*2.3. Comparison of 1H-NMR and UHPLC Results*

1H-NMR makes it possible to highlight the overall profile of the compounds present in the RYR DS in a single analysis of the crude extract. Indeed, not only all the monacolins, but also pigments (monascin and other pigments having the same skeleton), fatty acids (SFA and UFA), polyols (glycerol, sorbitol), glucose as well as various other products most often added to the formulation (piperine, carnitine, vitamins) are detected. The main drawback of the method is that the resonances identified are in most cases characteristic of a family of compounds and not of a specific compound as for example, monacolins with a hexahydronaphthalene ring and not only MK, or monascin and other pigments with the same skeleton and not the sole monascin. In contrast, the UHPLC-DAD-MS analysis allows a precise identification of the compounds, but like any separation technique, it requires much more time for implementation and especially the operating conditions used target certain structures of compounds, for example, here, monacolins and pigments.

As the 1H-NMR resonances quantified are specific of monacolin families and not of each monacolin, the monacolin amounts determined by 1H-NMR must be compared to those obtained by UHPLC-DAD-MS for the same group of monacolins. The relationships between the amounts measured from the quantification of the resonances at 5.84 ppm and 5.56 ppm in 27 and 28 formulations respectively, both characteristic of all the monacolin structures except dihydromonacolins (TotalM-DiMK (because only this dihydromonacolin was detected by UHPLC)) (Table 3), and those obtained by UHPLC-DAD-MS for the same set of monacolins (Table 5) were very good, as demonstrated by slopes near 1, y-intercepts close to zero and correlation coefficients (r2) greater than 0.99 for the two linear regression equations as well as p-values of 0.94 and 0.66 (Table S3). A similar very good correlation between NMR and UHPLC values was obtained when considering the mean concentrations measured from the two NMR resonances (Table S3).

The comparison of the contents of monacolins in lactone form determined from the quantification of their characteristic resonances at 4.60 ppm and/or 4.25 ppm in 22 RYR DS to those obtained by UHPLC-DAD-MS for the same panel of monacolins also showed results in good agreement. Indeed, the linear regression equation of the 1H-NMR and UHPLC values displayed a slope of 0.983, a y-intercept of 0.013 and a correlation coefficient of 0.996, and the *p*-value was 0.50 (Table S3). The resonance at 5.33 ppm is characteristic of all the monacolins except MJ, MJA, ML, MLA and DiMK. Its quantification, which could only be performed on 17 RYR DS, led to values in agreement within ±11% with those obtained by UHPLC for the same set of monacolins for only six (35%) of them (Tables 3 and 5). The very weak relationship between the concentrations measured by the two methods was demonstrated by the low correlation coefficient (r<sup>2</sup> = 0.723) and the y-intercept far from zero of the linear regression equation as well as a *p*-value of 0.02 representative of a significant difference between 1H-NMR and UHPLC data (Table S3). Therefore, for the RYR DS analyzed in this study, this resonance is unsuitable for quantification, in contrast to what was observed in a previous 1H-NMR assay of five formulations marketed for German consumers [18].

In the same way, the resonances at 4.05 and 3.63 ppm, characteristic of the monacolins in hydroxyl acid form, cannot be accurately quantified due to their strong overlap with matrix signals. Indeed, all the assays tentatively performed led to values considerably higher than those obtained by UHPLC for the same type of monacolins (data not shown).

From all the 1H resonances characteristic of the various monacolin chemical structures, only those involving the hexahydronaphthalene ring (at 5.84 and 5.56 ppm) and the lactone ring (at 4.60 and 4.25 ppm) are appropriate for quantification without requiring curve deconvolution algorithms. It should be noted that the quantification of the resonances at 5.84 and 5.56 ppm was hindered by the LOQ of the technique for three formulations (28/31 formulations were quantified), and that of resonances at 4.60 or/and 4.25 ppm was additionally hampered by their overlap with matrix signals in some cases (only 22/31 formulations were quantified). To confirm that the 1H-NMR signals at 5.84 and 5.56 ppm can be used to quantify the monacolin content in the RYR formulations, we compared the data obtained to TotalM determined by UHPLC (Table S3). The good relationship between the two sets of values (linear regression equation with a slope of 1.00, a y-intercept at 0.113 and a correlation coefficient of 0.986 as well as a t-test *p*-value of 0.25) was not unexpected as DiMK (the sole dihydromonacolin detected in this study by UHPLC) represented ≈2.8% of the TotalM (range 0–9.9%) (Table 5). The presence of DiMK was not surprising as it is produced in small quantities during the fermentation process of rice with *Monascus* [1,23]. In conclusion, 1H-NMR can be considered as a convenient method to determine the TotalM content in RYR DS.

#### *2.4. Quality Control Issues*
