Alison Bretnall

Investigation and optimisation of the use of organic modifiers in micellar electrokinetic chromatography

Abstract The effect of organic modifiers (methanol, ethanol, propan-2-ol, butan-1-ol, butan-2-ol, acetone, methyl ethyl ketone and acetonitrile) on the micellar electrokinetic chromatographic (MEKC) resolution and migration time of seven model compounds has been investigated. The compounds used were all drugs reported to have cardiovascular antiarrhythmic activity, The organic modifiers have each been investiated at 5, 10 and 15% (v/v) of the electrophoretic buffer (100 mM borate buffer pH 8.1 containing 50 mM SDS as surfactant), to determine the optimum resolution and peak shape. The elution order under almost every condition corresponded to increasing molecular mass of the analyte drugs. Propan-2-ol at a concentration of 10% (v/v) gave optimum separation of the analytes. Replicate injections under these conditions gave excellent precision data for the migration time and corrected peak area. Other modifiers which gave baseline resolution of the analytes but less precise repeatability data were acetone and methyl ethyl ketone.

Investigation and optimisation of the use of micellar electrokinetic chromatography for the analysis of six cardiovascular drugs

A micellar electrokinetic chromatography method was optimised for the separation of the six cardiovascular drugs atenolol, nicardipine, nifedipine, diltiazem, verapamil, and amlodipine by investigating the effects of pH, sodium dodecyl sulphate (SDS) concentration, selection and concentration of organic modifier. An electrophoresis buffer of 100 mM borate pH 8.1 containing 50 mM SDS and 15% (v/v) acetone was found to provide the optimum separation with respect to resolution and migration time.

Selectivity of capillary electrophoresis for the analysis of cardiovascular drugs

Examples of several classes of cardiovascular drugs have been separated using capillary electrophoresis and its associated application, micellar electrokinetic chromatography. The therapeutic classes of drug investigated include β-blockers, antiarrhythmic, calcium channel antagonists and angiotensin converting enzyme inhibitors. The exceptional selectivity of capillary electrophoresis is demonstrated using conditions that separate some twenty cardiovascular drugs in a single mixture, including all the aforementioned classes of drugs. The compounds have unrelated structures and varying molecular masses and yet are still resolved using a single set of conditions. The application of such selectivity is discussed together with a comparison with high-performance liquid chromatography.

Micellar electrokinetic chromatography stability indicating assay and content uniformity determination for a cholesterol-lowering drug product

This study describes a specific, linear, precise, accurate and sensitive method for the determination of a developmental cholesterol-lowering drug formulated in capsules. The method can also determine two known hydrolytic degradants of the drug. Samples are dissolved in acetonitrile-phosphate buffer pH 4.5, diluted with water and assayed by micellar electrokinetic chromatography (MEKC) in a buffer containing 0.1 M borate-0.025 M SDS at 30 degrees C with an applied voltage of 25 kV. Detection is by UV absorbance at 200 nm. The method was cross validated by comparison with a gradient elution HPLC method. The MEKC method gave at least equivalent precision, accuracy and sensitivity to HPLC but was superior in the resolution of the known impurities and gave a considerably shorter analysis time. The method has been accepted as part of a regulatory submission to the US Food and Drug Administration (FDA).

11 – Validation of Analytical Test Methods

The generation of high-quality analytical data is imperative in the effective knowledge generation and development of new medicines. Analytical methods are the tools that generate data are used to make fundamental development decisions, and validation demonstrates that methods are fit for this purpose. This chapter describes the parameters that should be investigated during the development of a pharmaceutical drug, from early development to commercial product. As a product continues through development, increased knowledge of the chemical and physical properties of the drug and the drug product is gained. Consequently, the validation requirements of analytical procedures become more complex and comprehensive, and a suitable strategy is described. The method validation parameters described in regulatory and compendial guidance that generally apply to commercial products are discussed, together with current concepts such as quality by design of analytical methods, in silico tools, validation of automated procedures, and example acceptance criteria for chromatographic procedures.

Determination of 2-Hydroxypyridine-1-Oxide (HOPO) at sub-ppm levels using derivitization and gas chromatography with mass spectrometry detection (GCMS)

This work describes the development and validation of an analytical method to determine residual trace levels of 2-Hydroxypyridine-1-Oxide (HOPO) in an active pharmaceutical ingredient (API). A method was required to be specific and sensitive enough to determine sub-ppm levels of this reagent. The approach taken to use a derivitization step overcame two of the primary challenges associated with the analysis of HOPO. Firstly, HOPO can tautomerize and the derivitization step provides a single stable entity to monitor, and secondly, the reaction enhances the volatility of the analyte to facilitate the use of gas chromatography. Mass spectrometry detection provides both suitable specificity and sensitivity. This paper describes the method development and optimisation of the derivitization step, the chromatographic conditions and mass spectrometry detection, together with a summary of the validation of the method. The method has been demonstrated to be robust and suitable to determine HOPO levels in commercially manufactured API materials.