I have conducted a study on the fructose, glucose, and sucrose compound content in tomato flesh extracts. The obtained peak areas aligned with the chromatogram graph. However, the concentrations calculated using the linear equation produced gave a contradictory results. All method validation parameters showed positive results except for the actual sample concentrations. I am puzzled by this discrepancy and would greatly appreciate any explanation. All three regression from standard curve is near to 1 (0.98-0.99).
Hi,
It is very important to have the exact weights of the samples and the appropriate amount of extraction buffer. I would check if the standards for glucose, fructose and sucrose and see if they also run weirdly and give signals with different intensity. If that turns out to be the case, I would check the protocol. If everything proves correct, keep in mind that variability is very large with sugars. it would help if you compare the exact same part of the tomato flesh and avoid introducing any biases. Harvest samples at the same time if possible and don't let them lose water, etc. Even with all the precautions you would need to allow for 6-8 biological replicates for each type of sample you are investigating. Hope that helps!
Good luck!
Svetlana
Perhaps, you can recall the temperature in each experimental step, and the evaporative light scattering detector is relatively sensitive for temperature change.