Journal of Chromatography A, 1218 (2011) 74–82 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Orthogonal analytical screening for liquid chromatography–mass spectrometry method development and preparative scale-up Luis M. Font a,∗ , A. Fontana a , M.T. Galceran b , L. Iturrino a , V. Perez a a Enabling Analytical Technologies, Janssen Research & Development a Division of Janssen-Cilag S.A., c/Jarama 75, 45007 Toledo, Spain b Departament de Química Analítica, Facultat de Química, Universitat de Barcelona, Diagonal, 647, 3 a planta, 08028 Barcelona, Spain article info Article history: Received 22 February 2010 Received in revised form 1 October 2010 Accepted 26 October 2010 Available online 2 November 2010 Keywords: HPLC–MS HPLC method development Preparative HPLC–MS Orthogonal screening abstract An analytical HPLC–MS screening methodology has been developed to improve preparative RP-HPLC–MS purifications in medicinal chemistry laboratories. Although several approaches have been previously described to optimize analytical separations, none of them met our needs for the optimization of prepar- ative conditions. Our screening protocol is based on searching among several orthogonal conditions to find the optimum preparative separation. Five different buffer conditions, from low to high pH, two organic solvents, acetonitrile and methanol, and five stationary phases of different polarities and char- acteristics were used. The orthogonality of the system was demonstrated using both, a standard mixture and mixtures from synthesis. To carry out the screening one of the analytical “open access” HPLC–MS systems was modified to perform the analytical screening while maintaining the open-access function- ality for synthesis reaction monitoring. A software tool for automated sample programming and data reporting was also developed. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Preparative RP-HPLC–MS is one of the most powerful techniques currently used for the purification of synthesized compounds in medicinal chemistry laboratories. Today, the requirements for this technique are quite different from the initial demands of the combi- natorial chemistry libraries. Instruments and methodologies were first developed to satisfy the requirements of the purification of large series of compounds [1,2] and in this context, RP-HPLC–MS was able to remove the unwanted reagents or side products that other techniques such as scavenger resins [3], liquid–liquid [4] or solid phase extractions [5] were not able to eliminate. On the other hand, the compound requirements for pharmacological tests changed as we moved from the hit to lead (HTL) phase to the lead optimization (LO) phase in the drug discovery process. The num- ber of compounds in the lead optimization stages of the discovery process is lower compared to the HTL phase, but the information required for each compound is higher. For this reason, not only the purity but also the amount of purified compound is important since a larger quantity is required for the studies being performed at this stage [6,7]. This is reflected in the increasing importance of hav- ing an efficient chromatographic procedure to purify compounds of pharmacological interest [8,9]. ∗ Corresponding author. Tel.: +34 925 24 57 85; fax: +34 925 24 57 71. E-mail address: lfont@its.jnj.com (L.M. Font). To separate mixtures of a wide range of polarities it is quite common to use standard generic gradients which can be applied efficiently and allow for increased laboratory throughput [1]. In general, the results are satisfactory and compounds are recovered with a high purity. However, frequently standard methods do not deliver sufficient separation for some crude mixtures. For instance, the presence of diastereoisomers, regioisomers and/or impurities which have small chemical differences compared to the main com- pound often results in closely eluting peaks in the chromatogram which are not well resolved with standard methods. Moreover, in recent years, the increased degree of automation and performance in normal phase low pressure purification systems has allowed the purification of many compounds by this technique in our labo- ratory. As a result, most compounds purified by our preparative RP-HPLC–MS instruments are the most challenging samples not resolved by standard methods. The strategy of HPLC method development used in medici- nal chemistry laboratories must fulfill several conditions. The first is that the methodology should lead to orthogonal results. This means that the use of different experimental conditions such as different columns or mobile phases, should give different retention times and elution orders in order to provide the desired separation [10]. With reversed phase HPLC different orthogonal experimental conditions can be achieved, as well as conditions for method devel- opment, such as changing the organic phase, the pH, the stationary phase and the temperature [11,12]. In order to find the optimal separation method for a given sam- ple the screening of a wide range of conditions must be performed. 0021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2010.10.102