Development of a chromatographic bioreactor based on immobilized beta-glucuronidase on monolithic support for the determination of dextromethorphan and dextrorphan in human urine.
We here reported the development and application of an immobilized enzyme reactor (IMER) based on beta-glucuronidase to the on-line determination of urinary molar ratios of dextromethorphan (DOMe)/dextrorphan (DOH) for the assessment of the metabolic activity of CYP2D6, a genetically variable isoform of cytochrome P-450 (CYP). beta-Glucuronidase was immobilized on an HPLC monolithic aminopropyl silica support. Catalytic activity and stability of the chromatographic reactor were evaluated using 8-hydroxyquinoline glucuronide (8-HQG) as substrate. The IMER was coupled through a switching valve to a reversed-phase column (C8) for the simultaneous determination of dextromethorphan and its main metabolite dextrorphan. On purpose a selective reversed-phase ion pair HPLC method coupled with fluorescence detection was developed. Urine samples were first centrifuged to remove insoluble materials and then aliquots of the supernatants were injected into the coupled-column analyser. Linearity, precision and accuracy of the method were established. The method reliability was verified by comparing our data with previous data of a phenotyping study carried out by the Poison Control Centre of Pavia-Clinical Toxicology Division.
Calleri
E
E
Department of Pharmaceutical Chemistry, University of Pavia, Via Taramelli 12, Pavia I-27100, Italy. enrica.calleri@unipv.it
Marrubini
G
G
Massolini
G
G
Lubda
D
D
de Fazio
S S
SS
Furlanetto
S
S
Wainer
I W
IW
Manzo
L
L
Caccialanza
G
G
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism; D003915:Dextromethorphan / Q000378:metabolism / Q000652:urine*; D003917:Dextrorphan / Q000378:metabolism / Q000652:urine*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005966:Glucuronidase / Q000378:metabolism*; D006801:Humans; D015394:Molecular Structure; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004800:Enzymes, Immobilized; D003917:Dextrorphan; D003915:Dextromethorphan; D019389:Cytochrome P-450 CYP2D6; D005966:Glucuronidase