FACTORS THAT INFLUENCE THE RECOVERY OF HYDROPHOBIC PEPTIDES DURING LC-MS SAMPLE HANDLING

Significance of Topic

Non-specific adsorption of peptides and proteins to sample containers can compromise LC-MS assay sensitivity, accuracy and reproducibility. Hydrophobic peptides are particularly prone to loss during sample storage and handling. Optimizing container selection, solvent composition and storage conditions is essential to preserve analyte recovery and ensure reliable quantitative results.

Study Objectives and Overview

This work evaluates factors influencing hydrophobic peptide recovery during LC-MS sample handling. The goals are to identify key causes of analyte loss and propose a systematic workflow to minimize non-specific binding (NSB) without relying on blocking agents that may interfere with downstream analysis.

Methodology and Instrumentation

Samples of model peptides (desmopressin, teriparatide, glucagon, insulin, melittin) were prepared in 80:20 water/acetonitrile with 0.2% TFA. Recovery experiments compared treated and untreated containers over 24–47 h at 4 °C and room temperature. LC-MS conditions:

• Liquid Chromatography: ACQUITY UPLC I-Class, fixed-loop injector
• Column: CORTECS C18+, 90 Å, 1.6 µm, 2.1×50 mm, 55 °C
• Mobile Phases: A = 0.1% formic acid in water; B = 0.1% formic acid in acetonitrile
• Gradient: 15→45% B in 1.2 min, wash at 95% B (0.5 min), re-equilibration (0.6 min)
• Flow Rate: 0.5 mL/min; Injection Volume: 10 µL
• Mass Spectrometry: Xevo TQ-S with Universal Source

Main Results and Discussion

Step 1: Container Selection

• Glass vials caused complete loss of hydrophobic peptides despite surface treatments.
• Standard polypropylene containers showed poor recovery for hydrophobic analytes.
• Specialty low-adsorption vials (e.g. QuanRecovery) maintained >95% recovery, with greatest benefit for highly hydrophobic melittin.

Step 2: Solvent Composition

• Increasing acetonitrile reduced hydrophobic interactions in polypropylene vials, improving recovery but risking chromatographic retention issues.
• QuanRecovery plates achieved maximal teriparatide recovery without solvent modification.

Step 3: Storage Conditions

• Peptide recovery decreased sharply above ∼10 °C in standard low-bind plates.
• QuanRecovery containers preserved >90% recovery for melittin and glucagon even at room temperature over 47 h.

Benefits and Practical Applications

Adopting appropriate containers and compatible solvents safeguards peptide integrity prior to LC-MS injection. This approach enhances quantitative accuracy, extends method robustness and reduces the need for carrier proteins or blocking agents that can complicate analysis.

Future Trends and Potential Applications

Advances in container surface engineering will further minimize NSB across a wider range of biomolecules. Integration of inert materials with automated sample handling can streamline high-throughput proteomic workflows. Combining optimized sample storage with novel chromatography chemistries promises greater sensitivity for low-abundance targets.

Conclusion

Non-specific peptide adsorption during sample handling is a critical source of LC-MS variability. A three-step strategy—selecting low-adsorption containers, tuning solvent composition and controlling storage conditions—effectively prevents analyte loss without blocking agents. Implementing these practices improves assay sensitivity, reproducibility and quantitative confidence.

References

1. Jung MC. Achieving Maximum Protein and Peptide Recovery, Sensitivity, and Reproducibility Using QuanRecovery Vials and Plates. Waters White Paper 720006543EN (2019).
2. Bobaly B, Sipko E, Fekete J. Challenges in liquid chromatographic characterization of proteins. J Chromatogr B Analyt Technol Biomed Life Sci. 2016;1032:3–22.
3. Rabe M, Verdes D, Seeger S. Understanding protein adsorption phenomena at solid surfaces. Adv Colloid Interface Sci. 2011;162:87–106.