Abstract
Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports.
Highlights
Amino acid analysis (AAA) seems to lose some importance over the recent years
We suggest the use of AAAA for aromatic amino acid analysis
Only aromatic amino acids are detected by this setup in the time window between 8 and 28 min
Summary
Amino acid analysis (AAA) seems to lose some importance over the recent years. One of the reasons might be the fact that AAA needs sophisticated analytical devices and remained a complex, relatively expensive, and nontrivial task. A very good separation performance is required to avoid peak overlap. Another traditional option is postcolumn derivatization, which needs ion-exchange columns run with highly optimized buffers and dedicated postcolumn derivatization devices. Gas-chromatographic (GC) techniques have been applied for amino acid analysis for a long time [9]. This approach always requires one or several derivatization steps to make the analytes sufficiently volatile [10]. LC-MS/MS-based methods often do not need any derivatization [15]; they do not achieve complete chromatographic separation in many cases; they heavily rely on the mass-spectrometric selectivity. A more recent development is the use of ICP-MS [16,17,18,19,20] and the detection of the sulphur-containing amino acids
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