Troobleshooting LCMS THC-COOH in urine

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

8 posts Page 1 of 1
Hi
My first post.
Hope someone can give me some tips on issue i have with LCMS analysis of THC-COOH in urine.
I am doing alkaline hydrolysis with ammoniumhydroxide 25% (60uL amhyd, 120 uL IS (THCA-d9 in water), 240 uL urine or spiked urine calibrator). So no SPE, dilute and shoot method.

I have done ion suppression and I had 40% suppression compaird to blank prepparation (ie water instead of urine) and THCA and THCAd9 behaves similar.

However when i spike THCA and IS to urine and check revovery agains blank prepparation THCA and IS do not behave similar between urines from different co-workes (subj healthy, meds and no meds, not using cannabis).
For exampel matrix X IS respons down 41% THCA only 17% and matrix P IS down 34% and THCA 27%.

Compairing the different urine matrixes to each other shows that for one matrix the IS can be higher but THCA can be lower than for an other urine matrix.

This affects the slope of my calibration curve. I have noted a slope difference of 30% which off course will affect my results proportionally and I get very high LOT to LOT variations.

Has anyone seen this effect or have any tips or ideas that could help me? SPE off cours could be one tip but trying to sett up method performed at several labs in Sweden.

Appologies for any misspellings or grammar errors since English is not my first language.

Sincerly
Pär
Welcome to the forum.

I don't have a lot of experience with urine analysis, but I can imagine differences in urine composition from different persons. I am not sure if you are looking at suppression or recovery differences here. If you want to look at suppression only, you should look at the differences in signal when the analytes / IS are spiked AFTER the sample preparation for both a blank and a matrix sample.

For a matrix effect, matrix has to co-elute. That's why an IS co-eluting with the analyte is so important in LCMS to correct for matrix effects. Your IS has 9 deuteriums, and the more deuteriums, the more tendency to elute earlier in RPLC than the corresponding analyte. If this retention difference is significant, this can explain the 30% difference in suppression between the analyte and the IS. I saw this behavior before using an IS with 8 deuteriums that was only 50% overlapping with the analyte. You could try to tweak the LC run to make them co-elute. Alternatively, if you have the sensitivity, you dilute everything (or inject a lower volume).

This seems to be a good reference for what you're doing.
I don't know for sure how deuterium behaves in alkaline solutions but I have had times in acidic solutions where the deuterium will be replaced by hydrogen so the recovery of the native analyte looks higher and the deuterated analog is low.

What purpose does the sodium hydroxide serve in the preparation?
The past is there to guide us into the future, not to dwell in.
Hi Rndirk
and thanks a lot for your tips.

With ion suppression I actually meant post column infusion. Sorry for the poor details.

You are completely right. Of course I should have spiked post preparation so no effects from the prepp it self would confuse me. I´ll do that straight away. Stupid of me.

Actually your previous experience is valid also for me. I don’t have 100% coelution of THCA and THCAd9. Tried to calculate it but tricky. Have approx. 70% overlapping area.

I`ll try your tips and read the article.
Hi James
and thanks for replying.
I´ll look in to this. I have also had some concerns of what’s happening in the vial.
There are several articles on alkaline hydrolysis of THCA-glucoronide.

I ‘am not using sodium hydroxide but ammonium hydroxide.
It has to be added to hydrolyze the THCA-glucuronide excreted in urine back to THCA. It is the first analyt of choice since it was historically hard to access THCA- glucuronide standards I think. Cut of values are based on THCA.
Förbrylium wrote:
Hi James
and thanks for replying.
I´ll look in to this. I have also had some concerns of what’s happening in the vial.
There are several articles on alkaline hydrolysis of THCA-glucoronide.

I ‘am not using sodium hydroxide but ammonium hydroxide.
It has to be added to hydrolyze the THCA-glucuronide excreted in urine back to THCA. It is the first analyt of choice since it was historically hard to access THCA- glucuronide standards I think. Cut of values are based on THCA.


Makes sense to me now, thanks.

Would it be possible to capture the target analytes on a solid phase extraction cartridge from the extract, wash with water to remove most of the matrix then elute with solvent for a cleaner sample to inject?
The past is there to guide us into the future, not to dwell in.
James_Ball wrote:
Förbrylium wrote:
Hi James
and thanks for replying.
I´ll look in to this. I have also had some concerns of what’s happening in the vial.
There are several articles on alkaline hydrolysis of THCA-glucoronide.

I ‘am not using sodium hydroxide but ammonium hydroxide.
It has to be added to hydrolyze the THCA-glucuronide excreted in urine back to THCA. It is the first analyt of choice since it was historically hard to access THCA- glucuronide standards I think. Cut of values are based on THCA.


Makes sense to me now, thanks.

Would it be possible to capture the target analytes on a solid phase extraction cartridge from the extract, wash with water to remove most of the matrix then elute with solvent for a cleaner sample to inject?



Yes absolutely SPE would be possible and there are applications on this. But at this time we are trying to get dilute and shoot working since others are doing it and we want to keep it simple. But of course if it just won’t work we will shift focus to SPE.
If alkaline hydrolysis would be the problem (deuterium - hydrogen exchange like James described), there's an alternative (and milder) method to hydrolyze glucuronide metabolites using enzymes. Here's some information.

I would suggest to start by looking if it's the sample preparation that's giving recovery differences, or it's really matrix effects due to co-elution with matrix.

One more thing: the article I mentioned earlier neutralizes the alkaline extract and adds organic solvent to precipitate and get rid of a bunch of stuff. Is this something that you do as well, or do you just inject the alkaline solution?
8 posts Page 1 of 1

Who is online

In total there are 14 users online :: 0 registered, 0 hidden and 14 guests (based on users active over the past 5 minutes)
Most users ever online was 599 on Tue Sep 18, 2018 9:27 am

Users browsing this forum: No registered users and 14 guests

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry