Human liver microsomes

Stock solutions of the studied compounds were made at a concentration of 1 mM in DMSO:MeOH mixture (10∶90 v/v) to prevent their precipitation. For biotransformation incubations, the solution of compounds was diluted to 10 µM with the presence of 1 mM of NADPH (Sigma-Aldrich, St. Louis, MO, USA) in a potassium phosphate buffer (0.1 M, pH 7.4). Incubation was carried out in thermoblock (Labnet International, Edison, NJ, USA) at 37°C and started with the addition of pooled human liver microsomes with a final concentration of 0.53 mg/mL (Sigma-Aldrich, St. Louis, MO, USA). Directly after HLM was added and at 120 min of incubation the reaction was ended by the addition of an equal volume of ice-cold methanol with 0.1% (v/v) of formic acid. The samples were immediately centrifuged (10 min at 11700 g) and the resulting supernatant was directly analyzed or kept at −80°C until LC-MS or LC-QTOF-MS analysis.
Additionally, chemical stability (negative control) in biotransformation experiment conditions was performed (phosphate buffer, 37°C, 2 h) to evaluate possible instability, unrelated to the activity of microsomes (including putative isomerization).

The metabolic stability of 4NSG was examined in the presence of PBS (pH 7.4) and Human liver microsomes as previously described [25 (link), 26 (link)]. Human liver microsomes were obtained from Sigma-Aldrich. 4NSG (10 µM) was spiked into PBS, or liver microsomes (1 mg protein/mL) and incubated in a shaking water bath at 37 °C for 8 h. Aliquots (100 µL) were taken after 0, 15, 30, 60, 120, 180, 220 and 240 min of incubation and immediately pretreated with an ice-cold methanolic solution including the internal standard, vortexed for 3 min and centrifuged (12,000 × g, 10 min). Supernatants were stored at -80 C until required for analysis [25 (link), 26 (link)].

Uridine 5′-diphosphoglucuronic acid trisodium salt (UDPGA Na), Tris buffer (PH = 7.5) and human liver microsomes (HLM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). AZT, FCZ, azidothymidine-glucuronide (AZTG), PBS buffer and potassium hydroxide were purchased from Melone Pharmaceutical Co., Ltd. (Dalian, China). Alamethicin, bovine serum albumin (BSA) and magnesium chloride were purchased from J&K Scientific Ltd. (Beijing, China). Perchloric acid (HCLO₄) was purchased from the hazardous compound platform of Fudan University. Distilled and deionized water was purified from Milli-Q water system (Millipore, Molsheim, France). Other reagents were of analytical grade.

Amoxicillin (AMOX) was supplied by Sigma-Aldrich (Schnelldorf, Germany). Human liver microsomes (HLM), glucose-6-phosphate, β-NADP⁺, glucose-6-phosphate dehydrogenase and uridine 5′-diphosphoglucuronic acid triammonium salt (UDPGA) were obtained from Sigma-Aldrich (Schnelldorf, Germany). Other chemicals of analytical quality (acetonitrile, formic acid) were purchased from Merck (Darmstadt, Germany). Water was obtained by means of Milli-Q RG apparatus by Millipore (Millipore Intertech, Bedford, MA, USA) in our laboratory.