Two questions, two instruments
Every batch record for an acylated GLP-1-family peptide carries two headline results, and buyers routinely read them as one. The RP-HPLC figure answers a quantity question: of the material that eluted and absorbed UV light, what share belongs to the main peak? The mass-spectrometry result answers an identity question: does that main peak have the mass the molecular formula predicts?
The two measurements fail in different ways. A chromatogram with one clean peak can belong to the wrong molecule; a spectrum that matches the theoretical mass can come from a sample that is largely something else.
This guide covers what each instrument does for peptides such as tirzepatide, retatrutide and cagrilintide, whose fatty-diacid side chain complicates both measurements. It then shows how to read the two results on a published Pepti record, and what a QA function can add once a 1 kit (10 vials) sample is on the bench.
RP-HPLC for acylated peptides
Column, gradient and modifier
The purity method on a GLP-1 peptide record is reversed-phase chromatography on a C18 column. The sample is dissolved in a weak diluent, loaded onto the column and eluted with a gradient from mostly water to a rising fraction of acetonitrile. An acid modifier, usually trifluoroacetic or formic acid, in both solvents holds the peptide in one protonation state and sharpens the peaks.
The more hydrophobic a species, the more acetonitrile it takes to move it off the alkyl-bonded stationary phase and the later it elutes.
Detection and the area-percent calculation
The detector is a UV absorbance cell after the column, set in the short-wavelength region where the peptide backbone itself absorbs, so that every peptide species gives a signal roughly in proportion to its number of amide bonds. The longer-wavelength region where only aromatic side chains absorb would under-report an impurity that has lost a tyrosine or tryptophan.
The software integrates every peak above a threshold, sums the areas and reports the main peak's area as a fraction of the total. That fraction is the purity figure. It is a ratio of areas inside one chromatogram: no weighing, no reference standard, no knowledge of what the peaks are.
Why the fatty-diacid side chain changes the picture
Semaglutide carries a C18 diacid; tirzepatide, retatrutide and cagrilintide carry a C20 diacid, attached to a lysine through a linker. The chain is far more hydrophobic than any natural residue, so the acylated peptide elutes well after its bare backbone, and a des-acyl impurity that lacks the chain separates easily.
The impurities that matter keep the side chain. An oxidized methionine or tryptophan (+16 Da), a deamidated asparagine or glutamine (+1 Da), a sequence missing one terminal residue, a diastereomer with one inverted residue: each still carries the same lipid, so each has nearly the same retention as the main peak.
The result is a main peak with shoulders and small peaks on either flank, often not resolved to baseline. The purity figure then depends on how the software splits those shoulders from the main peak, so the integration table matters as much as the number. A steep gradient, or a column that has lost efficiency, merges the shoulders into the main peak and reports a higher purity than a slower gradient on a fresh column. Expect a re-test in your own laboratory to move the figure; compare chromatogram shape before reading anything into a small difference.
What HPLC area-percent does not measure
Because area-percent is a ratio of UV signals inside one run, anything that gives no UV signal, or does not reach the detector at all, is invisible to it.
- Water. A lyophilized peptide retains bound water. It has no backbone absorbance and adds nothing to the area total.
- Counter-ion. The peptide is isolated as a salt, typically trifluoroacetate or acetate. The anion is not counted, although it can be a meaningful share of the powder's mass.
- Non-chromophoric material. Inorganic salts and any non-absorbing additive pass through unseen; a powder that is mostly salt can still show a high purity.
- Net peptide content. Area-percent has no axis for absolute amount. It cannot say whether a vial labeled 30 mg holds that mass of peptide.
- Co-eluting isomers. A sequence isomer or a D-amino-acid epimer that elutes under the main peak is counted as main peak.
Purity and content are two results, not one
Purity is the share of the eluting, absorbing material that is the main component. Content is the absolute mass of that component in the container, measured against a reference standard or by an independent method such as amino acid analysis. The two move independently: a high-purity peptide can be under-filled, and a correctly filled vial can carry a low purity.
Janoshik Analytical report #210105 shows the distinction on one sample. The laboratory tested a retatrutide 20 mg sample and reported two separate figures: 19.52 mg content and 99.837% purity. The second is an area-percent from the chromatogram; the first is a quantity measured against a standard. Neither could have been derived from the other. The report, analyzed 2026-10-07, is verifiable at verify.janoshik.com; it is a sample-specific result for one submitted vial and not a claim about any other product or lot.
