Carbamoylated azithromycin incorporated zirconia hybrid monolith for enantioseparation of acidic chiral drugs using non-aqueous capillary electrochromatography.

Carbamoylated derivatives of two antibiotics, namely, clindamycin phosphate (CLIP) and erythromycin (ERY) were successfully employed as co-precursors, in combination of zirconium tetrabutoxide as a precursor, to prepare chiral organic-zirconia hybrid monoliths (i.e., CLIP-ZHMs and ERY-ZHMs, respectively) via a single-step in-situ sol-gel approach in our previous works. Their superiority over chiral organic-zirconia/silica monoliths, prepared by post-modification approach, in terms of better enantioresolution and enhanced stability inspired us to prepare ZHMs based on an another antibiotic, azithromycin (i.e., AZI-ZHMs). Monolithic columns were employed for capillary electrochromatographic enantioseparation of acidic chiral drugs in mobile phases consisting of acetonitrile (ACN) and methanol (MeOH) as organic modifiers, and acetic acid (AcOH) and triethylamine (TEA) as electrolytes. The effects of composition of mobile phase and applied voltage on chiral separation were investigated by using ketoprofen as a representative analyte. Baseline resolutions were obtained for six acidic drugs in mobile phase consisting of 80/20 (v/v) ACN/MeOH with 300mM AcOH and 10mM TEA at a 10kV applied voltage and 25°C capillary temperature. The relative standard deviations for resolution values regarding column to column and batch to batch repeatability were less than 2.5% (for n=3) under optimized conditions, indicating satisfactory stability of the columns and reproducibility of the column preparation process.

Application of rifampicin as a chiral selector for enantioresolution of basic drugs using capillary electrophoresis.

Rifampicin, a member of rifamycin sub-class of antibiotics which belongs to the naphthalenic ansamycin class of antibiotics, has a characteristic ansa structure, i.e., a ring structure or chromophore spanned by an aliphatic chain. The present work was designed to evaluate its potential as a chiral selector (CS) as its structure consisting of nine stereogenic centers, an aromatic moiety and several functional groups (i.e., one imine, one amide, one acetoxy residue, two aliphatic hydroxyl and three phenolic hydroxyl groups) was expected to instigate multiple enantioselective interactions, namely, hydrogen bonding and inclusion complexation with chiral analytes, and therefore resulting in efficient enantioseparations. Systematic experiments were performed to investigate the effects of concentration of CS, composition of background electrolyte (BGE) and applied voltage on chiral separation. Enantiomers of propranolol and metoprolol were baseline resolved using a BGE consisting of 20mM CS and 50/50 (v/v) iso-propanol/phosphate buffer (100mM, pH 7.0) whereas for enantiomers of sertraline, a BGE consisting of 23mM CS and 40/60 (v/v) iso-propanol/phosphate buffer (100mM, pH 7.0) resulted in baseline resolutions.

Enantioseparation of basic chiral drugs on a carbamoylated erythromycin-zirconia hybrid monolith using capillary electrochromatography.

An organic-inorganic hybrid monolithic column was prepared within the confines of a capillary via a single-step in situ sol-gel approach using zirconium tetrabutoxide as a precursor to compose the inorganic backbone and 3-triethoxysilylpropyl carbamoylated derivative of erythromycin (TEOSPC-ERY) as a co-precursor to introduce the organic chiral selector moiety in the zirconia backbone. The resulting carbamoylated ERY-zirconia hybrid monolith (ERY-ZHM) showed homogeneous morphology with well-defined through pores and was tightly connected with the inner wall of the capillary. The column was employed for capillary electrochromatographic enantioseparation of six basic chiral drugs in mobile phases (MPs) consisting of acetonitrile (ACN) and triethylammonium acetate (TEAA) buffer. The effects of composition of MP and applied voltage on chiral separation were investigated by using propranolol as a representative analyte. The highest resolution (Rs=3.33) was obtained with a MP consisting of 10/90 (v/v) ACN/TEAA buffer (10mM, pH 7), 10 kV applied voltage and 25°C capillary temperature. The relative standard deviations for resolution values regarding run to run, day to day, column to column and batch to batch repeatability were 0.41%, 0.89%, 1.80% and 2.26% (for n=3), respectively, indicating satisfactory stability of columns and reproducibility of column preparation process.

Enantioseparation of basic chiral compounds on a clindamycin phosphate-silica/zirconia hybrid monolith by capillary electrochromatography.

An organic-inorganic silica/zirconia hybrid monolithic capillary column was prepared by sol-gel process in a fused-silica capillary by using triethoxysilylpropylcarbamate (TEOSPC) derivative of clindamycin phosphate (CLIP) as a chiral selector. A sol solution consisting of 6 × 10(-3)M of polyethylene glycol, 1 M of water, 2M of acetic acid and 0.04/0.96 ratio of CLIP-TEOSPC/Zr-Bu resulted in homogeneous monolith having well defined through-pores and tightly anchored to the capillary wall. The column was employed for capillary electrochromatographic enantioseparation of eight basic chiral drugs in mobile phases consisting of acetonitrile, methanol and ammonium acetate (AA, as the electrolyte). Effects of the compositions of solvents and electrolyte in the mobile phase, applied voltage and capillary temperature on chiral separation were investigated. The highest resolution values were obtained with mobile phases consisting of 40/60 MeOH/ACN and 100 mM AA (for citalopram, Tröger's base, indapamide, metoprolol, cetirizine and atropine) and 35/65 MeOH/ACN and 100 mM AA (for sertraline and propranolol) using -10 kV applied voltage at 25 °C.

Normal and polar-organic-phase high-performance liquid chromatographic enantioresolution of omeprazole, rabeprazole, lansoprazole and pantoprazole using monochloro-methylated cellulose-based chiral stationary phase and determination of dexrabeprazole.

Enantioresolution of four anti-ulcer drugs (chiral sulfoxides), namely, omeprazole, rabeprazole, lansoprazole and pantoprazole, was carried out by high-performance liquid chromatography using a polysaccharide-based chiral stationary phase consisting of monochloromethylated cellulose (Lux cellulose-2) under normal and polar-organic-phase conditions with ultraviolet detection at 285 nm. The method was validated for linearity, accuracy, precision, robustness and limit of detection. The optimized enantioresolution method was compared for both the elution modes. The optimized method was further utilized to check the enantiomeric purity of dexrabeprazole.

Application of antibiotics as chiral selectors for capillary electrophoretic enantioseparation of pharmaceuticals: a review.

Recent years have witnessed several new trends in chiral separation, for example, the enantiorecognition ability of several new antibiotics has been explored using capillary electrophoresis (CE) prior to HPLC; antibiotics have been employed as chiral selectors (CSs) in a nonaqueous CE (NACE) mode; and several new detection techniques (namely, capacitively coupled contactless conductivity detection) have been used in combination with CE for quantification of enantiomers. On account of these emerging trends, this article aims to review the application of various classes of antibiotics for CE enantioseparation of pharmaceuticals. A detailed account of the basic factors affecting enantioseparation, certain limitations of antibiotics as CSs and strategies to mitigate them, and advantages of NACE while using antibiotics as CSs has also been presented.

Penicillin G as a novel chiral selector in capillary electrophoresis.

The penicillin sub-class of ß-lactam antibiotics has not been examined for its enantiodiscriminating abilities in capillary electrophoresis (CE) until date. The present work was therefore designed to evaluate penicillin G potassium salt (PenG) as an ion-pair chiral selector (CS) using CE for its several attributes, namely, high solubility in water and lower alcohols, structure allowing multiple interactions with analytes and cost-effectiveness. Systematic experiments were performed to investigate the effect of composition of background electrolyte, applied voltage and capillary temperature on chiral separation. Baseline resolutions of enantiomers of five basic chiral drugs (namely, darifenacin, citalopram, sertraline, propranolol and metoprolol) were attained using a background electrolyte composed of water:methanol (90:10, v/v) and consisting of 10.7 or 16.1mM CS at 20°C using an applied voltage of 5kV.

Amino acids as chiral selectors in enantioresolution by liquid chromatography.

Amino acids are unique in terms of their structural features and multidimensional uses. With their simple structures and the ready availability of both enantiomers, amino acids not only serve as a chiral pool for synthesis but also provide an inexpensive pool for resolution studies. There has been no attempt to review the application of amino acids as chiral selectors for chromatographic enantioresolution of pharmaceuticals and other compounds. The present paper deals with application of l-amino acids and complexes of l-amino acids with a metal ion, particularly Cu(II), as an impregnating reagent in thin-layer chromatography or as a chiral ligand exchange reagent or a chiral mobile phase additive in both thin-layer chromatography and high-performance liquid chromatography. Enantiomeric resolution of ß-blockers, nonsteroidal anti-inflammatories, amino acids (and their derivatives) and certain other compounds is discussed.

