SELECTION OF ROBUST COMBINATIONS OF EXTRACTION LIQUID COMPOSITION AND INTERNAL STANDARD - MONTE-CARLO SIMULATION OF IMPROVEMENT OF ASSAY-METHODS WITH LIQUID-LIQUID-EXTRACTION PRIOR TO HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

The liquid-liquid extraction of a mixture of sulphonamides was achieved to examine the correlation between the experimental errors in the recoveries. Also, the impact of the composition of the extraction liquid was investigated. Six sulphonamides were repeatedly extracted simultaneously with ten different extraction liquids and determined with a reversed-phase high-performance liquid chromatographic (HPLC) system. The means, standard deviations and covariances (or correlations) of the recoveries were calculated. These data showed that correlation between the extraction of two or more structurally related compounds depends strongly on the extraction liquid composition used: the selection of an appropriate extraction liquid is very important for the development of accurate and reproducible assay methods. Selection of improper extraction liquids may introduce errors in internal standard calibration that are larger than the errors in external standard calibration. The selection of a suitable internal standard is also very important for the development of accurate and reproducible assay methods. Even compounds that are structurally related to the analyte may demonstrate completely different extraction behaviour. Selection of a proper internal standard and an accurate extraction liquid increases the accuracy and precision of the method. To investigate the influence on routine analysis, the data were used to simulate 50 analytical runs (calibration graphs with quality control samples) for each sulphonamide separately with external and internal standard calibration. In the latter option the other five sulphonamides were all tested as internal standards. This was done for all extraction liquids used. The results of these simulations demonstrate great differences between different extraction liquid compositions and internal standards for a given analyte. Also the calibration method (internal or external calibration) was found to be very important. Circumstances have been observed where external standard calibration gives better analysis results than internal standard calibration. The method described here can be applied for the selection of a suitable internal standard and extraction liquid for sample preparation by liquid-liquid extraction prior to HPLC.