Systematic development and validation of stability indicating micellar electrokinetic chromatography methods for early stage drug candidates.

Suitable methods for assessment of purity, potency and stability of new drug substances and drug products are required to be rapidly developed and validated to provide appropriate data for early project development decisions. In order to routinely provide methods of consistent and suitable quality to meet increasingly aggressive timelines, systematic approaches to both develop and validate analytical technologies have been developed. Systematic approaches to evaluate separation parameters such as buffer pH, buffer ionic strength, surfactant concentration, organic modifiers, organic modifier concentration, applied voltage and temperature were evaluated for an early stage drug candidate. Techniques to improve method precision and ruggedness were also examined. Finally, the validation results from the micellar electrokinetic chromatography method utilizing an internal standard were compared against the simultaneously developed high-performance liquid chromatography method.

Aspects of column fabrication for packed capillary electrochromatography.

Various parameters have been evaluated to develop a process for optimization of column manufacture for packed capillary electrochromatography (CEC). Spherisorb ODS-1 was packed into 75 microm I.D. capillaries to establish a standard set of packing conditions to afford high-performance columns free of voids. Numerous silica-based packing materials including porous and non-porous reversed-phase and ion-exchange phases were employed to evaluate the applicability of the standard conditions. Success of column manufacture and performance demonstrate a relationship to the colligative properties of the packing materials under the applied conditions. Frequently encountered difficulties arising from inadequate column conditioning and void formation in the packed bed are identified and discussed.

Evaluation of 1.5 microM reversed phase nonporous silica in packed capillary electrochromatography and application in pharmaceutical analysis.

Reversed-phase nonporous silica (RP-NPS) of 1.5 microm dp is employed to demonstrate rapid and efficient separations in packed capillary electrochromatography (CEC). Two methods for packing capillaries and two techniques to manufacture frits used to hold the packing in place are evaluated for their effect upon separation performance using polyaromatic hydrocarbons (PAHs) and polar neutral pharmaceutical compounds. Attention is given to conditioning of the packed capillaries for high efficiency separations without necessity for sodium dodecyl sulfate (SDS). Separation conditions for the nonporous materials were modified from those previously determined on porous reversed-phase silica. Feasibility for method development and validation of a parent pharmaceutical compound and related impurities in the range of 0.1-120% of a 5 mg/mL concentration was assessed and reported. An approach to improving detection sensitivity through use of large-bore capillaries is briefly discussed.

Method development in pharmaceutical analysis employing capillary electrochromatography.

Capillary electrochromatography (CEC) has been employed to explore method development for a series of structurally related polar neutral compounds of pharmaceutical relevance. Capillaries with dimensions of 75 microm ID x 25 cm length (34.5 cm total) were packed with Spherisorb ODS-1, Hypersil phenyl, and Hypersil MOS (all 3 microm particles) and were compared in the reversed-phase mode in order to determine which phase provided the best initial performance and thus serve as the phase of choice for additional method development experiments. The various separation parameters examined for their effect on efficiency, k', resolution, and linear velocity included percent and type of organic modifier, buffer concentration, voltage, and temperature. All separations were conducted with an acidic mobile phase (aqueous mobile phase component, pH 3.0). The separation efficiencies obtained were on the order of 200,000-260,000 plates/m, which equates to reduced plate heights of 1.22 for columns packed with Spherisorb ODS-1. Repeatable column-to-column separation performance was demonstrated.

Resolution of acylated dipeptide stereoisomers by capillary electrophoresis using sulfobutylether derivatized beta-cyclodextrin.

The separation of enantiomerically and diastereomerically related stereoisomers of acylated Asp-Phe dipeptides was explored using capillary electrophoresis (CE). This series of dipeptides included the alpha-L,L parent compound and the three other potential Asp containing stereoisomers (alpha-D,D, alpha-L,D, and alpha-D,L), as well the four possible isoAsp containing stereoisomers (beta-L,L, beta-D,D, beta-L,D and beta-D,L). The separation of these substances was explored using both neutral and charged cyclodextrins as the stereoisomer selector added to the running electrolyte. The major experimental parameters investigated included pH, the cyclodextrin type, and the cyclodextrin concentration. Due to differences in the pKa values of the carboxylic acid groups, adjustment of the separation buffer to between pH 3.0 and 4.0 provided for sufficient electrophoretic mobility differences to result in excellent separations of the diastereomerically related peptides in this pH region. The resolution of the enantiomerically related peptide stereoisomers was accomplished using low concentrations (1 mM) of the anionic cyclodextrin derivative, sulfobutylether-beta-cyclodextrin (SBE-beta-CD). This negatively charged cyclodextrin was found to be superior for the resolution of the enantiomerically related peptides as compared to native beta-cyclodextrin or the neutral derivatives, dimethyl beta-cyclodextrin and hydroxypropyl beta-cyclodextrin. An alternative approach using anionic or neutral surfactants in conjunction with the SBE-beta-CDs was also explored and found to be successful but problematic.