Chromatography Forum

Hi everyone, I would really be grateful if you could throw some light on this issue.

I am currently trying to add canabis (THC and its metabolite THC-COOH) into an existing method I have for drugs of abuse. All my compounds elute before 7.2 minutes on a C8 column. When I inject THC and THC-COOH in mrm mode they elute at approx 8.2 minutes each but peak shape is very broad and splitting. I inject samples of human urine 1/20 diluted with methanol:water (50:50)

My gradient is as follows:
min Flow rate ul/min A % B%
0 1000 95 5
1.5 1000 95 5
6.0 1000 5 95
8.0 1000 5 95
8.1 1000 0 100
10 1000 0 100
10.5 1000 95 5
12.5 1000 95 5

I would be grateful if you could give me advice on where to proceed without having to choose another column. The analysis is carried out on an applied biosystems QTRAP doing mrm to epi experiments in positive mode.

Mobile A=1900:100 (Water:methanol +25 mM ammonium acetate)
Mobile B = 1959:40:1( Methanol: 2-Propanol: Formic Acid)

What is the simpliest way to improve peak shape based on above?

Many thanks in advance
Emiedoo

Below is an application for LC-MS/MS (3200 Q Trap)
of THC and THC-COOH using Unison US-C18:

http://www.silvertonesciences.com/files/TI338E.pdf

You can try those same experimental conditions.

If you're trying to add THC and metabolite to a method that is also quantitating or looking for other drugs at the same time, the peak splitting would be difficult to solve.

Without changing the column, your options would be to modify the chromatography (ie. shallower gradient across longer time, or mobile phase) or the way you dilute your sample.

I would start by acidifying mobile phase A...

Try using 0.1% Formic Acid in DI Water and 0.1% Formic Acid in Acetonitrile as your mobile phases. Also, try a C18 column.

We have a couple of QTraps, but do not use them for THC analysis. Our blood THC and metabolite analysis is done on a Waters Quattro Premier XE. Urine is still on a GC/MS but will be switching to UPLC/MS/MS soon.

And the advice from above regarding adding THC and THC-COOH to an existing method with other drugs is spot on. It is probably not the best to include these analytes in analyses with other drugs. It is better to make a more specific method for the particular analytes. THC/THC-COOH are very sticky compounds and behave differently than alot of the other drugs of abuse.

I would suggest to make a isocraic step a little bit lower than 5/95, and deffinately the pH of the eluents should be as low as possible (use the same pH modifier);
Also using 2- propanol in the eluent makes it more complicated - try without it.
The peak shape is also dependant on the solvent you use for disolving the sample at the point of injection - try to be closer to the initial eluent composition.

K.I.Z engineer

Since the pKa of THC is probably greater than 9, I'm not sure if acidifying mobile phase A would make much difference. It would also probably cause significant changes in the chromatography of the other compounds. Your problem is likely caused by using an injection solvent that is much stronger than your initial mobile phase. Try injecting a smaller volume and see if that helps. If it helps, then the problem could be solved by using a lower percentage of methanol in your injection solvent and/or injecting a smaller volume. If it doesn't help, then you will have to make changes in your mobile phase. Acidifying solvent A is a good start. It seems kind of strange to have ammonium acetate in A and formic acid in B.

I think you're better off developing a fast 2-3 minute method separately to quantify THC (as in the paper above) on top of the 12.5 minute run you're doing for the other drugs.

Why do you dilute with methanol water 1:1? Just dilute with water and your peaks will be fine...

I think that THC is totaly insoluble in water. it would be realy nice to be able to to disolve the extract only in water, but it would definately affect the quantification of the analysis.

If it is "totally insoluble" in water, how do you think you will be able to find it in urine?

okay You're right, sory: poorly soluble

Injecting the sample in methanol:water 1:1 is giving you problems with the peak shape and prevents you from getting quantitation results. Injecting in water/buffer will give you normal peaks, and no problems with quantitation.
Try it...