Chapter 5 - The Use of Proton NMR as an Alternative for the Amino Acid Analysis as Identity Test for Peptides

Abstract

Using amino acid analysis (AAA), the relative amount (ratio) and absolute content of amino acids in a peptide can be determined. However, the use of proton NMR was proposed as an identity test for peptides. This chapter evaluates the usage of proton NMR as an alternative to AAA as an identity test for peptides. Proton NMR can readily distinguish and identify all peptides currently described in the European Pharmacopoeia. There are eight peptides currently mentioned in the Ph. Eur. that can be distinguished and identified by proton NMR spectra obtained at 400 MHz. Identification is straightforward and simple by comparison with the standard of the corresponding peptide without any calculations. Even peptides differing very slightly can be readily distinguished. Proton and Carbon-13 NMR spectra readily distinguish all 20 naturally occurring amino acids and unnatural amino acids present in synthetic peptides. A proton NMR spectrum of a peptide depends primarily on the relative amounts and identity of amino acids present in the sequence. This information concerning the identity of a peptide is the same as that obtained from an AAA. From a scientific point of view, NMR is a suitable alternative for AAA. A buffer is used to increase reproducibility of the spectra by reducing the pH effects on the chemical shifts and to prevent aggregation. The use of NMR as an identity test for larger peptides may give problems as spectra become less well resolved due to both the increasing number of resonances and the broader signals as a consequence of slower tumbling rates because of molecular size and, in some cases, aggregation.