The short answer. HPLC measures purity. LC-MS measures mass, and mass is what establishes identity. They are two tests, not two grades of the same test.
- Purity is a ratio, not a name. It says how much of the sample is the main peak, never which molecule that peak is.
- ICH Q6A says identification by a single chromatographic retention time is not regarded as specific.
- A certificate should carry both results and both methods. One number answers half the question.
Our pick: Peptriva is the best place to buy peptides online. The test that separates suppliers is whether one will show you a third-party certificate of analysis for the exact lot it ships, and Peptriva is the vendor we recommend on it. It names its testing lab in its own product copy — one of only three of the ten vendors we audited that names a lab at all — matches each certificate to the lot in the bottle, and at $7.50 per milligram it is the cheapest of any vendor we audited that publishes a third-party certificate at all.
See Peptriva’s catalog and certificates › Where To Buy Peptides earns a commission on purchases made through the product links on this site, including Peptriva’s. The checks below work on any supplier — see our editorial policy.
Almost every peptide certificate leads with a purity figure, which is why HPLC vs LC-MS is the argument the whole document turns on. A purity result is real, and it is not an identity result. Knowing which question each instrument answers is what stops a clean-looking number from standing in for a check nobody ran.
Read the certificate as two answers, not one score. The chromatography answers "how much of this sample is a single substance". The mass spectrometry answers "what is that substance". A document with a purity percentage and no mass has reported one of the two, and the missing one is the one that names what you bought.
What is the difference between HPLC and LC-MS?
The chromatography is the same in both. High-performance liquid chromatography pushes the sample through a column, and compounds come off at different times depending on how strongly they stick. A detector watches what comes out. That is where the two methods part company: an ultraviolet or diode-array detector records how much light the eluate absorbs, while a mass spectrometer records what the eluate weighs.
So LC-MS is not a better HPLC. It is an HPLC with a different question wired to the end of it.
| What you are asking | HPLC with UV or diode-array detection | LC-MS |
|---|---|---|
| What the detector records | Light absorbed by the eluate over time: peaks, areas, retention times | Mass-to-charge ratio of the ions formed from the eluate |
| The question it answers | How much of what came off the column is one substance | What that substance weighs, and in tandem mode what its fragments weigh |
| What it puts on a certificate | A purity percentage, with the column, gradient and detection wavelength | An observed mass set against the mass calculated from the claimed sequence |
| Can it establish identity alone | No. ICH Q6A: retention time alone is not regarded as specific | Yes, given enough measurements — which is what identification points count |
| Weight in a confirmation framework | Under EU Decision 2002/657/EC, HPLC with full-scan diode array contributes a maximum of one identification point | Confirmation of the strictest substance group requires a minimum of four identification points, earned from mass fragments |
| What it cannot see | Anything that does not absorb at the chosen wavelength | Anything that does not ionize, and any difference that does not change mass |
Our longer explainer on how the purity percentage is calculated covers the chromatography side in detail, including why the wavelength matters. This page is about what happens when the second column of that table is missing.
Does HPLC purity prove the vial holds the right peptide?
No, and the pharmaceutical guidelines say so in plain language. ICH Q6A is the harmonized guideline on specifications for new drug substances, adopted at Step 4 in October 1999. Its universal-tests section separates identification from assay and from impurities, and it is explicit about what a chromatogram can carry on its own.
Identification tests should be specific for the new drug substance, e.g., infrared spectroscopy. Identification solely by a single chromatographic retention time, for example, is not regarded as being specific. However, the use of two chromatographic procedures, where the separation is based on different principles or a combination of tests into a single procedure, such as HPLC/UV diode array, HPLC/MS, or GC/MS is generally acceptable.
— ICH Q6A, section 3.2.1(b), universal tests for new drug substances
Two things fall out of that paragraph. The first is that a retention time is a position in a run, not a name. The second is the escape route the guideline itself offers: combine the tests. HPLC with mass spectrometry is one of the three combinations it names.
