LC/MS/MS Analysis of Synthetic Cannabinoid Metabolites in Urine – The Saga Begins

In my last post back in February, I outlined an LC/MS/MS method for synthetic cannabinoids in herbal incense, and received some great comments. A couple of the comments were inquiries about a method for synthetic cannabinoid metabolites, and I promised to post a blog about a metabolites method. After much labor and head-scratching, and with the help of my colleagues Jack Cochran and Ty Kahler, as well as some valuable input from Paul Kennedy at Cayman Chemical, at long last, here is the first of several posts on Restek’s work with synthetic cannabinoid metabolites. Since a blog is not the format for presenting in-depth analytical data, the posts about this method will be purposed to discuss the method development process and the interesting results I have seen so far. A formal applications note will be published on this method soon, but in the spirit of staying current, I’d like to start some discussion on this evolving method now. In other words, I’m asking you, my fellow chromatographers to once again weigh in with more great comments. If you’re feeling a little shy (and that’s okay!), feel free to contact me directly by clicking on my name at the top of the post for all the information I have currently for this method. I intentionally excluded some details from this post because although initial results look promising, the method is truly a work in progress, and I feel that I should discuss its implementation in depth with anyone who wants to try it rather than just chuck it over the wall for public consumption. That being said, I would be more than happy to assist with the implementation of this method in a ‘real’ lab to put it through its paces with a larger body of samples. 

I feel that the title for this post is fitting, because the development of this method has become a saga that began in November with the development of an initial chromatographic method for metabolites of JWH-018 and JWH-073. The saga picked up again a few weeks ago when I was able to get my hands on some authentic samples to test with my method. Without boring you to tears with the details, in short, I ended up with a final method similar to the one that I initially developed in November, but with an SPE extraction step included, and a change in mobile phase. Figure 1 shows a 10 ng/mL calibrator in urine, and Figure 2 shows chromatograms from two patient samples.  

Probably one of the first things you’ll notice in the figures below is that the chromatograms for the patient samples end at about 2.6 minutes, as opposed to 8.5 minutes for the standard. This is because the only metabolites I observed from the several patient samples I analyzed were JWH-018 N-hydroxypentyl, JWH-018 N-pentanoic acid, and JWH-073 N-butanoic acid, which elute early, and nobody wants to look at 6 minutes of empty chromatogram. I also observed at least one, and possibly two suspected unknown metabolites. One of the obvious unknowns is noted in Figure 2. The specified unknown peak appears at varying levels in all of the positive samples, but is not present at any detectable level in 5 blank controls. I suppose now would be a good time to thank the 4 employees at Restek who supported the development of this method by providing me with control samples when I approached them unannounced with an empty sample container. Further exploration of these unknown metabolites will be a future post unto itself.

 

 

Since all of the observed known and unknown metabolites that appear at significant levels in authentic samples elute very early and with little resolution between them, they lend themselves nicely to an optimized method specific to these compounds. Although the method can be optimized, my initial LOD, linearity, and QC results for all of the metabolites in this post are pretty good, if I do say so myself (LOQ = 1ng/mL, S/N for quant MRM > 50 at LOQ, > 15:1 S/N for first qual MRMs, > 3:1 S/N for second qual MRMs, r > 0.9990). Since I’m not a toxicologist, and my results from authentic samples are very limited, I wanted to share a method for a more comprehensive panel of metabolites before presenting an optimized method in the hopes that some toxicologists and fellow chromatographers will add their expertise to the discussion.

As I said earlier, this blog is a great way for chemists here at Restek to share our most current chromatographic knowledge, but the exchange of information can go both ways. Through feedback from readers who work in a ‘real life’ production and method development setting, we here at Restek can potentially develop better methods to serve the needs of  a production laboratory. For those of you who would like to post a comment or question, don’t be surprised that your post doesn’t appear immediately on the blog. Due to incessant spamming, all incoming comments have to be approved before they are posted live. That being said, I’ll approve any comment -positive or negative – from any reader who would like to discuss this method, so fire away!

3 Responses to “LC/MS/MS Analysis of Synthetic Cannabinoid Metabolites in Urine – The Saga Begins”

  1. Michael Herrera says:

    Amanda,

    Thank you for the blog post. I am interested in methodology as well. Did you look at any techniques for hydrolysis of the sample to cleave any glucuronides? Particularly interested as you didn’t mention seeing any of the hydroxyindoles in patient samples and wondering if they were tied up in glucuronides.

    Regards,

    Mike

  2. Hi Mike,

    That’s a good question! I did hydrolyze my samples prior to extraction. The method I used was as follows:

    – Add 1mL solution of beta-glucuronidase from keyhole limpet (Sigma-Aldrich cat# G8132) to 1mL urine. Solution is prepared at a concentration of 5000 Fishman units/mL in 100mM ammonium acetate buffer (pH = 5.0)
    – Incubate at 60°C for 3 hours

    This hydrolysis method works pretty well for other applications, but since there are no glucuronide standards out there, I couldn’t test the hydrolysis efficiency. That being said, I would think that both the hydroxyalkyls and the hydroxyindoles would be conjugated, and I was able to detect the hydroxyalkyls.

    I tried some neutral loss experiments to see if I could directly determine the glucuronides, but I had no luck and went ahead with the rest of my method development. That’s actually something I want to revisit in the future.

    Also stay tuned for a follow-up post containing the complete details of this method. I told myself I’d write it today, but I’m a master procrastinator, so it will probably be posted tomorrow.

    Thanks again!

  3. […] The Restek interest in these compounds is analytical, and my colleague Amanda Rigdon has done some very good LC-MS/MS work on the analysis of synthetic cannabis and its metabolites in urine. […]

Leave a Reply