Mass Spec question
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I’m a beginner. I’m researching Tirzepatide. I look at the COAs and see that the Tirzepatide is eluting at 15 min. I’ve read it should elute at 4-8 minutes. So why can’t I find any? Do you guys know of a lab that sells Tirzepatide that elutes at 4-8 minutes ?
Thanks in advance.
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That’s pretty far from a beginner question. I would guess that the elution time differential is most likely caused by a procedural variation during testing, rather than the compound itself. If you can share the COA, maybe someone can shed a better light on it.
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This elution time, as I understand, heavily dependent on the equipment used and the calibration. The labs have the reference tables they use to compare, HPLC test is mostly about the purity, so they are more concerned about the number of peaks and the slopes. MS charts shows the mass of detected molecules, but only a few labs show it, which is a shame. They just want you to trust them they saw it.
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Compare these 2 MS charts for Tirz and Reta.
Charge State Tirzepatide m/z (Avg MW ~4,813.5 Da) Retatrutide m/z (Avg MW ~4,731.3 Da) Key Difference / Analytical Impact [M+3H]³⁺ 1605.5 1578.1 Shifting outside some narrow standard triple-quad mass limits. [M+4H]⁴⁺ 1204.1 1183.8 Base Peak for both; easiest to use for compound tracking. -
Thank you for responding!
The COA is from Onyx. https://onyxresearch.shop/wp-content/uploads/2025/07/Onyx2606230397.pdf
The other information is from research gate. https://www.researchgate.net/figure/Chromatogram-of-optimized-method_fig2_372427976
I’m new to this. I don’t understand everything I’m reading. Is the Onxy Tirzepatide not good? But it seems like every vendor shows the same thing. And the research articles show something different.
Another research paper: https://www.ijpsjournal.com/article/Analytical+Method+Development+and+Validation+of+Tirzepatide+in+Bulk+and+Pharmaceutical+Formulation+by+RPHPLC
Here’s another COA.
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I personally think you are overthinking this… and trying to find a Vendor that sells a peptide with exact specs you want will be impossible. There are only a few manufacturers that actually make this stuff in China, every place is getting it from one of them. That is why when we see a bad test result from one vendor, a few more pop up with the same bad results- they get it from the same place…
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Another couple of things to consider. A regulatory lab, or in house lab generally test one or maybe a few samples at a time, so they have the luxury of adjusting the settings more specifically. A private commercial lab loads 120is vials of different compounds into their test trays. They have to set the test parameters to work for all of them without changing column temps and such. For them the time to test the complete tray is key, since there are several trays waiting in line. Look at the two test screencaps I attached. Same batch, from the same vendor and manufacturer, different labs. So I agree with you, and with @commander. You are doing diligent research, but probably overthinking it. There’s being meticulous, and then there’s “picking fly shit out of black pepper”.
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Disclosure first: I work for Peptriva Research (research-use-only compounds), and I can't help with anything use-related. Testing questions are fair game though.
The way I explain this to people: mass spec and HPLC are answering two different questions, and vendors love to blur them together.
LC-MS tells you whether the molecule is what the label claims. It matches the molecular weight against the expected sequence, and that's it. A passing mass spec says nothing about how much of the vial is actually that molecule.
The purity % everyone quotes comes from HPLC, which is basically asking how much of the chromatogram is the main peak. Thing is, stuff like endotoxin doesn't show up there at all, which is why a decent COA lists LAL and microbial as separate tests instead of hiding behind one purity number.
So if a COA only shows one of these, you're getting half an answer. You want identity, purity, the contaminant screens, and a lot number that matches your vial. If you've got a specific COA you're squinting at, post it or DM me and I'll walk through it.
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Disclosure first: I work for Peptriva Research (research-use-only compounds), and I can't help with anything use-related. Testing questions are fair game though.
The way I explain this to people: mass spec and HPLC are answering two different questions, and vendors love to blur them together.
LC-MS tells you whether the molecule is what the label claims. It matches the molecular weight against the expected sequence, and that's it. A passing mass spec says nothing about how much of the vial is actually that molecule.
The purity % everyone quotes comes from HPLC, which is basically asking how much of the chromatogram is the main peak. Thing is, stuff like endotoxin doesn't show up there at all, which is why a decent COA lists LAL and microbial as separate tests instead of hiding behind one purity number.
So if a COA only shows one of these, you're getting half an answer. You want identity, purity, the contaminant screens, and a lot number that matches your vial. If you've got a specific COA you're squinting at, post it or DM me and I'll walk through it.
@mitchelldemeter OK, maybe you can answer me this LC-MS question. For example, during synthesis of EDR they messed up and made it in reverse: RDE. Will it be detected or we will think it's EDR because the mass is the same?
Or, in a longer peptide chain, if 2 amino acids are swapped because of operator error. Sometimes it is possible, as I understand. The total number of amino acids in the chain will be the same, just the order is different. Can it be detected?
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