Using single-isomer octa(6-O-sulfo)-γ-cyclodextrin for fast capillary zone electrophoretic enantioseparation of pindolol: Determination of complexation constants, software-assisted optimization, and method validation.

The present study describes a rapid and effective capillary electrophoresis (CE) method for the enantioseparation of pindolol using single-isomer octa(6-O-sulfo)-γ-cyclodextrin. The complexation parameters were determined under neutral and high pH conditions to identify optimal separation conditions using a theoretical model. Baseline separation of pindolol enantiomers was achieved within 6 min in a sodium/MOPS buffer, pH 7.2, with a selector concentration of 6 mM. The method was validated according to the ICH guidelines using imidazole as an internal standard. Low limits of detection and quantification were found, specifically 1.2 µg/mL and 4 µg/mL (0.6 µg/mL and 2 µg/mL per enantiomer), respectively. The calibration curves showed good linearity, with a coefficient of determination R2 ≥ 0.999 over a 5 - 55 µg/mL concentration range and over a 50 - 300 µg/mL concentration range of the racemic mixture. The relative standard deviations (%RSD) of intra-day and inter-day precision were lower than 8% at LOQ level, lower than 3% at 50 µg/mL level and lower than 1.5% at 300 µg/mL level. Accuracy ranged from 95 to 103% (106% at LOQ level). The proposed method was successfully tested on a medical formulation of Visken® Sandoz intravenous solution and Visken® Teofarma pills for oral use.

Chiral separations by non-aqueous capillary electrophoresis in DMSO-based background electrolytes.

Capillary electrophoresis (CE) is a powerful technique for enantioseparations due to its high separation efficiency, high versatility, speed of analysis and low consumption of samples and reagents. Non-aqueous capillary electrophoresis (NACE) appears as a promising technique to perform enantioseparations when the drugs, chiral selectors or samples are non-water soluble. Chiral separations have been performed by NACE mainly using alcoholic solvents as BGEs, with problems of current breakdowns and changes in the BGE composition, due to their high volatility. In this work, the suitability of DMSO as BGE in NACE has been evaluated. Different experimental variables affecting the enantioresolution of three drugs have been evaluated, finally achieving complete enantioresolution of two drugs (verapamil, Rs=1.5 and pindolol, Rs=2.0) and partial resolution of the third one (fenfluramine, Rs=1.2). DMSO has been demonstrated to be a good alternative to methanolic BGEs in NACE.

Electrically assisted capillary liquid chromatography using a silica monolithic column.

A silica monolithic capillary column was linked to an open capillary of the same internal diameter via a Teflon sleeve to form a duplex column to investigate the combination of chromatography and electrophoresis in the mode of electrically assisted capillary liquid chromatography (eCLC). Using a commercial CE instrument with an 8.5 cm long, 100 microm i.d. reversed phase silica monolithic section and a window 1.5 cm beyond the end of this in a 21.5 cm open section, a minimum plate height of 9 microm was obtained in capillary liquid chromatography (CLC) mode at a low driving pressure of 50 psi. In eCLC mode, high speed and high resolution separations of acidic and basic compounds were achieved with selectivity tuning based on the flexible combination of pressure (0-100 psi) and voltage. Taking advantage of the excellent permeability of silica monolithic columns, use of a step flow gradient enabled elution of compounds with different charge state.

Use of vancomycin chiral stationary phase for the enantiomeric resolution of basic and acidic compounds by nano-liquid chromatography.

