title: Rapid and sensitive drug metabolism studies by SU-8 microchip capillary electrophoresis-electrospray ionization mass spectrometry.
abstract: Monolithically integrated, polymer (SU-8) microchips comprising an electrophoretic separation unit, a sheath flow interface, and an electrospray ionization (ESI) emitter were developed to improve the speed and throughput of metabolism research. Validation of the microchip method was performed using bufuralol 1-hydroxylation via CYP450 enzymes as the model reaction. The metabolite, 1-hydroxybufuralol, was easily separated from the substrate (R(s)=0.5) with very good detection sensitivity (LOD=9.3nM), linearity (range: 50-500nM, r(2)=0.9997), and repeatability (RSD(Area)=10.3%, RSD(Migrationtime)=2.5% at 80nM concentration without internal standard). The kinetic parameters of bufuralol 1-hydroxylation determined by the microchip capillary electrophoresis (CE)-ESI/mass spectrometry (MS) method, were comparable to the values presented in literature as well as to the values determined by in-house liquid chromatography (LC)-UV. In addition to enzyme kinetics, metabolic profiling was demonstrated using authentic urine samples from healthy volunteers after intake of either tramadol or paracetamol. As a result, six metabolites of tramadol and four metabolites of paracetamol, including both phase I oxidation products and phase II conjugation products, were detected and separated from each other within 30-35s. Before analysis, the urine samples were pre-treated with on-chip, on-line liquid-phase microextraction (LPME) and the results were compared to those obtained from urine samples pre-treated with conventional C18 solid-phase extraction (SPE, off-chip cartridges). On the basis of our results, the SU-8 CE-ESI/MS microchips incorporating on-chip sample pre-treatment, injection, separation, and ESI/MS detection were proven as efficient and versatile tools for drug metabolism research.
authors.affiliation: Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5 E, 00014 University of Helsinki, Finland.
authors.forename: Nina
authors.initials: N
authors.lastname: Nordman
authors.forename: Tiina
authors.initials: T
authors.lastname: Sikanen
authors.forename: Maria-Elisa
authors.initials: ME
authors.lastname: Moilanen
authors.forename: Susanna
authors.initials: S
authors.lastname: Aura
authors.forename: Tapio
authors.initials: T
authors.lastname: Kotiaho
authors.forename: Sami
authors.initials: S
authors.lastname: Franssila
authors.forename: Risto
authors.initials: R
authors.lastname: Kostiainen
mesh_terms: D000082:Acetaminophen / Q000378:metabolism* / Q000652:urine; D000085:Acetates; D005591:Chemical Fractionation; D046209:Electrophoresis, Microchip / Q000379:methods*; D004852:Epoxy Compounds / Q000737:chemistry*; D004983:Ethanolamines / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D000432:Methanol; D008956:Models, Chemical; D011108:Polymers / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014147:Tramadol / Q000378:metabolism* / Q000652:urine
chemical_list: D000085:Acetates; D004852:Epoxy Compounds; D004983:Ethanolamines; D011108:Polymers; C520895:SU-8 compound; D000082:Acetaminophen; D014147:Tramadol; C010831:bufuralol; C018824:ammonium acetate; D000432:Methanol