3.5. Quantification of Thymol and Carvacrol by GC-MS

GC-MS analyses were carried out to determine thymol or carvacrol content within the composites. For the preparation of the samples, the composites were dispersed in ethanol at a concentration of 1 mg/mL (mass of the composite/volume of ethanol). In the ethanol extracting medium, the internal standard was also added, at a concentration of 250 μg/mL. Thymol was used as an internal standard for carvacrol and, symmetrically, carvacrol was used as an internal standard for thymol. The calibration curves obtained demonstrated an R² value always higher than 0.997 units. Each calibration level was fortified with the internal standard at a constant concentration of 2.5 μg/mL. For each sample, three extraction replicates were produced. The extraction was favored by vigorous vortexing, followed by sonication for 5 min. Subsequently, the mixture was subjected to centrifugation for 10 min at 4500 rpm. The supernatant, containing the extracted thymol or carvacrol, was then diluted by a factor of 1:100, followed by characterization by GC-MS.

A gas chromatographic method was developed to guarantee a rapid and efficient separation of the two isomers. The gas chromatographic separation involved the use of a 30 m Elite 5-MS column (Perkin Elmer), with an internal diameter of 250 μm and a stationary phase thickness of 0.25 μm. The separation was managed by the Clarus 680 gas chromatograph (Perkin Elmer), operating in isothermal mode for a total duration of 8 min at a temperature of 135 °C. Helium was used as the mobile phase at a flow of 1 mL/min, adopting a split ratio of 1:10. The sample injection took place manually, with reference to a sample volume equal to 1 μL. The injection chamber was maintained at a temperature of 350 °C for the entire duration of the gas chromatographic separation. The detection of the target analytes occurred by mass spectrometry, using a single quadrupole Clarus SQ 8 T (Perkin Elmer) spectrometer. Specifically, single ion monitoring (SIM) mode was used to maximize the sensitivity of the analytical method. For both carvacrol and thymol, the m/z 135 ion, resulting from the neutral loss of a methyl radical from the molecular ion ([M − CH₃]⁺), was used as a quantifier ion. For both isomers, in fact, this signal corresponded to the base peak within the relevant EI-MS spectra. The molecular ion (m/z 150), however, was used as a qualifier ion. The operation of the quadrupole provided dwell time and inter-channel delay values equal to 0.1 s and 0.02 s, respectively. Furthermore, a solvent cut time of 5 min was set. The ionization of the eluting species occurred by electron ionization (EI). The source operated in positive mode.

For each composite, the loading content and loading efficiency percentages (LC% and LE%, respectively) were calculated as follows:

where m_{Loaded AM} and m_{Expected AM} are the masses of the active molecule (thymol or carvacrol) measured after the composite preparation and of thymol or carvacrol added during the preparation, respectively, while the m_composite was the mass of the analyzed composite.

In addition, in vitro release tests were performed, in triplicate, immersing 5 mg of the composites in 5 mL of distilled water up to 7 days at 37°. Then, thymol or carvacrol released amounts were detected by GC-MS at 24 h and 7 days after an extraction with hexane.