3.4.2. Quantification

The quantification was performed on the characteristic 1H-NMR signals (CH=, CH-OH and CH-OR) of the different monacolin families (monacolins in lactone and hydroxyl acid forms, dehydromonacolins and dihydromonacolins). The amount of compound (mg) per dosage unit is calculated from the following equation:

$$Q = \frac{A\_i}{A\_{TSP}} \times \frac{Nb\_{TSP}}{Nb\_i} \times C\_{TSP} \times \frac{V\_2}{V\_1} \times V\_t \times M \times \frac{m\_t}{m} \tag{1}$$

with *Ai* and *ATSP* being the integrated areas of the characteristic NMR signal(s) of the monacolins to be quantified (*i*) and of the TSP signal respectively, *Nbi* and *NbTSP* the number of protons contributing to the signal of the analyte and of TSP (9 protons), *CTSP* the concentration of TSP in the solution analyzed, *V*<sup>1</sup> the volume of supernatant analyzed, *V*<sup>2</sup> the volume of solution analyzed (*V*<sup>1</sup> + *VTSP* (30 μL)), *Vt* the volume used to dissolve the sample (1 mL), *M* the molecular weight of the analyte, *mt* and *m* the weights of the dosage unit (tablet or powder from capsule) and of the sample analyzed. As NMR

did not allow to characterize individually all the monacolins present in RYR, the molecular weight considered was that of MK, the main one. This led to an approximation less than ±5% for MKA, DeMK, MX, DiMK and CP but higher for other monacolins, +10% for MN, +16% for MJA, ≈ +20% for MJ and MLA, +25% for ML and +29% for DeML (see Figure 2 for chemical structures). Nevertheless, all the monacolins whose molecular weight differs of more than 5% relative to MK, are very minor. From their amounts determined by UHPLC, we found that the approximation on their molecular weights led to an increase of the total content of monacolins determined by NMR of less than 3% (<1% for 22 DS, between 1 and 2.2% for 8 others, and 2.6% for one formulation).
