Brian J. Clark

Comparison of various cosolvency models for calculating solute solubility in water-cosolvent mixtures.

Previously published cosolvency models are critically evaluated in terms of their ability to mathematically correlate solute solubility in binary solvent mixtures as a function of solvent composition. Computational results show that the accuracy of the models is improved by increasing the number of curve-fit parameters. However, the curve-fit parameters of several models are limited. The combined nearly ideal binary solvent/Redlich-Kister, CNIBS/R-K, was found to be the best solution model in terms of its ability to describe the experimental solubility in mixed solvents. Also resented is an extension of the mixture response surface model. The extension was found to improve the correlational ability of the original model.

Current applications in the analysis of pharmaceuticals by capillary electrophoresis. I

Abstract Part I covered the use of capillary electrophoresis (CE) for main component determinations, stoichiometric assays, analysis of vitamins and regulatory aspects of pharmaceutical analysis. This second article examines the use of CE in the determination of drug-related impurities, chiral separations and the expanding use of capillary electrochromatography (CEC) within the pharmaceutical industry. Impurity determinations are probably the principal role of CE within pharmaceutical analysis and represent a challenge to both the selectivity and sensitivity capabilities of the technique. The main component and structurally-related impurities often have very similar chemical properties which place great requirements on the selectivity necessary. Detection limits of 0.1% area/area are widely accepted as a minimum requirement for a related impurity determination method and this is possible by CE. The use of a number of chiral selective electrolyte additives including proteins, carbohydrates, crown ethers and antibiotics are reviewed. Validation reports of chiral CE methods are discussed in comparison to high performance liquid chromatography methods. This review also examines the increasing use of CEC in the analysis of chiral compounds.

Selective and quantitative analysis of 4-hydroxybenzoate preservatives by microemulsion electrokinetic chromatography

A microemulsion electrokinetic chromatography (MEEKC) method has been developed and validated for the determination of 4-hydroxybenzoates and their impurities. These materials are commonly known as parabens and are widely used as preservatives in foods, cosmetics and pharmaceuticals. The method was shown to be selective and quantitative for the methyl, ethyl, propyl and butyl esters of 4-hydroxybenzoic acid. An internal standard, 4-hydroxyacetophenone, was employed to improve injection precision and detector linearity. In addition, 4-hydroxybenzoic acid, the major degradent, could also be monitored at the 0.1% (m/m) level. The method was successfully validated for assay and detection of the impurities in 4-hydroxybenzoic acid methyl ester and 4-hydroxybenzoic acid propyl ester samples and for the determination of 4-hydroxybenzoic acid methyl ester in a liquid pharmaceutical formulation. The determination of paraben content by MEEKC in a liquid sample was consistent with HPLC analysis. This work is the first reported validated MEEKC method and shows that the methodology can be successfully implemented into routine quality control testing.

The effect of operating variables in microemulsion electrokinetic capillary chromatography

SummaryMicroemulsion electrokinetic capillary chromatography (MEEKC) is similar to micellar electrokinetic chromatography (MEKC) in that it separates neutral solutes based on their chromatographic retention factors. In MEEKC solutes partition between the aqueous phase and oil droplets, which are moving through the solution. The background to MEEKC is described including novel approaches to method development and optimisation. In this case water-immiscible octane forms minute oil droplets that are coated with SDS and butan-1-ol. The effects were evaluated using a test-mixture containing nine components of insoluble and soluble acids, bases and neutrals. Selectivity has been adjusted by use of a large number of factors including organic solvent, co-surfactant, urea, temperature, cyclodextrins, ion-pair reagent. Previous reports on the selectivity in MEEKC have concentrated only on neutral solutes. Separation selectivity was drastically changed with addition of alcohol such as butan-1-ol, propan-2-ol, cyclodextrin or using a low pH buffer. Microemulsion preparation process or filtration of the buffer did not affect the separation. The separation was largely unaffected by the use of methanol or acetonitrile, surfactant concentration, buffer type, oil type, sample diluent or type of the counter-ion. Migration times were dramatically altered with the use of ion-pair reagent and buffer concentration. It was also demonstrated that temperature variations alter the migration time but not the selectivity.

The use of a short-end injection procedure to achieve improved performance in capillary electrophoresis

SummaryMinimum capillary lengths on commercial instruments are fixed and cannot be decreased further. To effectively reduce the capillary length used for separation the sample can be injected from the end of the capillary nearest the detector. This procedure is known as a ‘short-end’ injection and can reduces analysis times by at least two-thirds compared to conventional injections. The time reduction benefits are shown in rapid separations of basic drugs, drug-related impurities and chiral compounds. Short-end injections, in combination with both increased electrolyte strength and reduced voltage are an effective approach to reducing the detrimental impact of high sample solution ionic strength. They can also lead to improved resolution by increasing stacking effects and reducing peak tailing. Peak area and migration time precision obtained are shown to be equivalent to those obtained for conventional injection procedures. It is concluded that short-end injections should be considered for routine operation as they are a useful means of reducing analysis time, increasing sensitivity, decreasing buffer depletion effects. They also allow use of higher electrolyte strengths which can improve resolution and reduce peak tailing, and can overcome significant problems which occur when analysing samples containing high salt contents.

