Enhanced LC-MS/MS Platform to Address Common Challenges During the Analytical Evaluation of Modern Peptide and Oligonucleotide Therapeutics

Significance of the topic

Oligonucleotide and peptide therapeutics are rapidly expanding classes of drug molecules with chemical modifications that improve potency and pharmacokinetics but introduce analytical challenges for LC-MS/MS bioanalysis. High charge density, increased lipophilicity and chemical modifications lead to nonspecific surface binding, carryover, in-source fragmentation and complex collision-induced dissociation patterns; at the same time, higher potency reduces required doses and lowers biological concentrations, driving demand for more sensitive, robust LC-MS/MS workflows.

Objectives and overview of the study

This work evaluated an enhanced LC-MS/MS platform designed to mitigate common issues encountered when quantifying modern peptides and oligonucleotides. Study goals included: (1) increase analytical sensitivity, (2) reduce nonspecific binding and carryover, (3) streamline MRM and source optimization, and (4) demonstrate the approach on representative lipophilic peptides (semaglutide, liraglutide, tirzepatide, mazdutide) and a model siRNA duplex (sense and antisense strands). The study compared a BioInert (XSInert) flow path against a traditional stainless-steel (SUS) flow path using identical MS hardware and LC method templates.

Methods and methodology

Key methodological elements:

• MS platform: Shimadzu LCMS-8060RX triple quadrupole mass spectrometer operated with ESI (probe position set to +2 mm).
• LC systems: comparison between an XSInert (BioInert) UHPLC flow path and a standard stainless-steel LC-40 series flow path.
• Columns: reversed-phase columns (Shim-pack Velox C18 and Scepter Claris C18-300) selected according to peptide/oligo separations.
• Mobile phase chemistry: peptides—water/ACN with 1% formic acid; oligonucleotides—volatile ion-pairing systems using HFIP/TEA mixtures (50 mM HFIP/16.3 mM TEA in water or 25 mM HFIP/8.15 mM TEA in 50:50 MeOH:water) to enable desalting and chromatography of charged oligos.
• Flow and injection: peptide method at 0.3 mL/min, oligonucleotide method at 0.2 mL/min; injection volumes ~5–25 µL depending on assay.
• MRM and source optimization: automated optimization using LabSolutions Connect MRM to tune MRM transitions (multiple charge states) and interface parameters (interface voltage, focus voltage, CID gas pressure, collision energy).
• Data acquisition and processing: LabSolutions software for acquisition and LabSolutions Insight LCMS for data processing.

Used instrumentation

Instrumentation and software explicitly used in the study:

• Shimadzu LCMS-8060RX triple quadrupole mass spectrometer.
• Shimadzu LC platforms with XSInert (BioInert) flow path and LC-40 series (stainless-steel) flow path.
• Shim-pack Velox C18 and Scepter Claris C18-300 columns (various dimensions).
• LabSolutions Connect MRM for automated MRM and source optimization.
• LabSolutions Insight LCMS for data processing.

Main results and discussion

Highlights of analytical performance and observations:

• Sensitivity: The XSInert (BioInert) flow path produced increased signal and improved sensitivity for all tested lipophilic peptides relative to the stainless-steel flow path. Peptide limits of detection on the XSInert path reached ~0.1–0.25 ppb for several analytes (e.g., semaglutide <0.1 ppb), with quantitative ranges beginning as low as 0.1–0.5 ppb up to the higher ppb ranges depending on the peptide. Oligonucleotide responses were largely similar between the two flow paths, indicating a smaller impact of bioinert surfaces on these charged analytes under the tested conditions.
• Carryover: For peptide assays, the XSInert flow path reduced carryover substantially—approximately 50% reduction in the first blank after a high concentration injection compared with the SUS path and carryover <0.5% after three blank injections. Oligonucleotide assays showed minimal carryover on both flow paths.
• MRM and source optimization: Automated Connect MRM optimization identified CID gas pressure, interface voltage and focus voltage as the most influential parameters on signal intensity across peptides and oligos; collision energy optimization across multiple transitions enabled selection of robust quantitative transitions. Graphics from optimization runs (example shown for the +3 charge state of tirzepatide) facilitated selection of optimal settings.
• Calibration and linearity: Calibration curves for all analytes showed excellent linearity (R2 ≥ 0.99). Many peptides exhibited lower limits of quantitation on the XSInert system compared with stainless steel.
• Fragmentation considerations: The study notes increased in-source fragmentation and complex collision cell fragmentation for modified oligos and lipophilic peptides, reinforcing the importance of careful source/interface tuning and monitoring multiple charge states and confirmatory transitions.

Benefits and practical applications of the method

Practical advantages demonstrated:

• Reduced nonspecific adsorption and carryover for lipophilic peptides by minimizing stainless-steel exposure in the flow path, improving assay cleanliness and reducing the need for aggressive rinsing.
• Enhanced sensitivity for low-level peptide quantitation, supporting bioanalysis of potent therapeutics at low biological concentrations.
• Automated MRM/source optimization saves time and standardizes method development, enabling systematic tuning of collision energy, CID gas pressure and interface voltages across multiple charge states.
• Compatibility with routine LC-MS/MS triple quadrupole workflows and existing chromatographic chemistries (ion-pairing for oligonucleotides and reversed-phase for peptides).

Future trends and potential applications

Projected developments and opportunities in LC-MS/MS bioanalysis of oligonucleotide and peptide therapeutics:

• Broader adoption of bioinert flow paths, coatings and alternative materials to minimize adsorption of modified peptides and conjugated oligonucleotides.
• Further integration of automated optimization tools (MRM/source tuning) into routine method development and QA/QC pipelines to accelerate assay validation and robustness testing.
• Expansion of microflow/nanoLC and improved ion sources to further boost sensitivity for ultra-low concentration analytes.
• Improved collision cell designs and data acquisition strategies (e.g., parallel reaction monitoring or enhanced product-ion scanning on high-resolution instruments) to address complex fragmentation patterns of chemically modified molecules.
• Refinement of chromatographic chemistries for oligonucleotides that balance ion-pairing efficacy, MS-compatibility and surface interaction minimization.
• Application of these advancements to diverse matrices (plasma, tissue homogenates) and to hybrid modalities such as peptide–oligonucleotide conjugates.

Conclusions

The study demonstrates that minimizing stainless-steel surfaces in LC flow paths (XSInert/BioInert) can materially improve LC-MS/MS performance for lipophilic peptides by increasing signal and reducing carryover, while oligonucleotide assays showed less dependence on flow-path material under tested conditions. Automated MRM and source optimization (Connect MRM) effectively identified critical parameters (CID gas pressure, interface/focus voltage, collision energy) that control sensitivity. The combined approach—bioinert plumbing plus systematic MS optimization—provides a practical workflow to enhance sensitivity, robustness and throughput for contemporary peptide and oligonucleotide therapeutic bioanalysis.