P.M.J. Coenegracht

Comparison of optimization methods in reversed-phase high-performance liquid chromatography using mixture designs and multi-criteria decision making

Abstract The optimization of different mobile phase systems is compared using statistical mixture designs. Isoeluotropic and non-isoeluotropic ternary and quaternary mobile phase mixtures have been optimized with regard to selectivity and analysis time. For a sample consisting of six benzene derivatives, the logarithm of the capacity factor of each solute is modelled in a full quaternary mobile phase system consisting of water and three organic modifiers. From the models, different separation criteria such as the resolution or selectivity of the worst separated pair of peaks, i.e. , the Min Res or Min Alpha, respectively, are calculated. Also, the capacity factor of the last peak, the Max k , can be predicted as a measure of analysis time. Response surfaces that show the different criteria as function of the different mobile phase systems re plotted and evaluated by multi-criteria decision making (MCDM). It is concluded that the optimization of isoeluotropic ternary mobile phase systems consisting of mixtures of two pseudo-components of equal solvent strength is inferior to that of iseluotropic quaternary mobile phase systems consisting of mixtures of three pseudo-components of equal solvent strength or to that of non-isoeluotropic ternary systems consisting of mixtures of water and two organic modifiers. The latter two optimization methods do not guarantee that the global maximum with respect to the Min Res of the full quaternary system consisting of mixtures of water and three organic modifiers will be found. On the one hand, the isoeluotropic quaternary system does not use variation of the water fraction to influence the selectivity. On the other, a mixture of three modifiers and water may provide a wider range of selectivity than mixture of two modifiers and water. Both optimization methods predict and realize a good separation of a test sample in a short analysis time provided that the solvent strength of the isoeluotropic quaternary system is properly chosen.

Comparison of several curve resolution methods for drug impurity profiling using high-performance liquid chromatography with diode array detection

The performance of five curve resolution methods was compared systematically for the identification and quantification of impurities in drug impurity profiling. These methods are alternating least-squares (ALS) with either random or iterative key-set factor analysis (IKSFA) initialisation, iterative target transformation factor analysis (ITTFA), evolving factor analysis (EFA), and heuristic evolving latent projections (HELP). Real and simulated high-performance liquid chromatography diode array detection (HPLC-DAD) data were obtained for drug mixtures containing one main compound and two impurities. The elution order of the main compound and the impurities was varied. Furthermore, resolutions were varied from 0.56 to 3.36 and impurity levels from 30% down to 0.1%. For simulated data, ALS with IKSFA initialisation and HELP perform better than ITTFA and EFA, which perform better than ALS with random initialisation. ITTFA works better than EFA for almost completely separated data, while the opposite is true for moderately or strongly overlapping data. Only ALS with IKSFA initialisation and HELP were found to resolve the required 0.1% level for moderately overlapping data. For real data, comparison of the methods provides similar results. ITTFA performs clearly better than EFA. However, none of the curve resolution methods can identify or quantify impurities at the required 0.1% level. The results for real data are worse than for simulated data because of heteroscedasticity, nonlinearity, and the acquisition resolution of the A/D-converter.

Multivariate characterization of solvent strength and solvent selectivity in reversed-phase high-performance liquid chromatography

Abstract Principal component analysis was used to determine the dimensionality and structure of three data sets consisting of the capacity factors of eleven to twenty different solutes measured in nine different mobile phase compositions consisting of water and methanol and/or acetonitrile on three reversed-phase columns. Principal component analysis showed that two principal components could account for the total variance in the data and that the percentage variance explained by the first principal component (about 80–95%) was much greater than the percentage explained by the second principal component, but that the percentage depended strongly on the choice of solutes for the sample. The first principal component could be associated with solvent strength and solvent strength selectivity and the second principal component with modifier selectivity. Solutes that showed strong modifier selectivity could be distinguished from solutes that have almost zero modifier selectivity, which could be useful for the definition of an empirical solvent strength scale.

Chemometrics in bioanalytical sample preparation : a fractionated combined mixture and factorial design for the modelling of the recovery of five tricyclic amines from plasma after liquid-liquid extraction prior to high-performance liquid chromatography

A general systematic approach is described for the chemometric modelling of liquid-liquid extraction data of drugs from biological fluids. Extraction solvents were selected from Snyders solvent selectivity triangle: methyl tert.-butyl ether, methylene chloride and chloroform. The composition of a mixture of the three extraction solvents was varied and the extraction yield (recovery) of a group of tricyclic amines was measured at all compositions selected. Two process variables, the extraction time and the extraction intensity, were varied simultaneously with the mixture variables to study their influence and their interaction with the mixture composition. The combined mixture and factorial design statistical techniques obtained in this way enabled the recovery to be modelled as a function of both the composition of the extraction liquid and the process variables. The models were assessed with regard to both descriptive and predictive capacities. The results showed that structurally related compounds may demonstrate different partitioning behaviour with regard to both mixture variables and process variables. It was concluded that mixtures of solvents result in higher extraction efficiencies for the amines. A positive effect on the extraction efficiency was demonstrated by the extraction intensity process variable and extraction time. A positive effect on the extraction efficiency was demonstrated by an interaction between extraction intensity and time. Mixture models in which process variables were introduced were recognized as being very suitable for modelling liquid-liquid extraction systems.

