Research paper Method transfer for fast liquid chromatography in pharmaceutical analysis: Application to short columns packed with small particle. Part II: Gradient experiments Davy Guillarme, Dao T.T. Nguyen, Serge Rudaz, Jean-Luc Veuthey * Laboratory of Analytical Pharmaceutical Chemistry, University of Geneva, University of Lausanne, Geneva, Switzerland Received 29 January 2007; accepted in revised form 28 June 2007 Available online 6 July 2007 Abstract Liquid chromatography (LC) is currently considered as the gold standard in pharmaceutical analysis. Today, there is an increasing need for fast and ultra-fast methods with good efficiency and resolution for achieving separations in a few minutes or even seconds. A previous article (i.e. method transfer for fast LC in pharmaceutical analysis. Part I: isocratic separation) described a simple methodology for performing a successful method transfer from conventional LC to fast and ultra-fast LC in isocratic mode. However, for performing complex separations, the gradient mode is often preferred. Thus, this article reports transfer rules for chromatographic separations in gradient mode. The methodology was applied for the impurity profiling of pharmaceutical compounds, following two strategies. A first approach, using short columns (20–50 mm) packed with 3.5 lm particles and optimized HPLC instrumentation (with reduced extra-column and dwell volumes), was applied for the separation of a pharmaceutical drug and eight related impurities. Special attention was paid to the dwell (gradient delay) volume, which causes the most detrimental effect for transferring a gradient method. Therefore, the dwell volume was simultaneously decreased with the column dead volume. Under optimal conditions, it was possible to reduce the anal- ysis time by a factor of 10, with an acceptable loss in resolution since the column length reduction is less critical in gradient than isocratic mode. The second tested approach was Ultra Performance Liquid Chromatography (UPLC), where sub-2 lm particles were used simulta- neously with very high pressures (up to 1000 bar). A complex pharmaceutical mixture containing 12 compounds was separated in only 1.5 min allowing a reduction of the analysis time by a factor of 15 in comparison to a conventional method, with similar peak capacity. Ó 2007 Elsevier B.V. All rights reserved. Keywords: High speed liquid chromatography; Method transfer; Gradient mode; Short columns; UPLC; Fast separation 1. Introduction In a previous article [1], general rules were described for transferring, in isocratic mode, a conventional method to fast- or ultra-fast liquid chromatography (LC) in pharma- ceutical analysis. It was shown that among the different tested approaches, the use of short columns (i.e. 5 cm length) packed with 1.7 lm particles was the best strategy to significantly reduce the analysis time while maintaining efficiency and, therefore, resolution constant. However, it was also demonstrated that dedicated instrumentation was necessary, with low extra-column volumes and high detection acquisition rates. Furthermore, very small particles induce a large back pressure (Darcy’s law) and the equipment must be also compatible with the high gen- erated pressure. As shown previously [1], fast- and ultra- fast separations were achieved by Ultra Performance Liquid Chromatography (UPLC) compatible with short columns packed with 1.7 lm particles and high pressure (1000 bar). 0939-6411/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejpb.2007.06.018 * Corresponding author. Laboratory of Analytical Pharmaceutical Chemistry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, boulevard d’Yvoy 20, 1211 Geneva 4, Switzerland. E-mail address: jean-luc.veuthey@pharm.unige.ch (J.-L. Veuthey). www.elsevier.com/locate/ejpb Available online at www.sciencedirect.com European Journal of Pharmaceutics and Biopharmaceutics 68 (2008) 430–440