Human k2edta plasma

NHC and NHCtp, as well as their respective internal standards, β-D-N⁴-hydroxycytidine-¹³C₅ (NHC-IS) and β-D-N⁴-hydroxycytidine-triphosphate-¹³C₅ (NHCtp-IS), were generously provided by Ridgeback Therapeutics (Miami, FL) through an agreement with the Emory Institute for Drug Development (Atlanta, GA). All compounds arrived with certificates of analysis. Purities were determined by HPLC-UV and ¹H NMR; purities of NHC and NHC-IS were 99.7% and 96.1%, respectively; purities of NHCtp and NHCtp-IS were 97.2% and 90.2%, respectively. Chemical structures for NHC and NHCtp are shown in Fig. 1. Drug-free human K₂EDTA plasma and isolated human PBMC at a final concentration of approximately 100 × 10⁶ cells per donor were acquired from BioIVT (Hicksville, NY). PBMC were lysed in 70% methanol to achieve a final lysate concentration of 2 × 10⁶ cells/mL.Fig. 1

Structures and molecular weights of (A) NHC (C₉H₁₃N₃O₆) and (B) NHCtp (C₉H₁₆N₃O₁₅P₃). The asterisk (*) denotes position of isotopically labeled atoms for internal standards.

Optima™ LC/MS-grade water, methanol, acetonitrile, water with 0.1% formic acid, acetic acid, and reagent-grade 14.8 M ammonium hydroxide were acquired from Fisher Scientific (Fair Lawn, NJ). Reagent-grade formic acid and 10 M ammonium formate were purchased from Sigma-Aldrich (St. Louis, MO).