Re: quantification using a calibration curve
Posted: Fri Jan 19, 2018 10:57 pm
Thank you very much for the information.
Bravo! I will never understand clients so wrapped up in LOQ's and LOD's when all the samples have ppm levels in them. When you are pushing the sensitivity of the instrument they may very well be appropriate but I'm not shooting a 50 ppt standard in a study where the low level is 10 ppm just to give someone a low LOQ. It makes no sense and jeopardizes the success of the study.What I have found for best peak shapes is to have the dilution made in the beginning mobile phase, this makes sure there is no mixing problems with the injected sample and the mobile phase that is entering the column at that time.
The more aqueous the better normally. If you have a beginning mobile phase of 90% aqueous and inject a sample that is 75% methanol, some of the analytes will ride that solvent slug through the column and give a large peak early in the chromatogram, then the portion that is retained will give another peak in the gradient. This is known as "Peak Surfing" as a portion of the analyte rides through the stationary phase like a surfer on a wave.
If you always do a dilution on the sample, then the LOD and LOQ will have to take into account that dilution amount, so there is no need to calibrate to the lowest possible amount that can be detected on the instrument if the sample will always have a high level detect. If the sample will always be 100ppm then you don't need to calibrate to 0.000001ppm just because the instrument can see it(for an example). You could easily calibrate as 10ppm, 50ppm, 100ppm, 200ppm, and 500ppm to satisfy the needs of the test. If there could be some samples that will be non-detect even when not diluted then you need to know the limits of the instrument and procedure.
As far as the dilution issue, yes the more diluted usually the better for peak shape. However, when you begin to push instrumental sensitivity that peak shape will begin to deteriorate regardless and especially in an isocratic run. To have this issue when your samples all have ppm levels in them is in my opinion foolish.
I typically define my LOQ as the product of the concentration of the low level standard concentration and the dilution factor of the matrix blank sample. For SANCO studies I then state that if defining the LOQ based on the concentration of the lowest matrix fortification sample that achieved an acceptable recovery the LOQ is x. Usually that first LOQ is about half the second so I can report results for those QC's down in the range of 50% of nominal.