Instructions & Troubleshooting for HybridSPE®-Phospholipid (PL) 96-well Plates & Cartridges

Significance of the Topic

Phospholipid contamination in biological matrices is a major source of ion suppression in LC-MS and LC-MS/MS analyses of small molecules. The HybridSPE-PL platform offers a streamlined approach to remove both phospholipids and precipitated proteins, improving method sensitivity, reproducibility and throughput in bioanalysis.

Objectives and Overview of the Study

This application note presents the design and performance of HybridSPE-PL 96-well plates and cartridges for plasma and serum cleanup. Two workflows are described: an in-well precipitation method integrating protein removal and phospholipid trapping in a single step, and an off-line precipitation approach separating protein precipitation and SPE cleanup.

Methodology and Instrumentation

Samples are spiked with internal standard, then combined with acidified organic solvent (1% formic acid in acetonitrile) at a 1:3 ratio to precipitate proteins. After centrifugation or direct in-well mixing, supernatant is passed through zirconia-functionalized SPE media under vacuum. The high-affinity Lewis acid-base interaction between zirconia ions and phosphonate groups ensures efficient phospholipid retention. Two formats accommodate different volumes: a small-volume plate (20–40 µL sample) and standard plate (100–300 µL).

Used Instrumentation

• HybridSPE-PL 96-well plates (standard and small volume) and cartridges
• Vacuum manifold capable of –10 to –15 in. Hg
• Centrifuge (3 000 rpm, 2–5 min)
• Oscillating mixer or robotic liquid handler for in-well precipitation
• LC-MS or LC-MS/MS system for direct analysis of eluate

Main Results and Discussion

Under recommended conditions, >98% of endogenous phospholipids are removed, yielding clear extracts ready for LC-MS/MS without evaporation or reconstitution. Typical sample losses range from 30 µL (small-volume plate) to 90 µL (cartridge). Troubleshooting strategies address analytes with low recovery:

• Basic compounds: use 1% ammonium formate in methanol to reduce silanol interactions and HILIC retention.
• Chelator-type acids: condition phase with 0.5% citric acid in acetonitrile to inhibit analyte binding.

Benefits and Practical Applications

• Rapid cleanup in 4 min of vacuum application
• Minimal sample handling and no evaporation step
• Compatibility with high-throughput robotics
• Improved assay sensitivity and reproducibility

Future Trends and Potential Applications

Further integration with automated liquid handlers and sample tracking will enhance throughput. Adaptation to other biological matrices (urine, tissue homogenates) and coupling with ultrafast LC-MS gradients will expand method versatility. Development of new SPE chemistries may target additional interferences beyond phospholipids.

Conclusion

The HybridSPE-PL workflow effectively addresses phospholipid and protein interferences in plasma and serum, delivering cleaner extracts and more reliable LC-MS data. Its simplicity, speed and compatibility with high-throughput platforms make it a valuable tool for bioanalytical laboratories.