A purity figure answers how uniform a sample is. It does not answer whether the correct compound is present at all. That second question belongs to mass spectrometry. This page explains how LC-MS works, what a mass spectrum states about a peptide and where the method reaches its limits.
Two methods in one instrument
LC-MS is a coupling. The first part, liquid chromatography, separates the sample so that the components leave the column one after another. The second part, mass spectrometry, takes over the column outlet and determines the mass-to-charge ratio for each eluting component.
The gain lies in the assignment: one obtains not only the information that something elutes at a particular point in time, but also which mass this something has. A peak in the chromatogram is thereby given a chemical assignment instead of merely a retention time.
Ionisation: how a peptide enters the gas phase
Peptides are large, polar and non-volatile. In order to be recorded by mass spectrometry they must be ionised gently and transferred into the gas phase. In peptide analysis, electrospray ionisation (ESI) is the standard method: the liquid stream is nebulised at a capillary under high voltage, the solvent evaporates and charged molecular ions remain.
A characteristic of ESI is multiple charging. Peptides frequently take up several protons and therefore appear not as a single signal but as a series of signals with different charge states. The actual molecular mass is reconstructed computationally from this series, a step referred to as deconvolution.
A signal at m/z 800 can be a singly charged molecule of about 799 Da or a triply charged one of about 2397 Da. Only consideration of the entire charge series and its spacings allows an unambiguous assignment. A mass spectrum without a stated charge number is therefore only interpretable to a limited extent.
Comparison with the calculated mass
The theoretical molecular mass can be calculated exactly from the amino acid sequence. The comparison with the measured value is the actual proof of identity. Four terms must be distinguished for this:
- Monoisotopic mass – mass using the most frequent isotope of each element. Common with high-resolution instruments.
- Average mass – averaged over the natural isotope distribution. Common with instruments of lower resolution.
- Calculated mass – derived from the molecular formula or sequence, usually designated calculated or theoretical in the report.
- Measured mass – reconstructed from the spectrum, usually designated found or observed.
A comparison of calculated and found only becomes meaningful once it is clear whether monoisotopic or average masses are meant. If the reference values are mixed, apparent deviations arise that are purely problems of presentation. The figure in ppm denotes the relative mass deviation: 5 ppm at 2000 Da means an absolute difference of 0.01 Da.
What LC-MS does not answer
- Isobaric species. Compounds of the same mass but different structure, for example epimerisation products or transposed sequence sections, cannot be distinguished by mass alone. Chromatographic separation or fragmentation (MS/MS) helps here.
- Quantities. The signal intensity depends strongly on the ionisability of the respective substance. A mass spectrum is an instrument of identity, not an instrument of quantity.
- Non-ionisable constituents. Salts, water and some counter-ions provide no usable signal.
- The complete sequence. A matching mass confirms the molecular formula within the measurement tolerance. Sequence variants with identical composition have the same mass; proof of sequence requires fragmentation experiments.
Common misinterpretations
- Concluding the quantity proportion from the signal height.
- Treating a matching molecular mass as proof of sequence.
- Comparing monoisotopic and average mass against each other.
- Regarding LC-MS as a substitute for the chromatographic purity determination. The purity figure continues to be derived from the area ratio.
Documentation context
A mass spectrometric result is only as robust as its documentation. It becomes verifiable when the ionisation method, mass reference, calculated and measured mass as well as a tolerance figure are available and the measurement was demonstrably carried out on the same batch as the other tests. How a certificate of analysis is read section by section is described in Reading a COA correctly. The link between document and container is covered by Batch testing and traceability. Batch-related documents are published under Analytics & Certificates insofar as corresponding real documents are available.
Related guides
- HPLC in peptide analysis
- HPLC vs. LC-MS: differences in peptide analysis
- Understanding peptide purity
- Research Compound Library
For laboratory research only. This text serves as technical information and contains no medical, diagnostic or therapeutic statements, no dosages and no application instructions for humans.