Reading a certificate of analysis: what HPLC purity means

Synthetic peptides are often accompanied by a certificate of analysis: a short document reporting test results for a particular batch. The most prominent figure is usually an HPLC purity percentage. This article explains what a certificate of analysis can document, what each common result means, and why a single purity figure should not be read as a complete characterisation of the material in a vial.

This article describes certificates of analysis in general analytical terms. It does not describe the documentation or testing of any particular supplier, including ATOM PHARMA. The principles of HPLC and mass spectrometry are explained in our article on analytical methods for synthetic peptides.

What a certificate of analysis is

A certificate of analysis records the results of specified tests on one batch of material, usually alongside the acceptance criteria the results are judged against. For regulated peptide medicines, specifications cover three broad questions: identity, purity and assay, meaning the actual content of the substance. Even among pharmacopoeias, these specifications are not fully harmonised^[1]. Outside regulated medicines, there is no single standard format. What a certificate contains, and how the tests were done, varies.

A certificate is therefore a summary, not the data. It reports conclusions from analyses whose methods, raw chromatograms and spectra are usually not shown.

The anatomy of a certificate

Chromatographic purity

In reversed-phase HPLC, the sample is separated on a column and detected, usually by ultraviolet (UV) absorbance. Chromatographic purity is the area of the main peak as a percentage of the total area of all integrated peaks. Its value depends on the method used, including the column, mobile phase, gradient, detection wavelength and integration settings^[2].

Three assumptions sit behind the number.

• Equal detector response. Using peak area as a proxy for amount assumes that every component absorbs UV light equally. In reality, impurities formed by insertion, truncation, deamidation, oxidation and other processes can respond differently from the main peptide. Assuming a relative response factor of 1 by default can overestimate or underestimate impurities^[3].
• Complete separation. Anything that elutes under the main peak is counted as the main peptide. This matters most for isomers. Compounds with the same mass-to-charge ratio are not readily told apart by mass spectrometry, so they must be separated chromatographically. Assessing main-peak purity rigorously can require a second, orthogonal separation^[4].
• Everything is detected. Water, counter-ions, inorganic material and residual solvents are measured by other methods, if at all^[5]. They do not reduce a UV purity figure.

A purity percentage is therefore a relative measure of UV-absorbing, separable components under one method. It is not a measure of how much of the powder is the intended peptide.

Retention time and reference standards

Retention time is the time a compound takes to pass through the column. On its own, it is weak evidence of identity, because different compounds can elute at similar times. It becomes more informative when the sample is compared with a well-characterised reference standard run under the same conditions.

Establishing such a standard is itself demanding. A United States Pharmacopeia (USP) report describes several steps. Peptide reference standards were characterised with NMR, mass spectrometry and chromatography for identity, and with HPLC and gas chromatography for content and impurities. Their value was assigned by a mass-balance approach that integrated results from several laboratories. The authors noted that features such as chiral or isobaric amino acids may need additional techniques for full characterisation^[6].

Mass spectrometry and identity

Mass spectrometry measures mass-to-charge ratio. A measured mass matching the expected sequence is good evidence that the main component has the right composition. It cannot, however, distinguish isomers that share the same mass^[4]. It also cannot detect D-amino acids substituted for their natural L-forms. D-isomers can enter as impurities in starting materials, form during synthesis, and in some cases form during shelf life. Detecting them requires a dedicated chiral method^[7]. Sequence errors that preserve mass may need fragmentation experiments or other orthogonal techniques to detect.

Identity, purity and content are different questions

A high purity figure says nothing about identity, and a correct mass says nothing about content.

Water, solvents and counter-ions

A lyophilised synthetic peptide is rarely pure peptide by mass. Counter-ions such as trifluoroacetate can remain from synthesis and purification^[8]. Water and residual solvents are also usually present.

A detailed purity assignment for synthetic oxytocin illustrates the scale. Using liquid chromatography with high-resolution mass spectrometry, the analysts identified 21 structurally related impurities. They measured water by Karl Fischer titration, counter-ions by ion chromatography, inorganic elements by mass spectrometry and volatile organic compounds by gas chromatography. The assigned value was 796.5 mg of oxytocin per gram, meaning that about a fifth of the material's mass was something else^[5]. Methods for determining content also differ in precision. A multi-laboratory comparison of HPLC assay, quantitative NMR and amino acid analysis for oxytocin found different levels of variability between laboratories^[9].

Counter-ions are not always inert in experiments. Trifluoroacetate at low concentrations reduced the proliferation of cultured osteoblasts and chondrocytes. When trifluoroacetate and hydrochloride salts of the same peptides were compared, the trifluoroacetate forms consistently gave less proliferation. This led to a real effect being missed, or an antiproliferative effect being wrongly attributed^[10]. A certificate that does not report the salt form leaves this question open.

When a high purity figure is not enough

Small amounts of the wrong material can matter more than the percentage suggests. In one investigation, peptides from two custom peptide suppliers gave false-positive results in T-cell assays. One peptide was found to contain about 1% by weight of an unrelated viral peptide commonly used in immunology research. That contamination was enough to produce responses that looked antigen-specific^[11]. In another study, obestatin samples from five manufacturers were tested. One was a different peptide altogether, and two-thirds of the others did not meet the purity needed for experimental use^[12].

Batch traceability

A certificate describes one batch at one time. Its value depends on traceability: a clear link from the batch number on the certificate to the material being used, and from the results to the methods and data behind them. Testing records the state of the batch on the date of analysis, not after later storage, shipping or handling. Recommendations for peptide standards used in mass spectrometry emphasise careful characterisation, storage and handling, and transparent documentation^[13].

Summary

A certificate of analysis is a useful summary of tests on one batch, but it is not complete characterisation. HPLC purity is a relative measure of UV-detectable, separable components under a particular method. It depends on assumptions about detector response and separation. Mass spectrometry supports identity but cannot detect isomers or D-amino acids of identical mass. Neither measures how much of the powder is peptide, which requires a content determination accounting for water, solvents and counter-ions. Reading a certificate well means knowing which of these questions it answers and which it leaves open.