In situ detection of warfarin using time-correlated single-photon counting Annika M. Rosengren a , Björn C.G. Karlsson a , Inga Näslund b,  , Per Ola Andersson b , Ian A. Nicholls a,c,⇑ a Bioorganic and Biophysical Chemistry Laboratory, School of Natural Sciences, Linnaeus University, SE-391 82 Kalmar, Sweden b Swedish Defence Research Agency, FOI, CBRN Defence and Security, SE-901 82 Umeå, Sweden c Department of Biochemistry and Organic Chemistry Laboratory, Uppsala University, SE-751 23 Uppsala, Sweden article info Article history: Received 15 February 2011 Available online 24 February 2011 Keywords: Warfarin Time resolved fluorescence spectroscopy Time correlated single photon counting Human serum albumin Sudlow I Molecularly imprinted polymer MIP abstract Here we report on a novel method for the direct in situ measurement of specific isomeric forms of the anticoagulant warfarin using time correlated single-photon counting (TCSPC) spectroscopy in conjunc- tion with synthetic Sudlow I binding site receptors. The method is highly robust over the clinically sig- nificant concentration range, and demonstrates the potential of the binding site mimics in conjunction with the spectroscopic strategy employed here for the determination of this important pharmaceutical in clinical or even environmental samples. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction The coumarin derivative warfarin is widely used as an anticoag- ulant drug in the treatment and prevention of thrombolic disorders such as myocardial infarction and stroke [1]. Due to the narrow therapeutic window of warfarin, variability in dose–response and interactions with other compounds, careful monitoring of the ef- fect of drug dosage must be performed [2]. Despite its widespread use and the difficulties in adapting dosage to clinical function, no assay involving the direct detection of this anticoagulant is cur- rently used in general clinical practice. In principle, the fluoro- phoric nature of coumarins should allow for a fluorescence spectroscopy-based monitoring of warfarin. Recent efforts by us [3–5] using a series of theoretical and spectroscopic studies have highlighted the complex nature of warfarin, and in particular the medium dependent isomerization of this drug (see Chart 1), per- haps illustrating why spectroscopy-based methods for the direct determination of warfarin have not been forthcoming. The unrav- eling of the relationship between molecular environment, isomeric distribution and the spectroscopic characteristics of the coumarin derivative has afforded us the possibility for developing detection methods, which can potentially discriminate between warfarin in various states, e.g. bound to protein [3], polymeric synthetic recep- tors [6], and as a general molecular probe [7]. Here we combine the recognition characteristics of a polymeric synthetic receptor system, a human serum albumin Sudlow I binding site mimic [6], with warfarin’s molecular environment- sensitive fluorescence emission signal [3] to provide a basis for a robust technique for warfarin determination. The detection strat- egy described here could have impact for both clinical and even environmental monitoring [8]. 2. Materials and methods 2.1. Chemicals Racemic warfarin (3-(a-acetonylbenzyl)-4-hydroxycoumarin, min. 98%) was purchased from Sigma–Aldrich (St. Louis, MO, USA). All solvents were of analytical grade and used as received. 2.2. Fluorescence spectroscopy The warfarin imprinted (MIP) and the corresponding reference (REF) methacrylic acid–ethylene dimethacrylate co-polymers used were prepared as previously reported [6]. Prior to fluorescence measurements, polymer particles were allowed to swell for at least 20 h in acetonitrile (5 mg mL 1 ). All fluo- rescence spectroscopic measurements were typically performed 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.02.103 Abbreviations: TCSPC, time correlated single photon counting; MIP, molecularly imprinted polymer; REF, reference polymer. ⇑ Corresponding author at: Bioorganic and Biophysical Chemistry Laboratory, School of Natural Sciences, Linnaeus University, SE-391 82 Kalmar, Sweden. Fax: +46 480 446262. E-mail addresses: ian.a.nicholls@bioorg.uu.se, ian.nicholls@lnu.se (I.A. Nicholls).   Passed away November 23, 2009. Biochemical and Biophysical Research Communications 407 (2011) 60–62 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc