## 2 Materials and Methods
### LEO Residuals in Subcutaneous Fat and Muscle  
A sample of lean meat (LL muscle) and a sample of subcutaneous fat (from the thigh region) were taken at the slaughter plant. Samples were immediately refrigerated and transferred to the DIMEVET laboratories, where they were finely minced and stored in plastic tubes that were sealed under vacuum and preserved at \(-20\)\({}^{\circ}\)C pending the subsequent analysis, which was carried out within 1 month in the laboratories of Department of Life Sciences (University of Modena and Reggio Emilia).  
Chemicals used in this analysis were linalool analytical standard (Sigma-Aldrich, Milan, Italy, CAS No. 78-70-6) and paraffin oil (Sigma-Aldrich, Milan, Italy, CAS No. 8012-95-1).  
HS-SPME was performed using a manual holder (Supelco, Milan, Italy) and a stableflex 50/30 \(\upmu\)m divinylbenzene-carboxen-polydimethylsiloxane (DVB-CAR-PDMS; Supelco, Milan, Italy) fiber. The coating was 1 cm long. Before GC analysis, the fiber was conditioned in the injector of the GC system, according to the instructions provided by the manufacturer.  
About 1.5 g of the sample was weighed in a 15 mL glass vial (O.D. \(\times\) H 21 mm \(\times\) 70 mm), and 1 mL of paraffine was added. The vial was closed with a PTFE/silicone septum cap and rolled up with parafilm to avoid any gas leak. The sample was shredded in a magnetic stirrer for 5 min to destroy the tissues and cells of lean meat and subcutaneous fat. The sample was incubated in an ultrasonic bath for 15 min at 50 \({}^{\circ}\)C to saturate the headspace.  
After the saturation time, the SPME fiber was exposed for 15 min, at the same temperature. Then, the fiber was manually transferred to the GC inlet for the desorption step in splitless mode for 5 min at 250 \({}^{\circ}\)C.  
The GC analyses were carried out on a 7820 A gas chromatograph (Agilent Technologies, Milan, Italy) coupled with a flame ionization detector (FID). Compounds were separated on an Agilent Technologies HP-5 cross-linked poly-5% diphenyl-95% dimethyl polysiloxane (30 m \(\times\) 0.32 mm i.d., 0.25 \(\upmu\)m film thickness) capillary column.  
The column temperature was initially set at 70 \({}^{\circ}\)C for 2 min, then increased at a rate of 2 \({}^{\circ}\)C/min up to 100 \({}^{\circ}\)C and held for 2 min, then ramped to 250 \({}^{\circ}\)C at 5 \({}^{\circ}\)C/min rate and held for 2 min. Helium was used as the carrier gas, at a flow rate of 1 mL/min. The injector and detector temperatures were set at 250 and 300 \({}^{\circ}\)C, respectively. The analyses were performed in triplicate.  
To evaluate linalool as a marker of LEO in animal tissues, the calibration curve was created by analyzing the different concentrations of linalool standard dissolved in the control samples. Paraffin was selected as a diluent for linalool to prepare the solutions at different concentrations. The paraffin was analyzed via HS-SPME, and no volatile compounds were detected in the chromatographic analysis.  
The correlation of linalool concentration and areas of the GC peaks is y = 26 \(\times\) x \(-\) 1.35, R\({}^{2}\) = 0.972. The limit of determination (LOD) has been calculated for both pig samples (lard and muscle), and it is related to a linalool concentration of less than 0.25 nL/g.