In this paper we studied the potentiality of nano-liquid chromatography (nano-LC) for the enantiomeric resolution of both basic and acidic compounds of pharmaceutical interest using a vancomycin modified silica stationary phase. Experiments were carried out in a fused silica capillary of 75 microm I.D. packed with chiral modified silica particles of 5 microm diameter, the detection, was done on-line at 195 nm. Enantiomeric resolution of alprenolol, atenolol, metoprolol, oxprenolol, pindolol, propranolol (basic compounds) and some acidic analytes, namely 2-[(5'-benzoyl-2'-hydroxy)phenyl]propionic acid (DF1738Y), 2-[(4'-benzoyloxy-2'-hydroxy)phenyl]propionic acid (DF1770Y), ketoprofen, indoprofen and suprofen was studied by nano-LC utilizing mobile phases containing methanol-acetonitrile-ammonium formate or acetate. The effect of mobile phase composition (buffer type and concentration, organic modifier type and concentration) on chiral resolution (Rs), retention factor (k) and retention time (tR) was also investigated. Good enantiomeric resolution was achieved for basic compounds utilizing the mobile phase containing 90% (MeCN-MeOH)/5% water/5% of 100 mM ammonium acetate pH 4.5. Acidic compounds such as DF1738Y and DF1770Y were better resolved at lower pH 3.5 while ketoprofen, indoprofen and suprofen exhibited the highest resolution at pH 4.5; in this case the mobile phase contained MeOH or MeCN (90%), 5% buffer and 5% of water. The nano-LC method was validated using R-(+)-propranolol as an internal standard finding good repeatability, detection limit, correlation coefficient and recovery and applied to the assay of a pharmaceutical formulation containing a racemic mixture of metoprolol.

Enantioselectivity of structurally modified poly(sodium undecenoyl-L-leucinate) by insertion of Triton X-102 surfactant molecules.

The effectiveness of Triton X-102 (TX-102), as a structural modifier of the polymeric surfactant sodium undecanoyl-L-leucinate (L-SUL) was investigated for enhanced enantiomeric recognition of various chiral compounds in micellar electrokinetic chromatography (MEKC). Increasing concentrations of TX-102 were separately added into the micellar solutions of L-SUL and then polymerized to form poly-L-SUL. The resulting polymers were purified by use of 3500 molecular-weight-cutoff (MWCO) dialysis membranes. Fluorescence and pulsed field gradient-nuclear magnetic resonance (PFG-NMR) techniques were used to elucidate the structural effects of TX-102 on poly-L-SUL. Evaluation of data from fluorescence measurements suggested an increase in polarity with increasing concentration of TX-102. However, the polarity decreased at higher concentrations of TX-102. Evaluation of data from PFG-NMR suggested an increase in hydrodynamic radius upon increasing the concentration of TX-102. The racemates of coumarinic and phenythiohydantoin amino acid derivatives, and pindolol were used as test analytes in MEKC. A notable increase in resolution and capacity factors of the test analytes was observed when the modified poly-L-SUL was used in MEKC measurements. Examination of the data obtained from fluorescence, PFG-NMR, and MEKC suggests a strong correlation between the polarity and the hydrodynamic radii of TX-102 modified micelles and the enantiomeric resolution of the test analytes.

Enantioseparations by capillary electro-chromatography: differences exhibited by normal- and reversed-phase versions of polysaccharide stationary phases.

The influence of using normal-phase and reversed-phase versions of four commercial polysaccharide stationary phases on chiral separations was investigated with capillary electrochromatography (CEC). Both versions of the stationary phases, Chiralcel OD, OJ, and Chiralpak AD, AS were tested for the separation of two basic, two acidic, a bifunctional, and a neutral compound. Different background electrolytes were used, two at low pH for the acid, bifunctional and neutral substances, and three at high pH for the basic, bifunctional and neutral ones. This setup allowed evaluating differences between both stationary-phase versions and between mobile-phase compositions on a chiral separation. Duplicate CEC columns of each stationary phase were in-house prepared and tested, giving information about the intercolumn reproducibility. In general, reversed-phase versions of the current commercial polysaccharide stationary phases are found to be best for reversed-phase CEC, even though at high pH no significant differences were seen between both versions. Most differences were observed at low pH. For acidic compounds, it was seen that an ammonium formate electrolyte performed best, which is also an excellent electrolyte if coupling with mass spectrometry is desired. For basic, bifunctional and neutral compounds, no significant differences between the three tested electrolytes were observed at high pH. Here, a phosphate buffer is preferred as electrolyte because of its buffering capacities. However, if coupling to mass spectrometry is wanted, the more volatile ammonium bicarbonate electrolyte can be used as an alternative.

