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:
- Identify which curve is the one done with standard additions.
- If the internal standard is 25 uM, what is the approximate relative response factor in this experiment?
- 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?
- The 2 lines basically have the same slope. Would you expect that? Why or why not?
- The standard addition curve was performed in a plasma sample. What is the concentration of the ascorbyl-HNE adduct in the plasma?
- 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?
- 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.