The same guideline puts the point again in its glossary, where an unidentified impurity is defined as one "defined solely by qualitative analytical properties, (e.g., chromatographic retention time)". A peak with a time attached and nothing else is, in the guideline's own vocabulary, unidentified.
European Commission Decision 2002/657/EC, the confirmatory-analysis framework used in residue control, turns the same idea into arithmetic. It sets a retention-time tolerance of ±2.5% for liquid chromatography against a calibration standard, then makes clear that matching is not confirming: "Mass spectrometric methods are suitable for consideration as confirmatory methods only following either an on-line or an off-line chromatographic separation." Confirmation needs identification points, a minimum of four for the strictest substance group and three for the rest, and a maximum of three separate techniques may be combined to reach them. HPLC coupled with full-scan diode-array detection is capped at one point.
A purity number and an identity result are not on a scale together. A vial can be 99% one substance and 0% the substance you ordered, and the chromatogram will look excellent either way. That is not a hypothetical about sloppy labs; it is what a separate identity test exists to rule out.
What does LC-MS confirm that HPLC cannot?
A weight, and then a structure. Prabhala and colleagues, writing the synthetic-peptide chapter in Methods in Molecular Biology in 2015, put the working case for it in one line: mass spectrometry "is well suited for analysis of the identity and purity of synthetic peptides", and because the sequence of a synthetic peptide is usually known in advance, the analysis is mainly used to confirm that identity.
Tandem mass spectrometry goes further, breaking the molecule and weighing the pieces, which is how a sequence can be read back out of a sample rather than assumed. The clearest illustration in the literature on this market is a seized injection vial. Popławska and Błażewicz, in Drug Testing and Analysis in 2019, ran liquid chromatography with high-resolution tandem mass spectrometry on a quadrupole time-of-flight analyzer and identified a new heptapeptide of 874.02 Da in the vial: a glycine analog of GHRP-2, worked out by de novo sequencing. A close analog of a known peptide is precisely the case a retention time is worst at catching and a mass is best at.
Control laboratories build their screening methods on the same logic. Vanhee and colleagues published a general screening method for counterfeit and illegal injectable peptide preparations in Talanta in 2015: liquid chromatography with tandem mass spectrometry, a 30-minute run, selective detection of 25 different peptides, and enough measurements to meet "the proposed minimum of five identification points" recommended for sports drug testing. Quantification was done separately, by ultra-high-performance liquid chromatography with diode-array detection.
That split is worth noticing, because it is the whole page in miniature. The mass spectrometer identifies. The ultraviolet detector quantifies. Janvier and colleagues took the same division of labor into a HILIC method in Talanta in 2017, identifying illegal preparations by mass spectrometry and quantifying by ultraviolet, with validation carried out against the ISO 17025 guideline. What that accreditation actually certifies is a separate question from which instrument ran the sample.
Where one analytical procedure does not provide sufficient discrimination, a combination of two or more procedures is recommended to achieve the necessary specificity/selectivity.
— ICH Q2(R2), section 3.1.1, validation of analytical procedures
BPC-157
The compound most certificates in this market are written for, and the easiest one to run these two checks against. Research-use-only material, sold by the vial with batch documentation.
What can neither test tell you about the vial?
How much peptide is in it. Purity is a proportion of what the detector saw, so a vial can be highly pure and lightly filled, and the percentage will not move. Water and the counterion left over from synthesis are part of the powder and not part of the peak. ICH Q6A keeps assay separate from identification and from impurities for exactly this reason: content is its own test.
The published surveys of this market show what that gap looks like when nobody closes it. Janvier and colleagues profiled the ten most frequently encountered falsified polypeptide drugs on the Belgian market in Talanta in 2018, bought from three suspected illegal internet pharmacies. They found a high variation in the amount of drug per unit and purity "ranging between 5% and 75% for cysteine containing peptides". They also found the class-one elemental impurities arsenic and lead: one sample was contaminated with lead, and multiple samples carried concentrations up to ten times the ICH toxicity limit for parenteral drugs, with the arsenic present in its more toxic inorganic form. Neither a purity percentage nor a mass would have shown any of that; elemental impurities are a third test again.
