Y. Vander Heyden

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.

Assessment of new constraints applied to the alternating least squares method

Abstract The introduction of constraints in multivariate curve resolution methods, such as the Alternating Least Squares (ALS), is commonly used to limit the span of possible solutions, guiding the iterative process to a final result as close as possible to the true situation. In the present work, two modifications of the unimodality constraint and a new constraint for chromatographic concentration profiles related to the prevention of fronting have been checked. Simulated data sets as well as real data have been used to evaluate the effect of these new constraints in the resolution results. The parameters measured to assess the goodness of the constraints are related to the recovery of the concentration profiles and the quality of the data fit.

Screening approach for chiral separation of pharmaceuticals: Part II. Reversed-phase liquid chromatography

A screening strategy for the rapid separation of drug enantiomers by reversed-phase liquid chromatography was developed using three cellulose/amylose stationary phases. The key point to achieve enantioselectivity is the control of the compound ionisation. Only two mobile phases, i.e. an acidic phosphate buffer (pH 2.0) containing a chaotropic salt (KPF6) and a borate buffer (pH 9.0) mixed with acetonitrile, are used in the proposed strategy. This strategy was successfully applied to a set of 37 diverse chiral pharmaceuticals. Satisfactory enantioselectivity was achieved for 89% of them.

Ruggedness tests on the high-performance liquid chromatography assay of the United States Pharmacopeia XXII for tetracycline hydrochloride. A comparison of experimental designs and statistical interpretations

Ruggedness tests were performed for the high-performance liquid Chromatographic method for the determination of tetracycline hydrochloride from the United States Pharmacopeia XXII using Plackett-Burman and fractional factorial designs. Different statistical interpretation criteria for both kinds of designs were compared. The interpretation criteria for the Plackett-Burman designs were based on the use of dummy factors in the design and for the fractional factorial designs on two-factor interaction effects. The statistical interpretation criteria were compared with graphical ones (e.g., normal probability plots). The results of the designs (the ruggedness tests) were interpreted based on the above criteria. Other potential alternatives for statistical interpretation were also examined. Two different types of stationary phases (C8 and C18) were tested and compared.

Raman spectroscopy as a process analytical technology (PAT) tool for the in-line monitoring and understanding of a powder blending process

The aim of this study is to propose a strategy to implement a PAT system in the blending step of pharmaceutical production processes. It was examined whether Raman spectroscopy can be used as PAT tool for the in-line and real-time endpoint monitoring and understanding of a powder blending process. A screening design was used to identify and understand the significant effects of two process variables (blending speed and loading of the blender) and of a formulation variable (concentration of active pharmaceutical ingredient (API): diltiazem hydrochloride) upon the required blending time (response variable). Interactions between the variables were investigated as well. A Soft Independent Modelling of Class Analogy (SIMCA) model was developed to determine the homogeneity of the blends in-line and real-time using Raman spectroscopy in combination with a fiber optical immersion probe. One blending experiment was monitored using Raman and NIR spectroscopy simultaneously. This was done to verify whether two independent monitoring tools can confirm each others endpoint conclusions. The analysis of the experimental design results showed that the measured endpoints were excessively rounded due to the large measurement intervals relative to the first blending times. This resulted in effects and critical effects which cannot be interpreted properly. To be able to study the effects properly, the ratio between the blending times and the measurement intervals should be sufficiently high. In this study, it anyway was demonstrated that Raman spectroscopy is a suitable PAT tool for the endpoint control of a powder blending process. Raman spectroscopy not only allowed in-line and real-time monitoring of the blend homogeneity, but also helped to understand the process better in combination with experimental design. Furthermore, the correctness of the Raman endpoint conclusions was demonstrated for one process by using a second independent endpoint monitoring tool (NIR spectroscopy). Hence, the use of two independent techniques for the control of one response variable not only means a mutual confirmation of both methods, but also provides a higher certainty in the determined endpoint.

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.

