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Acid hydrolysis in 6 N HCl at 110 °C for 24 hours breaks the peptide bonds so bound amino acids can be measured. It destroys tryptophan, converts asparagine and glutamine to aspartate and glutamate, and partly loses serine, threonine and cystine, so results are reported with those limits.
What acid hydrolysis is
Most amino acids in a protein, peptide or protein powder are bound in peptide bonds and invisible to a free amino acid analysis. Acid hydrolysis breaks those bonds so the amino acids can be measured. The harmonized pharmacopoeial method uses 6 N hydrochloric acid containing 0.1–1.0% phenol, at 110 °C for 24 hours, in vacuum or an inert atmosphere to prevent oxidation 1. Phenol protects tyrosine from halogenation 1.
What hydrolysis destroys or converts
| Amino acid | What happens in 6 N HCl | How it is reported |
|---|---|---|
| Tryptophan | Destroyed 1 | Not reported |
| Asparagine, glutamine | Deamidated to aspartate and glutamate 1 | As Asx and Glx |
| Serine, threonine | Partly destroyed 1 | Reported, wider uncertainty |
| Methionine | May oxidize 1 | Reported, wider uncertainty |
| Cystine, cysteine | Recovery usually poor 1 | Not reported in a standard hydrolysis |
| Valine, isoleucine | Val–Ile, Ile–Ile and Val–Val bonds only partly cleaved in 24 h 1 | Reported; can read slightly low |
The pharmacopoeial text notes that these losses limit quantitation to 17 amino acids 1.
Getting closer to the true composition
When accuracy matters more than cost, hydrolysis is run at several time points. Serine and threonine are extrapolated back to zero time, and valine and isoleucine are read at their plateau 1. In one study, modeling 10 hydrolysis intervals from 2 to 141 hours gave a mean recovery of 100% (range 94–110%) for egg-white lysozyme, while a single 24-hour hydrolysis gave inaccurate estimates; cysteic acid was lost faster than serine 2.
Cysteine and methionine need oxidation first
To measure cysteine and methionine after hydrolysis, they are oxidized first. Performic acid converts cysteine to cysteic acid and methionine to methionine sulfone 1. Hydrolysis with 0.20% sodium azide oxidized 87–100% of cysteines to cysteic acid 3. These are separate preparations; ask us if you need them.
How much sample
Analytically, very little protein is needed: pre-column derivatization methods generally need 0.5–1.0 µg of protein per analysis 1. In practice we ask for at least 200 µL of a liquid or 100 mg of a solid so the sample can be weighed accurately and run again if needed. See total amino acid analysis for what we report and the price.
How we run it
We hydrolyze in 6 N hydrochloric acid at 110 °C for 24 hours, then derivatize the hydrolysate with AQC and separate it by reversed-phase HPLC with UV detection, the same chemistry as our free amino acid analysis. Results are reported as g per 100 g of sample and as mole percent, with the chromatogram and QC for every sample. For the difference between the two measurements, see free vs total amino acids.
Key papers
- Amino Acid Determination (harmonized pharmacopoeial text, B-01, Rev. 1 Corr. 1) Pharmacopoeial Discussion Group, November 2024Cited for: Most amino acids in a protein, peptide or protein powder are bound in peptide bonds and invisible to a free amino acid analysis.
- Correction for amino acid loss during acid hydrolysis of a purified protein Darragh AJ, Garrick DJ, Moughan PJ, Hendriks WH, Analytical Biochemistry, 1996Cited for: When accuracy matters more than cost, hydrolysis is run at several time points.
- Quantification of cysteine residues following oxidation to cysteic acid in the presence of sodium azide Manneberg M, Lahm HW, Fountoulakis M, Analytical Biochemistry, 1995Cited for: To measure cysteine and methionine after hydrolysis, they are oxidized first.
- High-pressure hydrolysis: A rapid sample preparation strategy for amino acid analysis of complex biological matrices Journal of Chromatography A, 2026
Latest research
New papers on this topic, newest first. We check PubMed every month; last checked October 2026.
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Derivatization agents for LC-MS analysis of amino acids: effects of core structure and functional groups
Analytical and bioanalytical chemistry
Compared pyridine-, quinoline- and isoquinoline-based derivatization agents for LC-MS amino acid analysis; NHS esters stayed stable over a year, and 6-CiQ-NHS performed best.
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A Rapid and Validated Reversed-Phase High-Performance Liquid Chromatographic Method with Pre-Column Derivatization and Fluorescence Detection for Quantification of Twenty Amino Acids in Total Parenteral Nutrition Solutions
Journal of chromatographic science
An AQC pre-column derivatization HPLC-fluorescence method separated all 20 amino acids in 18 minutes and was validated per ICH Q2(R2) for parenteral nutrition QC.
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Clinical validation of a liquid chromatography single quadrupole mass spectrometry (LC-MS) method using Waters Kairos™ Amino Acid Kit reagents
Clinical chemistry and laboratory medicine
An LC-MS method using Kairos kit reagents with AccQ-Tag Ultra derivatization ran in 19 minutes, with under 2% mean bias versus ion-exchange chromatography.
Key references
- Amino Acid Determination (harmonized pharmacopoeial text, B-01, Rev. 1 Corr. 1) — Pharmacopoeial Discussion Group, November 2024.
- Correction for amino acid loss during acid hydrolysis of a purified protein — Darragh AJ, Garrick DJ, Moughan PJ, Hendriks WH, Analytical Biochemistry, 1996.
- Quantification of cysteine residues following oxidation to cysteic acid in the presence of sodium azide — Manneberg M, Lahm HW, Fountoulakis M, Analytical Biochemistry, 1995.
- High-pressure hydrolysis: A rapid sample preparation strategy for amino acid analysis of complex biological matrices — Journal of Chromatography A, 2026.
OpenChemWorks Laboratory · Reviewed by the OpenChemWorks laboratory · Updated October 7, 2026