HPLC enantioresolution of (R,S)-baclofen using three newly synthesized dichloro-s-triazine reagents having amines and five others having amino acids as chiral auxiliaries.

Enantioresolution of (R,S)-baclofen was accomplished using a newly synthesized set of three chiral derivatizing reagents (CDRs) having amines [(S)-(-)-α,4-dimethylbenzylamine, (-)-cis-myrtanylamine and (R)-(-)-1-cyclohexylethylamine] as chiral auxiliaries in cyanuric chloride and another set of five CDRs having amino acids (L-Leu, D-Phg, L-Val, L-Met and L-Ala) as chiral auxiliaries. These eight CDRs were used for synthesis of diastereomers of (R,S)-baclofen under microwave irradiation. The diastereomers were separated on a reversed-phase C(18) column using mixtures of methanol with aqueous trifluoroacetic acid with UV detection at 230 nm. Chromatographic data obtained for the two sets of diastereomers were compared among themselves and among the two groups. The method was validated for limit of detection, linearity, accuracy and precision.

Application of cyanuric chloride-based six new chiral derivatizing reagents having amino acids and amino acid amides as chiral auxiliaries for enantioresolution of proteinogenic amino acids by reversed-phase high-performance liquid chromatography.

Six dichloro-s-triazine (DCT) reagents having L-Leu, D-Phg, L-Val, L-Met, L-Ala and L-Met-NH(2) as chiral auxiliaries in cyanuric chloride were introduced for enantioseparation of 13 proteinogenic amino acids. Four other DCTs and six monochloro-s-triazine (MCT) reagents having amino acid amides as chiral auxiliaries were also synthesized. These 16 chiral derivatizing reagents (CDRs) were used for synthesis of diastereomers of all the 13 analytes using microwave irradiation, which were resolved by reversed-phase high-performance liquid chromatography (RP-HPLC) using C18 column and gradient eluting mixture of aqueous TFA and acetonitrile with UV detection at 230 nm. It required only 60-90 s for derivatization using microwave irradiation. Better resolution and lower retention times were observed for the diastereomers prepared with CDRs having amino acids as chiral auxiliaries as compared to counterparts prepared with reagents having amino acid amides as chiral auxiliaries. As the best resolution of all the 13 analytes was observed for their diastereomers prepared using the DCT reagent having L-Leu as chiral auxiliary, this CDR was further employed for derivatization of Lys, Tyr, His and Arg followed by RP-HPLC analysis of resulting diastereomers. The results are discussed in light of acid and amide groups of chiral auxiliaries constituting CDRs, electronegativities of the atoms of achiral moieties constituting CDRs and hydrophobicities of side chains of amino acids constituting CDRs and analytes.

Enantioresolution of five ß-blockers by reversed-phase high-performance liquid chromatography using fifteen chiral derivatizing reagents having amino acids or their amides as chiral auxiliaries on a cyanuric chloride platform.

Enantioseparation of five ß-blockers, namely, (R,S)-atenolol, (R,S)-propranolol, (R,S)-bisoprolol, (R,S)-metoprolol and (R,S)-carvedilol, was achieved as their diastereomers prepared with chiral derivatizing reagents (CDRs) synthesized on a cyanuric chloride platform. Fifteen CDRs were synthesized by nucleophilic substitution of the Cl atom in cyanuric chloride or its 6-methoxy derivative with amino acids (namely, L-Leu, L-Val, D-Phg, L-Met and L-Ala) or their amides as chiral auxiliaries. The diastereomers were synthesized under microwave irradiation for 70 or 100 s at 85% power. Separation of diastereomers was carried out on a C(18) column and gradient eluting mixtures of methanol with aqueous trifluoroacetic acid with UV detection at 230 nm. Separation efficiencies of the reagents were compared on the basis of effect of chiral auxiliaries (i.e. amino acids or amino acid amides) and achiral substituents (i.e. chlorine or methoxy group) in the CDRs. The method was validated for detection limit, linearity, accuracy and precision.

Indirect enantioresolution of (R,S)-mexiletine by reversed-phase high-performance liquid chromatography via diastereomerization with [(S,S)-O,O'-di-p-toluoyl tartaric acid anhydride], (S)-naproxen and nine chiral reagents synthesized as variants of Marfey's reagent.

