Variable Ion Ratios in LCMS

[strobbia]

Hi everyone,

I am developing a method for analysis of paraquat and diquat (2+ quats) by LCMS using a HILIC column to avoid ion pairing.

There are two major ions present for each compound in the MS spectrum assigned to [M-H]+ and a [M]+. my problem is that the ratio of these differs signifiacntly across my calibration range.

A plot of [M]+. / [M-H]+ vs. concentration is logarithmic. I was hoping somebody could help me with the significance of this

It was my understanding that ion ratios were generally stable and were not concentration dependent.

Has anyone else observed anything similar??

Thanks

Stephen

[MassSpecM]

ok here we go - my first reply to try and help (i'm still quite new so to all the experts - help if i'm completely off the mark)

2 quick questions -
what ionisation mode
what are the actual masses of [M]+ and [M-H]+

if you look at the structure of paraquat the chlorine is only an ionic bond - so you usually wont see the chlorine (Br in diquat) in ESI mode.

also there is not really a H that can fall off under the mild conditions of ESI.

[strobbia]

Thanks for replying

Ionisation is with positive ESI

For paraquat [M]+. is at 186 and [M-H]+ is at 185

Diquat is 184 and 183

We are not sure if the H is lost from the methyl group or the ring

We don't see any impact from Cl or Br (dissolved in solution)

[MassSpecM]

ok thanks! just checking about the chlorine!

usually when you loose a H you get a negative ion (wont be visible in positive mode) and the fact that you get a [M]+ instead of [M+H]+ (the normal process in ESI) is a bit puzzling.
but i have seen some electron capture processes in APCI giving a [M]+ instead of [M+H]+ so its possible?

it might be that the one species ionizes better than the other and you get a kind of ion suppression occuring at higher concentrations.

[richiekichi]

I work with these compounds fairly regularly and like you, use HILIC coupled with ESI LCMS.

I do not see any variation in ion ratio across the standard range that I'm working with.

[strobbia]

Hi richiekichi

We have run into quite a few problems whilst developing this method, I was wondering if you could give me some more information on the method you use (column, LC solvents, ESI settings etc) if it's not restricted within your organisation.

If you could email me at strobbia@hotmail.com it would be greatly appreciated.

Thanks

Stephen

[richiekichi]

These application notes should provide you with a useful starting point with regard to your instrument conditions.

http://chromatographyonline.findanalyti ... rticle.pdf

http://www.chem.agilent.com/Library/app ... 7220EN.pdf

If you don't mind me asking, what type of samples are you looking to develop a method for?

[strobbia]

Thanks richiekichi, I had seen those methods and we are doing something similar to the waters application. We are quite a long way down the track with the method development, I jsut cant explain a few of the things were are seeing like the variable ion ratios. We are extracting them from blood and urine.

Stephen

[richiekichi]

I haven't observed any variation in the ion ratio in the range 500-10 ppb.

You've checked your standard solvent/mobile phase/blank matrix for interferences?

Have you cleaned your ion source since noticing this effect? It's possible that deposits on your entrance lens or skimmer might be causing this instability in your mass ratio.

[strobbia]

Yep it's definately a real effect, no interferences coming from elsewhere and the change in ratio has been relatively reproducable over my last 5 calibration curves.

Do you see both the 185 and 186 ions in your method?

We are using a QTOF and quanting in single MS mode, I realise that most methods would probably be on QQQ's and that only one ion would be selected for fragmentation.

We are working in a slightly higher concentration range, about 0.2 to 5 ug/ml

[lmh]

You asked about the chemistry. I am no chemist, but I'd guess:

Paraquat is a strong electron acceptor. That's how it works as a herbicide. It's a strong acceptor because it can stabilise a radical electron because it is so delocalised. It is obviously a very exceptional chemical because it's forming a radical in electrospray (as well as a conventional non-radical ion).

In order to form the radical ion, another unpaired electron has to be taken away by something else. The leaving group that you already have is HCl, which can't take a spare electron. It's therefore most likely that the spare electron is transfered to another paraquat molecule. So you have kinetics for formation of the radical ion that involve two paraquats (one to ionise directly, one to snaffle a spare electron), not one. This means it will have different concentration dependency to a simple unimolecular ionisation.

But this is all just a guess from someone with little or no chemistry.

[richiekichi]

Our target ion for paraquat is 93 (C6H7N, the fragment resulting from fission of the bond between the two pyridine rings). 186 and 185 are used as qualifier ions.