aldehyde quntitation..hydrazone formation? - (Jul/20/2007 )
Hi all,
I am trying to monitor aldehye to alcoholreduction in my assay but because my aldehyde alredy has some alcohol contamination I need to quantitate either. Does anybody know of a reaction to quantitate aldehyde or alcohol. I know that aldehydes form UV absorbing hydarazone with 2,4 Dinitrophenylhydrazine but cannot figure out how to set up this . Plz help
DNPH is used to analyse keto acids in urine. One of the interferants is aldehyde. Heating eliminates this interference. Presence of keto acid is seen by a yellowish precipitate. This can be measured as a change in turbidity. You want to measure the aldehyde, though, so you need to blank out any keto reaction unless you can be sure your samples aren't from ketotic subjects. You could "fix" the aldehyde using tris hydroxymethyle aminomethane. Then test for keto acids and subtract from total leaving you the aldehyde contribution.
As for the alcohol issue. Usually an enzymatic method is used to measure the formation of aldehyde from alcohol. Alcohol dehydrogenase is the enzyme and since the reaction proceeds via NAD to NADH conversion it can be monitored spectroscopically at 340nm. Usually, its the forward reaction that interests toxicology labs but I wnder if you would focus on the reverse reaction. Use NADH in acidic conditions and you should drive the reaction from aldehyde to alcohol. You'll need a solution blank and a set of standards made up with known concentrations.
You can buy kits for this assay that can be done manually or on autoanalysers (as in clinical labs). If you had access to and loads of money to throw at the analysis, chuck everything into a GC set up.
Hope that helps. Looks a load of fun.
Hey
Thanx for replying but ketoacids??? My reaction is aldehyde to alcohol and I need to quantitate either.. I will be using cellextract or purified protein and since my substrate will be provided in excess even if there is some keto acid background it sholuld be low. ive already done NADPH assay reduction assay. HOW ABOUT STAINING tlc WITH EXTRACTED PRODUCT WITH dnph...iF THE ADDUCT IS YELLOW i CAN CONFIRM MY nadph RESULT WITH A UV based assay for this adduct formation decrease. DO you know how to set it up ..any reference?
As for the alcohol issue. Usually an enzymatic method is used to measure the formation of aldehyde from alcohol. Alcohol dehydrogenase is the enzyme and since the reaction proceeds via NAD to NADH conversion it can be monitored spectroscopically at 340nm. Usually, its the forward reaction that interests toxicology labs but I wnder if you would focus on the reverse reaction. Use NADH in acidic conditions and you should drive the reaction from aldehyde to alcohol. You'll need a solution blank and a set of standards made up with known concentrations.
You can buy kits for this assay that can be done manually or on autoanalysers (as in clinical labs). If you had access to and loads of money to throw at the analysis, chuck everything into a GC set up.
Hope that helps. Looks a load of fun.
Yes, a bit obscure I know but that's what I know DNPH is used for (that and ascorbic acid). The keto acids cause a condition called Maple Syrup Usine Disease (MSUD). I suppose its the other side of the coin that you are working on: aldehydes by DNPH showing interference from keto acids whereas we watch for aldehyde interference in analysis of keto acids.
The method notes I have use 0.4g/dL DNPH in HCl (1mol/L). Mix with equal quantities of sample. Wait 5 mins and read absorbance. These notes are for the keto acid analysis, though, so you may still need to tweek things for aldehyde.