ASTM E3324-22 PDF

SIGNIFICANCE AND USE 5.1 Liposomes are vesicles of nanoscale dimensions, composed of lipid bilayers, which are used for various diagnostic and therapeutic applications (9). The growing interest in liposomal formulations in the delivery of various drugs, antisense oligonucleotides, cloned genes, or recombinant proteins by the biopharmaceutical industry, warrants QC and thorough characterization of the constituent lipids. Lipid structure, composition, and concentration are key attributes in determining the quality and efficacy of a liposomal drug product as they influence the stability of liposomes, drug loading, release kinetics, biodistribution, and pharmacokinetic properties (9). Cholesterol modulates the lipid membrane fluidity, elasticity, and permeability; hence, it plays a key role in controlled drug release and increased stability of the liposome (10). 5.2 This test method provides a rapid and reliable protocol for the determination of cholesterol, DSPE-PEG 2000, and HSPC in liposomal formulations using UHPLC-TQMS. Assessment of the stability of the analytes in terms of their degradation profiles is not included in this test method (11). This test method will benefit the biopharmaceutical industry in ascertaining quality assessment of liposomal formulations and monitoring batch-to-batch consistency for large-scale production, thereby facilitating safe and efficient drug development and regulatory review. 5.3 UHPLC-MS/MS measurements are analytically more sensitive and specific for lipid analysis compared to other contemporary techniques using universal detectors, such as a charged aerosol or an evaporative light-scattering detector. For liposomes, MS/MS has further advantages over ultraviolet detectors, as lipids lack chromophores for detection. In this test method, TQMS has been used as the MS/MS technique of choice because of its high selectivity, sensitivity, S/N, accuracy, and broad linear range of quantitation, thereby allowing reproducible quantitation of the analytes,... SCOPE 1.1 This 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). 1.2 This 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). 1.3 This 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. 1.4 This test method includes calibration and standardization, sample preparation, UHPLC-TQMS instrumentation, potential interferences, method validation with acceptance criteria, sample analysis, and data reporting. 1.5 The 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. 1.6 This 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. 1.7 All observed and calculated values shall conform to the guidelines for significant digits and rounding as established in Practice D6026. 1.8 Units—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. 1.9 Th...