Journal of Pharmaceutical and Biomedical Analysis 36 (2004) 249–255 UV spectroscopy and reverse-phase HPLC as novel methods to determine Capreomycin of liposomal fomulations Carlo Rossi a , Giuseppe Fardella a , Ione Chiappini b , Luana Perioli a , Claudia Vescovi a , Maurizio Ricci a,∗ , Stefano Giovagnoli a , Stefania Scuota b a Department of Chimica e Tecnologia del Farmaco, Via del Liceo 1, Universit` a degli Studi di Perugia, 06123 Perugia, Italy b Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Via G. Salvemini 1, 06100 Perugia, Italy Received 12 January 2004; received in revised form 21 June 2004; accepted 22 June 2004 Available online 6 August 2004 Abstract Capreomycin (CS) is an antitubercular drug active against several Mycobacterium strains, in particular, against M. Avium. In spite of its activity, it is considered a second line drug because it can induce severe renal and hepatic damages when administered as free drug. However, it is possible to employ drug delivery systems, such as liposomes, to reduce the toxicity of the peptide without loss of its biological activity. For this purpose, appropriately validated time and money saving analytical methods are needed for a careful capreomycin dosage. In the present paper, UV spectroscopy and a reverse-phase HPLC (RP-HPLC) were investigated as alternative methods for capreomycin quantitative analysis. These techniques were validated against the USP XXVI microbiological turbidimetric assay and the normal-phase HPLC (NP-HPLC) method reported in the British Pharmacopoeia 2003. The results obtained showed that either UV spectrophotometry or RP-HPLC are techniques having higher accuracy and reproducibility with respect to the microbiological assay. Moreover, the RP-HPLC method provided improved performances if compared to NP-HPLC. In fact, RP-HPLC showed: (i) enhanced sensitivity and (ii) increased resolution. Thus we propose RP-HPLC and UV as valid alternative methods to the conventional procedures for capreomycin quantitative analysis. © 2004 Elsevier B.V. All rights reserved. Keywords: Liposomes; Capreomycin; RP-HPLC; NP-HPLC; Spectrophotometric analysis; Microbiological turbidimetric assay 1. Introduction Capreomycin (CS) is an antitubercular drug produced by fermentation from Streptomyces capreolus and first described in the 1960s [1]. CS is characterized by a cyclic pentapeptide structure and it exists as a mixture of four active components, namely IA, IB, IIA, IIB, distributed as follows: 90% IA and IB forms and 10% IIA and IIB forms (Fig. 1). This peptide shows a high bacteriostatic activity both in vitro and in vivo against Mycobacterium tuberculosis, M. Avium, M. bovis, and M. kansasii. Recent studies demonstrated that only 10% of ∗ Corresponding author. Tel.: +39 075 5855125; fax: +39 075 5855163. E-mail address: riky@unipg.it (M. Ricci). the 46 drug resistant strains of M. Avium isolated from Italian patients were resistant to CS [2]. In spite of its biological activity, CS is considered a second-line drug and it can be used in the treatment of tu- berculosis when multi-resistance to conventional drugs, such as isoniazid and ethambutol, is developed [3]. In fact this peptide can induce progressive renal damages and severe ab- normalities in liver functions. Rare cases of hypersensitivity reactions are also reported [4,5]. The current strategy for reducing the toxicity of available drugs, without losing their biological activity, is to entrap these molecules inside drug delivery systems able to slowly release the drug over long periods of time. Consequently, a selection of suitable CS carriers was nec- essary. The attention was focused on liposomes as potential 0731-7085/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2004.06.015