Open almost any certificate of analysis (COA) for a research peptide and two acronyms usually sit near the top: HPLC and MS. They are often printed side by side, as if they were two ways of saying the same thing. They are not. Each one answers a different question, and each one is blind to something the other can see.
This guide explains what high-performance liquid chromatography (HPLC) and mass spectrometry (MS) each measure, what each can and cannot prove about the material in a vial, and why a COA that shows only one of them leaves an important question open.
The short answer
- HPLC answers "how much of this sample is the main component?" It separates the sample and reports the main peak as a share of everything the detector saw. That share is the purity figure.
- Mass spectrometry answers "what is the main component?" It measures the mass of the molecules and compares it with the mass the sequence predicts. That comparison is the identity check.
A sample can pass one and fail the other. A very pure sample of the wrong peptide passes HPLC and fails MS. The correct peptide mixed with a lot of related impurities passes MS and fails HPLC. You need both to know you have the right molecule at a useful level of purity.
What HPLC does
In HPLC, a dissolved sample is pumped under pressure through a column packed with fine particles. Different components interact with the packing to different degrees, so they leave the column at different times. A detector, usually ultraviolet (UV), records a signal as each component comes out. The output is a chromatogram: a trace with a peak for each separated component.
For peptides, the UV detector is usually set around 210–220 nm, where the peptide backbone absorbs. The purity figure on a COA is normally the area of the main peak divided by the total area of all detected peaks, expressed as a percentage. This is called area-percent purity.
What an HPLC purity figure proves
- That, under the stated method, one component accounts for a given share of the UV signal.
- That the other detected components (impurities) are present at small levels, if the minor peaks are small.
- That the lot looks consistent with previous lots, if the retention time and peak pattern match.
What HPLC does not prove
- Identity. HPLC does not tell you what the main peak is. It tells you the main peak is large. A different peptide with similar chromatographic behavior would look much the same.
- Quantity. Area-percent purity describes the proportion of the UV signal, not the mass of peptide in the vial. Water and counterions such as trifluoroacetate (TFA) or acetate are part of the powder's mass, but they do not show up as peaks in the same way. Our explainer on peptide purity testing covers this gap in detail. Related reading: Peptide Purity Testing Explained: Area-Percent, Net Peptide Content, and Why They Differ.
- Anything the detector cannot see. Inorganic salts and metals do not absorb UV at peptide wavelengths, so they are invisible to a standard HPLC-UV purity run.
What mass spectrometry does
A mass spectrometer turns molecules into charged ions and measures their mass-to-charge ratio (m/z). For peptides, the most common setup couples liquid chromatography to the mass spectrometer (LC-MS), so the sample is separated first and each separated component is then weighed.
Every peptide sequence has a theoretical mass that can be calculated from its amino acids. The identity check compares that theoretical mass with the observed mass. Peptides often pick up more than one proton when they are ionized, so a spectrum may show the same molecule at several m/z values, labeled as charge states such as [M+2H]²⁺ or [M+3H]³⁺. A good report labels these and shows how the observed mass was derived.
What an MS identity check proves
- That the main component has the mass the sequence predicts, within the instrument's accuracy.
- With tandem mass spectrometry (MS/MS), which breaks the molecule into fragments, that the fragment pattern fits the expected sequence.
- That specific impurities, such as a sequence missing one amino acid, can be named rather than just counted.
What MS does not prove
- Purity on its own. A mass spectrum can show that the right molecule is present without telling you how much of the sample it makes up. Ionization efficiency varies between molecules, so peak height in a spectrum is not a reliable purity measure.
- Isomers. Molecules with the same mass but a different arrangement, such as a peptide containing one amino acid in its mirror-image form, can give the same observed mass. Specialist methods are needed to separate them.
That last point is not academic. A 2023 paper from the United States Pharmacopeial Convention (USP) and the UK's Medicines and Healthcare products Regulatory Agency on peptide reference standards notes that some peptide features, "such as chiral or isobaric amino acids, may require additional techniques to ensure a full characterization" (McCarthy et al., Pharmaceutical Research, 2023; DOI: 10.1007/s11095-023-03493-1). The same paper describes identity testing by NMR, mass spectrometry and chromatography, and assessment of peptide content and impurities by HPLC and GC. In other words, even reference-standard laboratories use several methods together, because no single one covers everything.
