Calibration Curve and Sample Potency Application


I am operating Chemstation on OpenLabs with a Agilent 1220 Infinity II. My question revolves around the equation used to calculate sample potency based on the standard curve I have created. My procedure (standards used 1, 5, 10, 50, 100 µg/mL instead) is as follows

My standard curve is seemingly fine, great r^2? However, I have a sample of known potency 25%, when I run it through based on my calibration curve I get 45% potency. I am wondering where this difference could be coming from? I know the calibration curve slope is to shallow for the equation to return a correct potency on known potency samples (my slope for THCA is around 6, while in the calibration results of the link above it is 24). I am wondering why mine differs so much?

There is a custom calculation at the end to get total potency = (THCA*0.877)+THC9

I have the ceramic homogenizers in the tube with the sample and methanol at step 1/2 of Sample preparation for flower.

My processing method: Curve Calculation "from average per level", RF definition "Response per amount", Concentration calculation amount * multipliers / dilution factors. (Multipliers = 20, 20. Dilution factors = 0.2, 10000). Based on the method linked above. There are no multipliers or dilution factors on the calibration curve injections.

If anyone has any insight or if there is further information I can provide I would be pleased to reply. 

Thanks in advance.


  • 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,


  • Jacob,

    I am new to chemstation software and trying to learn it. I am having the same problem using the same method. I am testing hemp. Did you figure out why you were getting incorrect potency? Also, are you using (Multipliers = 20, 20. Dilution factors = 0.2, 10000)? In report is ESTD% good?

    Please give advise if possible.

    Thanks in advance.


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