Development of square-wave adsorptive stripping voltammetric method for determination of acebutolol in pharmaceutical formulations and biological fluids
© Al-Ghamdi et al 2012
Received: 21 November 2011
Accepted: 21 February 2012
Published: 21 February 2012
A validated simple, rapid, sensitive and specific square-wave voltammetric technique is described for the determination of acebutolol (AC) following its accumulation onto a hanging mercury drop electrode in a Britton-Robinson universal buffer of pH 7.5. The optimal procedural conditions were: accumulation potential Eacc = - 0.8 V versus Ag/AgCl/KCl, accumulation duration tacc = 30 s, pulse-amplitude = 70 mV, scan rate = 100 mV/s, frequency = 30 Hz, surface area of the working electrode = 0.6 mm2 and the convection rate = 2000 rpm. Under these optimized conditions, the adsorptive stripping voltammetry (AdSV) peak current was proportional over the concentration range 5 × 10-7 - 6 × 10-6 M (r = 0.999). Recoveries for acebutolol from human plasma and urine were in the range 97-103% and 96-104% respectively. The method proved to be precise (intra-day precision expressed as %RSD in human plasma ranged from 2.9 - 3.2% and inter-day precision expressed as %RSD ranged from 3.4 - 3.8%) and accurate (intra-day accuracies expressed as % error in human urine ranged from -3.3 - 2.8% and inter-day accuracies ranged from -3.3 - 1.7%). The limit of quantitation (LOQ) and limit of detection (LOD) for acebutolol were 1.7 × 10-7 and 5 × 10-7 M, respectively. Possible interferences by substances usually present in the pharmaceutical formulations were investigated with a mean recovery of 101.6 ± 0.64%. Results of the developed square-wave adsorptive stripping voltammetry (SW-AdSV) method were comparable with those obtained by reference analytical method.
Acebotolol, (RS)-N-(3-acetyl-4-[2-hydroxy-3-(propan-2-ylamino)propoxy]phenyl)butanamide is a cardioselective, lipophilic β-adrenoreceptor blocking agent with mild intrinsic sympathomimetric activity. It is therefore more suitable than non cardioselective β-blockers, if a patient with asthma or chronic obstructive pulmonary disease needs treatment with a β-blocker. It is marketed in tablets form for oral admininistration .Various techniques have been concerned with the development of rapid and sensitive methods for the separation, identification or determination of AC and others β-blockers in human urine. These techniques included high performance liquid chromatography (HPLC) [2, 3], HPLC-mass spectrometry(MS) , gas chromatography-mass spectrometry (GC-MS)  and capillary electrophoresis (CE) . On the other hand, existing publications concerning the individual determination of AC in pharmaceutical preparations were based on, spectrophotometry [7–10], spectrofluorimetry , thin layer chromatography (TLC) , HPLC [9–11], GC  and CE . The analytical methods reported for chiral separation of AC included CE [13–19] and HPLC [20–25]. Since, some of these methods required expensive equipment(s) and/or special treatment.
Adsorptive cathodic stripping voltammetry has been shown to be an efficient electroanalytical technique for the determination of sub-nanomolar levels of a wide range of drugs that have an interfacial adsorptive character onto the working electrode surface. It usually involves a simple accumulation step, and most of the excipients used do not interfere in the subsequent determination of drugs . The technique is easy to use, saves of time and costs, low detection limit, high accuracy, wide concentration range, applicability to colored and turbid solution. According to our knowledge, there are only two reported papers for the determination of AC by the electrochemical method based on potentiometry [27, 28]. The first reported method  was a coated wire electrode for some β-blockers (AC one of them) and calcium blockers has been suggested based on the use of dinonylnaphthaline sulphonic acid as ion exchanger material, the selectivity behavior was accurately predicted from calculated distribution coefficient constant for each drug. The second method  based on the use of ion-association complexes of AC with tetraphenylborate and phosphomolybdate as exchange sites in a polyvinyl chloride matrix. Therefore, there has been no report concerning the determination of AC by voltammetric method. The proposed method is a highly sensitive, simple, fast and accurate method with lower detection limits for the determination of AC in human plasma and urine.
