title: Systematic evaluation of commercially available ultra-high performance liquid chromatography columns for drug metabolite profiling: optimization of chromatographic peak capacity.
abstract: The present study investigated the practical use of modern ultra-high performance liquid chromatography (UHPLC) separation techniques for drug metabolite profiling, aiming to develop a widely applicable, high-throughput, easy-to-use chromatographic method, with a high chromatographic resolution to accommodate simultaneous qualitative and quantitative analysis of small-molecule drugs and metabolites in biological matrices. To this end, first the UHPLC system volume and variance were evaluated. Then, a mixture of 17 drugs and various metabolites (molecular mass of 151-749Da, logP of -1.04 to 6.7), was injected on six sub-2μm particle columns. Five newest generation core shell technology columns were compared and tested against one column packed with porous particles. Two aqueous (pH 2.7 and 6.8) and two organic mobile phases were evaluated, first with the same flow and temperature and subsequently at each column's individual limit of temperature and pressure. The results demonstrated that pre-column dead volume had negligible influence on the peak capacity and shape. In contrast, a decrease in post-column volume of 57% resulted in a substantial (47%) increase in median peak capacity and significantly improved peak shape. When the various combinations of stationary and mobile phases were used at the same flow rate (0.5mL/min) and temperature (45°C), limited differences were observed between the median peak capacities, with a maximum of 26%. At higher flow though (up to 0.9mL/min), a maximum difference of almost 40% in median peak capacity was found between columns. The finally selected combination of solid-core particle column and mobile phase composition was chosen for its selectivity, peak capacity, wide applicability and peak shape. The developed method was applied to rat hepatocyte samples incubated with the drug buspirone and demonstrated to provide a similar chromatographic resolution, but a 6 times higher signal-to-noise ratio than a more traditional UHPLC metabolite profiling method using a fully porous particle packed column, within one third of the analysis time. In conclusion, a widely applicable, selective and fast chromatographic method was developed that can be applied to perform drug metabolite profiling in the timeframe of a quantitative analysis. It is envisioned that this method will in future be used for simultaneous qualitative and quantitative analysis and can therefore be considered a first important step in the Quan/Qual workflow.
authors.affiliation: Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands. Electronic address: a.c.dubbelman@lacdr.leidenuniv.nl.
authors.forename: Anne-Charlotte
authors.initials: AC
authors.lastname: Dubbelman
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, Janssen R&D, Turnhoutseweg 30, 2340 Beerse, Belgium.
authors.forename: Filip
authors.initials: F
authors.lastname: Cuyckens
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, Janssen R&D, Turnhoutseweg 30, 2340 Beerse, Belgium.
authors.forename: Lieve
authors.initials: L
authors.lastname: Dillen
authors.affiliation: Pharmacokinetics, Dynamics and Metabolism, Janssen R&D, Turnhoutseweg 30, 2340 Beerse, Belgium.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Gross
authors.affiliation: Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hankemeier
authors.affiliation: Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands; Pharmacokinetics, Dynamics and Metabolism, Janssen R&D, Turnhoutseweg 30, 2340 Beerse, Belgium.
authors.forename: Rob J
authors.initials: RJ
authors.lastname: Vreeken
mesh_terms: D000818:Animals; D002065:Buspirone / Q000032:analysis / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods; D022781:Hepatocytes / Q000378:metabolism; D010316:Particle Size; D016062:Porosity; D011312:Pressure; D051381:Rats; D059629:Signal-To-Noise Ratio
chemical_list: D002065:Buspirone
keywords: Cortecs; Kinetex; Peak capacity; Quan/Qual; Solid-core particles; UHPLC