Hey guys,
Long time reader/first time poster. I have had an in-house HPLC for lab testing for a few months and have ran ~100 samples and 5 successful calibration curves so far. Today I prepped a new calibration curve and a new mobile phase (20mM Ammonium Formate/0.1% Formic Acid). When I ran the calibration curve, the THCA peak was absent on all of the standards and the recovery for d9-THC was ~180% for the 6 calibration points. The rest of the analytes were right on. It appears that THCA is decarboxylating on column but I can’t figure out why.
I’ve tried running old calibration standards that I know have THCA and they are also showing no THCA. All standards are capped and have been stored in freezer when not being ran. Methanol Blank injections are clean and I’ve checked the pH of the mobile phase to make sure that it isn’t too low (pH=2.93).
I am stumped. Any suggestions on what the problem is?
Hmm, if it weren’t for the second part about you re-running old standards (kudos to you for being wise enough to save your old standards after running the curve!), I would have said occam’s razor suggests you simple prepped the wrong standard and ran a THC standard instead of THCA and it never was the acid to begin with.
But, unless you misread a label twice or your standards were mislabeled twice…it sounds like maybe you really are observing the conversion of THCA to THC. I dont necessarily have an answer for you, but I will say (as I’ve said a few times before) is that protic solvents (like methanol, ethanol, IPA) can potentiate decarb. However, its not instantaneous. If your THCA standard was in methanol…and sat on a “shelf” somewhere for a while or maybe somewhere along the line sat at room temperature for weeks and weeks…you might see some conversion in your standard. I don’t think this answers your question unfortunately, just offering something I DO know that has a small possibility of being related…
Oh duh, also your oven temperature on the LC definitely matters - especially if you’re in methanol for the reasons I mentioned previously. May I ask your oven temperature for you method?
I actually have seen failure of columns to read the acids, based on issues with buffer/pH changes in the media. Water washing helps it. But that causes you to register no cannabinoid acids at all, hence my question if you can still read CBDA
The stock standard has been out of the freezer maybe 10 minutes since I opened 2 months ago. I am going to switch the guard column this morning, thanks for the suggestion. I haven’t reconditioned the column and don’t know how to do that . What would you recommend?
That’s really strange, don’t know any reason you’d see decarb on only one cannabinoid. Nonetheless, a long rinse with 5% methanol, 95% DI water may do some good.
is it possible the thca standard was improperly shipped? or improperly labeled?
I have had some year old acid standarda that have still not fully decarb themselves but who knows maybe it got hot while being shipped, were the cold packs still cold when you got the standards?
If your cbda is still good and testing correctly the only logical thing i can think of is the thca standard is bad, or mislabeled.
If you wanna send me a ready to inject 2ml vial ill gladly run it on our hplc to see if we can at least rule out the standard or the machine.
Here is a decent cleaning/regeneration method for reverse-phase C18 columns. If you are not using a reverse-phase C18 column, DO NOT do it this way.
Flush your reverse-phase C18 column with 20 column volumes each of the following HPLC -grade solvents (e.g., 80 mL total for a 4.6 x 250 mm column). I typically run my column cleaning flushes at 1 or 2 mL/min. I find that cleaning reverse-phase C18 columns works a bit better in a column oven set in the range of 35-55C.
I don’t see this being an HPLC issue, but rather a calibrant/standard issue. If this was an HPLC issue, I am assuming that all the others acids will experience the same anomaly. Are you using the same standard mix you have used for your previous 5 calibration runs?
The standards I purchase have MSDS that state cannabinoids need to be stored at -40C.
When I run isopropanol in my cleaning procedure, I never run it at more than 1 mL/min and I keep a very close eye on pressure. If any issues with pressure, I quickly drop to 0.25-5 mL/min. If that does not solve the problem, I stop using the isopropanol for my cleaning procedure.
Something else a lot of people don’t know is you aren’t supposed to store your columns in buffered mobile phase. You’re supposed store them in unbuffered (DI water) mobile phase and only use buffer during the actual batch run. This rule has been universally ignored in almost every lab I’ve been to, and as a result a simple DI rinse and then instituting this changed storage procedure often leads to dramatic improvements in a columns ability to read acidic or basic compounds.
It’s unlikely that anything would cause THCA in a picture to totally decarboxylate without seeing any decarboxylation of CBDA and CBGA.
Did your retention times shift, especially the retention times of the acids? That could be caused by having a different pH mobile phase and your THC and THCA could end up co-eluting.
What kind of detector does your HPLC have? If you can get a full spectrum of your THC peak what does it look like?
. What would you recommend?