Chromatography Forum

Dear Guys
i work recently on developing method for analysis of tramadol and his metabolites with acetaminophen in human plasma. i face a problem with autosampler stability although i use deteurated internal standard for all drugs especially for acetaminophen. my general note that the behavior of internal standard response differ from the drug.
the chromatography is the following
mobile phase 75% of 10mM ammonium acetate+0.05%FA :25 % ACN
column ACE 150*4.6 mm 5um
System: Quattro Premier MS/MS
is there any suggestions

I know dueterated internal standards should behave the same as the drug. If the drug response drops, the IS response should drop as well.
One reason that it may not is if your IS is not an exact "copy" of your analyte but differs a little in structure. I remember having a batch of Dueterated IS for a compound that they made incorrectly. The result was a IS compound with the correct mass but with a fragmentation pattern that did not make sense.

Do you use several reference solutions of fixed concentrations in pure solution spaced throughout your batch? Reference solutions prepared in the same solution as your samples?

Do you see the Drug/IS area ratio vary with time?
Do you see a trend (it goes up with time or down with time)?

Something you might do in your next batch:
You might want to inject a Standard curve of pure solutions only to see if something in the extracts is causing the problem. (not bio-extracts) .
Prepare a reference solution containing your analytes and Internal Standards. Something with concentrations at about the mid point of your analytical range.and distribute it to 20 or 30 vials. Space the references throughout your batch.
1. Does your curve in pure solutions work?
2. Do the ratios of analytes/IS remain more or less constant through the run (Try peak areas if peak heights are a problem).

What are your samples prepared in? Acidic solution?
Have you tried non-acidic, just plain organic/water?

Alp