Separation of basic drug enantiomers by capillary electrophoresis using chicken alpha1-acid glycoprotein: insight into chiral recognition mechanism.

Recombinant chicken alpha(1)-acid glycoprotein (alpha(1)-AGP) was prepared by the Escherichia coli expression system and completely deglycosylated alpha(1)-AGP (cd-alpha(1)-AGP) was obtained by treatments of native alpha(1)-AGP with a mixture of endoglycosidase and N-glycosidase. The average molecular masses of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP were estimated to be about 29 200, 21 700 and 20 700, respectively, by matrix-assisted laser desorption-time of flight-mass spectrometry. We compared the chiral recognition ability of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP using them as chiral selectors in capillary electrophoresis. The chicken alpha(1)-AGP showed higher resolution for eperisone, pindolol and tolperisone than cd-alpha(1)-AGP or recombinant alpha(1)-AGP. Recombinant alpha(1)-AGP still showed chiral recognition for three basic drugs tested. By addition of propranolol as a competitor in the separation solution in CE, no enantioseparations of three basic drugs were observed with chicken alpha(1)-AGP, cd-alpha(1)-AGP or recombinant alpha(1)-AGP. These results reveal that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition ability, and that the chiral recognition site(s) for basic drugs exists on the protein domain.

Separation of basic drug enantiomers by capillary electrophoresis using ovoglycoprotein as a chiral selector: comparison of chiral resolution ability of ovoglycoprotein and completely deglycosylated ovoglycoprotein.

Separations of basic drug enantiomers by capillary electrophoresis (CE) using ovoglycoprotein (OGCHI) as a chiral selector are described. The effects of running buffer pH and 2-propanol content on the migration times and resolution of basic drug enantiomers were examined using a linear polyacrylamide-coated capillary. High resolution of basic drug enantiomers was attained using a mixture of 50 mM sodium phosphate buffer (pH 4.5-6.0) and 2-propanol (5-30%) including 50 microM OGCHI. It was found that ionic and hydrophobic interactions could work for the recognition of basic drug enantiomers. Further, we compared the chiral resolution ability of OGCHI with that of completely deglycosylated OGCHI (cd-OGCHI) using them as chiral selectors in CE. OGCHI showed higher resolution for basic drug enantiomers tested than cd-OGCHI. The results suggest that the chiral recognition site(s) for OGCHI exists on the protein domain of OGCHI.

Automated sample preparation techniques for the determination of drug enantiomers in biological fluids using liquid chromatography with chiral stationary phases.

The determination of drug enantiomers has become of prime importance in the field of pharmaceutical and biomedical analysis. Liquid chromatography (LC) is one of the most frequently used techniques for achieving the separation and quantitation of the enantiomers of drug compounds. In the bioanalytical field, the integrated systems present an interesting alternative to time-consuming sample preparation techniques such as liquid-liquid extraction. Solid phase extraction (SPE) on disposable cartridges, dialysis or column switching are sample preparation techniques that can be fully automated and applied to enantioselective analysis in biological fluids. The selection of the most appropriate LC mode and chiral stationary phase for enantioseparations in bioanalysis is discussed and some aspects of these automated sample preparation procedures are compared, such as selectivity, detectability, elution of the analytes from the extraction sorbent, sample volume and analyte stability.

Comparison of two extraction methods for determination of propranolol and furosemide in human plasma by mixed-mode chromatography.

