(W1130-09-58) Qualitative and Quantitative Analysis of Lipids in Exparel ^® Injectable Liposomal Drug Formulation

Purpose: Application of liposomal formulations in drug delivery has gained popularity in recent times because of distinct advantages over traditional drug formulations in drug distribution. Exparel^® is a multivesicular liposomal formulation (MVL) of bupivacaine, which provides sustained release at the site of injection resulting in prolong anesthetic capabilities. Since the lipid composition leads to its unique structure, the quantification of major lipid constituents and identification of possible minor lipids, lipid degradation products, and cholesterol oxidation products are required for a complete evaluation of the formulation.

Methods: In this study, a UHPLC-HRMS (QTOF) method was developed to identify and quantify API (bupivacaine), major lipids (Figure 1), minor lipids, and oxidation products in two batches of Exparel^® injectable liposomal emulsion. Lipid components were separated using a reversed phase UHPLC method and detected using ESI/QTOF (electron spray ionization) MS technique. The Premier Biosoft SimLipid^® software was used for the identification of the separated lipids using their MS/MS spectra data provided in the software lipid database. The composition of identified lipids was quantitatively or semi-quantitatively determined with the commercially available lipid standards. We also developed chromatographic methods to quantify the bupivacaine, its enantiomers, cholesterol, and cholesterol oxidation products present in the formulation.

Results: The two different batches of Exparel^® were analyzed for both major and minor lipids (other than 1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-Dierucoyl-sn-glycero-3-phosphocholine (DEPC) and tricapyrlin) as indicated in Figure 2. We identified 24 minor lipids and two cholesterol oxidation products in the formulation. Out of the 24 minor lipids identified, 22 were phospholipids and the others were triacylglycerols. These minor lipids constituted approximately 4.26% and 3.70% of all lipids in Batch 1 and Batch 2, respectively. Considering just the minor lipids, 34.73% were phosphocholine (PC) lipids in Batch 1, while Batch 2 had 33.72% as PC lipids. The phosphoglycerol (PG) minor lipid amounts were 19.20% and 21.24%, respectively, for Batch 1 and Batch 2, while the total phosphotidylethanolamine (PE) minor lipids were 6.87% and 7.20% compared to the total minor lipid content. The content of triacylgycerols (TAG) were 17.90% and 19.54%, respectively, in Batches 1 and 2 when considering the amount of total minor lipids. Batch 1 showed a 10.69% of phosphatidic acid (PA) and 10.57% of phosphoserine (PS) minor lipids, while Batch 2 exhibited 10.08% and 8.24% minor lipid content in those two lipid categories. The minor lipid content among different lipid classes were almost the same.

Conclusion: We have successfully developed a set of LCMS-HRMS methods for the separation, identification, and quantitation of the API (bupivacaine), its enantiomers, major and minor lipids, cholesterols, and cholesterol oxidation products of the Exparel^® multivesicular liposomal drug formulation. However, we assume that this knowledge could be useful when developing generic formulations and it is required the reverse engineering of the reference product to completely understand the origin of these minor lipids in the formulation composition.

References: (1) Banstola, B.; Gamage, P. L.; Jiang, W.; Mudalige, T. Analysis of phospholipids and triacylglycerols in intravenous lipid emulsions. Journal of Pharmaceutical and Biomedical Analysis 2023, 222, 115112. DOI: https://doi.org/10.1016/j.jpba.2022.115112
(2) 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) with collaborations among ORA/ORS, and CDER/OGD/ORS. Dr. Prabhath Gamage was supported in part by an appointment to the Research Participation Program at the U.S. Food and Drug Administration administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. The opinions expressed in this abstract are those of the authors. The opinions should not be interpreted as current or future policy of the U.S. Food & Drug Administration or any other agency of the U.S. government. The mention of manufacturers or trade names are for experimental clarity and does not constitute product endorsement.

Major constituents of liposomal Exparel®

Ion chromatogram of minor lipids Phosphatidic acid (PA), Phosphoglycerols (PG), Phosphocholines (PC), Phosphotidylethanolamine (PE), Phosphocholines, Phosphoserines (PS) and Triacylglycerols (TG) with their retention times found in Exparel®