Purity, HPLC, and mass spectrometry: two measurements, two questions

Every supplier displays a purity figure on the page. The figure alone is not what matters: the question is which measurement underlies it and what that measurement can establish. Two analyses answer two different questions, and both are required. Below: how to read a chromatogram and a mass spectrum, and where the limitations of a report lie.

What a percentage actually expresses

Purity is determined using HPLC. The sample passes under pressure through a column that separates components based on how strongly they bind to the column material. For peptides, this is almost always reversed-phase HPLC: a non-polar column and a mobile phase in which the proportion of organic solvent gradually increases. What elutes from the column first binds weakly; what elutes later binds more strongly. A UV detector at the end registers what passes through, usually at a wavelength where the peptide bond absorbs. The review by Mant and colleagues (2007) describes this setup as the standard for analysis and purification of synthetic peptides.

The result is a chromatogram with peaks. Purity is the area of the main peak divided by the total peak area. If it states 99.4%, that means: of everything the detector registered, 99.4% was a single substance. Note what is absent from that definition: which substance.

Reading a chromatogram

The horizontal axis shows time in minutes; the vertical axis shows detector signal intensity. Three parameters are worth examining.

Retention time. The moment at which the apex of a peak appears. Under identical conditions (column, mobile phase, gradient, temperature), the same substance elutes from the column at approximately the same time. When compared to a reference standard, matching retention time is an indication of identity, but not proof: two different peptides can elute from the column virtually simultaneously.

Peak area. Purity is calculated from the area under the peaks, not from peak height. A narrow tall peak and a broad low peak can have the same area. Software establishes a baseline and integrates every peak above a defined threshold; where that baseline is positioned and which minor peaks are integrated directly influences the figure in the final decimal place.

Peak shape. A good main peak is narrow and symmetrical. A shoulder, tailing, or a split peak indicates that another substance co-elutes under the main peak. That is the inherent limitation of any HPLC percentage: an impurity that co-elutes precisely with the peptide is counted as part of the main peak. Purity is therefore only as reliable as the chromatographic separation.

Minor peaks immediately preceding or following the main peak are often related peptides. The review paper by D'Hondt and colleagues (2014) outlines known variants, including deletion or insertion sequences, oxidation, deamidation, and stereoisomeric variants. How peptides are synthesized and why such variants arise is covered in What are peptides.

Why identity requires a separate measurement

Mass spectrometry ionizes the molecule and measures the mass-to-charge ratio. This generates a numerical value that can be compared against the theoretical mass of the expected compound.

For BPC-157, that expected mass is 1419.5 g/mol; for the TB-500 fragment Ac-LKKTETQ, it is 889.0 g/mol. If the measured mass corresponds to the expected mass, this is strong evidence of the intended substance. If it deviates, a different compound is present, regardless of chromatographic purity.

Not every laboratory confirms identity via mass spectrometry. Some reports, including current reports from BTLabs, utilize FTIR, an infrared spectroscopic technique where the sample spectrum must display the characteristic absorption bands of the peptide. A step-by-step breakdown of such documents is detailed in How to read a BTLabs batch report.

Reading a mass spectrum

The horizontal axis of a mass spectrum displays m/z, the mass divided by charge; the vertical axis shows relative intensity. Under electrospray ionization, the standard technique for peptides, a molecule acquires one or more protons. Consequently, the same peptide is frequently observed at multiple positions.

Take BPC-157. The monoisotopic mass, calculated from the sequence, is approximately 1418.7. With one additional proton, the [M+H]+ ion is expected around m/z 1419.7; with two protons, the [M+2H]2+ ion appears around m/z 710.4, as the mass is divided by two. Both signals correspond to the identical molecule.

Note also the difference between average and monoisotopic mass. The average mass (1419.5 g/mol) accounts for natural isotopic distribution; the monoisotopic mass calculates using only the lightest stable isotopes. A report should specify which value is reported. The acceptable tolerance between measured and expected mass depends on instrumentation: on lower-resolution instruments, a few tenths of a mass unit; on high-resolution instruments, within a few ppm.

