A Certificate of Analysis that reports HPLC purity and mass-spectrometry identity has answered two questions: is the material mostly the intended peptide, and is that peptide the right molecule. Two US Pharmacopeia general chapters, <1503> and <1504>, set out how many more questions a complete quality picture actually requires, and push part of that picture upstream to the raw materials a peptide is assembled from before synthesis even starts.
What USP <1503> actually covers
USP General Chapter <1503>, Quality Attributes of Synthetic Peptide Drug Substances, is an informational chapter that lays out the quality attributes relevant to synthetic peptide drug substances: manufacturing method, raw materials, general characteristics and specification, peptide content and assay, impurities and related compounds, microbiological contamination, bacterial endotoxins, and a set of specific tests tied to how the peptide was made. It explicitly excludes finished drug product attributes and recombinant (biologic) production routes; its focus is the chemically synthesised peptide substance itself, made by solid-phase or liquid-phase peptide synthesis (SPPS or LPPS).
A 2023 peer-reviewed survey of the reference-standards landscape for synthetic peptide therapeutics, published in Pharmaceutical Research (McCarthy et al., PMID 36949371, DOI 10.1007/s11095-023-03493-1), groups the attributes a full characterisation should cover into primary sequence, secondary structure, oligomer or aggregation state, and impurities and degradation products. On the impurity side alone, the paper walks through several categories that a single purity percentage does not distinguish: related peptide impurities measured by HPLC, chiral or enantiomeric impurities confirmed by GC-MS, residual solvents by gas chromatography, the synthesis counterions acetic acid and trifluoroacetic acid, and inorganic residue by residue-on-ignition testing. Each of these maps to its own USP general chapter: water determination under <921>, acetic acid and TFA content under <503> and <503.1>, residual solvents under <467>, residue on ignition under <281>, and thermal behaviour under <891>. None of that is visible in a report that states only "purity: 98.5% by HPLC" and "identity confirmed by LC-MS."
The gap standard testing leaves: chiral purity
The clearest example of an attribute the two headline tests miss is chiral purity. Peptide synthesis builds a chain from amino acid building blocks, and every amino acid except glycine has a chiral centre. An L-amino acid that racemises, or a D-amino acid impurity carried in from a starting material, produces a peptide diastereomer with the same molecular formula and very nearly the same mass as the intended L-peptide. Mass spectrometry identity confirmation, which matches observed to theoretical monoisotopic mass, cannot distinguish the two: a D-substituted peptide and its correct L-form are effectively isobaric. Reversed-phase HPLC purity does not reliably separate them either, since the isomers frequently co-elute under standard conditions.
Detecting a chiral impurity takes a different, dedicated method: acid hydrolysis of the peptide back to its constituent amino acids, chemical derivatisation (using a reagent such as Marfey's reagent or o-phthalaldehyde), and separation by chiral gas chromatography-mass spectrometry or chiral HPLC. This is chiral amino acid analysis, a distinct assay from both the reversed-phase HPLC used for organic purity and the amino acid analysis under USP <1052> used to establish net peptide content. A COA silent on chiral purity has simply not run this test, which is a different statement from the material being free of D-amino acid content.
USP <1504>: grading the inputs, not just the output
USP General Chapter <1504>, Quality Attributes of Starting Materials for the Chemical Synthesis of Therapeutic Peptides, extends the same logic one step further back, to the protected amino acid derivatives, mainly Fmoc-protected amino acids, that solid-phase synthesis assembles into a peptide chain. The chapter is explicit that it is meant to be used alongside <1503>: a finished peptide's impurity profile is shaped substantially by the purity of the starting materials, and problems introduced at that stage, including D-isomer contamination in the amino acid building block itself, are not guaranteed to be removed by whatever purification the finished peptide later receives.
This is a genuinely different question from anything a finished-product COA can answer. A buyer or researcher evaluating a peptide has no direct way to inspect the starting materials that went into it; what <1504> does is give manufacturers a defined minimum specification (assay, chiral purity, related substances, residual solvents, and related attributes) to hold those inputs to, so that the input side of the process is not simply unexamined.
Why this matters under a verifiability standard
None of this is a claim that any particular peptide sample contains chiral impurities, degraded counterions, or contaminated starting materials. The point is narrower and more useful: <1503> and <1504> describe a wider set of quality attributes than the two tests, purity by HPLC and identity by mass spectrometry, that most buyer-facing COAs default to, and some of those attributes (chiral purity chief among them) require an assay that neither of the default tests can substitute for. Whether a report was produced by a named laboratory or one that redacts a supplier's identity has no bearing on any of this; what matters is which specific attributes were actually tested, against which method, and whether that data appears on the document at all. A report that lists residual solvents and counterion content alongside purity and identity has simply covered more of the <1503> attribute list than one that has not, independent of anything about the vendor's transparency.
Further reading
The chromatographic method behind the purity figure most COAs lead with is covered in HPLC for peptides, and how identity confirmation by mass spectrometry works, and where its limits are, is covered in mass spectrometry for peptide identity verification. Net peptide content by hydrolysis and amino acid analysis is explained in the USP <1052> article, a related but distinct assay from the chiral analysis discussed here.
For the standards themselves, the primary references are USP General Chapter <1503> and USP General Chapter <1504> from the United States Pharmacopeial Convention, and McCarthy et al., "Reference Standards to Support Quality of Synthetic Peptide Therapeutics," Pharmaceutical Research (2023), PMID 36949371, DOI 10.1007/s11095-023-03493-1.