Dilution Effect in Multichannel Liquid-Handling System Equipped with Fixed Tips: Problems and Solutions for Bioanalytical Sample Preparation

Abstract

This paper presents an in-depth investigation of the dilution effect from the liquid handlers equipped with fixed tips resulting from mixing with residues of the system liquid used to wash the inside and outside of the fixed tips. The impact of the dilution in bioanalytical sample preparation was evaluated. The liquid-transfer steps, where the dilution effect should be minimized or eliminated in bioanalytical sample preparation, were the sample dilution, and the standard (STD) and quality control (QC) sample preparation steps. The level of the dilution effect for the red dye was quantified by using the dual-dye photometric measurement. A significant dilution effect was observed for the red dye, indicating that without a proper liquid-transfer strategy to minimize it, it would be impossible to prepare serially diluted STD and QC samples because of the cumulative dilution effect. The impact on the bioanalytical results was substantial, especially when a multistep dilution scheme was used for the sample dilutions. A unique liquid-transfer strategy was designed in our lab to minimize the dilution effect for the sample dilution. The effectiveness of this strategy was demonstrated using the dual-dye photometric measurement and by generating a successful STD curve preparation of a model compound, Compound A, in dog plasma for an LC/MS/MS assay. With this strategy, high-quality bioanalytical sample preparation without the dilution effect could be achieved by using the liquid handlers equipped with fixed tips.