Dean Ellison

A strategic approach to the development of capillary electrophoresis chiral methods for pharmaceutical basic compounds using sulfated cyclodextrins

Enantioseparations of basic pharmaceutical compounds were investigated using different types of sulfated cyclodextrins as chiral selectors. A general strategy for method development was described, together with enantiomeric separations of a number of pharmaceutical related compounds. Based on this strategy, systematic method development approaches for several selected compounds were performed by modifying method parameters, such as the concentration of the chiral selectors, buffer pH, type of organic modifiers, buffer type, temperature and applied voltage. The results of the investigation elucidated the separation mechanism. Many practical aspects were also discussed through several specific examples in order to demonstrate how to develop and validate a precise, sensitive, accurate and rugged separation.

Mechanistic study of enantiomeric recognition with native γ-cyclodextrin by capillary electrophoresis, reversed-phase liquid chromatography, nuclear magnetic resonance spectroscopy, electrospray mass spectrometry and circular dichroism techniques

The possible mechanisms for the chiral recognition of 2(S)-(3,5-bis-trifluoromethyl-phenyl)-2-[3(S)-(4-fluorophenyl)-4-(1H-[1,2,4]triazol-3-ylmethyl)-morpholin-2(R)-yloxy]-ethanol (compound A) and its enantiomer with native gamma-cyclodextrin (gamma-CD) were investigated using capillary electrophoresis (CE), reversed-phase liquid chromatography (RPLC), proton (1H), fluorine (19F) and carbon (13C) nuclear magnetic resonance spectroscopy (NMR), electrospray mass spectrometry (ESI-MS) and circular dichroism (CD). All experiments provided clear evidence of the formation of diastereomeric complexes between the enantiomers and gamma-CD. Proton, fluorine and carbon NMR spectra suggested that both aromatic rings, with mono-fluoro and bis-tri-fluoro functional groups, on the guest molecule were partially included into the cavity of the gamma-CD. ESI-MS spectra indicated that the diastereomeric complexes have a 1:1 stoichiometric ratio. The binding constants of the diastereomeric complexes obtained by CE, RPLC and CD were compared. The effects of the gamma-CD concentration, organic modifiers and temperature on the CE-chiral separation were also investigated.

Anodic N-dearylation of 2-azetidinones

Abstract N-Methoxyphenyl-2-azetidinones are dearylated under mild conditions via anodic oxidation.

Comparison of capillary electrophoresis and reversed-phase liquid chromatography for determination of the enantiomeric purity of an M3 antagonist.

The chiral separation of an M3 antagonist was investigated using capillary electrophoresis (CE) with various sulfated cyclodextrins and by reversed-phase liquid chromatography with derivatized cellulose, derivatized amylose, and two protein stationary phases. Operational parameters for each technique, such as the concentration of the chiral selectors, background electrolyte (or mobile phase) pH and type, organic modifiers, injection mode and temperature were varied in order to achieve a desired elution order and to meet a 0.1% limit of quantitation (LOQ) criteria. Based on the advantages and disadvantages of each technique, a practical CE method using sulfated gamma-cyclodextrin was selected. The method was validated in terms of linearity, LOQ, accuracy, ruggedness and precision.

Use of a Quality-by-Design approach to justify removal of the HPLC weight % assay from routine API stability testing protocols

Due to the high method variability (typically > or = 0.5%, based on a literature survey and internal Merck experience) encountered in the HPLC weight percent (%) assays of various active pharmaceutical ingredients (APIs), it is proposed that the routine use of the test in stability studies should be discouraged on the basis that it is frequently not sufficiently precise to yield results that are stability-indicating. The high method variability of HPLC weight % methods is not consistent with the current ICH practice of reporting impurities/degradation products down to the 0.05% level, and it can lead to erroneous out-of-specification (OOS) results that are due to experimental error and are not attributable to API degradation. For the vast majority of cases, the HPLC impurity profile provides much better (earlier and more sensitive) detection of low-level degradation products. Based on these observations, a Quality-by-Design (QbD) approach is proposed to phase out the HPLC weight % assay from routine API stability testing protocols.

Examination of rofecoxib solution decomposition under alkaline and photolytic stress conditions

Rofecoxib is a highly active and selective cyclo-oxygenase II inhibitor. A stability-indicating method for the assay of rofecoxib has been developed using reverse-phase high-performance liquid chromatography (HPLC). Stress testing of rofecoxib was conducted during the method development and validation. HPLC analysis of rofecoxib solutions stressed under alkaline and photolytic conditions revealed the presence of several degradates. Two main degradates were determined to be the cyclization product formed by photo-cyclization and the dicarboxylate formed by ring opening in the presence of base and oxygen. The identities of these degradates were confirmed by comparison of UV spectra and HPLC retention time with the independently synthesized products. The mechanistic pathways for the formation of these degradates are discussed. Further improvement of the HPLC methods ruggedness has been made based on these studies.

ENANTIORECOGNITION MECHANISMS FOR DERIVATIZED CELLULOSE UNDER REVERSED PHASE CONDITIONS

The chiral recognition mechanism for a derivatized polysaccharide chiral stationary phase (CSP) under reversed phase conditions was investigated (Chiralcel OJ-R). While enantiorecognition mechanisms have been proposed for these types of phases under normal phase conditions, it is unlikely that they would apply to reversed phase conditions due to the high polarity of the eluent. Indandiol was utilized as the main probe. The effect of organic modifier, eluent anion, and temperature on enantioselectivity for indandiol was determined. Additionally, a number of structurally related probes were also investigated to define the role of various functional groups on enantioselectivity. The investigation revealed that hydrogen bonding did not have a gross direct effect on enantiorecognition but may contribute through its effect on the tertiary structure of the CSP. It appears that inclusion is the major contributor to enantioselectivity with the size and shape of the probe being the determinant as to the extent of enantioselectivity rather than electrostatic interactions involving the functional groups of the solute.

Enantioseparation of a protease inhibitor, indinavir, by subcritical fluid chromatography

Indinavir is a protease inhibitor which possesses five chiral centers. Enantioseparation of indinavir and its enantiomer was performed on an amylose type stationary phase, Chiralpak® AD, under normal-phase HPLC and subcritical fluid chromatography conditions. Under the utilized chromatographic conditions, it is believed that the leading interactions are hydrogen bonding between one or both hydroxyl groups of the solute with the carbonyl group of the carbamate. An inclusion mechanism appears to control the chiral recognition. The effect of various modifiers, pressure, and temperature were investigated.

On-line monitoring of the distillates of a solvent switch process by near-infrared spectroscopy

An on-line fiber-optic near-infrared spectroscopic method has been developed for real-time monitoring of distillate composition as a reliable way of assessing the degree of a solvent switch process from methanol and n-propanol to isopropyl acetate. Multivariate statistical methods of Multiple Linear Regression and Partial Least Squares were evaluated for quantitative determination of the total alcohol concentrations in the distillate samples. Robust models were obtained by incorporating temperature variance into the calibration set. Reliable prediction of total alcohol concentration was successfully achieved by near-infrared spectroscopy as judged by a comparison with gas chromatography data.

Liquid chromatographic determination of the known low level impurities in lovastatin bulk drug: An application of high—low chromatography

High-low chromatography in combination with gradient elution and multiple wavelength detection is a rapid and sensitive technique that can be used to assay low level impurities in lovastatin. The method is reproducible and can be applied to samples containing as little as 0.1% of any known impurity.