When EDTA solution is added to the 300 µl of RBC prior to extraction a considerable amount of pure water is added (21,6 µmol of EDTA, equals to 216 µl of EDTA water solution) with it. Therefore, the increase in ATP signal could also be due to the better lysis of RBCs as pure water causes osmotic imbalance and RBCs to burst, thus making more of ATP, ADP and AMP available for extraction. This was confirmed by adding 216 µl of pure water to 300 µl of RBC prior to the extraction. When the reconstituted RBC extract was treated with EDTA prior to adding it to the NMR tube (EDTA concentration in the NMR tube 1.1mM), similar ATP signal intensities were observed. Meaning that the lysis of RBCs is important to be able to fully extract ATP, ADP and AMP. However, the signal resolution was better in the RBC sample where EDTA was added prior to extraction (Figure , SI). In NMR metabolomics studies, biofluids are buffered with 100 to 200 mM buffers. Given the increase of ATP signals resulting from better lysis of RBCs, the buffering capacity of the Et3N/Et3N-HCl buffer was evaluated, by adding 20 mM, 40 mM or 60 mM of buffer to the EDTA treated blood extract (Figure ). Based on the work with RBC, 300 µl of the plasma fraction of the blood was treated with 3.6 µmol of EDTA prior to extraction and analyzed under optimal nh-PHIP conditions for ATP using 20 mM or 40 mM of Et3N buffer (see SI). Various amounts of plasma extract (corresponding to 50 µl up to 300 µl of plasma) were added to the NMR tube, but only nicotinamide was identified and detected (see SI). Various plasma ATP levels have been reported in the literature, ranging from as low as 20 nM to as high as 10,000 nM.