Amino acid analysis of mammalian cell culture medium by liquid chromatography with UV and fluorescence detection and derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate

Significance of the topic

The analysis of free amino acids in mammalian cell culture media is critical for monitoring cell metabolism, optimizing nutrient supplementation, and maximizing protein therapeutic yields. A reliable, cost-effective method that delivers high sensitivity, reproducibility, and long-term stability supports both research and industrial bioprocessing workflows.

Objectives and overview of the study

This application note presents a one-year performance evaluation of amino acid quantification in CHO cell culture supernatants using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and reversed-phase UHPLC with UV and fluorescence detection. Key goals included:

• Simplifying analysis without the need for mass spectrometry.
• Demonstrating robust retention time and peak area reproducibility over weeks and months.
• Assessing column longevity under daily injection routines.

Methodology and instrumentation

Samples of serum-free CHO bioreactor supernatant were thawed, centrifuged and diluted 100-fold in ultrapure water. Standards covering 22 amino acids were prepared at eight concentration levels (0–250 pmol/µL) with an internal check at 100 pmol/µL. Derivatization was carried out by mixing 10 µL of sample or standard with 70 µL borate buffer (50 mM, pH 8.5) and 20 µL AQC reagent (4 mg/mL in acetonitrile), followed by heating at 55 °C for 10 minutes.

Used instrumentation:

• Thermo Scientific Vanquish Flex UHPLC system (Binary or Quaternary pump).
• Acclaim Vanquish C18 column, 2.1×150 mm, 2.2 µm.
• Vanquish Variable Wavelength UV/Vis Detector (260 nm) and optional Fluorescence Detector (Ex 266 nm/Em 473 nm).
• Chromeleon 7.2 CDS for data acquisition and processing.

Mobile phases comprised 50 mM ammonium formate (pH 2.9) and acetonitrile. A shallow gradient over 18 minutes (binary system) separated 22 derivatized amino acids. A high-throughput variant on Vanquish Horizon reduced run time to under 9 minutes.

Main results and discussion

The method yielded excellent calibration linearity (R² > 0.999 for most amino acids), high accuracy (< 5% error) and tight reproducibility (retention time RSD < 0.2%, peak area RSD < 0.5%). Over 4.5 months, histidine retention time RSD was 1.0% and area RSD was 3.5%. Column lifetime exceeded 1300 injections on average (up to 2200) before resolution of the critical Arg/Gly pair fell below USP specifications. Sample quantification at days 7, 9 and 11 in a CHO bioreactor reflected expected nutrient uptake and metabolite patterns.

Benefits and practical applications

• Cost-effective alternative to mass spectrometry-based assays.
• Robust quantification of 22 primary and secondary amino acids in bioprocess monitoring.
• Long column life and minimal maintenance improve laboratory throughput and reduce consumable costs.
• Ready-to-use eWorkflows simplify method transfer across Vanquish UHPLC platforms.

Future trends and applications

Potential developments include integrating rapid UHPLC protocols with high-resolution mass spectrometry for deeper metabolomic profiling, automating on-line monitoring directly in bioreactors, and extending derivatization strategies to peptides and protein hydrolysates. Miniaturized flow cells and microfluidic UHPLC systems may further reduce sample consumption and analysis time.

Conclusion

The AQC pre-column derivatization method combined with Vanquish UHPLC offers a simple, sensitive and durable solution for routine amino acid analysis in mammalian cell culture media. Its reproducibility, extended column lifetime and flexibility across various UHPLC configurations make it well suited for both research and industrial applications.

References

1. Cohen, S. A. and Michaud, D. P. Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for hydrolysate amino acids analysis via HPLC. Anal. Biochem. 1993, 211, 279–287.
2. Reverter, M.; Lundh, T.; Lindberg, J. E. Determination of free amino acids in pig plasma by pre-column derivatization with AQC and HPLC. J. Chromatogr. B 1998, 696, 1–8.
3. HPLC Method Transfer Calculator, Thermo Fisher Scientific, available online.