Journal of Chromatography A, 1217 (2010) 1024–1032 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Extending the use of “Inverted Chirality Columns Approach” for enantiomeric excess determination in absence of reference samples: Application to a water-soluble camptothecin derivative Elena Badaloni a , Walter Cabri a , Alessia Ciogli b , Ilaria D’Acquarica b , Roberto Deias a , Francesco Gasparrini b,∗ , Fabrizio Giorgi a , Dorina Kotoni b , Claudio Villani b a Analytical Development, R&D Department, sigma-tau S.p.A., Via Pontina km 30,400, 00040 Pomezia, Italy b Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy article info Article history: Available online 17 October 2009 Keywords: Inverted Chirality Columns Approach (ICCA) Elution order reversal Anticancer drugs Camptothecin (CPT) family Namitecan (ST1968) abstract The aim of the present study was to extend the use of the “Inverted Chirality Columns Approach (ICCA)” previously developed for the identification and quantitation of the trace enantiomer in highly enriched samples of the camptothecin (CPT) family of drugs to a novel water-soluble CPT derivative, namely namitecan (ST1968), currently undergoing phase I clinical trials as anticancer agent. Namitecan, identi- fied from a series of hydrophilic 7-oxyiminomethyl derivatives, contains a free terminal amino group, which traditionally hampers the analysis under normal-phase HPLC conditions. Nevertheless, commer- cially available Pirkle-type chiral stationary phases (CSPs) available in both the enantiomeric forms (i.e., having the same bound selector with opposite configuration) mainly operate under normal-phase HPLC conditions. For this reason, namitecan was pre-column N-protected with isocyanates A–D and their sulfur analogues E–H to reduce its polarity by converting the amino group into a fragment compatible with the chiral recognition mechanism (i.e., ureido and thioureido groups). Once the optimal columns system and derivatizing agents were selected, an original enantioselective HPLC–MS/MS technique was developed on the Whelk-O1 CSPs. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Two years ago we described an original enantioselective HPLC–MS/MS technique for the identification and quantitation of the trace enantiomer in highly enriched samples of the camp- tothecin (CPT) family of drugs, even in absence of reference samples [1]. The approach we developed, designated as “Inverted Chiral- ity Columns Approach (ICCA)”, is based on the reversal of the elution order of a given enantiomeric pair [2] as a result of the columns switching, under identical chemical conditions. This tech- nique, which is not available when naturally occurring selectors such as polysaccharides or proteins are employed, is very useful for enantiomeric trace analysis when the minor enantiomer follows the major one and is partially hidden by the tailing of the lead- ing enantiomer: on the chiral stationary phase (CSP) with opposite configuration the trace enantiomer is eluted first, thus enabling a more precise and accurate quantitation by peak area integration [3]. ICCA was developed to respond to important incoming needs in the field of pharmaceutical analysis: (i) the complete assignment of the ∗ Corresponding author. Tel.: +39 06 49912776; fax: +39 06 49912780. E-mail address: francesco.gasparrini@uniroma1.it (F. Gasparrini). stereoisomeric composition of chiral drugs [4,5], including natural products and synthetic intermediates, as established by the Inter- national Conference on Harmonization (ICH) guidelines [6]; in such cases, however, only one enantiomer is available as reference, and the racemate could be prepared by means of expensive and mul- tistep, time-consuming total syntheses; (ii) extreme enantiomeric excesses (ee > 99%) must be estimated with large accuracy, which is a critical point in any of the available techniques [4]; (iii) low lim- its of detection (LODs) and quantitation (LOQs) in highly enriched samples contained in complex mixtures are strongly required to yield an unequivocal peak identification. Application of the ICCA to semi-synthetic derivatives of CPT endowed with anticancer activ- ity [1] clearly showed its selectivity and specificity in the accurate determination of extreme ee, even when only one enantiomer was available. The success of the approach was assured by the com- bination of Pirkle-type CSPs with multistage mass spectrometry (APCI-MS/MS) detection. The accomplished familiarity in the ICCA technique prompted us to approach a novel water-soluble CPT derivative, namely namite- can (ST1968) [7–10], currently undergoing phase I clinical trials as anticancer agent [11]. The CPT family of drugs appear to have a unique mechanism of action: they inhibit the nuclear enzyme topoisomerase I (topo I), by forming a ternary complex with topo 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.10.035