Observed vs theoretical mass: what an LC-MS identity result confirms

The certificates in our library report identity by LC-MS with two numbers: a molecular weight in g/mol, and the ion the laboratory detected, such as “664.8 [M+H]”. The two aren't meant to be equal, and they rarely agree to the decimal. This page shows how to get from one to the other, why small differences occur, and what a matching mass does and doesn't establish. The worked examples use figures copied from our certificates.

What the instrument measures

LC-MS pairs liquid chromatography, which separates the components of a sample, with a mass spectrometer, which measures ions. The spectrometer can only measure charged particles, so a molecule (M) first picks up a charge, usually by gaining one or more protons (H⁺). The instrument then reports the mass-to-charge ratio, m/z: the ion's mass divided by its charge. A proton's mass, on the same scale as a molecular weight, is 1.00728.

• [M+H]⁺ is the molecule plus one proton, with one charge. Expected m/z: molecular weight + 1.00728.
• [M+2H]²⁺ carries two protons and two charges: (molecular weight + 2 × 1.00728) ÷ 2. [M+3H]³⁺ is (molecular weight + 3 × 1.00728) ÷ 3, and so on.

Our certificates print these without the charge, as [M+H], [M+2H], [M+3H] and so on.

Worked examples

With one proton the arithmetic is a single addition. NAD+ 1000 mg (lot NAD1000VAS0826) prints 663.43 g/mol: 663.43 + 1.00728 = 664.44, and the certificate reports 664.8, 0.36 higher.

A larger molecule picks up several protons, so it reads at a fraction of its mass: VIP 10 mg (VIP10VAS0426), printed at 3326.8 g/mol, is reported at 1664.7 with two protons and 1109.8 with three. Every example, from the molecular weight each certificate prints:

The six differences run from 0.14 to 0.51. Klotho Frag's reading can be compared only with the weight its certificate prints, since neither the product name nor the certificate gives a sequence.

Why the numbers differ by a few tenths

• Average versus monoisotopic mass. A printed molecular weight averages over the natural mix of isotopes, such as carbon-12 and the heavier carbon-13. The spectrometer measures individual molecules, and the lightest form, the monoisotopic mass, sits below the average; the gap grows with the size of the molecule. For ARA-290 the monoisotopic [M+H] is 1257.61 and the average 1258.31, and the reported 1257.9 falls between them. Where a reading lands depends on the instrument's resolution and how it picks the peak.
• Calibration and rounding. Every mass spectrometer has a calibration and a mass accuracy, and these certificates report ions to one decimal place. NAD+ reads above both values: its monoisotopic [M+H] is 664.12, so 664.8 is 0.68 above it and 0.36 above the average. The certificates don't name the instrument or state its mass accuracy or an acceptance window, so they don't say how large a difference the laboratory accepts.
• Which structure the weight was calculated for. VIP's printed 3326.8 matches the figure PubChem gives for a structure ending in a free acid; native VIP ends in an amide, at 3325.8 g/mol. Divided by the charge, that one-unit difference shrinks to 0.5 at [M+2H] and 0.33 at [M+3H], about the size of the offsets themselves, so these ions can't tell the two forms apart (see the VIP certificate guide).

Adducts: ions that aren't [M+H]

A molecule can also take its charge from a metal ion. Sodium is a common one: [M+Na]⁺ reads at the molecular weight plus 22.99, about 22 above [M+H]⁺. For KPV that would be near 342.43 + 22.99 = 365.42. Potassium, [M+K]⁺, reads about 38 above [M+H]⁺. Traces of sodium from solvents or glassware are enough to form these ions, so a peak about 22 above the expected ion may be the same molecule carrying sodium rather than a different compound, and it doesn't show that the solid was a sodium salt. The results tables on the certificates above list protonated ions only.

What an identity result establishes

A detected ion within a few tenths of the expected value supports the identity of the main component: the sample contains a substance with the mass the laboratory expected. It doesn't establish:

• Purity. How strongly a substance shows up depends on how readily it ionizes, so ion signals don't measure proportions. UV purity is a separate measurement on the same certificate; see how peptide purity is measured.
• Sequence. The same atoms in a different order have the same mass: Lys-Pro-Val and Val-Pro-Lys read alike, and so do forms built from D- rather than L-amino acids.
• Net peptide content. The ion comes from the molecule alone, so it says nothing about how much of the powder's weight that molecule makes up.
• Counterion or water. Acetate, trifluoroacetate, sodium or water in the solid don't change the reading. Our NAD+ certificates print the free acid's 663.43 g/mol, and the detected ion can't show which salt form, if any, was tested.
• Anything about a particular vial. Like every result on a certificate, it describes the sample the laboratory tested from one lot.