Chromatography Forum

Hi,
I was wondering if anyone might be able to point me in the right direction on this. I have to analyze samples of rat tissue for a uronic acid by LC-ES-MS, negative mode. I am using a polystyrene-divinylbenze sulfonate column with 0.1% acetic acid mobile phase. I don't have an isotope available for use as an internal standard, and also I have not yet found a non-natural commercially available compound that would elute very closely. Can anyone recommend a specialty supplier that might have something that I could use? If I don't have any luck then how reliable is quantitation without an internal standard if you prepare standards in the sample matrix?
Any thoughts would be greatly appreciated...
Brent

If your internal standard doesn't coelute, it may not correct for ion suppression anyway, and only correct for autosampler variation (and possibly extraction efficiency depending on your terminology).

We also have to be careful about terminology. Some people spike a compound (similar as possible to the analyte) into the matrix prior to extraction, and call this the internal standard. Others call this the surrogate, and spike a separate internal standard into the final sample extract.

Without the former kind (surrogate), you won't be correcting for variations in your extraction. You could run some replicate recovery experiments to convince yourself that your extraction is reproducible. With some methods, I have seen very little variation in ISTD peak area. Meaning that if I reprocessed the data as external-standard, I would get substantially the same results. This is not the case with all methods, however.

I was thinking along the lines of a surrogate to correct for variations due to extraction. Actually it's more of a precipitation than an extraction. I think I may just have to get a compound as similar as possible while doing an external calibration since I can prepare standards in the sample matrices.

As MG said above you do not need an "internal" standard if you do not need to correct for the extraction/precipitation errors (this off course includes any errors due to pippeting/dilutions steps you might have); if the extraction yield remains constant I would say you do not need any inetrnal standard at all: either you have no suppresion or it is constant (unlikely). To check this you can spike an already extracted/precipitated blank solution and compare it with a solvent spiked (not extracted) standard.
If you just have to pp and inject, chances are that a surrogate standard is superfluous.
Regards
JNF