一种通过 H 和 P NMR 光谱法对脂质体中脂质成分进行相对和绝对定量的简便方法。

A convenient method for the relative and absolute quantification of lipid components in liposomes by H- and P NMR-spectroscopy.

机构信息

Department of Chemistry, College of the Pacific, University of the Pacific, 3601 Pacific Avenue, Stockton, CA 95211, USA.

出版信息

Chem Phys Lipids. 2024 Jul;261:105395. doi: 10.1016/j.chemphyslip.2024.105395. Epub 2024 Apr 12.

DOI:10.1016/j.chemphyslip.2024.105395

PMID:38615786

Abstract

OBJECTIVE

Liposomes are promising delivery systems for pharmaceutical applications and have been used in medicine in the recent past. Preparation of liposomes requires reliable characterization and quantification of the phospholipid components for which the traditional cumbersome molybdate method is used frequently. The objective was to improve relative and absolute quantification of lipid components from liposomes.

METHODS

A reliable method for quantification of lipid composition in liposome formulations in the 1-10 μmol range with H- and P NMR spectroscopy at 600 MHz has been developed. The method is based on three crystalline small-molecule standards (PhPO, (Tol)PO, and PhPO) in CDCl.

RESULTS

Excellent calibration linearity and chemical stability of the standards was observed. The method was tested in blind fashion on liposomes containing POPC, PEG-ceramide and a pH-sensitive trans-aminocyclohexanol-based amphiphile (TACH). Relative quantification (percentage of components) as well as determination of absolute lipid amount was possible with excellent reproducibility with an average error of 5%. Quantification (triplicate) was accomplished in 15 min based on H NMR and in 1 h based on P NMR. Very little change in mixture composition was observed over multiple preparative steps.

CONCLUSION

Liposome preparations containing POPC, POPE, DOPC, DPPC, TACH, and PEG-ceramide can be reliably characterized and quantified by H NMR and P NMR spectroscopy at 600 MHz in the μmol range.

摘要

目的

脂质体是有前途的药物传递系统，在最近的医学中得到了应用。脂质体的制备需要对磷脂成分进行可靠的表征和定量，而传统的繁琐钼酸盐法经常被用于这种情况。本研究的目的是改进脂质体中脂质成分的相对和绝对定量方法。

方法

开发了一种在 600 MHz 下使用 H 和 P NMR 光谱对 1-10 μmol 范围内脂质体制剂中脂质组成进行可靠定量的方法。该方法基于 CDCl 中的三种结晶小分子标准品（PhPO、（Tol）PO 和 PhPO）。

结果

观察到标准品具有极好的校准线性和化学稳定性。该方法以含有 POPC、PEG-神经酰胺和 pH 敏感的反式氨基环己醇基两性分子（TACH）的脂质体进行了盲法测试。相对定量（成分百分比）以及绝对脂质量的测定均可实现，重复性极好，平均误差为 5%。基于 H NMR，定量（重复 3 次）可在 15 分钟内完成，而基于 P NMR 则可在 1 小时内完成。在多个制备步骤中，混合物组成的变化很小。