Session: Advances in Mass Spectrometry:

Earlier studies from our lab identified acid ceramidase as a novel target for glioblastoma. While acid ceramidase inhibitors are able to kill the tumor cell lines in vitro, a little is known about their blood brain barrier (BBB) permeability which is the most important consideration for glioblastoma drug development. Parallel artificial membrane permeability assay (PAMPA) is a technique that mimics passive and transcellular transmission which is the principal mechanism of BBB permeability for small molecules. PAMPA kit is composed of two parts, the donor plate, and the acceptor plate, and estimates the permeability from donor to acceptor plate over an incubation period. An important consideration while doing PAMPA is to use multiple controls ranging from low to high BBB permeability in order to evaluate the membrane integrity and validation of study results. PAMPA samples from acceptor and donor wells are analyzed using either ultraviolet-visible (UV-Vis) spectrophotometry or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although, the UV-Vis spectrophotometry is an easier, cost effective and faster technique, it can falsely report a result if the test compounds are unstable and multiple compounds in the solution absorb light at the same wavelength. To overcome this limitation, we developed a reliable LC-MS/MS method for the quantification of controls (ranitidine, naproxen, verapamil) and acid ceramidase inhibitors (carmofur, N-oleoylethanolamine, ARN14988). The compounds are retained on an XBridge BEH C18 XP column (100 mm × 3 mm, 2.5 µm) column with a run time of nine minutes. The multiple reaction monitoring (MRM) transitions for the compounds were m/z 256.26 to m/z 129.00 for carmofur, m/z 264.10 to m/z 147.10 for ARN14988, m/z 326.35 to m/z 62.05 for N-oleoylethanolamine. The developed methods were evaluated in terms of selectivity, linearity, carryover, intra-day and inter-day accuracy, precision, and stability. The method shows adequate sensitivity with application to PAMPA and work as complement to UV-Vis spectrophotometry.

Shama Mirza

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