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A Reliable HPLC-ELSD Method for Determination of Cholesterol, Phosphatidylcholine, Lysophosphatidylcholine Content and the Stability of a Liposomal Formulation

[ Vol. 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*   Pages 623 - 632 ( 10 )

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.

Keywords:

HPLC-ELSD, LC-MS/MS, phospholipid, degradation, drug delivery systems, lipid-based formulation.

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

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