Journal of Pharmaceutical and Biomedical Analysis 49 (2009) 540–546 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba Short communication Development and validation of HPLC method for the determination of Cyclosporin A and its impurities in Neoral ® capsules and its generic versions F. Nunes Bonifacio a , M. Giocanti b,c , J.P. Reynier a , B. Lacarelle b,c , A. Nicolay d,e,f,g,∗ a Laboratoire de Pharmacie Galénique, Faculté de Pharmacie, 13385 Marseille cedex 05, France b Fédération de Pharmacologie, AP-HM Hôpital La Timone, Marseille F-13385, France c Unité FRE-CNRS 2737, Faculté de Pharmacie, Marseille F-13385, France d INSERM, U476, Nutriments lipidiques et Prévention des Maladies Métaboliques, Marseille F-13385, France e INRA, UMR1260, Univ Méditerranée Aix-Marseille 1 et 2, Marseille F-13385, France f Laboratoire de Chimie Analytique, Faculté de Pharmacie, 27 boulevard Jean Moulin, Marseille F-13385, France g IPHM, Marseille F-13385, France article info Article history: Received 8 July 2008 Received in revised form 13 November 2008 Accepted 14 November 2008 Available online 27 November 2008 Keywords: Cyclosporin A HPLC Impurities Neoral ® Generic versions abstract Cyclosporin A (CyA) is a cornerstone immunosuppressant for the prophylaxis against allograft rejection after organ transplantation. The most widely prescribed CyA formulation is Neoral ® soft gelatine cap- sules (Novartis Pharmaceuticals, Basel, Switzerland). After Novartis patent expiration, several generic formulations have been developed. In this paper, a simple and reliable HPLC method was developed and validated for the evaluation of four CyA degradation products (ID-005-95, CyH, IsoCyH and IsoCyA) and two related compounds (CyB and CyG) aimed for the quality control of Neoral ® capsules and its generic formulations. In a second step, the validated method was then compared to the USP assay method for capsules, where some of the mentioned impurities were not adequately resolved from the CyA peak. Isocratic elution at a flow rate of 1.0 mL min -1 was employed on a Lichrospher RP-18 (4 mm × 250 mm; 5 m) analytical column maintained at 75 ◦ C with a tetrahydrofuran:phosphoric acid (0.05 M) (44:56, v/v) as mobile phase. The chromatograms were recorded using a Hewlett Packard 1100 chromatographic system. The UV detection wavelength was performed at 220nm and 10 L of sample was injected. The developed method was validated in terms of selectivity, linearity, precision, accuracy, limit of detection and limit of quantitation. The validate method was successfully applied to commercial cap- sules, Neoral ® and generic versions. Therefore, the proposed method is suitable for the simultaneous determination of CyA as well as its major impurities. © 2008 Elsevier B.V. All rights reserved. 1. Introduction The immunosuppressive properties of Cyclosporin A (CyA) have been widely explored clinically, predominantly in the prophylaxis against allograft rejection after organ transplantation and in the treatment of certain auto-immune diseases [1,2]. In addition to this predominant use in transplantation, CyA has also been indicated for a number of new clinical applications, like the reversal of multi- drug resistance, anti-malarial, herpes virus infection, rheumatoid arthritis, type I diabetes, and also as a potent anti-human immun- odeficiency virus 1 (HIV-1) agent [3,4]. CyA is a cyclic undecapeptide produced by submerged cul- ture fermentation with a number of congeners differing by ∗ Corresponding author at: INSERM U476, INRA, UMR1260, Univ Méditerranée Aix-Marseille 1 et 2, Marseille F-13385, France. Tel.: +33 491835697. E-mail address: alain.nicolay@univmed.fr (A. Nicolay). one to three amino acids [5]. CyA are typically available from multiple sources, and each source may have a different manufac- turing process. The result of a fermentation process is generally a mixture of products having closely related structural and/or physical properties, and the purification of this mixture is the most important stage to acquire a product in a substantially purified form. Therefore, it is essential that the dosage-form manufacturer evaluate each supplier’s drug substance impurity profiles. According to the current rules (e.g., US Food and Drugs Admin- istration and European Pharmacopoeia), CyA is produced with a quality >98.5% [6,7]. In this respect, we should expect that the sum of all impurities do not exceed 1.5%, and that this amount might be respected no matter who produced the CyA. This is particu- larly interesting in view of some results indicating that industrial fungal strains also produced cyclosporins which were previ- ously detected among human trace metabolites ([Leu 4 ]Cs = AM4N, [Leu 9 ]CS = AM9N) [8–10]. 0731-7085/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2008.11.027