Amino acids serve not only as building blocks of proteins but also as cellular signaling molecules involved in regulating gene expression and the cascade of protein phosphorylation. Additionally, amino acids function as the primary energy source in the intestines and serve as precursors for the synthesis of neurotransmitters, polyamines, and nitric oxide. Quantitative analysis of amino acids is crucial for disease diagnosis and understanding the impact of different nutritional states on physiological functions.
Amino acid analysis methods involve determining the content of free amino acids in peptides or protein samples. Various methods are employed in the clinical diagnosis of amino acid metabolism disorders, biomedical research, biotechnology, food science, and other fields. Commonly used methods include precolumn derivatization-ion exchange chromatography, gas chromatography, liquid chromatography, and capillary electrophoresis.
In the past decades, amino acid analysis primarily relied on precolumn derivatization-ion exchange chromatography and precolumn derivatization-liquid chromatography using ortho-phthalaldehyde (OPA) or fluorenylmethyloxycarbonyl (FMOC) for separation, followed by detection using photometric methods. Although precolumn derivatization-ion exchange chromatography offers high reproducibility, it is time-consuming and costly. On the other hand, precolumn derivatization-liquid chromatography is sensitive and rapid but lacks strong specificity for the analytes. Moreover, both methods cannot differentiate between 14N and 15N-labeled amino acids, limiting their utility for isotope-labeled quantification.
With the advancement of modern amino acid analysis techniques, mass spectrometry has emerged as a vital tool, providing high selectivity and sensitivity in amino acid analysis.
Gas Chromatography-Mass Spectrometry (GC-MS)
Gas Chromatography-Mass Spectrometry (GC-MS) has found application in amino acid analysis since the 1970s. GC-MS, a crucial method for analyzing volatile or semi-volatile small molecules, has some limitations in detecting non-volatile and high-molecular-weight metabolites. Amino acid analysis using GC necessitates derivatization, with primary methods including silylation, alkylation, and acylation. Silylation, primarily achieved through N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and N-(tert-Butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) under dry conditions or esterification/acylation, is the predominant method. However, not all silylated amino acid derivatives are stable; for instance, arginine derivatives may decompose into ornithine and glutamic acid, with the latter transforming into pyroglutamic acid. Additionally, the presence of moisture in the sample during the derivatization process significantly affects the structure of the derivatives. While silylation is widely used, it suffers from the drawback of a single analyte corresponding to multiple by-products.
To overcome these limitations, researchers have employed chloroformate for indirect alkylation or acylation derivatization. The indirect alkylation reaction between amino acids and chloroformate can take place directly in the liquid phase without the need to remove proteins beforehand. Amino acid acylation and esterification derivatization are completed through the reaction with anhydrides/alcohols such as pentafluoropropionic anhydride and isopropanol or with chloroformate under conditions with ethanol and pyridine. The reaction between amino acids and derivatization reagents is rapid, and the derivatives are directly introduced into the mass spectrometer after organic acid extraction.
GC-MS analysis offers high sensitivity, good resolution, and repeatability, making it suitable for amino acid quantification in diseases, plants, and the identification of amino acid diastereomers.
Liquid Chromatography-Mass Spectrometry
Liquid Chromatography-Mass Spectrometry (LC-MS) is extensively employed in amino acid analysis, specifically in the pre-column derivatization-liquid chromatography-optical detection method. Various derivatization reagents are utilized, including ortho-phthalaldehyde (OPA), dansyl chloride (dansyl-cl), 2,4-dinitrofluorobenzene (DNFB), phenyl isothiocyanate (PITC), fluorescein isothiocyanate (FITC), chloroformates, and amino-benzoic acid esters.
While this method demonstrates good repeatability and high sensitivity, its specificity is not robust. Analysis uncertainties may arise from the presence of non-protein amino acids or compounds with similar retention times in complex biological samples. Additionally, optical detection methods are not suitable for analyzing samples containing isotopes.
In recent years, High-Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) has gained popularity for amino acid analysis due to its high specificity, broad applicability to both large and small molecules, and flexibility. Common liquid chromatography-mass spectrometry techniques for amino acid analysis include Ion Pair Liquid Chromatography Tandem Mass Spectrometry (IP-LC-MS/MS), Isobaric Tag for Relative and Absolute Quantitation-Liquid Chromatography Tandem Mass Spectrometry (iTRAQ-LC-MS/MS), and Hydrophilic Interaction Chromatography-Mass Spectrometry (HILIC-MS).
