Title:A Reliable HPLC-ELSD Method for Determination of Cholesterol, Phosphatidylcholine, Lysophosphatidylcholine Content and the Stability of a Liposomal Formulation
VOLUME: 16 ISSUE: 5
Author(s):Naiara Ieza Gallo de Magalhães Benedetti, Danillo Fabrini Maciel Costa Veloso, Thais Leite Nascimento, Danielle Guimarães Almeida Diniz, Lorena Maione-Silva and Eliana Martins Lima*
Affiliation:Laboratorio de Nanotecnologia Farmaceutica e Sistemas de Liberacao de Farmacos, Faculdade de Farmacia, Universidade Federal de Goias - UFG, Goiania, Goias, Laboratorio de Nanotecnologia Farmaceutica e Sistemas de Liberacao de Farmacos, Faculdade de Farmacia, Universidade Federal de Goias - UFG, Goiania, Goias, Laboratorio de Nanotecnologia Farmaceutica e Sistemas de Liberacao de Farmacos, Faculdade de Farmacia, Universidade Federal de Goias - UFG, Goiania, Goias, Laboratorio de Nanotecnologia Farmaceutica e Sistemas de Liberacao de Farmacos, Faculdade de Farmacia, Universidade Federal de Goias - UFG, Goiania, Goias, Laboratorio de Nanotecnologia Farmaceutica e Sistemas de Liberacao de Farmacos, Faculdade de Farmacia, Universidade Federal de Goias - UFG, Goiania, Goias, Laboratorio de Nanotecnologia Farmaceutica e Sistemas de Liberacao de Farmacos, Faculdade de Farmacia, Universidade Federal de Goias - UFG, Goiania, Goias
Keywords:HPLC-ELSD, LC-MS/MS, phospholipid, degradation, drug delivery systems, lipid-based formulation.
Abstract:
Background: Liposomes continue to play an important role in drug delivery research due to
their ability to improve transport and targeting of a wide range of active molecules. Analysis of liposomal
components is a key point in the characterization and evaluation of formulation stability. The aim
of this work was to develop and validate an HPLC-ELSD method for the characterization and quality
control of liposomes.
Methods: HPLC-ELSD method was validated by assessing selectivity, linearity, precision, accuracy,
limit of detection and quantitation. The mobile phase consisted of a 0.1% (v/v) of trifluoroacetic acid
(TFA) and methanol in gradient elution. Initial rate was 20:80 (0.1% TFA: methanol), with a ramp
reaching 100% methanol. HPLC-MS/MS was used to confirm the presence of the fatty acid mixture in
the analyzed lipids, as well as sub-products generated under pre-determined conditions in the stability
study.
Results: A HPLC-ELSD method has been developed to detect and measure cholesterol, phosphatidylcholine
and lysophosphatidylcholine. High specificity, sensitivity and linearity within the predetermined
range for all the compounds analyzed (R2>0.99) were obtained. Accuracy and precision results for all
the compounds were within the acceptance limit of ≤5% and 90-110%, respectively. Mass spectrometry
results showed complementary information about the phospholipid composition to evaluate the degree
of degradation of liposomes over different storage conditions.
Conclusion: The method was successfully applied as a quality control tool for the analysis of a wide
range of lipids, present in liposomal formulations. HPLC-MS/MS was used to ensure complete elucidation
of the lipid components and the detected lyso-forms.