Calibration curve is backwards!

Empower2018

Im trying to understand calibration curves when you batch process a sample set and I have a problem- I have two components A and B. I run the sample set as "modified throughout" in the wizard so 5 samples followed by 1 standard, and a final Quantitate line etc and that's fine when all my standards are at the same concentration. Even for my system suit standard which is at a low concentration, this curve is wiped by the "Clear Calibration" line before my first standard used for quantitation. Component A has the same concentration throughout (my units in the component editor is mcg/mL) and my amounts are fine but for Component B my values vary:

I want the first 15 samples quantified by the low strength standard, then I add a clear calibration line directly after Quantitate and start another "Modified throughout" set-up to include the high strength standard which has different component B values in the component editor but im getting weird results- the curve for the first 15 samples is backwards with a negative intercept- the response values are correct but out of order- the first response value corresponds to the third standard and so on. The only way the cal curve looks right for component B is when all values in the component editor for Component B are the exact same. 

My colleague told me that the bracketing set up of "Modified Throughout" only works if ALL standards used for quantitation are the same value in component editor, is this correct? Im used to a bracketing sliding with overlap set up so I wouldn't have thought Empower has an issue doing modified set up twice in one sample set, any thoughts?

HeatherLongden

You can use multiple levels, each with different amounts if you wanted. Running the samples before the standards is a bit odd. You'll need to be sure not to process the samples until the std you want to use has been run.

Modified Overall, if I'm thinking of the right term, will create one big std curve of everything, and only then will process the samples.(samples will be set to 'Dont process and report' and only processed at the end of the sample set using all standards. They can be the same or different concentration standards, averaged by None, Level or Amount depending how you set it.

I wonder if you first samples were processed before your standard so were using maybe a blank?

FYI.. the only good easy way to do the dual processing for Assay level standards and then Impurity Level standards is to have two processing methods, with different integration parameters..  the cal curves are kind of velcroed/attached to the processing method.

• So process the high conc standards with an Assay PM, then process the samples with that PM.
• Next process the low level standards with an Impurity PM, and then reprocess the Samples again, using the Impurity PM. MANY customer use this approach.

ydan1977

Hi

could you include a screen shot of the calibration curve

right click on calibration table and click 'point information'

is this for quantifying a dual active ?

Empower2018

I made sure that all the standards were the same in my component editor and reprocessed and now the calibration curve looks ok. I thought that if you were running different standard values in the same run, Empower would have no issue separating quantitation as long as you put in a  clear calibration line and started the whole sequence of Standard, Samples X6, Standard etc again, putting in clear labels for what standards and samples to use. Unless im missing something. is the layout of Modified Overall (average standards throughout) only to be used for standards of the same value?

Empower2018

Thanks for the explanation Heather. We have to inject a standard every 10 samples as per our validated test method to account for drift or minor changes in flow and temperature through the run time. I also tested the Modified Overall today on a set of samples with the same concentration of standards and you don't even need labels for the standards since the concentration is the same. But I see what you mean by using labels if the concentrations were different. 

HeatherLongden

You should be able to easily use different concentrations of standards, in ANY Standard / Calibration pattern.
These can be Averaged by Level (if you entered levels) and slightly different Amounts will be averaged to ONE point. Or you can average by Amount (when only those standard component amounts which are identical are averaged) OR you can average by None, so all points are put in the curve.
Is there a chance you Averaged by Level? and did not enter any Levels ( or enetered the SAME Level as metadata fro the Standards injections? So it tried to average low and high standards into a single point?