Dear Skyline support,

I am working to create a standard PFAS Skyline method that can be used to quantify a fixed list of 40 PFAS molecules. Ideally, the goal is that the method can be used with different sets of raw data, only with adjustments in RT if needed.

We have 40 unique PFAS analytes, 24 extracted internal standards (EIS), and 9 non-extracted internal standards (NIS). The EIS are added prior to sample extraction. The NIS are added after the extraction is completed, but before injection into the LC/MS system.

Per EPA guidelines, the quantification of PFAS analytes is done with respect to EIS, while the quantification of EIS is done with respect to NIS. Please see attachment "equations-for-eis-nis" for the equations that relate these quantities.

Each analyte is referenced to one EIS, while each EIS is referenced to one NIS. Please see "attachment-2" for the detailed list of these references.

1. Is it possible in Skyline to do quantification this way (i.e., for each analyte, specify that I want the analyte to be calculated based on an EIS, and I want that EIS to be calculated based on an NIS).

2. You will see in attachment 2 that certain EIS may be used as reference for multiple analytes. For example, PFOS-13C8 is an EIS that is used as reference for 5 analytes. Is there a way to enforce these settings directly in the Skyline, or would I have to do this via the transition list?

Please let me know if you require further information. Thank you so much!

Best,

Kyle Nguyen
UNC-Chapel Hill