HPLC and LC-MS: what does each method measure?

Certificates of analysis often mention HPLC and LC-MS. These techniques provide different types of information about the same sample. This guide mainly compares HPLC with UV detection and LC-MS. LC-MS also uses liquid chromatography: the distinction partly lies in the detector and the question the laboratory is investigating.

HPLC met UV-detectie en LC-MS vergeleken

Vraag	HPLC met UV-de­tec­tie	LC-MS
Wat wordt ge­me­ten?	UV-res­pons van ge­schei­den com­po­nen­ten tegen de tijd.	Massa-la­ding­ver­hou­din­gen van ionen, ge­kop­peld aan de chro­ma­to­gra­fi­sche schei­ding.
Wat helpt dit be­oor­de­len?	Het piek­pa­troon en, bin­nen de ge­bruik­te me­tho­de, re­la­tie­ve piek­op­per­vlak­ken.	Of ge­me­ten ionen pas­sen bij de ver­wach­te massa en la­dings­toe­stan­den.
Wat volgt er niet van­zelf uit?	Eén piek of een hoog per­cen­ta­ge be­wijst op zich­zelf geen iden­ti­teit of hoe­veel­heid in mil­li­gram.	Een pas­sen­de massa be­wijst op zich­zelf niet de vol­le­di­ge ami­no­zuur­volg­or­de.

Een kwantitatieve uitkomst vraagt bij beide technieken een geschikte meetopzet. Lees het gerapporteerde resultaat, de eenheid en de kalibratie; leid de hoeveelheid niet af uit alleen een grafiek of methodenaam.

What does HPLC do?

HPLC stands for High Performance Liquid Chromatography. A liquid carries the sample through a column. Compounds travel through it differently and can therefore be separated. A detector measures what leaves the column; the chromatogram plots the measured signal against time.

Source: Waters — how HPLC and detectors work

With UV detection, the signal depends on the wavelength selected. Also consider how well the peaks are separated. Compounds leaving the column at almost the same time can produce overlapping peaks. A single prominent peak is therefore not, by itself, a complete identification of a peptide.

Lees verder over hoe u een chromatogram beoordeelt, met aandacht voor assen, pieken en de grenzen van de interpretatie.

What does LC-MS add?

LC-MS couples the separation to mass spectrometry. The mass spectrometer measures the mass-to-charge ratio of ions, written as m/z. A peptide can carry multiple charges, so different m/z signals may belong to the same peptide. The laboratory takes those charges into account when comparing the results with the expected molecular mass.

Source: Waters — peptide analysis with UV and mass detection

A matching mass supports identification, but it is not the same as establishing the complete amino acid sequence. Fragmentation analysis, such as MS/MS, can provide additional structural information. Ask which identity checks were actually performed; the label LC-MS alone does not give the full answer.

Source: Waters — peptide mapping and structural information

What does this tell you about purity and quantity?

An HPLC purity percentage may be based on the area of the assigned peptide peak relative to the peaks included in the calculation. Check which calculation and detection method were used. That percentage does not automatically describe the entire contents of the vial: water and salts, for example, are not accounted for in the same way in a UV peptide analysis.

A report showing 99% chromatographic purity therefore does not, by itself, prove that a vial contains the number of milligrams stated on its label. Purity and measured quantity are separate measurements.

Source: Sigma-Aldrich — peptide purity and peptide content

Both HPLC and LC-MS can form part of a quantitative assay. This requires an appropriate measurement approach, such as calibration with standards containing known amounts. Look in the report for the quantitative result, its unit and the basis of the calculation. A graph or method name does not replace those details.

Source: Agilent — quantitation and calibration

In identiteit, zuiverheid en gemeten inhoud leest u waarom deze resultaten afzonderlijke vragen beantwoorden.

Five checks for your report

1. Check that the product, variant and batch number match your documentation.
2. Check which technique and detector were used; do not assume additional tests were performed.
3. Keep reported identity, purity and quantity separate.
4. Check the units, test date and any explanation of discrepancies.
5. Ask the laboratory or supplier for clarification if a result is unclear.

This guide helps you read a report; it is not an assessment of a specific Novogin batch. Do not infer a sterility result or suitability for human use from an HPLC or LC-MS label. Draw conclusions only about properties that were actually tested and reported.

Further reading and reports

Read also: how do you read a COA?

Controleer ook of het analyserapport bij de actuele batch hoort.

Browse available reports in the batch library

Would you like to compare product specifications alongside this explanation? Explore research peptides for professional research.

This knowledge base explains analytical documentation for laboratory research. It does not provide advice on dosing, administration or medical use.

Bronnen

• Waters — How Does High Performance Liquid Chromatography Work?: scheiding, detectoren en chromatogrammen.
• Waters — Mass-Directed Isolation of Synthetic Peptides Using the Waters SQ Detector 2 (2024): massa-ladingverhoudingen en meerdere ladingstoestanden.
• Waters — Peptide Mapping and Analysis: structuurinformatie in een peptide-mappingworkflow; dit is niet automatisch dezelfde test als ieder LC-MS-rapport.
• Sigma-Aldrich — Peptide Sample Amount Determination: peptidezuiverheid en peptidegehalte.
• Agilent OpenLab CDS 2.8 — Glossary: raadpleeg Calibration en Calibration standard voor de relatie tussen instrumentrespons en een bekende hoeveelheid.