Detection of Adenosine Phosphates in Blood by nh-PHIP

Hyperpolarization techniques are rapidly advancing and beginning to make an impact on chemical analysis by NMR spectroscopy, bringing noticeable benefits in sensitivity, resolution, and acquisition time. Herein, we apply non-hydrogenative parahydrogen-induced polarization (nh-PHIP), a parahydrogen-based technique, to the analysis of blood, the most commonly used biological sample matrix in medical diagnostics and metabolomics. The systematic development of nh-PHIP detection of ATP, ADP, and AMP in the red blood cell (RBC) fraction of blood is presented. Efficient RBC lysis with water before extraction with a methanol-chloroform mixture are essential steps to yield extracts compatible with nh-PHIP. Detection of adenosine phosphates in the extracts requires the removal of interfering Mg²⁺ cations from the ATP-Mg complex. Addition of the Et₃N/Et₃N-HCl organic buffer helped to maintain the protonation/deprotonation state of the analytes in the NMR sample. This work presents the first nh-PHIP spectra of both the RBC and plasma fractions of blood, a significant step toward sensitivity-enhanced blood-based NMR metabolomics.