Instrumentation and Chemicals
All adsorptive stripping measurements were carried out with 797 VA Computrace (Metrohm, Switzerland) in connection with Dell computer and controlled by (VA computrace 2.0) control software. Stripping voltammograms were obtained via a HP color laserjet CP 1215 printer. A conventional three electrode system was used in the hanging mercury drop electrode (HMDE) mode. pH values were measured with Hanna pH 211 (Romania made). Biohit adjustable micropipette (AU), and Brand adjustable micropipette (Germany), were used to measure microliter volumes of the standard solutions. All chemicals used were of analytical reagent grade and were used without further purification. (±) Acebutolol hydrochloride was obtained from Sigma Chemical Co. (St Louis, MO, USA). AC stock solution of 1 × 10-2 mol L-1 was prepared by dissolving the appropriate amount of AC in methanol in 25 ml volumetric flask and this stock solution was stored in the dark. Britton-Robinson (B-R) supporting buffer (pH 7.5, 0.04 M in each constituent) was prepared by dissolving 2.47 g of boric acid (Winlab, UK) in 500 ml distilled water containing 2.3 ml of glacial acetic acid (BDH, UK) and then adding 2.7 ml of ortho-phosphoric acid (Riedal-deHaen, Germany) and diluting to one liter with distilled water. In addition, phosphate supporting buffer [0.1 M NaH2PO4 (Winlab, UK) and 0.1 M H3PO4] was prepared by dissolving 12 g of NaH2PO4 and 6.78 g of H3PO4 in 1000 ml distilled water. Acetate supporting buffer (0.02 M in each constituent) was prepared by dissolving 1.68 g of sodium acetate (Winlab, UK) in 500 ml distilled water containing 1.12 ml of acetic acid and diluting to one liter with distilled water. Finally, carbonate supporting buffer (0.1 M in each constituent) was prepared by dissolving 10.6 g of sodium carbonate (BDH, UK) and 8.4 g of sodium hydrogen carbonate (Winlab, UK) in one liter distilled water.
Procedures and Analysis
Analysis of Standard AC
The general procedure adopted for obtaining square wave adsorptive stripping voltammograms was as follows: A 10 ml aliquot of B-R supporting buffer (unless otherwise stated) at desired pH was pipetted in a clean and dry voltammetric cell and the required standard solutions of AC were added. The test solutions were purged with nitrogen for 5 min initially, while the solution was stirred. The accumulation potential of - 0.8 V vs. Ag/AgCl was applied to a new mercury drop while the solution was stirred for 30 s. Following the preconcentration period, the stripping was stopped and after 20 s had elapsed, cathodic scans were carried out over the range 0.0 to -1.7 V. All measurements were made at room temperature.
Analysis of AC in Tablets
Twenty tablets of Sectral® (Alexandria Pharm. & Chem. Ind. Co., Egypt) labeled to contain 200 mg AC per tablet were powdered. An adequate amount of the homogenous powder, corresponding to 5 × 10-6 M, was accurately weighed and transferred into a calibrated flask and then dissolved in 25 ml of methanol by sonication for 10 min, followed by mechanical shaking for 10 min and lastly centrifuged for 5 min at 10,000 rpm. A portion of the clear solution was diluted with the supporting electrolyte to achieve the desired concentration. Then AC was quantified by means of the proposed stripping voltammetric procedure.
Analysis of AC in Spiked Human Plasma and Urine
Accurately measured aliquots of AC solutions were pipette into centrifugation tubes containing 300 μl human plasma and/or urine, then vortex were done for 5 min. Into each tube, 0.5 ml of acetontrile, 0.1 ml NaOH (0.1 M), 0.5 ml ZnSO4.7 H2O (5% w/v) were added, where most of the interfering substances (mainly proteins) were simply removed and eliminated by precipitation, then centrifuged for 30 min at 3500 rpm . The clear supernatant layer was filtered through 0.45 μm Milli-pore filter. A 0.1 ml volume of the supernatant liquor was transferred into the voltammetric cell then completed to a 10 ml volume with a pH 7.5 B-R universal buffer. Then AC was quantified by means of the proposed stripping voltammetric procedure.
