A certificate of analysis for a research peptide typically carries two numbers: a purity percentage from high-performance liquid chromatography, and a mass from a mass spectrometer confirming the molecule is the one the label names. Both are real measurements. Neither answers the question most readers think they answer.
This page sets out what each assay in the peptide toolkit measures, what it cannot measure, and where the gap between them sits.
The four questions, and the four different assays
Peptide analysis is not one test with a score. It is a set of measurements answering separate questions, and a result from one does not substitute for another.
| Question | Assay | What the result is |
|---|---|---|
| Is this the right molecule? | Mass spectrometry | The observed mass, compared with the target sequence's expected mass |
| How clean is it, relative to other peptides? | Analytical HPLC | The target's share of total peak area |
| How much of the powder is peptide? | Amino acid analysis or elemental CHN analysis | Net peptide content, as a percentage of weighed mass |
| What is the non-peptide mass made of? | Counterion content, Karl Fischer water, ion chromatography | Counterion, water and salt fractions |
The first two are the ones a research COA normally carries. The third is the one that converts a vial's stated milligrams into an actual quantity of peptide.
Purity is a ratio inside the peptide fraction
HPLC separates the target peptide from the other peptide-like species produced during synthesis and detects them as they elute. The purity figure is the target peak's share of the total peak area.
The species it separates from are close relatives. FDA's own assessment of generic teriparatide worked from 34 observed impurities, and the list is a catalogue of synthesis defects rather than of foreign matter: amino-acid deletions and duplications, N-terminal and C-terminal truncations, N-terminal extensions, and side-chain oxidation, succinimide formation, dehydration and acetylation. That work was published in Frontiers in Immunology on 8 December 2025 (doi:10.3389/fimmu.2025.1730346), and the impurities came from internal FDA studies rather than from applicants' submissions.
Two things follow from what purity is.
First, it is silent about everything that is not a peptide. Water, residual solvent, salt and counterion are not the target peak and not the impurity peaks; they sit outside the measurement.
Second, a single aggregate figure discards the structure the regulated market requires. FDA's stated position for certain generic synthetic peptides is that a new impurity above 0.5% of the active ingredient is not acceptable, and one between 0.10% and 0.5% must be identified, characterized and justified as not affecting safety and efficacy — with comparative immunogenicity risk testing where relevant. The framework is per-impurity. "99% pure" is precisely the aggregation that framework exists to prevent.
Mass spectrometry answers identity, not quantity
A mass spectrum tells you the mass of what is there. Compared against the sequence's calculated mass, it is a strong check on identity: a deletion, a truncation or a wrong residue shifts the mass and shows up.
It does not weigh the vial's contents. Two preparations whose spectra are indistinguishable can differ substantially in how much peptide a milligram of powder contains, because the difference sits in material the target's mass peak does not represent.
Net peptide content is the measurement that converts milligrams into peptide
This is the one most often missing, and it is the one that decides what a stated quantity means.
A lyophilized peptide powder is a mixture. AnaSpec, a peptide manufacturer, describes the gross weight as "the total weight of all components present in the lyophilized powder", including "the peptide of interest, any peptide impurities, water, residual solvents, and counterion". Net peptide content is the fraction that is peptide. AnaSpec's own worked example is 75%.
The counterion is the largest systematic contributor, and it is predictable from the sequence. Each basic residue carries a counterion; with trifluoroacetate, the usual counterion after reversed-phase purification, each charge adds roughly 114 g/mol of mass that is not peptide. AnaSpec states the relationship directly: the higher the peptide's charge, the lower its peptide content. A highly basic sequence can therefore be exceptionally pure by HPLC and still be well under 80% peptide by weight.
Determining it takes a different instrument. Amino acid analysis hydrolyses the peptide and quantifies the released residues — it needs little material, and AnaSpec describes its accuracy as limited. Elemental CHN analysis is more accurate and needs milligrams. Where the counterion is trifluoroacetate or acetate, nitrogen is the element used, chosen so the counterion cannot contribute to the peptide figure it is supposed to be excluded from.
The gap, stated plainly
AnaSpec publishes the quality-control panel it runs on research-grade peptides. On that table, for both custom and catalogue research grade, the standard tests are appearance, purity by percentage peak area on analytical HPLC, and identity by mass spectrometry. Listed as optional: peptide sequence confirmation, counterion content, metal content, endotoxin, bioburden.
Read that as a set. The two measurements that are always run establish what the molecule is and how clean it is among peptides. The measurements that would establish how much peptide is in the vial are the ones sold as extras.
This is not an accusation of concealment — the panel is published, and the manufacturer explains the distinction on the same page. It is a structural observation about what a research-grade certificate is for. It was never designed to underwrite a quantity claim, and read as one it overstates what has been measured.
The practical consequence runs straight into the dosage cluster. Every figure expressed as a mass — the milligram quantities on the dosage index, the arithmetic on how to reconstitute peptides — assumes that a vial labelled 5 mg contains 5 mg of peptide. On a research-grade certificate, that assumption is untested rather than confirmed.
What a report can and cannot be called
The distinction between accreditation and certification, and what a scope of accreditation covers, is set out on third-party testing explained; what a certificate of analysis is and how to read one is on how to read a COA. The short version relevant here: a test result is only as meaningful as the method named beside it, and a document that reports a purity percentage without naming the method, the column, the gradient and the detection wavelength has reported a number rather than a measurement.
The regulated framework is currently moving
One thing worth dating, because it changed recently. On 28 July 2026, FDA withdrew its May 2021 guidance ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs of rDNA Origin, stating that it "no longer reflects FDA's current scientific thinking", and published revised draft product-specific guidances in its place. Those cover five areas: submission of recombinantly, synthetically or semi-synthetically produced peptides as ANDAs, innate immune response testing, impurity thresholds, higher order structure assessment, and biological activity assessment. Seventeen peptide products are named, including semaglutide, tirzepatide, liraglutide, glucagon, teriparatide, calcitonin salmon, dasiglucagon, pegcetacoplan and vosoritide.
Anyone citing the 2021 guidance as current is citing a withdrawn document. The analytical principles in it did not become wrong on that date, but the agency's stated position on how they apply is now the revised drafts.
The one-line summary
Purity says how clean the peptide fraction is. Mass spectrometry says whether it is the right molecule. Neither says how much peptide is in the vial, and the assay that does — amino acid analysis or elemental analysis, reported as net peptide content — is the one a standard research panel leaves out.