Journal of Chromatography B, 799 (2004) 149–155 Improved liquid chromatographic method for mitoxantrone quantification in mouse plasma and tissues to study the pharmacokinetics of a liposome entrapped mitoxantrone formulation Jenifer L. Johnson, Ateeq Ahmad, Sumsullah Khan, Yue-Fen Wang, Aqel W. Abu-Qare, Jennifer E. Ayoub, Allen Zhang, Imran Ahmad ∗ Pharmacokinetics, Safety, and Efficacy Department, NeoPharm Inc., Research and Development, 1850 Lakeside Drive, Waukegan, IL 60085, USA Received 27 March 2003; received in revised form 14 October 2003; accepted 20 October 2003 Abstract A simple, rapid HPLC method for quantification of mitoxantrone in mouse plasma and tissue homogenates in the presence of a liposome entrapped mitoxantrone formulation (LEM-ETU) is described. Sample preparation is achieved by protein precipitation of 100 l plasma or 200 l tissue homogenate with an equal volume of methanol containing 0.5 M hydrochloric acid:acetonitrile (90:10, v/v). Ametantrone is used as the internal standard (i.s.). Mitoxantrone and i.s. are separated on a C18 reversed phase HPLC column, and quantified by their absorbance at 655 nm. In plasma, the standard curve is linear from 5 to 1000 ng/ml, and the precision (%CV) and accuracy (percentage of nominal concentration) are within 10%. In mouse tissue (heart, kidney, liver, lung, and spleen) homogenates (5%, w/v), the standard curve is linear from 25 to 2000 ng/ml, with acceptable precision and accuracy. The method was used to successfully quantify mitoxantrone in mouse plasma and tissue samples to support a pharmacokinetic study of LEM-ETU in mice. © 2003 Elsevier B.V. All rights reserved. Keywords: Pharmacokinetics; Mitoxantrone 1. Introduction Mitoxantrone (Fig. 1) has been used extensively as a component of chemotherapeutic regimens for a number of fatal diseases, including leukemia, lymphoma, cancers of the breast and prostate, and to treat multiple sclerosis [1–4]. While it has been shown to be efficacious and is better-tolerated than other treatments, its use is limited by unwanted side effects, particularly dose-related cardiomy- opathy [1–4]. In an effort to reduce the toxicity of mitox- antrone to normal cells, liposome entrapped formulations have been developed [3,5,6]. Liposomes have shown great promise for increasing the therapeutic efficacy of anticancer drugs. Many liposomes passively target the drug, accumulating in tumor tissue due to enhanced permeability of the tumor vascular system [7]. Liposomal delivery thereby decreases drug toxicity to ∗ Corresponding author. Tel.: +1-847-887-0800; fax: +1-847-887-9281. E-mail address: imran@neophrm.com (I. Ahmad). normal tissues, and may outmaneuver the mechanisms of multi-drug resistance by tumor cells [7,8]. Preclinical stud- ies of our liposome entrapped mitoxantrone formulation (LEM) have shown that it has improved pharmacokinet- ics and tissue distribution, and greater efficacy than free mitoxantrone [6]. Recently, we have developed a well char- acterized, easy-to-use liposome entrapped formulation of mitoxantrone (LEM-ETU) using our NeoLipid TM platform. To support pharmacokinetics and tissue distribution studies of the LEM-ETU formulation, we have developed a simple and rapid assay for quantification of mitoxantrone in mouse plasma and tissues in the presence of LEM-ETU. Various methods have been published describing pro- cedures for the extraction of mitoxantrone from plasma [9–13] and tissue homogenates [14]. HPLC using C18 reverse-phase columns is the most common means of separation, along with visible absorbance near 655 nm as the method of detection. Most of these methods require time-consuming sample preparation, including liquid–liquid extraction [14], SPE [12], and protein precipitation fol- lowed by solid-phase extraction [12] or by online column 1570-0232/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2003.10.034