtures were tested as mobile phases, varying both the nature of the alcohol and its percentage in the mobile phase. Different n-hexane/alcohol mixtures were tested as mobile phase and the best results were obtained by using a mobile phase composed of n-hexane and 2-propanol (80:20, v/v).

A facile and efficient single-step approach for the fabrication of vancomycin functionalized polymer-based monolith as chiral stationary phase for nano-liquid chromatography.

A facile single-step preparation strategy for fabricating vancomycin functionalized organic polymer-based monolith within 100µm fused-silica capillary was developed. The synthetic chiral functional monomer, i.e 2-isocyanatoethyl methacrylate (ICNEML) derivative of vancomycin, was co-polymerized with the cross-linker ethylene dimethacrylate (EDMA) in the presence of methanol and dimethyl sulfoxide as the selected porogens. The co-polymerization conditions were systematically optimized in order to obtain satisfactory column performance. Adequate permeability, stability and column morphology were observed for the optimized poly(ICNEML-vancomycin-co-EDMA) monolith. A series of chiral drugs were evaluated on the monolith in either polar organic-phase or reversed-phase modes. After the optimization of separation conditions, baseline or partial enantioseparation were obtained for series of drugs including thalidomide, colchicine, carteolol, salbutamol, clenbuterol and several other ß-blockers. The proposed single-step approach not only resulted in a vancomycin functionalized organic polymer-based monolith with acceptable performance, but also significantly simplified the preparation procedure by reducing time and labor.

Synthesis and evaluation of polymeric continuous bed (monolithic) reversed-phase gradient stationary phases for capillary liquid chromatography and capillary electrochromatography.

There is a demand of novel high resolution separation media for separation of complex mixtures, particularly biological samples. One of the most flexible techniques for development of new separation media currently is synthesis of the continuous bed (monolithic) stationary phases. In this study the capillary format gradient stationary phases were formed using continuous bed (monolith) polymerization in situ. Different reversed-phase stationary phase gradients were tailored and their resolution using capillary liquid chromatography and capillary electrochromatography at isocratic mobile phase conditions was evaluated. It is demonstrated, that efficiency and resolution of the gradient stationary phases can be substantially increased comparing to the common (isotropic) stationary phases. The proposed formation approach of the gradient stationary phase is reproducible and compatible with the capillary format or microchip format separations. It can be easily automated for the separation optimizations or mass production of the capillary columns or chips.

Evaluation of novel amylose and cellulose-based chiral stationary phases for the stereoisomer separation of flavanones by means of nano-liquid chromatography.

Three polysaccharide-based chiral stationary phases, Sepapak(®) 1, Sepapak(®) 2 and Sepapak(®) 3 have been evaluated in the present work for the stereoisomer separation of a group of 12 flavonoids including flavanones (flavanone, 4'-methoxyflavanone, 6-methoxyflavanone, 7-methoxyflavanone, 2'-hydroxyflavanone, 4'-hydroxyflavanone, 6-hydroxyflavanone, 7-hydroxyflavanone, hesperetin, naringenin) and flavanone glycosides (hesperidin, naringin) by nano-liquid chromatography (nano-LC). The behaviour of these chiral stationary phases (CSPs) towards the selected compounds was studied in capillary columns (100µm internal diameter (i.d.)) packed with the above mentioned CSPs using polar organic, reversed and normal elution modes. The influence of nature and composition of the mobile phase in terms of concentration and type of organic modifier, buffer type and water content (reversed phase elution mode) on the enantioresolution (R(s)), retention factor (k) and enantioselectivity (α) was evaluated. Sepapak(®) 3 showed the best chromatographic results in terms of enantioresolution, enantioselectivity and short analysis time, employing a polar organic phase mode. A mixture of methanol/isopropanol (20/80, v/v) as mobile phase enabled the chiral separation of eight flavanones with enantioresolution factor (R(s)) in the range 1.15-4.18. The same analytes were also resolved employing reversed and normal phase modes with mixtures of methanol/water and hexane/ethanol at different ratios as mobile phases, respectively. Loss in resolution for some compounds, broaden peaks and longer analysis times were observed with these last two chromatographic elution modes. Afterwards, a comparison with the other two CSPs was performed. A lower discrimination ability of Sepapak(®) 1 and Sepapak(®) 2 towards all the studied flavanoids was observed. However, Sepapak(®) 1 allowed the separation of naringenin enantiomers and naringin stereoisomers in polar organic phase which were not resolved with the other two CSPs. The nature of the chiral selector was found to be of utmost importance for the resolution of the selected compounds. Indeed, significant differences in enantioresolution among the three tested CSPs were observed. With regard to the only few data reported in the literature for the resolution of this class of compounds using polysaccharide-based CSPs by high performance liquid chromatography (HPLC), the results obtained in this study by means of nano-LC showed higher (R(s)) values and shorter analysis time.

