Hi,
I have read numerous papers about using (Trimethylsilyl)diazomethane (TMSD) to derivitize/methylate the negatively charged phosphate groups present in bisphosphonate compounds for LC-MS/MS or HPLC analysis.
I know that TMSD needs to be stored under nitrogen, however I am unsure if the derivitization reaction needs to be performed in an inert nitrogen environement or not? This step is not clear from the papers I have read. The procedure to methylate bisphosphonates appears relatively simple:
1. Evaporate solvent using SpeedVac or add directly to eluant from SPE
2. Use a 20:80 (TMSD:Methanol) ratio (1mL), let reaction sit for 1hr at RT.
3. Evaporate under nitrogen stream at 40C.
4. Resuspend in mobile phase for LC-MS/MS
Does anyone have experience working with TMSD or diazomethane? What do I need to know about TMSD other than what is provided on an SDS sheet?
Do steps 2-4 need to be carried out in an inert environment? Which steps can be done in "air?"
Any help is greatly appreciated!
The reaction is very quick so inert atmosphere is not necessary. However, it never hurts to conduct a reaction under nitrogen atmosphere (unless O2 is needed in reaction which is not for TMSdiazomethane derivatization).
Greg's answer is actually perfect. What I can add is that this reaction is effective to carboxylic acids not to carboxylates. I usually add TMSCHN2 in ether (yellow solution) to an alcoholic solution of a carboxylic acid at 0 oC, till the colorless solution turns pale yellow, followed by evaporation.
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