Assay using an internal standard- can you use Amount?
Im running a method with two actives, one in 210nm channel the other 235nm channel and I spike an internal std in my standards and samples at the same concentration. I weigh out my tablets and run them as per method. Although my method states to calculate assay amount of each active in the sample using a formula similar to the amount calculation in Empower, its missing the correction factor of potency of the working standard in the formula. Heres the formula:
(Sample peak area * Area of Internal Std in working standard solution* Standard weight * Sample Volume * 1000)/(Average Standard peak area*Area of Internal Standard in sample solution*standard volume*Sample weight).
Based on above, I put sample volume into Dilution, sampleweight into the SampleWeight field, I activate Internal Std peak in the processing methods for the active peaks, then in the component editor I put 1.00 for the Internal standard for all samples and standards and for my two actives I work out the concentration in mcg/ml of standard, correcting for purity, and put this figure in the standards section of my component editor.
There is no reference to potency of the working std here even though other assay methods I use do reference it and I can use Amount in Empower to calculate assay. Can amount be used for assay samples that use internal standards? Or is it better to just transfer the above calculation into an excel spreadsheet to match the formula? I feel im missing something small here as surely Empower can do assays with internal standards...
By the way its very possible the method needs to be updated to include potency, its not a very well explained method at all!
(Sample peak area * Area of Internal Std in working standard solution* Standard weight * Sample Volume * 1000)/(Average Standard peak area*Area of Internal Standard in sample solution*standard volume*Sample weight).
Based on above, I put sample volume into Dilution, sampleweight into the SampleWeight field, I activate Internal Std peak in the processing methods for the active peaks, then in the component editor I put 1.00 for the Internal standard for all samples and standards and for my two actives I work out the concentration in mcg/ml of standard, correcting for purity, and put this figure in the standards section of my component editor.
There is no reference to potency of the working std here even though other assay methods I use do reference it and I can use Amount in Empower to calculate assay. Can amount be used for assay samples that use internal standards? Or is it better to just transfer the above calculation into an excel spreadsheet to match the formula? I feel im missing something small here as surely Empower can do assays with internal standards...
By the way its very possible the method needs to be updated to include potency, its not a very well explained method at all!
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Best Answer
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Internal standards can certainly be used to generate amount values for your sample's APIs within Empower with no need to use Excel. In my experience, the issue with using an internal standard centers around the standard preparation's dilution scheme. We've had methods where someone might prepare the API stock in 50 mL, but the IS might be prepared in 100 mL or, due to desired working standard concentrations, one of them had to have an intermediate standard which changes the dilution scheme for the API vs IS. Right away, I'd have difficulty using weights for my API and IS amounts in the components tables as my dilution value has to be the same for both (applied to the injection, not the components) and clearly that is not possible. As a result, I had to configure the component values as concentrations which then already include the potency and dilution. With the proper configuration in your processing method, Empower has no trouble calculating sample amounts for the API by accounting for the internal standard values.
Ultimately, the formula you listed seems to have an error of some sort. Either account for the purity in that calculation as you indicate or change it to concentrations which then already account for purity.0
