title: Development and validation of the first assay method coupling liquid chromatography with chemiluminescence for the simultaneous determination of menadione and its thioether conjugates in rat plasma.
abstract: Menadione (2-methyl-1,4-naphthoquinone, MQ), a component of multivitamin drugs with antihemorrhagic, antineoplastic, and antimalarial activity, is frequently used to investigate quinone-induced cytotoxicity. The formation of MQ conjugates with glutathione (GSH) by Michael addition and subsequent biotransformation to yield N-acetyl-l-cysteine conjugates is believed to be an important detoxification process. However, the resulting conjugates, 2-methyl-3-(glutathione-S-yl)-1,4-naphthoquinone (MQ-GS) and 2-methyl-3-(N-acetyl-l-cysteine-S-yl)-1,4-naphthoquinone (MQ-NAC), retain the ability to redox cycle and to arylate cellular nucleophiles. Although the nephrotoxicity and hepatotoxicity of MQ-thiol conjugates have been reported in vitro, methods for their determination in vivo have yet to be published. Herein, a highly sensitive, simple, and selective HPLC-chemiluminescence (HPLC-CL) coupled method is reported, allowing for the first time the simultaneous determination of MQ, MQ-GS, and MQ-NAC in rat plasma after MQ administration. Our method exploits the unique redox characteristics of MQ, MQ-GS, and MQ-NAC to react with dithiothreitol (DTT) to liberate reactive oxygen species (ROS) which are detected by a CL assay using luminol as a CL probe. To verify the proposed mechanism, MQ-GS and MQ-NAC were synthetically prepared. Specimen preparation involved solid-phase extraction on an Oasis HLB cartridge followed by isocratic elution on an ODS column. No interference from endogenous substances was detected. Linearity was observed in the range of 5-120 nM for MQ-GS and MQ-NAC and 10-240 nM for MQ, with detection limits (S/N of 3) of 1.4, 0.8, and 128 fmol for MQ-GS, MQ-NAC, and MQ, respectively. The application of our method reported here is the first to extensively study the stability and reversibility of thiol-quinones.
authors.affiliation: Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki University , 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
authors.forename: Mohamed Saleh
authors.initials: MS
authors.lastname: Elgawish
authors.forename: Chikako
authors.initials: C
authors.lastname: Shimomai
authors.forename: Naoya
authors.initials: N
authors.lastname: Kishikawa
authors.forename: Kaname
authors.initials: K
authors.lastname: Ohyama
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Wada
authors.forename: Naotaka
authors.initials: N
authors.lastname: Kuroda
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D049449:Luminescence; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013440:Sulfides / Q000097:blood* / Q000737:chemistry*; D024483:Vitamin K 3 / Q000031:analogs & derivatives / Q000097:blood* / Q000737:chemistry*
chemical_list: D013440:Sulfides; D024483:Vitamin K 3