A mass deviation can often be identified. A shift of approximately +16 corresponds to oxidation, approximately +1 to deamidation, and −18 to the loss of water. A discrepancy matching the molecular weight of an amino acid residue, such as −57 for glycine or −97 for proline, indicates a deletion sequence.

Adducts and background signals

Not every secondary signal in a mass spectrum represents an impurity in the sample. During ionization, other ions readily associate with the peptide, forming adducts. The most common are sodium, appearing at approximately +22 relative to [M+H]+, potassium at approximately +38, and ammonium at approximately +17. For BPC-157, a sodium adduct is expected around m/z 1441.7.

The review by Keller and colleagues (2008) compiles these interfering signals and background ions in mass spectrometry, ranging from adducts to plasticizers and solvent clusters. The rule of thumb: if an additional peak appears at a known, fixed mass offset from the primary ion, it is likely an adduct of the same molecule rather than a separate chemical entity.

The impact of an impurity on experimental measurements

In laboratory research, this is not a theoretical consideration. A sample of 90% purity assumed to be 99% yields a working concentration that deviates 10% from calculation. This shifts concentration-response curves without an obvious cause.

More problematic is an impurity that possesses biological or chemical activity within the test model. In that scenario, the observed response represents two distinct substances attributed to one. This is not merely experimental noise, but an invalid measurement. That this occurs in commercial settings was demonstrated by Verbeke and colleagues (2015): among synthetic peptides sold as minimum 95% pure, less than half met that threshold upon independent re-analysis.

Degradation after receipt must also be considered. Oxidation and deamidation do not occur solely during synthesis, but also during storage, particularly in solution; see Storing peptides.

What we publish, and what it does not represent

For batches where analytical testing has been completed, we publish the complete report from BTLabs, including the lot number, allowing cross-referencing between the physical vial and the document. Using the report number and verification link, the report can be confirmed directly with the issuing laboratory. If a product does not yet have an uploaded report, it is marked "Batch report to follow". All documentation is available on Lab reports.

A lab report is verifiable analytical data, not a guarantee. It reflects the composition of the tested vial at the time of measurement. Sterility, endotoxins, and counter-ions are not covered unless specifically tested. It does not state biological activity and does not constitute instructions for use; scientific literature is available for reference, referenced on each product page with corresponding PMIDs.

Frequently asked questions

Is high purity sufficient to establish identity?

No. HPLC purity indicates what fraction of the detected sample consists of a single substance, not which substance it is. A separate identity assay is required, such as mass spectrometry or FTIR.

Why does the same peptide appear at multiple m/z values in a mass spectrum?

A peptide can accept one or more protons and form adducts with cations such as sodium or potassium. Each of these states produces a distinct signal. If these signals align with established mass offsets, they correspond to the same molecule.

What is the acceptable deviation between measured and expected mass?

This depends on the instrument resolution and whether the report uses average or monoisotopic mass. For low-resolution instruments, a deviation of a few tenths of a mass unit is standard; for high-resolution systems, within several ppm. Larger shifts matching specific chemical deltas, such as +16 or −18, indicate related structural variants.

What does retention time signify?

It is the time required for a substance to elute from the column. Under identical chromatographic conditions, this time is reproducible, facilitating comparison against a reference standard. In isolation, retention time does not establish chemical identity.

References

1. Mant CT, et al. HPLC analysis and purification of peptides. Methods in Molecular Biology, 2007. https://pubmed.ncbi.nlm.nih.gov/18604941/
2. D'Hondt M, et al. Related impurities in peptide medicines. Journal of Pharmaceutical and Biomedical Analysis, 2014. https://pubmed.ncbi.nlm.nih.gov/25044089/
3. Keller BO, Sui J, Young AB, Whittal RM. Interferences and contaminants encountered in modern mass spectrometry. Analytica Chimica Acta, 2008. https://pubmed.ncbi.nlm.nih.gov/18790129/
4. Verbeke F, Wynendaele E, Braet S, D'Hondt M, De Spiegeleer B. Quality evaluation of synthetic quorum sensing peptides used in R&D. Journal of Pharmaceutical Analysis, 2015. https://pubmed.ncbi.nlm.nih.gov/29403929/