An isocratic high performance liquid chromatographic method is described for the determination of the beta-adrenergic blocking drug, propranolol, and the diuretic, furosemide, in human plasma. The two compounds and the internal standard were extracted from plasma using a two-step extraction technique. Propranolol and pindolol (internal standard) were first extracted from alkaline plasma into diethyl ether; this was followed by extraction of furosemide into acidified ether: hexane (65:35). The two extracts were then combined and evaporated under nitrogen, and the reconstituted residues were analysed on a C18/SCX reversed-phase/cation exchange column with a mobile phase of acetonitrile: 0.1 M sodium acetate pH 4 (33:67). The drugs and the internal standard were detected by UV absorption at 230 nm. The drugs were also extracted from plasma by a column-switching technique utilizing a ten-port valve. The drug compounds were retained on a C18 pre-column. A comparison of RSD for within-batch (intra-assay) and between-batch (inter-assay) runs for both methods was carried out, the liquid/liquid extraction method giving better recovery values. The calibration graphs were linear from 25-300 ng ml-1 for furosemide and 50-400 ng ml-1 for propranolol. Recovery values were > 90.0% by liquid/liquid extraction and > 76.0% by column switching.

Lambda-carrageenan: a novel chiral selector for capillary electrophoresis.

Lambda-carrageenan, a linear high molecular weight sulfated polysaccharide, has been employed as a chiral selector in capillary electrophoresis for the separation of enantiomers of weakly basic pharmaceutical compounds. The racemic compounds that were enantioresolved included propranolol, pindolol, tryptophanol, laudanosine and laudanosoline. In addition, the diastereomeric pair of cinchonine and cinchonidine were also resolved. Method conditions such as buffer pH, electrolyte concentration, column temperature, and chiral selector concentration were found to be important for improvement of enantioselectivity.

A rapid high yield synthesis of no-carrier-added (-)-[123I]iodocyanopindolol.

A convenient and efficient radiosynthesis of no-carrier-added [123I]labeled (-)iodocyanopindolol, (-)-[123I]ICYP, a high affinity beta adrenergic antagonist, is described. (-)-[123I]ICYP was synthesized by a modified chloramine-T radioiodination of (-)cyanopindolol followed by a novel reversed-phase HPLC purification that provided the radiopharmaceutical as a directly injectable solution. The total synthesis time was typically less than 45 min and provided (-)-[123I]ICYP in a 59% radiochemical yield (not corrected for decay). In view of its high affinity for the beta adrenergic receptor, (-)-[123I]ICYP is a potentially useful probe for SPECT evaluation of cardiac adrenergic receptor density.

Separation of the enantiomers of beta-receptor blocking agents and other cationic drugs using a CHIRAL-AGP column. Binding properties and characterization of immobilized alpha 1-acid glycoprotein.

The effect of the immobilization procedure on the conformation of alpha 1-acid glycoprotein (AGP) was investigated by recording the fluorescence spectra of native and immobilized AGP. A 20-nm red shift was obtained for the immobilized form of AGP compared with the emission maximum of 338 nm obtained for native AGP. This demonstrates that the tryptophan residues are exposed on the protein surface after immobilization, indicating that the immobilized form of AGP has a more unfolded structure than the native AGP. The effect of N,N-dimethyloctylamine on the enantioselectivity for some fentiazine derivatives, observed with immobilized AGP, was equal to that obtained with AGP as a chiral complexing agent in the mobile phase. This demonstrates that even though the immobilization procedure affects the conformation of the protein there still exist large similarities between native and immobilized AGP concerning chiral recognition. The adsorption isotherm of (-)-terodiline was studied by use of the breakthrough technique. The adsorption isotherm indicates that (-)-terodiline is adsorbed to one site with high affinity and at least one more site with lower affinity. It was also observed that the enantiomers of amines, acids and non-protolytic compounds compete with the cationic compound, (-)-terodiline, for binding to the same sites. The beta-receptor blocking agents atenolol, metoprolol, pindolol, alprenolol, oxprenolol and propranolol were resolved on a CHIRAL-AGP column. The retention and enantioselectivity are highly influenced by the structure of the solute and the nature of the uncharged mobile phase additives. Separation factors of 1.2-1.8 were obtained for the beta-blockers under the studied conditions.