## 4 Applications of \({}^{31}\)P qNMR for Phosphorus-Containing Compounds
### In the Field of Pharmaceuticals  
\({}^{31}\)P qNMR provides precise, real-time, and direct drug quantification, supporting reliable in vitro/in vivo correlations. [PERSON] et al. [9] designed a targeted \({}^{31}\)P qNMR approach for the direct real-time measurement of therapeutic molecules in biological fluids. The results indicate that the method is simple and accurate and can quantify phosphorus-containing drug molecules from various biological fluids, such as vaginal fluid mimetics, semen mimetics, and human plasma.  
Moreover, [PERSON] et al. [48] introduced an innovative qNMR approach for quantifying total aluminum and phosphorus in formulated vaccine products. This method was applied to a commercial adjuvanted combination vaccine as well as individual adsorbed antigen drug substances used in its production. With minimal sample preparation, this method delivers rapid, quantitative analysis, making it ideal for routine monitoring of vaccine adjuvant and formulation processes. The qNMR technique is well-suited for optimizing and controlling vaccine formulations. Its adaptability to various aluminum phosphate adjuavants highlights its potential for broader use in vaccine development and manufacturing. Offering a faster, simpler alternative to conventional techniques, it enables the simultaneous analysis of free and total phosphate in a single workflow, reduces preparation complexity, and enhances accuracy.  
[PERSON] et al. [49] utilized \({}^{31}\)P NMR to track malignant human mammary epithelial cells simultaneously during treatment with the anti-inflammatory drug indomethacin. Additionally, [PERSON] et al. [50] employed \({}^{31}\)P qNMR and \({}^{1}\)H qNMR to assess the purity of the anti-hepatitis C virus medication sofosbuvir, finding that the results from \({}^{31}\)P qNMR were consistent with those obtained from \({}^{1}\)H qNMR. Two solvents were evaluated, and these findings highlight that DMSO-d\({}_{6}\) is a more suitable solvent for \({}^{31}\)P qNMR analysis. The study validates the efficacy of \({}^{31}\)P qNMR for determining the purity of organophosphorus compounds, particularly complex substances. Non-protic solvents, like DMSO-d\({}_{6}\), are recommended for analyzing compounds with exchangeable protons to avoid deuterium exchange effects. The development of new reference standards and higher-sensitivity probes for \({}^{31}\)P qNMR is suggested to expand its applicability. This study provides a robust framework for applying qNMR in pharmaceutical development and quality control, especially for complex organic compounds.  
Phosphorus, present in all nucleotide units, has an abundant isotope that is NMR-active. Past \({}^{31}\)P NMR studies on oligonucleotides have focused on peak assignment, chemical identification through the sensitivity of \({}^{31}\)P chemical shifts to the phosphor chemical bond, and backbone conformation analysis. The \({}^{31}\)P NMR could be applied to small-molecule drugs like cyclophosphamide. The capability of using \({}^{31}\)P NMR for accurate quantification of oligonucleotide drugs was recently demonstrated using an internal reference standard in a mixture with a small interference siRNA drug substance and product. [PERSON] and coworkers [52] report an external referenced qNMR method using \({}^{31}\)P to directly characterize various representative oligonucleotide drug products in formulation, independent of any chemical modifications of phosphorothioate or phosphorodiamidate morpholino oligomer, duplex formation of siRNA, or process-associated ions (Figure 6). The \({}^{31}\)P apparent T1 values were estimated by varying recycle delay from 0.1 s to 3 s. The accuracy of this measurement technique was better than the UV method, and its simplicity was much higher than the UV method.