The molecule a report is describing
Semaglutide is a synthetic peptide of 31 amino acids built on the GLP-1(7–37) sequence. Three changes separate it from that parent, and each one leaves a mark on the analytical record:
- One Aib residue. The second residue (position 8 in GLP-1 numbering) is alpha-aminoisobutyric acid (Aib) instead of alanine. Aib carries two methyl groups on its alpha carbon. It is sterically crowded and couples slowly during synthesis.
- An arginine where a lysine used to be. Position 34 is arginine. The parent sequence has two lysines; semaglutide keeps only one.
- A lipidated lysine. That remaining lysine (position 26 in GLP-1 numbering, residue 20 of 31) carries a C-18 fatty diacid joined through a gamma-glutamic acid and two 8-amino-3,6-dioxaoctanoic acid linker units.
Written out, with the acylated lysine marked: H-Aib-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K*-E-F-I-A-W-L-V-R-G-R-G-OH. Unlike tirzepatide, which ends in an amide, semaglutide ends in a free carboxylic acid. That changes its net charge and is one of the small details that distinguish the two on a column and in a mass spectrum.
Taking the side chain apart
The side chain is the single most important feature for anyone reading a semaglutide report. It is built from three pieces, and each has a defined mass increment once it is joined into the molecule:
| Piece | Chemistry | Mass added (approx.) |
|---|---|---|
| C-18 diacid | 17-carboxyheptadecanoyl: an 18-carbon chain with a carboxylic acid at each end, one end joined as an amide. | 296.4 Da |
| Gamma-glutamic acid | Glutamic acid linked through its side-chain (gamma) carboxyl rather than its backbone carboxyl. | 129.1 Da |
| Two linker units | 8-amino-3,6-dioxaoctanoic acid, a short ethylene-glycol spacer, used twice. | 2 × 145.2 Da |
| Total | The complete side chain on lysine 26 | about 716 Da |
That is roughly 17 percent of the whole molecule. Subtract it and the unmodified backbone (Aib8, Arg34 GLP-1(7–37)) comes out near 3398 Da, which matches the parent peptide's mass plus one extra CH2 for Aib and the lysine-to-arginine swap. The arithmetic is worth doing once, because it is exactly what lets a reader interpret an unexpected peak on a mass spectrum: a signal about 145 Da light is a molecule missing one linker unit, and a signal about 716 Da light is the bare backbone.
What the side chain does to the HPLC trace
Reversed-phase HPLC separates molecules by how strongly they stick to a hydrophobic stationary phase. An 18-carbon chain sticks hard. On a C18 or C8 column semaglutide elutes much later than an unmodified peptide of similar length, and that has three practical consequences for a chromatogram:
- Side-chain variants spread out. Molecules missing part of the side chain lose hydrophobicity and elute earlier. The bare backbone elutes far earlier still. That is useful: these impurities are usually well separated from the main peak instead of hiding under it.
- Backbone variants crowd in. A by-product that differs only in the backbone, such as a single deletion or an isomerized residue, keeps the whole side chain. Its retention is dominated by the same fatty chain, so it can elute very close to the main peak or on its shoulder.
- Peak shape matters. Lipidated peptides can self-associate in solution. Method conditions (organic modifier, column temperature, ion-pairing agent) are chosen to keep the peak sharp. A broad or tailing main peak is a reason to look at the method section, not just the percentage.
Purity is normally calculated at 214 or 220 nm, where the detector responds to peptide bonds. The figure is the main peak's area as a share of all detected peak area under that one method. It is relative: water, counter-ions and anything that co-elutes with the main peak are not separated out. A report that prints the chromatogram lets you check the baseline on both sides of the main peak, which is where the close backbone variants would appear. Our guide to reading an HPLC chromatogram covers what shoulders and small adjacent peaks mean.
What the side chain does to the mass result
At about 4.1 kDa, semaglutide does not show up in an electrospray spectrum as one peak at 4113. It picks up several protons and appears as a series of multiply charged ions. Working from the average mass, the expected signals fall at approximately:
| Charge state | Expected m/z (from 4113.6 average) |
|---|---|
| [M+3H]3+ | 1372.2 |
| [M+4H]4+ | 1029.4 |
| [M+5H]5+ | 823.7 |
Software deconvolutes the series back to one neutral mass, and that is the number a report should print. Two details help when reading it. First, average and monoisotopic masses differ by about 2.5 Da at this size (the monoisotopic mass is near 4111), so the report should say which convention it uses. Second, a strong result gives the expected value, the observed value and the acceptance window; "conforms" with no numbers is weaker evidence than the numbers.
The side chain makes the mass result unusually informative. Every partial side chain has its own distinct mass deficit, so an intact-mass measurement can separate a complete molecule from one that is missing a linker unit, the glutamic acid spacer or the whole chain. Where mass is weak is the same place it is weak for every peptide: two molecules with the same formula have the same mass. The trade-off is laid out in HPLC purity and mass-spectrometry identity, explained.
