Purity, endotoxins and heavy metals in peptides

By the Clean Peptides Editorial Team · Published 30 September 2026

Two analyses are standard for synthetic peptides: HPLC for purity and mass spectrometry (MS) for identity. In addition, there are further tests: net peptide content, endotoxins (with the LAL or rFC test), heavy metals and other elements (with ICP-MS or ICP-OES), residual solvents and the counterion trifluoroacetate (TFA). The certificate of analysis (COA) shows which tests were carried out on a batch. If a test is not listed there, assume it was not performed.

Purity, content and identity

Purity is measured by HPLC. The result is the area of the main peak as a percentage of all the peaks detected. ‘99% pure’ therefore means that 99% of the UV signal comes from the correct peptide; it says nothing about how much of the powder is peptide.

The net peptide content answers that second question: what proportion of the powder you weigh out is actually peptide? Lyophilised peptide powder contains not only the peptide but also water and counterions, such as trifluoroacetate (TFA) from HPLC purification [5]. The content is determined separately, for example by amino acid analysis, and the water content often by the Karl Fischer method. This matters for researchers: if you calculate a concentration from the weighed amount of powder without this figure, you will overestimate it.

Identity is confirmed by mass spectrometry. The measured mass should match the theoretical mass of the peptide. How to read HPLC and MS results on a certificate is explained in How to read a certificate of analysis (COA).

Endotoxins: the LAL test and recombinant factor C

Endotoxins are lipopolysaccharides (LPS) from the cell wall of Gram-negative bacteria. They are released when bacteria divide and when they die. In the blood, they trigger a strong inflammatory response from the immune system, which is why the pharmaceutical industry tests for them as standard [1]. They can also interfere with lab research: a cellular response to endotoxin may be wrongly attributed to the peptide under study.

LAL test. The most widely used test uses an extract of horseshoe crab blood cells (Limulus amebocyte lysate). Endotoxin triggers a measurable clotting reaction in this extract [1].
Recombinant factor C (rFC). An alternative that does not use animal blood. It uses a lab-produced (recombinant) version of factor C, a horseshoe crab clotting enzyme. Because rFC does not include the factor G pathway, it responds more specifically to endotoxin. In comparative studies, its results agreed closely with those of the LAL test [2].

The result is expressed in EU (endotoxin units) per milligram or per millilitre. Note that some solutions can mask endotoxin, causing a test to underestimate it, a phenomenon known as low endotoxin recovery [1]. A reliable endotoxin test is therefore validated for the specific product.

Heavy metals and other elements: ICP-MS

In medicines, these are now referred to as ‘elemental impurities’. According to the international guideline ICH Q3D, they can originate from catalysts used in synthesis, from contact with manufacturing equipment or packaging, or from other components [4]. The guideline divides elements into classes. Class 1 consists of arsenic, cadmium, mercury and lead: toxic elements that are rarely or never used in manufacturing [4].

In the past, a general ‘heavy metals test’ was carried out. Since ICH Q3D, this has been replaced by a risk assessment and measurements using techniques based on inductively coupled plasma: ICP-OES and ICP-MS [3]. ICP-MS is the more sensitive of the two and is needed above all to accurately determine low concentrations of arsenic and lead [3]. A certificate should state the measured value and the technique used for each element.

Residual solvents and TFA

Residual solvents are volatile organic compounds that are used or formed during manufacturing and are not completely removed. The ICH Q3C guideline divides them into three classes: class 1 (to be avoided), class 2 (to be limited; this includes acetonitrile) and class 3 (low toxicity, such as ethanol and acetic acid). They are usually measured by gas chromatography [4].

TFA (trifluoroacetic acid) is used as a solvent in HPLC purification. As a result, purified peptides are often obtained as TFA salts [5]. In a classic study, low concentrations of TFA inhibited the growth of cultured bone and cartilage cells. This could mask an effect of the peptide itself, or suggest one that was not there. The researchers recommended converting peptides intended for cell experiments into the hydrochloride or a similar salt [5]. If a certificate does not state the salt form, ask for it.

What a good certificate of analysis states

The batch number, matching the number on the label, plus the date of analysis and the name of the laboratory.
Purity by HPLC, preferably with the chromatogram and the wavelength used.
Identity by MS: measured versus theoretical mass.
The salt form (for example TFA or acetate) and, if determined, the net peptide content and the water content.
For every additional test, such as endotoxins, elements or solvents: the method, the unit and the measured value.

At Clean Peptides, every vial and pen has a batch number on its label. Have a question about the analysis of a product you have ordered? Please contact us and quote the batch number.

Products and further reading

Products

You will find the full range in the shop, including BPC-157 10 mg, TB-500 10 mg, GHK-Cu 100 mg and MOTS-c 10 mg.

Sources

Gorman A, Golovanov AP. Lipopolysaccharide Structure and the Phenomenon of Low Endotoxin Recovery. European Journal of Pharmaceutics and Biopharmaceutics 2022;180:289–307. doi:10.1016/j.ejpb.2022.10.006
Bolden J, Knutsen C, Levin J, et al. Currently Available Recombinant Alternatives to Horseshoe Crab Blood Lysates: Are They Comparable for the Detection of Environmental Bacterial Endotoxins? A Review. PDA Journal of Pharmaceutical Science and Technology 2020;74(5):602–611. doi:10.5731/pdajpst.2020.012187
Janchevska K, Stafilov T, Memed-Sejfulah S, et al. ICH Q3D based elemental impurities study in liquid pharmaceutical dosage form with high daily intake – comparative analysis by ICP-OES and ICP-MS. Drug Development and Industrial Pharmacy 2020;46(3):456–461. doi:10.1080/03639045.2020.1724136
ICH. Guideline for elemental impurities Q3D(R2) (2022) and Impurities: guideline for residual solvents Q3C(R9) (2024). Q3D(R2) · Q3C(R9) (accessed 30 September 2026)
Cornish J, Callon KE, Lin CQ, et al. Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes. American Journal of Physiology 1999;277(5):E779–E783. doi:10.1152/ajpendo.1999.277.5.E779

For laboratory research only. Clean Peptides products are not intended for use in humans or animals. This article provides general information and is not medical advice.

Purity, endotoxins and heavy metals: what is tested in peptides?