Peptide content (revision 11)

Peptide content, sometimes called peptide net content, is the proportion of the mass of a preparation that is the peptide itself. Everything else in the vial — water taken up during and after lyophilisation, the counterion left by the purification step, residual solvent, and any excipient — is excluded from the numerator and included in the denominator.^[1]

Content is the determination that makes a mass statement possible. A purchaser reconstituting a nominally 5 mg vial and calculating a concentration is implicitly assuming a content figure, and in the ordinary case the assumption made is 100%, which is never correct for a lyophilised peptide salt. Typical content for such material falls between roughly 70% and 90%, so the assumption is wrong by an amount that matters.^[1]

The relationship between content and area percent purity is the single most-misread pairing in research-peptide documentation. They are independent quantities: purity is a ratio among the species that eluted, and content is a fraction of the vial. A certificate reporting 99% purity and 83% content is internally consistent, and the two figures together say considerably more than either says alone.

For a lyophilised peptide purified by reverse-phase chromatography and freeze-dried from an aqueous acetonitrile mobile phase, four components account for essentially all of the non-peptide mass.

The acetonitrile carried over from preparative purification is the residual solvent that dominates in practice; it is classified for toxicity and given a concentration limit under the harmonised residual-solvent guideline, and it is determined by headspace gas chromatography rather than inferred.^[2]

The counterion share is the term that surprises people. Trifluoroacetic acid is used in the mobile phase of the purification, and a basic peptide leaves that step as its trifluoroacetate salt with one counterion per basic site. For a peptide with several basic residues and a modest molecular mass, the counterion can be a fifth of the dry weight.^[1]

Water is the term that varies most between vials of the same lot, because a lyophilised cake takes up moisture from the atmosphere on any exposure. That is what the desiccant sachet in a well-packed consignment is for, and it is why a content figure carries a date.

Two routes are in ordinary use and they are not equivalent.

Direct determination. Amino acid analysis hydrolyses the peptide and quantifies the released residues against a calibrated standard, giving the peptide mass directly. It is the reference approach and it is slow and comparatively expensive. Quantitative nitrogen determination is a faster direct route and is less specific, because any nitrogen-containing impurity contributes.