With this in mind, we examined the correlation between palladium concentrations and fluorescence intensities. First, with 180 μM TFP, 30 μM RAE, 1.0 mM NaBH4, 200 mM NH4OAc in 5:17:78 DMSO/H2O/EtOH, the reactions were carried out for 1 h in the 0-2000 nM palladium range (Figure ), revealing that the method was quantitative up to 250 nM. By lowering the NaBH4 concentration from 1 mM to 0.5 mM, the dynamic range broadened, at the expense of sensitivity (Figure ). The sensitivity could be improved by the second addition of NaBH4 (stop-and-go) to increase the NaBH4 concentration to 19 mM. To omit the second addition of NaBH4 for procedural simplicity, we started the reactions with 20 mM NaBH4, with the expectation that the data at earlier points might be more linear because less substrate (RAE) would be consumed. However, even the 5-min data point showed a non-linear trend (Figure ). These data were reproducible; Figure shows the result under the same conditions on a different day. This figure also compares the current method with our previous method, indicating that the current method is 54 times more sensitive in the 0-32 nM palladium concentration range. We hypothesized that this reaction might follow Michaelis-Menten kinetics because there may be preequilibrium among palladium, TFP, and RAE to form the catalyst-substrate complex. To test this hypothesis, we measured the velocity as a function of substrate concentration (Figure ). To slow the reaction to accurately measure fluorescence intensities for the first several minutes, we lowered the NaBH4 concentration to 0.5 mM. Indeed, we observed the saturation kinetics, indicating that the palladium-catalyzed deallylation fits Michaelis-Menten kinetics. Validation of the method. The purpose of implementing a high throughput screening method for palladium is not to accurately measure the absolute concentrations of palladium in APIs but to estimate the relative concentrations of palladium when process chemists screen for dozens of scavenging methods. One of major challenges is to semiquantify palladium at as low as 10 ppm and as high as 2000 ppm. We must consider the solubility of APIs and stability of palladium when solutions of APIs are prepared for analysis. If an API substance (1 mg) with X ppm palladium in the solid state is dissolved in 500 mM HCl in 1:4 DMSO/H2O (1.0 mL), the resultant concentrations are 1 mg API/mL and X ppb (9.4X nM) palladium. When this solution (20 μL) is added to a reaction mixture (180 μL) in a well, the palladium concentration is 0.1X ppb (0.94X nM). Therefore, when the palladium concentration in the well is determined to be Y nM, the palladium content in the original solid API is Y/0.94 ppm.