Peptide purity testing: HPLC and mass spectrometry explained

Why purity and identity are separate questions

A research peptide certificate usually reports two different things. Identity answers “is this the molecule we think it is?” and is normally confirmed by mass spectrometry. Purity answers “how much of what is detected is the target peptide, rather than related impurities?” and is normally estimated by high-performance liquid chromatography (HPLC). A sample can have the right identity but modest purity, or a high HPLC purity figure for a peak that turns out to be the wrong molecule, so both measurements are needed.

Pharmacopoeial monographs for peptide medicines treat identification, purity (related substances) and assay as distinct specifications, and a 2009 review comparing European and US pharmacopoeial approaches proposed a consistent structure built around exactly those headings [2]. Research-grade material is not made to medicinal standards, but the same concepts are the most useful way to read any peptide certificate.

How most peptides are made – and where impurities come from

Most peptides today are manufactured by solid-phase peptide synthesis (SPPS), in which amino acids are added one at a time to a growing chain anchored to a resin [1][4]. Each coupling and deprotection step is very efficient, but not perfectly so, and small inefficiencies accumulate over the length of the sequence.

A 2014 review of related impurities in peptide medicines grouped the typical by-products into recognisable families [1]:

• Deletion sequences – a residue is missing because a deprotection or coupling step was incomplete.
• Insertion sequences – an extra residue is present, associated with excess amino-acid reagent.
• Diastereomers – racemisation of a residue during synthesis gives a molecule with the same mass but different stereochemistry.
• Protection adducts – side-chain protecting groups that were not fully removed.
• Oxidation products and dimers or oligomers.
• Degradation products such as diketopiperazine, pyroglutamate and succinimide forms.

The review also notes that such impurities can influence early functional studies and lead to erroneous conclusions [1] – the practical reason purity matters to a laboratory.

Reversed-phase HPLC: what the purity figure means

In reversed-phase HPLC the sample is injected onto a column packed with a hydrophobic stationary phase and eluted with a gradient of increasing organic solvent, typically acetonitrile in water with an acidic modifier. Molecules separate according to their hydrophobicity, and a UV detector – commonly set around 214–220 nm, where the peptide bond absorbs – records each as a peak.

The purity figure on a certificate is normally an area percentage: the area of the main peak divided by the total area of all integrated peaks. Several consequences follow:

• It describes UV-absorbing, peptide-related material. Water, residual salts and counter-ions are not captured by this number.
• Impurities that co-elute with the main peak – including some diastereomers – may be counted as the main peak.
• The figure depends on the method: column, gradient, wavelength and integration settings. A chromatogram printed with the result lets you see how well the main peak is resolved.

For this reason HPLC purity is not the same as peptide content (sometimes called net peptide content), which is the proportion of the weighed powder that is peptide rather than water and counter-ion. Pharmacopoeial approaches handle purity and assay as separate tests for the same reason [2].

Mass spectrometry: confirming identity

Mass spectrometry measures the mass-to-charge ratio of ions formed from the sample. Electrospray ionisation (ESI) usually produces several multiply-charged ions for a peptide, from which the molecular mass is calculated; MALDI typically shows a dominant singly-charged ion. Either way, the certificate should report an observed mass that agrees with the theoretical mass calculated from the sequence, within the stated accuracy of the instrument.

A matching mass is strong evidence of identity but not proof of a single isomer: diastereomers and some sequence variants share the same mass. When HPLC and mass spectrometry are combined (LC–MS), each chromatographic peak can be assigned a mass, which is the most informative way to see what the minor impurity peaks are.

Counter-ions: the TFA question

Peptides purified by reversed-phase HPLC are frequently isolated as trifluoroacetate (TFA) salts, because trifluoroacetic acid is used in the mobile phase [3]. TFA counter-ions are a form of related material the impurity review also lists [1].

This is relevant to cell-based work. A 1999 study found that TFA at 10⁻⁸ to 10⁻⁷ M reduced cell numbers and thymidine incorporation in osteoblast cultures, with similar effects in chondrocytes; TFA salts of several peptides gave consistently lower proliferation than the hydrochloride salts [3]. The authors recommended converting peptides to a hydrochloride or biologically equivalent salt before assessing biological effects in culture [3]. Where the counter-ion matters to an experiment, it should be specified when ordering and checked on the certificate.

A quick checklist

• Does the lot number on the certificate match the lot printed on the vial?
• Is the observed mass consistent with the calculated mass for the stated sequence?
• Is an HPLC chromatogram provided, with the integration method and wavelength stated?
• Is the counter-ion stated, and is it appropriate for the assay?
• Is the test date recent relative to the material’s storage history?

Every Britpep lot is listed in our COA checker by the batch number on the label. Our quality and testing page describes how lots are released, and the companion article on reading a certificate of analysis walks through a typical report field by field.

References

1. D'Hondt M, Bracke N, Taevernier L et al.. Related impurities in peptide medicines. J Pharm Biomed Anal. 2014; 101:2-30. doi:10.1016/j.jpba.2014.06.012. PubMed 25044089
2. Vergote V, Burvenich C, Van de Wiele C et al.. Quality specifications for peptide drugs: a regulatory-pharmaceutical approach. J Pept Sci. 2009; 15:697-710. doi:10.1002/psc.1167. PubMed 19750489
3. Cornish J, Callon KE, Lin CQ et al.. Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes. Am J Physiol. 1999; 277:E779-83. doi:10.1152/ajpendo.1999.277.5.E779. PubMed 10567002
4. Lau JL, Dunn MK. Therapeutic peptides: Historical perspectives, current development trends, and future directions. Bioorg Med Chem. 2018; 26:2700-2707. doi:10.1016/j.bmc.2017.06.052. PubMed 28720325

References were checked against PubMed and official UK sources at the time of writing. Summaries describe what each source reports; they are not claims about Britpep products.