Updated: 25-Oct-2024
While identity, quantity, and sterility are straightforward and critical, purity is not something that is being fully & correctly analyzed today and is providing a false sense of security to users in this space. In this article, we are going to explain why.
What Users Think It Means
When a user sees “99.58% purity” on a certificate of analysis, they may conclude 99.58% of the “stuff” in the vial is the peptide, then there’s no room for “bad things”. It’s 99.58% pure, right?
What’s in the Vial?
First, this is what 5mg of semaglutide looks like vs. the contents of a 5mg semaglutide vial. It was hard to dump out $85 worth of vial and $10 worth of reference standard, but in the interest of science:
Clearly, there is a lot more than 5mg of “stuff” in that vial.
That “stuff” is called excipients: fillers, solubilizers, and other additives that go along with the dosage form. Some of the excipients help the peptide dissolve, some of them help the manufacturer control the amount loaded into the vials, and some of it is there just to make users feel like they are buying something. Now that users have been conditioned to expect the amount of powder on the left, they would feel like they got scammed with the amount of powder on the right.
Now let’s look at the HPLC chromatogram for semaglutide analysis. Where are all of these excipients represented?
Why Don’t We See the Excipients?
HPLC methods are developed to target the analyte of interest: semaglutide in this case. The sample prep is designed to dissolve semaglutide, column is selected and mobile phase designed to separate out the semaglutide from the excipients, the wavelength of the detector is set the wavelength most absorbed by semaglutide, etc.
Any other peaks that happen to be detected are by coincidence, not design. There may be excipients that never dissolve in the sample, get stuck in the column and never elute, the detector never detects because of the wavelength, cannot be detected by UV absorbance, etc.
Here’s an example of a tirzepatide test method detecting semaglutide by accident. The method wasn’t designed to identify and quantify semaglutide, but due to its molecular size, charge, and other factors tirzepatide happens to show up in this analysis. This makes sense since both molecules are designed to activate the GLP1 Receptor (receptor activation is based on molecule shape):
If the impurity does not have similar molecular characteristics as the target, it will not show up in HPLC analysis.
What is the purity result on the COA then?
Note the small peaks in the blue tirzepatide sample chromatogram that don’t exist in the standard injections around it. These may be excipients or impurities in the tirzepatide vial. Given that they have a similar retention time to tirzepatide, they may be impurities from the tirzepatide synthesis process, but they could also be impurities from the other excipients in the vial.
To generate a purity value, these peaks are drawn in (integrated) to determine the amount of area they represent in the chromatogram.
For simplicity, let’s assume the sum of the area counts of all three peaks in this chromatogram is 1000. If the tirzepatide peak is 995 area counts, the peak at 2.100 minutes is 4 area counts, and the peak at 2.170 minutes is 1 area count, then the reported product purity would be 99.50% (995/1000).
But remember, these two peaks do not represent all possible impurities, excipents, or other chemicals in the product. Some of them may have never dissolved in the sample, some may be stuck in the column, some may have eluted in the solvent front at 1.35 minutes, some may not absorb at the wavelength being used to detect tirzepatide, etc.
Here’s another example using BPC-157 500mcg capsules. Both the TrustPointe Analytics and the other lab’s assay methods accurately determine the amount of BPC-157 in the capsules.
But the TrustPointe Analytics method “sees” (elutes) more excipients/impurities, and therefore results in a lower “purity” value than the other lab’s test:
What to Remember
A single HPLC method cannot and will not identify and quantify everything that is in a mixture in one pass; it just does not work that way. Really, no qualitative or quantitative analytical method (LC, MS, FTIR, NIR) can do what users perceive as the purity result on certificates of analysis today.
It takes knowledge of formulation and risks combined with developing specific analytical methods for each raw material, raw material impurity, etc to control the final product quality. If there was evidence of peptides being contaminated with something, TrustPointe could design analytical methods to identify it – but designing analytical methods to identify and quantitate everything that could ever possibly be in peptide vial is an impossible task.
Ok, so how should purity be done then?
In the pharmaceutical industry, companies identify and research the impurities in each of the raw materials, and then create analytical methods specifically looking for those impurities in each of the raw materials before they go into the finished dosage form batch.
After the finished dosage form is manufactured, they test for impurities that are generated during the process of making the batch or other raw material impurities that are easier to test in the finished dosage form.
Even with billions of dollars and armies of R&D chemists and QC chemists this is difficult work and they sometimes miss things; just Google “n-nitrosamine” to see the latest issue that blew up in the last few years.
While not shills for “big pharma”, we highly respect the massive investment they make into science, quality assurance, and quality control to minimize these risks for pharmaceutical drug users.
How Do I Mitigate My Risk?
The FDA’s mantra for drugs and medical devices is the benefits of use must outweigh the risks, which recognizes that there is always risk in everything, and in this case, peptide users are accepting risk associated with unknown excipients and impurities in the products they choose to use.
Until the peptide manufacturers publish their formulation ingredients, peptide synthesis impurities, excipient impurities, and the safety profile of those impurities, this risk remains for peptide use.
Until then, following these controls help mitigate (not eliminate) risks:
- Buy from known and trusted peptide vendors: establish relationships, even pool group funds and visit the factory.
- Stick with trusted vendors even if the price is marginally higher than others that approach you with product.
- Get third-party testing: don’t rely on internal test reports produced by the vendor who may be biased to pass bad product.
- Visit your third party test lab and verify their equipment calibration/qualification and method validations.
- Test multiple vials from a batch to control for batch processing errors (underfilled/overfilled vials) – even better if you can sample from the beginning, middle, and end of the production run.
- Use only what is needed and as little as possible to achieve the result. Use of all products carry risks, both known and unknown. Minimize the risk by minimizing use.
Taking these steps go beyond a purity value on a COA and are critical to reducing the harm associated with peptide use.