Talanta 83 (2011) 1037–1049 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta The use of experimental design in the development of an HPLC–ECD method for the analysis of captopril Sandile M. Khamanga, Roderick B. Walker ∗ Faculty of Pharmacy, Rhodes University, Grahamstown, 6140, South Africa article info Article history: Received 6 October 2010 Received in revised form 10 November 2010 Accepted 10 November 2010 Available online 18 November 2010 Keywords: High performance liquid chromatography–electrochemical detection (HPLC–ECD) Central composite design (CCD) Oxidative-screen Captopril abstract An accurate, sensitive and specific high performance liquid chromatography–electrochemical detection (HPLC–ECD) method that was developed and validated for captopril (CPT) is presented. Separation was achieved using a Phenomenex ® Luna 5 m (C 18 ) column and a mobile phase comprised of phosphate buffer (adjusted to pH 3.0): acetonitrile in a ratio of 70:30 (v/v). Detection was accomplished using a full scan multi channel ESA Coulometric detector in the “oxidative-screen” mode with the upstream electrode (E 1 ) set at +600 mV and the downstream (analytical) electrode (E 2 ) set at +950 mV, while the potential of the guard cell was maintained at +1050 mV. The detector gain was set at 300. Experimental design using central composite design (CCD) was used to facilitate method development. Mobile phase pH, molarity and concentration of acetonitrile (ACN) were considered the critical factors to be studied to establish the retention time of CPT and cyclizine (CYC) that was used as the internal standard. Twenty experiments including centre points were undertaken and a quadratic model was derived for the retention time for CPT using the experimental data. The method was validated for linearity, accuracy, precision, limits of quantitation and detection, as per the ICH guidelines. The system was found to produce sharp and well- resolved peaks for CPT and CYC with retention times of 3.08 and 7.56 min, respectively. Linear regression analysis for the calibration curve showed a good linear relationship with a regression coefficient of 0.978 in the concentration range of 2–70 g/mL. The linear regression equation was y = 0.0131x + 0.0275. The limits of detection (LOQ) and quantitation (LOD) were found to be 2.27 and 0.6 g/mL, respectively. The method was used to analyze CPT in tablets. The wide range for linearity, accuracy, sensitivity, short reten- tion time and composition of the mobile phase indicated that this method is better for the quantification of CPT than the pharmacopoeial methods. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Captopril (CPT) is an orally active antihypertensive agent [1] and has been widely used for the treatment of hypertension and con- gestive heart failure and it acts as a potent and specific inhibitor of angiotensin converting enzyme (ACE) [2–4]. CPT is designated chemically as 1-[3-mercapto-2-(S)-methyl-1-oxopropyl]-S (L) pro- line [5,6] with an empirical formula of C 9 H 15 NO 3 S and molecular weight of 217.3 (Fig. 1) [6]. Despite the fact that CPT has two stere- ogenic centres, the molecule was developed and is marketed as a single enantiomer, as only one of the four possible isomers can bind with the active site of ACE [6]. It has been reported that the biological activity resides mainly in S-captopril while R-captopril possesses non-ACE inhibiting activity [7]. CPT has a pKa in the range of 2.5–3.5 and is ionized at physiological pH. The pKa and ionization ∗ Corresponding author at: Faculty of Pharmacy, Rhodes University, P.O. Box 94, Grahamstown, 6140, South Africa. Tel.: +27 46 603 8398; fax: +27 46 636 1205. E-mail address: R.B.Walker@ru.ac.za (R.B. Walker). of the secondary amine present in the dicarboxylate chain depends on the adjacent functional group and whether it is in the product or active form [8]. Several methods have been reported for the determination of CPT in a variety of matrices, including high performance liquid chromatography (HPLC) [9–13], capillary zone electrophore- sis (CZE) [14], gas chromatography (GC) [15,16] and gas chromatography–mass spectroscopy (GC–MS) [17]. The use of elec- trochemical detection (ECD) for the analysis of CPT using sequential injection analysis [18] and in rat serum, liver and kidney samples [19] has also been reported. CPT exhibits low UV absorptivity and is a relatively unstable molecule making the assay of dosage forms of CPT very difficult [20]. As a result, a pre- or post-column derivatization procedure is normally required to ensure accurate analysis of CPT resulting in an increased cost and complexity for the quantitation of CPT. However, some derivatives are either not stable [21] or their accu- rate measurement is hampered by the presence of excess reagent and products from side reactions that may cause inaccuracies in quantitation [22,23]. 0039-9140/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2010.11.025