Dilution factor and internal standards

Basic questions from students; resources for projects and reports.

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Hello,

I have been racking my brain over the use of internal standards in quantifying some of my samples. First I'll describe the sample preparation, bare with me, I'll get to the problem. I added 1 uL of an internal standard solution containing 18.75 uM 18O-labeled nicotinamide to 20 uL of milk. I then acidified the milk, incubated it on ice for some time, centrifuged to clarify the supernatant, then dried the sample down. I resuspended in 25 uL. I then set up a standard curve from 0.05 uM to 30 uM for nicotinamide. I created a 5X stock of each and added 5 uL to 20 uL containing 1 uL of the same internal standard mixture used above to prepare my samples. Therefore, the 5 uL of 150 uM solution should produce a final concentration of 30 uM. I then plotted the analyte-to-internal standard ratio on the y-axis and the standard solution concentrations on the x-axis. I quantified the amount of nicotinamide in the milk products by dividing the analyte-to-internal standard ration by the slope of the line of best fit. Now, the problem is this, when I quantify the amount of nicotinamide in my sample the concentrations are with 1 uM. However, I know from the literature that it should be 9.8 uM. Am I making a simple math error here? I thought that once internal standards are added, there is no need for dilution factor corrections as long as the amount of internal standard contained in the sample is the same concentration in the standards. Can anyone see a problem in my work flow here?
When you analyze the final injection solution and receive the result of 1uM for the sample, that is a concentration of 1uM of that 25uL solution. There was only an initial amount of 20uL of the milk used, so you do have a dilution factor of 1.25 to take your value back to the concentration that was present in the milk to begin with. Dilution factors are used to account for what happens to the sample from what it starts as to what it is analyzed as.

This does not explain the discrepancy that you are seeing. As these are samples, and you only have the literature results from someone else's work, what is to say that your samples aren't really only 1 (or 1.25uM)? It might be in your best interest to spike a milk sample with nicotinamide at say the 10uM concentration you're expecting, and then put it through the process and see if you return a result of 11uM (showing recovery of your spike plus the initial 1uM present) or a result of 2uM (showing that you only recovered 1uM from a 10uM spike). If you return 11uM, then your sample really started at 1uM it would seem. If you return 2uM it only showed an increase of 1uM return from the 10uM spike then there is some fuzzy math, or else a 90% loss in your sample treatment process.

Paul
EPA has a guideline for calibration and quantification in chromatographic analysis listed here

http://www.epa.gov/waste/hazard/testmet ... /8000b.pdf

Section 7.4.2.2 will give the equations for calculating the calibration by internal standard method

Section 7.10.2 will give the equations for calculating the concentration of results by internal standard method

These should work no matter what the units or volumes used are, just plug in the correct units and concentrations of the knowns to calculate the unknown values.
The past is there to guide us into the future, not to dwell in.
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