ESI mass spectrometry: from charge envelope to neutral mass
The identity result comes from electrospray ionization. The peptide solution is sprayed from a charged needle into the instrument, and the peptide picks up protons on its basic residues and amino terminus as it enters the analyzer. A peptide of this size carries several protons at once, so it appears not as one peak but as an envelope of peaks, one per charge state.
Reading the envelope
The analyzer measures mass-to-charge ratio, written m/z, not mass. For the GLP-1-family peptides the envelope is usually dominated by the z = 3, 4, 5 states: the same molecule appears at several m/z values, each equal to the neutral mass plus the attached protons, divided by the charge. Adjacent charge states are spaced predictably, and the software uses that spacing to work back to a single neutral mass, a step called deconvolution. That deconvoluted mass is what the record compares with the theoretical value computed from the formula.
| Compound | Formula | Average mass | Residues and lipid |
|---|---|---|---|
| Semaglutide | C187H291N45O59 | 4114 g/mol | 31 residues, C18 diacid |
| Tirzepatide | C225H348N48O68 | 4813 g/mol | 39 residues, C20 diacid |
| Retatrutide | C221H342N46O68 | 4731 g/mol | 39 residues, C20 diacid |
| Cagrilintide | C194H312N54O59S2 | 4409 g/mol | 37 residues, one disulfide, C20 diacid |
Average and monoisotopic mass
The values above are average masses, computed from the natural isotope mixture of each element. A high-resolution instrument that resolves the isotope peaks within a charge state can instead report the monoisotopic mass, computed from the lightest isotope of each element only; for a molecule of this size it is a few daltons below the average. Know which convention the record used before comparing a found mass with the table, otherwise a correct result looks like a mismatch. For cagrilintide the disulfide matters too: the intended, oxidized form is two hydrogen atoms lighter than the reduced chain.
What MS cannot do alone
A found mass that agrees with the theoretical mass proves that the dominant species in the spray has the right elemental composition. It does not prove that the sample is pure, that the residues are in the right order, or that every residue has the intended stereochemistry.
Isobaric impurities
Some impurities shift the mass enough to see. Oxidation adds +16 Da, deamidation adds +1 Da, water loss or aspartimide rearrangement gives −18 Da, and a missing residue removes that residue's full mass. A +1 Da shift sits inside the main isotope cluster and needs a high-resolution instrument to call with confidence.
Other impurities shift nothing. A sequence isomer, where two residues have swapped places, has the identical formula and mass. A D-amino-acid substitution from racemization during synthesis is likewise invisible, and so is a positional isomer with the diacid chain on the wrong lysine. These are what a well-resolved chromatogram or a chiral amino-acid method exists for; mass alone will pass them.
Quantitation and salt form
Peak heights in an ESI spectrum are not proportional to amounts: species ionize with different efficiency, salts suppress the signal unevenly, and the charge-state distribution shifts with source conditions. A small-looking impurity peak says nothing reliable about how much is present. The counter-ion is stripped during ionization, so trifluoroacetate and acetate salts give the same found mass, and water is not seen at all.
LC-MS as the combined view
Running the mass spectrometer as a second detector after the UV cell, on the same chromatographic run, joins the two answers. Each integrated peak now has a mass attached: the main peak is confirmed as the intended molecule, the shoulders on its flanks are assigned as +16 Da, +1 Da, −18 Da or truncated species, and an unknown peak is flagged for follow-up.
The combined view also catches both failure modes from the opening section: identity confirmed on a minor peak shows up at once, and a main peak inflated by a steep gradient carries a broader mass trace than it should.
| RP-HPLC area-percent | ESI mass spectrometry | |
|---|---|---|
| Question answered | How much of the eluting, absorbing material is the main peak | Whether the main peak has the predicted mass |
| Sees | Every UV-absorbing species that elutes, in proportion to amide-bond count | Elemental composition of each ionized species |
| Does not see | Water, counter-ion, salts, non-eluting material, co-eluting isomers | Sequence isomers, epimers, positional acylation isomers, relative amounts, salt form |
| Depends on | Column, gradient, modifier, detection region, integration settings | Resolution, calibration, deconvolution settings, average or monoisotopic convention |
When to ask for the chromatogram and the spectrum
Two numbers on a certificate are a summary of two images. Ask for the images when qualifying a new source, receiving a first lot of a new compound, seeing a figure outside the supplier's published range, or planning a re-test. The useful set is the chromatogram with baseline and integration marks, the integration table, the raw spectrum showing the charge envelope, the deconvoluted spectrum with the found mass, and a method summary: column, gradient, modifier, detection region.
