జర్నల్ ఆఫ్ ఎనలిటికల్ & బయోఅనలిటికల్ టెక్నిక్స్

నైరూప్య

A Standard Addition Method Utilizing an Endogenous Substance as an Internal Standard for Quantitating Arabinofuranosylguanosine 5’-Triphosphate in Human Peripheral Blood Mononuclear Cells by Lc-Ms/Ms

Yoshiyuki Minamide, Harue Igarashi, Akira Wakamatsu and Shinobu Kudoh

A highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for quantitation of arabinofuranosylguanosine 5’-triphosphate (ara-GTP) in human peripheral blood mononuclear cells (PBMC). The determination of ara-GTP in PBMC was validated using a standard addition method with the human Tlymphoblastoid cell line as an alternative blank matrix. Ara-GTP was extracted with methanol/250 mmol/L ammonium carbonate solution (7/3, v/v) from the cells at a density of 106 cells per 0.5 mL. Extracts were subjected to LC-MS/ MS using a TurboIon spray interface and selected reaction monitoring with the transitions of m/z 524 to m/z 152 for quantitation. Endogenous guanosine triphosphate in the extract was used as an internal standard. Separation of the analytes was achieved on a porous graphitic carbon column (100 mm length × 2.1 mm i.d., 5 μm particle size) by isocratic elution with 250 mmol/L ammonium carbonate buffer (pH 9.5)/water/acetonitrile (40/51.5/8.5, v/v/v) at a flow rate of 0.2 mL/min. The method was validated in the range of 2–250 pg/mL. The pharmacokinetic profile of ara-GTP in PBMC in a Phase I clinical study of nelarabine in relapsed or refractory T-ALL/T-LBL patients was successfully determined using this method.