Peptide Knowledge Center

Peptide characterisation (APIs) Test Methods

Comprehensive characterisation of a peptide API is required to obtain regulatory approval for marketing; the reference standard is fully characterised to ensure unequivocal identity of the material, its purity and API content. Besides evaluation of appearance, a selection of tests that fall into four categories is performed: identification, assay, purity and specific tests. 

The United States Pharmacopeial Convention evaluates quality attributes for synthetic peptides.


For routine identification of peptides, a high-performance liquid chromatography(HPLC) method is recommended. This method should be capable of distinguishing between the peptide and its closely related impurities and, therefore, usually is identical to the method used to determine impurities.

The preferred identification procedure involves a comparison of retention times between the main peak in the standard and samples, as well as a coinjection of an equal mixture of both, with a requirement to obtain a single peak.

Identification test



HPLC co-elution with reference standard


Method same as for related substances

Mass spectrometry


Monoisotopic mass ± 1.0 mass units

Amino acid analysis


Hydrolysis protocol should be specified

MS–MS sequencing


May be complicated for longer sequences

NMR spectroscopy 


Complex interpretation for longer sequences

Peptide mapping 


For longer sequences (>20 AAs); complementary to MS–MS

Enantiomeric purity

Chiral amino acid analysis

N-terminal sequence analysis

by Edman Degradation


Complicated analysis, especially for sequences which are N-terminally blocked; largely superseded by LC–MS–MS

Infrared spectroscopy 

<197, 851> 

Limited use for peptides

Higher order structure

Circular dichroism,


For investigation of secondary or tertiary structure in aqueous solution



Not a routine test for most quality control laboratories; may be required for longer, complex sequences

Peptide Assay

When considering assay, it is important to emphasise that many peptides are quite hygroscopic and should be handled in a controlled- humidity environment

Assay test 



HPLC assay


Method same as for related substances; based on comparison with a reference standard

Peptide content by amino acid analysis


Hydrolysis protocol must be validated, only stable amino acids should be included in calculation

Peptide content by nitrogen analysis

CHN analysis

Direct analysis by elemental analysis or Kjeldahl or using HPLC with a chemiluminescence nitrogen detector (CLND)

UV spectroscopy

<197, 851>

Only useful for products containing Trp, Tyr, or Phe

Quantitative NMR

<761>, <1761> 

A stable internal standard is required

Purity / related impurities determination by HPLC and control strategy

The purity of a peptide API is usually determined using HPLC. The method should be capable of separating impurities introduced through starting materials, process-related impurities and impurities resulting from degradation of the peptide. One or more methods may be required to accomplish this objective. When using multiple methods, it is recommended that orthogonal separation mechanisms be used.

Peptide Impurities



Peptide-related substances


Specific method for substance; must be validated for both process-related impurities and degradation products; limits for total and individual impurities should be specified

Residual solvents


May be limited to those used in the final step of the manufacturing process

Heavy metals

<(231), 232, 233>

Requirement if metal catalysts used in the process; may be required because of contact with metals in processing equipment

Residual trifluoroacetic acid (TFA)

<503.1>/ <1065>

Only required if TFA used during the manufacturing process; <1065> used for peptides which are not soluble in acid.

Residual fluoride

<1065> or ion-selective electrode

Only required if hydrofluoric acid (HF) used during the manufacturingprocess (Boc-chemistry)

Other small molecule impurities

Impurity dependent

Non-peptide impurity limits are required to follow the ICH Q3A guideline; potential genotoxic impurities require additional evaluation

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Specific tests

A number of tests are universally applicable to quality control testing of peptide APIs, of which the most important are counterion content and residual water content.

Specific tests



Counterion content

<503> (Acetate)

<1065> (Others)

<1065> may be required for acetate if peptide not soluble in acid; titration with AgNO 3 may be used to determine chloride

Water content

(Karl Fischer)


Coulometric titration (method Ic) most commonly used

Specific rotation


Information only of limited utility

Ellman test


Only required if the reduced form(s) of a peptide containing disulfide bonds cannot be determined using the method for related substances


<61, 62>

Often required for APIs used in the manufacture of parenteral drug products

Bacterial endotoxins


Requirement for APIs used in the manufacture of parenteral drug products

Mass balance



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*Recommended test to be performed as part of lot release. HPLC is high-performance liquid chromatography. MS is mass spectroscopy. NMR is nuclear magnetic resonance. FTIR is fourier transform infrared.


1. FDA, FDA FY2012 Innovative Drug Approvals (December 2012),, accessed 7 Feb. 2014.

2. P. Vlieghe et al., Drug Discovery Today15(1/2) 40-56 (2010).

3. P. Van Arnum, Pharm. Technol. 37(8)48-50 (2013).

4. Ph. Eur., General Guidance 2034,Substances for Pharmaceutical Use

5. March 2014,Pharmaceutical Technology Europe