Chromatography Forum

Dear all
I'm trying to develop a LC-MS/MS method to calculate concentration of total homocysteine in plasma samples, but I'm in trouble!
well: I use deuterated IS: d8 homocystine (50µM in CH3OH with 1% HCl)and the reduction step has been perfomed with DTT (500 mM in H2O solution ) for 15' T ambient (Flow solvent H20/Acetonitrile)
the scan is MRM with their specific transition
after optimization of parameters of natural Hcy, I've begun to study the method with acqueous samples :I added in a vial natural homocysteine 50 µl (50 µM in H2O) + 50 µl d8 Homocystine (50 µM that will give, after reduction, the d4 Homocysteine 100µM)+50 µl DTT , after injection in MS/Ms system I've got a strange behaviour of the analytes :
the intensityes of D4 homocysteine is very,very lower respect of the natural homocysteine one !!!
insted the intensityes of the natural is very good
I thought IS intensityes must be more or less two time more!
Why??
the ionization behaviour of a deuterated is not the same (I mean proportional) of the the natural?There same problem with reduction-reoxidation , ionization or.......?
I need help to understand where is my mistake!!!
I've also change the reductor with TCEP but it caused seriuos ion suppression

thanks in advance to all of you
M.C.

I had samilar problem when I tried to use D8-compound as IS. My case is worse, I didn't see any d8 signal.(only a simple solution preparation, no reduction! I post this question some time earlier on this forum). Our coclusion is that the compound lost its D8, but We condn't know why. We finally give up without clear conclusion.

I would guess that in your case the problem is in your reduction step. Have you checked the transition 277->140 to verify that you do not have any of the original D8 Homocystine? A high background could also indicate on column degredation.

Another possibility is that during the reduction you have exchange of H with D... I do not know where the D are positioned in the molecule and if this exchange is possible...