You really need to get known samples of GHK, GHK-Cu, and Mannitol, and run those on the setup you have.
I agree. I just have not been able to find any reference standards for GHK or GHK-Cu. I could have a Chinese supplier send me a sample. However, that is not really a trusted reference.
Do you know for sure that the patent was using isocratic elution?
No, it was gradient. However, my chemist was supposed to a calculate the average concentration over that time; which she just told me she messed up. I did not purchase the more expensive gradient module for our HPLC, unfortunately. Some day I will.
We are the manufacture of GHK and GHK-Cu with lab located in China, based on our experience, you need to adjust analytic method in 2 different ways.
1. Mobile phase ratio. 30% Acetonitrile is too strong for strong polar compound GHK-Cu , that is the reason you got almost no retention time on column. The suitable mobile phase could be from 0.1% to 30% solvent B in 30minutes, you can check our result in #299; What the WO patent want to express is linear increase from 0 to 30% rathar than constant 30% solvent B.
2. Injection amount. your sample maybe too concentrated. usually the best resuly can be obtained when absorption value is around 1000(sharp peak) which can be easily obtained by dilute sample or inject less volumn.
Fantastic! That is exactly what I needed. My chemist was supposed adjust the acetonitrile concentration to 5.5%, to account for the difference between gradient and isocratic. She screwed that up. That might account for the retention time difference.
As I explained in #299, the first peak is Cu ion, the 2nd peak is GHK, you can't see GHK-Cu peak due to the exist of TFA in mobile phase(GHK-Cu destryed in strong acid environment)
Would .1% TFA be strong enough to destroy the GHK-Cu that quickly? If that is the case, then we would have to use acetonitrile without TFA in it. I would need to order that from Sigma. The patent I linked was an HPLC test for just GHK. They were not testing GHK-Cu. So that would explain why they did not worry about the TFA.
So it looks like it might be GHK-Cu, GHK, and Mannitol. We would need to get a reference standard to be absolutely sure on it. If anyone finds a reference standard, you can send it to us and we can run the test again. I will have to order some acetonitrile without TFA from Sigma if we do it. Just let me know ahead of time.