Calibration Curve and Sample Potency Application

There are multiple possible issues that you could be running into so I can't point out something specific, but one way you could definitely find out is by pulling the data over into excel and doing the calculation yourself and finding where numbers start to differ. Below I've listed several possibilities.

1. How many injections/samples of each standard did you do? If you are doing multiple injections from one sample of each standard and the standards are in a relatively volatile solvent or the run time is long, evaporation before later injections can be modulating the areas, which will skew your curve. To resolve this, I recommend doing one injection from each standard and if you want to do multiple from the same level then prepare multiple vials of the standard instead of telling it to do multiple injections from the same. Personally, I prefer using the "from each point" option so that the R^2 value reflects the total residuals rather than the residual from the mean of each level. This may cause your calibration curves to change depending on how many injections there are at each level because the curve optimization depends on a different calculation (for one injection it should be the same, for two fairly close, for three and above it can be wildly different).

2. I noticed that your slope is about 4 times less than the slope in the linked results. Is it a coincidence that your potency is 25% (one fourth)? This could be playing a role, but I don't know enough about your calculations to say for sure. I will note that slope difference here likely is not a factor because different detectors and integration methods will cause this to vary significantly anyway.

3. Are your calibration curves set to ignore the origin and have no weighting like in the procedure?

4. How sure are you that 25% is what it should be?

5. If I'm understanding the report properly, the potency % is the "total potency" divided by the "total CBD" times 100. This means that if your number is wrong, then you have four different values that can be causing it. You should check the values of THC9, THCA, CBD, and CBDA and their calibration curves if they have them.

6. Are your calibration curves being made in the same sequence? If so, are you clearing all calibration on the first calibration standard? You could be pulling in old data that was done with a different column, study, detector, etcetera and therefore could be skewing your curves.

7. Do your sample chromatograms look messy? How about the peaks? Your integration method may not be optimal for what you're seeing. Perhaps you're getting extra peaks that are being included in your analyte peaks. Perhaps peaks are being integrated incorrectly due to unusual baseline behavior.

8. Have you run fortified controls of the extraction procedure? Perhaps you (or your analysts) have poor pipetting technique and are pulling over an extra 40 uL of supernatant. Perhaps your glassware, pipettes, homogenizers or other equipment aren't being cleaned properly and are giving you extra analyte because of this. Perhaps the balance you are using for your initial weight of flower is uncalibrated. Perhaps the procedure isn't being followed exactly and some measurements are wrong, which is leading to incorrect results. 

9. Are your reference standards pure and fresh? Perhaps your calibration curves are off due to degradation or purity issues

10. Are you properly equilibrating your column before running the samples? How about including blanks to make sure the column gets cleaned out when it may be necessary. These can affect your area counts.

11. Is that 0.877 in the total potency calculation applicable in your case?

For all of these, I recommend approaching the easiest and least time-consuming ones first. Those are usually the calculations. If you rule out the calculations as the problem, start ruling out the others.

Best of luck,

Cole

There are multiple possible issues that you could be running into so I can't point out something specific, but one way you could definitely find out is by pulling the data over into excel and doing the calculation yourself and finding where numbers start to differ. Below I've listed several possibilities.

1. How many injections/samples of each standard did you do? If you are doing multiple injections from one sample of each standard and the standards are in a relatively volatile solvent or the run time is long, evaporation before later injections can be modulating the areas, which will skew your curve. To resolve this, I recommend doing one injection from each standard and if you want to do multiple from the same level then prepare multiple vials of the standard instead of telling it to do multiple injections from the same. Personally, I prefer using the "from each point" option so that the R^2 value reflects the total residuals rather than the residual from the mean of each level. This may cause your calibration curves to change depending on how many injections there are at each level because the curve optimization depends on a different calculation (for one injection it should be the same, for two fairly close, for three and above it can be wildly different).

2. I noticed that your slope is about 4 times less than the slope in the linked results. Is it a coincidence that your potency is 25% (one fourth)? This could be playing a role, but I don't know enough about your calculations to say for sure. I will note that slope difference here likely is not a factor because different detectors and integration methods will cause this to vary significantly anyway.

3. Are your calibration curves set to ignore the origin and have no weighting like in the procedure?

4. How sure are you that 25% is what it should be?

5. If I'm understanding the report properly, the potency % is the "total potency" divided by the "total CBD" times 100. This means that if your number is wrong, then you have four different values that can be causing it. You should check the values of THC9, THCA, CBD, and CBDA and their calibration curves if they have them.

6. Are your calibration curves being made in the same sequence? If so, are you clearing all calibration on the first calibration standard? You could be pulling in old data that was done with a different column, study, detector, etcetera and therefore could be skewing your curves.

7. Do your sample chromatograms look messy? How about the peaks? Your integration method may not be optimal for what you're seeing. Perhaps you're getting extra peaks that are being included in your analyte peaks. Perhaps peaks are being integrated incorrectly due to unusual baseline behavior.

8. Have you run fortified controls of the extraction procedure? Perhaps you (or your analysts) have poor pipetting technique and are pulling over an extra 40 uL of supernatant. Perhaps your glassware, pipettes, homogenizers or other equipment aren't being cleaned properly and are giving you extra analyte because of this. Perhaps the balance you are using for your initial weight of flower is uncalibrated. Perhaps the procedure isn't being followed exactly and some measurements are wrong, which is leading to incorrect results. 

9. Are your reference standards pure and fresh? Perhaps your calibration curves are off due to degradation or purity issues

10. Are you properly equilibrating your column before running the samples? How about including blanks to make sure the column gets cleaned out when it may be necessary. These can affect your area counts.

11. Is that 0.877 in the total potency calculation applicable in your case?

For all of these, I recommend approaching the easiest and least time-consuming ones first. Those are usually the calculations. If you rule out the calculations as the problem, start ruling out the others.

Best of luck,

Cole