(T0930-11-61) Elevating Liposomal Formulation Analysis: An Advanced CE-C4D Method for Quantification of Ionic Excipients in Liposomal Doxorubicin

Purpose: Liposomes serve as effective drug delivery vehicles, offering reduced toxicity, prolonged blood circulation, controlled release, and a means of delivering water-insoluble active pharmaceutical ingredients (APIs). The approval of the first liposomal doxorubicin (DOX) HCl formulation, Doxil® by the U. S. Food and Drug Administration (FDA) in 1995, marked a significant milestone. In the case of DOX liposomal formulations, maintaining intraliposomal (NH₄)₂SO₄ concentrations over 200 mM is critical in generating DOX sulfate nanorod crystals and sustained release of DOX. Existing analytical methods for quantifying intraliposomal and external ionic excipients often involve the separation of liposomes by filtration. This approach is time-consuming and may yield erroneous results due to liposome rupture and ion binding to filtering media. We developed and validated a method using capillary electrophoresis coupled with capacitively coupled contactless conductivity detector (CE-C4D) for the direct quantitation of total and external ionic excipients in doxorubicin hydrochloride liposomal formulations without additional separation steps, providing a more efficient and accurate means of assessing cations and anions within the formulations.

Methods: CE-C4D was used for selective quantitation of extra liposomal and total inorganic ions in liposomal formulations. CE experiments were conducted using an Agilent CE 7100 system equipped with an eDAQ Capacitively Coupled Contactless Conductivity Detector with data acquisition frequency 16.67 Hz. The experiments utilized a Polyamide-coated fused silica capillary (25 µm internal diameter, 52 cm total length). The optimized Background electrolyte solution (BGE) comprised sucrose, 2-(N-Morpholino) ethanesulfonic acid (MES), 18-crown-6 (18-C-6), and L-histidine at pH 6.00 ± 0.05. Prior to analysis, the capillary was conditioned with the background electrolyte solution. Sample hydrodynamic injection was employed. For cation mode, a positive polarity voltage of 30 kV was applied for 4 minutes, while for anion mode, a negative polarity voltage of 30 kV was applied for 5 minutes. For external liposomal inorganic ions quantification, the doxorubicin formulation was spiked with the internal standard, lithium fluoride (LiF). Total liposomal inorganic ions were determined by disrupting the liposome bilayer. The doxorubicin formulation was mixed with methanol, sonicated to break the lipid bilayer, and LiF was added. Intra-liposomal inorganic ions were calculated by subtracting external ion concentrations from total ion concentrations. All measurements were conducted in triplicates, and results were reported as mean ± standard deviation (SD).

Results: The cation and anion analyses conducted under normal and negative polarities, respectively, revealed distinct migration orders of NH₄⁺, K⁺, Na⁺, Li⁺ for cations under +30kV (Figure 1A) and Cl^-, SO₄^2-, F^- for anions under -30kV voltage (Figure 1B). Baseline separation, with a high peak resolution, was consistently achieved for all ions at all tested concentrations. While increase in MES (2-(N-Morpholino) ethanesulfonic acid) concentration in the buffer led to enhanced peak resolution and symmetry in cations, the effect on anions were complex. Specifically, the resolution between SO₄^2- and F^- decreased as MES concentration increased. This observation can be attributed to the differential impact of MES concentration on monovalent and divalent ions. To optimize resolution while maintain adequate separation, a final buffer concentration of 100 mM MES was chosen. Experiments examining capillary diameter sensitivity revealed a proportional ratio of 8:4:1 for capillaries with diameters of 25 µm, 50 µm, and 75 µm in a 50 mM MES buffer. Analyses of hydrodynamic injections further revealed a reduction in resolution as injection volumes increased. The addition of the 18-C-6 complexing agent effectively separated NH₄⁺ and K⁺ ions, with concentrations at both 2.5 mM and 3.5 mM proving suitable for analysis. The correlation coefficients (R²) for this method surpass 0.99, with limit of detection (LOD) values consistently below 3.0 µM for all analyzed ions. Application of the optimized CE-C4D method to liposomal doxorubicin formulations allowed successful determination of total, and external ion concentrations. External NH₄⁺ concentrations ranged from 3.5-5.3 mM, and external SO₄^2- concentrations ranged from 0.2-1.3 mM (Table 1). Notably, all five FDA-approved doxorubicin HCl liposome formulations contained an excess of 200 mM ammonium sulfate, crucial for generating rod-like doxorubicin crystals and maintaining liposomal stability.

Conclusion: The study presents a validated CE-C4D method for selectively quantifying total and extra liposomal ionic excipients in DOX liposomal formulations without the need for additional separation steps. This method offers a valuable tool for reverse engineering of the formulation as well as batch-to-batch analysis of the formulations.

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.


Figure 1 CE-C4D ion separation, A) cations (NH4+, K+, Na+ and Li+) with normal polarity; and B) anions (Cl-, SO42- and F-) using reverse polarity.


Table 1 The total, external, and intra-liposomal ions in DOXIL® and its generic versions