Capillary electrochromatography as a new tool to assess drug affinity for membrane phospholipids.

This work proposes a new capillary electrochromatography (CEC) method for determination of drug partition in membrane phospholipids. CEC experiments were carried out in a 100 µm (ID) fused-silica capillary, partially packed with a chromatographic phospholipid stationary phase, so-called Immobilized Artificial Membrane, IAM.PC.DD2. The observed retention values were corrected by both the electro-osmotic and electrophoretic mobility values, measured by capillary electrophoresis (CE) experiments, assuming the values of the logarithms of "chromatographic" affinity factors, log k(CEC) as indexes of affinity for phospholipids. Analogously to biochromatography, all the values were determined with a totally aqueous mobile phase, or extrapolated to 100% aqueous buffer. The analytes were 16 structurally unrelated compounds, of basic, neutral, and acidic nature. To evaluate the effectiveness of CEC data to describe partition in phospholipids, log k(CEC) were related to both log P and log k(w)(IAM) values. log P are the lipophilicity values expressed as the logarithms of n-octanol/water partition coefficients and log k(w)(IAM) are the retention data measured by High Performance Liquid Chromatography (HPLC) on an IAM.PC.DD2 column, assumed as the reference values for phospholipid affinity. Phospholipid affinity scale by CEC related to that achieved by HPLC, but only if two different subclasses were considered separately, i.e. protonated and unprotonated analytes; indeed, all the compounds protonated at the experimental pH value (7.0) were retained stronger in CEC than in HPLC. This discrepancy may be due to the use of different buffers in CEC and HPLC since, to avoid the occurrence of a high current, the eluent in CEC experiments was of different composition and lower ionic strength than in HPLC. CEC analyses were faster and required lower amounts of both solvent and stationary phase than HPLC; moreover, with the exception of only three analytes, all analyses were performed with 100% aqueous eluents avoiding time-consuming and tedious extrapolation procedures.

Polyethylenimine-modified metal oxides for fabrication of packed capillary columns for capillary electrochromatography and capillary liquid chromatography.

The need for novel packing materials in both capillary electrochromatography (CEC) and capillary liquid chromatography (CLC) is apparent and the development towards more selective, application-oriented chromatographic phases is under progress world-wide. In this study we have synthesized new polyethyleneimine (PEI) functionalized Mn(2)O(3), SiO(2), SnO(2), and ZrO(2) particles for the fabrication of packed capillary columns for CEC and CLC. The nanocasting approach was successful for the preparation of functionalized metal oxide materials with a controlled porosity and morphology. PEI functionalization was done using ethyleneimine monomers to create particles which are positively charged in aqueous solution below pH 9. This functionalization allowed the possibility to have both hydrophobic (due to its alkyl chain) and ionic interactions (due to positively charged amino groups) with selected compounds. For comparison aminopropyl-functionalized silica was also synthesized and tested. Both slurry pressure and electrokinetic packing procedures used gave similar results, but fast sedimentation of the material caused some problems during the packing. The high stability and wide pH range of PEI-functionalized SiO(2) material, with potential for hydrophobic and electrostatic interactions, proved to be useful for the CEC and CLC separation of some model acidic and neutral compounds.

Control of EOF in CE by different ways of application of radial electric field.

Various ways of application of radial electric field for the control of electrokinetic potential and EOF in a home-made device for CE are presented. The device comprises three high-voltage power supplies, which are used to form a radial electric field across the fused-silica capillary wall. One power supply provides the internal electric field - a driving force for electrophoretic migration of charged analytes and for the EOF. Two power supplies are connected to the ends of the outer low-conductivity polymeric coating, which is formed by the dispersion of insoluble conductive copolymer of aniline and p-phenylendiamine in polystyrene matrix (dissolved in N-methylpyrrolidone) attached to the original outer polyimide coating of the capillary. They are able to constitute the external longitudinal electric field with variable values of electric potential at both ends of the outer coating. The potential gradient between the external and internal electric field is perpendicular to the capillary wall and forms a radial electric field across the capillary wall, which affects the electrokinetic potential at the solid-liquid interface and EOF inside the capillary. The developed device and methodology has been applied for the analysis of both chiral and achiral molecules such as terbutaline enantiomers and oligopeptides (diglycine and triglycine). The effect of magnitude, orientation, and different ways of application of the radial electric field on the flow rate of the EOF and on the speed, efficiency, and resolution of CZE separations of the above analytes in the internally noncoated fused-silica capillaries have been evaluated.