I am tasked with developing a method for determining enzyme activity of 2 transaminase enzymes using LCMS. The reaction principle is simple enough, load up substrate and coenzyme with sample (serum) --> monitor for set period --> quench reaction with high organic --> quantitate amount of product made or substrate disappearance to then calculate enzyme activity.

My question is, is it possible to directly monitor the transamination products (or disappearing substrate) given that the reaction is double arrowed? Also, most reference method use 'couple reactions' and monitor color changes over period of time for quantifying enzyme activity but some of these enzymes that are 'coupled' ALSO have a double arrow reaction. How can one confidently quantify enzyme activity for reversible enzyme reactions?!

Any thoughts or help would be greatly appreciated!