LC-MS combines liquid chromatography with mass spectrometry. For a peptide identity question, check whether the report describes intact mass, fragmentation or another comparison, and which alternatives the method can distinguish.
Read the ion label before the number
A mass spectrometer measures ions by mass-to-charge ratio, written m/z. In [M+2H]2+, M represents the neutral molecule and the label describes two added protons and a charge of +2. More than one charge state can come from the same molecule.Waters MS primer (opens in a new tab)
In an illustrative protonated-ion calculation, m/z 501.0073 at charge +2 corresponds to a neutral mass of approximately 1,000.0000 Da after subtracting two proton masses. This assumes the stated ion composition; [M+Na]+ describes a different adduct. A reconstructed neutral or zero-charge mass is a calculation from ion data.Waters MS primer (opens in a new tab)NIST — 2022 CODATA constants (opens in a new tab)
An adduct label describes an observed ion. Its signal intensity is not a measurement of the bulk salt composition of the vial. Check the instrument method and ion assignment before comparing an observed value with a formula-based mass.Waters MS primer (opens in a new tab)
Match the question to the evidence
| Structural question | Additional evidence to look for |
|---|---|
| Residue order or deletion position | Sequence-resolving information and its coverage |
| Leucine versus isoleucine | A method shown to distinguish the relevant same-mass alternatives |
| D/L configuration | A method addressing the stereochemical distinction |
| Disulfide pairing | Evidence identifying the relevant connectivity |
| Terminal or side-chain modification position | Localising evidence, rather than mass difference alone |
Tandem mass spectrometry examines fragments and can add sequence information, but the actual coverage and assignments matter. In the illustrative sequences ALA and AIA, replacing leucine with isoleucine preserves intact mass; specialised methods are needed to distinguish that alternative. A report that merely names LC-MS does not establish that those methods were used.Waters tandem-MS primer (opens in a new tab)Waters 720008287 (opens in a new tab)
Read an actual identity conclusion
The supplied Semax report records an LC-HRMS identity conclusion of conformity with a reference. Quote that conclusion with its report identifier and method. The report’s separate 99.18% chromatographic purity result and 9.98 mg content result address different questions.Semax report (PDF) (opens in a new tab)
For an unresolved identity detail, ask which reference, mass convention, ion assignments and discriminating evidence supported the conclusion. Repeated agreement with a signal is different from demonstrating that plausible alternatives were distinguished.ICH analytical selectivity (opens in a new tab)
Sources and further detail
- Waters — What is mass spectrometry and how does it work? (opens in a new tab)
Instrument primer; ion measurement, mass-to-charge ratio and interpretation of spectra.
- Waters — Mass spectrometry instruments and tools (opens in a new tab)
Instrument primer on tandem analysis and fragmentation. Not a specification for an unreported assay.
- Waters — Application note 720008287 (2024) (opens in a new tab)
Leucine/isoleucine discrimination demonstrated with the specified method; no unreported assay is implied.
- Foley and colleagues — Disulfide mapping by partial reduction and alkylation (opens in a new tab)
Disulfide-mapping study, DOI 10.1016/j.ab.2008.02.025; used only for connectivity evidence.
- IUPAC Blue Book — Amino acids and peptides (opens in a new tab)
Sections P-103.1.3 and P-103.3: stereodescriptors and peptide representation. Cysteine prevents a universal L = S shortcut.
- Supplied Semax 10 mg certificate (opens in a new tab)
MA-SEM-0041, revision 01; task DMAS3J7; lot SEM-41-3J7. Source of the example, figures and document-specific observations. The cited PDF was re-read on 3 October 2026 to check this transcription; its issuer and findings were not independently authenticated.
- ICH — Q2(R2): Validation of analytical procedures (opens in a new tab)
Section 3.1 and glossary on specificity/selectivity. Used for the question of analytical discrimination, not verification of a Novum method.
- NIST — 2022 CODATA constants (opens in a new tab)
Proton mass in unified atomic mass units. Examples round it to 1.007276 Da or 1.0073 Da as stated; exact ion assignments require the appropriate mass convention.
- Goodlett and colleagues — Peptide chiral purity determination (opens in a new tab)
J Chromatogr A 707, 233–244 (1995), DOI 10.1016/0021-9673(95)00352-n. Primary deuterated-hydrolysis and Marfey-derivatisation study.
Revised from the existing source dossier on 3 October 2026. Supplied-report values are document transcriptions, not independent authentication or new measurements. Source notes identify the scope of each reference; this article has not undergone independent scientific peer review.