LC-MS detection: how to read the m/z and adducts

Q: Why does the main peak appear 22 units above [M+H]+?

It is almost always the sodium adduct [M+Na]+, which sits about 21.98 units above [M+H]+ (the difference between sodium and a proton). Sodium is everywhere in glassware and solvents. A strong [M+Na]+ is not an error, but if it dominates you may want to clean up the mobile phase or add a modifier that favors [M+H]+.

What the mass detector adds

A UV detector tells you that something eluted at 7.84 minutes and absorbs light. A mass detector tells you that this something weighs 166 dalton and has the formula of an ethylparaben. That is the difference: the MS adds a second dimension of identity, the mass, almost independent of retention time. Two peaks that co-elute and that a UV cannot separate, the MS tells apart if their masses differ.

The price of that information is that the MS is more demanding: it only detects what it manages to ionize, and what it reports is not the mass of the molecule but that of an ion. Understanding that step, from neutral molecule to ion, is the whole secret to reading a spectrum.

What m/z is, exactly

The horizontal axis of a mass spectrum is the m/z: the ion's mass divided by its number of charges (z). For the small molecules typical of LC-MS, z is almost always 1, so the m/z equals the ion's mass. When large proteins or peptides appear you can see z = 2, 3 or more ([M+2H]²⁺), and then the m/z comes out at half, a third, and so on, of the real mass: that is why a 15,000 Da protein shows up at low m/z, as a series of multiply charged peaks.

Another key detail: the mass the MS uses is the monoisotopic mass, the most abundant isotope of each element (¹²C, ¹H, ¹⁶O...), not the average molecular mass from the periodic table. For caffeine, the average mass is 194.19, but the monoisotopic mass is 194.0804. A high-resolution instrument works with the second.

Ionization sets the polarity

Before it can weigh anything, the source has to charge the molecule. The three common LC-MS sources are:

ESI+ (positive electrospray): hands protons to the gas phase. Favors basic compounds (amines, most drugs), which come out as [M+H]⁺.
ESI- (negative electrospray): favors acidic compounds (carboxylic acids, phenols, sulfates), which come out as [M-H]^-.
APCI: atmospheric-pressure chemical ionization, for weakly polar or neutral compounds that electrospray ionizes poorly.

The rule of thumb: base, positive; acid, negative. Choosing the wrong polarity is the number-one cause of a blank spectrum: the analyte was there, but in that polarity it does not ionize. Many methods switch between both polarities in the same run so nothing is missed.

Adducts: the molecule rarely travels alone

Here is the point that confuses people most at first. The main peak is almost never at the mass of the molecule (M). It is shifted, because the ion is M plus (or minus) something. That something is the adduct. The most common ones:

[M+H]⁺: protonated, mass +1.007. The bread and butter of ESI+.
[M-H]^-: deprotonated, mass -1.007. The equivalent in ESI-.
[M+Na]⁺: sodium adduct, +22.99 (about +22 relative to [M+H]⁺). Very common from sodium in glassware and solvents.
[M+K]⁺: potassium, +38.96.
[M+NH₄]⁺: ammonium, +18.03, when the mobile phase carries an ammonium buffer.
[M+HCOO]^- or [M+Cl]^-: formate or chloride, typical adducts in negative mode.
[M+H-H₂O]⁺: water loss in the source, -18. Not a real adduct, but early fragmentation.
[2M+H]⁺: dimer, two molecules in one ion. Shows up at high concentration.

Reading a spectrum is largely this game: you see a peak, subtract the adduct and step back to the mass of M. If the big peak is at 217 and you suspect sodium, 217 minus 22.99 is 194: caffeine.

How to compute the m/z from the formula

If you know the molecular formula, the ion's m/z comes out with simple arithmetic. First you add up the monoisotopic mass of every atom; then you apply the adduct. Example with caffeine, C₈H₁₀N₄O₂:

Monoisotopic mass of M: 194.0804 Da.
[M+H]⁺ = 194.0804 + 1.0073 = 195.0877.
[M-H]^- = 194.0804 - 1.0073 = 193.0731.
[M+Na]⁺ = 194.0804 + 22.9892 = 217.0696.

Note that for [M+H]⁺ you add the mass of a proton (1.0073), not that of a whole hydrogen atom (1.0078): the difference is the electron the ion is missing. At low resolution it does not matter, but at high resolution those millidalton matter to confirm a formula.

