Journal of Chromatography B, 809 (2004) 81–86 Determination of the cyclic depsipeptide FK228, a histone deacetylase inhibitor, by liquid chromatography–mass spectrometry Kyunghwa Hwang a , Richard L. Piekarz b , Susan E. Bates b , William D. Figg a,∗ , Alex Sparreboom a a Clinical Pharmacology Research Core, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA b Cancer Therapeutics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA Received 21 April 2004; received in revised form 3 June 2004; accepted 7 June 2004 Available online 26 June 2004 Abstract An analytical method was developed for the quantitative determination of the novel histone deacetylase inhibitor, depsipeptide FK228 (formerly FR901228; NSC 630176), in human plasma. Calibration curves were constructed in the range of 0.5–100 ng/ml, and were analyzed using a weight factor proportional to the nominal concentration. Sample pretreatment involved a liquid–liquid extraction with ethyl acetate using 500 l aliquots of plasma. The analyte was separated on a column (50 mm × 4.6 mm i.d.) packed with 3.5 m C8 material, and eluted with methanol—10 mM ammonium formate (55:45; v/v; pH 8). The column effluent was monitored by mass spectrometry with electrospray ionization. The values for precision and accuracy were always ≤7.88% and <3.33% relative error, respectively. The method was successfully applied to examine the pharmacokinetics of FK228 in a cancer patient. © 2004 Elsevier B.V. All rights reserved. Keywords: Cyclic depsipeptide; FK228 1. Introduction Acetylation and deacetylation of histones plays a ma- jor role in the regulation of gene transcription and in the modulation of chromatin structure [1]. The state of acetylation of these nucleosome core proteins is deter- mined by two classes of enzymes with opposing activity, which are referred to as histone acetyl transferases and histone deacetylases (HDACs). During the last decade, various agents have been identified that inhibit HDAC activity and induce cell growth arrest, differentiation and/or apoptotic cell death [2]. These agents belong to diverse structural classes and include short-chain fatty acids, hydroxamic acids, synthetic benzamides, and cer- tain cyclic tetrapeptides. In the latter group, cyclic dep- sipeptide FK228 (formerly FR901228, NSC 630176; (E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]4,21 -diisopropyl-2- oxa - 12,13 - dithia-5,8,20,23-tetraazabicyclo[8,7,6]tricos-16- ∗ Corresponding author. Tel.: +1-301-402-3623; fax: +1-301-402-8606. E-mail address: wdfigg@helix.nih.gov (W.D. Figg). ene-3,6,9,19,22-pentanone; Fig. 1) is a novel, highly po- tent histone deacetylase inhibitor [3], which was first iso- lated from the fermentation broth of Chromobacterium violaceum [4]. This agent induces expression of the cyclin-dependent kinase inhibitor p21 WAF1 [5–7] through ataxia telangiectasia-mutated-related protein kinase [8], and has also shown antiproliferative activity in various in vitro and in vivo models for human solid tumors [9,10] and chronic lymphocytic leukemia [11,12]. Clinical trials of FK228 in patients with refractory solid tumors and hematological malignancies are currently ongo- ing [13,14]. Preliminary data indicate that FK228 is a po- tentially effective agent for the treatment of peripheral and cutaneous T-cell lymphoma [15] as well as renal cell car- cinoma [14]. It has been suggested that, in vivo, FK228 may act as a prodrug that requires glutathione-mediated in- tracellular activation [16,17]. As part of a project to fur- ther assess the disposition and the pharmacodynamic pro- file of FK228, we report here on the development and val- idation of an analytical method that allows the determina- tion of the drug at low concentrations in human plasma samples. 1570-0232/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2004.06.007