**3. Materials and Methods**

#### *3.1. Red Yeast Rice Dietary Supplements*

Thirty-one RYR DS bought mainly on internet web sites (24) but also from local health food stores (7) between September 2013 and June 2014 were analyzed before their expiration date with UHPLC and 1H-NMR techniques. RYR products were formulated as capsules (20) or tablets (11). All samples with their name, origin, form, batch number, expiration date and RYR extract content are listed in Table 1.

#### *3.2. Chemicals and Reagents*

Authentic standards of lovastatin (MK), citrinin and monascin were purchased from Sigma Aldrich (St. Louis, MO, USA) and those of DeMK, CP and DiMK from TRC (North York, ON, Canada). All other chemicals and reagents used as well the NMR reference for internal chemical shift and quantification (sodium 2,2,3,3-tetradeutero-3-(trimethylsilyl) propanoate (TSP)) were supplied from Sigma Aldrich (St. Louis, MO, USA). Deuterated solvents were obtained from Euriso-Top (91194 Saint Aubin, France). MKA was prepared by hydrolyzing a solution of standard lovastatin in acetonitrile:water (CH3CN:H2O) or deuterated acetonitrile:deuterated water (CD3CN:D2O) 80:20 *v*/*v* (3.7 mg mL<sup>−</sup>1) with a 1M NaOH or NaOD solution under the optimized conditions described in literature [12]. The complete conversion of the lactone form (MK) to its acidic form (MKA) was confirmed by HPLC-MS as the [M + H]<sup>+</sup> peak of MK at *m*/*z* 405 disappeared and the peak of MKA at *m*/*z* 423 was sole detected. Demineralized water was obtained with a Milli-Q system Purelab flex Veolia Waters.

#### *3.3. Choice of Extraction Solvent and Preparation of Samples for Analyses*

The extraction of monacolins from RYR bulk powders with various solvents such as CH3OH, ethanol: water mixtures, CH3CN or ethyl acetate has been extensively described, the best results being obtained with CH3OH or ethanol:water 75:25 [11–13,47,48]. Two extraction solvents were tested in this study, CH3OH and CH3CN:H2O 80:20 (because the MK solubility in CH3CN is higher than in ethanol, 28 and 16 mg mL−<sup>1</sup> respectively) and led to the almost same extraction recovery for all compounds, as measured by UHPLC-DAD. In this study, all sample extractions were performed with the mixture CH3CN:H2O (or CD3CN:D2O).

For the qualitative 1H-NMR analysis, around 100 mg of the powdered RYR samples were mixed with 1 mL of CD3CN:D2O (80:20 *v*/*v*) under vortex agitation for 1 min and then sonicated for 10 min. The suspension was then centrifuged (5 min, 3000 rpm) and 700 μL of the supernatant analyzed. TSP as internal chemical shift (δ) reference was added before NMR recording.

For the quantitative 1H-NMR analysis, between 20 and 100 mg of powdered sample was exactly weighed and mixed with 1 mL of CD3CN:D2O (80:20 *v*/*v*) under magnetic stirring during 20 min, then sonicated for 10 min. After centrifugation (5 min, 3000 rpm), 30 μL of a 10.0 mM solution of TSP were added to 800 μL of supernatant and the resulting solution was analyzed. The final concentration of TSP was 0.36 mM.

The efficacy of this single-step extraction procedure was demonstrated in samples with low and high contents of monacolins. Around 30, 85 and 90 mg accurately weighed of three different powdered samples (respectively DS **13**, **22** and **5**) were extracted as described above, except that after centrifugation, the whole supernatant was carefully collected and analyzed by 1H-NMR. The exactly weighed residual wet pellet was re-extracted with the same protocol than above and the supernatant analyzed using 1H-NMR. In the second extraction, the amounts of monacolins found were respectively ≈ 4.7%, 8.0% and 14.0% of that measured in the first extract. However, the solvent present in the wet pellet represented respectively at least (as the total weight of the powdered sample used was subtracted from the pellet weight) ≈ 5.4%, 8.3% and 14.0% of the initial solvent weight (1 mL = 905.48 mg). The amount of monacolins extracted was thus directly proportional to the amount of solvent remaining in the pellet. So, all the monacolins were dissolved in the solvent (1 mL) used in the single-step extraction procedure and were thus quantified.

For the quantitative analysis by UHPLC, between 10 and 100 mg of each powdered sample was exactly weighed and mixed with 1 mL of CH3CN:H2O (80:20 *v*/*v*) under magnetic stirring during 20 min, then sonicated for 10 min. The suspension was then centrifuged (5 min, 3000 rpm). The supernatant was filtered through a 0.45 μm pore size filter before the injection.
