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

Nevertheless, the unambiguous determination of compound identity required the additional use of HR-MS and HR-MS/MS. For instance, the UV absorption spectrum of the compound eluting at tR 3.89 min with λmax at 230, 239 and 247 nm was characteristic of monacolins. Its protonated molecular ion [M + H]<sup>+</sup> at *m*/*z* 363.2159 and sodium adduct ion [M + Na]<sup>+</sup> at *m*/*z* 385.1988 were in agreement with respectively the calculated masses 363.2171 of C21H31O5 (relative mass error (RME) −3.3 ppm) and 385.1991 of C21H30O5Na (RME −0.8 ppm) suggesting a C21H30O5 molecular formula (Table 4). Moreover, the MS/MS product ions obtained were characteristic of the fragmentation pathway of monacolins (Table 4) [13,14,26]. So, the compound detected was a monacolin derivative that might be monacolin N (MN) or dehydromonacolin N hydroxyl acid (DeMNA) recently described by Li et al. [26]. We propose that it was MN based on the fact that its tR at −0.16 min with respect to monacolin L in hydroxyl acid form (MLA) was in agreement with the value given by Li et al. for MN (−0.22 min) and not for DeMNA (+1.72 min). Moreover, DeMNA was detected only at trace level using nanoflow HPLC-Chip-MS whereas MN was also detected by conventional HPLC-MS which is much less sensitive [26].

The identification of the compound eluting at 9.78 min is another example of the interest of coupling tR, UV-Vis profile and HR-MS spectra. This compound was not detected at 238 nm but only by its UHPLC-MS profile exhibiting a molecular ion [M + H]<sup>+</sup> at *m*/*z* 407. Among the known monacolins, two have a nominal mass of 406: DiMK (C24H38O5) with an exact mass for [M + H]<sup>+</sup> at *m*/*z* 407.2798 and monacolin M (MM; C23H34O6) with an exact mass for [M + H]<sup>+</sup> at *m*/*z* 407.2434. The measured mass of the [M + H]<sup>+</sup> ion at *m*/*z* 407.2796 indicated that the compound was DiMK. Furthermore, its MS/MS fragmentation pattern gave the same ions than MK but with 2 more *m*/*z* units (305, 287, 269 ... compared to 303, 285, 267 ... ) confirming that a double bond of the fused ring was hydrogenated (Table 4), whereas MS/MS fragmentation behavior of MM is identical to that of MK [26]. Also, MM eluted between MJ and MLA with a relative tR versus MJ of 1.2–1.3 [13,26] and hence, in our UHPLC conditions, MM would be expected at 2.4–2.6 min and not at 9.78 min. The incorrect assignment of this compound to MM by Li et al. in 2004 [10] was based on the mountain-like UV spectrum centred at 238 nm characteristic of classical monacolins and on the MS [M + H]<sup>+</sup> ion at *m*/*z* 407, but the UV absorption and the [M + H]<sup>+</sup> peak did not correspond to the same compound. Indeed, the compound with an UV absorbance at 238 nm was very probably dehydromonacolin L (DeML), a monacolin analogue described for the first time in 2011 [37] whose tR is close to that of DiMK (Table 4) [26].

Monacolins and pigments identified in each RYR formulation are listed in Table 5. UHPLC UV-Vis and MS profiles of five characteristic RYR DS, one with only monacolins (formulation **13**), two with mainly monacolins (formulations **10** and **30**) and two with more pigments than monacolins (formulations **12** and **16**) are illustrated in Figure 5 (the UHPLC UV-Vis and MS profiles of nine other RYR DS analyzed are shown in Figure S2). MK was detected in all the samples analyzed and MKA and DeMK in respectively 28 and 29 DS. MLA, ML, CP, DiMK and DeML were found in 18–22 DS, MJ in 11 and MN in 8. The other monacolins were observed only in few samples: one for monacolin J in hydroxyl acid form (MJA) and two for monacolin X (MX). MM cannot be found in any RYR DS. The mycotoxin citrinin was only detected at trace level in two samples. Among the yellow azaphilone pigments, monascin, monascuspiloin and monaphilone B were identified respectively in 18, 15 and 21 of the RYR DS analyzed, whereas ankaflavin, monaphilone A, monasfluore A and B were found in few samples (6, 4, 5 and 3 respectively). The red azaphilone pigments, rubropunctamine, monascorubramine and PP-R, were identified in 11, 4 and 2 samples, respectively.

#### 2.2.2. Quantitative Analysis of Monacolins

The LOD and LOQ of standard lovastatin, established for SNR 3 and 10, were respectively 0.4 <sup>×</sup> 10−<sup>3</sup> mg mL−<sup>1</sup> and 1.5 <sup>×</sup> 10−<sup>3</sup> mg mL−<sup>1</sup> corresponding to 0.4 <sup>μ</sup>g and 1.5 <sup>μ</sup>g in the 10 to 100 mg of powdered.


