Amino acid analysis (revision 21)

Amino acid analysis hydrolyses a peptide into its constituent amino acids and quantifies each. It yields two things a chromatographic purity determination cannot: the residue composition, which supports identity, and an absolute peptide content, which supports quantity.^[1]

It is one of the few methods that gives content without requiring a reference standard of the peptide itself, because the standards required are the individual amino acids.^[2] This makes it particularly valuable for compounds with no marketed product and therefore no compendial standard.^[3]

The method is destructive, laborious and subject to well-characterised systematic errors, which is why it is used to qualify a standard or to establish content on a lot rather than as a routine release test.^[1]

A weighed sample is hydrolysed, conventionally in 6 M hydrochloric acid at 110 °C for 24 hours under vacuum or inert atmosphere. The hydrolysate is derivatised and the amino acids separated and quantified by chromatography against standards of each.^[1]

The molar ratios obtained are compared with those expected from the sequence. Agreement supports identity, though it does not establish sequence order — a permutation of the same residues gives the same composition, which is why composition is complementary to mass and fragmentation rather than a substitute for them.

Absolute content follows from the total recovered amino acid mass against the sample mass taken. This is the figure that makes the method valuable for quantitation, and it is unaffected by water, counterion or residual solvent, all of which contribute to the weighed mass and none of which yields amino acids.^[3]

These are not defects of a particular laboratory; they are properties of acid hydrolysis and are handled by convention. Serine and threonine losses are corrected by hydrolysing for several durations and extrapolating to zero time; valine and isoleucine by extending hydrolysis. Both corrections are established procedures rather than ad hoc adjustments.^[1][4]

The practical consequence is that an amino acid analysis result should be read with the hydrolysis conditions in view, and that residues known to be destroyed are simply not evidence either way. A composition report that lists tryptophan at its theoretical value from a standard hydrolysis is reporting something that did not happen.

Amino acid analysis appears on peptide certificates less often than purity or water, and more often on custom-synthesis documentation than on catalogue material. Where it appears, it is generally the strongest single determination on the document, because it addresses content directly.^[3]

Its absence is not evidence of a problem. It is a laborious method, and content can also be established by quantitative nitrogen determination or by chromatographic assay against a qualified standard where one exists.

For investigational compounds without a reference standard, however, it is one of very few routes to an absolute content figure, and its absence leaves content resting on subtraction of water, counterion and solvent from unity — an arithmetic estimate rather than a measurement. See Peptide content.^[1]

• Peptide content
• Reference standard
• Mass spectrometry
• Trifluoroacetate counterion
• Certificate of analysis