Amino acids are key organic compounds that play important roles in several biological processes, including protein synthesis, cell signaling, and cellular metabolism. Due to their great importance, the analysis of amino acids generates lots of interest.
Not only are amino acids the building blocks of proteins, but they are also the precursors of many biomarkers, have shown antioxidant properties, and are used in dietary supplements. Various analytical techniques have been used for the analysis of amino acids, including capillary electrophoresis, nuclear magnetic resonance, gas, and liquid chromatography.
Introduction to Amino Acid Analysis
Chromatography methods are the most common choice. However, due to the high polarity, low volatility, and absence of strong chromophores, amino acid chromatography faces some challenges, and the identification and separation of amino acids is far from straightforward.
Amino acid chromatography ranges from thin-layer chromatography (TLC), gas chromatography (GC), and liquid chromatography (LC) – including high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC).
Detection methods are based on fluorescence or spectrophotometric detectors and mass spectrometry. Because most amino acids are unable to absorb light, as well as being of low molecular weight, pre- or post-column derivatization is needed in most cases to make amino acids detectable.
The Evolution of Chromatographic Techniques
Ion chromatography was the first technique used for amino acid analysis in the mid-nineties. The amino acid content determination via ion exchange chromatography after post-column derivatization with ninhydrin was groundbreaking. However, the separation with this technique is slow and takes 60-150 minutes, limiting sample throughput.
Hydrophilic interaction liquid chromatography (HILIC) is a common technique used for native amino acids. HILIC utilizes a polar stationary phase (i.e., silica, sulfobetaine) and a mobile phase consisting of two components – a non-polar solvent and a low amount of water. A partition formed between the two mobile phase components and the hydrophilic interactions between the water layer and polar compounds allows for their retention.
HPLC advancements have allowed important breakthroughs in amino acid chromatography, making HPLC one of the methods of choice for the analysis and separation of complex mixtures of amino acids.
Key benefits include shorter analysis time (down to a few minutes), greater resolution, and higher sensitivity, which translate into the ability to perform high-throughput analysis and quantify compounds that might be present at low levels.
Over the years, an increasing number of novel stationary phases have been developed, enhancing HPLC sensitivity and specificity, and the technique has also been combined with several detection methods.
The most commonly used method for amino acid analysis is reversed-phase (RP)-HPLC, where the stationary phase is chemically modified to become non-polar. In general, RP columns come in a variety of materials, length, internal diameter, and particle size. The mobile phase consists of mixtures of organic solvents and water.
Derivatization procedures are still needed in most cases, with only a few methods reported for the analysis of underivatized amino acids. However, dedicated columns for amino acid separations without derivatization are starting to appear on the market.
The Emergence of UPLC in Amino Acid Research
The evolution of UPLC has led to significant improvements in analysis time and efficiency, making it an attractive alternative to traditional HPLC methods in molecular research and amino acid analysis.
The average particle size for UPLC is 1.7 μm (versus 5 μm for ordinary columns), and pressures are up to 15,000 psi (rather than 5000 psi). This allows faster flow rates whilst preserving the separation efficiency, consequently increasing the sample throughput of analysis.
This emerging technique, for instance, allowed the quantitative analysis of underivatized amino acids in body fluids better than other more established methods. It also proved to be a powerful tool for the quantification of free amino acid and total amino acid profiles in complex matrixes such as royal jelly.
Integration of Mass Spectrometry
Since fluorescence or spectrophotometric methods are unable to differentiate amino acids based on the detector signal, effective chromatographic separation is essential to ensure correct identification and quantification.
The integration of mass spectrometry (MS) as a detection method has brought several advantages to the analysis of amino acids, including higher sensitivity and selectivity. MS detectors measure charged molecules and can resolve ions based on their m/z ratio, therefore differentiating between co-eluting species (except for constitutional isomers or enantiomers), making chromatographic separation less problematic.
Tandem mass spectrometry (MS/MS) adds another level of improvement in analyte identification through the fragmentation of molecules, resulting in higher specificity and reproducibility. Both MS and MS/MS are excellent detection methods for amino acid chromatography. However, they often lead to higher equipment costs and require trained personnel.
Novel Chromatography Media and Column Technologies
Current developments in amino acid analysis are associated with the introduction of new reagents and columns, equipment miniaturization, and the use of multidimensional separations. Chromatographic miniaturization uses special columns with sub 2 µm particles in the stationary phase.
This requires specialized pumps and equipment for high pressures (up to 18.000 PSI). The advantages include faster run times, narrower peaks and, better resolution, and minimized matrix effects. Miniaturization also reduces the amount of sample needed for analysis.
Two-dimensional LC (2D-LC) is also advancing the field of amino acid analysis, where better separations are achieved by coupling two LC techniques. 2D-LC takes advantage of orthogonal separation mechanisms by utilizing two columns, where the eluate from the first column enters the second column, increasing the resolution between peaks that are typically difficult to resolve.
In conclusion, chromatography plays a critical role in the analysis of amino acids, allowing the separation and quantification of individual amino acids in complex mixtures. The development of new chromatographic techniques, such as UPLC, as well as the integration of mass spectrometry, has led to significant improvements in analysis time, efficiency, and accuracy.
Further advances in the development of chromatography media and column technologies are also contributing to improving amino acid analysis, opening up new avenues for research and allowing for a deeper understanding of the role of amino acids.
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