Comparison of Phencylidine and Prazepam as Internal Standards in Medical Cannabis Potency Analysis with GC-FID

Recently in the ChromaBLOGraphy posts below I proposed the use of Phencylidine (PCP) as an internal standard (ISTD) for cannabinoids analysis with GC-FID when using the Rxi-35Sil MS GC column.  I demonstrated that the RSD% of Average Response Factors (Avg RFs) and Correlation Coefficients (CCs) for the calibration curves generated using the ISTD technique were excellent, and that ISTD calibration/quantification could reduce quantitative error associated with sample introduction into the GC.

Possible Internal Standards for Medical Cannabis Potency Testing by GC

Calibration Curves for Cannabinoids Based on PCP Internal Standard – Medical Cannabis GC-FID

Internal Standard versus External Standard Quantification in Medical Cannabis Potency Analysis with GC-FID

You may remember that in addition to PCP, I added Prazepam to each calibration standard (see chromatogram below).  Prazepam worked about as well as PCP as an ISTD, with slightly larger RSD% values for select cannabinoids and very slightly lower CCs (see table below).  The absolute RF differences for cannabinoids using ISTDs PCP or Prazepam are explained simply by having larger peak areas for PCP versus Prazepam, a function of the better FID response for PCP.

So…should we use two internal standards for this work?  We could.  One strategy is to use PCP as an ISTD for earlier eluting cannabinoids (e.g., CBDV, THCV, CBC, CBD) and Prazepam for later eluting cannabinoids (e.g., delta-8-THC, delta-9-THC, CBN, CBG).  Why?  It’s possible that PCP response is more representative for the more volatile cannabinoids (earlier eluters) and that Prazepam is better for the less volatile compounds, although the current data set is not definitive in that regard.

My preference at this point?  Consider PCP as an internal standard for all the cannabinoids and Prazepam as a potential indicator for determining when to change the GC inlet liner.  Prazepam, as the last eluting compound and potentially the least volatile, should be an early indicator of non-volatile “dirt”, like chlorophyll,  building up over repeated cannabis extract injections and eventually leading to poor quantitative transfer of cannabinoids from the GC inlet to the GC column.  Think of Prazepam as a “control chart” compound that upon declining in area count in a continuous fashion signals the need for GC inlet maintenance.

In summary, the addition of a combination of PCP and Prazepam to every cannabinoids standard and every cannabis extract prior to analysis will lead to better potency determinations when using GC-FID.

Chromatogram with ISTDsDifferent ISTDs Table

3 Responses to “Comparison of Phencylidine and Prazepam as Internal Standards in Medical Cannabis Potency Analysis with GC-FID”

  1. Matthew says:

    Jack, how would you recommend introducing an ISTD such as this to an actual sample extract? For these blog posts, did you add PCP/prazepam standard solutions directly to aliquots of the cannabinoid mixed standard? You’d have to have done this to have proportional solvent loss in the ‘sit over the weekend on the GC’ test right? If so, wouldn’t this require going through absolutely ridiculous volumes of standard? I.e., in my lab we’re currently working with 1 ml aliquots of extract, in the usual range of 1000 ng/ul. Mixing half a ml of extract and half a ml of standard for every sample clearly isn’t workable (even vial inserts would only slightly reduce this). Or are you co-injecting at the autosampler? Thanks!

  2. Jack Cochran says:

    Hi Matthew:

    I typically introduce the internal standard (ISTD) as a negligible amount (by volume, I mean) to the standard/sample. An example would be for some PAH work I was doing last week where I added 5 µL of a concentrated stock of an ISTD mix to 1 mL. I use a 10 µL syringe to deliver the small volume and I squirt it under the surface of the liquid. For all practical purposes, the final volume is still 1 mL. In my example, I had calibration points at 1, 2, 5, 10, 20, 50, and 100 ppb and my ISTD concentration was 20 ppb in ALL of those standards. It was also, through addition, at 20 ppb in all the samples I analyzed. Remember to make sure that your internal standard elutes clear and free of any analytes of interest, unless you’re using a mass spectrometer and then just make sure any coeluting compounds aren’t isobaric.

    Another way to accomplish this as you note is through the use of micro-inserts, and I use that very often. 5µL added to say 50µL allows you to use a less concentrated ISTD (still, it should be in the range of the concentration for the calibration standards like my example above). Remember to add 5µL of the same ISTD to each and every standard and sample. In this case the volumetric change is larger, but “it all comes out in the wash” since the same volume is added to every sample. Using micro inserts for standards and samples allows you to preserve the bulk of them for additional standard prep later, reanalyses of samples.

    Another thing I do is dilute my original calibration standards down from 1000. I am typically working with a mixed standard in the 100-200 range, and even lower for calibration curves. Of course this means you may have to dilute your samples too, which is a tradeoff, but dilution preserves valuable standards. The FID is super sensitive; I’ve shown results down to 10 pg on column for cannabinoids. It’s also super linear, our most linear detector, so being outside the calibration range on the upper end for samples versus standards should not be a complete disaster.

    In summary, I think ISTDs are good practice. At the very least they let you know about poor injections, and they also help quantitative accuracy. Thank you so much for reading the blog post and asking questions!

    JC

  3. […] such as 4-bromofluorobenzene could be a suggested IS for chlorobenzene analysis. In the case of medical cannabis potency testing, my colleague Jack Cochran used compounds somewhat similar in structure and behavior to the […]

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