Updated: 05-Jul-2026
Identification by Retention Time (ID by RT) is one of the standard methods of identifying a drug per the United States Pharmacopeia (USP). 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 compound will elute (or work its way through the column) in more or less time depending on the molecular size, shape, charge, the stationary phase, mobile phase, pH, temperature, and more. The “retention time” is the measurement of how long the compound 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 the molecular size, shape, charge, of tirzepatide and how it interacts with the HPLC method parameters.
In addition to visually being different, we perform a calculation called Identification 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 USP 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.
The closest eluting compounds we've found so far in our testing is retatrutide running on our tirzepatide method, and that still fails the Identification by Retention Time calculation and specification limit.
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 TrustPointe's 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).
What about MS? Isn't it better?
Mass spec is great, and in this space, mass spec is performed initially by the company synthesizing the peptide. No compounder is buying a compound at $1000/gram without seeing a mass spec result.
In general quality control checks where we're looking for batching mix-ups (e.g. wrong peptide powder was added to the solution) or labeling/vial mix ups, HPLC is sufficient. Repeating mass spec for identification isn't bad, it's just like buying a Ferrari to never drive it faster than 40 mph.
Doing a deep dive investigation on an issue where a peptide was synthesized incorrectly, identifying the structure of a peptide impurity, or trying to determine the identification of an unknown compound - that is where mass spec really shines. But most people just want to know if a peptide good or bad, and don't want to spend money to determine why it's bad (we spent $3500 on one simple impurity investigation).
Mass spec certainly doesn't hurt, but we don't see a ton of value for it in this space. We'd rather invest our time in validating our methods for accuracy and precision, doing forced degradation studies to ensure we can resolve peptide degradation peaks from the main peptide peak, etc. It also helps us maintain our fast turnaround times, especially as peptide labs struggle with capacity issues.
We have your back.
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.
