Increasing Sensitivity and Minimizing Sample Volume for the Quantification of Therapeutic and Endogenous Cyclic Peptides in Plasma Using ionKey/MS

Significance of the Topic

The quantification of cyclic therapeutic and endogenous peptides in plasma is critical for pharmacokinetic, toxicokinetic and bioanalytical studies. Increased use of peptide drugs demands methods that achieve low picogram-per-milliliter sensitivity while conserving limited sample volumes.

Objectives and Study Overview

This work aimed to enhance sensitivity and reduce plasma volume requirements for measuring desmopressin, vasopressin and octreotide. The approach integrated mixed-mode μElution SPE with microfluidic LC and triple-quadrupole mass spectrometry to achieve sub-pg/mL limits of quantification.

Methodology and Used Instrumentation

The sample preparation employed a 96-well Oasis WCX μElution plate for mixed-mode solid-phase extraction. Human plasma (25–200 µL) was acidified with 4 % phosphoric acid, loaded onto the SPE plate, washed with ammonium hydroxide and acetonitrile, then eluted in minimal solvent. Chromatography used an ACQUITY UPLC M-Class system configured with ionKey Source and ionKey HSS T3 150 µm × 100 mm separation device. A trap and back-flush strategy delivered analytes to the column under a 0.3 µL/min flow and a linear gradient from 2 % to 50 % acetonitrile. Detection was performed on a Xevo TQ-S mass spectrometer using ESI positive mode. Data acquisition and quantification were controlled by MassLynx 4.1 and TargetLynx software.

Main Results and Discussion

The method achieved linear quantification from 1 to 2000 pg/mL (r2 > 0.99) for all three peptides. Limits of quantification below 1 pg/mL were obtained using 100 µL plasma, and 2.5 pg/mL detection was demonstrated from only 25 µL. Microfluidic LC yielded narrow peak widths (< 4.5 s) and excellent separation from endogenous interferences. Mixed-mode SPE provided selective cleanup and reduced sample matrix effects. The ionKey/MS system offered up to an order of magnitude gain in sensitivity over conventional 2.1 mm analytical scale assays, enabling smaller injection and sample volumes.

Benefits and Practical Applications

This workflow offers:

• Ultra-sensitive peptide quantification with minimal plasma consumption
• High throughput processing in a 96-well format with reduced solvent use
• Automatable extraction and chromatographic robustness
• Enhanced accuracy, precision and dynamic range for bioanalysis and PK/PD studies

Future Trends and Opportunities

Emerging directions include:

• Further miniaturization of sample preparation and LC interfaces
• High-throughput screening of large peptide libraries in discovery settings
• Integration with high-resolution MS and ion mobility for complex isobaric separation
• Extension to other biologics, including antibodies and large peptides at fg/mL levels

Conclusion

The combined use of mixed-mode μElution SPE and ionKey/MS microfluidic LC–MS/MS enables reliable and reproducible quantification of cyclic peptides down to the sub-pg/mL level from minimal plasma volumes. This approach meets the growing demand for sensitive, low-volume bioanalytical assays in drug development.

Reference

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