ASTM E 3324 : 2022

1.1This test method describes the determination of lipid components in liposomal formulations, which includes sample solubilization in methanol followed by separation of the analytes using ultra-high-performance liquid chromatography (UHPLC) and detection with tandem mass-spectrometry (MS/MS). This test method adheres to multiple reaction monitoring (MRM) mass spectrometry on a triple quadrupole mass spectrometer (TQMS).

1.2This test method is specific for liposomal formulations containing cholesterol, 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE- PEG 2000), and hydrogenated (soy) L-α-phosphatidylcholine (HSPC).

1.3This test method is applicable to report the absolute concentrations of cholesterol, DSPE-PEG 2000, and HSPC and their ratio (DSPE-PEG 2000: HSPC: cholesterol) in liposomal formulations. Assessment of the stability of the analytes in terms of their degradation as a result of oxidation or hydrolysis is beyond the scope of this test method.

1.4This test method includes calibration and standardization, sample preparation, UHPLC-TQMS instrumentation, potential interferences, method validation with acceptance criteria, sample analysis, and data reporting.

1.5The detection limits for cholesterol, DSPE-PEG 2000, and HSPC using this test method are 5.3, 0.5, and 0.5 ng/g, respectively. In addition, the quantitation limits for cholesterol, DSPE-PEG 2000, and HSPC are 10.6, 0.8, and 0.5 ng/g, respectively.

1.6This test method is intended for concentration ranges of 8-1600 ng/g for cholesterol, and of 2-400 ng/g for DSPE-PEG 2000 and HSPC.

1.7All observed and calculated values shall conform to the guidelines for significant digits and rounding as established in Practice D6026.

1.8Units—The values stated in SI units are to be regarded as the standard. Where appropriate, c.g.s units in addition to SI units are included in this standard.

1.9This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.

1.10This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.