A. Jardy

Single-column anion chromatography with indirect uv detection using pyromellitate buffers as eluents

Abstract Ion chromatography can be conveniently carried out with conventional high-performance liquid chromatographic equipment and classical ion exchangers using a single column and a UV-absorbing eluent ion. Solute peaks are detected as vacancy peaks (corresponding to a decrease in eluent absorbance). Benzenepolycarboxylate buffers such as those obtained from pyromellitic acid are used as eluents for anion analysis; owing to the four carboxylic acid groups, the eluent ion charge varies with pH, and the eluting power can be adjusted within a wide range. A linear relationship holds between the logarithm of the solute capacity factor and the logarithm of the concentration of the eluting species; the slope observed is - x/y, here x and y are the charges on the solute and eluent anion, respectively. At a constant analysis time, the best sensitivity is achieved when the initial absorbance of the eluent (determined by the detection wavelength) is high but its concentration is low, and therefore its charge is high enough to give the required eluting power. Within the linearity range, quantitative analysis requires only one calibration graph when peak-area measurements are performed. The detection limits are about 2–5 ng injected and compare favourably with other detection modes, considering the simplicity of the method. Various examples of separations of inorganic and organic anions are given.

Methodology for transfer of liquid chromatography methods based on statistical considerations

Abstract An important task in the pharmaceutical industry today is analytical transfer. However, no actual guidelines are available today. It is for this reason that we decided to devise a rigorous method using statistic exploitation of results. The statistical technique used is ANOVA (analysis of variance). We chose to treat the case of quantitative analysis in LC but the methodology could easily be adapted to other analytical techniques. The criteria of the transfer validation could be formulated thus: “for each response of interest, the new laboratory must produce results that are not significantly different from those of the reference entity”, or more explicitly by: “the new laboratory must have dispersion characteristics compatible with those of the reference entity and must exhibit no bias”. While compatibility of precision can easily be assessed, the test of absence of bias requires that certified materials be available. Since certified materials can only be obtained through an inter-laboratory study, it means that the reference entity is necessarily a pool of laboratories. Using a single laboratory instead of a pool would not allow a distinction to be drawn between a bias and an inter-laboratory dispersion, which would lead to abnormal transfer failure. The methodology developed was then used on an example. The last part deals with the situation where certified materials are suspected to be slightly degraded. It is explained how such a case, likely to be encountered in pharmaceutical products stored over a long period, can be handled without re-starting the study from scratch.

Study of the Linear Range in HPLC Analyses with UV Detection: Methodology and Experimental Application to the Influence of the Analyte UV Spectrum

Determination of the linear range is one of the main concerns in validation of an HPLC analysis method. It is particularly important since single point calibration will be then used routinely. We proposed an iterative methodology to handle this problem. The idea was, at each step, to test statistically whether the following point belonged to the same regression line. The methodology was then used to evaluate quantitatively the effect on linear range of a shift in detection wavelength or of the detector bandwidth. Although experimental results were globally in accordance with spectroscopic theory, magnitudes observed were rather large. So the linear range could vary by a factor of over 2 with changes in conditions that remained within the range of current practical values. Changes in detection wavelength were limited to about fifteen nm around λmax and the detector used was considered to be representative of modern high-performance UV detectors. The question of how to take consequences in method validation into account was raised. The solution proposed recommended that the validation was undertaken in conditions as close as possible to those where the method would be conducted routinely. This means with the same instrumentation and on the product of interest for analyses.

Dependence of HETP upon resin particle size and solution flow-rate in high-speed ion-exchange chromatography

Abstract In order to obtain a method for selecting the optimum experimental conditions for achieving difficult separations in high-speed ion-exchange chromatography, the dependence of the height equivalent to a theoretical plate (HETP) on the solution flow-rate and resin particle size was studied. The HETP has been measured for the nickel—zinc ion exchange (permutation front) at different flow-rates and for severla fractions of variable sizes obtained by elutriation from the same variety of a commercially available cationic resin, Dowex 50W-X8 (−400 mesh). For a given particle diameter, dp, the HETP (H) was found to vary with the solution flow-rate, u, according to the equation where α depends on the particle size: it decrease as dp increases, e.g., from 0.9 to 0.4 when dp varies from 20 to 100 μm, tending to a limiting value which was found to be dependent on the exchange reaction studied. On the other hand, at a constant flow-rate, H was found to vary with dp according to the equation where β depends on the solution flow-rate: it decreases slowly as u increases. The influence of these parameters on the column efficiency, and of the pressure drop that results from using high flow-rates and very small particles, are discussed.

VALIDATION OF INTERNAL NORMALIZATION FOR IMPURITY ASSAYS

The simplicity of the internal normalization made it a very attractive method. Yet, because of its restrictive applicability requirements, internal normalization is not widely implemented in HPLC quantitative analysis. Basically, applicability requirements are that all the solutes must not only be eluted and detected but must also present similar behavior toward the detection system. Ideally, response factors should be identical for all the solutes or, in practice, of the same order of magnitude. The methodology developed to validate, in a rigorous way, internal normalization was based on the use of a statistical tool called analysis of covariance (ANACOVA). ANACOVA is more or less similar to ANOVA but can manage a continuous variable, like for example, concentration. So, it is possible to use it to compare calibration curves of all the different solutes present in a sample, for example, the main product and its impurity. After having checked that for the main product the response factor was the same around the target concentration of the HPLC method, and at low concentration, it was then possible to make comparison with impurity behavior, and to determine whether the use of the response factor was necessary or not. Eventually, ANACOVA enabled the validation of internal normalization by assessing that all the solutes presented required behavior. This methodology was successfully applied to an actual example of liquid chromatography quantitative analysis, taken from the pharmaceutical industry. In this case, internal normalization for impurity assays of an anticytomegalovirus drug substance was validated after response factor correction.

Removal of Arsenical By-Products from Chemical Warfare Destruction Effluents

Within the context of the chemical warfare destruction (April, 29th 1997 convention), the removal of arsenic, which is highly toxic, from the lewisite hydrolysis effluents has been studied. A process in two steps is presented, which is compatible with industrial and economical constraints.