by
lmh » Tue Dec 22, 2020 11:13 am
This is probably the most important post you will ever read in your life:
With an extraction procedure like that, the way to calculate the dilution factors is this:
Draw a little diagram of the entire extraction procedure. Draw little tubes, and arrows pointing from one stage to the next, showing what volume you took out, what volume you put in, where everything is.
You can work one way or the other. You can either start at the beginning with 5 gr of sample, or you can start at the end with a final result of X units fumonisin (more about units later!).
If you start at the beginning, work out how much original ground material corresponds to each solution you make, all the way through. You start with 5g extracted to 25mL. You dilute it in all sorts of ways, and add other volumes, and I think (but please check) that by the end you have 60uL of derivatised solution equivalent to 0.03g of your original ground material. You inject 30uL of this, so you're injection came from 0.015g of ground material.
Let's assume you do your calibration curve in mg/mL. If your result from injecting a sample (that contained fumonisins from 0.015g ground material) came out at X mg/mL, we know that you had 0.030X mg of fumonisin derived from 15 mg ground material, so it's a fairly simple matter to work out the ppm by weight (0.030X / 15 * a million)
If you start at the end, with a result of X units of fumonisin, you'll find it much easier if the units are something easy to handle. I don't recommend ppm because it gets really mind-twisting when you have a dried-down stage in there! If your calibration curve is in mg/mL fumonisin, you can easily work backwards by working out how many mg (ug, ng...) fumonisin the original 30uL injection contained... so how much did the entire 60uL derivatisation reaction contain? (twice as much), so how much did the 400uL redissolved dried stuff contain?... so how much was sitting in the dry pellet... how much was eluted from the Vicam column... etc. etc. right back to how much, in mg, was present in the original 5g of material that you extracted.
Do dilution calculations this way, and you will rarely, if ever, get them wrong.
(Now I'm going to look a right wally when someone points out that I have indeed made a mistake, but my excuse is I did this really quickly on the back of an envelope that wasn't quite big enough).
(but further to the last comment, yes, there are lots of ways to lose the analyte in an extraction like this, so an internal standard is a good idea. If you don't have access to an internal standard, at least run spiked experiments where you extract material in parallel, some of it spiked with the authentic product, and then check that at the end of the extraction, you get back what you expected. If your recovery is 90-110% you can be very happy. If it's 15% you probably need to panic!