title: Simple chromatographic method for simultaneous analyses of phosphatidylcholine, lysophosphatidylcholine, and free fatty acids.
abstract: This study describes a simple chromatographic method for the simultaneous analyses of phosphatidylcholine (PC) and its hydrolytic degradation products: lysophosphatidylcholine (LPC) and free fatty acids (FFA). Quantitative determination of PC, LPC, and FFA is essential in order to assure safety and to accurately assess the shelf life of phospholipid-containing products. A single-run normal-phase high-performance liquid chromatography (HPLC) with evaporative light scattering detector has been developed. The method utilizes an Allsphere silica analytical column and a gradient elution with mobile phases consisting of chloroform: chloroform-methanol (70:30%, v/v) and chloroform-methanol-water-ammonia (45:45:9.5:0.5%, v/v/v/v). The method adequately resolves PC, LPC, and FFA within a run time of 25 min. The quantitative analysis of PC and LPC has been achieved with external standard method. The free fatty acids were analyzed as a group using linoleic acid as representative standard. Linear calibration curves were obtained for PC (1.64-16.3 μg, r(2) = 0.9991) and LPC (0.6-5.0 μg, r(2) = 0.9966), while a logarithmic calibration curve was obtained for linoleic acid (1.1-5.8 μg, r(2) = 0.9967). The detection and quantification limits of LPC and FFA were 0.04 and 0.1 μg, respectively. As a means of validating the applicability of the assay to pharmaceutical products, PC liposome was subjected to alkaline hydrolytic degradation. Quantitative HPLC analysis showed that 97% of the total mass balance for PC could be accounted for in liposome formulation. The overall results show that the HPLC method could be a useful tool for chromatographic analysis, stability studies, and formulation characterization of phospholipid-based pharmaceuticals.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40506, USA. aeme222@uky.edu
authors.forename: Abebe Endale
authors.initials: AE
authors.lastname: Mengesha
authors.forename: Paul M
authors.initials: PM
authors.lastname: Bummer
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods*; D045424:Complex Mixtures / Q000032:analysis; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005230:Fatty Acids, Nonesterified / Q000032:analysis*; D008244:Lysophosphatidylcholines / Q000032:analysis*; D010713:Phosphatidylcholines / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D045424:Complex Mixtures; D005230:Fatty Acids, Nonesterified; D008244:Lysophosphatidylcholines; D010713:Phosphatidylcholines