The current manuscript reports the first capillary electrophoresismethod for the separation and quantification of metoprolol (MET)and hydrochlorothiazide (HCT) in their combined dosage form. METand HCT were detected at 240 and 214 nm, respectively, using aphotodiode array detector. The univariate approach was used foroptimizing voltage, injection time and capillary temperature. Thefactorial design with response surface plots, as a multivariate ap-proach, was used to study the effect of buffer concentration andpH on resolution, peak area and migration time. The optimum condi-tions were 50 mmol/L phosphate at pH 9.5, injection time 10.0 s,voltage 25 kV and capillary temperature 258C. The method waslinear in the range of 2.5–250 mg/mL for both drugs with correl-ation coefficients above 0.9997. Additionally, acceptable recoveryof the contents of MET and HCT in their formulations (96.0–100.3%) with acceptable precision (1.38–2.60 %) were achieved.Moreover, the limits of detection of MET and HCT were 0.02 and0.01 mg/mL, respectively, which were suitable for pharmaceuticalanalysis.IntroductionThe preparation of new combinations of medicines, as well asthe requirements of modern industrial-scale pharmaceuticalanalysis, requires researchers to develop new and efficientmethods for multi-quantification with separation procedures.High-performance liquid chromatography (HPLC) is a domin-ant separation technique, especially in pharmaceutical analysis.However, HPLC does not always fulfil all of the requirements ofmodern industrial-scale pharmaceutical analysis with respect toreagent consumption, analysis time, instrumentation cost andinstrumentation simplicity. Capillary electrophoresis (CE), as amicrofluidic-based technology, is simpler, faster, less expensive,consumes less reagent and is more efficient in separation thanHPLC (1, 2). Hence, the extensive utilization of CE to generatecomplementary and alternative methods of pharmaceutical ana-lysis is desirable.To improve the therapy of cardiovascular diseases, many me-dicinal substances are used in combined dosage forms, as inthe case of metoprolol (MET) and hydrochlorothiazide (HCT)(3–5). MET is a b-1 selective adrenoceptor antagonist. It iswidely used for the treatment of mild to moderate hyperten-sion and angina pectoris (6). MET is chemically known as6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulphonamide1,1-dioxide (Figure 1A). HCT is one of the oldest thiazidediuretics used to treat hypertension (7). HCT is chemicallyknown as 2-propanol, 1-[4-(2-methoxyethyl) phenoxy]-3-[(1-methylethyl) amino]-,(+)-,butanedioate (Figure 1B).The British Pharmacopoeia (8) and the United StatesPharmacopeia (9) have addressed assay methods for MET andHCT in their single formulation. However, an official methodfor their simultaneous quantification in combined dosage formshas not yet been developed. Moreover, despite the widespreaduse of the combination of MET and HCT, few validated assaymethods have been proposed using some analytical techniques.Regarding this issue, two reversed-phase HPLC methods withultraviolet (UV) detection have been developed (10, 11).Another HPLC method with mass spectrometric detection hasalso been developed (12). Moreover, derivative spectrophoto-metric methods for simultaneous quantification of MET andHCT in pharmaceutical preparations have also been reported(13–15).On the other hand, many CE methods have been reportedfor the assay of MET or HCT with other drugs. Among thosemethods, CE and micellar electrokinetic capillary chromato-graphic (MEKC) (16) have been applied for the separation andquantification of HCT with six angiotensin-II-receptor antago-nists (ARAII). In the CE method, a simple electrolyte of60 mmol/L phosphate at pH 2.5 was adopted, while the MEKCmethod applied 55 mmol/L phosphate buffer at pH 6.5 with15 mmol/L sodium dodecyl sulfate (SDS). The authors reportedthat the CE and MEKC methods were suitable for the qualita-tive and quantitative determination of combined HCT–ARAIIsin pharmaceutical formulations. In another CE method (17),MET, atenolol and esmolol were separated and quantified withelectrochemiluminescence detection. In that method,poly-b-cyclodextrin was added to the running buffer of20 mmol/L phosphate at pH 10.0 to improve the separation.This method was applied to quantitatively determine MET inits pharmaceutical preparation and to monitor its pharmacokin-etics in the human body. Another study proposed the couplingof CE with a capacitively coupled contactless conductivity de-tector for the determination of a single analysis of MET, chlor-pheniramine, clomiphene, catharanthine and vinorelbine andtheir counter-ions (18). Dual-opposite end injection was usedto hydrodynamically introduce the analytes at each end of thecapillary (18). In another study, CE with laser-induced fluores-cence detection was used for the assay of MET in rabbit plasma(19). In addition, CE and capillary electrochromatography