Abstract
Phospholipids and their derivatives represent a broad range of multifunctional substances used as excipients or active ingredients by different industries due to their natural origin and unique properties. A fast and reliable quantification as well as comprehensive stability evaluation are of major importance in the process of development and quality control of lipid-based systems. Therefore, the present study is focused on the development and validation of a rapid ultra-high performance liquid chromatography – charged aerosol detector based (UHPLC-CAD) method for simultaneous detection of a multitude of natural and synthetic lipids, (charged) phospholipids, lipophilic fluorescent markers and their possible degradation products. Twenty-two compounds were characterized by a strong linear response of the detector (R2 > 0.97). Moreover, remarkable limits of detection (≤10 μg mL−1) and limits of quantification (≤25 μg mL−1) associated with a consistent reproducibility were achieved for all tested molecules. The performance of the analytical method was demonstrated by analyzing the lipid composition (after different production stages and photodegradation) of both bupivacaine loaded liposomes and a Doxil®-like formulation. The newly developed method combines a rapid, comprehensive, and efficient quantification with minimal economic effort and ecologic consequences, meeting the requirements of modern analytical processes and offering a broad range of possible applications in various industrial sectors and scientific laboratories.
Highlights
Phospholipids and their derivatives represent a broad range of multifunctional substances used as excipients or active ingredients by different manufacturing branches due to their unique properties and their natural origin
With the aim of obtaining an efficient, not mass spectrometer (MS)-coupled profiling strategy for a multitude of phospholipids-based products, we developed a high-performance liquid chromatography (HPLC)-DAD-charged aerosol detectors (CAD) method that would enable a remarkably rapid and simultaneous detection of routine components oflipid mixtures and of liposomal components
For a better overview they are separated by headgroup or origin: synthetic phosphatidylcholines (Figure 2a), natural phosphatidyl choline blends SPC and EPC (Figure 2b), sphingomyelin and hydrogenated SPC (Figure 2c), negatively charged phospholipids with phosphatic acid headgroup (Figure 2d), negatively charged phospholipids with phosphatidylglycerol headgroup (Figure 2e), negatively charged phospholipids with phosphatidylserine headgroup (Figure 2f), fatty acids (Figure 2g) and lipophilic fluorescent tracers detected with the DAD (Figure 2h)
Summary
Phospholipids and their derivatives represent a broad range of multifunctional substances used as excipients or active ingredients by different manufacturing branches due to their unique properties and their natural origin. The major focus in liposome technology shifted from a plain quest for improved bioavailability of poorly water-soluble drugs to a multifaceted approach aimed either at enhancing the therapeutic efficacy by a specific targeting or by a controlled and prolonged drug release To meet this goal a large variety of (semi-)synthetic components such as charged phospholipids, polyethylenglycol (PEG-) derivates, surfactants and other specific long-chained lipids like non-exchangeable membrane staining indocarbocyanine dyes (DiO, DiD; vide infra) could be introduced in the formulation, resulting in very complex compositions [10,11]. While the Bartlett assay is based on the analysis of inorganic phosphate occurring after the oxidation of phospholipids with perchloric acid and the subsequent reaction with ammonium molybdate, the Stewart assay enables to detect only organic phosphate after complexing phospholipids with ammonium ferrothiocyanate in chloroform Both methods require different preparation steps and are prone to an inter-user variability and a high error rate. Being a definite differentiation of the individual element of a multicomponent liposomal formulation by means of GC not achievable, this technique results unsuitable for quality control and process monitoring
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