How does your peak area of the sample compare to the peak areas of the standard injections? If the area falls within the calibration then you have an error in your input into the Empower software. If the area is outside of the calibration standards range then you have an error in either your standard dilutions or your sample dilution. Could you give details of the areas as well as sample and standard preparations please.

Did you dilute the sample before injecting? if yes, did you incorporated the dilution factor in your calculation set up? You have to add the dilution factor in ur calculation to get the correct result.

You can also manually plot the cal. curve in MS excel and calculate ur unknown using slope and y-Intercept. If u get the same result as in the HPLC sofware, then u may suspect sample processing error

have you checked the solubility of your active?

does the active substance, like a standardized extract, have any matrices?

One of the most common errors at the time of making the dilutions of your curve, I do not know the procedure you perform, however it is appropriate to perform them separately and also check the misibility of your  analyte in the matrix. Another problem may be that the analysis units in the software are modified.

The fact that you are off by a factor of 10 implies that this is a calculation error and/or missing dilution factor.

I would start with the standards first then work my way to the sample. Looks like you are trying to make a standard curve with different injections volumes. I would check to see what the instrument is calibrated for, in terms of injection range. Then I would plot out the inj volume vs the area then reveiw the data. Using the numbers above I would the precision of the lowest limit.

Dear Shea,

I agree with Stephen.

I would also add that your range of calibration curve standards is too wide. You are attempting to make quantitations at the far low part of this wide spanning calibration curve.

Kind regards,

Ade Kujore

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Prepare a standard at your expected sample concentration and inject the same amount of both the standard and sample. Your peak areas should be comparable. if not, you might not be extracting the analyte from the sample matrix.

Just one more thing, besides every comments above: did you use the same solvent (methanol) in your sample? Sometimes, if your solvent is more strong than mobile phase, the analyte desapears or reduce signal

Why are you calibrating an order of magnitude above your expected concentration? Bracket your nominal concentration; i.e.. if your expected concentration is 29-30 mg/100 mL then run your calibration curve from 5 to 50 mg/100mL. I agree with Stephen that you most likely have a dilution factor missing, but that doesn't alter the fact that you aren't calibrating your system correctly.

I strongly agree with Stephen. How did know what you are expecting, is the sample a reference material or spiked sample? If it's reference material, then try to populate the calibration points at the lower end, just as Mark proposed and your calculation manually and then using the software. But if a recovery work, then try preparing a standard concentration where you are expecting and compare the sample with. If they are comparable, then probably, the error may be coming from the curve, but if not then your extraction was not effective and efficient.