The role of universal detection in modern liquid chromatography

Significance of Topic

Universal detectors in liquid chromatography provide a uniform response for all eluting compounds except the mobile phase. This broad response is vital in applications where selective detectors are inadequate, such as quantification of nonvolatile analytes, impurity profiling and emerging lipid nanoparticle therapeutics.

Objectives and Study Overview

The study reviews universal detection techniques and illustrates the use of charged aerosol detection (CAD) coupled with ultra-high performance liquid chromatography (UHPLC) to characterize lipid nanoparticle (LNP) formulations used in mRNA therapeutics.

Methodology and Instrumentation

Universal detectors compared include:

• Refractive Index Detector (RID): simple and cost-effective but limited to isocratic methods and temperature control.
• Evaporative Light Scattering Detector (ELSD): medium sensitivity, compatible with many solvents but limited linearity.
• Charged Aerosol Detector (CAD): broad dynamic range, uniform response and compatibility with volatile mobile phases.
• Mass Spectrometry (MS): highest sensitivity and specificity, requires ionizable compounds and complex maintenance.
• Flame Ionization Detector (FID): universal for hydrocarbons, low sensitivity for polar analytes.

The UHPLC-CAD method for LNP analysis employed:

• Vanquish UHPLC system with Charged Aerosol Detector.
• Accucore C30 column (3.0 × 100 mm, 2.6 μm).
• Gradient of mobile phase A (0.1% formic acid in 50:50 acetonitrile/water) and mobile phase B (0.1% formic acid in 60:30:10 isopropanol/acetonitrile/water) at 0.9 mL/min.
• Column temperature 50 °C, post-column cooler 40 °C, CAD evaporator 35 °C, gas resolution mode Analytical.
• Quadratic calibration for each lipid standard with coefficients > 0.999.

Main Results and Discussion

The comparative evaluation highlighted CAD as a versatile detector with medium-high sensitivity, good linearity and ease of use. UHPLC-CAD separation resolved key lipid components—ionizable cationic lipid, PEGylated lipid, phospholipid and cholesterol—in under 12 minutes. Calibration curves demonstrated high precision over the analytical range. The method enables accurate quantification of nonchromophoric lipids essential for consistent LNP formulation.

Benefits and Practical Applications

• Uniform detection for lipids lacking chromophores or ionizable groups.
• Robust quality control tool for vaccine formulation and process monitoring.
• Complementary to UV and MS, offering enhanced confidentiality of data and simplified solvent requirements.

Future Trends and Possibilities

Advances may include integration of CAD with high-resolution MS for simultaneous structural and quantitative analysis, development of more volatile mobile phase additives to broaden detector compatibility, and automation in nanoparticle characterization for regulatory compliance in gene and mRNA therapies.

Conclusion

Universal detection in liquid chromatography, particularly using CAD, delivers comprehensive, reliable data for diverse analytes. The UHPLC-CAD approach for LNP analysis addresses current demands in pharmaceutical development, ensuring precise lipid quantification in emerging mRNA vaccine platforms.

Reference

• Heidorn M. The role of universal detection in modern liquid chromatography. 2022.
• Schoenmaker L. et al. mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. Int J Pharmacol. 2021;601:120586.
• Zatsepin TS. et al. Lipid nanoparticles for targeted siRNA delivery. Int J Nanomedicine. 2016.