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.