Evaluating LC methods for enhanced charged aerosol detector response: a case study using underivatized amino acids

Significance of the topic

Analysis of underivatized amino acids is crucial for pharmaceutical quality control, yet conventional derivatization approaches are time‐consuming and costly. Charged aerosol detection (CAD) offers a universal, label‐free means to quantify polar, non‐volatile analytes, enabling simultaneous impurity profiling.

Objectives and study overview

This study compares ion‐pair chromatography (IPC) and hydrophilic interaction chromatography (HILIC) coupled to CAD for profiling branched‐chain amino acids (BCAA: leucine, isoleucine, valine) and their impurities. Key performance indicators include selectivity, sensitivity, limit of quantitation, linearity, and analysis time.

Methodology and instrumentation

Two chromatographic setups were implemented: IPC using HFBA/TFA reagents with a polar‐embedded column and gradient elution, and HILIC employing an amide‐bonded core‐shell column under isocratic high‐organic conditions. Samples and standards were prepared in aqueous or ACN‐rich mobile phase blends. CAD parameters were standardized at 50 °C evaporation, 5 s filter, and 10 Hz data acquisition.

Used Instrumentation

• Thermo Scientific Vanquish Flex UHPLC system with Binary Pump F, Split Sampler FT, and Column Compartment H
• Charged Aerosol Detector H and Corona 1010 nitrogen generator
• Accucore 150 Amide HILIC column and Acclaim Polar Advantage II column
• Thermo Scientific Chromeleon CDS for data acquisition and analysis

Main results and discussion

Both methods separated BCAA and related impurities within 16–20 min. HILIC‐CAD achieved approximately threefold higher signal‐to‐noise and half the LOQs of IPC‐CAD. IPC required higher sample concentrations and exhibited retention variability due to ion‐pair equilibrium. The core‐shell amide HILIC column minimized background noise and improved method robustness.

Benefits and practical applications

• Simplified workflow without derivatization reduces analysis time and cost
• Universal CAD response enables detection of non‐chromophoric and semi‐volatile impurities
• HILIC‐CAD is suited for low‐level impurity profiling in pharmaceutical QC

Future trends and possible applications

Innovations in column coatings and mobile phase additives will further enhance HILIC stability and sensitivity. Coupling CAD with high-resolution mass spectrometry could expand analyte identification. Broader application to peptides, organic acids, and metabolomic targets is expected.

Conclusion

HILIC combined with CAD outperforms IPC for underivatized BCAA impurity analysis, offering greater sensitivity, lower detection limits, and robust performance. The simplified protocol represents a viable alternative to derivatization‐based methods in pharmaceutical analytics.

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