Isolation and Mass Spectrometric Analysis of Cyclosporine Metabolites Isolation of Metabolites AM19 and AM1c9. The CsA secondary metabolites AM19 and AM1c9

CsA, bovine serum albumin, and β-nicotinamide adenine dinucleotide 2’-phosphate reduced tetrasodium salt hydrate (NADPH), were purchased from Sigma-Aldrich (St. Louis, MO). Cyclosporine D (internal standard for clinical study) and cyclosporine B (internal standard for in vitro study) were purchased from LKT Laboratories (St. Paul, MN). The CsA metabolites used for calibration curves and quality control were generated using a bioreactor approach (see below). Blank (outdated) human plasma and blood were purchased from Puget Sound Blood Center (Seattle, WA). Methanol (Optima grade), acetonitrile (Optima grade), ethyl acetate (Optima grade), ammonium acetate, hydrochloric acid, sodium hydroxide, potassium phosphate monobasic, potassium phosphate dibasic, and silanized glass LC inserts were purchased from Fisher Scientific (Santa Clara, CA). Methyl tert-butyl ether (J.T. Baker) was purchased from VWR International (USA). CYP3A4 + b5 SupersomesTM (Catalog No. 456202) and human CYP3A5 + b5 Supersomes TM (Catalog No.456256) were purchased from BD Biosciences (Woburn, MA).