title: Solid Phase Extraction as an Innovative Separation Method for Measuring Free and Entrapped Drug in Lipid Nanoparticles.
abstract: OBJECTIVE
  Contrary to physical characterization techniques for nanopharmaceuticals (shape, size and zeta-potential), the techniques to quantify the free and the entrapped drug remain very few and difficult to transpose in routine analytical laboratories. The application of Solid Phase Extraction (SPE) technique was investigated to overcome this challenge.
  
  
  METHODS
  The separation of free and entrapped drug by SPE was quantitatively validated by High Performance Liquid Chromatography. The developed protocol was implemented to characterize cyclosporine A-loaded 120 nm-sized lipid nanoparticles (LNPs, Lipidot®) dispersed in aqueous buffer. The colloidal stability was assessed by Dynamic Light Scattering (DLS).
  
  
  RESULTS
  Validation experiments demonstrated suitable linearity, repeatability, accuracy and specificity to quantify residual free, entrapped and total drug. For the investigated LNPs, the method revealed a very limited shelflife of the formulation when stored in an aqueous buffer at 5°C and even more at elevated temperature. Nevertheless, the DLS measurements confirmed the stability of nanoparticles during SPE in a suitable concentration range.
  
  
  CONCLUSIONS
  SPE, when successfully validated, represents a valuable tool for drug development and quality control purposes of lipid-based nanopharmaceuticals in an industrial environment.
authors.affiliation: PHAST GmbH, Kardinal-Wendel-Str. 16, D-66424, Homburg, Germany. alexis.guillot@phast.com.|Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) Helmholtz Center for Infection Research (HZI), Department DDEL, Saarland University, Campus A41, D-66123, Saarbrücken, Germany. alexis.guillot@phast.com.|PHAST Development GmbH & Co. KG, Byk-Gulden-Str. 2, Campus Konstanz, D-78467, Constance, Germany. alexis.guillot@phast.com.
authors.forename: Alexis
authors.initials: A
authors.lastname: Guillot
authors.affiliation: Univ Grenoble Alpes, F-38000, Grenoble, France.|CEA - LETI Minatec, Technologies for Healthcare and Biology Division, 17, rue des Martyrs, F-38054, Grenoble Cedex 09, France.
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Couffin
authors.affiliation: Univ Grenoble Alpes, F-38000, Grenoble, France.|CEA - LETI Minatec, Technologies for Healthcare and Biology Division, 17, rue des Martyrs, F-38054, Grenoble Cedex 09, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: Sejean
authors.affiliation: Univ Grenoble Alpes, F-38000, Grenoble, France.|CEA - LETI Minatec, Technologies for Healthcare and Biology Division, 17, rue des Martyrs, F-38054, Grenoble Cedex 09, France.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Navarro
authors.affiliation: PHAST GmbH, Kardinal-Wendel-Str. 16, D-66424, Homburg, Germany.|, Joseph-Haydn-Str. 41, D-66125, Saarbrücken, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Limberger
authors.affiliation: Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) Helmholtz Center for Infection Research (HZI), Department DDEL, Saarland University, Campus A41, D-66123, Saarbrücken, Germany.|Department of Pharmacy, Saarland University, Campus A41, D-66123, Saarbrücken, Germany.
authors.forename: Claus-Michael
authors.initials: CM
authors.lastname: Lehr
mesh_terms: D002851:Chromatography, High Pressure Liquid; D016572:Cyclosporine / Q000008:administration & dosage / Q000737:chemistry / Q000302:isolation & purification*; D004337:Drug Carriers / Q000737:chemistry*; D004355:Drug Stability; D007166:Immunosuppressive Agents / Q000008:administration & dosage / Q000737:chemistry / Q000302:isolation & purification*; D008055:Lipids / Q000737:chemistry*; D053758:Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D052616:Solid Phase Extraction / Q000379:methods*
chemical_list: D004337:Drug Carriers; D007166:Immunosuppressive Agents; D008055:Lipids; D016572:Cyclosporine
keywords: cyclosporine; lipid nanoemulsion; nanoparticles; separation techniques; solid-phase extraction