Selection of robust combinations of extraction liquid composition and internal standard: Monte Carlo simulation of improvement of assay methods with liquid—liquid extraction prior to high-performance liquid chromatography

Abstract The liquid—liquid extraction of a mixture of sulphonamides was achieved to examine the correlation between the experimental errors in the recoveries. Also, the impact of the composition of the extraction liquid was investigated. Six sulphonamides were repeatedly extracted simultaneously with ten different extraction liquids and determined with a reversed-phase high-performance liquid chromatographic (HPLC) system. The means, standard deviations and covariances (or correlations) of the recoveries were calculated. These data showed that correlation between the extraction of two or more structurally related compounds depends strongly on the extraction liquid composition used: the selection of an appropriate extraction liquid is very important for the development of accurate and reproducible assay methods. Selection of improper extraction liquids may introduce errors in internal standard calibration that are larger than the errors in external standard calibration. The selection of a suitable internal standard is also very important for the development of accurate and reproducible assay methods. Even compounds that are structurally related to the analyte may demonstrate completely different extraction behaviour. Selection of a proper internal standard and an accurate extraction liquid increases the accuracy and precision of the method. To investigate the influence on routine analysis, the data were used to simulate 50 analytical runs (calibration graphs with quality control samples) for each sulphonamide separately with external and internal standard calibration. In the latter option the other five sulphonamides were all tested as internal standards. This was done for all extraction liquids used. The results of these simulations demonstrate great differences between different extraction liquid compositions and internal standards for a given analyte. Also the calibration method (internal or external calibration) was found to be very important. Circumstances have been observed where external standard calibration gives better analysis results than internal standard calibration. The method described here can be applied for the selection of a suitable standard and extraction liquid for sample preparation by liquid—liquid extraction prior to HPLC.

Resolution optimisation in micellar electrokinetic chromatography using empirical models

Theoretical and empirical models can be used to model the migration or separation characteristics in micellar electrokinetic chromatography in order to optimise the resolution. In this paper only empirical models were used, because it is easier and more straightforward to obtain these models. Several empirical approaches for the optimisation of the resolution were compared in order to determine which response should be modelled preferably. The use of models of the effective mobility in combination with average plate numbers proved to be the most suitable approach to optimisation of the resolution, because the relative prediction errors of the models of the effective mobility were a factor of 2-4 smaller than the relative prediction errors of the models of the apparent mobility. Moreover for the least separated peak pair the resolutions based on the models of the apparent and effective mobility showed relative prediction errors that were approximately a factor of 2 smaller than the relative prediction errors of the resolutions based on the models of the resolution and separation factor. The predictions of the separation factor based on the different models generally showed lower prediction errors than the predictions of the corresponding resolutions. Although the relative prediction errors were large, particularly for closely migrating compounds, the empirical approach will probably lead to the optimum separation buffer composition.

Optimization of a novel two-phase potentiometric titration method of barbiturates with mercury (II). Part II: Experimental evaluation.

A two-phase potentiometric titration procedure of barbiturates with mercury(II) was developed. The composition of the formed mercury-barbiturate complexes was elucidated by infrared spectrometry.In the new titration procedure the barbituric acid derivative is dissolved in an aqueous borate buffer. An organic phase consisting of chloroform and benzyl alcohol is added, and the vigorously stirred contents of the titration vessel are titrated by a mercury(II) nitrate solution. In the potentiometric determination of the end-point a rotating mercury electrode is used as an indicator electrode that also serves as an efficient stirrer.The two-phase procedure was compared with a one-phase mercurimetric potentiometric titration in borate buffer and with the potentiometric titration by sodium hydroxide in an ethanol-water solution. The proposed two-phase procedure is superior to both methods because lower concentrations of barbiturates (10−3-10−4M) can be determined successfully. The one-phase procedure suffers from systematic errors, while the titration with sodium hydroxide is less precise at concentration levels of the barbiturates prevailing in content uniformity studies. By the two-phase procedure the direct titration of phenobarbital and mephobarbital in a dry mix of tablet excipients was possible with a relative standard deviation smaller than 1.5 percent.

Robustness testing of an optimized reversed-phase high-performance liquid chromatographic system for the separation of six sulphonamides using the rule

Abstract In a previous investigation, the composition of the mobile phase for the reversed-phase HPLC separation of twelve sulphonamides was optimized. The pred