Case Study 2: Practical Analytical Considerations for Conducting In Vitro Enzyme Kinetic Studies.

Characterization of enzyme kinetics in an experiment is dependent on measurement of a change in concentration of either the substrate (loss of parent) or the product (formation of metabolite). Modern analytical techniques such as ultrahigh pressure liquid chromatography, high resolution mass spectrometry, etc. have allowed accurate characterization of minute changes in concentration. Therefore, complex kinetic data such as a sigmoidal phase at low substrate concentrations or terminal half-life in a PK curve can be evaluated by stretching the limits of analytical quantification. This chapter presents some elementary dos and don'ts and provides insight into some of the underlying principles for utilizing the best possible analytical techniques when investigating enzyme kinetics. The objective of this case study is to answer the following questions: (a) Why is it necessary to determine lower and upper limits of quantification (LLOQ and ULOQ, respectively) of a bioanalytical assay, specifically for enzyme kinetic assays? How do you utilize LLOQ and ULOQ to correctly interpret your kinetic data? (b) Why should one use a linear fit and not a quadratic fit for standard curves? (c) Is quantification of an analyte possible without a reference standard? Can one assume equal signal intensities regardless of analytical technique (MS, UV)? (d) In the absence of reference standards, can you still determine kinetic constants? (e) With the need to keep substrate depletion at less than 20% for linearity assumptions, does bioanalytical variability matter? (f) What buffer do you use for your enzyme systems? How do you choose your buffer ? Does choice of bioanalytical methods (LC, MS) dictate your choice of buffer ?