Ultrasensitive Quantification Assay for Oxytocin in Human Plasma Using the ionKey/MS System

Significance of the Topic

This work addresses the need for ultrasensitive and reliable quantification of therapeutic peptides in human plasma. Oxytocin, a cyclic nine–amino-acid peptide present at low picogram-per-milliliter levels, poses analytical challenges for bioanalysis. Achieving limits of quantification comparable to immunoassays, while maintaining the speed, precision, and robustness of LC-MRM methods, is critical for pharmacokinetic, clinical, and quality-control studies.

Objectives and Overview of the Study

The study aimed to develop and validate a fast, robust LC-MRM assay for oxytocin in human plasma using the integrated ionKey/MS System. Key goals included reaching a lower limit of quantification (LLOQ) of 10 pg/mL, establishing linearity over three orders of magnitude, confirming assay precision and minimal carryover, and demonstrating practical throughput in a 96-well format.

Methodology and Used Instrumentation

Sample Preparation and Cleanup:

• Spike 200 µL K2-EDTA human plasma with oxytocin standards (10–20 000 pg/mL) and 100 pg/mL U-labeled internal standard.
• Protein precipitation with acetonitrile (1:1 dilution), centrifugation, dilution with 4% phosphoric acid.
• Solid-phase extraction on Oasis HLB 96-well plates (conditioning, equilibration, load, wash, elute, and dilution steps).

Chromatography and Detection:

• Waters ACQUITY UPLC M-Class system with ionKey BEH C18 iKey separation device.
• Mobile phase: 0.1% formic acid in water and acetonitrile; flow rate 1–5 µL/min; 8-minute gradient.
• MS/MS on Waters Xevo TQ-S with ionKey source, positive ESI mode; optimized MRM transition m/z 1007.4 → 723.3 for oxytocin.
• Data processing using MassLynx, TargetLynx Application Manager.

Main Results and Discussion

The optimized assay achieved an LLOQ of 10 pg/mL with signal approximately twice that of endogenous oxytocin background. Linearity was demonstrated from 10 to 20 000 pg/mL (r2 = 0.998). Intra-run precision (RSD) remained below 15% across all levels. Carryover following a 20 ng/mL injection was 0.02%, well below the LLOQ signal. Protein precipitation combined with SPE effectively removed matrix components, and protein-binding studies confirmed no significant oxytocin loss.

Benefits and Practical Applications

This workflow offers:

• Ultrasensitive detection matching or exceeding ELISA performance without affinity enrichment.
• Reduced development time, improved precision, and assay robustness.
• Lower solvent and consumable costs due to microflow operation (1–5 µL/min) and small injection volumes.
• High throughput via 96-well plate format suitable for pharmacokinetic and clinical studies.

Future Trends and Potential Applications

Emerging microfluidic LC-MS platforms will further enhance sensitivity and throughput for peptide therapeutics. Integration with automated sample preparation and multiplexed MRM panels can expand to other low-abundance biomarkers. Coupling microflow separation with high-resolution MS or ion mobility may improve specificity and broaden applications in clinical proteomics.

Conclusion

A fast, sensitive, and robust LC-MRM assay for oxytocin in human plasma was developed on the ionKey/MS System. It achieved an LLOQ of 10 pg/mL, excellent linearity, low carryover, and high precision. The microflow approach reduces operational costs and supports high-throughput bioanalysis of peptide therapeutics.

Reference

1. Burd JM, Weightman DR, Baylis PH. Quantitative determination of oxytocin in plasma by immunoassay. J Immunoassay. 1985;6(3):227–243.
2. Fisher LA, Fernstrom JD. Measurement of oxytocin in human plasma by radioimmunoassay. Life Sci. 1981;28(13):1471–1481.
3. Cool DR, DeBroose D. LC-MS measurement of oxytocin in plasma. J Chromatogr B. 2003;792(2):375–380.
4. Zhang G et al. Two-dimensional LC-MS/MS quantification of oxytocin in human plasma. Anal Biochem. 2011;416(1):45–52.
5. Sobhi HR et al. Two-dimensional LC-MS/MS with large-volume injection for oxytocin. J Chromatogr A. 2011;1218(47):8536–8543.
6. Mabrouk OS, Kennedy RT. Endogenous oxytocin quantification by mass spectrometry. J Neurosci Methods. 2012;209(1):127–133.