Effect of mobile phase pH on liquid chromatography retention of mepartricin related compounds and impurities as support to the structural investigation by liquid chromatography-mass spectrometry

: Mepartricin is a semisynthetic polyene macrolide with antifungal and anti-protozoal activities, and it is widely used for the treatment of benign prostatic hyperplasia. Mepartricin is produced by synthetic methyl esterification of the more toxic partricin, and its activity is due to a complex of related compounds. Among them, the main ones are mepartricin B and mepartricin A which are characterized by the presence of a primary and a secondary amine group, respectively. In this work a previously reported HPLC-UV method was properly modified to make it MS-compatible. The selected conditions entail the use of a C18 reverse phase column, and a mobile phase composed by ammonium formate and acetonitrile, with the addition of heptafluorobutyric acid as modifier. The developed method was applied to the characterization of a mepartricin reference standard and a mepartricin experimental batch. All the UV responding peaks, 30 for the standard and 21 for the experimental batch, were successfully detected by MS, allowing to define their m/z values and acquire their fragmentation spectra. For the structural elucidation of isobaric species and, in particular, the identification of toxic partricin-related impurities, the presence of differently ionisable chemical groups was considered, as partricins contain free caboxy-groups, while mepartricins represent their estherified counterparts. A deep study of the effect of mobile phase pH on the chromatographic retention of partricin and mepartricin related compounds was performed in the pH range 2.5-6.5. This study allowed to successfully cluster all the detected species and asses, in the considered batch, the absence of other partricin-related impurities in addition to partricin B and partricin A.