HPLC and Mass Spectrometry Purity Testing Explained

Two techniques sit behind almost every peptide certificate of analysis: high-performance liquid chromatography (HPLC) and mass spectrometry (MS). They answer different questions, and understanding both makes the numbers on a COA much easier to trust.

HPLC: how pure is the sample?

In HPLC a small amount of dissolved sample is pushed under high pressure through a column packed with fine particles. Different molecules travel through at different speeds, so the sample separates into its components. A detector at the end, usually measuring ultraviolet light at about 214 nm where the peptide bond absorbs, draws a trace called a chromatogram: a series of peaks, one per component.

The tallest peak should be the target peptide. Purity is the area of that peak divided by the total area of all peaks, expressed as a percentage. If the main peak accounts for 99.5% of the total area, the report says 99.5% purity. The smaller peaks are impurities such as truncated sequences, deletion products or leftover protecting groups.

What 99% does and does not mean

A 99% HPLC purity means 99% of the detected UV-absorbing material is the target peptide. It does not mean the vial contains 99% peptide by weight, because lyophilized powder also holds water and counter-ions such as acetate. That is why the report has a separate assay or net peptide content value, in milligrams.

Mass spectrometry: is it the right molecule?

HPLC says how many components there are but not what they are. Mass spectrometry fills the gap. The sample is ionized, and the instrument measures the mass-to-charge ratio of the ions. The measured molecular weight is compared with the expected one for the intended sequence. A match is strong evidence of identity, and a mismatch is an immediate red flag. LC-MS combines both steps: a liquid chromatograph feeding a mass spectrometer.

Other tests you may see

• ICP-MS measures trace metals such as arsenic, cadmium, lead and mercury.
• LAL endotoxin testing detects bacterial endotoxins, reported in endotoxin units per milligram.
• USP <71> sterility testing checks for microbial growth.

Limits of the methods

Different HPLC methods can give slightly different purity values for the same sample because they separate impurities differently, so the method and detector should be stated on the report. And any test describes the sample that was analyzed. For that reason, reports note the lot, the date and the tested strength, and you should compare them with your vial. Our guide How to Read a Peptide Certificate of Analysis turns this knowledge into a checklist, and the news item How HPLC purity figures appear on Ascend reports shows examples. Research use only.