3.5.1. Instrumentation

The liquid chromatographic system was a Waters Acquity UPLC-DAD-SQD (Ultra Performance Liquid Chromatography with Diode Array Detector and Single Quadrupole Detector). It consists of the following modular components: a binary pump, an automatic sample injector with two 48-well trays, a column oven, a diode array detector and a simple quadrupole detector with ESI. All the analyses were performed using ESI ionization with the following settings: positive mode, electrospray source temperature 135 ◦C, desolvatation temperature 300 ◦C, capillary voltage 2.8 kV, cone voltage 3 V, extractor voltage 2.0 V and RF lens voltage 0.1 V. The full scan mass spectra were acquired over a range of *m*/*z* 100–1000. The separations were achieved on a Kinetex C18 (100 mm × 2.1 mm, particle size 1.7 μm) column. The mobile phase consisted of water with 0.02% formic acid (solvent A) and acetonitrile with 0.02% formic acid (solvent B) at a flow rate of 0.6 mL min<sup>−</sup>1. The temperature of the column oven was set at 40 ◦C. The chromatographic analysis consisted of an isocratic elution with a 65% A/35% B mixture for 0.5 min followed by a linear gradient program: from 65% A/35% B to 35% A/65% B between 0.5 and 15 min and finally to 0% A/100% B over 3 min. After each run, the percentage of solvents ramped to their initial composition in 1 min and then the column was re-equilibrated for 2 min. The UV detection and quantification were performed at 238 nm and UV-Vis spectra were recorded within a range of 200–800 nm. The data acquisition and processing were done with the Empower 2 software.

#### 3.5.2. Validation Procedure

The UHPLC-UV method described was validated in terms of system suitability, linearity, precision, sensitivity (LOD, LOQ) and specificity.

The system suitability tests to ensure the reproducibility of the chromatographic system were performed by injecting six times 1 μL of a solution of standard lovastatin. The RSD found was 0.2% for a 0.42 mg mL−<sup>1</sup> solution and 1.5% for a 4.2 <sup>×</sup> <sup>10</sup>−<sup>3</sup> mg mL−<sup>1</sup> solution and was acceptable as it was less than 2% [13].

The linearity of the UHPLC-UV assay was tested for eight concentration levels of standard lovastatin in the range 4.18 <sup>×</sup> <sup>10</sup><sup>−</sup>3–0.42 mg mL−<sup>1</sup> in CH3CN:H2O (80:20 *<sup>v</sup>*/*v*). The correlation coefficient value (r2 = 0.9993) of the calibration curve obtained by plotting the peak areas versus concentrations indicated satisfactory linearity of the method in the range studied. All the monacolins with a characteristic UV maximum absorption peak at ≈238 nm were quantified using the calibration curve established for standard lovastatin and their amounts in mg calculated considering their respective molecular weights. DiMK was not detected at this wavelength and was thus quantified by MS from its [MH]<sup>+</sup> peak area by comparison to that of MK whose amount was previously determined by UV, considering that their ionization efficiencies were similar.

Each solution of extracted RYR sample was analyzed three times. Two independently prepared samples were analyzed for 18 RYR DS and three for the other 13.

The specificity of the method, under the conditions described above, was verified using the chromatographic peak purity tool included in the Empower 2 software and showed no co-elution between peaks of monacolins and those of the complex matrix.
