## 3 Overview of 31P qNMR for Phosphorus Compounds  
Phosphorus, a key element in phosphoric acid esters and DNA molecules, plays a crucial role in physiological processes. It is vital for metabolic regulation, as phosphorylation and dephosphorylation reactions occur rapidly at the protein level. Investigating physiological pathways, metabolomics, kinetics, disease processes, and biomarker identification constitutes a major focus of contemporary research. [10]. Various analytical techniques are employed to analyze phosphorylation and dephosphorylation processes, offering both qualitative and quantitative detection. These methods include fluorescence, electrophoresis, label-free approaches like flow cytometry, stable isotope labeling, and mass spectrometry [11]. While these methods have certain advantages and challenges, the exploration of alternative quantification approaches remains a central focus. Phosphorylated compounds such as metabolites can be measured by nuclear magnetic resonance spectroscopy [12]. 1H NMR spectroscopy of liver extracts is highly sensitive but challenging to interpret due to the full complexity of metabolic pathways and significant signal overlap, combined with strong background signals from water. This complexity makes it difficult to extract clear and quantifiable data from liver extracts using 1H NMR, even when focusing specifically on phosphorylated metabolites. In contrast, 31P NMR offers significant advantages, including relatively high sensitivity and a broader signal dispersion that minimizes signal overlap [13]. Quantitative 31P NMR spectroscopy has been utilized to study phosphorylated metabolites in liver extracts [14]. Only a limited number of methods can utilize a universal reference standard for quantification. Quantitative nuclear magnetic resonance (qNMR) stands out by enabling direct comparison of the analyte's signal with an internal or external reference standard. When primary reference materials are employed, qNMR delivers highly precise measurements with minimal uncertainty, while also facilitating the certification of reference materials for 1H or other nuclei such as 31P. Over recent decades, the significance of qNMR has grown substantially [15; 16; 17; 18]. As qNMR expands into new areas, including metabolomics, environmental analysis, and physiological research, the range of substances requiring analysis and the development of certified reference materials has broadened considerably. This includes complex biomolecules, proteins, and metabolites. These developments present growing challenges for 1H qNMR, the most commonly utilized method in qNMR, requiring enhanced reliability, improved precision, and accurate quantification of increasingly complex molecules [19]. The development of 31P NMR has opened new application areas for studying phosphorus-containing compounds and 31P qNMR has many advantages, making it an important qNMR method. Both 1H and 31P are spin-1/2 nuclei with high natural abundance. In molecular structures, the number of phosphorus atoms is typically low, leading to only one or a few signals in the entire nuclear magnetic resonance spectrum. This minimizes the  
Figure 3: Principle of nuclear magnetic resonance. Copyright (2024) Mariabaias network. risk of overlap between the signals of the sample and the standard in mixtures. The broad chemical shift range of \({}^{31}\)P NMR (approximately 2000 ppm) allows for better separation of signals [20]. Moreover, commonly used deuterated NMR solvents lack \({}^{31}\)P, eliminating interference from residual solvent or water peaks. Quantitative NMR (qNMR) studies focusing on \({}^{31}\)P have been previously reported [21; 22]. Quantitative nuclear magnetic resonance (qNMR) spectroscopy operates on the principle that the area under an NMR signal is directly proportional to the number of nuclei contributing to that resonance. This proportionality enables highly precise quantification, as the integrated signal intensity reflects the concentration of the corresponding nuclear species.