This is a topic that's really been baffling for me. I've been trying to run some calibration curves and quantify compounds in some samples on a GC/MS (5977B MSD/ 8890 GC) under SIM settings. I know the concentrations of some of the compounds in the samples based on previous HPLC data.
To start off, I was unsure how much I should dilute my samples in order to get them in the ideal range on the HPLC. Some compounds are rather abundant and some are not. I've done a series of dilutions of the known samples with a full injection as well as 1:20, 1:100, 1:200 and 1:400 dilutions. I then attempted to quantify the compounds to see what worked best.
My hope was to get them all onto the calibration curve, and in general I can get them all there using the 1:100 dilution, but the compounds that fall in the lowest two calibrators don't seem to give me good concentrations. Looking at the batch table in Quant, it generally shows accuracy values of ~200% for the next to last calibrator and ~300-400% for the final calibrator. Knowing this I find that I need to analyze many of the compounds at 1:20 dilution and the rest at 1:100. And even then, some of the compounds in 1:20 fall into these lower points and don't give good data. But sometimes I do have points that fall in this range that do seem to give good results, I just don't entirely understand why (dumb luck?), and when I'm working with compounds where I don't know the concentration, how would I know when I could trust the data?
This got me thinking about LOD and LOQ, and many of the discussions I've read leave me quite confused. Some use a formula of LOD = SD*3.3/slope, and the LOQ is 10x that. When I try this it gives me values that essentially makes compounds that fall in the lower half of the calibration curve unusable. Even when I know many of these points do seem to be giving me accurate data. So I'm either doing the math wrong, or I don't understand what it means.
I found other sources that say to look at the signal to noise ratio. If I'm interpreting this correctly, I searched for my compounds in a blank. If the blank gave a reading of 9x10^1 then I would multiple this by 3 and I would get 2.7 x 10^2. And the LOQ would be 10 times that, or 2.7 x 10^3. Based on this I would look at the size of the peaks and see if they are over this measurement. When they are, I could trust that the compound is really there and/or I could quantify it. But when I use this technique to screen against my data sometimes it seems to work and sometimes it doesn't.
The LOD and LOQ do seem like things that I should be able to quantify directly in Quant, but I can't seem to figure it out. I can get columns in my batch table to include these, but I can't get it to populate with numbers. If anyone knows how to do that I would appreciate that.
Anyway, if anyone can help me figure this all out I would very much appreciate your help.
