Chromatography Forum

I am analysing rat plasma samples for SN38 by LC-MS using protein precipitation with ACN (3× plasma volume, CPT as IS) on a C18 column. The method works fine for calibration (LOQ 0.1 ng/mL, linear range) and used to work for unknown plasma samples as well.

Now, unknown samples show multiple peaks (1.2, 2.6, 2.8 min) instead of a single sharp SN38 peak at 2.8 min. This does not occur with spiked plasma standards. Acidification to favour the lactone form only improves it slightly.

Has anyone encountered this issue before?

Thanks,
Ali

My first thought is that your current plasma samples are from a different rat population or species, and hence have a different metabolic profile.

Can you provide details on the same/different UV and [M+H]+ or MRM transitions for the three peaks that are detected?

Regards,
JMB

edit: Is there any evidence of ring-opening?

Hello JMB,

Yes, they are from a different rat population. All peaks are detected at the 393.1 → 349.1 transition. I suspect an in-source carboxylate-to-lactone transition (by losing a water molecule) or deglucuronidation. However, in many published papers measuring SN38 plasma levels in animal or human samples, such issues or any measures to prevent them are not described in the methods.

Thanks,
Ali

Are you sure it's not just some other chemical altogether? Loss of 44 isn't a particularly uncommon loss from many molecules, and it's a small molecule where there are likely to be large numbers of near-isobaric things. I find most of my new MRMs have multiple peaks in chromatograms in real samples. That's one of the reasons why chromatography is so very useful!