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Solid Phase Extraction as an Innovative Separation Method for Measuring Free and Entrapped Drug in Lipid Nanoparticles

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ABSTRACT

Purpose

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.

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Abbreviations

ACN:

Acetonitrile

API:

Active pharmaceutical ingredient

CSA:

Cyclosporine A

CV:

Coefficient of variation

DAD:

Diode Array Detector

DLS:

Dynamic Light Scattering

EDCD:

Entrapped Drug Content Determination

F0:

Fraction 0

F1:

Fraction 1

F2:

Fraction 2

FDCD:

Free Drug Content Determination

HPLC:

High Performance Liquid Chromatographie

ICH:

International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use

LNP:

Lipid nanoparticle

LOD:

Limit of Detection

LOQ:

Limit of Quantification

MeOH:

Methanol

MWCO:

Molecular Weight Cut-Off

n. a.:

Not available/not applicable

NP:

Nanoparticle

PBS:

Phosphate Buffered Saline

PdI:

Polydispersity Index

PEG:

Polyethylene Glycol

R2 :

Determination coefficient

RP:

Reverse Phase

rpm:

Revolutions per minute

RSD:

Relative Standard Deviation

SPE:

Solid Phase Extraction

TDCD:

Total Drug Content Determination

TFA:

Trifluoroacetic acid 0.5% (v/v)

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ACKNOWLEDGMENTS AND DISCLOSURES

This work was sponsored by the German Federal Ministry of Education and Research and financially supported by the EuroNanoMed Project Delivering Nanopharmaceuticals through Biological Barriers ‘BiBa’ (ERA-Net EuroNanoMed Project Number 13N11846).

The authors thank Christina Draheim for performing the DLS preliminary tests and Johannes Krämer for providing the facility and equipment necessary for the investigations.

Author information

Author notes

  1. Alexis Guillot

    Present address: PHAST Development GmbH & Co. KG, Byk-Gulden-Str. 2, Campus Konstanz, D-78467, Constance, Germany

  2. Markus Limberger

    Present address: , Joseph-Haydn-Str. 41, D-66125, Saarbrücken, Germany

Authors and Affiliations

  1. PHAST GmbH, Kardinal-Wendel-Str. 16, D-66424, Homburg, Germany

    Alexis Guillot & Markus Limberger

  2. 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 & Claus-Michael Lehr

  3. Univ Grenoble Alpes, F-38000, Grenoble, France

    Anne-Claude Couffin, Xavier Sejean & Fabrice Navarro

  4. CEA – LETI Minatec, Technologies for Healthcare and Biology Division, 17, rue des Martyrs, F-38054, Grenoble Cedex 09, France

    Anne-Claude Couffin, Xavier Sejean & Fabrice Navarro

  5. Department of Pharmacy, Saarland University, Campus A41, D-66123, Saarbrücken, Germany

    Claus-Michael Lehr

Authors
  1. Alexis Guillot
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  2. Anne-Claude Couffin
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  3. Xavier Sejean
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  4. Fabrice Navarro
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  5. Markus Limberger
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Corresponding author

Correspondence to Alexis Guillot.

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Guillot, A., Couffin, AC., Sejean, X. et al. Solid Phase Extraction as an Innovative Separation Method for Measuring Free and Entrapped Drug in Lipid Nanoparticles. Pharm Res 32, 3999–4009 (2015). https://doi.org/10.1007/s11095-015-1761-8

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  • DOI: https://doi.org/10.1007/s11095-015-1761-8

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