Extraction of Oxytocin and Vasopressin from Serum Using EVOLUTE® EXPRESS ABN Prior to LC-MS/MS Analysis

Significance of the Topic

Accurate measurement of oxytocin and vasopressin in serum is essential for clinical diagnostics and therapeutic monitoring. These neuropeptides play key roles in social behavior, stress response, and water homeostasis, making sensitive and reproducible analytical methods critical for research and quality control.

Study Objectives and Overview

This application note presents a streamlined solid-phase extraction (SPE) protocol using a 96-well EVOLUTE EXPRESS ABN sorbent for the extraction of oxytocin and vasopressin from serum prior to LC-MS/MS analysis. The method is designed to deliver high recovery, low matrix interference, and compatibility with high-throughput workflows.

Methodology and Instrumentation

Sample preparation involved spiking serum with peptides (0.2–500 ng/mL), dilution with 1% formic acid, and SPE via a load-wash-elute sequence on EVOLUTE EXPRESS ABN plates. Optional conditioning with methanol and formic acid enhanced reproducibility. Chromatographic separation used a Waters ACQUITY I-Class UPLC system equipped with an ACE C18-300 column (50 × 2.1 mm, 1.7 µm) under a gradient of 0.1% formic acid in water and acetonitrile. Detection was performed on a Xevo TQ-S triple quadrupole mass spectrometer in positive electrospray MRM mode with optimized cone voltages and collision energies.

Instrumental Setup

• EVOLUTE EXPRESS ABN 30 mg 96-well SPE plate
• Waters ACQUITY I-Class UPLC with ACE C18-300 column
• Xevo TQ-S triple quadrupole MS with ESI interface
• Biotage SPE Dry 96 evaporator

Main Results and Discussion

The method achieved recoveries greater than 70% with RSDs below 10% across the calibration range (0.2–500 ng/mL). Calibration curves demonstrated linearity (r2 > 0.99) and minimal endogenous interference. Post-column infusion and phospholipid profiling confirmed effective removal of proteins and lipids, reducing ion suppression. Both direct injection and evaporation/reconstitution approaches provided comparable results, with slightly lower suppression observed after reconstitution.

Benefits and Practical Applications of the Method

• High-throughput extraction using 96-well format
• Robust peptide recoveries and precision
• Efficient removal of proteins and phospholipids
• Flexible workflow: direct injection or concentration

Future Trends and Potential Applications

This SPE-LC-MS/MS approach can be extended to other polar peptides and hormones, with potential integration into automated SPE systems and high-resolution mass spectrometry. Development of greener solvents and micro-SPE formats may further enhance sustainability and sensitivity.

Conclusion

The described SPE-LC-MS/MS protocol offers a reliable, high-throughput solution for quantifying oxytocin and vasopressin in serum, combining simple sample preparation with efficient matrix cleanup and excellent analytical performance.

References

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