Practical tip: ask for the chromatogram and spectrum as exports from the instrument software, not as values retyped into a table. An export carries the integration marks, axis scales and acquisition date; retyping is where transcription errors enter a certificate.
How to read the two results on a Pepti record
Open the public record for lot PEP260722-TR30 at peptioem.com/coa/tirzepatide-30mg-pep260722-tr30: a tirzepatide 30 mg lot manufactured by Pepti, Guangzhou, made 2026-07-22 and tested 2026-07-29. The HPLC result is 99.7%: the share of the integrated UV area that belongs to the main peak, on the method stated on the record.
The identity line is a separate result. Compare the found mass with the theoretical average mass for tirzepatide, 4813 g/mol from C225H348N48O68, allowing for the average-versus-monoisotopic convention. Agreement tells you the main component is tirzepatide; it neither adds to nor subtracts from the purity figure.
Placing a lot within the published range
The Pepti COA library held 76 public entries as of 2026-10-19. Across those records the published HPLC ranges are: semaglutide 98.7%–99.6% over 7 records; tirzepatide 98.4%–99.7% over 9 records; retatrutide 98.72%–99.55% over 9 records; cagrilintide 99.2% on 2 records, both at that value; mazdutide 99.12%–99.37% over 2 records; survodutide has no public record yet. The PEP260722-TR30 result sits at the top of the tirzepatide range.
The range is the context for any new lot. A new tirzepatide certificate inside 98.4%–99.7% is consistent with the published history; a value well outside it, in either direction, is a reason to ask for the chromatogram before accepting the number. The same logic applies to a retatrutide record against 98.72%–99.55% or a cagrilintide record against 99.2%. The full set of records and the release procedure behind them are on the quality and batch records page.
For research use only. Not for human or veterinary use.
Pepti does not present itself as a cGMP drug-substance (API) manufacturer or CDMO and does not publish a GMP certificate or DMF. The two results on a record are analytical statements about one lot, on the methods stated.
Additional assays on a received sample
Once a 1 kit (10 vials) sample is on your bench, the two certificate results can be checked and extended. Each assay below covers a gap that neither area-percent nor mass identity closes; each is described by what it measures, since acceptance limits belong in your own specification.
Amino acid analysis and net peptide content
A weighed portion of powder is hydrolyzed to free amino acids, which are quantified against calibrated standards. Summing the recovered residues gives the mass of peptide in the portion, independent of UV response, counter-ion or water; dividing by the mass weighed in gives net peptide content. The residue ratios also serve as a coarse composition check against the sequence.
Karl Fischer water
Coulometric Karl Fischer titration measures water directly, by its reaction with iodine, on the small quantities a lyophilized vial provides. It accounts for the share of the powder's mass that is bound water, and a vial that has taken up water after sealing shows it here first.
Counter-ion
The anion balancing the peptide's basic residues is measured by ion chromatography or, for trifluoroacetate, by fluorine NMR. The result is the mass fraction of the powder that is salt rather than peptide, and it confirms which salt form was supplied. With water and amino acid analysis it closes the mass balance.
Residual solvent
Headspace gas chromatography detects volatile solvents carried over from purification and lyophilization, such as acetonitrile or alcohols. Neither HPLC nor MS reports them.
Orthogonal chromatography
A second purity run on a different column chemistry or at a different pH changes the elution order and can separate species that co-eluted on the certificate method. For lipidated peptides this is often the most informative addition a QA function can make. Allocate the vials in the kit across the destructive tests before opening any, so that intact material remains for retention.
Sources and verification
- Pepti batch record PEP260722-TR30 (tirzepatide 30 mg) — HPLC result 99.7%, manufactured 2026-07-22, tested 2026-07-29
- Pepti batch record PEP260819-RT20 (retatrutide 20 mg) — HPLC result 99.26%, manufactured 2026-08-19, tested 2026-08-22
- Janoshik Analytical report #210105 — Retatrutide 20 mg sample: 19.52 mg content, 99.837% purity, analyzed 2026-10-07; sample-specific result
- PubChem CID 166567236 (tirzepatide) — Molecular formula C225H348N48O68 and computed molecular weight
- PubChem CID 171934787 (retatrutide) — Molecular formula C221H342N46O68 and computed molecular weight
- PubChem CID 171397054 (cagrilintide) — Molecular formula C194H312N54O59S2, one disulfide
- PubChem CID 56843331 (semaglutide) — Molecular formula C187H291N45O59