Retatrutide vs tirzepatide vs semaglutide: how their analytical reports differ

Why compare three reports at all

Retatrutide, tirzepatide and semaglutide are often mentioned in the same breath, and their certificates of analysis look almost interchangeable at a glance. Each lists HPLC purity, an identity result, a lot and a date.

That works only up to a point. All three are long synthetic peptides carrying a fatty-acid side chain, so they share a family of analytical problems. But they differ in length, in the size of the fatty chain, in how the chain ends and in how many unusual residues they contain. Those details shift where impurities elute, how large the mass gaps between the target and its by-products are, and how much an identity result really proves.

The three molecules side by side

The formula and molecular weight for retatrutide are left blank on purpose: our retatrutide listing publishes the CAS number but not a formula or weight, and a mass check should always be made against the expected value the testing laboratory prints on that lot's report.

Length: 31 residues versus 39

Solid-phase synthesis builds a peptide one residue at a time. Each coupling is efficient but never perfect, and the small losses compound. The characteristic impurity of a long synthesis is the deletion sequence: a chain that is complete except for one missing internal residue. It is chemically close to the target, so it tends to elute near the main peak.

Semaglutide's 31 residues mean eight fewer couplings. That does not make a semaglutide lot automatically cleaner; the specific synthesis run and purification matter more than the count. It does mean the two longer molecules have more places for a deletion to occur, and the region just before and after the main peak on the chromatogram deserves a closer look. Deletions at the hindered Aib positions are the classic example: Aib carries two methyl groups on its alpha carbon and couples slowly, and a missing Aib shows up as a mass about 85 Da light.

Side chain: C-18 versus C-20

The fatty diacid is the single feature that makes these molecules behave so differently from ordinary peptides on a column. In reversed-phase HPLC it dominates retention, so all three elute much later than unmodified peptides of similar length. The longer C-20 chain on tirzepatide and retatrutide is two carbons longer than semaglutide's C-18, which adds hydrophobicity on top of an already long backbone.

The side chain also accounts for a large, predictable share of the mass. On semaglutide the full chain (C-18 diacid, gamma-glutamic acid and two 8-amino-3,6-dioxaoctanoic acid linker units) adds about 716 Da, roughly 17 percent of the molecule. On tirzepatide the C-20 version adds about 744 Da, roughly 15 percent. Because each building block has a defined mass, a partly built side chain leaves a recognizable gap in a mass spectrum:

Side-chain variants are the easy case: they separate well and they have distinct masses. The hard case is a backbone variant that keeps the whole side chain. The same fatty chain dominates its retention, so it can sit on the main peak's shoulder. That is why a chromatogram is worth more than a bare percentage. Our guide to reading an HPLC chromatogram shows what shoulders and adjacent peaks look like.

Ends and isomers: where mass stops helping

Tirzepatide and retatrutide end in an amide; semaglutide ends in a free acid. The difference is under 1 Da, too small to read from an intact-mass result at this size, but it changes net charge and therefore retention.

The bigger blind spot is the isomer. Anything with the same formula has the same mass. Tirzepatide has two lysines, so a side chain attached to the wrong one gives a molecule that an intact-mass result cannot tell apart from the target. Semaglutide has only one lysine, which removes that particular risk, but its N-terminal amine is still an alternative attachment site. For any of the three, only chromatography, or a peptide map that digests the molecule and locates the side chain, can separate same-mass isomers. The trade-offs between the two kinds of evidence are set out in HPLC purity and mass-spectrometry identity, explained.

What the mass result should look like

At 4 to 5 kDa none of these molecules appears as one peak at its molecular weight in an electrospray spectrum. Each picks up several protons and shows a ladder of multiply charged ions that software deconvolutes back to one neutral mass. Working from the average masses:

For retatrutide, read the observed mass against the expected value printed on the report itself. In every case a strong identity line gives the expected value, the observed value and whether the figure is average or monoisotopic; at this size the two conventions differ by roughly 2.5 to 3 Da. "Conforms" with no numbers is weaker evidence than the numbers.

Purity figures are not a league table

It is natural to line up three purity percentages and rank them. The figures do not support that. Each is the main peak's area as a share of all detected peak area under one method: one column, one gradient, one detection wavelength. A different method on the same sample can give a different figure, and a figure for one compound says nothing about another.

A more useful comparison is what each report lets you check: a named method, a printed chromatogram, and an identity method able to catch the impurities that molecule is prone to.

Two kinds of identity, and why one lot has both

Reports for these materials use one of two identity methods. An HPLC retention-time match shows that the main peak elutes at the same time as a reference standard run under the same conditions; it is a single dimension of evidence, and a close relative with similar hydrophobicity could elute at nearly the same time. LC-MS adds an observed mass, which rules out most deletion sequences and missing side-chain pieces but not same-mass isomers. The two are complementary, which is why guidance written for pharmaceutical peptides recommends combining orthogonal methods. Retatrutide lot RT-2502-A, described below, has one report of each kind; the tirzepatide and semaglutide lots described below were identified by LC-MS.

Net content: the separate question

Lyophilized peptide powder is never pure peptide by weight; it also holds bound water and counter-ions from purification. Purity does not address this. A net-content line, with its method named, does, and it should be read as its own measurement rather than a second purity figure. Background is in net peptide content and label weight.

How Arctic documents the three

The COA library lists every published lot report with its verification code. For the three 10 mg listings discussed here:

• Retatrutide 10mg, lot RT-2502-A. Two independent reports. ILS Laboratories (certificate COA-2026-6LOOFG, tested August 10, 2026): 99.83% by HPLC, identity by HPLC retention-time match against a reference standard. Freedom Diagnostics (reported October 15, 2026): 99.86% by HPLC-UV, identity confirmed by LC-MS, plus the other lines shown on that page.
• Tirzepatide 10mg, lot TZ-2510-A. Freedom Diagnostics (reported November 1, 2026): 99.83% by HPLC-UV, identity confirmed by LC-MS, net content reported. No endotoxin test on this report.
• Semaglutide 10mg, lot SEMA-2510-A. Freedom Diagnostics (reported November 1, 2026): 99.91% by HPLC-UV, identity confirmed by LC-MS, net content reported. No endotoxin test on this report.

Each report describes the sample of its own lot. It does not describe other lots or strengths, and it does not establish any attribute it does not list. Match the lot printed on the vial to the report, and check the code on the laboratory's own lookup page as described in how to verify a certificate of analysis.

A reading checklist that works for all three

1. The report names the compound, the strength and one lot identifier, and that lot matches the vial label exactly.
2. An independent laboratory issued it, with a code you can check on that laboratory's own site.
3. Purity comes with a named method and a printed chromatogram. Look just before and after the main peak, where backbone variants carrying the full side chain would sit.
4. Identity names its method. For LC-MS, the observed mass agrees with the expected value and there are no strong signals at the side-chain gaps listed above.
5. Any net-content figure names its method and is read separately from purity.
6. Nothing on one compound's report is read as evidence about another compound, lot or strength.

The molecule-by-molecule versions of this page go further: what a retatrutide report has to show, what a tirzepatide lot report has to show and semaglutide's C-18 side chain on HPLC and mass.

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