# NMR Analysis of Pulegone in Food Products
## 2 Results and Discussion  
### Method Development and Optimization  
#### 2.1.1 Influence of the Solvent on the Pulegone Signals  
Pulegone is insoluble in water and exhibits a n-octanol/water partition coefficient lg(\(K_{\text{OW}}\)) of 3.08 [23,24]. It can be concluded that lipophilic solvents are suitable for the dissolution of pulegone. In this study, NMR spectra of pulgeone were obtained in a series of solvents, including CDCl\({}_{3}\), methanol-\(d_{4}\) (MeOD), MeOD with CDCl\({}_{3}\) (1:1; _v/v_, 1:4; _v/v_, 4:1; _v/v_), cyclohexane-\(d_{12}\), and xylene-\(d_{10}\) with CDCl\({}_{3}\) (1:50; _v/v_, 1:20; _v/v_). A comparison of the spectra of pure pulgeone in different solvents reveals that the signals are separated to varying degrees of success and that some signals are shifted. Figure 1 shows a comparison between the \({}^{1}\)H spectrum of pulgeone in CDCl\({}_{3}\) and that of pulgeone in MeOD. The use of MeOD resulted in the detection of an additional signal at \(\delta_{\rm H}\) = 1.98 ppm, which was not present in the spectrum obtained using CDCl\({}_{3}\). However, the separation between the doublet at \(\delta_{\rm H}\) = 1.94 ppm and the multiplet at \(\delta_{\rm H}\) = 1.89 ppm is less effective when MeOD is used as the solvent, compared to using CDCl\({}_{3}\). Figure 2 shows the pulgeone spectra at MeOD/CDCl\({}_{3}\) ratios 1:4 (_v/v_), 1:1 (_v/v_), and 4:1 (_v/v_). The separation of the signals in the range \(\delta_{\rm H}\) = 1.85 to 2.00 ppm differed between the three spectra shown. The multiplet at \(\delta_{\rm H}\) = 1.90 ppm is less distinctly separated from the doublet at \(\delta_{\rm H}\) = 1.96 ppm at a mixing ratio of 4:1 (_v/v_) than at a mixing ratio of 1:1 (_v/v_) and 1:4 (_v/v_). The aforementioned multiplet is most effectively separated from the doublet at a mixing ratio of 1:4 (_v/v_). In the spectrum with a mixing ratio of 1:4 (_v/v_), the separation between the multiplet at \(\delta_{\rm H}\) = 2.00 ppm and the doublet at \(\delta_{\rm H}\) = 1.98 ppm was the least distinct compared to the other mixing ratios. The optimal separation between the multiplet at \(\delta_{\rm H}\) = 2.00 ppm and the doublet at \(\delta_{\rm H}\) = 1.98 ppm was observed at a ratio of 4:1. The quality of the separation between the multiplet at \(\delta_{\rm H}\) = 2.00 ppm and the doublet at \(\delta_{\rm H}\) = 1.98 ppm, as well as between the doublet at \(\delta_{\rm H}\) = 1.98 ppm and the multiplet at \(\delta_{\rm H}\) = 1.90 ppm, was intermediate between those of the other mixing ratios at a mixing ratio of 1:1 (_v/v_). In the spectrum of the MeOD/CDCl\({}_{3}\)-mixture in a ratio of 1:1 (_v/v_), the signals in the range  
Figure 1: Comparison of \({}^{1}\)H NMR spectra of pulgeone in chloroform-\(d_{1}\) (CDCl\({}_{3}\)) (upper panel, blue line) and pulgeone in methanol-\(d_{4}\) (MeOD) (lower panel, red line). The inset shows a detailed enlargement of the range \(\delta_{\rm H}\) = 1.81–2.04 ppm for a better view of the differences between the spectra. \(\delta_{\rm H}\) = 1.85 to 2.00 ppm were more intense than those in the spectrum with only MeOD as the solvent (see Figure 1). This difference in signal intensity likely arises from the different solubility properties of pulegone in the two solvent systems rather than from spectroscopic effects, as the same concentration was used for both measurements. In addition, the signals are better separated. The pulegone signals from future samples can be superimposed by resonances from other matrix constituents, and the CDCl\({}_{3}\)/MeOD-mixture (1:1, _v/v_) offers the best dispersion of pulegone resonances. Thus, further samples were prepared using a solvent mixture of MeOD and CDCl\({}_{3}\) at a 1:1 (_v/v_) ratio. As illustrated in Figure 2, signal shifts are observed for all other signals.