Simple Approach for the Removal of Phospholipids in Small Molecule Quantitative Bioanalysis Using Ostro Sample Preparation Plates

Importance of the Topic

The presence of phospholipids and proteins in biological matrices can cause ion suppression, MS source contamination and reduced assay sensitivity and robustness. Efficient removal of these matrix components is critical to maintain instrument performance, extend maintenance intervals and obtain reliable quantitative data in small molecule LC-MS bioanalysis.

Objectives and Study Overview

This application note evaluates a simple generic protocol using Ostro™ Pass-through Sample Preparation Plates for plasma phospholipid and protein removal, compared to conventional organic solvent protein precipitation. Gefitinib, a small molecule oncology drug, is used as a model analyte to demonstrate method performance.

Methodology and Instrumentation

Mouse plasma samples were spiked with gefitinib and internal standard, then processed by:

• Conventional protein precipitation: 10 µL plasma + 40 µL methanol/1% formic acid, centrifugation, dilution.
• Ostro plate protocol: 10 µL plasma + 40 µL methanol with internal standard + 200 µL methanol/1% formic acid, aspirated through the Ostro plate under vacuum, followed by dilution.

Used instrumentation:

• Waters ACQUITY UPLC I-Class PLUS System
• ACQUITY UPLC BEH C18 Column (1.7 µm, 2.1 × 100 mm)
• Waters Xevo TQ-S micro Triple Quadrupole MS in positive ESI MRM mode
• Data acquired with MassLynx™ v4.2 and processed with TargetLynx™

Main Results and Discussion

LC-MS chromatograms monitoring phospholipid precursors (m/z 184) showed a marked reduction in phospholipid signal using the Ostro plate compared to organic solvent precipitation. Summed spectra between 3.4 and 4.8 min revealed that lysophosphatidylcholines, phosphatidylcholines and sphingomyelin signals were effectively removed by the Ostro procedure, whereas they remained abundant in the solvent-precipitated samples. Calibration of gefitinib in mouse plasma over 15–7500 ng/mL was linear (r2 = 0.9988) with no loss of analyte on the plate.

Benefits and Practical Applications

The Ostro plate workflow:

• Provides rapid, simple sample cleanup without extensive method development
• Removes phospholipids and proteins to minimize ion suppression and instrument downtime
• Maintains analyte recovery and assay sensitivity across a wide concentration range
• Is readily adaptable to high-throughput drug discovery and development assays

Future Trends and Potential Applications

Ongoing and future opportunities include:

• Extension to other drug classes and biological matrices
• Integration with automated platforms for higher sample throughput
• Combination with high-resolution MS for untargeted lipidomic profiling
• Development of tailored sorbents for enhanced selectivity

Conclusion

Ostro Pass-through Sample Preparation Plates offer a streamlined, efficient solution for the removal of phospholipids and proteins from plasma, enabling robust, sensitive and reproducible small molecule bioanalysis by LC-MS. The generic protocol eliminates the need for extensive method development while preserving analyte integrity.

Reference

• Lei M, Gan W, Sun Y. HPLC-MS/MS analysis of peramivir in rat plasma: elimination of matrix effect using phospholipid-removal solid-phase extraction. Biomed Chromatogr. 2018;32(3).
• An G, Bach T, Abdallah I, Nalbant D. Aspects of matrix and analyte effects in clinical pharmacokinetic sample analyses using LC-ESI/MS/MS: two case examples. Pharm Biomed Anal. 2020;183:113135.
• McKillop D, Partridge EA, Hutchison M, et al. Pharmacokinetics of gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, in rat and dog. Xenobiotica. 2004;34(10):901-915.