A peptide certificate of analysis (CoA) is the analytical record for one lot of a peptide: it names the compound and lot, lists each test performed with its method and acceptance limit, and states the value measured. The core lines are HPLC purity and identity by mass spectrometry; a fuller certificate adds net peptide content, counter-ion, water and endotoxin. Read one by first matching the lot number to your vial, then checking each line for its method, its limit and its measured value.
This post goes through a certificate line by line, explains what each measurement can and cannot tell you, and ends with a checklist.
The header: what and which lot
The top of a certificate identifies the material. Each field is checkable against something else.
| Field | What to check |
|---|---|
| Product name | Matches the label on the vial |
| Sequence | Written out in full, including modifications (acetylation, amidation, D-residues) |
| Molecular formula | Consistent with the sequence and any modifications |
| Molecular weight (theoretical) | Matches the formula; states whether it is average or monoisotopic |
| CAS number | Where one exists, matches PubChem or the originating literature |
| Lot number | Identical to the lot number on your vial |
| Date of analysis or manufacture | Plausible relative to when you received the material |
| Storage condition | For a lyophilized peptide, usually frozen and protected from light |
| Approval | A signature or name of the person who reviewed and released the certificate |
The lot number is the most important field on the page. A certificate describes the lot that was tested and is not a claim about any other lot of the same compound. A certificate with no lot number, or with a lot number that does not match the vial, tells you nothing about the material in your hands.
Appearance
Usually the first test line, and the simplest: a visual description such as "white to off-white lyophilized powder". It is a real check, since discoloration or a collapsed, glassy cake can signal moisture uptake or degradation, but it is qualitative. The post on lyophilized peptides explains what a normal cake looks like and what collapse and meltback look like.
HPLC purity
The purity line is the number most people read first. It comes from reversed-phase HPLC with UV detection, normally at 210 to 220 nm where the peptide bond absorbs, and is calculated as the area of the principal peak as a percentage of the total integrated area of the chromatogram.
What to look for on this line:
- A stated method: reversed-phase HPLC, ideally with column type, gradient and run time.
- A stated wavelength: 214 nm or 220 nm is standard.
- An acceptance limit and a measured value: for example "≥ 98.0%" and "99.1%". A measured value without a limit tells you what was found but not what was required.
- The chromatogram itself, with an integration table listing each band's retention time and area.
Purity says how much of the peptide-related material is the target sequence. It does not say how much of the powder is peptide, and it cannot tell a correct sequence from a wrong one of similar hydrophobicity. How HPLC measures peptide purity covers the method and its limits in detail.
Mass spectrometry: identity
Identity is shown by measuring the molecular mass of the main component and comparing it with the mass calculated from the sequence. Two ionization methods are common.
| Electrospray ionization (ESI) | MALDI | |
|---|---|---|
| Sample introduction | Usually coupled to liquid chromatography (LC-MS) | Sample dried with a matrix on a target plate |
| Typical ions | Several charge states: [M+H]⁺, [M+2H]²⁺, [M+3H]³⁺ | Mostly singly charged: [M+H]⁺ |
| What the spectrum shows | A series of m/z signals that are deconvoluted to one mass | One dominant m/z close to M + 1 |
How to check the number. The spectrum reports mass-to-charge ratio (m/z), not mass. For a peptide carrying z protons, the neutral mass is M = z × (m/z) − z × 1.00728, where 1.00728 Da is the mass of a proton. For example, a hypothetical peptide with a calculated average mass of 1,500.0 Da would be expected to show [M+2H]²⁺ at about m/z 751.0 and [M+3H]³⁺ at about m/z 501.0.
Average versus monoisotopic. The monoisotopic mass assumes every atom is its lightest isotope; the average mass weights all isotopes by natural abundance. A high-resolution instrument reports monoisotopic values; a lower-resolution one reports something close to the average. The certificate should say which it is comparing, because for a peptide of a few kilodaltons the two differ by roughly a dalton or more.
How close is close enough. That depends on the instrument. A study from FDA's laboratory on peptide quality control using high-resolution LC-MS reported measured masses within 5 to 10 ppm of theoretical values, and used tandem MS (MS/MS) fragmentation to confirm sequence as well as mass. A lower-resolution instrument has a wider tolerance, which the certificate should state.
What mass spectrometry does not prove. A correct mass rules out most wrong sequences, but not all. Isomers have identical mass: a D-residue in place of an L-residue, or a swap of two residues, will not show up in intact mass. MS/MS sequencing and HPLC together close most of that gap.
Net peptide content
Net peptide content (NPC) is the percentage of the powder's mass that is peptide. The rest is mostly counter-ion and water. Bachem's quality control guide defines it as the percentage of peptides relative to non-peptidic material and notes that it is not equivalent to purity.
