A QUANTITATIVE HPLC DETECTION METHOD FOR WHI-P154 [4-(3′-BROMO-4′-HYDROXYLPHENYL)-AMINO-6,7-DIMETHOXYQUINAZOLINE

Abstract

WHI-P154 [4-(3′-bromo-4′hydroxylphenyl)-amino-6,7 di-methoxyquinazoline] is a novel anti-tumor agent with unique cytotoxic activity against human glioblastoma cells (Clin. Cancer. Res. 4:1405-1414, 1998). Further development of WHI-P154 will require detailed pharmacodynamic studies in preclinical animal models. Therefore, we established a sensitive and accurate high performance liquid chromatography (HPLC)-based quantitative detection method for WHI-P154. This method allows the measurement of WHI-P154 levels in plasma, as well as in target human glioblastoma cells. Plasma and cell lysates were extracted with chloroform, dried with nitrogen gas and reconstituted in methanol : water (9:1, v/v). An aliquot was injected into a Hewlett Packard HPLC system employing a 250 × 4mm Lichrospher 100, RP-18 (5 μm) analytical column in conjunction with a 4 × 4 mm Lichrospher 100, RP-18 (5 μm) guard column. The eluted compounds were detected by a diode array detector set at a wavelength of 335 nm. Acetonitrile/water containing 0.1% trifluoroacetic acid and 0.1% triethylamine (28:72, v/v) was used as a mobile phase. The average extraction recovery of WHI-P154 was 78.3% for plasma and 96.0% for U373 human glioblastoma cells. The assay was linear (r>0.999) within the concentration range of 0.1 – 20 μM in 100 μL plasma and within the quantity range of 0.025 – 5 nmol per 2.5 million U373 glioblastoma cells. The intra- and inter-assay variabilities were less than 6% and the lowest detection limit of WHI-P154 was 0.05 μM in plasma and 0.01 nmol in U373 cells, respectively. The practical utility of this new HPLC method was confirmed in pilot pharmacokinetic studies using rats as well as cellular uptake studies using U373 human glioblastoma cells.