Amino acid analysis – AAA in short – is performed to quantitatively determine the number of amino acid residues in a peptide and quantify the overall peptide content.
We employ an internal standard method (enantiomer labelling, ELAB) to account for sample diversity and analyte discrimination during e.g. sample preparation, derivatisation, separation and detection.
Here, a defined amount of a substance, chemically and physically as similar to the analyte as possible, is added to the sample. Thus, sample and standard are co-prepared and co-analysed. Hereby both moieties experience the exact same environment and are therefore discriminated in the exact same manor.
In our case, the enantiomer of an optically active analyte (amino acid) is added as the standard. For our AAA method, a mixture of D-amino acids with defined composition is used.
Ensuing sample preparation and derivatisation, the separation is performed on an optically active phase which separates all chiral components.
Using the enantiomer as internal standard gives the following advantages:
If the sample itself contains a certain amount of the exact same enantiomer which would be used as the standard, these amounts are determined in an analysis without addition of standard and taken into account during quantitative calculations.
For quantitative analysis of amino acids, we offer two variants of enantiomer labelling technique. The decision which variant is appropriate will depend on the type of sample, the requirements placed on accuracy of determination and regulatory aspects.
If the peptide content should be determined on the basis of quantitation of amino acids, the sample is initially kept under desiccating conditions to account for residual water content within the sample.
Two samples are prepared. A first sample is hydrolysed. A portion of the hydrolysate is used to determine the enantiomeric purity of each of the constituent amino acids. A standard is not added to this aliquot. The obtained values represent the optical purity after hydrolysis and account for racemization during hydrolysis. A second sample is spiked with enantiomer-standard. This is done prior to sample clean-up, hydrolysis and derivatisation. The amino acids used as an internal standard (enantiomer standard) are calibrated against certified and traceable standards from the National Institute of Standards and Technology (NIST).
Some matrices could lead to decomposition or very low yield during derivatization procedure. Additionally, coelutions can be possible. High concentration of e.g. mannitol or buffers should be removed before derivatization. We are able to offer standard clean up by solid phase extraction on ion exchange or filtration depending on the matrix.
SPE via ion exchange: For the amino acids after hydrolysis it is to prove if they can be retarded sufficiently and released quantitatively. The recovery depends on the matrix. Cleanup via Microcolumns (SPE) should be applied in order to improve the numbers of amino acids which can be determined.
If the sample is known to be pure in respect of the enantiomeric purity, the determination of optical purity will be skipped and is set to 100%. Please note, this method is not applicable for analyses under GMP guideline due to the potential error cause by unexpected racemization of the peptide either during hydrolysis or derivatisation.
Some matrices could lead to decomposition or very low yield during derivatization procedure. Additionally, coelutions can be possible. High concentration of e.g. mannitol or buffers should be removed before derivatization. We are able to offer standard clean up by solid phase extraction on ion exchange or filtration depending on the matrix.
SPE via ion exchange: For the amino acids after hydrolysis it is to prove if they can be retarded sufficiently and released quantitatively. The recovery depends on the matrix. Cleanup via Microcolumns (SPE) should be applied in order to improve the numbers of amino acids which can be determined.