Improving the Cleanliness of DBS Extracts using the Ostro Pass-through 96-well Sample Preparation Plate and Single Step Method

Importance of the Topic

DBS analysis provides a cost-effective and minimally invasive approach for bioanalysis, reducing blood volume requirements, simplifying sample collection, storage and transport.
Nonetheless, endogenous phospholipids from dried blood spots often accumulate in LC-MS/MS systems, causing matrix effects that compromise sensitivity, reproducibility and column lifetime.
The development of a streamlined, single-step extraction that effectively removes these interferences is crucial to enhance assay robustness and throughput.

Objectives and Study Overview

This work compares a traditional in-tube DBS extraction with an in-well, single-step method using the Ostro Pass-through 96-well Sample Preparation Plate.
Key aims include demonstrating phospholipid removal efficiency, preserving analyte recovery for risperidone and 9-OH risperidone, and validating method sensitivity and precision.

Methodology

Three-millimeter DBS punches containing risperidone, its hydroxylated metabolite and clozapine (internal standard) were prepared on Whatman DMPK-C cards.
Traditional extraction employed 250 µL of 5% water in methanol in tubes, vortex mixing, centrifugation and transfer.
The Ostro plate method performed in-well extraction by adding DBS punches and 250 µL of 5% water in methanol directly to each well, followed by vortexing and vacuum elution.
Extracts were diluted 1:1 with water and injected without evaporation.

Used Instrumentation

• Waters ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm)
• Waters Xevo TQ-S triple quadrupole mass spectrometer, ESI positive mode
• Ostro Pass-through 96-well Sample Preparation Plate

Main Results and Discussion

The Ostro plate removed over 99.9% of five monitored phospholipids compared to traditional extraction, as indicated by summed area counts.
Total ion current chromatograms illustrated negligible residual phospholipids and no column build-up across multiple injections with the Ostro method, in contrast to substantial accumulation using the in-tube approach.
Semi-validation showed QC concentrations within ±15% of nominal values and linear calibration from 0.05 to 50 ng/mL (R2 ≥ 0.99).
Lower limits of quantification of 0.05 ng/mL were achieved for both risperidone and 9-OH risperidone.

Benefits and Practical Applications

• Single-step, in-well workflow reduces preparation time and hands-on steps.
• High phospholipid removal enhances method robustness, reproducibility and column life.
• Direct injection eliminates evaporation and reconstitution losses, improving sensitivity.
• 96-well format enables high throughput for pharmaceutical bioanalysis and clinical studies.

Future Trends and Potential Applications

The integration of phospholipid-removal plates with automated liquid-handling systems may further boost throughput and consistency.
Expanding this approach to multi-analyte panels and alternative sample matrices (e.g., dried plasma spots) will broaden its applicability.
Continued miniaturization and greener solvent systems could enhance sustainability and reduce cost in high-volume testing.

Conclusion

The single-step Ostro Pass-through 96-well method delivers rapid, high-throughput DBS extraction with exceptional phospholipid removal (>99.9%), robust sensitivity and reduced column fouling.
This streamlined workflow offers a compelling alternative to traditional in-tube protocols for LC-MS/MS bioanalysis.