HPLC/UV/MS method application for the separation of obeticholic acid and its related compounds in development process and quality control.

An HPLC method with UV and electrospray ionization - mass spectrometry (ESI-MS) detection was developed for the separation and determination of obeticholic acid (OBE) and its related compounds in development process and quality control. OBE and its related compounds were classified into three major group based on the mass spectra profiles: (A) those containing a hydroxyl group at position 3 and 7, (B) those containing a hydroxyl group and/or carbonyl group at position 3, hydrogen, ethyl or ethylidene group at position 6 and a hydroxyl group and/or carbonyl group at position 7, and (C) those containing carbonyl groups at position 3 and 7. ESI-MS ionization of OBE and its related compounds often produced intense adduct ions [M+H+98]+ and/or [M+H+196]+ that were identified as the adduct ions of phosphoric acid ([M+H+H3PO4]+ and [M+H+2H3PO4]+) originating from the mobile phase. The separation on HPLC system was accomplished using stationary phase based on XSelect CSH C18 (3.0×150mm×2.5µm) and a linear gradient elution using acetonitrile and 0.05% of o-phosphoric acid. The condition of chromatographic system was set as follows: flow rate 0.7mL/min, temperature 45°C and UV detection at 192nm. The separation of the 19 compounds was finished in less than 18min (including equilibration time). The HPLC/UV method was partially validated according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines in terms of repeatability, selectivity, linearity and limit of quantification and detection.

Identification, characterization, synthesis and HPLC quantification of new process-related impurities and degradation products in retigabine.

Two new impurities were described and determined using gradient HPLC method with UV detection in retigabine (RET). Using LC-HRMS, NMR and IR analysis the impurities were identified as RET-dimer I: diethyl {4,4'-diamino-6,6'-bis[(4-fluorobenzyl)amino]biphenyl-3,3'-diyl}biscarbamate and RET-dimer II: ethyl {2-amino-5-[{2-amino-4-[(4-fluorobenzyl) amino] phenyl} (ethoxycarbonyl) amino]-4-[(4-fluorobenzyl)amino] phenyl}carbamate. Reference standards of these impurities were synthesized followed by semipreparative HPLC purification. The mechanism of the formation of these impurities is also discussed. An HPLC method was optimized in order to separate, selectively detect and quantify all process-related impurities and degradation products of RET. The presented method, which was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ) and selectivity is very quick (less than 11min including re-equilibration time) and therefore highly suitable for routine analysis of RET related substances as well as stability studies.