Journal of Chromatography B, 932 (2013) 144–151 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B j ourna l h o mepa ge: www.elsevier.com/locate/chromb Development and validation of a simple and sensitive HPLC–UV method for the determination of captopril in human plasma using a new derivatizing reagent 2-naphthyl propiolate Noushin Rastkari a,d , Mehdi Khoobi b , Abbas Shafiee b , Mohammad Reza Khoshayand c , Reza Ahmadkhaniha d,∗ a Center for Air Pollution Research (CAPR), Institute for Environmental Research (IER), Tehran University of Medical Sciences, Tehran 1417613151, Iran b Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran c Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14174, Iran d Department of Human Ecology, School of Public Health, Tehran University of Medical Sciences, Tehran 1417613151, Iran a r t i c l e i n f o Article history: Received 9 March 2013 Accepted 12 June 2013 Available online 19 June 2013 Keywords: Captopril 2-Naphthyl propiolate Optimization Pre-column derivatization Plasma HPLC a b s t r a c t In this study, a simple, sensitive and reliable HPLC–UV method applying rapid sample preparation tech- nique for the determination of captopril in human plasma was developed and validated. The method is based on pre-column derivatization of captopril and 2-propene-1-thiol (internal standard) with a new reagent 2-naphthyl propiolate. Sample clean-up, derivatization and extraction were carried out in two steps, totally less than 30 min. The extracts were chromatographed on a C18 column (5 m, 150 mm × 4.6 mm i.d.). The mobile phase consisted of methanol (75%, v/v) and phosphate buffer (25%, pH = 8, 0.01 M). UV detection was performed at 290 nm. To obtain the best reaction yield, the factors that could influence the derivatization process, including the concentration of derivatization reagent, pH of sample solution and temperature were investigated in detail and optimized using Box–Behnken response surface methodology. Under optimized conditions the average extraction recovery of captopril and internal standard were >86%. The achieved lower limit of quantification (LLOQ) was 3 ng/mL; the assay exhibited a linear dynamic range of 3–2000 ng/mL with correlation coefficient (r 2 ) of ≥0.99. The precision was satisfactory in the whole calibration range with RSD of 5.9–12.4% (accuracy: from 97.5% to 93.6%) and of 6.4–12.8% (accuracy: from 97.3% to 95.2%) for intra- and inter-assay, respectively. The method stability was confirmed in a series of experiments including: freeze–thaw, short- and long-term stability testing. Lastly, the developed method was successfully applied to the bioequivalence study of captopril administrated as a single oral dose (50 mg) to 12 healthy male volunteers. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Captopril (CAP) is a specific and competitive inhibitor of angiotensin-I converting enzyme that was used primarily for the treatment of hypertension and some types of congestive heart fail- ure. Due to captopril pharmacological relevance, many analytical methods have been developed and reported for its determina- tion in biological samples, including chromatographic methods [1–11], voltammetric sensors based on carbon nanotubes [12], kinetic spectrophotometry [13], colorimetry [14], fluorimetry [15], ∗ Corresponding author. Tel.: +98 21 88978395; fax: +98 21 88978398. E-mail addresses: nr rastkari@yahoo.com (N. Rastkari), mehdi.khoobi@gmail.com (M. Khoobi), ashafiee@ams.ac.ir (A. Shafiee), khoshayand@tums.ac.ir (M.R. Khoshayand), r-ahmadkhaniha@tums.ac.ir, ahmadkha@razi.tums.ac.ir (R. Ahmadkhaniha). spectrophotometry [13,16,17], atomic absorption spectrometry [18] and FT-Raman spectrometry [19]. Amongst these, the most commonly used approaches involve chromatographic methods using derivatization of the analyte prior (pre-column) or after a separation step (post-column) [1–5,9,10]. The main goals of deriva- tization procedures are (a) to enhance the stability of CAP in sample matrices, (b) to increase sensitivity of the method by attaching suit- able chromophore or fluorophore moieties and (c) to improve its chromatographic properties [9,20,21]. Most of the reported deriva- tization methods are based on pre-column derivatization which has the advantage of stabilizing the analyte in biological medium. Although many reagents have been synthesized and used for the determination of CAP in biological specimens, but due to the draw- backs of the already existing methods and the medical significance of CAP, development of new reagents for highly sensitive detec- tion is still continuing [22]. Propiolate esters have recently been introduced as highly effective reagents in alkyl thiol derivatization 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.06.019