1.1 本试验方法描述了脂质体制剂中脂质成分的测定，包括样品在甲醇中溶解，然后使用超高效液相色谱（UHPLC）分离分析物，并使用串联质谱（MS/MS）进行检测。本试验方法遵循三重四极质谱仪（TQMS）上的多反应监测（MRM）质谱。 1.2 本试验方法适用于含有胆固醇、1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺的脂质体制剂- N-[甲氧基（聚乙二醇）-2000]（DSPE-PEG 2000）和氢化（大豆）L-α-磷脂酰胆碱（HSPC）。 1.3 本试验方法适用于报告脂质体制剂中胆固醇、DSPE-PEG 2000和HSPC的绝对浓度及其比率（DSPE-PEG 2000:HSPC:胆固醇）。分析物因氧化或水解而降解的稳定性评估不在本试验方法的范围内。 1.4 该测试方法包括校准和标准化、样品制备、UHPLC-TQMS仪器、潜在干扰、采用验收标准的方法验证、样品分析和数据报告。 1.5 使用该试验方法对胆固醇、DSPE-PEG 2000和HSPC的检测限分别为5.3、0.5和0.5纳克/克。此外，胆固醇、DSPE-PEG 2000和HSPC的定量限分别为10.6、0.8和0.5纳克/克。 1.6 本试验方法适用于胆固醇的浓度范围为8-1600纳克/克，DSPE-PEG 2000和HSPC的浓度范围为2-400纳克/克。 1.7 所有观察值和计算值应符合实践中确定的有效数字和舍入准则 D6026 . 1.8 单位- 以国际单位制表示的数值应视为标准。 在适当情况下，本标准包括国际单位制以外的通用单位制。 1.9 本标准并非旨在解决与其使用相关的所有安全问题（如有）。本标准的用户有责任在使用前制定适当的安全、健康和环境实践，并确定监管限制的适用性。 1.10 本国际标准是根据世界贸易组织技术性贸易壁垒（TBT）委员会发布的《关于制定国际标准、指南和建议的原则的决定》中确立的国际公认标准化原则制定的。 ====意义和用途====== 5.1 脂质体是纳米尺寸的囊泡，由脂质双层组成，用于各种诊断和治疗应用 ( 9 ) . 生物制药行业对脂质体制剂在输送各种药物、反义寡核苷酸、克隆基因或重组蛋白方面的兴趣日益浓厚，这就保证了组成脂质的质量控制和彻底表征。脂质体的结构、组成和浓度是决定脂质体药物产品质量和疗效的关键属性，因为它们影响脂质体的稳定性、载药量、释放动力学、生物分布和药代动力学特性 ( 9 ) . 胆固醇调节脂质膜的流动性、弹性和通透性；因此，它在控制药物释放和提高脂质体稳定性方面起着关键作用 ( 10 ) . 5.2 该试验方法为使用UHPLC-TQMS测定脂质体制剂中的胆固醇、DSPE-PEG 2000和热休克蛋白C提供了快速可靠的方案。本试验方法不包括根据降解曲线评估分析物的稳定性 ( 11 ) . 该测试方法将有助于生物制药行业确定脂质体制剂的质量评估和监测批次- 批量生产的一致性，从而促进安全有效的药物开发和监管审查。 5.3 与使用通用检测器（如带电气溶胶或蒸发光散射检测器）的其他当代技术相比，超高纯液相色谱-质谱/质谱测量在分析上更为敏感，对脂质分析更具特异性。对于脂质体，MS/MS比紫外线检测器有更多的优势，因为脂质体缺乏用于检测的生色团。在本试验方法中，由于其高选择性、灵敏度、信噪比、准确性和广泛的定量线性范围，TQMS已被用作首选的MS/MS技术，从而允许对分析物进行可重复的定量，尤其是在低浓度下。 5.4 根据现行《良好制造规范》[21 CFR 211.194（a）（2）]，用户需要验证试验方法在实际使用条件下的适用性。验证应评估产品基质试验方法的适用性、从产品基质中回收分析物、色谱条件和色谱柱的适用性、检测器信号响应的适当性、特异性、检测和定量限、准确性和精密度。如果使用不同的色谱柱、电离方法或质量分析仪，用户可能需要优化方法参数并交叉验证。

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 This 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. 1.10 This 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. ====== 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, especially at low concentrations. 5.4 According to the Current Good Manufacturing Practice regulations [21 CFR 211.194(a)(2)], users are required to verify the suitability of the test method under actual conditions of use. Validation should assess the suitability of the test method for the product matrix, recovery of the analytes from the product matrix, suitability of chromatographic conditions and column, appropriateness of the detector signal response, specificity, limit of detection and quantitation, accuracy, and precision. The user may need to optimize method parameters and cross validate if a different chromatography column, ionization method, or mass analyzer is used.