A.mphetamine Selectivity LCMS

Hi all, I recently encountered a problem with an LCMS screening method we have for forensic toxicology drugs of abuse in urine. I used the ForTox database supplied with our Agilent 6470 to generate my MRM database, currently at 220+ MRMs. Amongst these is Amphetamine where I used, 136/91.1, and 136/65, all very standard, and widely used MRMs for this compound. However, I am getting false positives for Amphetamine from the Urine samples I inject. I know they are false positives because (a) Amphetamine is being detected in our in-house urine blanks, (b) it's being detected in every single specimen I've tested, very unlikely, (c) it's being detected in external proficiency test samples which do not contain Amphetamine, (d) the tR is every so slightly different, 3.4mins instead of 3.3mins.


However, this interfering peak is a bit of a problem for us since it is a screening method we will end up having to confirm/reject every positive hit. I've tried to separate out the two compounds chromatographipally by providing a very gentle gradient in the affected region, but so far no success. I've had limited success by doing two things which might be of interest to people here. Firstly, I dropped the fragmentor voltage from 150V to 75V for all amphetamine MRMs, this had the effect of increasing the sensitivity of my actual Amphetamine peak dramatically. Interestingly it also seemed to create two species of the interferring peak, one at 3.4mins, but a much more intense peak at 3.5mins, which might be enough to provide the selectivity we'd like to see. Secondly, I added the 136/119 MRM which I had done before, but until I dropped the fragmentor voltage this 119 mass was not giving a strong response. So, ultimately, thanks to dropping the fragmentor voltage I end up with a squeaky clean 136/119 MRM, and a slightly "dirty" 136/91, and 136/65 set of MRMs, each with 3 peaks, , a significant peak at 3.3mins, a small peak at 3.4mins, and large peak at 3.5mins, the 3.3 min peak can be integrated in to ignore the others.


My question is, has anyone experienced analytes endogenous to urine that interferred with Amphetamine before, specifically the MRMs mentions above. What did it turn out to be, and how did you get around it?



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