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Abstract: Bioanalytical method development largely depends on the experience and the preference of the developer. Mathematical models could help in selecting the proper conditions to develop a selective and robust method, using liquid chromatography, liquid–liquid extraction, solid phase extraction and protein precipitation. This paper reviews the literature providing relevant equations and algorithms to model LC based bianalytical methods for the quantification of small molecules. By using the cited references, it will be possible to build models to describe the analytical methods either as an approximate impression or in a detailed way, incorporating many experimental variables. Special attention has been paid to matrix effects, the most important issues in bioanalysis and possible solutions to handle these issues are discussed. By proper use of the proposed models a more structured method development is accomplished, resulting in a description of the method that could be used for future use to control the complete bioanalytical method. [Copyright &y& Elsevier]

Abstract: Solid phase extraction (SPE) is a widely used method for sample cleanup and sample concentration in bioanalytical sample preparation. A few methods to model the retention behaviour on SPE cartridges have been described previously but they are either not applicable to ionised species or are not suitable when using multiple wash and elution steps with solvents differing in volume, modifier concentration and acidity. Furthermore, these models were not applied to mixed mode SPE sorbents. In order to overcome these limitations a new SPE modelling algorithm was proposed. The retention behaviour was determined directly on the SPE cartridge by connecting the cartridge online with an HPLC system using a simple but suitable device that was developed and described. The results from these online experiments were used to model the elution behaviour using a quadratic retention function combined with an exponentially modified Gaussian peak shape model to predict analyte recovery under different wash and elution conditions. The validity of the proposed algorithm was tested using practical SPE experiments with an aqueous test mixture as well as with spiked human plasma. Different sequential wash and elution steps were performed using solvents differing in volume and composition. The predicted band shape and recoveries in each collected step were in good agreement with the results obtained from practical experiments. The proposed algorithm is very useful for the description of the SPE behaviour of the analytes on the actual used SPE cartridge and can be used in structural and automated SPE method development. [Copyright &y& Elsevier]

Abstract: pH adjustment in bioanalytical sample preparation concerning ionisable compounds is one of the most common sample treatments. This is often done by mixing an aliquot of the sample with a proper buffer adjusted to the proposed pH. The pH of the resulting mixture however, does not necessarily have to be the same as the pH of the used buffer due to the significant buffer capacity of the sample. Calculation methods from titration technology were adapted and applied to this problem. The acid–base characteristics of human blood plasma and serum samples were determined and used to calculate the pH of buffer–plasma mixtures. Based on these parameters and the characteristics of the used buffers, two alternative methods were described to prepare buffers that lead to the proposed pH when mixed in the right volume ratio with human plasma samples. The resulting pH of several mixtures of different buffers with human blood plasma were in good accordance with the calculated pH. The proposed calculation methods and recommended buffer preparation methods may lead to more robust bioanalytical methods. [Copyright &y& Elsevier]