Wednesday, April 13, 2016

How low can you go? LOD/LOQ for a Q Exactive PRMs!

Y'all have this unfortunately accurate level of insight into how my brain works. Start off with a cool paper that pushes the limits of detection for absolute quan in a Q Exactive, move to a limbo analogy, find out David Hasselhoff made an album called "Do the Limbo Dance" (probably only released for Germany...but I refuse to investigate further)!

The paper in question is by M Concheiro et al., and you can find it at PubMed here.  In this study these researchers are working on super low-level quantification for a deadly illegal drug in oral fluid. Turns out that previous detection methods yield a lot of false positives as even being in the same room with people using this nefarious substance can cause a person to test positive for it using the classical assays.

What's the solution? A better target AND more resolution, of course!

So these researchers work up a better method using the Q Exactive and targeted MS2 (which we now call Parallel Reaction Monitoring or PRM). They optimize the method by injecting controls into their complex matrix (oral fluid).

To get the best sensitivity (while maintaining robustness) they use microflow separation (looks like 20uL/min flow rates.

How'd they do? Pretty good. They find that the Q Exactive was linear for one of these compounds down to 15picograms/mL and they had CVs at all levels in the 10% range.

I investigated this one a little personally, cause I generally consider Q Exactive PRM sensitivity to be better than this. I've got a couple compounds I've gotten down to 2 picograms/mL with standard flow. One reason their numbers are lower than mine is that they had to work in negative mode here. Okay. That makes 15 seem pretty good. I haven't done a ton of QE negative quan, but on my old QTrap, I'd expect at least a 1-2 log drop if I had to go to that evil negative polarity. That makes sense to me.

Secondly (that's a word?), I think this group undercut themselves a little. If you look at the peaks, they are somewhere in the 15-20 second range at base. I heard from another researcher who had access to this QEs method that a maximum injection time for the PRM was 120ms.

When I'm trying to show of Orbitrap sensitivity I crank that maximum fill time up as high as I can get away with. If I assume a 15 second peak, I'd almost increase that injection time 10x. at 1200ms injection time I'd still get 12+ scans across the peak which meets the Case Western requirements for label free quan (the gold standard in my mind) and I'd be able to get a bunch more ions. I can't say that the sensitivity would go up 10x here, but I guarantee it wouldn't decrease.

Anywho, this is a nice paper showing 1) the benefits of microflow for sensitivity and 2) That you can go real low in small molecule detection and quan in matrix with a Q Exactive, so its a solid win in my book!  I leave you in the capable hands of THE HOFF.


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