Direct Analysis of Multicomponent Vaccine Adjuvants by HPLC with Charged Aerosol Detection

Significance of the Topic

Vaccines often require adjuvants to enhance immune response, reduce dosage, and prolong protection. Analytical methods that accurately quantify adjuvant components, even those lacking UV chromophores, are critical to ensure potency, purity, and stability during development and quality control of human and veterinary vaccines.

Objectives and Study Overview

This study aimed to develop a rapid, sensitive reversed-phase HPLC method coupled with charged aerosol detection (CAD) for direct analysis of multicomponent vaccine adjuvants. The method targets triterpenoid saponins, cholesterol, phospholipids (DPPC), and their degradation products without requiring UV-active moieties or pure primary standards.

Methodology and Instrumentation

Chromatographic separation was achieved on a 2.1 × 100 mm, 1.9 µm PFP column at 45 °C using a gradient of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile/reagent alcohol (10:90, mobile phase B). Flow rate was 0.5 mL/min, injection volume 2 µL, sample temperature 8 °C. Detection employed a Thermo Scientific Dionex Corona Veo RS charged aerosol detector (evaporation at 50 °C) and diode array detection at 210 nm for comparison.

Instrumentation

• Dionex UltiMate 3000 RSLC system with DGP-3600RS pump, SRD-3600 solvent rack with degasser, PS-3000TRS autosampler, and TCC-3000RS column compartment
• DAD 3000RS diode array detector
• Corona Veo RS charged aerosol detector
• Thermo Scientific Chromeleon CDS software v6.8

Main Results and Discussion

All adjuvant components and the Lyso-PC degradation product eluted within 12 minutes with baseline resolution. CAD provided uniform mass-based response, detecting nonvolatile species down to low µg/mL on-column levels, surpassing UV sensitivity and gradient compatibility. Ten replicate injections of 80 µg/mL standards yielded retention time RSD <0.1% and peak area RSD between 0.7% and 1.5%. Calibration over 1.5–400 µg/mL (quadratic fit) produced R² ≥0.9996 and limits of detection of 2–16 µg/mL for all analytes. CAD also quantified impurities (~3.2 µg/mL) without authentic standards.

Benefits and Practical Applications

• Universal detection of nonchromophoric vaccine adjuvants and impurities
• High sensitivity, precision, and gradient compatibility without complex MS instrumentation
• Accurate relative quantitation even without pure standards
• Rapid analysis suitable for routine QC of adjuvant strength, purity, and stability

Future Trends and Potential Applications

Integration of CAD-based HPLC methods into automated high-throughput workflows can accelerate adjuvant screening and formulation optimization. Extending this approach to lipid-based nanoparticles, novel saponin derivatives, and complex biologics will support emerging vaccine platforms. Coupling CAD with orthogonal detectors or MS may further enhance structural characterization and impurity profiling.

Conclusion

The developed HPLC-CAD method offers a precise, sensitive, and universal approach for direct analysis of multicomponent vaccine adjuvants. It overcomes limitations of UV detection and complex instrumentation, delivering reliable quantitation of active ingredients, degradation products, and impurities essential for vaccine development and quality control.

References

• Heidorn M., Martin M., Steiner F., Plante M., McLeod F. Towards Standard-Free Quantitative and Qualitative Analysis in Liquid Chromatography. Thermo Fisher Scientific Application Note, 2011.
• Pedersen G.K. et al. T-helper 1 cells elicited by H5N1 vaccination predict seroprotection. J. Infect. Dis. 2012;206(2):158–166.
• Picard M.D. et al. High-throughput proteomic screening identifies Chlamydia trachomatis antigens. Vaccine. 2012;30(29):4387–4393.