Ruggedness tests for a high-performance liquid Chromatographic assay: Comparison of an evaluation at two and three levels by using two-level Plackett-Burman designs

Ruggedness tests are performed on a high-performance liquid Chromatographic assay for a drug substance. For this purpose two-level Plackett-Burman designs are executed. The factors selected are tested at two and three levels. Significant effects of factors are determined graphically and statistically. For the latter purpose experimental error is estimated from dummy factor effects. The use of other error estimates, as for instance the variance from replicate nominal experiments, as an alternative for the dummies was also examined. The dummy factor effects were found suitable to estimate the experimental error in a Plackett-Burman design. Appropriate replicate injections at nominal levels could be an alternative. Testing at three levels gives more detailed information about the behaviour of a response in function of the factors. It was also found that not only the effects on qualitative responses should be considered but those on quantitative ones as well in order to be able to make a full interpretation concerning the ruggedness of the method.

Gas-chromatographic fatty-acid fingerprints and partial least squares modeling as a basis for the simultaneous determination of edible oil mixtures

Partial least squares modeling and gas-chromatographic fatty-acid fingerprints are reported as a method for the simultaneous determination of cottonseed, olive, soybean and sunflower edible oil mixtures. In this work, two sets of three- and four-component combinations of oils were prepared, hydrolyzed and the obtained free fatty acids analyzed by gas chromatography (GC) without any further derivatization. The normalized percentages of the myristic (14:0), palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18:3) acids were chromatographically measured in samples and used for constructing calibration matrix. The cross-validation method was used to select the number of factors and the proposed methods were validated by using two sets of synthetic oil mixture samples. The relative standard error for each oil in mixture samples was less than 10%. This approach allows determining possible adulteration in each of the four edible oils.

Classification and regression tree analysis for molecular descriptor selection and retention prediction in chromatographic quantitative structure-retention relationship studies.

The use of the classification and regression tree (CART) methodology was studied in a quantitative structure-retention relationship (QSRR) context on a data set consisting of the retentions of 83 structurally diverse drugs on a Unisphere PBD column, using isocratic elutions at pH 11.7. The response (dependent variable) in the tree models consisted of the predicted rention factor (log kw) of the solutes, while a set of 266 molecular descriptors was used as explanatory variables in the tree building. Molecular descriptors related to the hydrophobicity (log P and Hy) and the size (TPC) of the molecules were selected out of these 266 descriptors in order to describe and predict retention. Besides the above mentioned, CART was also able to select hydrogen-bonding and molecular complexity descriptors. Since these variables are expected from QSRR knowledge, it demonstrates the potential of CART as a methodology to understand retention in chromatographic systems. The potential of CART to predict retention and thus occasionally to select an appropriate system for a given mixture was also evaluated. Reasonably good prediction, i.e. only 9% serious misclassification, was observed. Moreover, some of the misclassifications probably are inherent to the data set applied.

Determining orthogonal chromatographic systems prior to the development of methods to characterise impurities in drug substances.

To define starting conditions for the development of methods to separate impurities from the active substance and from each other in drugs with an unknown impurity profile, the parallel application of generic orthogonal chromatographic systems could be useful. The possibilities to define orthogonal chromatographic systems were examined by calculation of the correlation coefficients between retention factors k for a set of 68 drugs on 11 systems, by visual evaluation of the selectivity differences, by using principal component analysis, by drawing color maps and evaluating dendrograms. A zirconia-based stationary phase coated with a polybutadiene (PBD) polymer and three silica-based phases (base-deactivated, polar-embedded and monolithic) were used. Besides the stationary phase, the influence of pH and of organic modifier, on the selectivity of a system were evaluated. The dendrograms of hierarchical clusters were found good aids to assess orthogonality of chromatographic systems. The PBD-zirconia phase/methanol/pH 2.5 system is found most orthogonal towards several silica-based systems, e.g. a base-deactivated C16 -amide silica/methanol/pH 2.5 system. The orthogonality was validated using cross-validation, and two other validation sets, i.e. a set of non-ionizable solutes and a mixture of a drug and its impurities.