Sub-Picogram Level Bio-Analytical Method for Quantification of Desmopressin in Human Plasma Using LCMS-8060NX

Significance of the topic

Desmopressin is a synthetic vasopressin analogue widely used in clinical practice for its antidiuretic properties. Accurate quantification at sub-picogram levels in human plasma is essential for pharmacokinetic, bioavailability and bioequivalence studies, as well as therapeutic drug monitoring. The development of a highly sensitive, robust and transferable LC-MS/MS method addresses the growing demand for precise peptide analysis in research and clinical laboratories.

Study objectives and overview

This work presents the development and partial validation of a novel bioanalytical method using the Shimadzu LCMS-8060NX system to quantify desmopressin in human plasma. Key goals included achieving a lower limit of quantification (LLOQ) of 0.25 pg/mL, ensuring reproducible results across laboratories and complying with major US FDA guidelines for method validation.

Methodology and Instrumentation

• Sample preparation: 300 µL human plasma mixed with extraction buffer, followed by solid-phase extraction (SPE) using C18 cartridges. Eluate was evaporated under nitrogen at 50 °C and reconstituted in 100 µL of 70:30 (v/v) 1 mM ammonium formate/methanol.
• Chromatography: Shim-pack GIST-HP C18-AQ column (50 × 2.1 mm, 1.9 µm) with a binary gradient of 1 mM ammonium formate in water (A) and methanol (B), flow rate 0.5 mL/min, column oven at 60 °C.
• Detection: Shimadzu LCMS-8060NX triple quadrupole mass spectrometer with heated ESI, UF-Qarray™ ion guide and IonFocus unit. Multiple reaction monitoring (MRM) in positive mode, transition m/z 535.0 → 328.0, collision energy −21 V.

Main results and discussion

• Linearity: calibration range 0.25–40 pg/mL, weighted (1/x²) regression r² > 0.99.
• Precision and accuracy: intra- and inter-day RSD ≤ 15% (≤ 20% at LLOQ); accuracy within ±15% of nominal values (±20% at LLOQ).
• Recovery: mean 76.7% with 4.8% RSD, consistent across low, mid and high QC levels.
• Matrix effect: negligible influence, matrix factors 1.05 (LQC) and 0.97 (HQC) with RSD ≤ 11.3%.
• No carry-over detected in blank injections following highest calibrator.

Benefits and practical applications of the method

• Enables pharmacokinetic and bioequivalence studies at sub-picogram sensitivity.
• Reduces plasma volume requirements, lowering sample consumption and instrument wear.
• Single-step SPE protocol enhances throughput and reproducibility across laboratories.

Future trends and potential applications

Advances in microflow LC-MS and automated SPE are expected to further lower detection limits and increase throughput. Integration with high-resolution mass spectrometry could broaden applications to other low-abundance peptides. The method’s robustness supports future use in clinical therapeutic drug monitoring, pediatric pharmacokinetics and comparative peptide pharmacology.

Conclusion

The described LC-MS/MS method on the Shimadzu LCMS-8060NX platform achieves exceptional sensitivity and robustness for desmopressin quantification in human plasma. Its validated performance and streamlined workflow make it well suited for bioequivalence, pharmacokinetic research and routine drug monitoring.

References

1. https://go.drugbank.com/drugs/DB00035 (accessed May 04, 2023)
2. https://www.accessdata.fda.gov/drugsatfda_docs/psg/Desmopressin%20acetate_%20oral%20tablet_%20RLD19955_Final%2008-17.pdf (accessed May 04, 2023)
3. https://www.fda.gov/media/71390/download (accessed May 04, 2023)