IP-LC-MS/MS can separate non-derivatized amino acids using a reverse-phase C18 column, with volatile IP reagents such as perfluorocarboxylic acids suitable for liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) detection.
Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) technology, introduced by Applied Biosystems, is an external isotope labeling technique. After reacting with a derivatization reagent, the analyte carries an isobaric tag. The reporter group within the tag undergoes collision-induced dissociation in tandem mass spectrometry, enabling detection. The slight difference in molecular mass between different reporter groups allows for amino acid quantification. While iTRAQ-LC-MS/MS has the advantage of providing an internal standard for each analyte, precision in quantifying sulfur-containing amino acids such as cysteine and methionine may be less accurate.
Select Services
For urine amino acid analysis, methods such as capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF-MS), 13C nuclear magnetic resonance spectroscopy (NMR), gas chromatography-mass spectrometry (GC-MS), and isotope dilution LC-MS/MS are currently employed.
HILIC utilizes a hydrophilic stationary phase, such as silica gel columns, polar polymer fillers, or ion-exchange adsorbents, in combination with a reverse-phase solvent system to separate polar substances. As a liquid chromatography mode for separating polar compounds, HILIC finds applications in drug analysis, metabolomics, and proteomics. HILIC's simple mobile phase composition, high separation efficiency, and compatibility with mass spectrometry make it a valuable tool in amino acid analysis.
Amino acid sequence identification by LC/MS (Zhao et al., 2020).
Capillary Electrophoresis (CE)
Capillary Electrophoresis (CE) is a separation technique that emerged in the 1980s, providing a low-cost, high-resolution, and highly sensitive micro-separation platform for the analysis of amino acids and their enantiomers, utilizing capillary electrophoresis separation and laser-induced fluorescence detection [42-43]. Amino acid analysis using capillary electrophoresis does not require derivatization, although the sensitivity of detection can be enhanced when amino acids are derivatized. Most derivatization reagents applicable to High-Performance Liquid Chromatography (HPLC), such as FMOC, NDA, OPA, and FITC, are also suitable for capillary electrophoresis methods [44]. Moreover, sample enrichment and chemical derivatization in capillary electrophoresis are easily integrated online, eliminating the need for complex offline sample handling steps, making automation straightforward. However, precise and reliable quantification of complex samples through photometric or electrochemical detection methods after CE separation is somewhat lacking.
CE-ESI-MS is a highly selective, sensitive, and reliable method for identifying amino acid spectra in complex biological samples with minimal sample processing. Mass spectrometric analysis of amino acids and their derivative compounds is typically conducted under strong acidic conditions in positive ion mode detection. With advancements in online sample enrichment, chemical derivatization, and ESI interface design, the resolution of amino acid isomers and the detection limits of low-concentration unstable amino thiol compounds have been further improved.
Comparison of Amino Acid Analysis Methods
| Method | Advantages | Disadvantages | LOD/LOQ (μmol/L) |
|---|---|---|---|
| Amino Acid Analyzer | Good repeatability | High cost, low sensitivity | - |
| GC-MS | Good repeatability | Not suitable for heat-sensitive amino acid derivatives; complex derivatization steps; cannot detect Arginine | LOD: 0.03~12 LOQ: 0.3~30 |
| LC-Methods with Optical Detection | Good repeatability, good linearity | Requires protein and amino acid derivatization; limited specificity; cannot differentiate co-eluting substances | LOQ: 5 |
| IP-LC-MS-MS | No derivatization required; good resolution for polar amino acids | Requires protein removal; may experience ion suppression; IP reagents may contaminate the analysis system | LOD: 3×10^-4~9×10^-4 |
| HILIC | No derivatization required; compatible with mass spectrometry; suitable for analyzing polar compounds | Requires protein removal; moderate repeatability; prone to ion suppression | LOD: 5, LOQ: 10 |
| iTRAQ | High resolution, fast separation, each analyte has an internal standard | Requires protein removal; poor recovery for sulfur-containing amino acids | LOQ: 2~10 |
| CE-MS | No derivatization required; small sample volume; requires protein removal; suitable for small sample quantities | - | LOD: 0.1~14 |
| Direct-infusion MS-MS | No sample preparation required; high throughput | Requires derivatization; cannot differentiate positional isomers | - |
| NMR | No need for separation and derivatization; requires small sample volume; strong quantitative capability | Low sensitivity; long analysis time | LOD: 20~312 |
Reference
- Zhao, Yan, Qi Zhao, and Qingyu Lu. "Purification, structural analysis, and stability of antioxidant peptides from purple wheat bran." BMC chemistry 14 (2020): 1-12.
