I am building a complicated quantitation method using Masshunter. I have 65 analytes and 15 internal standards. The concentration of my analytes ranges from 10ng/mL to 500ng/mL, depending on the analyte, while the concentration of my ISTDs ranges from 200ng/mL to 1,000ng/mL. When I build my quant method I assign calibration levels to my analytes as follows
L1 - 10ng/mL,
L2 - 20ng/mL,
L3 - 50ng/mL
L4 - 100ng/mL
L5 - 150ng/mL
L6 - 200ng/mL
L7 - 300ng/mL
L8 - 500ng/mL
I also apply calibration levels to my ISTDs which are all at higher levels, L10, L11, and L12
However, I get a validation error when attempting to Exit/Save my quant because there are instances where the concentration of the ISTD doesn't match the concentration of the analyte it's being referenced against. For example, I have ******** in my samples at 50ng/mL but ********-D3 is present at 200ng/mL. This means L3 analyte is referenced against L10 ISTD. If I assign them both as L3 (50ng/mL) then this technically "fixes" the problem, in the sense that the validation error clears, but there are two problems with this, 1, the concentration of ********-D3 is not 50ng/mL, it's 200ng/mL, and secondly, while this does clear the validation error for ********, but the problem re-appears elsewhere for other analytes at different calibrations levels L1, L2, L4 etc, that are also referenced against ********-D3.
Is there a quick and easy way around this problem?
Thanks in advance.
Actually, I managed to resolve this issue. I wasn't using Quant correctly at all. I have a single solution containing all my analytes at the cut-off concentration, and while the cut-off concentration is different for each analyte they should all be considered L1, same for the ISTDs. Once I assign L1 for everything I get a quant method that works, Cal curve, calculated concentrations etc. Hopefully this is helpful for someone else.