HPLC, Mass Spec, Endotoxin: What the HHS Peptide Testing Standard Asks a COA to Show

What testing does the HHS peptide policy require?

Condition 3 of the HHS policy requires the peptide bulk drug substance to come from an FDA-registered facility and to be “characterized and identified in a valid certificate of analysis (COA) that includes test results consistent with existing guidance-based criteria described by FDA”. HHS says the point is assurance that what a compounder receives “is what it claims to be and has been tested for identity, purity, potency, and the absence of harmful contaminants such as endotoxins, heavy metals, microbial contamination, and impurities” (HHS policy).

The policy attaches a testing table that lists each quality attribute, acceptable analytical methods, acceptance criteria and USP or ICH references. A second table lists acceptable “orthogonal” methods. This article explains what those tests measure in plain terms. It applies to bulk API for licensed 503A compounders, and it is also a useful reference for reading any peptide COA.

What does “orthogonal methods” mean?

The standard defines orthogonal methods as “at least two independent analytical procedures relying on distinct physicochemical principles” (HHS, footnote 3). It requires them for physicochemical properties, identity and impurities, and for larger peptides (eight amino acids or more) for higher-order structure, aggregates and biological activity.

Different methods have different blind spots. Mass spectrometry confirms that a molecule of the right mass is present, but it cannot always tell two sequences with the same composition apart. Amino-acid analysis confirms composition but not order. Tandem MS sequencing, Edman degradation or peptide mapping confirm order. Two methods that work on different principles make a misidentification much less likely.

Which tests are in the standard, and what does each one measure?

Source for every row: the HHS testing standard and its Table 1 (HHS policy). HHS notes that bioburden and endotoxin are critical quality attributes for injectables and that other dosage forms need tests appropriate to that form.

Why is “99% purity” not the same as “99% peptide”?

This is the most common misreading of a peptide COA. An HPLC purity figure is usually the share of the chromatogram’s peak area that belongs to the main peak. It shows how much of what the detector saw was the target peptide, compared with peptide-related impurities. It does not show:

• Net peptide content. A lyophilised peptide powder also contains counter-ions (acetate or TFA) and water. That is why the standard lists peptide content and counter-ion, TFA and water testing as separate attributes.
• Assay against a reference standard. The standard asks for HPLC compared with an established quantitative reference standard, which gives an amount, not just a ratio.
• Anything the HPLC detector cannot see. Endotoxins, microbes, metals and residual solvents need their own tests.

A vial can therefore show “99% purity” and still differ from its label quantity, carry a different salt form, or fail an endotoxin limit. The COA has to report those things separately for a reader to know.

What must the COA itself state?

The standard’s footnote 1 lists the information required on the API documentation (HHS policy):

• Bulk drug substance name (USAN, INN or IUPAC), structure, amino-acid sequence, molecular formula and weight
• Manufacturer’s corporate name, FDA Establishment Identifier (FEI) and address, plus any processing firm and repacker, and the testing laboratory
• Batch number, date of synthesis, date of release, expiry or retest date, and storage conditions
• Grade: “Intended for human drug compounding”, and the intended dosage form
• A statement of CGMP compliance, dated and signed by the quality unit
• For each test: the method name and number, and the method validation or verification report number, plus the reference standards used

Condition 4 adds that the physical location of the original manufacturer’s facility must be on the COA. A repacker or reseller must have validated both the manufacturer and the manufacturer’s COA. McDermott notes that this reaches into the supply chain beyond section 503A’s existing registration requirement (McDermott).

How does this compare with a typical research-market COA?

The middle column reflects the report formats we see when indexing labs in our testing facilities directory. It is a qualitative description, not a count. A research-market report can be accurate and still answer a narrower question than a compounding COA. Step-by-step checks for any COA are in how to verify a peptide COA, and what to publish on a COA page is in COA pages.

Who is this standard for?

Formally, it applies to the bulk peptide API that a 503A pharmacy, federal facility or physician uses under the HHS policy. It is the compounder’s condition to meet, and in practice the API manufacturer’s documentation to supply. It does not regulate research-use-only sellers, who are outside the policy altogether (RUO vs compounded).

It is also the first detailed public statement from HHS of what a fully characterised peptide API looks like. Before this, FDA’s category 2 notes for these peptides repeatedly cited “complexities with regard to peptide-related impurities and API characterization” (FDA). Testing labs, suppliers and buyers now have a public reference to compare reports against.

Sources

• HHS Enforcement Discretion Policy: conditions 3–4, Testing Standard for Peptides Used in Compounding, and Table 1 (PDF as reposted by Reason; hhs.gov blocked automated access)
• Federal Register, FR Doc. 2026-22192, 30 October 2026
• FDA, 503A bulk drug substances (COA and registration requirements) · FDA, category 2 safety notes
• McDermott Will & Schulte, 2 November 2026

Checked 4 November 2026. This is an explainer on analytical testing. It is not a quality certification of any product and not medical advice.

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