Analyte adhesion to LC vials: impact of surface chemistry on recovery at low concentrations

Importance of the topic

Non-specific binding (NSB) of proteins and peptides to sample containers is a pervasive source of error in liquid chromatography workflows, particularly as bioanalytical assays push toward ever-lower analyte concentrations. Losses to vial surfaces can produce biased, non-linear, and irreproducible recoveries that degrade sensitivity, precision, and accuracy. Understanding how vial surface chemistry and routine analytical parameters interact with analyte properties is therefore critical for robust method development in drug discovery and biotherapeutic analysis.

Goals and overview of the study

The study aimed to elucidate mechanisms of analyte adhesion to LC vial surfaces and demonstrate practical approaches to improve recovery at trace concentrations. Using a panel of peptides and proteins with diverse properties, the work compared conventional vial materials and low-adsorption technologies while surveying controllable method variables (autosampler temperature, diluent composition, storage time, and vial fill volume). The objective was to identify material- and method-level mitigation strategies that are implementable without major instrument changes.

Methodology

Analytical design and samples:

• A representative set of analytes: insulin, glucagon, semaglutide, Magainin 2, Mastoparan X, tryptic cytochrome c digest, NISTmAb, and trastuzumab.
• Concentration strategy: a high reference point (100–200 μg/mL) where adsorption is negligible and trace concentrations (varied per analyte) where adsorption effects become measurable.
• Diluents tested included 0.1% formic acid (FA) in water, 0.1% trifluoroacetic acid (TFA) in water, and mixtures with acetonitrile (0% vs 20% ACN in selected experiments).
• Design of experiments (DoE) variables: autosampler temperature (5 °C vs 25 °C), vial storage time (6, 12, 24 h), vial fill volume (1/3 vs 2/3), and vial material.

Vial materials compared:

• Unmodified borosilicate glass
• Silanized (deactivated) glass
• Standard polypropylene (PP)
• Vendor W low-adsorption vials (commercial comparator)
• Thermo Fisher SureSTART GOLD-Grade polypropylene (engineered surface with a silicon dioxide–based architecture)

Chromatography and detection:

• Thermo Scientific Vanquish UHPLC platform with diode array detection; Chromeleon CDS for data processing.
• Columns: Hypersil GOLD C18 Selectivity and Hypersil GOLD Peptide for small proteins/peptides; MAbPac RP Phenyl for monoclonal antibodies.
• Typical injection volume 10 μL, autosampler 5 °C, detector wavelengths 214 nm or 280 nm depending on analyte.

Instrumentation used

The core instrumentation and consumables included:

• Thermo Scientific Vanquish Flex UHPLC system (System Base Vanquish Duo, Vanquish Dual Pump F, Vanquish Split Sampler FT, Column Compartment H, Vanquish Diode Array Detector with semi-micro biocompatible flow cell).
• SureSTART vial products: various borosilicate glass and polypropylene microvials, silanized and GOLD-Grade polypropylene inserts and screw-top vials; AVCS screw caps with silicone/PTFE septa.
• Other: Hypersil GOLD peptide/C18 columns and MAbPac RP Phenyl column; standard LC solvents (FA/TFA in water and ACN), pipettes, vortex mixer.

Main results and discussion

Concentration dependence and general trends:

• At high analyte concentrations, surface sites tend to saturate and adsorption effects are masked; at trace levels the surface-to-analyte ratio increases and NSB becomes a major determinant of observed recovery.
• Recovery and reproducibility were strongly analyte-dependent, driven by molecular properties such as hydrophobic patches, fatty-acid conjugation (e.g., semaglutide), and net charge (e.g., cationic peptides).

Impact of vial material:

• Standard PP and silanized glass often showed lower recoveries for certain analytes (notably insulin and hydrophobic peptides) compared with engineered low-adsorption surfaces.
• GOLD-Grade polypropylene delivered the most consistent and often highest recoveries across a broad panel, maintaining detectable signals at the lowest tested concentrations where other materials failed or produced poor repeatability.
• Vendor W low-adsorption vials performed comparably for many peptides but exhibited subtle differences for specific fragments; in several cases GOLD-Grade PP yielded higher recovery for hydrophobic peptides.

