*3.4. 1H-NMR Analysis*

#### 3.4.1. Recording Conditions

For the qualitative analysis, the 1D 1H-NMR experiments were performed on a Bruker Avance 500 spectrometer (Bruker Biospin AG, Fallanden, Switzerland) equipped with a 5 mm 1H-optimized triple resonance NMR inverse helium-cooled probe (TCI CryoProbe) at 298K (standard 1H sensitivity for 0.1% ethylbenzene in CDCl3: SNR 4200). Typical acquisition parameters were as follows: spectral width 8000 Hz, 32K data points, pulse width 10.0 μs (flip angle 90◦), acquisition time 2.04 s, relaxation delay 1 s, number of scans 16, corresponding to a recording time of 0.8 min.

For the quantitative 1D 1H-NMR experiments, analyses were acquired on a Bruker Avance 400 spectrometer equipped with a TBO (Triple resonance Broadband Observe) probe at 298K (standard 1H sensitivity for 0.1% ethylbenzene in CDCl3: SNR 266). The spectra were recorded with the following parameters: spectral width 8000 Hz, 64K data points, pulse width 3.7 μs (flip angle ≈ 30◦), acquisition time 5.12 s, and relaxation delay 4.88 s. The number of scans was 64 or 512 for recording times of ≈11 or 85 min respectively. Three different samples of the same DS were analyzed.

T1 relaxation times of the methyl protons of TSP and those of the characteristic monacolin protons used for quantification (ethylenic protons of the hexahydronaphthalene ring (H6 and H4) and of the unsaturated lactone of dehydromonacolin derivatives (H22), and CH-OH proton of the lactone ring (H22)) (see Table 2 for proton numbering) were measured in RYR DS by the inversion-recovery pulse sequence method. Twenty delays from 0.001 to 30 s were used with an acquisition time of 2.56 sec and a relaxation delay of 30 s. The T1s found were between 1.6 and 2.2 s for monacolin derivatives and 4.5 s for TSP. All the 1H resonances were thus considered as fully relaxed since more than 98% of the signal intensity of the TSP protons was recovered in the recording conditions used.

The concentrations were calculated by comparing the signal areas of convenient protons of targeted compounds with those of TSP, the area of each NMR peak being directly proportional to the number of corresponding nuclei in fully relaxed recording conditions.

Data were processed with the Bruker TopSpin software 2.1 or 3.1 with one level of zero-filling and Fourier transformation after multiplying FIDs by an exponential line-broadening function of 0.3 Hz. Assignment of signals of MK, MKA, CP and DiMK was achieved with 1D 13C-NMR and 2D experiments (gCOSY, gHSQC and gHMBC).
