Imikhiqizo

Ukuze silinganise ngokuqondile ama-metabolites amaningana e-5-fluorouracil (5-FU) kanye nama-nucleotide amabili e-monophosphate, sithuthukise indlela yoqobo esekelwe ku-liquid chromatography–tandem mass spectrometry (LC-MS/MS).Lokhu kuhlolwa kuvumele ukunqunywa: (i) ukukhiqizwa kwe-intracellular ye-5-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP) kusuka ku-5-FU noma 5-fluoro-2'-deoxyuridine (5-FdUrd) , (ii) umthelela wokugxilisa kwe-5-FdUMP ku-intracellular 2′-deoxyuridine-5′-monophosphate (dUMP)/thymidine-5′-monophosphate (TMP) ratio, kanye (iii) nezinga lokuphuma kwe-5-FdUMP kanye 5-FU kusuka kumaseli akhuliswe abantu ngabathuthi be-ABC.Ngaphansi kwezimo zethu zokuhlola, amaseli afakwe i-5-FU noma i-5-FUrd.Khona-ke, amaprotheni amaselula adonswa yi-methanol.Le nqubo inikeze ukutholwa okuphezulu kokukhipha.Ukwengeza, ukukala ukukhishwa kwe-5-FU kanye ne-5-FdUMP kusuka kumaseli, senze ukulinganisa kwamanani alawa ma-molecule endaweni yesiko.Imidiya ijovwe ngokuqondile (i-5-FU) noma yahamba ngesigaba esiqinile kusetshenziswa i-Oasis Wax extraction cartridge (5-FdUMP).Ukuhlukaniswa kokuhlaziya kwenziwe kukholomu ye-dC18 Atlantis 3.5 μm, (100 mm × 2.1 mm id) enemodi ye-isocratic kusetshenziswa i-ammonium formate buffer/methanol/water (5/5/90, v/v) njengesigaba seselula.Isikhathi sokugijima asidluli ku-6.2 min.Ama-analyte ayenziwe ionized ku-interface ye-electrospray ngaphansi kwemodi ye-ion engalungile.Siqinisekise indlela phezu kobubanzi be-2.5-150 ng mL -1 ngokusho kwezinhlanganisela.Ukwehluka kwangaphakathi naphakathi kwe-assay bekungaphansi kuka-10% ezinsukwini eziyisikhombisa.Zonke izinhlanganisela bezizinzile kumaseli noma endaweni yesiko lapho amasampula egcinwe ku-−20 °C okungenani amasonto amabili, nangemuva kwemijikelezo emithathu yokuncibilika kweqhwa.Awukho umphumela we-matrix obonwe kuyo yomibili imidiya.