Analytical release testing for peptides
A peptide arrives as a white powder with a certificate of analysis. The certificate decides whether you can use the material for what you intend, and reading it properly takes a few minutes that save weeks. The tests below are the ones that carry information, in roughly the order they matter.
Identity and purity
Mass spectrometry confirms identity. Electrospray or MALDI against the calculated monoisotopic or average mass tells you the sequence assembled. What it does not resolve is anything isobaric: an epimer, a scrambled disulfide, or aspartimide-derived isoaspartate all carry the same mass as the product. A matching mass is necessary and not sufficient.
Analytical RP-HPLC gives the purity figure, usually as area percent at 214 nanometers. The wavelength matters, because 214 detects the amide backbone and therefore sees peptide-related impurities roughly in proportion to their size, while 280 only sees aromatic residues and will miss any impurity lacking tryptophan or tyrosine. A purity quoted at 280 on a peptide with one tyrosine is close to meaningless.
Ask which gradient produced the number. A steep gradient co-elutes closely related impurities into the main peak and inflates purity by several points. The honest comparison is the same gradient across every lot.
Content is not purity, and it is the number that bites
Peptide content, sometimes called net peptide content, is the fraction of the powder that is actually peptide. The rest is counterion, residual water and any inorganic salt. For a TFA salt of a basic peptide this can easily be twenty to thirty percent of the mass.
If you weigh out one milligram of powder at ninety-five percent purity and assume you have one milligram of peptide, you may have seven hundred micrograms. Every concentration, every EC50 and every dose derived from that weighing is wrong by the same factor, and it is wrong consistently, which is worse, because it looks reproducible.
Content is determined by amino acid analysis or by quantitative nitrogen determination. Many suppliers omit it unless asked, and quote purity alone. For anything where the absolute concentration matters, request it, or determine concentration in solution by UV absorbance against the calculated extinction coefficient, which sidesteps the weighing entirely for peptides containing tryptophan or tyrosine.
Counterion, water and residual solvent
The counterion should be stated. TFA is cytotoxic to cells in the tens of micromolar range and interferes with electrospray signal, so for cell work or for in vivo dosing the material should be supplied as the acetate or hydrochloride salt.
Water content by Karl Fischer matters for a peptide that will be weighed, stored long term or formulated. A hygroscopic lyophilized powder equilibrating with room air changes mass on the balance while you weigh it.
Residual solvent, mainly acetonitrile from the purification, belongs on the certificate for material entering biological work.
The tests that only appear when you ask
Bioburden and endotoxin are not on a standard research-grade certificate. Synthetic peptides are made in organic solvent and are not inherently sterile, and the lyophilization and vialing steps are where contamination enters. Cell culture and any in vivo work need both specified.
Disulfide connectivity for a peptide with more than two cysteines is not established by mass, because every regioisomer has the same mass. Confirming it requires proteolytic digestion and mapping of the resulting fragments, or comparison against a reference with known connectivity.
Chiral purity is likewise invisible to mass and usually invisible to a standard RP gradient. Where a D isomer at a single position would matter, it has to be a specified test rather than an assumption.
A certificate that lists mass, an RP trace at 214, content, counterion, water and endotoxin describes material you can make decisions on. A certificate listing mass and purity alone describes a powder.