Side by side
| Question | HPLC (UV) | Mass spectrometry (LC-MS) |
|---|---|---|
| Is it the right molecule? | No | Yes (observed vs. theoretical mass) |
| What share of the UV signal is the main peak? | Yes | Not reliably |
| Can it name a specific impurity? | Only by retention time, if a reference exists | Yes, often by mass |
| Does it see salts and metals? | No | Not in a standard peptide run |
| Does it tell you how many mg of peptide are in the vial? | No | No |
| What should the COA show? | Chromatogram + method + measured purity vs. spec | Spectrum + theoretical and observed mass |
Why control laboratories use both
Official control laboratories that test seized or suspect peptide products pair the two methods as a matter of routine. A 2015 method from Belgium's Official Medicines Control Laboratory used LC-MS/MS to screen for and identify 25 different peptides, and UHPLC with diode-array (UV) detection to quantify them. The method was validated to ISO 17025 requirements (Vanhee et al., Talanta, 2015; DOI: 10.1016/j.talanta.2015.04.022). Identity came from the mass spectrometer. Quantity came from the chromatograph.
The same institute's later study of the ten falsified peptide products seen most often on the Belgian market shows what happens when you look beyond the main peak. Alongside large variation in peptide amount and low purity for some products, the researchers found arsenic and lead (Janvier et al., Talanta, 2018; DOI: 10.1016/j.talanta.2018.06.023). Neither a standard HPLC purity run nor an LC-MS identity check would flag those elements. That takes a third technique, such as inductively coupled plasma mass spectrometry (ICP-MS).
How the two fit into a full characterization
The most complete published approach to assigning a peptide's purity uses many methods at once. A national metrology institute in China assigned an SI-traceable purity value to a synthetic peptide using a "mass balance" approach: LC with high-resolution tandem MS to identify and quantify 21 structurally related impurities, plus Karl Fischer titration for water, ion chromatography for counterions, ICP-MS for inorganic elements and headspace GC-MS for volatile compounds. The final assigned value was 796.5 mg of peptide per gram of material (Wang et al., Journal of Pharmaceutical and Biomedical Analysis, 2022; DOI: 10.1016/j.jpba.2021.114401).
That number is worth pausing on. Roughly a fifth of that carefully characterized material was not the peptide. A routine research-grade COA will not go that far, and it doesn't need to for most lab work. But it shows why "99% by HPLC" and "correct mass by MS" are the start of the story, not the end.
What to look for on a COA
When you read the analytical section of a COA, check for:
- An HPLC chromatogram, not just a typed percentage, with the method conditions (column, wavelength, gradient) stated.
- A measured purity value next to its specification, for example "≥ 98% spec / 99.1% found".
- A mass spectrum, with the theoretical mass, the observed mass and the charge states labeled.
- The lot number matching the number on your vial, plus the laboratory name and test date.
- Optional but valuable: net peptide content, water content and counterion content, which tell you how much of the powder is peptide.
If a COA shows HPLC only, you know the sample is dominated by one component, but not which one. If it shows MS only, you know the right molecule is present, but not how clean the sample is. Either way, it is worth asking the supplier for the missing half.
How Seratide presents it
Seratide prints a lot number on every vial and links each lot to a record on its Lot lookup page, laid out with HPLC purity against specification, identity by LC-MS, and endotoxin. The records on that page today are clearly labeled layout samples showing the format, with placeholder values. Issued laboratory reports replace them as each lot is analyzed. For background on how peptide sequences and masses are written, see our peptide basics page, or browse the research peptides shelf.
Sources
All sources below were opened and their PubMed abstracts read on 2026-09-29. Claims are limited to what the abstracts state.
- McCarthy D, et al. Reference Standards to Support Quality of Synthetic Peptide Therapeutics. Pharm Res. 2023;40(6):1317-1328. PubMed: https://pubmed.ncbi.nlm.nih.gov/36949371/ · DOI: https://doi.org/10.1007/s11095-023-03493-1
- Vanhee C, et al. Analysis of illegal peptide biopharmaceuticals frequently encountered by controlling agencies. Talanta. 2015;142:1-10. PubMed: https://pubmed.ncbi.nlm.nih.gov/26003685/ · DOI: https://doi.org/10.1016/j.talanta.2015.04.022
- Janvier S, et al. Impurity profiling of the most frequently encountered falsified polypeptide drugs on the Belgian market. Talanta. 2018;188:795-807. PubMed: https://pubmed.ncbi.nlm.nih.gov/30029448/ · DOI: https://doi.org/10.1016/j.talanta.2018.06.023
- Wang S, et al. Mass balance method for SI-traceable purity assignment of synthetic oxytocin. J Pharm Biomed Anal. 2022;207:114401. PubMed: https://pubmed.ncbi.nlm.nih.gov/34656934/ · DOI: https://doi.org/10.1016/j.jpba.2021.114401
For laboratory research use only.