## 4 Applications of \({}^{31}\)P qNMR for Phosphorus-Containing Compounds
### In the Field of Biology  
Quantitative \({}^{31}\)P NMR kinetic analysis is applicable to phosphate transfer reactions and has garnered widespread attention in biology, biotechnology, and biomedical sciences [56]. [PERSON] et al. [57] used phosphorus nuclear magnetic resonance (\({}^{31}\)P NMR) to determine the total phospholipid (PL) absolute concentration and PL spectrum. This technology is increasingly being used for the qualitative and quantitative analysis of PL in various biological matrices, including fish oil. Nevertheless, [PERSON] et al. [56] developed a continuous and quantitative \({}^{31}\)P NMR method for the analysis of (R)-5-hydroxyvaleric acid-5-phosphate formation. This method can be used to determine the efficacy of biocatalysts in the synthesis of enantiomerically pure (R)-mechanic acid-5-phosphate, as well as to develop biocatalytic processes. [PERSON] et al. [53] investigated the feasibility of quantifying siRNA content through a platform approach that integrates quantitative 31P nuclear magnetic resonance and the internal standard method. The platform method uses phosphooacetic acid as the internal reference standard, which shows no signal overlap or interaction with siRNA. The optimization includes adjusting the acquisition time to remove spectral artifacts and setting a relaxation delay based on longitudinal relaxation times for accurate signal integration. The method demonstrates high precision, with a %RSD of 0.9%, outperforming UV spectroscopy. \({}^{31}\)P-qNMR provides more accurate siRNA quantifications, closer to the theoretical mass balance (100% \(w/w\)) after adjusting for sodium and water content. \({}^{31}\)P qNMR avoids uncertainties related to extinction coefficients and dilution errors. The method is versatile and applicable to both drug substance and drug product formulations, with minimal differences in preparation protocols. Furthermore, \({}^{31}\)P qNMR offers structural insights into siRNA, confirming the backbone's theoretical group ratios. The \({}^{31}\)P qNMR platform method is a superior alternative to UV spectroscopy for siRNA quantification, offering greater accuracy, efficiency, and robustness. It represents a promising tool for analyzing complex oligonucleotides in pharmaceutical development and quality control. Figure 6: The external referenced qNMR method using \({}^{31}\)P to directly characterize various representative oligonucleotide drug products. Copyright (2024) American Chemical Society. Taking a different approach, [PERSON] and colleagues [54] employed a phosphormetabolomics strategy to gain deeper insights into the variations in phosphorus-containing metabolites associated with low sperm motility. They performed a comparative \({}^{31}\)P NMR analysis of human seminal plasma samples from asthenozoospermic and normozoospermic individuals (Figure 7). Quantitative \({}^{31}\)P NMR was crucial in detecting, identifying, and quantifying phosphorus-containing metabolites in human semen. It provided valuable insights into the metabolic differences between normozoospermic and asthenozoospermic individuals, offering potential biomarkers for diagnosing male infertility and enhancing the understanding of sperm energy metabolism and motility. Quantitative data analysis was achieved due to the direct correlation between signal intensity and analyte concentration. To enable comparison of metabolite levels across different samples, normalization was carried out. For seminal plasma and whole semen, metabolite quantities were normalized per volume and expressed in nmol/\(\mu\)L. In the case of spermatozoa analysis, metabolite abundance was normalized based on the number of cells, expressed as nmol per million cells. Interestingly, [PERSON] et al. [55] explored the use of low-field \({}^{31}\)P NMR with simple, maintenance-free benchtop spectrometers to analyze phospholipids in complex mixtures (Figure 8).