Sample preparation for urine THC-COOH in GC/MS

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

6 posts Page 1 of 1
Good morning everyone.
First I would like to emphasize that the world of chromatography is quite recent for me ( 1 year).

I would like to analyze THC-COOH in urine with a GC/MS but I have some issues below 100 ug/L. I can detect THC-COOH > 100 ug/L ( according to the immunoassay) but nothing below in urine samples.

All the solutions are new and analytical grade.

To explain this lack of sensitivity, I suspect an issu with my sample preparation or the injection.
Since I am not an expert at all in this field, I hope someone could tell me what am I doing wrong.

Here is my sample preparation:

1 mL urine
Add 125 uL KOH 12N and heat 15 min at 60°C for the hydrolysis
Cool the sample and add 450 uL of acetic acid in order to obtain a pH 4-5
Add 4 mL 1-chlorobutane
Vortex 2 minutes and centrifuge
Collect the organic phase into a 5 mL glass tube without taking any of the aqueous phase
Evaporate to dryness at 37°C under slow nitrogen flow

Residue is resuspended into 75 uL of BSTFA ( 1%TMCS) and heated at 60°C, 30 min for derivatization.

The solution is eventually transfered into a GCMS vial and 2 uL are injected into the GC/MS.

More informations:
GC: Agilent 7890 and MS: Agilent 5977
Column: HP-MS
Injection temperature: 250°C
Injection mode: splitless

The aquisition is not made in SIM mode but SCAN mode.
EM setting is set on gain factor
Autotune was recently made and liner changed.

Any help would be greatly appreciated :)
First, welcome to to the forum and thank you for providing plenty of information.

May I make the following observations

1. Someone on the forum will have far more experience in this analysis than me

2.. Someone please check my maths/arithmetic :-)

100 ug/L is 0.1ppm, or 0.1ug/ml or 100ng/ml
At this level 1ml of urine will contain 0.1ug or 100ng
After concentrating from 1ml to 75ul and with some very! approximations on derivative weight then you will have 1000ng of derivative in 100uL
Therefore in 2uL you will be injecting very approximately 20ng of derivative in splitless mode

3. was the liner change part of your troubleshooting or have you been previously successful with standards below 100ug/L.

4. Why are you not using SIM for detecting below this level of 100 ug/L?

From a quick Google search Ions should be for this derivative of THCCOOH m/z 473, 371, 488 ... bks067.pdf

5. Have you tried standard addition?

Dear Gom,
Thank you so much for the respond.

I forgot to mention that I'm trying to include this THC-COOH into a drug screening, that is why I want to stay in SCAN mode if possible.

According to the immunoassay, the concentration is 100 ng/mL.
Taking 1 mL of urine and if the LLE is 100% efficient ( which is probably not), I should obtain after evaporation 100 ng / 75 uL = 133 ng/ 100 uL and therefore approx. 2 ng for 2 uL.

Liner was never part of troubleshooting, it was just to mention that it was probably not an issue

4) I was so focused on the scan mode for screening purposes that I didn't consider it. According to your experience, is that possible to detect this compound < 100 ug/L using the SCAN mode or only the SIM mode would have the necessary sensitivity?
However, I am going to try the SIM mode. Thanks

Here is the thing about standards. I actually don't have any because I cannot buy them without special autorization for cannabinoids (this is on process).

By the way, don't hesitate to correct me if anything is wrong =)
You are quite correct.

I made an error :oops: and yes you are injecting approximately 2ng

Thank you for spotting that.

I have no experience of drugs of misuse analysis, I was rather hoping that others may have..

It would be interesting to hear how you get on with SIM. My feeling is that this is the way to go at levels below 100 ug/L

You imply that you can detect > 100 ug/L so possibly check and confirm the major identifier ions for SIM.

As for the the other target analytes, you can change your SIM acquisition during the run. It depends on what they are and if you know their retention times. (I seem to recall that you can even alternate between SIM and scan during the run with a slightly reduced sensitivity)

For quantitative analysis you will need standards

Do you any way of verifying the results from the immunoassay ?, if they are biased high you could be injecting much less than you think.

Do you get a linear peak are vs quantity plot above your 100 ng/ml ?

Peter Apps
I think it works! I still have to check if I properly handle the programm in SIM mode but it seems to work :)

I actually have no way of verifying the immunoassay ( since I am doing a GCMS qualitative screening and I cannot buy standards for the moment). However, I don't think result could change from single to double, even thout it is semi-quantitative immunoassay

In Scan Mode, I don't really have nice peaks but this is sufficient for the programm (thanks to the deconvolusion) to detect with high confidence THC-COOH ( when > 100 ug/L).

However, I am still wondering if I could go < 100 ug/L in scan mode.
That's something I am going to check if I get the pure substance one day or if an experimented GCMS drug screener could enlighten me =)
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