LCMS grade water, methanol, acetonitrile and acetic acid were purchased through Fisher Scientific. All synthetic standards for molecular analysis were purchased from MedChemExpress. Clarified lung homogenates were gamma-irradiated (2 megarads) for removal from biocontainment according to IBC-approved protocol41. Standard curves of MK-4482 and EIDD-1931 were made in lung homogenate from uninfected animals and subjected to irradiation to account for molecular degradation. Samples were prepared for analysis by adding 300 µL of methanol to 100 µL of homogenate and incubating at 4 oC for 30 min to precipitate macromolecules. Samples were centrifuged at 16,000g at 4 oC and the supernatant was transferred to a sample vial for LCMS analysis. Samples were separated by HILIC chromatography on a Sciex ExionLC™ AC system. Samples were injected onto a Waters XBridge® Amide column (130 Å, 3.5 µm, 3 mm × 100 mm) and eluted using a binary gradient from 95% acetonitrile, 0.8% acetic acid, 10 mM ammonium acetate to 50% acetonitrile, 0.8% acetic acid, 10 mM ammonium acetate over 8 min. Analytes were measured using a Sciex 5500 QTRAP® mass spectrometer in positive mode with electrospray ionization (CUR: 40, CAD: Med, ISV: 2500, Temp: 450, GS1: 50, GS2: 50). Multiple reaction monitoring (MRM) was performed using the optimized conditions in Supplementary Table 3. To ensure signal fidelity triggered spectra were compared back to synthetic standards. Previously published MRM signals for biological nucleosides were utilized to confirm minimal interference at the retention time of interest42. All analytes were quantified against an 8-point calibration curve of the respective synthetic standard prepared in the target matrix and processed in the same manner as experimental samples. Limit of quantification in lung homogenate after irradiation was 5 ng/mL for EIDD-1931 and 50 pg/mL for MK-4482.

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