Sample prep: hydrazine derivatization for LC-MS/MS analysis

[syntel]

Hi everyone,
Synthetic chemist, doing some proper analytical work here. So any tips/pointers is appreciated.
I'm trying to develop/optimize a quantitative method for analysis of diketones in plasma samples which require an excess of hydrazine to form the corresponding hydrazones for detection. For the reaction to go to completion, a large excess of hydrazine (aq) is used. However with those concentrations present some issues may present on the instrument (Waters Xevo TQ-S)... In the literature there seem to be some different approaches; evaporation of the sample mixture, which hopefully will remove the hydrazine, followed by re-dissolution in an appropriate solvent. Or simply just inject with the excess hydrazine present...

I'm a bit skeptical about injecting samples containing large amounts of hydrazine (1-3 mM), but is evaporation (via N2-stream, combined with slight heating) of the samples (sometimes followed by re-dissolution in MeOH and evaporation again) enough?

My current plan is basically:
1. To plasma, add internal standards (structurally similar diketones) and hydrazine solution.
2. Add CH3CN/MeOH to precipitate proteins.
3. Incubate to complete derivatization.
4. Centrifuge, remove aliquot for analysis.
5. Evaporate to dryness.
6. Re-dissolve in mobile phase.
7. Inject.

Thoughts?

[ChromKing]

Have you tried using cinnamaldehyde to derivatize the hydrazine? I am not sure if this will solve your problem as you will just be replacing the hydrazine with the conjugated form, but maybe it would separate it from your interested species, or if you are worried about the reactivity of hydrazine this might be the lesser of 2 evils.

[MSCHemist]

Most of the hydrazine (2,2,2-trifluoroethylhydrazine or other hydrazine derivatives) based derivatizations I've seen use headspace or headspace SPME GC/MS. Such as this one
http://www.sciencedirect.com/science/ar ... 3209006679


Here is an HPLC method using dansyl hydrazine.
http://www.ncbi.nlm.nih.gov/pubmed/6860411. I can't access it though.

Here is a good article from Thermo-Fisher on aldehyde/Ketone modification
https://www.thermofisher.com/us/en/home ... tones.html

[sav]

I've developed FIA-MS (flow-injection analysis) method for analysis succinylacetone in DBS (dry blood spot). The excess of hydrazine removed by speedVAC evaporation (45 C; 2 repeated steps with re-dissolution in MeOH).