Chromatography Forum

Hi,

I am working with a waters ACQUITY UPLC-TQD and am having issues with the consistency of the ratios between the quantifier and qualifier MRMs. The method I am working on is an opiates/opioids quant method that is analysing 41 different drugs, each drug has three transitions monitored (with the exception of two drugs).

The main problem that I am having is with 6-monoacetylmorphine (6-MAM) where the ratios of the quant transition and the two qualifier transitions are seemingly random and there is no consistency what-so-ever. Our tolerance is +- 20%.

The way that I have been measuring this is to do 5 injections each of a low, medium and high solution then taking the median and testing to make sure all of the ratios fall within 20% of this number. I am able to achieve acceptable results for most of the drugs in my assay by removing the results from the low solution but 6-MAM, morphine-3-glucuronide and morphine-6-glucuronide have issues with ion ratios at each of the three different concentrations.

I have tried removing 30 of the drugs from the assay to see if it is a case of asking the instrument to do more than it is capable of but this has resulted in very similar results.

Has anyone else had issues like this or know of a way to overcome these issues?

Any help would be greatly appreciated.

Thanks,

Jo

Hello Joanne,

My field of work is mostly the analysis of residues of veterinary drugs in animal tissues and urine. I use a Waters Acquity-TQD system.

I usually encounter that type of problems with MRM ratios. I don’t think it is a matter of too many transitions in the time segment of the MSFile; I have suffered from those issues with just two transitions in the time segment. In my experience they are very analyte/matrix dependent, although concentration is important too (in general the higher it is the fewer problems you’ll have). A strong sample work up (e.g. SPE) should help.

Another complementary approach is the use of a spiked sample as a “reference” sample for the MRM ratios. This sample would be affected by the same matrix effects as your sample so that the ratios should be more realistic.

In my field, under current EU legislation, 20 % tolerance is used for MRM ratios of 50% intensity (qualifier/quantifier) and over. For lower ratio intensities the tolerance is increased, up to 50% when the intensity is 10 % or lower. This is also the case in pesticide analysis. Maybe you can revise your target tolerance.

Hello Carras,

Sorry I took so long to reply. I did not know that I wouldn't receive notification of a reply (my bad). Thank you for your help. It is very useful to know that there is a greater tolerance in other fields for transitions with intensities lower than 10%. This makes it easier to justify relaxing the tolerance somewhat.

The solutions that I have been running have been pure solutions so no matrix effects should be encountered. I have found that the higher the concentration, the more consistent the ratios which makes me think it might be a sensitivity issue with the instrument. I have been experimenting with different MS methods and have found that if I run a method looking for 6MAM alone, the ion ratios are a lot more consistent which could be due to the fact that dwell times can be optimised and the instrument isn't trying to look for so many other transitions at the same time.

I think we may decide to use two different methods, one for quanting 6MAM and one for screening (this one will have a lot more relaxed criteria)

Thanks again,

Jo

I do lcms analysis similar to what you do.

It is normal to sometimes see a concentration dependence of the ratio, because different ions can experience varying amounts of matrix supression or even supression by other concentrated analytes in the sample if they start becoming significantly concentrated.

Its not super common, but I would say that I see a concentration variation in 2 or 3 out of 100 drugs that we do, but then again matrix effects are a significant factor in my case. Are there any other isobaric compounds to 6mam in your sample? do you see this ratio variation if you change the chromatography?

May I ask what transitions you are using for 6mam? have you tried alternative ones if this variation really bothers you,

Hi Peter,

Oxycodone, oxycodone-d6 and 6MAM-d6 all come out at approximately the same retention time as 6MAM. I have not tried changing the chromatography yet but it will be something I will try.

The transitions that I am using for 6MAM are 328.22>43.12 (Q1), 328.22>58.02 (Q2) and 328.22>165.1 (Q3). I have not tried any other transitions as yet. I will need to speak to my supervisor about changing them as he may not like the idea of not using the transitions that the instrument has optimised.

Thanks,

Jo

True.

I also work with a LC-MS/MS from Waters and sometimes I don't see the same ratio in a sample as in the fortified sample simply because they have too different concentrations.

If I simply dilute the sample into the concentration of the fortified sample, usually ion ratio check out just fine.

Not always do I see this effect though. But when I see it, getting the two at the same concentration works.