Development and validation of a simple and versatile method for the quantification of everolimus loaded in H-ferritin nanocages using UHPLC-MS/MS

Cristina Sottani, Elena Grignani, Serena Mazzucchelli, Arianna Bonizzi, Fabio Corsi, Sara Negri, Federica Prati, Enrica Calleri, Danilo Cottica

Research output: Contribution to journalArticlepeer-review


Everolimus (Eve) is an immunosuppressive macrolide that is being analyzed in various biological matrices and fluids. Its antitumor activity makes this drug suitable not only for organ transplantation but also for breast cancer treatments. In the attempt to reduce the incidence and severity of its side effects, Eve was loaded in H-ferritin (HFn), a natural biomolecule that is involved in specific cellular uptake pathways. Thus, Eve pre-complexed with Cu(II) and encapsulated in HFn resulted in an Eve nanoformulation, named HEve. The quantification of HEve was performed using a tailored pH-induced procedure to precipitate H-ferritin. This sample preparation was effective enough to reduce the ion suppression effect on the mass spectrometric responses of Eve in electrospray ionization (ESI). The ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-ESI-MS/MS) system operating in positive ionization mode showed to be a versatile technique in achieving more than 77 % recovery of Eve from the cytoplasmic compartment. This simple, selective and sensitive method enabled the quantification of Eve within the linear range of 2.5−100 ng/mL in matrix spiked with the isotope-labeled internal standard, EveD4. This method was validated according to FDA Guidance. The intracellular distribution of HEve and its accumulation at a cytoplasmic level were studied in breast cancer cell lines. As expected, HEve was more effective than free Eve on sensitive (i.e. BT474) and resistant cell lines, as a result of a better penetration into the target subcellular compartment.

Original languageEnglish
Article number113644
JournalJournal of Pharmaceutical and Biomedical Analysis
Publication statusPublished - Nov 30 2020


  • Cytoplasm
  • Everolimus
  • H-ferritin (HFn)
  • Nanopharmaceutical
  • Protein precipitation

ASJC Scopus subject areas

  • Analytical Chemistry
  • Pharmaceutical Science
  • Drug Discovery
  • Spectroscopy
  • Clinical Biochemistry


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