A convenient method for the relative and absolute quantification of lipid components in liposomes by 1H- and 31P NMR-spectroscopy

Abstract

ObjectiveLiposomes 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. MethodsA reliable method for quantification of lipid composition in liposome formulations in the 1–10 μmol range with 1H- and 31P NMR spectroscopy at 600 MHz has been developed. The method is based on three crystalline small-molecule standards (Ph3PO4, (Tol)3PO4, and Ph3PO) in CDCl3. ResultsExcellent 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).11Et3PO4 Triethyl phosphate, Ph3PO4 triphenyl phosphate, (Tol)3PO4 tritolyl phosphate, Ph3PO triphenylphosphine oxide, POPC 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine, DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DOPC 1,2-dioleyl-sn-glycero-3-phosphocholine, DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, POPE 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, PEG-ceramide N-palmitoyl-sphingosine-1-(succinyl[methoxy(polyethylene glycol)2000]), COSY correlation spectroscopy, TOCSY total correlation spectroscopy, HMBC heteronuclear multiple-bond correlation, TACH trans-aminocyclohexanol, ANTS 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt, DPX p-xylene-bis-pyridinium bromide, HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, pfg pulse-field gradient. 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 1H NMR and in 1 h based on 31P NMR. Very little change in mixture composition was observed over multiple preparative steps. ConclusionLiposome preparations containing POPC, POPE, DOPC, DPPC, TACH, and PEG-ceramide can be reliably characterized and quantified by 1H NMR and 31P NMR spectroscopy at 600 MHz in the μmol range.