Determination of Proteins and Carbohydrates by 2D HPLC (RPLC and HILIC) with Charged Aerosol and Ultraviolet Detection

Importance of the Topic

Monitoring proteins and carbohydrates in biologic formulations ensures product stability and safety
Carbohydrates stabilize proteins and excipients impact formulation performance
Simultaneous quantification supports quality control and formulation development

Study Objectives and Overview

Develop a 2D HPLC method combining reversed phase and HILIC for concurrent analysis of proteins and seven common carbohydrates
Evaluate sensitivity, linearity, precision and accuracy using UV and charged aerosol detection

Methodology and Instrumentation

• Sample preparation of bovine serum albumin and seven carbohydrates in deionized water with serial dilutions
• 2D HPLC setup featuring a polymeric C18 reversed phase column and a HILIC column interconnected by a 10 port valve and 500 µL sample loop
• Detector configuration including diode array detection at 214 and 280 nm for protein and Corona Veo charged aerosol detection for both analyte classes
• Calibration from low ng to µg on-column levels to determine limits of detection and quantitation

Main Results and Discussion

• Quantitative analysis achieved for BSA and seven carbohydrates with correlation coefficients above 0.997
• Limits of detection ranged from 4 to 9 ng on column for carbohydrates and the limit of quantitation varied from 14 to 29 ng
• Protein LOQ by CAD was 12 ng compared to 79 ng by UV at 280 nm, demonstrating over five-fold sensitivity gain
• Precision was high with relative standard deviations between 0.3 and 4.4% and recoveries between 97.5 and 101.9%

Benefits and Practical Applications

• Unified workflow allows simultaneous protein and carbohydrate determination in a single injection
• Charged aerosol detection extends sensitivity beyond traditional UV methods
• Applicable to quality control, stability testing and formulation optimization in biopharmaceutical laboratories

Future Trends and Applications

• Integration with mass spectrometry for detailed structural characterization
• Automation and high throughput for accelerated screening and process monitoring
• Extension to glycoprotein and peptide profiling in complex matrices
• Miniaturized and portable systems for on-site and real-time analysis

Conclusion

A robust 2D HPLC approach using reversed phase and HILIC along with UV and charged aerosol detection enables accurate, precise and sensitive simultaneous quantitation of proteins and carbohydrates in biologic formulations supporting quality assurance and research efforts