Fig. 2.

IC₅₀ data analysis and troubleshooting. Plots of percent control activity against inhibitor concentration (real compound data from HLM midazolam 1′-hydroxylase activity inhibition experiments analysed by LC-MS/MS). a Desired effect profile for IC₅₀ determination; b Solubility problems prevent use of highest concentration datapoints but IC₅₀ value can still be estimated (full range of enzyme activity used in IC₅₀ determination); c Data as in b, but data processed using measured activity asymptotes for this compound, resulting in an under-estimate of the IC₅₀ value; d Data difficult to interpret due to solubility limitation, “IC₅₀ >3 μM” or “IC₅₀ not determined” could be reported; e Solubility limit likely to be below lowest inhibitor concentration tested in the experiment. Care should be taken not to automatically assign an IC₅₀ value of >50 μM; f In vitro activation indicates inhibitor binding to enzyme at concentrations of 1 –10 μM exists but IC₅₀ value could not be determined. Inhibition should be assessed using alternative substrates