Effect of diluent organic content and pH modifiers:

• Adding organic modifier (20% ACN) reduced hydrophobic interactions and improved recovery for many analytes, making solvent composition a major mitigant for hydrophobic adsorption.
• Cationic peptides (Magainin 2, Mastoparan X) displayed strong pH-dependent adsorption: acidic diluent (0.1% TFA) suppressed surface electrostatic attraction to negatively charged glass surfaces and improved recovery, whereas neutral water diluent exacerbated loss—except in GOLD-Grade polypropylene where recovery remained robust regardless of diluent pH.

Temperature, storage time, and fill volume:

• Temperature effects were analyte-specific: increasing autosampler temperature sometimes improved desorption but could also reduce recovery or risk analyte instability; therefore temperature optimization requires analyte-specific assessment.
• Storage time in the autosampler produced measurable but generally smaller effects than concentration or material; ranking of vial performance remained stable over 24 h.
• Under the tested conditions (V-bottom vials), fill volume (1/3 vs 2/3) did not significantly influence recovery.

Peptide mapping and digest analysis:

• Trypsin digest of cytochrome c revealed that hydrophobic, later-eluting peptide fragments were selectively depleted when stored in standard polypropylene, sometimes falling below detection limits.
• GOLD-Grade polypropylene preserved these hydrophobic fragments, improving sequence coverage and the reliability of peptide-level quantitation.

Large proteins and monoclonal antibodies:

• For large mAbs (NISTmAb, trastuzumab) vial surface chemistry dominated recovery behaviour at low concentrations; GOLD-Grade polypropylene again provided superior recovery and injection-to-injection precision relative to conventional materials.

Practical benefits and applications

Key practical takeaways:

• Selection of vial material should be an explicit decision in method development, informed by analyte physicochemical properties rather than by historical lab practice.
• Combining modest method changes (use of acidic modifiers or small amounts of organic solvent) with engineered low-adsorption vials yields the most robust strategy for trace-level analyses.
• Surface-engineered vials such as GOLD-Grade polypropylene offer a cost-effective means to increase sensitivity, preserve low-abundance species, and improve reproducibility without hardware modification.

Applicable workflows:

• Bioanalysis in drug discovery (peptide therapeutics, small proteins), QC labs monitoring low-concentration analytes, peptide mapping for sequence coverage, and LC-MS workflows sensitive to analyte loss and MS adduct formation.

Future trends and potential applications

Anticipated developments and opportunities:

• Broader adoption of engineered low-adsorption surfaces across vial formats and plate geometries to support high-throughput automated workflows.
• Integration of surface characterization (contact angle, XPS, AFM) into vial development and selection to better correlate surface properties with analyte-specific adsorption behavior.
• Standardized qualification protocols that include vial material as a validation parameter for trace-level assays, reducing inter-laboratory variability.
• Development of tailored surface chemistries for particularly challenging analytes (highly hydrophobic conjugates, extreme basic peptides, or sticky mAbs) and expansion to materials compatible with downstream MS (minimizing adducts).
• Method packages that combine optimized vial chemistry, solvent composition, and autosampler management to streamline low-level protein/peptide analysis in regulated environments.

Conclusion

Surface-induced analyte loss is a major contributor to poor recovery and variability in low-concentration LC analyses of peptides and proteins. While method adjustments (organic content, pH, temperature) can mitigate specific interactions, surface-engineered vial technologies that address multiple adsorption mechanisms simultaneously provide the most consistent and broadly applicable improvement in recovery and precision. Selecting vial material appropriate to analyte class and method conditions is an essential and cost-effective step to enhance assay sensitivity and reliability.

References

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4. Kristensen K, Henriksen JR, Andresen TL. Adsorption of Cationic Peptides to Solid Surfaces of Glass and Plastic. PLoS ONE. 2015;10(5):e0122419.