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

**Table 5.**

Chemical fingerprints

 of the RYR dietary

supplements

 analyzed by UHPLC and quantitative

determination

 of the monacolins

 identified (mg per capsule or

citrinin; Mo: monascin; Moco:

7-(2-hydroxyethyl)-monascorubramine;

monascorubramine;

 Rubro:

 Mocusp: rubropunctamine.

monascuspiloin;

 Mof A, Mof B: monasfluore

 A, monasfluore

 B; Mop A, Mop B: monaphilone

 A, monaphilone

 B; PP-R:

145

**Figure 5.** UHPLC chromatograms with UV detection at 238 nm (**A1**–**A5**) and full scan MS profile in positive ESI mode (**B1**–**B5**) of five characteristic RYR DS. MJ, MN, ML, MK: monacolins J, N, L, K; MLA, MKA: monacolins L, K in hydroxyl acid forms; CP: compactin; DiMK: dihydromonacolin K; DeML, DeMK: dehydromonacolins L,K; Anka: ankaflavin; Cit: citrinin; Mo: monascin; Moco: monascorubramine; Mocusp: monascuspiloin; Mof B: monasfluore B; Mop A and Mop B: monaphilone A and monaphilone B; PP-R: 7-(2-hydroxyethyl)-monascorubramine; Rubro: rubropunctamine.

RYR formulation 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 ≈0.9 μg of lovastatin or other monacolins per capsule or tablet. The precision of the method was acceptable with a RSD for the replicates less than 2% (range 0.1%–1.9%). The overall average value of RSD was 5% ranging between 2% and 11%.

The amounts of the 12 monacolins identified in the 31 RYR DS are summarized in Table 5. There is a marked variability in total monacolins (TotalM), MK and MK + MKA contents per capsule or tablet with values ranging respectively between 0.07 and 23.84 mg, 0.07 and 23.18 mg and 0.07 and 23.80 mg. MK is by far the main monacolin in the 31 RYR analyzed representing 68% of TotalM (range 37–100%) and the sum MK + MKA representing 82% (range 52%–100%). As MKA is the active form of MK, the European Food Safety Authority (EFSA) considers that the effective MK content in RYR DS corresponds to the sum of both lactone and hydroxyl acid forms [38], which allows to get rid of the great variability of the ratio MK/MKA. Indeed, this ratio was comprised between 0.9 and 37 in 28 of the RYR DS analyzed in the present study and could not be determined for the three others due to the absence of MKA. These values are in agreement with literature data which report ranges from 0.4 to 85 [10,12,13,39] while the ratio varies between 1.5 and 2 in properly prepared RYR [1]. Minor monacolins represent on average 18% of TotalM, ranging from 0 to 47%. All these results fully confirm previous studies that showed that monacolin contents vary considerably in RYR end-use products, as they depend on the yeast strain and the fermentation process. For example, the HPLC analysis of 15 commercial tablets or capsules showed that TotalM ranges between 0.31 and 11.15 mg per 600 mg of RYR [39–42]. The literature review of the monacolins quantification established that MK represents 57% of TotalM (*n* = 26; range 0%–99%), MK + MKA 83% (*n* = 41; range 32%–100%), and the minor monacolins 17% (*n* = 41; range 0%–68%) [1,10,39–43].

Our own results and all the literature data show that to improve the effectiveness and safety of RYR products in lowering/regulating cholesterol levels, a precise quantification of their monacolin content should be mandatory. Indeed, the MK content indicated for some formulations (18 over the 31 tested) is not sufficient to know the effectiveness of RYR DS as the amount of MKA, which is largely variable, is not specified. However, it is unclear if the term MK used by the manufacturers refers to the sole MK or to the sum MK + MKA. The chemical structures of MK and MKA being different, this should be mentioned. Moreover, the content of minor monacolins should be taken into account as they represent at least ≈ 14% of TotalM in 65% of the RYR tested in our study (Table 5) and in 71% of the 41 RYR products reported in the literature [1,10,39–43]. Indeed, if MK after its transformation by liver metabolism into MKA has the highest HMG-CoA reductase inhibitory activity, the other secondary monacolins such as CP, MJ, ML or DiMK are also active although at a lower or much lower extent (except for DiMK). Relative to MK or DiMK activity stated at 1, those of the other monacolins are 0.5 for CP and dihydroCP, 0.15 for ML, 0.2 for dihydroML, MX and dihydroMX, and 0.04 for MJ [44]. The dehydromonacolins, often considered as inactive, also present a low activity (0.2 for DeMK [45]).
