Heavy metals and residual solvents in peptides

Why would a peptide vial contain metals or solvents at all?

Both come from the way synthetic peptides are made. The EMA guideline on synthetic peptides, in force from 1 June 2026, groups them as non-peptide impurities: "process reagents, by-products, residual solvents, elemental impurities and nitrosamines". It also notes that solid phase synthesis "requires extensive washing of the resin with solvents" (EMA, Guideline on the Development and Manufacture of Synthetic Peptides). Lesson 5 covered the other non-peptide parts of a powder, counterions and water.

Elements arrive by several routes. ICH Q3D names residual catalysts added on purpose during synthesis, interactions with processing equipment, and container and closure systems (ICH Q3D(R2)). For a vial, that last route means the glass and the rubber stopper as well as the powder.

What does ICH Q3D say about elemental impurities?

ICH Q3D is the international guideline for elements in medicines. Its scope explicitly includes "drug products containing synthetically produced polypeptides" (ICH Q3D(R2), section 2). It sets a permitted daily exposure (PDE) for each element of concern, sorts elements into classes, and asks manufacturers to test only where a risk assessment shows testing is the right control.

• Class 1: arsenic, cadmium, mercury and lead. Q3D calls them "human toxicants that have limited or no use in the manufacture of pharmaceuticals" and requires them to be evaluated across all sources and routes.
• Class 2A: cobalt, nickel and vanadium, which have a relatively high probability of turning up.
• Class 2B: elements such as palladium, platinum and silver, with a reduced probability of turning up; they may be left out of the risk assessment unless added on purpose, for example as a catalyst.
• Class 3: elements with relatively low oral toxicity, such as copper, that still need evaluating for parenteral and inhalation products.

Q3D Table A.2.1 gives separate oral and parenteral PDEs in micrograms per day. A few examples, copied from that table:

A PDE is a daily amount, not a concentration. To turn it into a limit per gram, Q3D's Option 1 assumes a product intake of no more than 10 grams a day. On that assumption the parenteral concentration limits in Table A.2.2 are 0.2 micrograms per gram for cadmium, 0.5 for lead, 1.5 for arsenic and 0.3 for mercury. Q3D also defines a control threshold of 30% of the PDE: if the total from all sources is consistently below it, extra controls are not required.

Where do USP <232> and <233> fit in?

According to the US FDA, "General Chapters <232> and <233> replaced General Chapter <231> Heavy Metals", and the revised <232> that aligns with ICH Q3D became official on 1 August 2017 (FDA, Elemental Impurities in Drug Products). Chapter <232> sets the limits; <233> sets out the procedures.

That matters when a certificate just says "heavy metals: conforms" with no element named. Q3D asks for a test that is "specific for each elemental impurity identified for control" unless otherwise justified. A published example digested the sample and validated an inductively coupled plasma mass spectrometry (ICP-MS) procedure for ten elements, including cadmium, lead, arsenic and mercury, against limits of 0.2, 0.5, 1.5 and 0.3 ppm respectively (Hao et al., Int J Anal Chem, 2022). The ICP-MS method page explains how the instrument works.

What does ICH Q3C say about residual solvents?

ICH Q3C defines residual solvents as "organic volatile chemicals that are used or produced in the manufacture of drug substances" and "are not completely removed by practical manufacturing techniques". It sorts them into three classes (ICH Q3C(R9)):

• Class 1, solvents to be avoided: known or strongly suspected human carcinogens and environmental hazards. Benzene is limited to 2 ppm.
• Class 2, solvents to be limited: each has its own PDE and a concentration limit under Option 1, which, like Q3D, assumes a product mass of 10 grams a day.
• Class 3, low toxic potential: amounts of 50 mg per day or less, "corresponding to 5000 ppm or 0.5% under Option 1", are acceptable without justification.

Trifluoroacetic acid is the counterion from lesson 5. Q3C lists it among solvents "for which no adequate toxicological data was found", so no PDE is given. The EMA peptide guideline handles it separately, asking for "residual ion content (TFA)" in the specification. A TFA figure on a certificate is therefore a counterion result, not a residual solvent result.

Q3C notes that residual solvents "are typically determined using chromatographic techniques such as gas chromatography", and that a non-specific loss on drying test may be used only when Class 3 solvents alone are present. The GC residual solvents method page covers headspace sampling and what the output looks like.

Why do most peptide certificates leave these out?

Certificates sellers share usually show identity, purity and sometimes amount. Elemental impurities and residual solvents each need a separate instrument and sample preparation, a digestion and ICP-MS run for metals and a GC run for solvents, each using material from a small vial, and neither changes the HPLC number buyers look for.

Regulated manufacturing works differently. The EMA guideline lists water content, residual solvents, elemental impurities "(e.g. in case of use of metal catalysts)", bacterial endotoxins and microbiological purity in its non-exhaustive list of tests a peptide specification may include. A research-market certificate seldom shows whether anyone asked which of them apply.

HPLC with UV detection sees molecules that absorb UV light and elute from the column. Metal ions and volatile solvents largely do not show up as peaks, so they are not in the purity denominator. In a mass balance study of synthetic glucagon, the inorganic ions measured by ICP-MS were each below 0.1% and could be ignored, while water and TFA were large enough to matter (Wang et al., Scientific Reports, 2020). That was one well-made reference material: it shows these quantities must be measured separately, not that they are always small.

How do I read these lines if a certificate has them?

Check which limit column was used: mercury and nickel differ tenfold between the oral and parenteral columns. And a "not detected" result is only as good as the method's reporting limit. If the certificate does not state it, you cannot tell whether nothing was there or the method could not see down to the limit.

Which compounds raise the question most?

Any synthetic peptide can carry residual solvent, because the synthesis and purification steps use them. Metal-containing compounds raise a different question. GHK-Cu is a copper complex by design, so copper is part of the product, not an impurity, and Q3D excludes "elements that are intentionally included in the drug product for therapeutic benefit". A copper result for it is closer to an amount measurement than a contamination check. For long sequences such as tirzepatide, every coupling step is followed by solvent washes, and the EMA guideline asks for the purge of every reagent and solvent to be addressed.

The which test tool helps decide which tests answer a particular question. For a certificate you already have, the COA checker asks whether these lines are present and what limit they were compared with.

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