One of the most deleterious solid brain tumors is Glioblastoma with more than 10,000 new cases reported annually in the United States. Regardless its aggressive multimodal treatment approaches, the overall survival is still poor. A previous study from our lab has shown ARN14988, an acid ceramidase inhibitor, to be more cytotoxic compared to FDA-approved, Temozolomide. This initial finding makes ARN14988 a desired candidate for further pharmacological assessment. Therefore, an analytical method is needed to quantify the drug concentration. We have developed and validated a sensitive, and reliable sample preparation and liquid chromatographic–tandem mass spectrometry (LC-MS/MS) method for quantitative determination of ARN14988 in rat plasma and tissues. In this method, the liquid-liquid extraction of ARN14988 from rat plasma or tissue was achieved using 5% ethyl acetate in hexane while the chromatographic separation was achieved with a reverse-phase C<sub>18</sub> column (100 X 2.1 mm, 1.7 µm). The mobile phase consisted of 10mM ammonium acetate at pH 5 and 0.1% formic acid in methanol with a gradient elution at flow rate of 0.3mL/min and runtime of 10 minutes. The samples were detected using triple quadrupole mass spectrometer with multiple reaction monitoring (MRM). The monitored transitions were m/z 391.20 to m/z 147.00 for ARN14988 and m/z 455.30 to m/z 165.00 for Verapamil HCl (internal standard) in positive electrospray ionization. The calibration curve for ARN14988 was linear over the range of 10 ng/mL – 5000 ng/mL (r<sup>2</sup>&gt;0.99) and with lower limit of quantification of 10 ng/mL. The selectivity, sensitivity, matrix effect, recovery, stability, inter-day and intra-day accuracy and precision were determined using four quality control samples: low (10 ng/mL), medium (100 ng/mL), high (1000 ng/mL) and ultra-high (5000ng/mL). This validated method is currently being used for the application of pharmacokinetic and drug distribution studies of ARN14988 in rats.

Development and validation of an LC-MS/MS method for the quantitative determination of  ARN14988, an acid ceramidase inhibitor, in rat model

ACS Spring Meeting 2021

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