Quantitative Analysis of Oxytocin in Rat Plasma Using LC/MS/MS

Significance of the Topic

Oxytocin plays a pivotal role in parturition and social behavior, influencing uterine contractions during labor and modulating interpersonal bonding. Accurate quantitation of oxytocin in biological matrices such as rat plasma supports research in reproductive physiology, neuroendocrinology, and pharmaceutical development.

Objectives and Overview of the Study

This study demonstrates a robust liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for quantifying oxytocin in rat plasma. Using a Shimadzu LCMS-8050 triple quadrupole system coupled with Nexera X2 UHPLC, the authors aimed to establish sensitivity, linearity, and accuracy of the assay.

Methodology and Instrumentation

Sample Preparation:

• Collected heparin-treated rat blood and centrifuged to isolate plasma.
• Performed protein precipitation by adding ten volumes of acetonitrile to plasma, followed by centrifugation and vacuum concentration.

Chromatographic Conditions (Nexera X2):

• Column: Shim-pack GISS-HP (2.1×50 mm, 3 µm)
• Mobile Phase A: Water; B: Methanol; gradient from 30 % to 50 % B over 5 min
• Flow Rate: 0.3 mL/min; Column Temp: 40 °C; Injection: 2 µL

MS/MS Conditions (LCMS-8050):

• Ionization: ESI in negative mode
• MRM Transition: m/z 1005.4 → 939.4 (CE 56 V)
• Gas Flows: Nebulizing 2.0 L/min; Drying 15.0 L/min; Heating 5.0 L/min; DL Temp: 150 °C; Block Heater: 500 °C; Interface: 350 °C

Main Results and Discussion

Calibration Curve:
Satisfactory linearity was observed over 0.1–500 ng/mL (r2 ≥ 0.9996) with an RSD of 8.3 % at the 0.1 ng/mL level.

Quantitation of Plasma Samples:
Unspiked rat plasma blank showed a detectable oxytocin signal at 0.23 ng/mL. A spike-and-recovery experiment at 2 ng/mL yielded a recovery rate of 91 %, indicating minimal matrix effects and high assay accuracy.

Benefits and Practical Applications

The developed LC/MS/MS method offers:

• High sensitivity enabling detection in low-volume plasma samples.
• Excellent reproducibility and quantitation over a wide dynamic range.
• Rapid sample throughput suitable for preclinical pharmacokinetic and biomarker studies.

Future Trends and Potential Applications

Advancements may include microflow LC to reduce solvent consumption, automated sample preparation to increase throughput, and expansion to human clinical samples. Coupling with stable-isotope internal standards and high-resolution MS could further improve specificity and sensitivity for peptide biomarkers.

Conclusion

This application note demonstrates a robust, sensitive, and accurate LC/MS/MS assay for oxytocin in rat plasma using Shimadzu instrumentation. The method’s linearity, precision, and recovery metrics support its utility in preclinical research and peptide quantitation workflows.

References

• Shimadzu Application News No. C211, Quantitative Analysis of Oxytocin in Rat Plasma, First Edition May 2020.