alendronate in LC-MS/MS

[alpesh]

dear group
i am working on triple quadrupole LC-MS/MS of applied biosystems (API 4000) and the analyte is alendronate sodium. the source is turbo ion spray. what i am observing is that very low intensity is coming in authentic standard. and upon extraction no peak is coming at all. we have tried all extraction technic. the LC is shimadzu. we have tried all column and all extraction cartdrige. but nothing is coming at all. we have tried every combination of mobile phase contatining methanol water and acetonitrile. if anybody has worked on alenronate on API instrument, any suggestions will be highly appreciated.

[sassman]

Hi alpesh,

How did you determine what mass to scan for? I would infuse the standard solution into the mass spectrometer to find out what mass your compound will show up at. This would best be done by infusing the standard into the LC stream using a tee and then into the mass spec. You probably need some kind of pH control for your mobile phase since alendronate is ionizable. If you adjust the pH to about 5 using ammonium acetate/formic acid, then I would expect alendronate to have a net negative charge (--+). Hope this helps.

[alpesh]

hi
thanx for suggestion. we have found parent and precursor in negative mode. but the sensitivity was less. all literature of alendronate told about derivatization of alendronate. can u tell me how to derivatize it or can it be done without derivatization.
best regards and thanx once again.
alpesh

[jag]

Hi Alpesh,
It is very difficult to analyse Alendronate by LC-MS/MS without derivatization. Biphosphonates show very low signal in ESI. You need to methylate the molecule with Diazomethane reagent then analyse methylated Alendronate by ESI positive. You will be surprised by seeing the signal in ESI after derivatization. Waters Atlantis HILIC column works best for chromatography of these class of compounds.

[alpesh]

thanx for suggestion. we have tried derivatiozation as has been told by you. but steel we have very low signal after derivatiozation.
can it be possible to check whether derivatization completed or not????
alpesh

[Bryan Evans]

What was the pH of the mobile phase you were using with
the underivatized alendronate?

[alpesh]

sir,
we have used entire range of pH form highly acidic to moderate basic i.e. form 2 to 9 pH of mobile phase. but as the derivatization itself in doubtful, we havent got much sensitivity in Mass. All the colums being used were of merck and ACE.
alpesh