What Each Test on a Peptide COA Detects, and What It Cannot
A certificate of analysis (COA), the lab’s report on one lot, can carry several kinds of test; this page covers six, and each one answers a single narrow question: mass spectrometry checks that the molecule has the expected mass, HPLC estimates what share of the detected material is the main component, a content test measures how much of the powder is peptide, the endotoxin test measures bacterial endotoxin activity, the sterility test looks for microbial growth in the units tested, and an elemental impurities test measures named metals. No line covers what the others measure, and each result is only as useful as its stated method, units, limit and lot number.
What does each test on a COA detect, and what can’t it tell you?
| Test (usual method) | What it detects | What it cannot tell you | A complete result line shows |
|---|---|---|---|
| Identity (mass spectrometry) | The mass of the molecules in the sample, compared with the mass expected for the labeled sequence | The exact sequence: chiral (mirror-image) or isobaric (same-mass) amino acids may need additional techniques (PMID 36949371) | Expected and observed mass, ideally with the spectrum |
| Purity (HPLC with UV detection) | The share of the detected signal in the main peak | Anything the detector does not see, and equal amounts of different molecules can give different signals (PMID 17539659) | Area percent, detection wavelength and the chromatogram |
| Content (net peptide content) | How much of the powder is peptide, for example by amino acid analysis (PMID 19937719) | Whether that peptide is the right one or how pure it is | Percent by weight and the method |
| Endotoxin (bacterial endotoxins test, LAL) | Endotoxin from gram-negative bacteria, using a reagent made from horseshoe crab blood cells (PDG Bacterial Endotoxins Test) | Pyrogens other than endotoxin, for which FDA says a different test may be more appropriate (FDA Pyrogen and Endotoxins Testing Q&A) | Technique, result in EU, the limit, and a pass or fail against it |
| Sterility (USP <71> methods) | Microbial growth in the units sampled (FDA aseptic processing guidance) | Whether units that were not sampled are free of microbes (FDA aseptic processing guidance) | Method, number of units tested, and the result |
| Heavy metals (elemental impurities, ICP-MS) | Named elements, each measured separately (ICH Q3D(R2) section 9) | The chemical form an element is in (ICH Q3D(R2) section 8) | Each element, the method, the result and the limit |
What do the identity and purity tests show?
Mass spectrometry measures the mass of the molecules in a sample. A match between the observed and the expected mass is good evidence that the main molecule is the labeled one. It is not proof of sequence, because two peptides with the same amino acids in a different order, or with an amino acid in its mirror-image form, have the same mass. Authors from the United States Pharmacopeial Convention wrote that features such as chiral or isobaric amino acids may require additional techniques for full characterization (PMID 36949371).
HPLC (high-performance liquid chromatography) separates a sample into its components and records each one as a peak. Purity is usually the main peak’s share of the total peak area. The UV detector does not respond equally to every molecule: one study at 214 nm measured tryptophan absorbing about 30 times as much light as the peptide bond (PMID 17539659). HPLC vs mass spectrometry for peptides covers both methods and how to read their charts.
What does a content result add?
Purity and content answer different questions. Purity asks how much of the detected material is the main component. Content, often called net peptide content, asks how much of the powder in the vial is peptide at all. A freeze-dried powder can also hold water and counterions, the charged partners a peptide carries when it is supplied as a salt. Peptides made by solid-phase methods and purified with trifluoroacetic acid are obtained as trifluoroacetate salts, and the authors of a 2025 study emphasized the importance of counterion quantification and specification in assays with synthetic peptides (PMID 40872554).
Content is measured by methods such as amino acid analysis, which breaks the peptide into its amino acids and measures each one (PMID 19937719). Producers of peptide reference standards describe a mass balance approach to assign a quantitative value to bulk peptide material (PMID 36949371). A powder can be 99% pure by HPLC and well below 99% peptide by weight, so the two figures should never be read as the same thing.
What does an LAL endotoxin test tell you about a lot?
Endotoxins are substances from gram-negative bacteria. The bacterial endotoxins test, harmonized between the US, European and Japanese pharmacopoeias and published in the US as USP chapter <85>, detects or measures them with amoebocyte lysate, an extract of horseshoe crab blood cells that reacts with endotoxin. That reagent is where the name LAL (Limulus amebocyte lysate) comes from. The harmonized text describes three techniques: gel-clot, which reads whether a gel forms; turbidimetric, which reads cloudiness; and chromogenic, which reads a color change. Results are expressed in endotoxin units (EU), and a limit is stated per milliliter, per milligram or per unit of biological activity.
A complete endotoxin line names the technique, the result in EU, and the limit the result was compared with. A “pass” with no number and no limit cannot be checked.
The test has limits worth knowing:
- It detects endotoxin only. FDA’s 2026 questions-and-answers guidance tells firms to assess the risk of non-endotoxin pyrogens and notes that a different test may be more appropriate if they may be present.
- The reagent can react to other things. The harmonized text notes that amoebocyte lysate reacts to some beta-glucans, a class of sugar polymers, as well as to endotoxin, and that lysate prepared to ignore glucans is available.
