LC–MS/MS analysis

The nirmatrelvir stock solutions were prepared in DMSO at 1 mg/ml and further diluted to obtain a working solution (WS) at 20 μg/ml. The drug JWH‐250 was used as internal standard. The internal standard working solutions (IS‐WS) was prepared at 20 ng/ml in methanol:acetonitrile (50:50, v/v) acidified with 0.1% formic acid.

Plasma of mice were collected, centrifuged at 10,621 g for 10 min, and incubated at 60°C for 30 min. The mixture of 30 μl of plasma, 105 μl of IS‐WS, and 15 μl of WS was vortex for 1 min and centrifuged at 12,500 g for 10 min at 4°C. The supernatant was collected and 100 μl were injected into the liquid chromatography tandem mass spectrometry (LC–MS/MS) system. The HPLC equipment consisted of an LC AC System from AB Sciex (Toronto, ON, Canada). A Triple Quadrupole Mass Spectrometer (API 2000) from AB‐Sciex (Toronto, ON, Canada) was used for detection. The analytes were separated using an Acquity UPLC BEH C18 1.7 μm Column (2.1 × 50 mm ID) from Waters. The mobile phases were (B) MeOH containing 0.2% formic acid and (A) water containing 0.1% formic acid, at a flow rate of 0.4 ml/min, and were entirely transferred into the mass spectrometer source. The gradient elution was as follows: increase in the organic phase from 10 to 100% in 2 min and after 1.5 min of 100% B, the column was led to the original conditions; equilibration of the column was achieved in 2 min. Both analytes were detected in positive ionization with a capillary voltage of 4,500 V, nebulizer gas (air) at 45 psi, turbo gas (nitrogen) at 70 psi, and 450°C. The other ion source parameters were set as follows: curtain gas (CUR) 25 psi; collision gas (CAD) 6 psi; declustering potential 80 V; and entrance potential 8 V. Instrument conditions optimization was performed by direct infusion and manual tuning. Data collection and elaboration were performed by means of Analyst 1.4 software (AB‐ Sciex). The quantitative data were acquired using multi reaction monitoring (MRM) mode. Two MRM transitions (precursor ion > fragment ion) were selected for the analytes. The parameters used for each analyte are listed in the Appendix Table S1.

The analytical method was validated according to FDA guidelines for bioanalytical method validation. Linearity, precision, accuracy, limits of detection (LODs), and limits of quantification (LOQ) were evaluated. Calibration standard solutions were prepared in blank plasma by spiking 15 μl of a standard mixture at appropriate concentration to 30 μl of plasma and by adding 105 μl of methanol:acetonitrile (50:50, v/v). Calibrators were then treated similarly to the animal samples. The calibration range was 2–750 ng/ml and the calibrators were prepared at nine level of concentration. Precision, recovery, and accuracy were evaluated at three level of concentrations (25, 100, and 750 ng/ml) and resulted within the acceptable limits. LOD was defined as the lowest concentration with a signal‐to‐noise (S/N) ratio greater than 3. LOQ was defined as the concentration at which both precision (RSD %) and accuracy were less than 20%. LOQ resulted to be 2 ng/ml, while LOD was 1 ng/ml for both analytes.