Eleven chiral derivatizing reagents (CDRs) were used for preparation of diastereomers of (R,S)-mexiletine containing a primary amino group in close proximity to the stereogenic center. One anhydride, namely [(S,S)-O,O'-di-p-toluoyl tartaric acid anhydride] was synthesized and (S)-naproxen was used as such as the chiral derivatizing reagent. The other nine CDRs were synthesized by substituting one of the fluorine atoms in 1,5-difluoro-2,4-dinitrobenzene with six amino acid amides and three amino acids. The diastereomers were separated by reversed-phase high-performance liquid chromatography. The method was validated for linearity, accuracy, limit of detection and limit of quantification. The limit of detection was found in the range of 10-30 pmol.

Reversed-phase high-performance liquid chromatographic separation of diastereomers of (R,S)-mexiletine prepared by microwave irradiation with four new chiral derivatizing reagents based on trichloro-s-triazine having amino acids as chiral auxiliaries and 10 others having amino acid amides.

A new series of chiral derivatizing reagents (CDRs) consisting of four dichloro-s-triazine reagents was synthesized by nucleophilic substitution of one chlorine atom in trichloro-s-triazine with amino acids, namely L-Leu, D-Phg, L-Val and L-Ala as chiral auxiliaries. Two other sets of CDRs consisting of four dichloro-s-triazine (DCT) and six monochloro-s-triazine (MCT) reagents were also prepared by nucleophilic substitution of chlorine atom(s) with different amino acid amides as chiral auxiliaries in trichloro-s-triazine and its 6-methoxy derivative, respectively. These 14 CDRs were used for the synthesis of diastereomers of (R,S)-mexiletine under microwave irradiation (i.e. 60s and 90 s at 85% power (of 800 W) using DCT and MCT reagents, respectively), which were resolved by reversed-phase high-performance liquid chromatography using C18 column and gradient eluting mixtures of methanol with aqueous trifluoroacetic acid (TFA) with UV detection at 230 nm. The resolution (R(s)), difference between retention times of resolved diastereomers (Δt) and retention factors (k) obtained for the three sets of diastereomers were compared among themselves and among the three groups. Explanations have been offered for longer retention times and better resolution of diastereomers prepared with DCT reagents in comparison of their MCT counterparts and, for the influence of hydrophobicity of the side chain R of the amino acid in the CDRs on retention times and resolution. The newly synthesized CDRs were observed to be superior as compared to their amide counterparts in terms of providing better resolution and cost effectiveness. The method was validated for limit of detection, linearity, accuracy and precision.

Microwave-assisted synthesis and reversed-phase high-performance liquid chromatographic separation of diastereomers of (R,S)-baclofen using ten chiral derivatizing reagents designed from trichloro-s-triazine.

Four dichloro-s-triazine (DCT) and five monochloro-s-triazine (MCT) chiral derivatizing reagents (CDRs) were synthesized by incorporating amino acid amide moieties as chiral auxiliaries in trichloro-s-triazine and its 6-methoxy derivative, respectively. Another MCT reagent was synthesized by substitution of two chlorine atoms with two different amino acid amides in trichloro-s-triazine. These reagents were used for synthesis of diastereomers of (R,S)-baclofen under microwave irradiation (i.e. 60 s at 85% power using DCT reagents and 90 s at 85% power using MCT reagents). The diastereomers were separated on a reversed-phase C18 column using mixtures of methanol with aqueous trifluoroacetic acid (TFA) with UV detection at 230 nm. The separation behavior in terms of retention times and resolutions obtained for the two sets of diastereomers prepared with DCT and MCT reagents were compared among themselves and among the two groups. Longer retention times and better resolutions were observed with DCT reagents as compared to MCT reagents. The calibration curves were linear for both (R)- and (S)-baclofen in the concentration range 50-500 µg/ml. The average regression was 0.999 for both (R)- and (S)-baclofen. The RSD for (R)-baclofen was 0.40-0.86% for intra-day precision and 0.60-1.40% for inter-day precision and these values for (S)-baclofen were 0.52-0.75% and 0.64-1.32%, respectively. The recovery was 97.2-98.9% for (R)- and 97.0-98.9% for (S)-baclofen. The limit of detection was 1.63 ng/ml and 1.52 ng/ml for (R)- and (S)-baclofen, respectively.