title: Identification of novel rapamycin derivatives as low-level impurities in active pharmaceutical ingredients.
abstract: We describe the identification of novel rapamycin derivatives present as low-level impurities in active pharmaceutical ingredients using an integrated, multidisciplinary approach. Rapamycin, a fermentation-derived natural product is itself used clinically and provides the starting material for several rapamycin analog drugs, typically used in oncology. LC-MS proved a sensitive means to analyze impurity profiles in batches of rapamycin. MS fragmentation was used to gain structural insight into these impurities, usually fermentation by-products, structurally very similar to rapamycin. In cases where MS fragmentation was unable to provide unambiguous structural identification, the impurities were isolated and purified using orthogonal HPLC methods. Using the higher mass sensitivity of small-volume NMR microprobes, submilligram amounts of isolated impurities were sufficient for further characterization by multidimensional NMR spectroscopy. Full assignment of the (1)H and (13)C NMR signals revealed the structure of these impurities at an atomic level. This systematic workflow enabled the identification of several novel rapamycin congeners from active pharmaceutical ingredient without the need for large-scale isolation of impurities. For illustration, two novel rapamycin derivatives are described in this study: 12-ethyl-rapamycin and 33-ethyl-rapamycin, which exemplify previously unreported modifications on the carbon skeleton of the rapamycin macrocycle. The methodologies described here can be of wide use for identification of closely related structures found; for example as fermentation by-products, metabolites or degradants of natural product-based drugs.
authors.affiliation: ARIAD Pharmaceuticals, Cambridge, MA 02139, USA. Stephan.Zech@ariad.com
authors.forename: Stephan G
authors.initials: SG
authors.lastname: Zech
authors.forename: Michael
authors.initials: M
authors.lastname: Carr
authors.forename: Qurish K
authors.initials: QK
authors.lastname: Mohemmad
authors.forename: Narayana I
authors.initials: NI
authors.lastname: Narasimhan
authors.forename: Christopher
authors.initials: C
authors.lastname: Murray
authors.forename: Leonard W
authors.initials: LW
authors.lastname: Rozamus
authors.forename: David C
authors.initials: DC
authors.lastname: Dalgarno
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D004340:Drug Contamination; D009682:Magnetic Resonance Spectroscopy; D004364:Pharmaceutical Preparations / Q000032:analysis / Q000737:chemistry*; D020123:Sirolimus / Q000737:chemistry* / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D004364:Pharmaceutical Preparations; D020123:Sirolimus