Analysis of Amino Acids in Spent Media Using Automated Precolumn Derivatization with the Agilent 1290 Infinity II Bio LC System

Significance of the topic

Amino acids are critical nutrients in cell culture media, directly influencing cell growth, metabolism and biopharmaceutical production. Monitoring their concentration in spent media prevents nutrient depletion, guides timely media replenishment and supports consistent culture performance. An efficient, automated analytical workflow enhances reproducibility and throughput in research, process development and quality control.

Objectives and overview

This study demonstrates a streamlined method for quantitative profiling of amino acid changes in spent cell culture media. HeLa and HepG2 cultures were sampled over 0, 16, 24, 48 and 72 hours. Automated precolumn derivatization was integrated with chromatographic separation on an Agilent 1290 Infinity II Bio LC system, coupled to both diode array (DAD) and fluorescence (FLD) detectors. A fast gradient extension was developed to shorten analysis time while preserving resolution.

Methodology and instrumentation

Sample preparation involved only 10-fold dilution of spent media with water and internal standard. Precolumn derivatization employed OPA for primary and FMOC for secondary amino acids, executed automatically by the multisampler injector program. Chromatography was performed on an Agilent AdvanceBio AAA column (3.0×100 mm, 2.7 µm) with a guard column.

Mobile phases:

• Phase A: 10 mM Na₂HPO₄, 10 mM Na₂B₄O₇, pH 8.2
• Phase B: acetonitrile:methanol:water (45:45:10, v/v/v)

Two gradient profiles were used:

• Gradient 1: 17 min runtime at 0.6 mL/min (2 % to 100 % B)
• Gradient 2: 11 min runtime at 1.2 mL/min (2 % to 100 % B)

Detection:

• DAD at 338 nm (ref. 390 nm)
• FLD at Ex/Em 345/455 nm switching to 265/315 nm at 12 min

Instrumentation used

• Agilent 1290 Infinity II Bio high-speed pump
• Agilent 1290 Infinity II Bio multisampler
• Agilent 1290 Infinity II multicolumn thermostat
• Agilent 1290 Infinity II diode array detector (variable slit)
• Agilent 1260 Infinity II fluorescence detector

Main results and discussion

Twenty amino acids plus two internal standards were well separated under both gradients. FLD provided superior sensitivity for most analytes; cystine required DAD detection due to low fluorescence yield. In spent media, key nutrients such as glutamine, serine, arginine and tyrosine showed decreasing trends with culture time, whereas glycine and alanine increased. Quantitative profiles over 0–72 h revealed metabolic phase–dependent consumption and production patterns in HeLa and HepG2 cells. The fast gradient achieved comparable chromatographic performance in under 11 minutes, suitable for high-throughput analysis.

Benefits and practical applications

The automated workflow minimizes manual handling errors and accelerates sample throughput. Simple dilution avoids complex extraction. Dual detection ensures reliable quantification across diverse amino acids. This approach supports media optimization, bioprocess monitoring, quality control and rapid decision-making in cell culture workflows.

Future trends and potential applications

Integration of online sampling and real-time LC analysis could enable continuous media monitoring in bioreactors. Coupling with mass spectrometry may extend coverage to broader metabolite classes. Further miniaturization and higher flow rates promise even faster assays. Machine-learning tools can mine time-course data for predictive control of nutrient feed strategies.

Conclusion

The described Agilent 1290 Infinity II Bio LC method delivers rapid, automated and robust analysis of amino acids in spent cell culture media. It combines automated precolumn derivatization, dual detection and a fast gradient to yield high sensitivity and throughput, facilitating effective monitoring and optimization of cell culture processes.

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