2.1.2. Quantitative 1H-NMR Analysis

With our experimental conditions, the limit of detection (LOD; for a signal-to-noise ratio (SNR) = 3) of monacolin derivatives in real samples was estimated between 4 <sup>×</sup> 10−<sup>5</sup> and 10−<sup>4</sup> M depending on the multiplicity of the targeted proton signal, corresponding respectively to 0.016 and 0.04 mg of monacolins (considered with the molecular weight of MK (404 g mol<sup>−</sup>1)) in the volume of solvent used for the extraction of 20 to 100 mg of RYR formulation. The limit of quantification (LOQ; SNR = 10) for the same signals was evaluated between 10−<sup>4</sup> and 3 <sup>×</sup> <sup>10</sup>−<sup>4</sup> M, corresponding respectively to 0.04 and 0.12 mg of monacolin derivatives in the amount of powdered RYR extracted. Considering the ratio between the mass of sample extracted and that of the formulation (tablet or powder in the capsule), the LOQ was estimated to be ≈0.25 mg of monacolin per capsule or tablet.


1H-NMR

1 and list of other



138

that the compound was mentioned on the label of the formulation.

The H5 resonance (6.01 ppm) was not used for the quantification of monacolins as it may also contain part of the signal (ddd) of the H23 of DeMK at 5.97 ppm and can be slightly overlapped with the H1 signal (qd) of monascin at 6.05 ppm. The H6 (5.84 ppm) and H4 (5.56 ppm) resonances are not hindered by signals of other molecules and they have been quantified respectively in 27 and 28 RYR DS as no monacolin signal was detected in formulations **11**, **12** and **29** whereas both resonances were observed in DS **16** where only the H4 signal could be quantified with an intensity at the LOQ level. The amount of monacolins in lactone form was determined from either H20 or H22 or both signals in only 22 RYR DS because of significant overlap with matrix signals in some formulations. It was possible to quantify the H1 signal (5.33 ppm) in only 17 formulations due to its overlap with the large resonance of the ethylenic protons of non-conjugated UFA at ≈5.36 ppm and maybe also of other compounds. The 2D 1H-13C HSQC-NMR experiment recorded for the DS **15** and illustrated in Figure 4 clearly shows these findings.

The intensity of the DeMK ddd resonance (H22 at 7.03 ppm) was low or very low and overlapped with matrix signals in most sample spectra and could not thus be quantified precisely. The dd resonance of DiMK at 5.69 ppm was not clearly detected in any formulation and could not thus be quantified. The signal intensities of monacolins in hydroxyl acid form (H22 at 4.05 ppm and H20 at 3.63 ppm) would have allowed their quantification in most of the formulations but their substantial overlap with the resonances of matrix compounds did not permit to obtain accurate values without curve deconvolution treatment.

The monacolin contents determined by 1H-NMR are reported in Table 3. For more clarity, the coefficient of variation (CV) and the relative standard deviations (RSD) are given in Table S2. The RSD of the 1H-NMR assays ranged between 1.5% and 13%. The relevance of the 1H-NMR data will be discussed later when compared to those obtained by UHPLC.

**Figure 4.** *Cont*.

**Figure 4.** 2D 1H-13C HSQC-NMR spectrum of the dietary supplement **15** recorded in CD3CN:D2O (80:20). (**A**) 1H enlarged region 5.2–6.1 ppm. (**B**) 1H enlarged region 3.98–4.72 ppm. The proton numbering of monacolins is given in Table 2.

#### *2.2. Chromatographic Analysis*

#### 2.2.1. Identification of Monacolins and Pigments

RYR formulations were traced by UHPLC-DAD-MS. Among the great number of compounds observed, 23 (12 monacolins and 11 azaphilones; see Figure 2 for their chemical structures) were identified by comparing with the literature their elution sequence, their UV-Vis and MS profiles as well as the accurate mass measurements of their parent and fragment ions in High Resolution Electrospray Ionization (HR-ESI) MS and MS/MS [13,26]. Moreover, the identification of MK, CP, MKA, DeMK, DiMK, citrinin and monascin was confirmed by comparing their UV-Vis and MS and MS/MS chromatographic profiles with those of the corresponding standards. The retention time (tR), UV-Vis λmax, accurate masses of parent ion and of its MS/MS major fragment ions for each compound identified are gathered in Table 4. We will not describe the process that allowed the identification of the 23 compounds but we will show the respective contributions of UV-Vis and HR-MS and MS/MS to determine their chemical structure through some selected examples.

The UV-Vis absorption spectra gave some indication on the chemical structure of the compounds detected. For example, all the monacolins with a conjugated hexahydronaphthalene ring displayed the same characteristic mountain-like UV spectrum with three maximum absorptions at ≈ 230, 238 and 246 nm (range 229–230, 238–239 and 244–247 nm) (Table 4) in accordance with literature data [1,10,27,28]). Due to the absence of conjugated double bonds, the dihydromonacolins with an octahydronaphthalene ring and so an UV maximum absorption band at ≈210 nm [1,20] were not detected at the wavelength used in this study (238 nm). In the same way, the yellow azaphilone pigments with the classical monascin-type chromophore showed specific profiles with high intensity peaks at 227–233 and 386–392 nm and a very low intensity band at 285–291 nm, while the red ones with the rubropunctamine-type chromophore presented specific absorbances at 251–252, 302–307, 412–423 and 525–530 nm (Table 4), all these values being in agreement with literature reports [29–34].


*Molecules* **2020**, *25*, 317

**Table 4.**

Retention times, UV-Vis

characteristics

 and accurate mass

measurements

 of the compounds

 observed and identified from the RYR dietary

supplements


#### **Table 4.** *Cont.*
