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Using GC-MS to identify and quantify dopamine in drosophila
Posted: Mon Mar 16, 2015 3:39 pm
by tim08
Hi,
To seperate out dopamine, most people appear to use LC with an electrochemical detector (which unfortunately I don't have access to). What would be the best way to derivatise dopamine for use in a db-5 column GC attached to a quadrupole MS? I have tried using TFAA which works fine for a dopamine standard but doesn't appear sensitive enough to look at dopamine in a crushed fly head matrix.
Thanks for any help!
Tim
Re: Using GC-MS to identify and quantify dopamine in drosoph
Posted: Mon Mar 16, 2015 11:26 pm
by tom jupille
You should be able to make the trimethylsilyl derivative using something like BSTFA or TMS-imidazole, but unless your TFA derivatization was incomplete, there's no reason to expect that the sensitivity by GC/MS would be any better.
Ultimately, detection limit is a matter of signal/noise ratio. If you weren't too high, you might be better off finding ways to decrease the noise rather than trying to increase the signal.
Re: Using GC-MS to identify and quantify dopamine in drosoph
Posted: Tue Mar 31, 2015 9:32 pm
by JMB
Since you are running GC/MS, I assume that you are using electron impact ionization (EI). This is a very "hard" (high-energy) ionization and will generally give a high degree of fragmentation of most organic molecules; the ion current is therefore spread amongst many m/z values, and none of them may be intense enough for low-level detection.
Dopamine from a crushed fly-head sounds like ultra trace-level analysis. I think that you need to go to Chemical ionization (CI), using initially CH4 as the reagent gas. This is a "soft" (low-energy) ionization process, and will give you primarily intense [M+H]+, lower intensity [M+C2H5]+ and lower still intensity [M+C3H5]+ ions. There will, IN GENERAL, be little to no fragmentation and most of the ion current will be carried by the [M+H]+ signal.
However, I suspect that you will ultimately have to go with electron capture negative ion chemical ionization (ECNICI) for best sensitivity; this will usually give M as a radical anion. Again, this can be performed with CH4 as the reagent gas, and the analyte molecules have to be derivatized with a group of high electron affinity (EA). Typical high EA groups contain halogen atoms (C6F5-, CF3CO-, etc).
There are at least a couple of good ref. books that give comprehensive reviews & detailed procedures for such derivatizations.
One is Blau & King, another is by Knapp et al.
If GC/MS is ultimately unsuccessful, HPLC with fluorescence detection may be required.
Good Luck & let us know if any technique is successful.