Journal of Chromatography B, 877 (2009) 2493–2498 Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb Determination of the antifungal agent posaconazole in human serum by HPLC with parallel column-switching technique Werner Neubauer a,b , Armin König c , Richard Bolek c , Rainer Trittler b , Monika Engelhardt a , Manfred Jung d , Klaus Kümmerer c,∗ a Department of Hematology & Oncology, University Medical Center Freiburg, Freiburg, Germany b Hospital Pharmacy, University Medical Center Freiburg, Freiburg, Germany c Department of Environmental Health Sciences, University Medical Center Freiburg, Freiburg, Germany d Institute of Pharmaceutical Sciences, University of Freiburg, Freiburg, Germany article info Article history: Received 13 February 2009 Accepted 12 June 2009 Available online 21 June 2009 Keywords: Posaconazole Bioavailability Serum HPLC Column-switching On-line sample preparation abstract Posaconazole is a new broad-spectrum antifungal agent that is currently only available as an oral suspen- sion and shows high intra- and inter-individual differences in oral bioavailibility. Pre-existing methods for the determination of the substance involve the use of internal standards or require a quite complicated and time-consuming sample pre-treatment. Our HPLC method is fast and fully-automated and there is no need for any manual sample pre-treatment. On-line transfer of posaconazole from the extraction column was followed by chromatographic separation on a C18 column and fluorescence detection ( ex : 261 nm,  em : 357nm). Retention time of posaconazole was about 11.7min, the lower limit of quantification was found to be 0.1mg/l. A linear calibration curve was obtained over the concentration range of 0.1–5mg/l using a 50 l sample (r 2 =0.999). The relative standard deviations of intra-day variations ranged from 2.3% to 9.4%,intra-day accuracy from 88.8% to 114.8%. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Invasive fungal infections (IFIs) are associated with high morbid- ity and mortality rates among patients with hematological diseases and in those undergoing hematopoietic stem cell transplantation [1–3]. Their incidence has increased significantly during the last 20 years and the current treatment options provide limited benefit [1,4–6]. Posaconazole (4-4-[4-(4-{(3R,5R)-5-(2,4-difluorophenyl)-5- (1H-1,2,4-triazol-1-ylmethyl)tetrahydro-3-furanyl}methoxy- phenyl)piperazino]phenyl-1-[(1S,2S)-1-ethyl-2-hydroxypropyl]- 4,5-dihydro-1H-1,2,4-triazol-5-one) (chemical structure in Fig. 1) is a new antifungal triazole used for the prophylaxis and therapy of invasive fungal infections [7,8]. It is a derivative of itraconazole with a high in vitro and in vivo activity against a number of molds and yeasts, including Candida and Aspergillus species, but also less common fungi, such as the Zygomycetes, Cryptococcus or fusarium species [9–14]. As all triazole antimycotics, the substance works by inhibiting the cytochrome P450 dependent lanosterol ∗ Corresponding author at: Department of Environmental Health Sciences, Uni- versity Medical Center Freiburg, Breisacher Str. 115B, D-79106 Freiburg i. Br., Germany. E-mail address: klaus.kuemmerer@uniklinik-freiburg.de (K. Kümmerer). 14--demethylase, which catalyses an essential step in ergosterol biosynthesis. So far, posaconazole is not available as an intravenous formu- lation and has to be given as an oral suspension. Comparable to itraconazole, its bioavailability can be augmented when given with food (area under the curve [AUC] about four times greater when administered with high-fat meals) [15]. A daily administration of 800 mg, given in two divided doses, resulted in a maximum expo- sure, but there are high intra- and inter-individual differences [16,17]. Oral exposure, for instance, is especially low in patients after bone marrow or peripheral blood stem cell transplantation. The American product information reports on a positive association between average posaconazole levels in the blood and the effi- cacy of antifungal prophylaxis, and a possible association between an increased risk of treatment failure with lower posaconazole concentrations is described [7]. An open-label multicenter study recently reported that higher plasma concentrations were asso- ciated with improved response rates in the treatment of invasive aspergillosis [18]. For these reasons, monitoring serum posacona- zole levels is recommended at least for patients with suspected poor oral absorption, patients with progressive disease under posaconazole therapy, patients on concomitant medications with significant drug interactions [19] and patients at risk for lower serum posaconazole levels (e.g. patients after bone marrow or peripheral blood stem cell transplantation). 1570-0232/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2009.06.022