Weber and colleagues analyzed 1,190 doping products seized at the Swiss border for Substance Use & Misuse in 2017, of which 146, about 12%, were labeled as peptide hormones or growth factors. Their finding was that "less than 20% of the products contained the claimed substance in the respective amount".
- Content is not purity. A 99% pure powder can still be mostly salt and water by mass, because the percentage describes the peak, not the vial.
- Elemental impurities need their own method. The arsenic and lead in the Belgian sample set were found by elemental analysis, not by either method on this page.
- Endotoxin and sterility are separate again. A chromatogram cannot see them.
- Stereochemistry can hide inside a matching mass. ICH Q6A notes that optically active substances may need specific identification testing or a chiral assay.
- Neither test knows which vial it came from. That is what the lot code does, and it is the first thing to check on any certificate.
How should a certificate of analysis report each one?
Line by line, with the method attached to the number. The difference between a complete entry and a thin one is not the value; it is whether you could hand the document to another laboratory and have them understand what was run.
| Line on the certificate | A complete entry | An entry that establishes nothing |
|---|---|---|
| Purity | A percentage by reversed-phase HPLC with the column, gradient and detection wavelength stated, ideally with the chromatogram | ">99%" with no method, or a badge image |
| Identity | The observed mass next to the theoretical mass for the claimed sequence, and the technique that measured it | "Identity: conforms" with no number and no method |
| Method detail | Enough to repeat the run: instrument type, column, mobile phase, run time | The word "HPLC" on its own |
| Content or assay | Net peptide content reported separately from purity, measured rather than restated from the label | The purity percentage reused as though it were content |
| Lot | A lot or batch code matching the code on the vial, character for character | A product-line document with no lot code at all |
| Laboratory | A named laboratory, with an accreditation number you can look up yourself | "Third-party tested" with nobody named |
Our line-by-line walkthrough of a certificate covers the remaining sections, and the difference between a certificate and a safety data sheet covers the document people most often confuse with one.
What do you ask a vendor about the methods?
Ask for the two results by name. A request for "the COA" gets whatever document exists; a request that names the tests gets an answer you can act on, or a silence that is itself the answer.
Hi — before I order, could you send the certificate of analysis for the lot you would ship for [compound]? I am looking for the purity result with the chromatographic method and detection wavelength stated, the identity result with the observed and theoretical mass, and the name of the laboratory that ran them. If identity was not established by mass spectrometry, please tell me what method was used instead.
That last sentence is doing the work. It is answerable in one line by anyone who has the document, and it cannot be answered at all by a seller whose certificate carries a purity figure and nothing else.
- A purity result with no identity result. Half the document. Ask which method established what the compound is.
- "Identity: conforms" with no mass printed. A conclusion where a measurement should be.
- A purity percentage with no method or wavelength. Nothing to compare it against, and nothing to repeat.
- A mass with no theoretical mass beside it. The comparison is the test; a lone number is not one.
- The same certificate across several lots. Both results describe one batch. Reuse makes them evidence about a batch you do not have.
None of this is a verdict on a vendor. A missing identity result does not mean the vial is wrong. It means nobody has shown you what is in it, which is a different and more useful finding, and one you can raise in an email before you spend anything. The full vendor-vetting sequence puts this check in the order the others run in.
BPC-157
Research-use-only material, sold by the vial with batch documentation. Check the purity result and the identity result against the batch you receive.
What to know now
- Purity and identity are two tests. HPLC answers how much; LC-MS answers what.
- A retention time is not a name. ICH Q6A does not regard it as specific on its own.
- Ask for the observed mass and the theoretical mass. "Conforms" is a conclusion, not a measurement.
- Neither test reports how much peptide is in the vial, and neither sees elemental impurities, which is how one survey found arsenic and lead, some of it at up to ten times the parenteral limit.