NPC is measured by amino acid analysis, which hydrolyzes the peptide and quantifies each amino acid, or by nitrogen content from elemental analysis. Many research-grade certificates do not include it, and its absence is common rather than suspicious, but it changes how the stated mass should be read.
A worked example, with invented figures:
| Line | Value |
|---|---|
| Powder mass in vial | 10.0 mg |
| Net peptide content | 80% |
| HPLC purity | 98% |
| Peptide mass (10.0 × 0.80) | 8.0 mg |
| Approximate target-sequence mass (8.0 × 0.98) | about 7.8 mg |
The last line is an approximation, because NPC counts all peptide material including peptide impurities, while HPLC purity is a UV area ratio rather than a mass ratio. It is still the right order of calculation when preparing an in-vitro stock solution by weight.
Counter-ion: TFA versus acetate
A peptide with basic groups (the N-terminus, lysine, arginine, histidine) exists as a salt, and the certificate should name the counter-ion.
Why TFA is the default. In Fmoc solid-phase synthesis, the finished peptide is cleaved from its resin with concentrated trifluoroacetic acid, and TFA is also the usual ion-pairing acid in reversed-phase purification. So a synthetic peptide is obtained as a trifluoroacetate salt unless it is deliberately exchanged. A 2025 study measured roughly 25% TFA by weight in its peptides before exchange.
Why it can matter. The same study summarizes reports that residual TFA can increase or inhibit cell proliferation in culture. For cell-based work, a counter-ion at a quarter of the powder's mass is not an inert bystander.
Exchange. TFA can be replaced with acetate or chloride by ion-exchange resin, by preparative HPLC with a different acid, or by treatment with dilute hydrochloric acid followed by lyophilization. The 2025 study found 10 mM HCl reduced TFA below 1% by weight in a single exchange.
| Counter-ion | Origin | How it is measured | Notes |
|---|---|---|---|
| Trifluoroacetate (TFA) | Cleavage from resin and HPLC purification | Ion chromatography, ¹⁹F NMR, HPLC with ELSD | The default unless exchanged |
| Acetate | Exchange step or acetic acid purification | Ion chromatography, HPLC | Common where TFA is unwanted |
| Chloride (HCl salt) | Exchange with dilute HCl | Ion chromatography | Another low-TFA option |
A certificate that states the counter-ion and its content lets you correct for it. One that states neither leaves the salt form unknown.
Water content
Lyophilized peptides still hold some water, and hygroscopic sequences absorb more once a vial is opened. Water is measured by Karl Fischer titration and reported as a percentage by weight. Together with counter-ion content, it accounts for most of the difference between powder mass and net peptide content.
Endotoxin
Bacterial endotoxins are lipopolysaccharides from the outer membrane of gram-negative bacteria. They are measured with the Limulus amebocyte lysate (LAL) test, in gel-clot, turbidimetric or chromogenic formats, and reported in endotoxin units (EU), usually as EU per mg of peptide. FDA's guidance on endotoxin testing refers to USP chapter 85, the bacterial endotoxins test.
Endotoxin is not removed by lyophilization and is not detected by HPLC or mass spectrometry. For cell-based assays, where endotoxin can itself provoke a response in some cell types, an endotoxin line is worth asking for. If a certificate has no endotoxin line, the lot was not tested for it.
Other lines you may see
- Residual solvents, by gas chromatography.
- Amino acid composition, from amino acid analysis, compared with the sequence.
- Sterility or bioburden, rarely on reagent certificates. A lyophilized research peptide is not sterile unless the certificate says it was tested.
- Peptide content by mass (m/m) in place of net peptide content, on some reports.
A checklist for any peptide certificate
| Check | Why |
|---|---|
| Lot number matches the vial | A certificate covers one lot only |
| Sequence and theoretical mass are consistent | A mismatch means an error somewhere on the page |
| Purity states method, wavelength, limit and value | A bare percentage cannot be interpreted |
| Chromatogram and integration table are attached or available | The figure can be checked against the trace |
| Mass spectrum shows the expected m/z values | Identity is confirmed, not asserted |
| Counter-ion is named | Salt form changes the mass of peptide per mg of powder |
| Net peptide content, water, endotoxin present or absent | Absent lines were not tested |
| Date and approval are present | The certificate has a date and a responsible reviewer |
An independent analysis is a different document from a supplier's certificate: it is commissioned by whoever sends the sample and covers only that sample and the tests ordered. What a Janoshik report shows explains how to read and verify one.
Certificates for Anhydrolabs lots
Anhydrolabs supplies each compound, for example ipamorelin, as a lyophilized powder in a vacuum-sealed vial and as 10-vial kits, with the lot number printed on every vial. To see the certificate for a lot, write to [email protected] with the lot number. Storage after delivery affects what any later analysis will find; the storage and handling page sets out the recommended conditions. All material is supplied for in-vitro research only, as set out in the research-use statement and in what research use only means.