Trying to establish in-house potency testing using HPLC and followed instructions from Agilent

Testing and Analytics
1

Title: I am having a lower CBD% than outside laboratory

Hello Future4200 community,

I have a question on potency testing results.

I am trying to establish in-house potency testing using HPLC and followed instructions from Agilent. I’ve got my calibration curve with good R^2 numbers, prepared my sample, and ran HPLC. I found that the potency results are lower than out-sourcing data. For example, CBD% in house was 84% where CBD% from outside lab was 94%. It is 10% difference. I will share with some of information on analysis and sample preparation. Please let me know what you think. Thank you.

Hemp oil concentrate HPLC analysis:

HPLC:
Agilent 1100 series
CDS Chemstation

10 Cannabinoid Standards:
THCV, CBD, CBG, CBDA, CBGA, CBN, 9-THC, 8-THC, CBC, THC-A

Calibration curve set up, 5 levels:
0.5 ug/mL, 25ug/mL, 50ug/mL, 100ug/mL, 150ug/mL
R^2 = 0.999 above

Sample prep:

  1. Using balance, weight empty 100mL volumetric flask. Record the weight.
  2. After heating oil concentrate, transfer 4 drops of concentrate using 2mL disposal pipette into 100mL volumetric flask.
  3. Record the final weight.
  4. Subtract final weight to empty flask weight to get sample weight. (100mg to 130mg)
  5. Add 30mL of HPLC grade MeOH.
  6. Swirl the flask to dissolve the oil. Dilute up to the mark.
  7. Transfer 1mL of this solution to 10mL volumetric flask and dilute with MeOH to the mark.

Sample Conc: about 100ug/mL

Things that I want to ask:

  1. We do not have a fancy analytical balance. The balance can measure only up to 0.1mg
    minimum weight it can measure is 10mg. I suspect that balance could be the source of the difference.

  2. I used micropipette to deliver 1mL. I planned to change to volumetric pipette.
    Could it be the source of getting lower % number?

  3. We do not have a vortex, sonicator, nor centrifuge. Does it make difference?

  4. I was suggested to use EtOH instead of MeOH. Having a different solvent could lead better sample prep?

Thank you for taking time to read my post.

Sincerly,

Sean

2

Not sure why this just showed up on the newest post feed but here’s a little help,

1st: Get a scale capable of accurately reading down to 0.XXX grams or 1mg. What scale do you currently have?

2nd, make sure you are diluting those samples correctly, this is the most common place for human error. Are you holding that pipette up to eye level when measuring (pretend the accuracy of this step is life and death and slow down.) Are you aligning the 1ml mark on your pipette to the top or bottom of the surface tension of the liquid? what grade is the volumetric flasks? A B or C?

Start here and then see how your results change with these two variables.

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3

Yeah I am not too sure why this popped up for me since its been a while since this is posted.

My question is how are you making your calibration curve? I don’t know how you can get a 150ug/mL calibration level since most manufacturers provide their cannabinoids in a concentration of 1mg/mL. If you are preparing your standards by adding all 10 of them together and creating a mix, the highest concentration you can actually make is 100ug/mL…

4

What system/method is the 3rd party lab running? Try adding an additional level to your cal curve on the high end. I find the best concentrations to prep for are within that 100-200ppm range.

Also when dealing with analytics it is critical that you have an analytical balance capable of reading down to the thousandth.

Do you perform pipettor checks? If not, you should incorporate this step into your daily balance calibration. Again, you will need an analytical balance to ensure you are measuring properly.

I prefer volumetric pipettes when I can use them because it reduces user error. You have no idea how many techs don’t know how to properly use a micropipettor.

The methanol should be fine for sample prep, that is standard in cannabis testing. Just ensure you are using HPLC grade solvents.

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