Tricyclics by UPLC/MS/MS

[mtorrey]

I am trying to set up a Tricyclic antidepressant method on a Waters Aquity UPLC/TQ and am having difficulty getting linear curves and wonder if anyone has set this up yet on UPLC/QQQ. All the free literature is for HPLC, very little of it use to me.

Right now I’m using a 2.1 x 100mm UPLC HSS T3 column, mobile phase is 2 mM ammonium acetate 0.1% FA (A) and Acetonitrile with 0.1% FA (B). If I do a 30:70 isocratic everything comes out in one peak. So I’m trying a gradient starting at 80:20 to 65:35 with flow rate of 0.6 mL/min. This was the gradient my clinical application specialist came up with, I’m afraid I’m quite ignorant when it comes to these gradients as I’m a GC man. All I know is temperature.

Anyway I cannot separate Imipramine from Nortriptyline (and nortriptyline D3) and for some reason the Imipramine peak dwarfs the Nortriptyline peak by a factor of 4 or 5. They (waters) tell me I don’t need separation because it’s MS/MS but from the reading I’ve been doing this is not good because of ion suppression which I think is going on here. So I’m wondering if there is a better column to use. One thing that is irritating is how much pressure it takes to push 80% water at 0.6 mL/min through this column. It is as high as 10,000. I’m afraid if I go to a 150 mm column I’ll go over the 15,000 psi limit.

Another question is I’m extracting my samples with Oasis MCX extraction cartridges. I’m wondering what strength aqueous phase should I reconstitute my samples after drying down the elute (80:20 or 65:35).

Sorry this is so long and hope it makes sense.

TIA
Mike

[Uwe Neue]

Mike,

There are several possibilities (I know people who have spend years of their lives trying to separate all the tricyclics. At acidic pH, nothing much will change, but if you go to neutral or alkaline pH, the pattern will change. This would change the relative retention of nortriptyline and imipramine. Your column is not the best choice for either of these approaches. You will need an ACQUITY UPLC BEH C18 column, if you choose this path.

You should reconstitute your sample in the starting composition of your gradient.

I do not think that a longer column will be the solution. I do not think that it will give you a sufficient advantage to solve the problem.

[mtorrey]

Thanks for the advice. I have a BEH I'm using for another procedure, I'll try an alkaline ph.

Mike