Journal of Chromatography A, 1101 (2006) 122–135 “Orthogonal” separations for reversed-phase liquid chromatography Jackson Pellett a , Patrick Lukulay a , Yun Mao b , William Bowen b , Robert Reed b , M. Ma c , R.C. Munger c , J.W. Dolan d , Loren Wrisley e , K. Medwid e , N.P. Toltl f , C.C. Chan f , M. Skibic g , Kallol Biswas g , Kevin A. Wells g , L.R. Snyder h,∗ a Pfizer Global Research & Development, Pharmaceutical Sciences, Analytical R&D, Michigan Laboratories, Ann Arbor, MI, USA b Merck & Co., Inc., Pharmaceutical Research & Development, Pharmaceutical Analysis and Control, West Point, PA 19486, USA c Amgen Inc., Analytical Sciences, One Amgen Center Drive, Thousand Oaks, CA 91320, USA d BASi Northwest Laboratory, McMinnville, OR 97128, USA e Wyeth Research, Pearl River, NY 10965, USA f Eli Lilly Canada, Toronto, Canada g Eli Lilly, Indianapolis, IN, USA h LC Resources, 26 Silverwood Ct., Orinda, CA 94563, USA Received 11 July 2005; received in revised form 27 September 2005; accepted 27 September 2005 Available online 19 October 2005 Abstract A general procedure is proposed for the rapid development of a reversed-phase liquid chromatographic (RP-LC) separation that is “orthogonal” to a pre-existing (“primary”) method for the RP-LC separation of a given sample. The procedure involves a change of the mobile-phase organic solvent (B-solvent), the replacement of the primary column by one of very different selectivity, and (only if necessary) a change in mobile phase pH or the use of a third column. Following the selection of the “orthogonal” B-solvent, column and mobile phase pH, further optimization of peak spacing and resolution can be achieved by varying separation temperature and either isocratic %B or gradient time. The relative “orthogonality” of the primary and “orthogonal” RP-LC methods is then evaluated from plots of retention for one method versus the other. The present procedure was used to develop “orthogonal” methods for nine routine RP-LC methods from six pharmaceutical analysis laboratories. The relative success of this approach can be judged from the results reported here. © 2005 Elsevier B.V. All rights reserved. Keywords: Reversed-phase liquid chromatography; Orthogonal method; Pharmaceutical analysis 1. Introduction When pharmaceutical samples are to be separated by reversed-phase liquid chromatography (RP-LC), a common con- cern is that an impurity or sample degradation peak may be overlooked—due to its being overlapped by a second (possi- bly larger) peak in the chromatogram. This has led to a need for so-called “orthogonal” separations; i.e., two separations of quite different selectivity, with marked changes in relative retention so that two peaks which are unresolved in one chromatogram will likely be separated in the second chromatogram. Note that the term “orthogonal” is presently used in the literature [1–4] and within the pharmaceutical industry to denote separations, which ∗ Corresponding author. simply provide differing relative retention. Other workers prefer to limit the use of “orthogonal” to systems where elution times can be treated as statistically independent [5]—a much more restrictive definition than is used by us. In the present paper, which deals with separations that are not statistically indepen- dent, we distinguish our use of “orthogonal” by enclosing the word in quotation marks. A hypothetical example of separation “orthogonality” is shown in Fig. 1. Assuming an original (primary) RP-LC method: (a) in which such a peak overlap occurs (peaks 6, 6a), an “orthogonal” separation (b) should have a high probability of disengaging the two peaks. The degree of “orthogonality” for two such separations is commonly characterized by the scatter of plots of retention for one separation versus the other (Fig. 1c). Isocratic elution is assumed in Fig. 1, but gradient separations are more common for measurements of sample impurities. 0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.09.080