- The sample can interfere. The harmonized text includes a test for interfering factors, which checks that the sample neither suppresses nor boosts the reaction. FDA’s guidance says endotoxin assays are subject to a variety of interferences related to the physical and chemical properties of the material tested. A lab should be able to say how it checked for this.
What does a sterility pass mean?
A sterility test places samples from the lot in growth media and watches for microbial growth. FDA’s guidance on aseptic processing names USP chapter <71> “Sterility Tests” as the principal source for sterility testing methods.
The same guidance states that sterility tests are limited in their ability to detect contamination because of the small sample size typically used. Its worked example: if a 10,000-unit lot with a 0.1 percent contamination level were tested using 20 units, there would be a 98 percent chance that the lot would pass. A sterility pass therefore describes the units that were tested. A complete line names the method, the number of units tested and the result.
What is ICP-MS heavy metals testing?
In the US Pharmacopeia, chapters <232> Elemental Impurities, Limits and <233> Elemental Impurities, Procedures replaced chapter <231> Heavy Metals, according to FDA’s 2018 guidance on elemental impurities. A 2018 paper described <233> as providing two compendial procedures, one using ICP-AES (inductively coupled plasma atomic emission spectrometry) and one using ICP-MS (inductively coupled plasma mass spectrometry), and noted the extremely low detection limits that ICP-MS affords (PMID 29414002).
ICH Q3D(R2), the international guideline on elemental impurities, places arsenic, cadmium, mercury and lead in Class 1, which it describes as human toxicants with limited or no use in pharmaceutical manufacturing. The guideline applies to drug products, so here it is useful only as a list of the elements a metals line should name. It says the test should be specific for each elemental impurity identified for control, and it allows total levels of each element to be used, with speciation (which chemical form an element is in) as separate information.
A complete metals line names each element, the method, the result with units and the limit. A line that says “heavy metals: pass” with no elements leaves the reader unable to tell what was measured.
Related guides
- How to Read a Peptide Certificate of Analysis, Field by Field
- Why lot traceability matters for research peptides
Last reviewed: 2026-10-20
References
- Bacterial Endotoxins Test, Q-06 Rev. 2, harmonised text and sign-off cover sheet. Pharmacopoeial Discussion Group. April 2026. https://www.pmda.go.jp/files/000280573.pdf
- Pyrogen and Endotoxins Testing: Questions and Answers (Edition 2). U.S. Food and Drug Administration. March 2026. https://www.fda.gov/media/83477/download
- Sterile Drug Products Produced by Aseptic Processing: Current Good Manufacturing Practice. U.S. Food and Drug Administration. September 2004. https://www.fda.gov/media/71026/download
- Elemental Impurities in Drug Products, Guidance for Industry. U.S. Food and Drug Administration. August 2018. https://www.fda.gov/media/98847/download
- Guideline for Elemental Impurities Q3D(R2). International Council for Harmonisation. April 2022. https://database.ich.org/sites/default/files/Q3D-R2_Guideline_Step4_2022_0308.pdf
- Reference Standards to Support Quality of Synthetic Peptide Therapeutics. Pharm Res. 2023. PMID: 36949371
- Prediction of molar extinction coefficients of proteins and peptides using UV absorption of the constituent amino acids at 214 nm to enable quantitative reverse phase high-performance liquid chromatography-mass spectrometry analysis. J Agric Food Chem. 2007. PMID: 17539659
- Amino acid analysis. Curr Protoc Protein Sci. 2009. PMID: 19937719
- Towards a Consensus for the Analysis and Exchange of TFA as a Counterion in Synthetic Peptides and Its Influence on Membrane Permeation. Pharmaceuticals (Basel). 2025. PMID: 40872554
- Study of the use of axial viewed inductively coupled plasma atomic emission spectrometry with ultrasonic nebulization for the determination of select elemental impurities in oral drug products. J Pharm Biomed Anal. 2018. PMID: 29414002
Frequently asked questions
What tests should a complete peptide COA include?
A full panel usually covers identity by mass spectrometry, purity by HPLC, peptide content, bacterial endotoxins, sterility and elemental impurities (heavy metals). Each line answers a different question, so a complete line names its method, its units, its result and the limit it was compared with.
What does an LAL endotoxin test tell you about a peptide lot?
It measures endotoxin activity from gram-negative bacteria in the sample tested, reported in endotoxin units (EU) against a stated limit. It does not detect pyrogens other than endotoxin, which FDA says may call for a different test, and it says nothing about identity, purity or sterility.
Does a sterility pass mean every vial in the lot is sterile?
No. A sterility test examines a sample of units. FDA guidance notes that sterility tests are limited in their ability to detect contamination because of the small sample size typically used, so a pass describes the units tested.
What is ICP-MS heavy metals testing?
ICP-MS (inductively coupled plasma mass spectrometry) is one of the methods used to measure individual elements, such as lead, arsenic, cadmium and mercury, at very low levels. In the US Pharmacopeia, the elemental impurities chapters replaced the older heavy metals chapter, and a complete result names each element, the method and the limit.