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Dear all,
I am facing a typical problem wherein I am observing change in concentration with increase in
dilution in urine.
My queries are
what are the factors influencing for this type of change?
what steps can be taken to resolve this problem?
for a given linearity how many folds samples can be diluted?
Does anybody faced this type of problem, if so please guide me in this regard
Regards
Sreekanth Dittakavi
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Sreekanth:
I presume you are seeing a non-linear change in concentration with urine dilution. This is a common
problem usually having to do with the aqueous solubility of your analyte in urine (an aqueous salt
matrix), analyte salting out and /or the composition of the glass/plasticware you use in processing
the sample. Adding a solublizer to the vessal the urine is collected in usually works. Some folks
use plasma or albumin. I recommend doing an experiment to show recovery across the concentration
ranges you will be working in.
Chris
Christopher J. Kemper, Ph.D.
Pharma Navigators, LLC
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If you LC-MS for detection & quantitation you'll need to check for ion suppression (in your case).
What do you use to dilute?
Best regards,
Frederik
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Dear Sreekanth,
It would help to know what kind of change in concentration occurs. Does it increase/decrease?
Also, how is the dilution performed? Using urine, using a solvent, how many steps, is there a trend
etc.
My next advice may help if the dilution is done using urine or aqueous solvent for dilution and if
the concentration in the diluted sample is lower than expected. If you haven't already done so, you
may want to verify if there is any non-specific binding. The compound could retain on anything,
from tips to storage containers and their caps. Each one of these needs to be tested separately.
For instance, in a test container, prepare a sample in urine at a concentration around the middle of
the curve. Using the same tip, aliquot 6x a small volume in an injection plate where the same
volume of organic solvent was added (higher than 1/1 urine/organic may not be 100% miscible).
Transfer the entire liquid from the test container to an identical test container and vortex-mix for
30 seconds. Transfer again to another identical test container and vortex-mix for 30 seconds.
Using the same tip used previously (which should be "saturated" at this point), aliquot 6x as
described above. The first aliquot is not reliable and the result can be ignored. Repeat again the
transfer 2x to generate a third point. If the measured response in the 3x6 aliquots shows a
downward trend, then non-specific binding can be suspected. Loss due to non-specific binding is
proportional to the ratio volume of liquid / surface of container. Filling no more than 10% of the
container's volume will help isolate this problem. If that is the problem, there are many
publications that show how it can be solved. This should be done starting with the collection
containers. Sometimes glass behaves better than plastic. If one type of material doesn't show
non-specific binding, let's say HDPE, please keep in mind that not all HDPE containers will work for
sure. Once a type of container is found to work without having to add any other substance, it should
be ordered from the same manufacturer as the one tested.
Another possibility could be the difference in miscibility between urine and some of the solvents
used during sample preparation (if applicable) from lot to lot of urine. For instance,
urine:acetonitrile 1:5 could be a homogeneous mixture for some lots, or create 2 distinct layers for
other lots. The compound of interest could distribute between the layers and depending on the depth
at which the tip or the injection needle reaches, it may aspirate different concentrations.
There is no limitation of how many folds samples can be diluted, as long as accuracy and precision
are demonstrated.
Good luck!
Ileana
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