Bdh aristar plus

Glacial acetic acid (BDH aristar^® Plus) was purchased from VWR (Radnor, PA, USA). Sodium hydroxide (Emsure^®), HPLC-grade water, methanol, acetonitrile and biosynthesis-grade dichloromethane (OmniSolv^®) were obtained from EMD Millipore Chemicals (Billerica, MA, USA). Hydroxocobalamin acetate (vitamin B_12a, OH-Cbl) was purchased from Hebei Huarong Pharmaceutical Co. (Hebei Province, China). Cyanocobalamin (CN-Cbl), dicyanocobinamide (DCC) and 5,6-dimethylbenzimidazole (DMBI) reference standards were purchased from Sigma-Aldrich Co. (St. louis, MO, USA). nitrocobalamin (NO₂-Cbl) was synthesized as described previously in the literature [13 ]. nitric oxide gas (99.5 %) was purchased from Praxair (Bethlehem, PA, USA).

Samples were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) on an X-Series 2 instrument (Thermo Scientific) using a 1 ml sample loop. Manganese, Fe, Cu, and Zn, were quantified using isotope standards for ⁵⁵Mn, ⁵⁷Fe, ⁶⁵Cu, and ⁶⁴Zn respectively. All samples were normalized to the ¹⁰³Rh signal. Iron was analyzed using collision cell technology to reduce polyatomic interferences. Operating conditions of the ICP-MS are presented in Table 1. Quantification was performed with external calibration curves (n ≥ 3) in 2% nitric acid at concentrations from 0.1 to 250 ng/ml. A standard metal mix (82026–108, BDH Aristar® Plus, VWR International) was used to quantitate all metals.