LC-MS small molecules
24 important questions on LC-MS small molecules
What is the most common approach of LC-MS in practice?
MS: triple quadrupole mass analyser (ms/ms) in combination with electrospray ionization (75%) and multiple reaction monitoring with fixed m/z values for first and third quadrupole
What is an advantage of a very narrow column?
What is a good mobile phase for reversed phase LC?
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Why is the coupling of LC and MS difficult?
What is the principle of ESI?
The thing is that the more ions you will have, the more sensitive the system is.
Which parameters play a role in how good the ESI will be?
- surface tension
- viscosity
- conductivity
- ionic strength
- dielectric constant
- pH
What is the rule of thumb for the influence of type of LC solvent?
Therefore the coupling of reversed-phase LD with ESI-MS is generally feasible.
For LC solvents with a low polarity (e.g. hexane) a post column addition of more polar solveent may help increase the ESI response. Therefore the coupling of normal-phase LC with ESI-MS is generally more difficult.
What is the influence of pH of LC solvent?
However, the best chromatographic performance with reversed-phase LC is achieved if the analytes are neutral in the LC solvent.
What does "wrong way around ionization" mean?
Neutral compounds easily pick up a charge. Ionization then probably takes place in the gas phase or at the liquid-gas interface of the solvent droplets.
What is the influence of additives in LC solvent?
What is the typical analytical procedure?
2) addition of internal standard
3) extraction
4) (derivatization)
5) chromatography
6) detection
The best concentration for the internal standard should be somewhere in the middle of the callibration range.
Where do internal standards for correct?
- extraction behaviour
- derivatization yield
- transfer and injection volumes
- chromatographic performance
- detection
It does not correct for:
- initial sample aliquoting (it was not present there yet)
- peak integration
What kind of internal standards can be used for LC-MS?
- a stable-isotope (STIL) form (most used nowadays)
typically there is one IS for each analyte.
What is the reason that there is so much more variability for UV than for MS/MS?
- wavelength
- quality/age of UV lamp
For MS/MS there are more factors important:
- the mass of the different quadrupoles
- the resolutions of the different quadrupoles
- the spray position
- the cleanliness of the the ion source.
- ion source temperature
- ion spray voltage
- sample composition
What is the most ideal correction?
- physico-chemical properties are essentially similar
- only difference is molecular mass
- same extraction recovery
- co-elution in HPLC
- simultaneous ionization in MS
Typical heavy atoms that are used are: deuterium, C13, N15 or O18
How can the signal from the analyte contribute to the signal of the internal standard?
The percentage and mass increase depends on the type of atom
In this way, the analyte may contribute to the signal of the STIL internal standard.
How can the contribution from the analyte to the signal of the internal standard be minimized?
- Selecting an internal standard with a proper (higher) mass.
What are the isotopes of the most important atoms?
- Carbon: C12 = 100%, C13 = 1,1%
- chlorine: Cl35 = 100%, Cl37 = 32,4%
- Bromine: Br79 = 100%, Br81= 97,5%
- Sulphur: S32: 100%, S33= 0,8%, S34 = 4,4%
How can the signal from the IS contribute to the signal from the analyte?
The acceptable contamination is 0,2 * LLOQ level.
There are 2 ways of reducing the contamination:
- inject less internal standard (ul)
- decrease the concentration of IS
What are the practical issues when there is signal contribution form the internal standard to the analyte?
- This will result in a lower MS response for internal standard when analyte concentration is increasing.
- This may affect the correction by the internal standard.
What could be a problem with LC-MS regarding the matrix?
- Therefore you constantly inject an amount of analyte to get a higher background signal. If endogenous compounds are competing with the analyte, you will see a drop in the spectrum. This can variate between individuals.
- The solution is to remove interferences e.g. by an improve sample extraction or play around with the mobile phase.
Which 2 different types of phospholipids do you have?
- Lysophospholipids: about 10% of plasma phospholipids
How to get rid off phospholipids?
2) protein precipitation (add acetonitrile)
What is the basic extraction technique that can be used to remove phospholipids?
- this phosphote group will bind to the zirconium of the column and the rest of the compounds will be separated from the phospholipids since only the column needs to be thrown away.
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