Your sample could show one clean peak that takes up most of your chromatogram. But still have impurities in it that the test didn’t detect. Or it could contain something that relates to your main peak and didn’t show on the chart either.
So what is purity really telling you about your sample? It depends on how the test was done and what was used to test for purity.
The following sections explain each test and what it can show about the sample.
What you’ll learn:
- What a Purity Percentage Really Means
- HPLC: Separating Peptides and Impurities
- Mass Spectrometry: Confirming Identity
- The Tests That Fill the Gaps
- How to Read a Peptide Test Report
- Putting the Results Together
What a Purity Percentage Really Means
A test lab will typically use the terms identity, purity, and peptide content. These can all relate to your sample, but mean different things.
Identity is determining if you found the desired peptide in your sample. Chromatographic purity is often reported as the proportion of integrated detector peak area attributed to the main peptide. Content will be determined by how much of your sample weight consists of your peptide of choice.
What’s the difference?
Well, a sample can have one major HPLC peak and contain a large amount of water and/or salts that your test method won’t detect. Just because your main peak fell within the expected retention time doesn’t mean the compound’s identity has been established.
Consider Loti Labs KPV as an example. The identity of this molecule will be lysine, proline, valine, in that order. It then needs to determine what chemical form we are looking for and what specific lot is being tested.
You will notice there is no percent for any of these statements. They each call for different tests to verify your results.
HPLC: Separating Peptides and Impurities
As mentioned earlier, an HPLC shows where the sample components elute. When performing our test we are using a liquid flow rate and pushing our sample through the column at the same time. This will cause your peptide(s) to elute off the column and display a peak on our chart.
This can help separate the target peptide from any of its impurities or byproducts from manufacturing. There could also be some form of degradation in the sample.
Knowing where the desired compound is, the other recorded peaks can then be examined.
When analyzing HPLC it’s important to note the following:
- Can the method resolve the target peak from impurities?
- What type of detector was used?
- How is your sample being integrated for % area?
- Did you compare your main peak to a standard?
All of these factors will come into play when trying to get the best information out of your HPLC run. Even with a flat baseline undetected impurities may still be present below the method’s detection limit.
Two compounds could even overlap and give you a false positive that there is only one compound. By changing your column or method, you may be able to separate these peaks, giving you more information on your sample.
When purity is derived from HPLC peak-area integration, label it as area percentage rather than whole-sample peptide content.
Mass Spectrometry: Confirming Identity
Once the peaks have been separated, how do you know that the desired peak is what you think it is?
Mass spectrometry provides evidence of the molecular weight of the desired compound. MS ionises the sample and measures mass-to-charge ratios to infer a molecular mass, the machine will give you a reading that you can compare to your desired molecular weight.
LC-MS allows you to couple both your HPLC results with mass spec to get a better understanding of what your main peak could consist of. If there is anything abnormal, it can point to impurities or degraded compounds that can affect your desired peak.
Tandem mass spectrometry (MS/MS) can provide additional structural information. This will help determine the sequence of the peptide. However, if you encounter a problem, structural isomers or stereochemical variants can require additional separation or analytical testing.
Again, you want to ensure your identity is being tested against your desired chemical form, and not a generic peptide name.
The Tests That Fill the Gaps
There are many more tests that can help when looking into the purity of samples.
Karl Fischer titration can measure water that does not appear in an ordinary HPLC peptide-area calculation. Other methods target different components:
- Ion chromatography: Can quantify counterions, including trifluoroacetate, when present.
- Gas chromatography: Can check for residual solvents from processing.
- Amino acid analysis: After controlled breakdown of the peptide, can support a peptide-content measurement.
- Chiral separation: Can help distinguish stereochemical variants that share the same mass.
This all comes down to what you are wanting to find out about your sample and how these impurities could alter your overall purity numbers.
In a 2026 study of a purified sublancin sample, researchers reported chromatographic purity above 99% but mass-balance purity of 78.60%.
These results were obtained for a specific sample, but are a great representation of how to view % Area vs Mass Balance in your sample.
Don’t rush to throw extra tests on your report, utilize what makes sense for the sample being analysed.
How to Read a Peptide Test Report
Your COA should provide more than just big numbers that state your purity level. Make sure the report identifies what is being tested, and on which sample its results apply to.
First, start by checking if the Batch # matches what you are reviewing. Then the date of the test, chemical form, and test method can be evaluated.
The report should establish if there is valid testing that backs up your claims. We should know what was used to test your purity, and if it is appropriate for the task.
Some tips when looking over your report:
- Does your lot match the product you purchased? Check for the proper ID of the sample, what it is, and what chemical form is being analysed.
- Can you tell how your test result was determined, what testing was performed to find that data?
- What unit is the data represented in? % Area, or a different method that gives you mass %?
- Are there any cut off numbers for what the test can analyze?
Your report can be as complex as the method that is used to get your desired data. The ICH Q2(R2) lists many attributes that are used to evaluate analytical methods. Such as, but not limited to, specificity, accuracy, precision, Limit of Quantitation, etc…
Having another lab perform testing on your product can be beneficial, but they still need to show proper data to back their claims on your product.
Putting the Results Together
When evaluating a good testing report, always remember what you are trying to find out about your product.
Testing purity of a sample will give you different information than knowing its identity or the mass percentage of the compound you are after. Knowing these factors can allow you to build a well informed testing method to represent what is in your product.