Assay methods for research peptides

Purity

Reverse-phase HPLC

A sample is pushed through a column packed with a non-polar stationary phase. Compounds that bind the phase more strongly take longer to come off, so the mixture separates in time. A detector, usually UV at 214 nm where the peptide bond absorbs, records what elutes and when. The output is a chromatogram: a baseline with peaks on it.

Purity is computed as main peak area ÷ total peak area, expressed as a percentage. That is the whole definition, and its limits follow directly from it:

• It is a proportion of what the detector saw. Anything that does not absorb at the chosen wavelength is invisible to it, including most salts and the counter-ion.
• An impurity that co-elutes with the main peak is counted as the main peak. Purity is only as good as the separation.
• It says nothing about how much peptide is in the vial. A vial containing 3 mg of peptide and a vial containing 8 mg can both report 99%.

A gradient, column and wavelength should accompany the number. A purity figure with no method behind it is not reproducible.

Identity

Liquid chromatography mass spectrometry

The separated sample is ionised, usually by electrospray, and the instrument measures mass-to-charge ratio. Peptides pick up multiple charges, so a single species appears as a family of peaks that deconvolute to one molecular weight. That observed mass is compared to the theoretical mass computed from the intended sequence.

A match within instrument tolerance establishes that the compound is what the label says. This is the assay that separates a real peptide from a mislabelled one, and it is the reason identity and purity are reported separately: a sample can be extremely pure and be the wrong molecule entirely.

What it does not establish: quantity, sterility, or whether a closely-related analogue differing by a small mass increment has been correctly resolved. Deamidation, for instance, shifts mass by roughly 1 Da and needs adequate resolution to catch.

Quantity

Net peptide content

A lyophilised vial is not pure peptide. It also contains counter-ions from the final purification step (commonly trifluoroacetate or acetate), residual water, and any excipient. Net peptide content, measured by amino acid analysis or nitrogen determination, states what fraction of the vial's mass is actually peptide.

This is the single most commonly omitted figure on a peptide certificate, and the one that most affects a calculated concentration. Purity and net content are independent. A lot reported at 99% HPLC purity may be 80% peptide by mass; the remaining 20% is counter-ion and water, which the HPLC detector never saw.

If a protocol depends on an accurate molar concentration, net peptide content is the number that governs it, not purity.

Moisture

Water content by Karl Fischer titration

Karl Fischer titration quantifies water specifically, rather than total volatiles as loss-on-drying would. Residual moisture matters because water is a reactant: it participates in hydrolysis of labile bonds and accelerates deamidation at asparagine and glutamine residues.

A low water figure is a necessary condition for a stable lyophilised powder, not a sufficient one. Temperature history and light exposure act independently of moisture.

Contamination

Bacterial endotoxin (LAL)

Endotoxin is lipopolysaccharide from the outer membrane of Gram-negative bacteria. It is heat-stable, survives conditions that kill the organism that produced it, and is quantified by Limulus amebocyte lysate assay in endotoxin units per milligram.

Endotoxin testing is not a sterility test. It detects one class of contaminant. A sample can report low endotoxin and still carry fungal contamination, non-endotoxin pyrogens, or viable Gram-positive organisms. Those require their own assays.