Introduction The benefits of using DBS for the determination of drug concentrations in whole blood have been widely reported.1,2 The technique continues to evolve and gain acceptance for DMPK support in the pharmaceutical industry. However, one of the disadvantages of the technique is the additional time and labor involved for the analyst during the sample preparation process when compared to traditional preparation techniques.

Here we report on an accurate and precise method for the semi-automated DBS extraction and HPLC/MS/MS analysis of Alprazolam, α-Hydroxyalprazolam, and Midazolam from human whole blood. The method shows that conventional liquid handling tools found in a bioanalytical laboratory can be used to semi-automate the DBS extraction. This technique includes adding internal standard/color indicating dye to the cards with a liquid handling instrument3, followed by extraction in the pipette tips of automated liquid handling instruments.