*3.7. Statistical Analysis*

Comparison of quantitative determination of the different monacolin sets obtained by 1H-NMR and UHPLC was performed (i) by simple linear regression and (ii) with the Wilcoxon signed-rank test, a non-parametric test that can be used as an alternative to the paired Student's t-test for matched pairs when the population cannot be assessed to be normally distributed; the *p*-values > 0.05 (95% confidence level) were considered as proofs of no significant difference between the values measured by the two methods.

#### **4. Conclusions**

This work demonstrated that 1H-NMR is a powerful method to establish rapidly (<1 min) the spectral signature of RYR DS and to afford the quantitative determination of their total monacolin content in a reasonable recording time (from ≈10 to 90 min depending on their concentration in the solution analyzed). The method was validated against UHPLC-DAD-MS, the gold standard technique for a detailed determination of all the monacolins existing in RYR products. Indeed, the quantification of the 1H resonances of the hexahydronaphthalene ring at 5.84 and 5.56 ppm, characteristic of all the monacolin families except the dihydomonacolins present in very low amounts, led to values in close agreement with those of all the monacolins, including dihydromonacolins, measured by UHPLC. These two resonances did not overlap with matrix signals in the RYR DS tested and their quantification is only limited by the sensitivity of the method (LOQ ≈ 0.25 mg per capsule or tablet in our recording conditions). Therefore, 1H-NMR can be considered as an accurate method for determining the total monacolin content of RYR DS, not only those of the main ingredients MK and MKA, but also of minor monacolins which are also active HMG-CoA reductase inhibitors although generally less potent, accounting for a non-negligible amount (mean of ≈ 18%) of TotalM. Taking into account the easy preparation of the RYR DS for the analysis (a simple extraction of capsule/tablet powder by an adequate deuterated solvent), 1H-NMR can be proposed as a high-throughput (thanks to commonly used sample changers) screening method for quality control of RYR formulations on the market. Indeed, the product labels are often incomplete and inappropriate: 42% of the RYR DS analyzed in this study did not provide information on the concentration of monacolins; when the label mentioned MK, it was not specified whether the amount of MK included or not MKA; the amount of the other minor monacolins was never indicated. The deviation between labelled and measured contents must also be emphasized: only 50% of the labelled formulations actually contained the declared amount of monacolin(s). In conclusion, health authorities should impose to manufacturers a strict control of

the quality of the RYR DS to assess the ability of the marketed RYR products in reducing/regulating cholesterol level.

**Supplementary Materials:** The following are available online, Table S1: 1H and 13C-NMR data (solvent: CD3CN:D2O 80:20) of monacolin K in lactone (MK) and hydroxyl acid (MKA) forms, compactin and dihydromonacolin K. Table S2: 1H-NMR quantitative determination of monacolins in RYR dietary supplements. Table S3: Comparison of monacolin amounts measured in RYR dietary supplements by 1H-NMR and UHPLC. Figure S1: 1H-NMR spectra (CD3CN:D2O (80:20)) of all the dietary supplements analyzed in the present study. Upper part: entire spectrum, lower part: enlarged downfield region (4–9 ppm). Figure S2: UHPLC chromatograms with UV detection at 238 nm (A) and full scan MS profile in positive ESI mode (B) of 9 RYR dietary supplements.

**Author Contributions:** Conceptualization, M.M.-M.; methodology, R.H., G.A., S.B., N.M.-F.; validation, R.H., G.A., S.B.; formal analysis, S.B.; investigation, R.H., G.A., S.B., N.M.-F.; writing—original draft preparation, R.H., V.G.; writing—review and editing, R.M., M.M.-M.; visualization, V.G., S.B., M.M.-M.; supervision, M.M.-M.; funding acquisition, M.M-M., V.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the French National Agency for the Safety of Medicines and Health Products (Agence Nationale de Sécurité du Médicament et des produits de santé: ANSM), grant AAP-2012-082, (convention ANSM/UPS n◦2012S071).

**Acknowledgments:** The authors wish to thank Isabelle Fabing from the SPCMIB lab and the Integrated Screening Platform of Toulouse for support with chromatography.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
