8 hydroxy 2 deoxyguanosine

Fresh royal jelly (RJ) from Apis mellifera L. was obtained from the Honey Bee Farm (Changhua, Taiwan). Fresh RJ was dissolved in 0.01 M phosphate buffered saline (pH 7.2) and heated in a water bath (90 °C) for 30 min to eliminate its enzymatic activity. It was then lyophilized and stored at −20 °C until use.
The enzymes used for protein hydrolysis were alcalase (2.4 Anson units (AU)/g) and flavourzyme (0.5 unit/g) (Sigma Chemicals Co., St. Louis, MO, USA), respectively. TBA (2-thiobarbituric acid, minimum 98%), 8-hydroxy-2′-deoxyguanosine, 2′-deoxyguanosine monohydrate (2′-dG; 99–100%), bleomycin sulphate and calf thymus DNA were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). All other chemicals and solvents used were of analytical grade.

Both 8-hydroxy-2′–deoxyguanosine (purity ≥ 98%) and 5-hydroxymethyluracil (purity 97%) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and 8-hydroxy-2′-deoxyguanosine-15N5 (purity 95%) was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). 8-hydroxyguanosine (purity ≥ 98%), 8-isoprostane (purity ≥ 99%), and 8-isoprostane-d4 (purity ≥ 99%) were obtained from Cayman Chemical (Ann Arbor, MI, USA). 8-hydroxyguanosine-13C,15N2 (purity 98%), 4-hydroxynonenal (purity ≥ 98%), and 4-hydroxynonenal-d3 (purity 97%) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Methanol (LC-MS grade) was purchased from Burdick and Jackson (Muskegon, MI, USA). Formic acid and ammonium hydroxide were obtained from Sigma-Aldrich (St. Louis, MO, USA). Strata-X-A cartridges (33 µm Polymeric Strong Anion, 200 mg/3 mL; 8B-S123-FBJ), analytical columns (Kinetex phenyl-hexyl column, 100 mm × 4.6 mm, 2.6 µm particle size; 00D-4495-E0), and Kinetex C18 column, (100 mm × 4.6 mm, 2.6 μm particle size, 00D-4462-E0) were purchased from Phenomenex (Torrance, CA, USA).