High-resolution mass spectrometry
HRMS
High-resolution mass spectrometry measures a molecule's mass precisely enough, often to within a few parts per million, and separates peaks finely enough to resolve its isotope pattern, so a peptide's identity can be confirmed far more tightly than with a nominal-mass instrument.
Planned, not yet offered
What it answers
- Whether the observed mass matches the theoretical mass to within a stated tolerance in parts per million
- Whether the isotope pattern fits the expected elemental composition
- Whether a near-isobaric variant is present, such as a deamidated form, that a nominal-mass instrument might blur
- With fragmentation, whether fragment masses fit the expected sequence with high confidence
What it cannot answer
- Purity or amount: ion signal still varies between molecules
- Whether two molecules with identical elemental composition (for example, a residue-order swap or a D-amino acid) are told apart by mass alone
- Water, counter-ion, salt, endotoxin, sterility, metals or solvents
- Anything about other vials or batches
What it outputs
- A high-resolution mass spectrum with resolved isotope peaks
- Observed accurate mass, usually to four or more decimal places
- Mass error against theory, in parts per million (ppm)
- Optional fragment spectra with accurate fragment masses
On a COA
An HRMS identity line typically shows the theoretical monoisotopic mass, the observed mass and the error in ppm, for example a row reading "observed", "calculated" and "Δ ppm". It should name the instrument type (Orbitrap, Q-TOF or FT-ICR) or at least "HRMS" or "accurate mass". The verdict is usually "conforms" when the error sits inside the laboratory's stated tolerance.
What makes a mass spectrometer high resolution?
Two separate qualities are often lumped together: resolving power (showing two close m/z values as two peaks rather than one hump) and mass accuracy (how close the measured value is to the true one). A review of Fourier transform mass spectrometry in Molecular and Cellular Proteomics defines mass accuracy as the ratio of the measurement error to the true m/z, usually quoted in parts per million, and resolving power as the ability to distinguish two peaks of slightly different m/z, measured at full width at half maximum (Scigelova and colleagues, Molecular and Cellular Proteomics, 2011).
A 2023 review of ultra-high-resolution MS for pharmaceutical analysis gives the working formula: resolving power equals m/z divided by the peak width at half height (Molecules, 2023).
A mass spectrum with a dashed marker at the expected mass and the observed peak sitting on it, labelled as a match within tolerance.
Why does ppm accuracy matter for a peptide?
Many molecules share the same whole-number mass, and fewer candidates fit as accuracy tightens. Scigelova and colleagues show this for tryptic peptides: the number of possible peptide candidates for a measured mass drops sharply as mass accuracy improves. In a small-molecule example (quercetin), they show that fragment mass deviations under 3 ppm, together with the fragmentation pattern, confirm the elemental composition of the starting compound.
Isotopes add a second check. Carbon, nitrogen, oxygen and sulfur each have heavier isotopes, so a peptide shows a cluster of peaks whose relative heights depend on the elemental formula. Kind and Fiehn showed that, without isotope information, even a hypothetical 0.1 ppm instrument could not give unique elemental compositions above about 186 daltons (far below any peptide) when C, H, N, S, O and P were allowed, and that isotope abundance patterns remove most wrongly assigned formulas (Kind and Fiehn, BMC Bioinformatics, 2007). A good HRMS identity check uses both accurate mass and isotope pattern.
Which instruments count as HRMS?
| Analyser | Principle | Trade-off |
|---|---|---|
| Time-of-flight (TOF, Q-TOF) | Flight time of accelerated ions down a tube | Fast acquisition, suits chromatography; resolving power lower than trap-based instruments |
| Orbitrap | Ions orbit a central electrode; their oscillation frequency gives m/z | Higher resolving power needs longer acquisition per scan; the 2023 review notes LC work often uses 60,000 to 70,000 FWHM as a compromise |
| FT-ICR | Ions circulate in a strong magnetic field; cyclotron frequency gives m/z | Highest resolving power, but slow, so many studies use direct infusion rather than chromatography |
Because FT-ICR is slow, the 2023 review notes that coupling it to chromatography means shortening the measurement and giving up resolving power. For a peptide identity check after liquid chromatography, a Q-TOF or Orbitrap is the usual choice.
Does high resolution prove the sequence?
Not by itself. Accurate mass narrows the elemental composition, but two peptides with the same amino acids in a different order have identical compositions and identical masses. So do peptides that differ only in the chirality of one residue. Fragmentation (MS/MS) or peptide mapping is needed to address order, and chiral methods are needed for D and L forms.
ICH Q2(R2) recognises the strength of the technique while keeping it in proportion. It notes that specificity can sometimes be justified by the technology itself, giving resolution of isotopes in mass spectrometry as an example, so additional experiments may not be needed where justified (ICH Q2(R2), 2023). That justification covers telling isotopes apart, not proving a sequence.
How is HRMS different from standard LC-MS on a certificate?
A nominal-mass result from a quadrupole LC-MS is usually given to one decimal place or as a whole number, which catches a gross swap. An HRMS line gives several decimal places and a ppm error, which matters when the alternative molecule is close in mass, such as a deamidated form of the peptide.
Does Tested.ph offer HRMS?
HRMS is planned but not yet offered; identity in the standard package is by LC-MS. Use the mass checker to compare an observed mass on any certificate with the theoretical value.
Sources
- Fourier transform mass spectrometry (Scigelova and colleagues, 2011), Molecular and Cellular Proteomics, via PubMed Central
- Seven Golden Rules for heuristic filtering of molecular formulas obtained by accurate mass spectrometry (Kind and Fiehn, 2007), BMC Bioinformatics, via PubMed Central
- Advances in Ultra-High-Resolution Mass Spectrometry for Pharmaceutical Analysis (2023), Molecules, via PubMed Central
- ICH Q2(R2) Validation of Analytical Procedures, International Council for Harmonisation
Last reviewed 6 Oct 2026.