OPA-MPA derivatizing agent for Gabapentin

[ghie malig]

Hi everyone!
I'm here again asking for your expertise. So far we've finished the cefuroxime method already. I am now working on Gabapentin. It involves pre-column derivatization with O-phthaldialdehyde - Mercaptopropionic acid as derivatizing agent. It's my first time to use OPA-MPA reagent and I'm not quite familiar with its stability and storage conditions. Has anyone here experienced working with this kind of reagent? Need your help. Thanks!

-ghie-

[Mark Tracy]

I'v done a fair amount of work with OPA/thiol systems, both pre- and post-column. These derivatives are moderately stable, 3-mercaptopropionic acid is better by far than the more common 2-mercaptoethanol derivative. Even so, for the best results, the time between reaction and analysis should be consistent. If at all possible, use an autosampler with sample prep capabilities. The reaction normally goes to completion in less than a minute at room temperature.

Unfortunately 3-MPA smells like old tennis shoes, but the reagent will be at pH 9 or so, and the 3-MPA will be mostly nonvolatile.

The thiol reagent oxidizes in air, and should be prepared fresh daily. Some formulas use separate thiol and OPA solutions, some combine them. Degassing the reagent will give you a head start on the oxidation process. If you arrange to keep it under nitrogen, it will last a week.

[ghie malig]

Thank you for your reply. It's true I cannot get a reproducible result yet using an injector. But when we did it manually, the results were constant. I think we're having problems with the auto-injector's program. I have another concern though, is the ratio of the analyte and OPA-MPA always 1:1? I've read that excess derivatizing agent will cause degradation of the derivative. Pls. enlighten me. I would like to also ask for some more techniques that may help me in handling the method. This is totally new to me. I'll appreciate any help. Thanks so much.


-ghie malig-

[Mark Tracy]

Yes, excess mercaptan can lead to stability problems. The optimum ratio needs to be empirically established.

Most autosamplers use the "draw and dispense" approach to mixing. If the solvents for sample and reagent are too different, say acetonitrile and borate buffer, mixing will be poor. The mix-in-the-needle systems have real trouble with mismatched solvents. You may need more mixing cycles or faster dispense rates. Try reversing the order for picking up the sample and reagent. Or change your sample prep to have an aqueous end product.

[ghie malig]

Hello Sir!
thanks for your reply. Pls. be patient with me, I still have so much questions to ask. When I'm using on-line pre-column derivatization, I'm not getting repeatable area for my analytes. The analytes are dissolved in aqueous solutions. My sample loop is 50 uL. I'm not so sure if I need to use a pretreatment loop because considering that my sample loop capacity is only 50 uL, my mixing volume needs to be 100 uL and dispensing volume is also 100 uL. I tried lowering down the volumes of mixing and dispensing equal to that of my sample loop's capacity, but, I did not get any peak at all. I always use fresh OPA-MPA reagents and fresh solutions too. So I think the problem is not my reagent. Pls. help!!! Thanks!

-ghie malig-

[Mark Tracy]

As a general rule, you should not draw more than 75% of the loop capacity because you will lose some sample out the back of the loop. (You can find the actual limit for your instrument by a series of injections af increasing volumes.)

Another common trick is to draw up 2--5 µL of air before picking up the sample or reagent; when you dispense the air blows out the needle. When mixing, draw some air first, then mix, but don't blow out the air until the last dispense stroke.

Try this:
draw 5 µL air, 15 µL reagent, 2 µL air, 15 µL sample.
dispense 32 µL into the mixing vial
3 x 32 µL mixing cycles
blow out the air to a waste vial, and wash the needle in the rinse vials
wait for the reaction
inject 25 µL of mixture