ATP, ADP of Pancreatic Islets via HPLC

[ashwanth]

We have a method set up for measuring the [ATP]/[ADP] ratio via HPLC and it's been working well so far. I just came across a problem now. The ADP peak is very small, which makes the ATP/ADP ratio drive up. But there's a bigger peak a minute/minute and a half before the ADP peak. Could this be the real ADP peak? I run spikes so I know which peaks are ATP and ADP. Would you think that the standard ATP or ADP would have a different retention time as compared to the ATP and ADP from actual samples (cellular)? All this is to find out if the small peak which I think is ADP is the correct peak and it's not the bigger peak. Anyone come across this?

[Mark Tracy]

The only way that could happen is if your standard is mis-labeled or decomposed. Your spike experiment proves that the early, large peak is not the same chemical substance as the standard.

[ashwanth]

I do understand that. But could it be that the standard is a purer forms of ADP than what is present in the sample which would result in a different retention time?

[ym3142]

I run spikes so I know which peaks are ATP and ADP.

if this spike was the mixture of sample and ADP standard then your conclusion should be valid. So did you see the peak area increase for the ADP peak in the ADP standard and the sample mixture/

[ashwanth]

yes, the spike is the mixture of the sample and the standard..along side I run just a sample, so where ever I see spikes, I know there is the molecule I'm looking for in the sample. Since the peaks are usually really small, the peak area increases but not a lot. So that wouldn't really help me much. Unless I decrease the amount of standard drastically that the small amount is a big difference. Does anyone have a method set up for ATP, ADP, etc? I wouldn't mind trying other optimal methods.
Thanks!