The impurities that are specific to this molecule
Because the structure is known, the likely failure modes are predictable. These are the ones a careful report, or a careful reader of one, keeps in mind:
| Impurity class | Why it arises | How it shows up |
|---|---|---|
| Incomplete side chain | One of the side-chain building blocks failed to couple. | Mass about 145 Da (one linker), 290 Da (both linkers) or 129 Da (glutamic acid) light; elutes earlier than the main peak. |
| Non-lipidated backbone | The side chain was never attached. | Mass near 3398 Da; elutes far earlier because most of its hydrophobicity is gone. |
| Wrong fatty-acid length | Shorter or longer diacids present in the acylating reagent. | Mass 28 Da light or heavy per CH2CH2 unit; retention shifts with chain length. |
| Side chain on the N-terminus | The N-terminal amine of histidine is the only other free amine in the molecule. | Same mass as the target. Only chromatography or peptide mapping separates it. |
| Deletion at Aib | A slow coupling at the hindered Aib position. | Mass 85 Da light; keeps the side chain, so it can elute close to the main peak. |
| Oxidation and deamidation | The single tryptophan oxidizes; the glutamine can deamidate. | Peaks 16 or 32 Da heavy (oxidation) or about 1 Da heavy (deamidation), usually near the main peak. |
One contrast with tirzepatide is worth noting. Tirzepatide has two lysines, so its side chain can end up on the wrong one, giving a same-mass isomer. Semaglutide's lysine-to-arginine swap removes that particular possibility, leaving the N-terminal amine as the main alternative site. The parallel analysis is in what a tirzepatide lot report has to show, and the same logic for a longer chain is in what a retatrutide report has to show.
Content: why the label weight is not the peptide weight
Lyophilized peptide powder is never pure peptide by weight. The solid also holds bound water and counter-ions left from purification. Purity says nothing about this: a sample can be highly pure by HPLC and still contain more or less peptide than the label weight. A separate net-content line, with its method named, answers that question, and it should be read as a measurement with its own uncertainty, not as a second purity figure. Background is in net peptide content and label weight.
A reading checklist for any semaglutide report
- The report names semaglutide, the strength on the vial and one lot identifier, and that lot matches the vial label exactly.
- An independent laboratory issued it, with an accession number or search code you can check on that laboratory's own lookup page.
- Purity comes with a named method and a printed chromatogram. Look at the region just before and after the main peak, where backbone variants with an intact side chain would sit.
- Identity is stated with its method. An LC-MS result should agree with 4113.6 g/mol average (or about 4111 monoisotopic) and should not show strong signals at the side-chain deficits listed above.
- Any net-content figure names its method and is read separately from purity.
- The report covers the tested sample only. It does not describe another lot or strength, and it does not establish anything it does not list, such as endotoxin.
How Arctic documents semaglutide
The Semaglutide 10mg listing has a lot-matched report in the COA library: lot SEMA-2510-A, issued by Freedom Diagnostics and reported November 1, 2026. It lists purity of 99.91% by HPLC-UV, identity confirmed by LC-MS, and a net-content figure marked report only. It does not include an endotoxin test. The page gives the laboratory's search code so the PDF can be checked on the laboratory's own lookup, as described in how to verify a certificate of analysis. Reports in the library come from two laboratories: ILS Laboratories reports confirm identity by HPLC retention-time match against a reference standard, and Freedom Diagnostics reports confirm identity by LC-MS. Related lipidated materials are listed as Tirzepatide 10mg and Retatrutide 10mg; for any of them, check that the lot on the report matches the lot printed on the vial.
Questions
What are semaglutide's CAS number, formula and molecular weight?
CAS 910463-68-2, molecular formula C187H291N45O59, average molecular weight 4113.6 g/mol. It is a 31-residue synthetic peptide with a free C-terminal acid, one alpha-aminoisobutyric acid (Aib) residue, and a C-18 fatty diacid attached through a gamma-glutamic acid and two short ethylene-glycol linker units to its only lysine.
How much of semaglutide's mass is the side chain?
About 716 Da of the 4113.6 g/mol total, roughly 17 percent. The 17-carboxyheptadecanoyl group adds about 296 Da, the gamma-glutamic acid about 129 Da and the two linker units about 145 Da each. The unmodified backbone alone would sit near 3398 Da.
Why does semaglutide elute so late on reversed-phase HPLC?
The C-18 diacid is a long hydrocarbon chain, and on a C18 or C8 column it dominates retention. Semaglutide therefore elutes far later than an unmodified peptide of the same length, and partly built side-chain variants elute measurably earlier than the full molecule.
What should an LC-MS identity result for semaglutide show?
A series of multiply charged ions, approximately m/z 1372.2 for the 3+ ion, 1029.4 for 4+ and 823.7 for 5+, that deconvolutes to a neutral mass in agreement with 4113.6 g/mol average (or about 4111 monoisotopic). A good report states the expected value, the observed value and which mass convention it uses.
Does Arctic publish a semaglutide lot report?
Yes. Lot SEMA-2510-A of the Semaglutide 10mg listing has a Freedom Diagnostics report in the COA library: purity 99.91 percent by HPLC-UV, identity confirmed by LC-MS, and a reported net content figure. It does not include an endotoxin test, and it describes that lot only.