Inetrnal Standard for LC-MS/MS
Posted: Mon Feb 07, 2005 4:58 pm
What are the general rules to pick an internal standard for LC-MS/MS? Are there any references available regarding this? Thank You.
I'm afraid I don't have any references handy, but I can tell you what we do. The best choice is an isotopically labeled version of the analyte. With deuterium labels, be sure they are in positions on the molecule where they won't exchange for H in solution. With carbon-13 labels, you won't have to worry about this. Also, make sure there are enough labels so that your unlabeled analyte won't interfere due to its naturally occurring isotopes. The minimum number of labels you need will therefore depend on what elements are present in your molecule.
Labeled compounds are expensive, and sometimes the project manager won't want to pay for it. In this case, I use the following general rules for the ISTD:
(1) Has similar functionality, so that it ionizes in the same mode and hopefully the same functional groups are responsible for the ionization.
(2) Elutes close to your analyte, or ideally it coelutes*.
(3) If it coelutes, it must have an MRM transition unique from the analyte.
*(4) Alternately, if you use an isomer of your analyte as ISTD that has the same MRM transitions, then it must not coelute with your analyte.
(5) If you are adding ISTD to your samples prior to preparation (some people call this a "surrogate"), then it should have similar extraction recoveries.
These rules are not set in stone and depend on your purpose in using the internal standard, but the more of these you satisfy, the better internal standard you will have.