A validated H-NMR method for quantitative analysis of DOTAP lipid in nanoliposomes containing soluble Leishmania antigen.

Leishmaniasis is a serious health problem that needs a suitable vaccine delivery system to control the disease. Cationic lipids such as 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) have been widely used in nanoliposomes' formulation to deliver antigen and adjuvant at the same time to induce protection against Leishmaniasis. Therefore, it is necessary to accurately quantify DOTAP concentration in the formulation and biological materials. Due to the poor UV absorbance of DOTAP, the use of the conventional HPLC-UV method was impossible. Currently, an evaporative light scattering detector (ELSD) or MS/MS detector in conjunction with HPLC is used to quantify DOTAP. These methods have several disadvantages, including time- consuming during extraction procedure and decrease or/and even remove some components of the formulation. According to the advantages of the quantitative H Nuclear Magnetic Resonance (H-NMR) spectroscopic method, a free extraction approach was developed to the assay of DOTAP in nanoliposomes containing Leishmania antigens. This method was carried out based on the relative ratio of signal integration of DOTAP [CH (CH-CH = CH-CH)] in δ 2 ppm to a definite amount of an internal standard called dimethyl sulfone (DMSO). The qH-NMR method showed good precision (intra-day RSD = 1.8 % and inter-day RSD = 2.5 %), linearity (in the ranges of 1.3-7.8 mg. mL with correlation coefficients at 1), repeatability (RSD ≤ 2.39 %), and stability (RSD ≤ 2.32 %) for the quantification of the DOTAP without any extraction method. Considering all the experiments conducted in this study, NMR can be a feasible alternative to other traditional techniques for the simultaneous quantification of lipids in liposome formulations.