I performed 10 repeat injections and separations of a single urine sample on a reverse phase HPLC-DAD system. Everything was kept constant (Flow rate 1ml/min, column temp 35C, injection volume 5uL). I used an Agilent Eclipse XBD-C18 5um, 4.6*150mm column and Water with 1% formic acid and methanol as by mobile phases. With a post run of 3 minutes.
The sample clean-up only involved centrifugation at 10 000G for 10 minutes.
Is it possible that the salts present in urine can affect my retention times?
If so how should I go to work to resolve this ?
do you see the RT shift with change in M/Z value, if yes then you are right that salts interact with metabolites and one metabolite exist is two forms i.e. with and without salt.
0.2 minutes, may or may not be significant.
Here is a link to an article that may assist you.
"HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it"; https://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
How about an air bubble in the injector of the HPLC? Sometimes the priming of the needle hepls to stabilize the system.
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