Please: [[ question related to the use of LC-MS ]]

[Newchromatographer]

HI, dear scientists please help me

I have been using LC-UV for many years. Unfortunately i have no experience in MS detector ( i.e., LC-MS . LC-MS/MS ETC) and now I have to work on them and need help through the following question:

1- I am familiar with LC-UV i.e, i see the separated peaks then i take the peak area and i do validation accordigly. Is the same thing happen in LC-MS because i don't need to do identification , i just want the peak area, in other way will LC-MS allow me to see seperated peaks and then will I be able to just take the peak area? is the validation such as LOD,LOQ etc same as LC-UV.

2- Interpretation is worrying me is it difficult, where can i start?

3- The problem is i developed a method in LC-UV, do you thing i can transfer it easily


Thanks

[tom jupille]

will LC-MS allow me to see separated peaks and then will I be able to just take the peak area? is the validation such as LOD,LOQ etc same as LC-UV.

Yes, for quantitation, you use peak area just as you do with UV detection. For straightforward MS detection, the issues are parallel to those in UV (in UV you have to choose a wavelength; in MS you have to choose a m/z), but the MS requires quite a bit more fine tuning.

Interpretation is worrying me is it difficult, where can i start?

If you're lucky it may not be an issue. With ESI you will hopefully see a single m/z for the pseudo-molecular ion. If you're unlucky, it can be a very big issue indeed. Check with mass-spec-training.com ; they have excellent on-line and self-paced courses in LC-MS and MS interpretation:
http://www.mass-spec-training.com/

The problem is i developed a method in LC-UV, do you thing i can transfer it easily.

Again, it depends how lucky you are. If you used volatile buffers (e.g., formic acid or ammonium formate) it may simply be a case of scaling to a smaller column. If you have anything non-volatile in the mobile phase (e.g., phosphate), that will have to be replaced. If you have non-volatile junk in the sample, you will have to modify your prep to get rid of it. Then you will have to adjust the MS parameters (voltages, temperatures, gas flow) as necessary.

[Newchromatographer]

Thanks Tom, thats perfectly answered.

[lmh]

The one thing you have to be aware of is that mass spec is much more sensitive to dirty spray-chambers and variation in sensitivity caused by the sample and gradual deterioration through a batch of samples. For this reason, for the really good quantitation, it is far more important to use internal standard calibration in LC-MS than it would be in LC-UV. If you cannot (because you are analysing things that are not commercially available, and perhaps only looking at peak areas) then it is vital you randomise your experiment appropriately, and keep in mind that peak area can be influenced by other chemicals.

You will come across the term "cosuppression". This is when two chemicals coelute, one is detected by the MS, but the other hinders the ionisation of the first, meaning that when you see a smaller peak, it could be either that the detected chemical is less abundant, or that the interfering chemical is more abundant!

[Newchromatographer]

Thanks lmh for the great advice.