Optimizing Sample Preparation Protocols for Extraction of a Panel of PROTACs Molecules From Biological Fluids

Importance of the Topic

Proteolysis targeting chimeras PROTACs are an emerging class of bifunctional molecules that harness the cell’s protein degradation machinery to eliminate disease related targets. Their relatively high molecular weight and unique structure present bioanalytical challenges in sample preparation and quantification. Advancing robust extraction protocols is essential for reliable pharmacokinetic and pharmacodynamic studies during drug discovery and clinical development.

Objectives and Overview

This study aimed to develop and optimize sample preparation workflows for a panel of six PROTAC molecules and related small drugs in biological fluids. The goals included systematic evaluation of common cleanup techniques, assessment of recovery yield, reduction of matrix effects, and maximization of phospholipid removal. Two distinct approaches were compared: generic fit-for-purpose workflows for discovery and high-sensitivity protocols for clinical assays.

Methodology and Instrumentation

The experimental design combined various sample cleanup formats with high-performance liquid chromatography tandem mass spectrometry LC-MS/MS analysis. Key instrumentation and materials included

• LC system ACQUITY Premier UPLC and HSS T3 column operated at 60 °C with 0.6 mL/min flow
• Mobile phases of aqueous 0.1% formic acid with ammonium formate and 95% acetonitrile mix
• Oasis PRiME HLB, Oasis WAX mixed mode and other protein and phospholipid removal plates
• Xevo TQ Absolute triple quadrupole mass spectrometer with positive ESI at 1 kV capillary voltage
• Data handling via MassLynx 4.2 and TargetLynx XS software

Main Results and Discussion

Comparative screening showed that most cleanup methods delivered approximately 60% recovery for PROTACs, while Oasis PRiME HLB and Ostro phospholipid removal plates achieved over 98% phospholipid clearance, leading to reduced matrix effects. For ultra-sensitive applications, the Oasis mixed mode WAX protocol yielded recoveries above 90% for PROTACs and near 100% for related small molecules in the neutral eluate fraction.

Benefits and Practical Applications

The optimized Oasis PRiME HLB workflow offers a streamlined solution for high-throughput discovery assays, balancing acceptable recovery with matrix cleanup. The mixed mode WAX method is ideally suited for development and clinical studies requiring maximum sensitivity from limited sample volumes.

Future Trends and Opportunities

Continued innovation in sample preparation sorbents and automated liquid handling will further enhance assay robustness and throughput. Integration of microfluidic extraction techniques and novel surface chemistries may reduce sample volumes and improve analyte stability. Advances in high-resolution mass spectrometry will support broader PROTAC profiling and multiplexed quantification in complex matrices.

Conclusion

This work establishes tailored protocols for PROTAC bioanalysis: Oasis PRiME HLB for generic screening and Oasis mixed mode WAX for assays demanding highest recoveries. Implementation of these methods will accelerate reliable data generation across drug discovery and clinical phases.

Reference

No external literature references were cited within the original document.