When the ATP standard solution was analyzed together with 20mM of Et3N/Et3N-HCl buffer a 4-fold increase in ATP-iridium complex hydride signal was observed (Figure ). The change in the NMR sample environment also caused the ATP signals to shift left. The addition of buffer did not affect the chemical shift of the hydride of nicotinamide complex. However, a similar increase in ATP signal was not observed, when 20 mM of the Et3N/Et3N-HCl buffer was added to the RBC extract, although the nicotinamide signal increased 6-fold (Figure , orange spectrum). The [(IMes)(H)2(mtz)2(ATP)] + complex hydride chemical shifts were similarly affected by the change in the chemical environment (Figure ) in the NMR tube as the signals shifted to the left, similar to Figure . These results confirm that the addition of EDTA and buffer is crucial for the successful detection of ATP in RBC extracts, but further optimization of the nhPHIP conditions is required. First, the the conditions for extracting ATP from RBC fraction of the blood were optimized. It is more convenient to add EDTA to the RBC prior to methanol/chloroform extraction, because EDTA complex with the metal cation can precipitate out. Although EDTA can be added to the RBC extract at the reconstitution stage (see SI Figure ), experiments showed that EDTA in the NMR tube cannot exceed the iridium catalyst concentration of 1.2 mM. However, this may not be a sufficient amount of EDTA to release all of the ATP found in the RBCs as blood contains a considerable amount of metal cations. Therefore, varying amounts of EDTA (1.8, 3.6, 7.2, 14.4, 21.6, 28.8 µmol of EDTA) were added to 300 ul of RBCs prior to the extraction (Figure ). 7.2 µmol of EDTA is optimal by the intensity of the ATP hydride signals in the 1D 1 H spectrum, but the two-dimensional nh-PHIP spectra (Figure ) 21.6 µmol of EDTA is better, because in addition to ATP and ADP, AMP signals were also detectable.