title: Comprehensive quantitative and qualitative liquid chromatography-radioisotope-mass spectrometry analysis for safety testing of tolbutamide metabolites without standard samples.
abstract: Liquid chromatography-radioisotope-mass spectrometry (LC-RI-MS) analysis was used to determine the structures of 12 (four previously unknown) (14) C-tolbutamide (TB) metabolites in rat biological samples (plasma, urine, bile, feces, and microsomes). The four novel metabolites are ω-carboxy TB, hydroxyl TB (HTB)-O-glucuronide, TB-ortho or meta-glutathion, and tolylsulphoaminocarbo-glutathion. In rat plasma, after oral administration of (14) C-TB at therapeutic dose (1 mg/kg) and microdose (1.67 µg/kg), the total RI and six metabolites [HTB, carboxy TB (CTB), M1: desbutyl TB, M2: ω-hydroxyl TB, M3: α-hydroxyl TB, and M4: ω-1-hydroxyl TB] were quantified by LC-RI-MS. The plasma concentrations were calculated using their response factors (MS-RI intensity ratio) without standard samples, and the area under the curve (AUC) of plasma concentration per time for evaluation of Safety Testing of Drug Metabolites (MIST) was calculated using the ratio of TB metabolites AUC/total RI AUC. The ratios were as follows: TB 94.5% and HTB 2.5% for the microdose (1.67 µg/kg) and TB 95.6%, HTB 0.96%, CTB 0.065%, M1 0.62%, M2 0.0035%, M3 0.077%, and M4 0.015% for the therapeutic dose (1 mg/kg). These values were less than 10% of the MIST criteria.
authors.affiliation: ADME & Tox. Research Institute, Sekisui Medical Company, Ltd., Tokai, Ibaraki 319-1182, Japan. tozuka-z@admet.sekisuimedical.jp
authors.forename: Zenzaburo
authors.initials: Z
authors.lastname: Tozuka
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Aoyama
authors.forename: Kohei
authors.initials: K
authors.lastname: Nozawa
authors.forename: Shoji
authors.initials: S
authors.lastname: Akita
authors.forename: Toshinari
authors.initials: T
authors.lastname: Oh-Hara
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Adachi
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Ninomiya
mesh_terms: D000818:Animals; D019540:Area Under Curve; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007004:Hypoglycemic Agents / Q000097:blood / Q000493:pharmacokinetics* / Q000652:urine; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008861:Microsomes / Q000378:metabolism; D011868:Radioisotopes / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014044:Tolbutamide / Q000097:blood / Q000493:pharmacokinetics* / Q000652:urine
chemical_list: D007004:Hypoglycemic Agents; D011868:Radioisotopes; D014044:Tolbutamide