Amino acid analysis

The determination and monitoring of essential nutrients within the cell are vital for maximising protein yield and ensuring a consistent synthesis process. Waters’ UPLC® Amino Acid Analysis (AAA) turnkey solution is designed to deliver accurate and highly efficient amino acid analysis.

The HPLC solution for amino acid analysis provides control over the key factors that typically cause variability in amino acid analysis results. From optimised equipment and controlled reagents to standardised software packages, methods, report templates and qualification tools, you are guided by a reliable methodology – a detailed approach that ensures the successful analysis of every sample.

The UPLC system for amino acid analysis combines cutting-edge technologies in the fields of instrumentation, chemistry, software, qualification tools and services – all specifically optimised for exceptional, method-driven performance. Developed by scientists for scientists, this solution is considered the best choice for the needs of your programme.

The value of amino acids

Amino acid analysis is essential in several fields of research and is a fundamental tool for food analysis. The application of such analysis imposes various requirements on the analytical method, as amino acids fulfil different roles.

Thanks to amino acid analysis by HPLC, you achieve superior resolution and sensitivity in a short analysis time:

The AccQ • Tag™ chemistry suite, used in the analysis of amino acids by HPLC, is a comprehensive and fully validated set of reagents, columns and eluents, optimised for use with ACQUITY UPLC systems, ACQUITY UPLC H-Class and ACQUITY UPLC H-Class Bio systems. Thanks to the excellent separation capability for all amino acids, you can identify peaks and determine their quantitative composition, which in turn ensures the accuracy of the results obtained.

This chemistry is based on the well-known and straightforward AccQ•Tag derivatisation method. Primary and secondary amino acids react with a single reagent, AccQ•Fluor™, during a high-throughput batch process, resulting in exceptionally stable derivatives. High-quality separation is achieved using pre-qualified AccQ•Tag Ultra UPLC columns and mobile phases. The derivatised amino acids are quantified at the sub-picomolar level by single-wavelength UV detection.

The validation services available for the analysis of amino acids by HPLC meet the most stringent regulatory requirements. Installation, operational and performance qualification (IQ / OQ / PQ) protocols ensure that every component of the system functions as intended. An additional performance qualification (PQ) specific to amino acid analysis is also available to verify system performance and ensure accurate results.

Performance: The solution for analysing amino acids by HPLC provides high resolution to ensure the reliability of the method, with good quantitative results. The short cycle time does not affect the accuracy of the results.

Effective, reliable analysis of food and feed: soya meal hydrolysate

Key applications of amino acid analysis: Two samples of hydrolysed soya meal were analysed. The top chromatogram shows the untreated sample, whilst the bottom one shows the results of the oxidation of formic acid. It is also worth noting the appearance of cysteine and methionine sulphonic acid in the oxidised sample.

For biopharmaceutical purposes

Accurate and reliable amino acid analysis provides key information for a range of research projects, as well as for routine quality control and process monitoring. In biochemical structural analysis, the amino acid composition of hydrolysed proteins is used to confirm the sequence and determine extinction coefficients. Unlike measurements of protein amino acids, free amino acids are important indicators of metabolic status and genetic origin. Such analyses are required for various applications, such as optimising cell culture conditions in the production of biopharmaceuticals, as well as monitoring industrial-scale fermentation processes.

The demand for these applications led to the invention of amino acid analysers and the continuous refinement of the technology over several decades. A comprehensive software solution was developed on a ‘turnkey’ basis. The methodology is based on a well-established derivatisation protocol followed by high-performance chromatographic separation. Amino acids are derivatised with 6-aminoquinolyl-N-hydroxysuccinimyl carbamate (AQC) under strongly aqueous conditions. The derivatives are separated using ultra-high-performance liquid chromatography (UPLC). The enhanced resolution of UPLC ensures a reliable chromatographic method that delivers accurate measurement results.

This method has been widely used in biopharmaceutical laboratories since its introduction in 2007. This ready-to-use solution for the analysis of amino acids by HPLC is now available for ACQUITY UPLC (binary solvent mixing) and ACQUITY UPLC H-Class and Bio- (quaternary mixing) H-Class, as well as detection protocols with documented linearity and accuracy. Continuous refinement of this application solution ensures the most convenient and efficient development of reliable amino acid analyses.

Amino acid analysis is important for various research projects, as well as for routine quality control and process monitoring. Successful analysis requires careful consideration of sample preparation, separation and detection of amino acids. The HPLC amino acid analysis system is designed to provide accurate quantification of a wide range of sample types. Amino acids are derivatised by reaction with 6-aminoquinolyl-N-hydroxysuccinimylcarbamate and separated by reverse-phase HPLC. The suitability of this method has been confirmed in several programmes.

The amino acid composition of acid-hydrolysed pure proteins was compared with the expected values derived from the known sequence. Amino acids, as metabolic indicators, were measured in growing cell cultures, various foodstuffs and beverages. The nutritional content of feed was also determined following hydrolysis. Optimising the processing for each sample type yielded more reliable results. The exceptional sensitivity of the derivatisation and separation methods simplified certain aspects of sample preparation, thereby facilitating the optimisation of others.

The use of microwave hydrolysis methods served as a practical addition to the overall analytical process for feed samples. Feed samples were subjected to acid hydrolysis, both with and without oxidation, as well as alkaline hydrolysis, to determine the complete amino acid profile and, consequently, the protein content. The sample processing throughput was significantly higher with this approach and was compatible with the analytical throughput of the system.

These experiments demonstrate the suitability of the sample preparation procedures and the amino acid analysis system.

Analysis of hydrolysate and cell culture standards

Analysis of cell culture: The top chromatogram shows the results of the analysis of the hydrolysate standard at 10 pmol/ml, whilst the lower chromatogram shows a standard mixture of a larger number of amino acids detected in the cell culture medium, also at 10 pmol/ml. Adapting the method requires only a change in the dilution of eluent A concentrate, which is added as part of the system’s total solution.

Direct analysis of cell cultures

Aliquots of the cell culture medium were taken directly from the bioreactor producing the biopharmaceutical protein. Samples were collected over a period of six days, and no sample preparation was required prior to the amino acid analysis. Trends in amino acid concentrations can be interpreted both as the cells’ response to growth and as a response to the amount consumed.