If a peptide hasn’t been tested by these three methods, you don’t know what’s in the vial.
HPLC tells you how pure it is. Mass spectrometry tells you if it’s the right compound. Endotoxin testing tells you whether it carries bacterial contamination that would invalidate biological assays. Together, they’re the foundation of any meaningful Certificate of Analysis.
This is a researcher’s guide. What each method actually measures, what its results look like, and what they tell you about the compound you bought.
HPLC: measuring purity
HPLC stands for High Performance Liquid Chromatography. It’s the standard analytical method for measuring the purity of a peptide.
The way it works: a small amount of the peptide is dissolved in solvent and pumped at high pressure through a column packed with a stationary phase. Different molecules interact with the stationary phase differently, so they travel through the column at different speeds. As they emerge from the other end, they pass through a detector that measures their concentration.
For research peptides, the detector is usually a UV detector. Peptide bonds absorb UV light at around 215 nm. As each compound elutes from the column, the detector registers an absorbance peak. The size of the peak is proportional to the amount of compound.
How to read an HPLC chromatogram
The output is a chromatogram: a graph with time on the x-axis and absorbance on the y-axis. A clean peptide produces one large peak, with small or no peaks at other retention times.
Purity is calculated by integrating the area under the main peak and dividing by the total area under all peaks in the chromatogram. A purity of 99.5% means the main peak accounts for 99.5% of the total UV-absorbing material that came through the detector.
What to look for on a peptide HPLC report:
- The chromatogram itself. Not just a number. The shape of the peak, the absence of shoulders, the baseline noise level, the presence or absence of impurity peaks.
- Retention time. The time at which the main peak elutes. Consistent retention time across batches is a sign of consistent compound identity.
- Method conditions. Column type, mobile phase, gradient, flow rate, detection wavelength. These define how purity was measured.
- The numeric purity result. Reported as a percentage with the analytical method specified.
A purity number without a chromatogram is a claim, not a measurement.
Mass spectrometry: confirming identity
HPLC measures purity. Mass spectrometry measures identity. Both are necessary, because purity without identity is purity of an unknown compound.
Mass spectrometry ionizes the peptide and measures the mass-to-charge ratio of the resulting ions. The output is a mass spectrum: a graph with mass-to-charge on the x-axis and intensity on the y-axis.
For a peptide of known sequence, the theoretical molecular weight can be calculated. The observed mass from the mass spectrometer should match the theoretical mass within a small error margin. If the masses don’t match, the compound in the vial is not the compound you ordered, regardless of what the purity number says.
How to read a mass spectrometry result on a peptide COA
What to look for:
- The theoretical molecular weight for the named compound
- The observed molecular weight from the mass spectrometer
- The error in parts per million (ppm) or in mass units (Da). For research peptides, mass accuracy within a few mass units is typical for the instruments used.
- The ionization method. ESI (electrospray ionization) and MALDI are the most common for peptides.
A clean mass spectrum shows the expected molecular ion as the dominant peak, often with adducts (sodium, potassium) and charge states (singly charged, doubly charged) at predictable masses.
If the dominant ion in the spectrum is not at the expected mass, the compound is misidentified. That’s a critical failure regardless of how the purity number reads.
Endotoxin testing: bacterial contamination
Endotoxins are lipopolysaccharides from the cell walls of gram-negative bacteria. They are pyrogenic, meaning they cause fever and inflammatory responses in mammals. Even trace amounts can interfere with biological assays, particularly anything involving cell culture, immune system measurements, or in-vivo work.
HPLC and mass spec don’t detect endotoxins. They have to be measured separately, using a specific assay.
The standard method is the LAL (Limulus Amebocyte Lysate) assay. LAL is derived from the blood of horseshoe crabs and clots in the presence of endotoxins. The assay is described in USP <85> (United States Pharmacopeia chapter 85) and is the same standard used in pharmaceutical quality control.
How to read endotoxin results on a peptide COA
Results are reported in endotoxin units (EU) per milligram of peptide or per milliliter of prepared solution. Pharmaceutical-grade thresholds are typically ≤ 0.05 EU/mL or comparable per-mg limits.
For research applications:
- In vitro cell culture work generally requires endotoxin-tested compounds
- In vivo work in animals requires low endotoxin material to avoid confounding the experiment
- Most chemical and biochemical characterization work does not require endotoxin testing
Not every peptide batch needs endotoxin testing. It’s a flagship-batch test for compounds where biological assays are part of the use case. ANKR adds endotoxin testing to flagship batches and discloses it on the certificate.
The full breakdown of what we test and when is at /testing/.
A note on net content
One more analytical method worth mentioning, because it gets ignored more than it should: net peptide content.
A peptide vial labeled 10 mg usually contains 10 mg of total material. But not all of that material is peptide. Some of it is salt counter-ions (TFA, acetate). Some of it is residual water. The actual peptide content can be anywhere from 60% to 95% of the label weight, depending on the synthesis and purification process.
Net content testing (typically by HPLC against a reference standard, or by amino acid analysis) measures how much of the labeled mass is actual peptide. For concentration-response work and any application that depends on accurate peptide quantification, net content is critical.
Most research peptide COAs don’t report net content. ANKR’s flagship batches do.
The three-method minimum
For a research peptide Certificate of Analysis to be defensible:
- HPLC with UV detection. Reports purity with a chromatogram and a percent number.
- Mass spectrometry. Confirms identity by matching observed to theoretical mass.
- Endotoxin testing (where applicable). Reports bacterial contamination in EU/mL or EU/mg using a USP-aligned method.
Without all three, you have an incomplete picture. With all three, you have a defensible foundation for your research.
Frequently asked
Why is HPLC the standard for peptide purity?
HPLC separates compounds by their interaction with a stationary phase, which makes it sensitive to small differences between peptides and their impurities. With UV detection at peptide-bond wavelengths, it provides a quantitative measurement of purity that can be reproduced across laboratories.
What does the mass spectrometry result tell me about a peptide?
The observed mass from a mass spectrometer confirms the molecular identity of the compound. If the observed mass matches the theoretical mass for the peptide sequence on the label, the compound is correctly identified. If it doesn’t match, the compound is something other than what was ordered.
Does every peptide batch need endotoxin testing?
No. Endotoxin testing matters for biological applications, particularly cell culture and in-vivo work. For chemical characterization, concentration-response screens against isolated targets, and many other uses, endotoxin testing isn’t necessary. Flagship batches intended for sensitive applications should have it.
What is USP <85>?
USP <85> is the chapter of the United States Pharmacopeia that describes the standard bacterial endotoxin test. It’s the same standard used in pharmaceutical quality control for injectable drugs. Research peptides tested to USP <85> have been evaluated against a pharmaceutical-grade threshold.
What is net peptide content and why does it matter?
Net peptide content is the actual mass of peptide in a labeled vial, separate from counter-ions, water, and other material. It can be substantially less than the label weight. For accurate concentration-response work and any application requiring precise peptide quantification, net content is critical and often overlooked.
See it in practice
The ANKR testing protocol, including which analytical methods we run on which batches, is documented at /testing/. The full lab report archive is searchable from there. Our complete operations are at /how-we-operate/.