Comprehensive Guide to Hydrolysis and Analysis of Amino Acids

The task of accurately separating, identifying, and quantitating amino acids in the research, development, and commercialization of food, feed, and biotherapeutic products is challenging. Common to these applications is the need for adequate sample hydrolysis, an important first step in the workflow for analysis of bound amino acids, such as peptides and proteins. Hydrolysis allows for the analysis of the released amino acids, which can be separated using either ion-exchange or reversed-phase chromatographic methods.

Given the challenges of the sample preparation workflow, this document was compiled to provide useful guidelines for sample hydrolysis. The material contained in this document serves only as a starting point for this process. Depending on the sample type and its complexity, modifications to the described hydrolysis methods may be required. For additional information or troubleshooting tips on hydrolysis or a specific procedure, you should contact the manufacturer of your hydrolysis instrument or the appropriate agency – the Association of Official Analytical Chemists (AOAC). Waters cannot provide guidance on specific sample types.

To analyze the hydrolyzed samples (released amino acids) by HPLC or UPLC, Waters provides many solutions that use pre-column derivatization so that amino acids can  be analyzed by optical detection. As this is an integral part of the workflow, we include brief introductions to Waters HPLC (AccQ•Tag) Method and UPLC Amino Acid Analysis (AccQ•Tag Ultra) Solution. This document does not contain information related to Waters Pico-Tag (phenyl isothiocyanate pre-column derivatization reagent) amino acid analysis method. Although the Pico-Tag method is still available, AccQ•Tag-based methods offer significant performance benefits and are recommended for new applications.

Note: This document is devoted to hydrolysis and amino acid analysis from purified proteins/peptides, foods, and feeds. It is not intended to specifically address the analysis of amino acids in samples of urine, plasma, total parenteral nutrition products, or cell culture media. For information on these applications, please visit www.waters.com/AAA and www.waters.com/Kairos.

General Analysis Guidelines and Recommendations

Given the ubiquitous nature of amino acids in the environment, background contamination can easily contribute 20 pmol or more of some amino acids to the desired sample analysis, particularly for serine (Ser) and glycine (Gly). The amount can be reduced by exercising special precautions. Alternatively, if enough sample is hydrolyzed to provide a minimum of 200 pmol per amino acid, the contribution of background contamination can be limited to less than 10%. Regardless, to take advantage of the sensitivity of modern derivatization techniques and chromatographic systems, which may have detection limits of 1 pmol or less, cleanliness and good laboratory techniques are critical. Sources of contamination are everywhere; dirty glassware, laboratory animals, airborne dust, fingerprints, and talcum powder are common offenders.

Although there is no single perfect system for improving the overall quality of low-level amino acid analyses, the following considerations and practices help:

• Pyrolyze all 6 x 50-mm hydrolysis tubes (p/n: WAT007571) overnight at 500 °C to provide the cleanest glass possible. After you remove the tubes from the cooled furnace, cover them with aluminum foil (cleaned with acetone) and store them in a covered area.
• Be aware of the potential introduction of contaminants from pipets, disposable tips, syringes, and other accessories used in the procedure.
• Prior to hydrolysis, perform all handling steps with clean, powderless gloves and clean forceps. Use of surgical masks may be appropriate.
• At all times, use fresh reagents. Every reagent can be a source of contamination. The best practice is to divide freshly opened bottles into smaller, clean containers.
• Check reagent quality weekly with control blank derivatizations.
• Always use the vapor-phase hydrolysis procedure when possible. Impure HCl can be the source of unreliable hydrolysis. Our most consistent results have been with constant- boiling HCl. High-purity, reagent-grade, concentrated HCI is probably not as clean, and it contains significantly higher levels of chlorine than the constant-boiling grade.
• Test water and buffer constituents for any contribution to background contamination as control samples. For example, laboratory, double-distilled water often contributes unacceptable levels of amino acids. For the best water quality, we highly recommend a purification system (18.2mΩ-cm water).
• A proper control blank for hydrolysis is a sample tube that has been treated exactly as a sample is treated. This includes the hydrolysis step.