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The use of experimental design in separation science

Dejaegher B, Vander Heyden Y

Acta Chromatographica

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2009

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Vol. 21, no. 2

161--201

EN

In this tutorial, the application of experimental designs in separation science is discussed. Method optimization is often divided into screening and optimization phases. In the screening step, many factors, potentially affecting the separation, are screened to identify those with the largest effects. These are then further examined in an optimization phase, to determine the best separation conditions. After optimizing the method, it should be validated, before use for quantitative purposes. Robustness testing is part of method validation and examines the effects on the responses of small changes in method conditions. During the first phase of method optimization and during robustness testing, so-called screening designs are usually applied, whereas during the second phase of optimization, response-surface designs are often used. In this tutorial, the different steps in the application of both types of design are explained and elaborated with some examples.

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Development and validation of an HPLC method with post-column derivatisation to assay n-acetylcysteine in plasma

Vander Heyden Y., Mangelings D., Van Brempt J., Spapen H.

Acta Chromatographica

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2004

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No. 14

149-164

EN

A quantitative reversed-phase HPLC method has been developed that enables determination of both low endogenous and high therapeutic concentrations of N-acetylcysteine (NAC) in plasma. The compound is detected fluorimetrically after derivatisation with ortho-phthalaldehyde in the presence of a primary amine. Validation of the method revealed injection and method repeata-bility were good. The linear range was adequate and the limit of quantifi-cation was between 0.4 and 0.6 žM. Recovery of N-acetylcysteine from plasma samples was also acceptable. This method was applied to plasma samples from patients with a clinical septic shock who had received very high doses of N-acetylcys-teine. Six samples were taken at different times after administration of N-acetylcysteine. The blood-concentration profiles obtained indicate the me-thod is suitable for following the evolution of NAC in plasma under these conditions and can therefore be used for pharmacokinetic profiling.

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Modelling of solute retention in normal-phase HPLC with the chemically bonded 3-cyanopropyl stationary phase

Prus W., Vander Heyden Y., Massart D. L., Kowalska T.

Acta Chromatographica

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1998

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No. 8

98-108

EN

We have investigated the applicability of two models of solute retention (i.e. Models I and II, devised by Kowalska for liquid chromato-graphy with alcohol-hydrocarbon mobile phases) for description of the chromatographic behavior of 25 test solutes in normal-phase (NP) HPLC systems with the 3-cyanopropyl stationary phase and 2-propanol-n-hexane as mobile phase. The test solutes employed were from five chemical classes, (i) an alkylbenzene, (ii) crown ethers, (iii) hydroxyaromatic and methylhydroxyaromatic compounds, (iv) naphthylamines and (v) quino-line and its derivatives, and thus differed considerably in molecular structure, and hence in their ability to compete with 2-propanol molecules for active sites (i.e. the -CN and ≡Si-OH groups) of the stationary phase, which is the quintessence of the so-called displacement mechanism of solute retention. The performance of Models I and II was evaluated in terms of the numerical values of the root-mean-squared error (RMSt), calculated for each employed test solute and our investigations showed that Models I and II are very well suited for description of the chromato-graphic behavior of solutes for which there is a non-linear empirical relationship between retention parameter (RF or lnk) and the composition of a binary mobile phase.