Extraction and analysis by HPLC of the novel compound BCH in formulations, cell culture, and animal tissues
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In order to carry out developmental studies with BCH [cholesteryl 1,12 dicarba-closo-dodecaborane 1-carboxylate] and its formulations, simple, efficient, and safe analytical techniques have been developed whereby BCH may be isolated and quantitated in support of formulation, cell culture, and animal studies. One method, suitable for the analysis of BCH in formulations and cell cultures involves evaporating the sample to dryness under vacuum, extracting the residue with 50:50 methanol:2-propanol (v:v), and analyzing the clear extract by isocratic non-aqueous reverse phase high performance liquid chromatography using ultraviolet absorbance detection (NARP-HPLC-UV). The analyte is eluted from a 5µm 150x4.6mm C-18 analytical column by a 50:50 methanol:2-propanol (v:v) mobile phase and detected by UV absorption at 202nm, the ë max of BCH. A second method, suitable for the analysis of BCH in rat plasma, liver, spleen and intestine utilizes lyophilization to dry the tissue followed by solvent extraction with 50:50 methanol:2-propanol (v:v). The extract is then analyzed by HPLC. The analyte is eluted from a 3.5µm 150x3.0mm C-18 narrow bore column by a linear gradient of methanol to 50:50 methanol:2-propanol (v:v) and detected by UV absorption at 202nm. These methods provide selectivity for BCH in liposomes, microemulsion, the 9-L glioma rat brain cancer cell line, SF763 and SF767 human glioblastoma multiforme cell lines, and rat plasma, liver, spleen, intestine, and brain. BCH is detectable and quantifiable and presents a linear response (R 2 >0.9) in such samples from approximately 100ng/mL to 40,000ng/mL. These methods have been successfully used in support of extensive liposome and microemulsion formulation development, drug uptake studies with cell cultures, and preliminary oral absorption and distribution studies conducted in rats.