Optimisation, validation and application of a capillary electrophoresis method for the determination of ranitidine hydrochloride and related substances

Ranitidine hydrochloride is an H2-antagonist which is widely prescribed for the treatment of peptic ulcers. The drug is marketed in a variety of dosage forms including tablets, syrups and injection solutions. A range of synthetic and degradative impurities of ranitidine are known and currently, these impurities are routinely determined using thin-layer chromatography (TLC). Alternatively a high-performance liquid chromatography (HPLC) method has also been employed in the assay of the pharmaceutical preparation. Unlike TLC, capillary electrophoresis (CE) offers the capability to quantify simultaneously both the active drug content and the levels of the related substances. The advantages of simplicity, selectivity, versatility and ease of use of CE offers a complementary separation technique to the established methods of HPLC and TLC in the determination of ranitidine and its related substances. This work represents a comprehensive evaluation of the performance of a developed CE method in the determination of drug-related impurities in both drug substance and various pharmaceutical formulations. The data obtained clearly shows that the performance of an optimised CE method can be equivalent in terms of sensitivity and precision to that of a HPLC method employed for a similar purpose and offers better selectivity against TLC and HPLC.

Determination of factors controlling the particle size in nanoemulsions using Artificial Neural Networks

The purpose of this study was to use Artificial Neural Networks (ANNs) in identifying factors, in addition to surfactant and internal phase content, that influence the particle size of nanoemulsions. The phase diagram and rheometric characteristics of a nanoemulsion system containing polysorbate 80, ethanol, medium chain triglycerides and normal saline loaded with budesonide were investigated. The particle size of samples of various compositions prepared using different rates and amounts of applied energy was measured. Data, divided into training, test and validation sets, were modelled by ANNs. The developed model was assessed and found to be of high quality. The model was then used to explore the effect of composition and processing factors on particle size of the nanoemulsion preparation. The study demonstrates the potential of ANNs in identifying critical parameters controlling preparation for this system, with the total amount of applied energy during preparation found to be the dominant factor in controlling the final particle size.

Determination of gentamicin in urine samples after inhalation by reversed-phase high-performance liquid chromatography using pre-column derivatisation with o-phthalaldehyde.

Gentamicin and netilmicin (internal standard) were extracted from urine using C18 solid-phase extraction cartridges (94.3% recovery) and then derivatised with o-phthalaldehyde and 3-mercaptopropionic acid. The derivative was stable for >6 h. The mobile phase methanol-glacial acetic acid-water (800:20:180, v/v), contained 0.02 M sodium heptanesulfonic acid, pH 3.4, and was passed at 1.0 ml min(-1) through a C18 column with fluorescence detection (excitation 340 nm, emission 418 nm). The four main components of gentamicin (C1, C1a, C2, C2a) and netilmicin, the internal standard, were separated. Using the C1a gentamicin peak, linearity was demonstrated from 0.5 to 10 microg ml(-1) and the limit of detection was 75 microg l(-1). Following 80-mg oral, 40-mg intravenous and 80-mg nebulised administration, the mean (SD) gentamicin urinary excretion was zero, 38.27 (0.96) and 1.93 (0.28) mg, respectively. Despite the relatively low lung deposition following inhalation of gentamicin the assay developed can be used to quantify the low urinary concentrations. Using this assay it should be possible to carry out urinary pharmacokinetic studies to identify the relative lung deposition of gentamicin following different methods of inhalation.

Resolution of the enantiomers of oxamniquine by capillary electrophoresis and high-performance liquid chromatography with cyclodextrins and heparin as chiral selectors.

The methods of separation of the enantiomers of the chiral drug oxamniquine are compared, between HPLC with either cyclodextrins and their related derivatives as chiral selectors in the mobile phase or immobilised in a chiral stationary phase (as Cyclobond I and II) and between capillary zone electrophoresis (CZE) where the cyclodextrins are added to the buffer solution. The HPLC experiments, which included structured method optimisation were largely unsuccessful in resolving the enantiomers, with the exception of when a Chiral-AGP protein stationary phase was introduced into the programme. However although this chiral stationary phase provided baseline resolution of the enantiomers the stability of the method was suspect to small changes in the pH (0.2 units). In contrast the CZE method developed for both cyclodextrins and their derivatives gave good resolution of the enantiomers and method stability (R.S.D. < 1%, n = 10 on precision). The basis of the interaction mechanism between selector and selectand was shown as a 1:2 relationship of cyclodextrin to analyte by NMR. In addition the polysaccharide, heparin was investigated as a chiral additive and excellent resolution of the oxamniquine was achieved with 3 mM heparin in 50 mM sodium dihydrogenphosphate (pH 3.0) as buffer in CZE, which also gave a stable procedure. This method allowed the detection of each of the enantiomers in the presence of the other down to 0.23% (m/m). The overall composition of the heparin material from different sources can however be slightly variable and this can result in small differences in resolution capability.

Approaches used in the reduction of the buffer electrolysis effects for routine capillary electrophoresis procedures in pharmaceutical analysis

Electrolysis of the electrolyte in the buffer reservoirs can occur when performing multiple assays in capillary electrophoresis (CE). This effect, which has been termed buffer depletion, results in a pH gradient being formed across the capillary. This gradient can effect both migration time, peak efficiency and selectivity and therefore can cause problems of reproducibility which leads to poor quantitative assays in pharmaceutical analysis. In this study the factors affecting the extent of buffer depletion were investigated. This involved performing a series of multiple assay runs where a number of factors were varied such as the use of dilute or concentrated buffer solutions and single or separate vials for pre-rinse and separation. In addition, the effect of the volume of buffer in the separation vials on the degree of depletion was also examined using standard (4 ml) and small volume (0.3 ml) CE vials. From these studies, it was observed that long multiple assay runs should not be carried out with small volumes of dilute buffer when pre-rinse and separation steps are from the same vial. The rate of electrolysis also increased when using wide-bore capillaries and at elevated temperatures. The use of short-end injections, zwitterionic buffers and reversing the polarity were found to appreciably reduce the effects of buffer depletion. From this work a number of recommendations are provided to maximise the number of acceptable separations that can be obtained in routine CE injection sequences.