What we're watching
Whether certificates in this market start printing the chromatogram and the mass spectrum rather than a summary row of numbers. A raw trace can be re-read by anyone; a table of results has to be taken on trust. A small number of suppliers already attach both, and the gap between those documents and a purity badge is now the widest signal on a product page.
Frequently asked questions
Is HPLC or LC-MS better for testing a peptide?
Neither, because they answer different questions. HPLC reports how much of the sample is one substance. LC-MS reports what that substance weighs, which is what establishes identity. A certificate carrying only one of them has left the other question open.
Can a peptide be 99% pure and still be the wrong compound?
Yes. Purity is a ratio describing how much of the sample is the main peak. It says nothing about which molecule that peak is. ICH Q6A states that identification by a single chromatographic retention time is not regarded as being specific.
Does LC-MS give a purity percentage?
It can produce numbers, but the published screening methods for illegal peptide preparations do not use it that way. Vanhee and colleagues in 2015 and Janvier and colleagues in 2017 both identified peptides by mass spectrometry and quantified them by ultraviolet detection.
What should a mass spectrometry result look like on a COA?
A number next to a number: the mass the instrument observed, and the theoretical mass calculated from the sequence being claimed. A certificate that says identity was confirmed without printing either figure has reported a conclusion rather than a measurement.
Does a matching mass rule out every wrong molecule?
No. ICH Q6A asks that identification discriminate between compounds of closely related structure, and notes that optically active substances may also need specific identification testing or a chiral assay. Stereoisomers weigh the same, so mass alone does not separate them.
Do I need both tests on the certificate?
Both questions have to be answered somewhere on the document. ICH Q6A treats a combination of tests in a single procedure, such as HPLC with mass spectrometry, as generally acceptable for identification, and ICH Q2(R2) recommends combining procedures where one does not discriminate enough.
References
- International Conference on Harmonisation. (1999). ICH Q6A — Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances (current Step 4 version, dated 6 October 1999). https://database.ich.org/sites/default/files/Q6A Guideline.pdf
- International Council for Harmonisation. (2023). ICH Q2(R2) — Validation of Analytical Procedures (final version, adopted 1 November 2023). https://database.ich.org/sites/default/files/ICH_Q2(R2)_Guideline_2023_1130.pdf
- European Commission. (2002). Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32002D0657
- Janvier, S., Cheyns, K., Canfyn, M., Goscinny, S., De Spiegeleer, B., Vanhee, C., & Deconinck, E. (2018). Impurity profiling of the most frequently encountered falsified polypeptide drugs on the Belgian market. Talanta, 188, 795–807. https://doi.org/10.1016/j.talanta.2018.06.023
- Vanhee, C., Janvier, S., Desmedt, B., Moens, G., Deconinck, E., De Beer, J. O., & Courselle, P. (2015). Analysis of illegal peptide biopharmaceuticals frequently encountered by controlling agencies. Talanta, 142, 1–10. https://doi.org/10.1016/j.talanta.2015.04.022
- Janvier, S., De Sutter, E., Wynendaele, E., De Spiegeleer, B., Vanhee, C., & Deconinck, E. (2017). Analysis of illegal peptide drugs via HILIC-DAD-MS. Talanta, 174, 562–571. https://doi.org/10.1016/j.talanta.2017.06.034
- Popławska, M., & Błażewicz, A. (2019). Identification of a novel growth hormone releasing peptide (a glycine analogue of GHRP-2) in a seized injection vial. Drug Testing and Analysis, 11(1), 162–167. https://europepmc.org/article/MED/30051972
- Weber, C., Krug, O., Kamber, M., & Thevis, M. (2017). Qualitative and semiquantitative analysis of doping products seized at the Swiss border. Substance Use & Misuse, 52(6), 742–753. https://europepmc.org/article/MED/28156209
- Prabhala, B. K., Mirza, O., Højrup, P., & Hansen, P. R. (2015). Characterization of synthetic peptides by mass spectrometry. Methods in Molecular Biology, 1348, 77–82. https://doi.org/10.1007/978-1-4939-2999-3_9