Reading the spectrum: precursor, base peak and isotopes

In a real spectrum you will see several signals. The precursor is the intact ion of the analyte (for example [M+H]⁺); the base peak is the tallest, taken as 100 % intensity. Around the precursor sits the isotope pattern: a peak at +1 (the ¹³C, taller the more carbon the molecule has) and, if there is chlorine or sulfur, a very visible peak at +2. Two chlorines give a +2 and +4 pattern so characteristic that it lets you see the compound has halogens with no further analysis.

Full scan, SIM and MRM: three ways to look

The same detector can acquire in three ways, with a clear trade-off between overview and sensitivity:

Full scan: records a whole m/z range. Good for identifying and for not missing the unexpected. Lower sensitivity per ion.
SIM: watches only one or a few m/z. You lose the overview, but gain sensitivity to quantify a known analyte.
MRM: only on a triple quadrupole (LC-MS/MS). It selects a precursor, fragments it and follows a specific fragment (the precursor-to-product transition). It is the most selective and sensitive: two mass filters in series remove almost all matrix noise, ideal for traces.

Common mistakes when reading a spectrum

Mistaking the sodium adduct for the analyte. A strong [M+Na]⁺ at +22 can be read as a different substance. Subtract the adduct before concluding.
Choosing the wrong polarity and believing the analyte is not there, when it simply does not ionize in that mode.
In-source fragmentation: seeing [M+H-H₂O]⁺ and taking it for the precursor. The real mass is 18 higher.
Ion suppression: in a dirty matrix, co-eluting with something abundant quenches the analyte signal even though it is present. That is why MRM and a good upstream separation still matter.

How PureAnalyt shows it

In the simulator, when you pick an LC-MS or LC-MS/MS detector, the results table adds an m/z (adduct) column: the m/z is computed from the analyte's molecular formula, and the adduct shown matches the chosen polarity (ESI+ gives [M+H]⁺, ESI- gives [M-H]^-). You can click a peak to see its spectrum, choose the acquisition mode (full scan, SIM or MRM) and the m/z range, and load a reference spectral library to identify by the fragment pattern. All without spending a single real injection.

Takeaways

The MS reports m/z (mass over charge); for small molecules z = 1, so the m/z is the ion's mass.
It only sees ions: base, positive ([M+H]⁺); acid, negative ([M-H]^-).
The peak is shifted by the adduct: subtract it to get back to the mass of M.
From the formula, the m/z is the monoisotopic mass plus the adduct (a proton = 1.007).
Full scan identifies, SIM quantifies, MRM gives maximum selectivity and sensitivity.

Frequently asked questions

What does m/z mean in mass spectrometry?

m/z is the ion's mass-to-charge ratio: its mass divided by the number of charges. For a singly charged ion (z = 1), the m/z equals the ion's mass. The mass used is the monoisotopic mass (the most abundant isotope of each element), not the average molecular mass from the periodic table.

What is an adduct, and why don't I see the molecule's exact mass?

The detector only sees ions, not neutral molecules. To become an ion, the molecule (M) gains or loses something: gaining a proton gives [M+H]⁺ (mass +1.007), losing one gives [M-H]^- (mass -1.007), and it can also pick up sodium [M+Na]⁺ (+22.99), potassium, ammonium or formate. That is why the peak is not at the mass of M, but shifted by the adduct.

How do I know whether to run positive (ESI+) or negative (ESI-) mode?

The analyte's chemistry decides. Basic compounds (amines, most drugs) ionize better in positive mode as [M+H]⁺. Acids (carboxylic acids, phenols, sulfates) ionize in negative mode as [M-H]^-. Weakly polar or neutral compounds usually prefer APCI. If you pick the wrong polarity, the analyte may give no signal at all.

What is the difference between full scan, SIM and MRM?

Full scan records a whole m/z range: good for identifying and seeing what is there. SIM watches only one or a few m/z: it loses the overview but gains sensitivity for quantifying. MRM (on a triple quadrupole, LC-MS/MS) follows a precursor-to-fragment transition: it is the most selective and sensitive, ideal for traces in complex matrices.

Why does the main peak appear 22 units above [M+H]+?

It is almost always the sodium adduct [M+Na]⁺, which sits about 21.98 units above [M+H]⁺ (the difference between sodium and a proton). Sodium is everywhere in glassware and solvents. A strong [M+Na]⁺ is not an error, but if it dominates you may want to clean up the mobile phase or add a modifier that favors [M+H]⁺.