A fully automated plasma protein precipitation sample preparation method for LC-MS/MS bioanalysis.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmacokinetics

Background:

• Protein precipitation is a crucial step in plasma sample preparation for drug analysis.
• Manual protein precipitation is labor-intensive and prone to variability.
• Complete automation of this process can enhance efficiency and reproducibility.

Purpose of the Study:

• To develop and validate a fully automated robotic system for plasma sample preparation via protein precipitation.
• To integrate all necessary peripheral devices for unattended operation.
• To assess the accuracy and precision of the automated method.

Main Methods:

• A Tecan Freedom EVO 200 liquid handling platform was integrated with peripheral devices including robotic arms, plate shakers, a temperature-controlled centrifuge, a plate sealer, and a piercing station.
• Automated preparation of plasma calibration and quality control samples from stock solutions.
• Protein precipitation achieved by adding acetonitrile, followed by automated centrifugation, sealing, piercing, and supernatant transfer.

Main Results:

• The automated system successfully produced sealed, injection-ready 96-well plates of plasma extracts.
• Excellent intra-day and inter-day precision (CV <5%) and acceptable accuracy (relative error <8%) were achieved.
• The system demonstrated flexibility for pharmacokinetic studies with varying sample numbers and time points.

Conclusions:

• This represents the first complete automation of the protein precipitation method for plasma sample analysis.
• The developed robotic system is robust, accurate, and precise, suitable for pharmacokinetic applications.
• Full automation significantly improves efficiency and reduces variability in plasma sample preparation.