(T1030-11-61) Comprehensive Composition Analysis of Doxorubicin HCl Liposomes

Purpose: Liposomal technologies have advanced over decades, serving as versatile carriers that have transformed cancer therapy with formulations like Doxil^® and its generic versions. These formulations deliver the active pharmaceutical ingredient (API) with enhanced efficacy and altered biodistribution, resulting in reduced toxicity. However, lipids are prone to degradation and oxidation. The objective of this study is to develop an analytical method to obtain comprehensive composition and degradation product profiles of doxorubicin HCl liposome formulations.

Methods: In this investigation, we explored high resolution mass spectrometry methods aimed at identifying and quantifying lipid degradation products, oxidation products, and lipid impurities in Doxil© and its generic formulations. Various reversed-phase UHPLC techniques were used for lipid component separation. Lipids and their derivatives (PCs, LPCs, LPEs, mPEG-DSPEs) were analyzed in positive ionization mode using an ACQUITY UPLC® CSH™ C18 column with an ESI ion source. Free fatty acids (FFAs) were separated in negative ionization mode with an ACQUITY UPLC® BEH™ C8 column. Cholesterols and their derivatives were analyzed in positive mode with an ACQUITY UPLC® CSH™ C18 column using an APCI ion source. Identification of these compounds was facilitated by Premier Biosoft SimLipid® software, cross-verifying with standards where available. Quantification was done through quantitative or semi-quantitative methods using commercial lipid standards. The composition of these identified lipids was determined, employing either quantitative or semi-quantitative methods with the aid of commercially available lipid standards.

Results: The detailed analysis of Doxil^® and its generic versions revealed a range of lipids, degradation products, oxidation products, and cholesterol derivatives present in varying quantities. In 1 ml of Doxil^®, there are approximately 16 mg of HSPC, MPEG2000-DSPE, and cholesterol, which closely matches the labelled value. Alongside the reported lipids and cholesterols, presence of PC(17:0_18:0) and PC(18:0_20:0) lipid impurities were identified via MS/MS fragmentation patterns (see Figure 1). Furthermore, 13 lipid degradation and oxidation products were identified, including 6 free fatty acids, 4 lyso-phospholipids, and 3 cholesterol derivatives (see Figure 2). The quantities of these phospholipid impurities are less than 2% (w/w) of the total phospholipids, with phospholipid degradation products such as LPC, LPEs, and FFAs also found in amounts less than 2% (w/w) across all Doxil^® and generic versions. Additionally, cholesterol derivatives were detected at levels less than 0.01% (w/w) of total phospholipids.

Conclusion: UHPLC-QTOF-ESI/APCI methods and SimLipid analysis were developed to identify and quantify key lipids, cholesterol, lipid degradation products, and cholesterol oxidation products, providing a comprehensive understanding of lipid profiles in Doxil^® and its generic formulations.

References: (1) Ansar, S. M.; Jiang, W.; Mudalige, T. Analysis of verteporfin liposomal formulations for phospholipids and phospholipid degradation products by liquid chromatography-mass spectrometry (LC-MS). Journal of Pharmaceutical and Biomedical Analysis 2022, 208, 114473. DOI: https://doi.org/10.1016/j.jpba.2021.114473.

Acknowledgements: These studies were conducted using the Nanotechnology Core Facility (NanoCore) located on the U.S. Food and Drug Administration’s Jefferson Laboratories campus (Jefferson, AR), which houses the FDA Office of Regulatory Affairs Arkansas Laboratory and the FDA National Center for Toxicological Research. This project was supported in part by appointment to the Research Participation Program at the Arkansas Laboratory/ Office of Regulatory Affairs, U.S. Food and Drug Administration, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and FDA.
Disclaimer: The views expressed in this document are those of the authors and should not be interpreted as the official opinion or policy of the U.S. Food and Drug Administration, Department of Health and Human Services, or any other agency or component of the U.S. government. The mention of trades names, commercial products, manufacturers, or organizations is for clarification of the methods used and should not be interpreted as an endorsement of a product or manufacturer.


MS/MS Fragmentation patterns and relevant chemical structures of protonated [M+H]+ (A) PC (17:0_18:0), and (B) PC (18:0_20:0)


Chromatograms of (A) lipids, and lyso-phospholipids and (B) Free fatty acid products in Doxorubicin HCl Liposomes