Quantitative 31P-NMR for Purity Determination of Sofosbuvir and Method Validation

Quantitative H-1-NMR (H-1-qNMR) is useful for determining the absolute purity of organic molecules; however, it is sometimes difficult to identify the target signal(s) for quantitation because of their overlap and complexity. Therefore, we focused on the P-31 nucleus because of the simplicity of its signals and previ-ously reported P-31-qNMR in D2O. Here we report P-31-qNMR of an organophosphorus compound, sofosbuvir (SOF), which is soluble in organic solvents. Phosphonoacetic acid (PAA) and 1,4-bis(trimethylsilyl)benzene-d(4) (1,4-BTMSB-d(4)) were used as reference standards for P-31-qNMR and H-1-qNMR, respectively, in methanol -d(4). The purity of SOF determined by P-31-qNMR was 100.63 +/- 0.95%, whereas that determined by H-1-qNMR was 99.07 +/- 0.50%. The average half bandwidths of the 31P signal of PAA and SOF were 3.38 +/- 2.39 and 2.22 +/- 0.19 Hz, respectively, suggesting that the T-2 relaxation time of the PAA signal was shorter than that of SOF and varied among test laboratories. This difference most likely arose from the instability in the chemical shift due to the deuterium exchange of the acidic protons of PAA, which decreased the integrated intensity of the PAA signal. Next, an aprotic solvent, dimethyl sulfoxide-d(6) (DMSO-d(6)), was used as the dissolving solvent with PAA and sodium 4,4-dimethyl-4-silapentanesulfonate-d(6) (DSS-d(6)) as reference standards for P-31-qNMR and H-1-qNMR, respectively. SOF purities determined by P-31-qNMR and H-1-qNMR were 99.10 +/- 0.30 and 99.44 +/- 0.29%, respectively. SOF purities determined by P-31-qNMR agreed with the established H-1-qNMR values, suggesting that an aprotic solvent is preferable for P-31-qNMR because it is unnecessary to consider the effect of deuterium exchange.