Journal of Chromatography A, 1218 (2011) 3648–3653 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Synthesis of (S)-naproxen-benzotriazole and its application as chiral derivatizing reagent for microwave-assisted synthesis and indirect high performance liquid chromatographic separation of diastereomers of penicillamine, cysteine and homocysteine Ravi Bhushan ∗ , Rituraj Dubey Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee-247667, India article info Article history: Received 13 January 2011 Received in revised form 28 March 2011 Accepted 6 April 2011 Available online 14 April 2011 Keywords: (S)-Naproxen Benzotriazole Penicillamine Cysteine Homocysteine High performance liquid chromatography Enantioseparation abstract (S)-Naproxen-benzotriazole was synthesized by the reaction of (S)-naproxen with 1H-benzotriazole using coupling reagent dicyclohexyl carbodiimide and 4-dimethylamino pyridine (DCC/DMAP). It was used as chiral derivatizing reagent for microwave irradiated synthesis of diastereomers of penicillamine, cysteine and homocysteine. The diastereomers were separated by reversed phase high performance liquid chromatography using gradient elution of triethylammonium phosphate (pH 3.5)–acetonitrile (30–65% within 30 min). The method was validated for accuracy, precision, and limit of detection. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Penicillamine (PenA), cysteine (Cys) and homocysteine (Homo- cys) are thiol group containing amino acids and are easily available pharmaceutically important compounds (Fig. 1). d-Isomer of PenA is pharmacologically active while l-isomer, found naturally, is toxic [1] and hence use of dl-PenA is restricted [2]. Certain pharmaceuti- cal applications and aspects of biological importance of PenA [3–7], Cys [8,9], and Homocys [10] are described in literature. The development of analytical methods for enantioseparation of pharmaceutically useful compounds becomes important and desir- able for control of their enantiomeric purity and to understand their pharmacokinetics and pharmacodynamics. The application of various chiral derivatizing reagents (CDRs) for enantioseparation has been advantageous due to availability of, excellent detection sensitivity and flexibility of chromatographic conditions required for separation of diastereomers on inexpensive achiral columns [11,12]. Different CDRs have been used for enantioseparation of PenA [13–18], Cys [17,19–23] and Homocys [17,20]. ∗ Corresponding author. Tel.: +91 1332 285795; fax: +91 1332 286202. E-mail address: rbushfcy@iitr.ernet.in (R. Bhushan). Enantiomeric mixture of derivatives of dl-PenA, prepared by cyclization with HCHO, was separated by ligand exchange approach both in thin layer chromatography (using ChiralPlate ® ) [24] and high-performance liquid chromatography (HPLC) [25]. Separa- tion of spiro-derivatives of dl-PenA, prepared by cyclization with ninhydrin, was made using Cu(II)-l-proline complex as chiral mobile phase additive in HPLC [26], while -cyclodextrin and -glycoprotein columns provided analytical and preparative enan- tioseparation [27] of spiro derivatives of dl-PenA and dl-Cys. Enantioresolution of Homocys has been achieved by HPLC as homocysteine thiolactone using different CSPs based on l- valine-3,5-dimethylanilide attached to monodisperse poly(glycidyl methacrylate-co-ethylene) beads [28], isopropyl-carbamate func- tionalized cyclofructan6 (IP-CF6) bonded to silica gel [29] and diphenyl substituted 1,1 ′ -binaphthyl crown ether [30]. Literature reveals that different N-acylbenzotriazoles have been synthesized via reaction of benzotriazole with different unsatu- rated carboxylic acids [31] and reaction of 1-(1-methanesulfonyl) benzotriazole with different carboxylic acids [32] and N-Boc-- amino acids [33]. These N-acylbenzotraizoles have been further utilized for N-acylation of different amino compounds [34–36]. (S)-naproxen provides a chiral platform having a UV absorb- ing chromophore to synthesize CDRs since it has a carboxylic 0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2011.04.016