title: Development of a quantification method for digoxin, a typical P-glycoprotein probe in clinical and non-clinical studies, using high performance liquid chromatography-tandem mass spectrometry: the usefulness of negative ionization mode to avoid competitive adduct-ion formation.
abstract: Highly sensitive and accurate liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for measuring digoxin (DGX), a typical P-glycoprotein probe, in human plasma, rat plasma, and rat brain. We extracted DGX and deuterium-labeled DGX (as internal standard) from sample fluids under basic conditions using acetonitrile and sodium chloride-saturated 0.1 mol/L sodium hydroxide. The upper organic layer was diluted with distilled water, and the resulting solution was injected into an LC/MS/MS system in negative ionization mode. Chromatographic separation was achieved on a C(18)-ODS column in the gradient mobile phase, which comprised 0.05% (w/v) ammonium carbonate (pH 9.0) and methanol at a flow rate of 0.7 mL/min. Regardless of the type of biological matrix, intra-day and inter-day validation tests demonstrated good linearity of calibration curves within ranges of 0.1-10 ng/mL for plasma and 0.5-50 ng/g for rat brain and gave excellent accuracy and precision of quality control samples at 4 concentration levels. Unlike existing methods, our approach uses negative ionization to avoid competitive adduct formation of DGX. Our method showed higher sensitivity and wider applicability to various types of biological matrices than existing methods. Our method will support clinical and preclinical investigation of in vivo P-glycoprotein functionality using DGX.
authors.affiliation: Drug Metabolism and Pharmacokinetic Research Laboratories, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan. Hirabayashi_Hideki@takeda.co.jp
authors.forename: Hideki
authors.initials: H
authors.lastname: Hirabayashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sugimoto
authors.forename: Shinichi
authors.initials: S
authors.lastname: Matsumoto
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Amano
authors.forename: Toshiya
authors.initials: T
authors.lastname: Moriwaki
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004077:Digoxin / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D004355:Drug Stability; D057230:Limit of Detection; D016014:Linear Models; D059625:Liquid-Liquid Extraction; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D004077:Digoxin