Improved Bioanalytical Performance Using a Simplified 2-Step Oasis™ PRiME HLB Protocol with an Ultra-Short UPLC Column for UPLC-MS/MS

Improved Bioanalytical Performance Using a Simplified 2-Step Oasis PRiME HLB Protocol with an Ultra-Short UPLC Column

Importance of the Topic

High-throughput bioanalysis demands sample preparation methods that balance speed, cleanliness and robustness. Residual phospholipids from plasma can cause ion suppression, column fouling and increased maintenance, compromising data quality and laboratory efficiency. Simplified solid-phase extraction (SPE) workflows that effectively remove matrix components are critical to sustain performance in discovery, development and regulated environments.

Objectives and Study Overview

This study compared a two-step Oasis PRiME HLB SPE protocol against conventional protein precipitation (PPT) using human plasma spiked with a panel of 14 diverse analytes. The workflow was paired with a novel ultra-short UPLC column (2.1 × 10 mm) to evaluate impacts on phospholipid clearance, system pressure, sensitivity, area count stability and column lifetime over thousands of injections.

Methodology and Instrumentation

A two-step “load and elute” SPE protocol reduced preparation time by 35 % versus PPT. Fourteen compounds varying in polarity and ionization properties were spiked into plasma. Chromatographic separation employed an ACQUITY Premier UPLC I-Class System with a prototype 2.5 µm HSS T3 2.1 × 10 mm column, 1 minute gradient (5–95 % B), 500 µL/min flow, 55 °C column temperature. MS detection used a Xevo TQ Absolute in positive ESI MRM mode. Instrument control and quantification were handled by MassLynx™ and TargetLynx™, respectively.

Used Instrumentation

• ACQUITY Premier UPLC I-Class System
• Prototype 2.5 µm HSS T3 2.1 × 10 mm UPLC Column
• Xevo TQ Absolute Triple Quadrupole Mass Spectrometer
• MassLynx™ v4.2 and TargetLynx™ v4.2 Software

Main Results and Discussion

Phospholipid Removal

• SPE achieved over 90 % reduction in residual phospholipid signal (m/z 184 precursor), compared to PPT.

System Pressure and Column Lifetime

• With PPT, column pressure rose sharply after ~1 000 injections and area counts dropped 70 % by 2 000 injections.
• SPE-prepared samples maintained stable pressure and consistent sensitivity over 5 000 injections, extending column lifetime by >2.5×.

Sensitivity and Signal Stability

• PPT showed a 45 % decrease in peak area after 500 injections.
• SPE yielded <7 % variability in area counts across 5 000 injections, reducing recalibration needs and downtime.

The ultra-short column delivered sharp peaks and high throughput without sacrificing data quality, supporting reliable one-minute methods in high-injection workflows.

Benefits and Practical Applications

• Significant phospholipid clearance enhances MS sensitivity and assay robustness.
• Rapid, automation-friendly SPE reduces hands-on time and accelerates sample throughput by 35 % versus PPT.
• Extended column lifetime and stable system pressure lower maintenance costs and downtime.
• One-minute UPLC methods enable fast cycle times in discovery and regulated labs.

Future Trends and Applications

Continued development of ultra-short columns with advanced particle technology promises even faster separations. Integration with robotic SPE workflows and online cleanup modules will further minimize manual steps. Advances in real-time monitoring and AI-driven method optimization can refine SPE and chromatographic conditions, improving laboratory agility. Expansion of SPE chemistries targeting emerging bioanalytical challenges (e.g., polar metabolites, lipids) will broaden applicability across omics and clinical diagnostics.

Conclusion

The simplified two-step Oasis PRiME HLB SPE protocol, when combined with an ultra-short UPLC column, offers a robust, fast and clean workflow for plasma bioanalysis. It delivers superior phospholipid removal, extended column lifetime and consistent sensitivity across thousands of injections, making it an ideal solution for high-throughput environments seeking to maximize data quality and laboratory efficiency.

References

1. Tanna N., Plummer C. Oasis PRiME HLB – The Fastest Way to Clean Samples. Waters Application Note 720008684, February 2025.