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Internal Standards and Standard Addition

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    Data from: John Sowell et al. PNAS 2004;101:17964-17969 (retracted)

    This is a graph of both an internal standard calibration curve and a standard addition calibration of a plasma sample (done with an internal standard) for an LC-MS experiment.  They were identifying Vitamin C (i.e. ascorbate) conjugates of lipid oxidation products.   Answer the following questions:

    1. Identify which curve is the one done with standard additions.




    1. If the internal standard is 25 uM, what is the approximate relative response factor in this experiment?





    1. The internal standard was added to a concentration of 25 uM. What does that tell you about the sensitivity of the assay: is it more sensitive to the ascorbyl-HNE or to the internal standard? About how much does the sensitivity vary?




    1. The 2 lines basically have the same slope. Would you expect that? Why or why not?





    1. The standard addition curve was performed in a plasma sample. What is the concentration of the ascorbyl-HNE adduct in the plasma?





    1. For the point marked 1 uM on the standard addition curve: what is the actual concentration of the ascorbyl-HNE adduct in the sample if you calculate it? Draw a horizontal line to the other internal standard curve. Does that match your calculation?




    1. Interestingly, this paper was actually retracted because they could not confirm the identity of the ascorbyl-HNE adduct in the real sample. What does that tell you about the danger of using only standard additions to identify the amount in a real sample? Discuss why these calibration curves could be misleading. 




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