J. Smeyers-Verbeke

Guidance for robustness/ruggedness tests in method validation.

This paper is intended to give guidance in setting-up and interpreting a robustness test. The different steps in a robustness test are discussed and illustrated with examples. The recommendations given for the different steps are based on approaches found in the literature, several case studies performed by the authors and discussions of the authors within a commission of the French SFSTP (Société Française des Sciences et Techniques Pharmaceutiques). In the end of the paper a worked-out example is given of a robustness test case study set up and interpreted according to the guidelines.

Validation of bioanalytical chromatographic methods

A strategy is discussed for the validation of chromatographic methods that are developed to quantify drugs in biological matrices. Both the validation terminology and the hypothesis testing are briefly reviewed. The emphasis is on the design of the experiments required to allow a reliable conclusion about acceptance or rejection of the bioanalytical method. In particular, it is explained how to evaluate the calibration line, devise experiments to estimate precision and bias and how to determine the stability of the analyte between the time of the sample collection and the analysis of the processed sample.

The chemical composition of idiopathic nonarteriosclerotic cerebral calcifications

Calculi from a case of cerebral idiopathic nonarteriosclerotic calcification (Fahrs disease) were examined. The stone consists of hydroxyapatite and possesses a typical structure: the calcification process seems to be initiated by the formation of small round bodies that are cemented to each other to form the final stone. Calcified vessels are also present, but seem to be a secondary effect. From a comparison with other calcifications, it is concluded that no pathologic significance should be attached to the relatively high levels of trace metals such as zinc, iron, copper, magnesium, lead, and others, with the possible exception of manganese. The organic matrix of the stone contains large quantities of protein. On hydrolysis of this fraction, an important unidentified ninhydrin-positive peak was found. No mucopolysac-charides were found.

Determination of system suitability limits with a robustness test

A robustness test was performed on a chromatographic method to identify and assay an active substance and two related compounds in film-coated tablet. For a number of responses the originally applied system suitability criteria were evaluated based on the results of the robustness test. Ambiguous situations can occur in situations where a method is found to be robust to assay the substances, as was the case here, but when system suitability criteria for some responses are violated. To avoid this, a proposal is made to define or re-define system suitability limits based on the results of the robustness test. From the effects found in the robustness test, the experimental conditions giving the worst result that still is acceptable and probable to occur are predicted and the system suitability limits are defined from replicated experiments in these conditions.

Simultaneous separation of ammonium and alkali, alkaline earth and transition metal ions in aqueous-organic media by capillary ion analysis

Abstract The simultaneous separation of the mono-, di- and trivalent cations NH+4, K+, Na+, Li+, Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Ni2+, Zn2+, Cu2+ and Cr3+ by capillary ion analysis was studied in a background electrolyte system composed of imidazole, 2-hydroxyisobutyric acid, 18-crown-6 and methanol. The effects of this system on the electroosmotic and electrophoretic mobility were studied in detail. A competitive complexation between 2- hydroxyisobutyric acid and sulphuric acid was observed which determined the cation mobility. Applying a central composite design, a quantitative description of the electrophoretic behaviour of the inorganic cations as a function of the system parameters was made and the optimum separation could be predicted. It was found in most instances that a linear model is sufficient for mobility modelling. Using a full factorial design, the main effects on the electroosmotic flow were estimated and methanol was found to be the dominating factor. The applicability of the capillary electrophoresis method was examined by separating ions in Chinese tea infusions. Five metal cations (K+, Na+, Ca2+, Mg2+ and Mn2+) were detected. A limit of detection at the μg l level could be achieved using electromigration injection.

Inter-laboratory studies in analytical chemistry

Inter-laboratory studies are performed with different aims and consequently require different evaluation methods and statistical treatment. The review considers method-performance studies (collaborative trials), laboratory-performance studies (proficiency tests), collaborative bias evaluation, inter-laboratory evaluation of to-be standard methods as well as certification studies for reference materials. Besides the classical evaluation methods using outlier tests, robust statistics and graphical methods are taken into account.

Study of parameters important in the spheronisation process

Abstract An experimental design was used to determine the influence of parameters that are important in the extrusion-spheronisation process. The parameters tested were water content of binary mixtures of Avicel PH 101 ® /water, spheroniser speed and spheronisation time. They appeared to have a significant influence on the quality of the spheres. By using a Pareto analysis, optimal parameter settings for water content, spheroniser speed and spheronising time were obtained.

Comparison of different approaches to estimate the uncertainty of a liquid chromatographic assay

A measurement result cannot be properly interpreted without knowledge about its uncertainty. Several concepts to estimate the uncertainty of a measurement result have been developed. Here, four different approaches for uncertainty estimation are compared on the example of the RP-high-performance liquid chromatography (HPLC) assay for tylosin for veterinary use: the guide to the expression of uncertainty in measurement (GUM) approach, which derives the uncertainty of a measurement result by combining the uncertainties related to the uncertainty sources of the measurement process; the top-down approach, which uses the reproducibility estimate from an inter-laboratory study as uncertainty estimate; an approach recently presented by Barwick and Ellison, which combines precision, trueness and robustness data to obtain an uncertainty estimate of the measurement result and finally a further approach, which directly estimates the measurement uncertainty from a robustness test. The comparison shows that the different approaches lead to comparable uncertainty estimates.

HPTLC methods to assay active ingredients in pharmaceutical formulations: A review of the method development and validation steps

High-performance thin-layer chromatography (HPTLC) is still increasingly finding its way in pharmaceutical analysis in some parts of the world. With the advancements in the stationary phases and the introduction of densitometers as detection equipment, the technique achieves for given applications a precision and trueness comparable to high-performance liquid chromatography (HPLC). In this review, the literature is surveyed for developed and validated HPTLC methods to assay active ingredients in pharmaceutical formulations published in the period 2005-2011. Procedures and approaches for method development, validation and quantitative assays are compared with the standard ways of conducting them. Applications of HPTLC in some other areas are also briefly highlighted.

The determination of Cu, Zn, and Mn in subcellular rat liver fractions.

Abstract An atomic absorption technique is used for the determination of Cu, Zn, and Mn in subcellular fractions of individual rat livers. In addition to the supernatant fluid a determination of the trace elements was also carried out in the rough and smooth microsomal subfractions. The results show that the concentration of Mn is highest in the nuclei and mitochondria and lowest in the supernatant, whereas, for Zn and Cu, the highest concentrations are found in the supernatant. The supernatant fraction was subjected to gel filtration to determine the amount of binding of Cu and Zn with the protein fraction of the supernatant fluid.