Nexera xr lc system

For detection and analysis of RDV and GS-441524, our previously validated HPLC with fluorescence detection was utilised, using a Shimadzu Nexera XR LC system (Rydalmere, NSW, Australia) (Kimble et al. 2023 (link)). The Waters X-Bridge C18, 5 µm, 150 × 4.6 mm (Dundas, NSW, Australia) was selected as the stationary phase with the column temperature maintained at 35 °C. Flow rate was set at 1.2 mL/min and analytes monitored with excitation and emission wavelengths of 250 nm and 475 nm, respectively. For screening of GS-704277, GS-441524 and RDV, a mixture of 20 mM of ammonium acetate (adjusted to pH 4.5 with acetic acid) with solutions of either 5% of acetonitrile (Mobile phase A) or 70% of acetonitrile (Mobile phase B) were prepared for the following gradient: 0–1 min (0–5% B); 1–2 min (5% B); 2–5 min (5–50% B); 5–10 min (50–90% B); 10–11 min (90% B); and 11–12 min (90–0%). Total run time was 15 min to equilibrate the condition; where retention times of GS-441524 and RDV were 4.78 and 9.95 min, respectively. For monitoring the RDV peak area for both in vitro t_1/2 and intrinsic clearance, isocratic mobile phase (45% MeCN in 20 mM of ammonium acetate, adjusted to pH 4.5 with acetic acid) was used.

For detection and analysis of GS441524, a Shimadzu Nexera XR LC system (Rydalmere, NSW, Australia) was used. For the mobile phase, a mixture of 20 mM of ammonium acetate (adjusted to pH 4.5 with acetic acid) with solutions of either 5% of acetonitrile (A) or 70% of acetonitrile (B) prepared for following gradient: 0–1 min (0–5% B), 1–2 min (5% B), 2–5 min (5–50% B), 5–10 min (50–90% B), 10–11 min (90% B), and 11–12 min (90–0%) at a flow rate of 1.2 mL/min. A total run time was 15 min in order to equilibrate the condition. After testing several columns (Synergi 4 µm, 150 × 4.6 mm, Hypersil ODS 5 µm, 4.6 mm × 150 mm, and Discovery® C18, 5 µm, 4.6 mm × 250 mm), the Waters X-Bridge C18, 5 µm, 150 × 4.6 mm (Dundas, NSW, Australia) was selected as the stationary phase with the column temperature maintained at 35 °C. A fluorescence detector (RF-20A xs) (Shimadzu, Rydalmere, NSW, Australia) was used to monitor GS-441524 with excitation and emission wavelengths of 250 nm and 475 nm, respectively.