I have been collecting urine from mice to analyze certain metabolites, but on my latest attempt, I'm getting huge variation in my IS.
Does anyone with some experience in HPLC-MS have any suggestions as to what might be going on, and how I can correct the problem?
Here's my procedure:
Collect urine, dilute 5x with ddH2O to bring up volume (sometimes the mice just don't need to go!), take 40 µL sample and combine with 10 µL Salicylic Acid and 4 µL of deuterated cystathionine (IS) (4.4 mM). Centrifuge sample to pellet precipitate and transfer supernatant to an autosampler vial. I don't run the instrument myself, I just hand over the samples at this point to be run, so I'm afraid I don't know much about the LC-MS protocol.
When I got my results, the variability in IS Area (deuterated cystathionine), was very high (in some cases 20 fold) and there was also some variability in retention time (0.15 min between the extremes).
I don't know how to account for this, or what to do about it. (It has been suggested that my IS may have been binding to proteins in the urine which were then precipitated?)
Any ideas or suggestions would be greatly appreciated!
Cheers
Hi Michael.
You did'nt explain your chromatographics conditions. Your problem could be ionic supression or with chromatographic conditions. You could verify ionic supression running a blank matrix and make direct standard or internal standard infusion, you should use a T conection.
I hope this help you
No HPLC or LC/MS info was provided so there is no way we can comment on that. Too many things come to mind which can cause variations and if you are not the person trained in how to analyze the samples, then no point going there for now.
First, you need to characterize the data (the observed problem). Is it caused by poor quality methods and analysis or is it a result of the matrix, absorption or something else? To answer the questions, let us start with the basics. Can you submit samples prepared the same way, but without the mouse urine? IOW: Can you submit controls with and without the IST present? That would at least provide you with a control to evaluate. * I would forget about analyzing the samples for now and instead resolve any observed problems with blanks and controls.
Hi,
in addition to what Bill Letter says you could try also in the previous processes, that is to say the extraction. Urine has a big amount of salts that interfere with ionization. So you could try to desalt the sample in order to reduce the ionic strength. As well you could try sonication afterwards to break up potential protein clotting. Nonetheless I do not see any step in your sample preparation method for protein precipitation.
Hope this will be useful
I don't think you can subcontract an analysis like that. At least, someone should be in a position to write up the complete procedure with information on the analytical development.
The problem is likely to be due to the matrix (salts, urea...), though there should not be much protein in urine (Olimpio's response). I have seen cases where people have been trying to measure peaks that are insufficiently retained on the column, so there is basically very little chromatographic separation.
An approach to reducing sample matrix problems that some of us have been talking about for years is to use LC column switching routinely, without even asking if it is necessary in a particular case.
Finally, judging from the amount of internal standard, you should not need MS here; any of the standard LC methods for amino acids should do. Sometimes MS is just convenient because the same instrumentation can handle a wide range of situations; if that is your case, it may be sufficient to greatly dilute the specimens, though I don't know why you need salicylic acid or at what concentration.
As stated above, running of quality controls, blanks and standard in this can help to point out problematic area
Many thanks for all of your responses.
Update: We have successfully measured blanks and various concentrations of IS control (no urine). This would seem to indicate that the variability is in some way caused by the urine matrix. The addition of salicylic acid caused a precipitate, which I assume consisted of some salts and proteins. I am trying some different methods of "cleaning up" the samples before running, to see if this yields better results. Thanks again for your suggestions and insights, I've learned a lot from tackling this problem.
Interesting note; mouse urine, unlike normal human urine, contains high protein levels. See: The Composition Of The Urine Of White Mice, I. A. Parfentjev, William A. Perlzweig, J. Biol. Chem. 1933, 100:551-555.
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