Results and Discussion
The Electrochemical Behavior of AC
Optimum Parameters and Experimental Conditions
Effect of Supporting Electrolyte and pH
Effect of Accumulation Time and Potential
Effect of Potential Sweep Conditions
Effect of Other Instrumental Variables
The influence of other operating parameters such as the size of the adsorption area (HMDE) and convection rate on the efficiency of the adsorption accumulation of AC was additionally checked. As expected, a linear enhancement for the electrochemical peak intensity was observed when the surface area of HMDE was increased over the range 0.15-0.6 mm2 drop size area. Besides, the SW-AdSV peak current can be maximized further by increasing the stirring rate of the rotating rod over the range 0-2000 rpm. Hence, for optimal sensitivity, 0.6 mm2 drop size and 2000 rpm stirring speed were selected.
In conclusion, for electroanalytical purposes, the optimized experimental conditions for SW-AdSV measurements of AC were accumulating for 30 s at -8.0 V preconcentration potential with stirring rate of 2000 rpm. These voltammetric measurements were carried out in Britton-Robinson buffer at pH 7.5. The applied potential was scanned at 100 mV s-1 with 30 Hz SW frequency rate and 70 mV pulse amplitude.
Validation of the Method
where Ip is the stripping voltammetric peak current in nano-amperes, C is AC concentration and r is the correlation coefficient.
Limit of quantification and limit of detection
The limits of detection (LOD) and limits of quantification (LOQ) were determined using the formula: LOD or LOQ = k S.D.a/b, where k = 3 for LOD and 10 for LOQ, S.D.a is the standard deviation of the intercept, and b is the slope. Also lower limit of detection (LOD) defined as the concentration of AC corresponding to the intersection of the extrapolated linear segment of the calibration graph which is 1.7 × 10-7 M.
This obtained sensitivity was significantly preferable than those reported for other analytical technique used for determination of AC such as potentiometric method  with 6 × 10-6 mol L-1.
Accuracy and precision data for acebutolol in spiked human plasma and urine by the proposed SW-AdSV method.
Experimental conc. (μg mL-1)
0.39 ± 0.012
0.98 ± 0.028
2.04 ± 0.065
0.29 ± 0.011
0.88 ± 0.024
1.85 ± 0.039
0.39 ± 0.012
0.97 ± 0.033
2.06 ± 0.072
0.29 ± 0.014
0.89 ± 0.031
1.83 ± 0.045
The ruggedness of the SW-AdSV method was evaluated by carrying out the analysis using two different analyst (operator) and different instruments on different days. The RSD of less than 2.5% were observed for repetitive measurements in three different day time periods using two different instruments and operators. The results indicate that the method is capable of producing results with high precision.
The robustness of the method was explained by the evaluation the influence of small variation of some of the most important procedure variables including pH, scan rate, accumulation potential and duration. Preliminary inspection of the results under various conditions suggested that the method is fairly robust, but the pH of the measuring solution should be 7.5.
The competitive co-adsorption interference was evaluated in the presence of various substances that are usually found in the pharmaceutical tablets and formulations. For these investigations, the interfering species were added at different concentrations (twice, 5-fold and 50-fold) higher than the concentration of AC (5 ×10-6mol L-1). The additions of filling materials (sucrose, lactose and cellulose), disintegrate agent (starch) and lubricants such as magnesium stearate caused no significant effects on the SW-AdSV response of AC. Hence, this compound may need not to be extracted from these tablet ingredients or additives prior to its determination in tablets.
Comparative determination of acebutolol tablet by the proposed SW- AdSV method and the reference potentiometric method.
Labeled content of Sectral® Tablet ∗ (200 mg acebutolol)
Square-wave adsorptive stripping voltammetric (SW-AdSV) method has been developed for the determination of acebutolol in biological fluids and pharmaceutical formulation for first time. The principal advantage of the proposed method over the reference potentiometric method is sensitivity and specificity. The proposed voltammetric technique has the advantages of being simpler, faster, more selective and more cost-effective than potentiometric procedure. The SW-AdSV method are rapid, requiring about 5 min to run sample, and involve no sample preparation other than dissolving, diluting and transferring an aliquot to the supporting electrolyte. The possibility of monitoring of the compound in human urine and plasma makes the voltammetric method useful for pharmacokinetic and pharmacodynamic purposes.
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project no. RGP-VPP-037.
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