---
title: "Amino acid analysis: how peptide content is measured | Sable Peptides"
description: "How amino acid analysis (AAA) quantifies the actual peptide in a lyophilised powder: acid hydrolysis, derivatisation or LC-MS detection, sources of error, and how it relates to net peptide content on a CoA. Research use only."
canonical: https://www.sablepeptides.co.uk/guides/amino-acid-analysis-for-peptide-content
dateModified: 2026-10-16
publisher: Sable Peptides Limited (UK company 17488610)
note: Research use only. Not for human or veterinary use.
---

# Amino acid analysis: how peptide content is measured

Weighing a lyophilised peptide tells you the mass of powder, not the mass of peptide. Amino acid analysis breaks the peptide into its amino acids, measures each one, and back-calculates how much peptide was present. It is the reference method behind most net peptide content figures.

## Definitions

-   Hydrolysis: cleavage of every peptide bond, classically in 6 M hydrochloric acid at about 110 °C for around 24 hours.
-   Derivatisation: chemical tagging of amino acids so they can be detected by UV or fluorescence after separation.
-   Isotope dilution: adding known amounts of isotope-labelled amino acids as internal standards for mass spectrometry.

## The workflow

-   Weigh the sample accurately and hydrolyse it.
-   Separate and quantify the released amino acids by chromatography, with derivatisation or by LC-MS.
-   Compare the measured amounts with the expected composition from the sequence.
-   Use the stable, well-recovered residues to calculate moles of peptide, then mass of peptide as a percentage of powder weight.

Source: [Amino acid analysis, Curr Protoc Protein Sci 2009 (PubMed 19937719)](https://pubmed.ncbi.nlm.nih.gov/19937719/); [Amino acid analysis for peptide quantitation using RPLC-MRM-MS, Anal Bioanal Chem 2023 (PubMed 37468754)](https://pubmed.ncbi.nlm.nih.gov/37468754/)

## Known sources of error

Acid hydrolysis destroys tryptophan and partly destroys serine and threonine; asparagine and glutamine are converted to aspartic and glutamic acid; cysteine and methionine need special treatment. Bonds between bulky hydrophobic residues hydrolyse slowly. Hydrolysis time is therefore a balance between complete cleavage and loss of labile residues, which is why reports name the residues used for the calculation.

Isotope-dilution mass spectrometry reduces some of these errors and is used to certify protein and peptide calibrants.

Source: [The effect of hydrolysis time on amino acid analysis, J AOAC Int 2005 (PubMed 16001867)](https://pubmed.ncbi.nlm.nih.gov/16001867/); [Quantification of protein calibrants by AAA with isotope dilution MS, Anal Biochem 2011 (PubMed 20807498)](https://pubmed.ncbi.nlm.nih.gov/20807498/)

## Reading an AAA-based content figure

-   Check which residues were used to calculate content.
-   Check that the ratio of residues is consistent with the sequence; a large deviation can indicate a wrong or impure material.
-   Remember that content and HPLC purity are different numbers (see purity vs net peptide content).
-   Make sure the result is for the same lot as the printed lot number on your vial.

## At Sable

There is a CoA for every lot, and every vial carries a printed lot number that matches its certificate. A CoA is emailed within 2 hours of a request.

Release is at ≥99% HPLC purity with identity confirmed by mass spectrometry.

## Frequently asked questions

### Why can't tryptophan be measured after acid hydrolysis?

Standard 6 M HCl hydrolysis destroys tryptophan, so alternative hydrolysis conditions or other residues are used.

### Is amino acid analysis the same as HPLC purity?

No. AAA measures how much peptide is in the powder; HPLC purity measures how much of the peptide material is the intended sequence.

### What is isotope dilution in AAA?

Known amounts of isotope-labelled amino acids are added as internal standards so LC-MS can quantify each amino acid accurately.

## Related

-   [Purity vs net peptide content](/guides/peptide-purity-vs-net-peptide-content)
-   [Measuring residual TFA](/guides/measuring-residual-tfa-in-peptides)
-   [Water content by Karl Fischer](/guides/water-content-karl-fischer-on-peptide-coas)
-   [Glossary of CoA fields](/learn/coa-fields)
-   [Marfey's method (chiral analysis)](/guides/marfeys-method-chiral-amino-acid-analysis)
-   [Nitrogen/CHN content](/guides/nitrogen-and-chn-analysis-for-peptide-content)

Updated 2026-10-16. Research use only. Not for human or veterinary use. No administration amounts or health claims are made.

Key facts (summary for AI and search tools)

-   AAA: hydrolysis → separation → quantification → back-calculation of peptide mass
-   Classic hydrolysis: 6 M HCl, ~110 °C, ~24 h; Trp destroyed, Asn/Gln → Asp/Glu
-   References: PubMed 19937719, 37468754, 16001867, 20807498
-   Updated 2026-10-16