Semaglutide testing: methods, impurity profile, salt forms and how published samples fail

What a semaglutide test shows

A semaglutide test should answer three questions with three results. LC-MS shows whether the main peptide has semaglutide’s mass. HPLC shows how pure it is and which impurities are present. A content assay shows how many mg of semaglutide, as free base, the vial holds. For vials meant for injection research, endotoxin and sterility are separate tests.

The semaglutide compound page lists the formula (C187H291N45O59), CAS number 910463-68-2 and the calculated ions an LC-MS run looks for: m/z 1371.3791 (3+), 1028.7861 (4+) and 823.2304 (5+). It also lists a recommended panel for each form: freeze-dried vial, liquid vial, pen cartridge and raw powder.

Semaglutide testing methods, step by step

A typical semaglutide test procedure at a lab runs in this order:

Step	Method	Result
Receipt	Photo of the vial, lot and seal logged	The vial the report covers
Identity	LC-MS: measured mass against 4111.1154 Da	Expected and observed mass, error in ppm
Purity	HPLC-UV: peak areas at a stated wavelength	% area, impurity peaks by retention time
Content	Quantitative HPLC against a reference standard	mg per vial as free base, % of label
Composition	Counter-ion and water (Karl Fischer)	TFA or acetate %, water %
Safety	Endotoxin (LAL) and Sterility (USP <71>, 14 days)	EU/mg and EU/vial; growth or no growth

ICH Q6A says retention time alone “is not regarded as being specific”. That is why identity rests on mass, and purity on the chromatogram. Each method covers a gap in the other.

How semaglutide samples fail in published data

Finnrick, a US platform that publishes HPLC results for samples from anonymous public submissions and its own purchases, is the largest public source. In its public data summary read on 4 October 2026, semaglutide had 373 published tests from 71 vendors, the first on 17 December 2024 and the latest on 20 September 2026. 250 passed, 119 failed and 4 were incomplete. About 32% of classifiable samples failed.

That is a smaller data set than retatrutide or tirzepatide, which each have thousands of published tests and fail rates of about 22% in the same summary. Finnrick’s default standard, Purity Focus, passes a sample at purity of at least 99.5% and a fill within 35% of the label claim. The counts do not say which requirement each sample missed. The guide to peptide fail rates compares all products.

The FDA warns that counterfeit GLP-1 drugs could contain the wrong ingredients, too little, too much or no active ingredient at all. Identity and content tests answer exactly those questions.

Semaglutide impurity profile

A semaglutide impurity shows up as an extra HPLC peak. LC-MS can put a mass on it. Typical mass shifts include +16 Da (oxidation) and +0.98 Da (deamidation).

A technical report from HALO, an HPLC column brand, shows what accurate-mass work looks like. It measured semaglutide at 4111.1027 Da against 4111.1154 Da predicted, an error of 3.09 ppm. In a compounded semaglutide sample, it also found a +12 Da modification at the N-terminal histidine, above the level in the reference product. A report that prints the expected mass, the observed mass and the ppm error lets you check this kind of result yourself.

Mass cannot see everything. Isomers with a D-amino acid in place of an L-amino acid have the same mass as semaglutide. A 2022 study in the Journal of Pharmaceutical and Biomedical Analysis examined these isomers. Only chromatographic separation shows them, so a semaglutide impurity profile needs both HPLC and LC-MS.

Salt forms: semaglutide sodium, acetate and free base

Semaglutide is sold as free base or as a salt, such as semaglutide sodium or semaglutide acetate. The FDA states that these salt forms are different active ingredients from those used in the authorised medicines. For testing, the salt form changes the weight of the powder, not the peptide.

Two things follow for a report:

• The content result must state its basis. Free base counts only semaglutide. A result on total powder reads higher.
• The salt form should be stated. A counter-ion test measures the share of TFA or acetate in the powder.

The guide on net peptide content explains why a vial’s powder weighs more than the peptide in it.

Mix-ups: cagrilintide sold as semaglutide

Semaglutide is often sold next to cagrilintide, and in blends with it. The two are easy to tell apart by mass: cagrilintide’s monoisotopic mass is 4406.25 Da, 295 Da above semaglutide. Retatrutide is 617 Da heavier, and tirzepatide 699 Da heavier.

Swaps between these peptides are a known risk, and buyers have reported vials sold as semaglutide that held cagrilintide. Retention time alone can miss such a swap; a mass match does not. If a label is in doubt, a blind GLP-1 identification asks the lab to name the peptide without seeing the label. The cagrilintide testing guide covers the other side of this mix-up.

Compounded semaglutide testing

Compounded semaglutide usually comes as a liquid in a vial or a pen cartridge, with other ingredients. The same core tests apply: identity, purity and content, plus endotoxin and sterility on separate units. Tell the lab the form and the listed ingredients. They change how the lab prepares the sample, and they can add peaks to the chromatogram that are not semaglutide impurities.

Some searches ask about a semaglutide ELISA test. ELISA is an antibody method used mostly on biological samples. It gives no purity result, so it does not replace LC-MS and HPLC for a vial.

What HK Analytical does for semaglutide

We test semaglutide with any mix of identity (LC-MS), purity (HPLC), content per vial, blind GLP-1 identification, endotoxin (LAL), Sterility (USP <71>, 14 days), residual solvents, counter-ion (TFA, acetate), water content (Karl Fischer) and vial-to-vial consistency. Results within days of receipt. Sterility (USP <71>, 14 days) is the one test that takes 14 days, because the method incubates for 14 days. The report states the salt form as received and gives content as free base. Every number comes with its raw data, in a signed record you can check on /verify, with our photo of the vial as received. See the example report or request a quote.