Is there a logical way to calculate this assay?

I have never worked with internal standards before, here is my question:

I have two actives (X and Y) derived across 2 different pda wavelength channels. Internal standard is spiked in with sample solutions. I need to calculate % Assay as follows- get the response of my active peak area/internal standard peak area. Multiply this by my dilution value and divide by (average response over two injections* Sampleweight). This result is divided by a Target and multiplied by 100 to return the final result of % Target. 

Now to me this seems like a pretty typical "Amount" calculation but I'm not sure how to set it up as its an internal standard. I was going to use custom fields for this but it seems like a lot of hassle if Empower can do this using a mix of the component editor, processing methods and sample set. Can anyone help with this?

Best Answers

  • MJSMJS
    Accepted Answer
    Do you intend to process the internal standard at the same wavelength as each of the two actives?  For example, active X is at 220 nm and active Y is at 250.  You integrate and quantitate the internal standard at both wavelengths, not just one of them?

    If you do, then the built-in internal standard fields apply and you shouldn't have any problem calculating amounts by utilizing the typical/built-in fields.  I think that the limitation is that Empower would not be able to calculate amounts if the internal is not in the same processing channel as the active and that would require you to create a rather cumbersome custom field, or, as discussed in other threads, utilize something like a timed event or alter your instrument method with an event to create a single channel for processing.

    You seem to have two things going on in your proposed formula description.  The first is calculating the amount of active and a resulting percentage and the second is an average of the two injections.  If possible for you, I'd highly suggest to just move the averaging to a report where you can simply group injection results based on sample name, label, etc. and calculate the mean there for reporting purposes.  It is then one less custom field.

    To address the first, I presume that you actually intend to use amount and not simply an area response comparison to the internal standard as using the area doesn't really get you anything without knowing the concentrations of the components and I'll also presume that you intend to process the internal at each of the respective actives' wavelengths.  So, quick basics for an internal standard (probably some useful stuff in the help with a little search would provide some good info as well):  Enter the standard amounts for both the actives and the internal standard in the sample set (note that you MUST enter the internal amounts for each sample as well or Empower can't determine the ratio and get you a sample amount).  In the processing method, components tab, choose the internal standard in the "internal std" column for the active(s)...so activeX has "internal" listed.  Cycle through your standards and Empower will generate a calibration curve for each active and internal.

    For each sample, Empower de-convolutes this by effectively calculating the amount of active in the sample from the area/ratio in the sample and the relationship in the calibration curves, multiplies by dilution, divides by sample weight to determine amount values for the active.  At this point, you simply need a formula to take this amount result and divide by your target and multiply by 100 (so, Amount/Target*100).

    If you really still need an average as a custom field, then you can do that with basic intersample calculations and the percentage calculation from above.  Something like "SAME.%..AVE(Amount)/Target*100" presuming you are injecting twice from the same row and not from separate rows in a sample set.

  • MJSMJS
    Accepted Answer
    When you say enter standard amounts for active and internal standards in sample set do you mean the concentration in the component editor?

    Yes, for Empower to auto-calculate "amount" using no custom fields.  The concentrations or stock wts/purity/etc. info in the component editor and by entering your sample weight/dilution in the sample set, they automatically get incorporated into the amount calculation.  For the standards, you'd be entering your active's values based on your stocks/dilution to working standard/etc. just like you do for a non-internal standard method (e.g. ActiveX: 10.251 µg/mL, ActiveY: 20.652 µg/mL, Internal: 9.895 µg/mL).  With the internal standard, however, you also need to enter the internal standard value for each of the samples as well (leaving the ActiveX and ActiveY blank).  I have a method where the standard and sample have differing amounts of internal standard even, so I'm entering 160 µg/mL for my bracket standard's internal where the samples are at 80 µg/mL for the internal.  Empower takes the values from the calibration and their ratio to then calculate the Active in the sample based on the ratio observed in the sample, effectively correcting for "loss" during sample prep/injection precision/etc. which is the whole point of using an internal of course.  I've attached the mathematical formula in my method for what Empower does to calculate the Amount.

    The specific formula you detail is different from using Empower's Amount field and the info above.  Your formula doesn't do anything to actually quantitate the amount of active as there is nothing in that calculation which incorporates your standard concentrations.  If we substitute units...
    Response=unitless as area/area cancels out
    dil factor= should be unitless
    Sample wt = mg for the sake of the example

    So, you basically have a ratio of the active to internal resulting in units of per mg sample as your output and then you multiply that ratio by the average ratio of the two injections with overall units of per mg?  Is there another step in your calculation that wasn't included above that does somehow incorporate standards?  I don't see how this is mathematically getting you what you seem to want which was a %Target as your Target would have to simply be a ratio of active to internal and I don't really understand how that is useful.

Answers

  • Hi MJS, thanks very much for the response. To answer your question, yes I'm intending to process the internal sample and sample at each wavelength so essentially two sets of results. When you say enter standard amounts for active and internal standards in sample set do you mean the concentration in the component editor? I already know the sampleweight and dilution values for the samples. The formula in the method is laid out as follows:

    Area of Active/Area of Internal standard = Response. 

    Response * Dil Factor/Sampleweight*Average Response of two injections. 

    Are the above calculations one and the same and covered by "Amount", I'm unsure about that? Thanks again for getting back to me. 
  • HI MJS, yes I am going to enter the standard concentrations into the comp editor as you describe- for Active X and Active Y in the brackets then for the internal standard only for the sample injections. 
    I was handed this method along with a big convoluted excel spreadsheet which I must attach actually to show you, and from a look at the excel sheet they are entering in areas, concentrations, averages, area of internal etc etc and I think they don't understand that Empower can do these functions inbuilt, pretty much as you laid out. Even I am finding it hard to follow as the method isn't written great. I will attach the spreadsheet and see what you make of it, if you have a chance to look at it. 

    Thanks for the reply, I'm sure Empower can do this calculation I just have to get all the pieces right so the desired calculation is nailed down!
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