Identification by Retention Time (RT) is one of the standard methods of identifying a drug per the United States Pharmacopeia. It relies on the uniqueness of a drug’s interaction with the stationary phase and mobile phase in HPLC testing. Like a snowflake, every molecule’s interaction with the HPLC system parameters will be unique to that molecule.
A drug will elute (or work its way through the column) differently depending on the molecule’s size, shape, charge, the stationary phase, mobile phase, pH, temperature, and more. The “retention time” is the measurement of how long the drug is retained in the column before it elutes.
This is a chromatogram of semaglutide on the TrustPointe Analytics test method for semaglutide. The retention time is approximately 4.5 minutes:
If a supplier sold a vial that contained mixed semaglutide and tirzepatide, and we injected it using our semaglutide method, we could easily tell that it is not semaglutide. Here’s why:
As you can see, the retention time of tirzepatide on this semaglutide method is 5.5 minutes, a minute longer than semaglutide. This is because of tirzepatide’s molecular size, shape, charge, and system parameters.
In addition to visually being different, we perform a calculation called ID by Retention time to compare retention times and know they are different.The sample retention time is divided by the average retention time of five reference standard injections. If we calculate it for this peak, it’s 0.82 and the acceptable range is 0.98 – 1.02. We know that the peak at 5.5 minutes is not semaglutide.
This is also true for our tirzepatide method. This is tirzepatide eluting at 2.2 minutes:
If semaglutide is injected on our tirzepatide method, it shows up as a tailing (misshapen) peak at 3.4 minutes, where tirzepatide elutes at 2.2 minutes:
To the naked eye, both semaglutide and tirzepatide are generic white powders. This is why analytical testing is so important.
But what if someone wants to sell fake semaglutide by finding a molecule that elutes at the exact same time as semaglutide?
It would be extremely challenging to find a molecule that behaves exactly the same way as semaglutide in this method and has exactly the same retention time. It would likely take more time and investment than just selling semaglutide itself.
And remember, that’s just for our HPLC method – there are hundreds of different HPLC methods out there with different parameters and this one molecule would have to perform exactly the same on all of those other methods too (highly unlikely).
In the off chance that this was possible and someone wanted to take the time and invest in doing it (extremely remote), we also perform a spectral comparison against the reference standard that would fail – but that’s for another post.
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At TrustPointe Analytics, we are passionate about quality and accuracy of our testing. We when we develop and validate our analytical methods, we are always considering risks to having inaccurate results and implementing measures to mitigate them. As always: Accuracy Matters. Results You Can Trust.