Fast and versatile analysis of liposome encapsulation efficiency by nanoParticle exclusion chromatography.

Liposomes are an attractive drug delivery platform for a wide variety of pharmaceutical molecules. Encapsulation efficiency, which refers to the amount of drug contained inside liposomes compared with the total amount of drug, is a critical quality attribute of liposome products, as the free drug in a liposomal formulation may cause toxicity or undesired biodistribution. The determination of encapsulation efficiency requires the measurement of at least two of the three drug populations: total drug, encapsulated drug and free drug. However, direct measurement of the encapsulated drug and free drug remains a challenging analytical task. Nanoparticle exclusion chromatography (nPEC), an emerging high-performance liquid chromatography (HPLC) technique, has shown great potential in separating and quantifying the free drug in liposomal formulations. In this study, nPEC was systematically evaluated for two representative liposomal formulations containing either hydrophilic or hydrophobic small molecule drugs. It is reported for the first time that the insoluble free drug suspended in the aqueous formulation can be directly measured by nPEC. This free drug in the suspension sample was quantified with excellent accuracy and precision. On the other hand, the total drug measurement from dissociated liposomes was confirmed by the benchmark methodology of reversed phase liquid chromatography (RPLC). The facile quantitation of free and total drug in the liposome formulation enables the fast and accurate determination of the encapsulation efficiency, which can be used to guide the formulation development and characterize the product quality.

Simultaneous achiral-chiral analysis of pharmaceutical compounds using two-dimensional reversed phase liquid chromatography-supercritical fluid chromatography.

A new interface was designed to enable the coupling of reversed phase liquid chromatography (RPLC) and supercritical fluid chromatography (SFC). This online two-dimensional chromatographic system utilizing RPLC in the first dimension and SFC in the second was developed to achieve simultaneous achiral and chiral analysis of pharmaceutical compounds. The interface consists of an eight-port, dual-position switching valve with small volume C-18 trapping columns. The peaks of interest eluting from the first RPLC dimension column were effectively focused as sharp concentration pulses on small volume C-18 trapping column/s and then injected onto the second dimension SFC column. The first dimension RPLC separation provides the achiral purity result, and the second dimension SFC separation provides the chiral purity result (enantiomeric excess). The results are quantitative enabling simultaneous achiral, chiral analysis of compounds. The interface design and proof of concept demonstration are presented. Additionally, comparative studies to conventional SFC and case studies of the applications of 2D LC-SFC in pharmaceutical analysis is presented.

Assessing stability-indicating methods for coelution by two-dimensional liquid chromatography with mass spectrometric detection.

Chromatographic analysis of trace organic impurities/degradants coeluting in the midst of active pharmaceutical ingredient can be challenging given similarities in their structures and differences in their relative levels/intensities. Conventional detection techniques such as diode array detection and mass spectrometry are often inadequate to detect/identify these residual coeluting impurities and could result in a false negative. Application of two-dimensional chromatography to address/evaluate coelution in conventional chromatography is presented. Areas of interest, usually corresponding to the main component, are transferred to secondary column/s for further separation termed as pseudocomprehensive two-dimensional liquid chromatography. Coelution, if any, in the rest of the chromatogram is monitored using conventional detectors. In this work, the use of similar and complementary phases in both dimensions is presented. The use of the same phase in both dimensions to resolve coeluting impurities (especially in the front and tail of the main component differing by orders of magnitude) is an easier alternative to finding complementary column/s, as hydrophobicity dominates reversed-phase separation. The same phase separation is practical as relative levels of impurities and main component in some transferred fractions are comparable enabling their separation. The results were confirmed using mass spectrometry. This work has significant bearing as a method assessment tool in pharmaceutical and other industries.