Voltammetric Determination of Azidothymidine ... - Wiley Online Library

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Voltammetric Determination of Azidothymidine Using Silver Solid Amalgam Electrodes Karolina Peckova´,a* Toma´sˇ Navra´til,b Bogdan Yosypchuk,b Josino Costa Moreira,c Katia Christina Leandro,d Jirˇ Bareka a

Charles University in Prague, Faculty of Science, Department of Analytical Chemistry, UNESCO Laboratory of Environmental Electrochemistry, Albertov 6, CZ-128 43 Prague 2, Czech Republic *e-mail: [email protected] b J. Heyrovsky´ Institute of Physical Chemistry AS CR, v.v.i., Dolejsˇkova 3, CZ-182 23 Prague 8, Czech Republic c CESTEH/ENSP/FIOCRUZ, Rua Leopoldo Bulhoes, 1480 Manguinhos, 21041-210 Rio de Janeiro, Brazil d National Institute of Quality Control in Health (INCQS/FIOCRUZ), Av. Brazil, 4365, 21040-900 Rio de Janeiro, Brazil Received: April 23, 2009 Accepted: June 4, 2009 Abstract A new simple and direct electroanalytical method was developed for the determination of azidothymidine in commercial pharmaceutical preparations. It is based on differential pulse voltammetry at silver solid amalgam electrode with polished surface (p-AgSAE) or surface modified by mercury meniscus (m-AgSAE). The electroreduction of azidothymidine in basic media at these electrodes gives rise to one irreversible cathodic peak. Its potential in 0.05 mol L1 borate buffer, pH 9.3 at ca. 1050 mV is comparable to that using hanging mercury drop electrode (HMDE). Achieved limits of quantitation are in the 107 mol L1 concentration range for both amalgam electrodes. According to the procedure based on the standard addition technique, the recoveries of known amounts of azidothymidine contained in pharmaceutical preparations available in capsules were 101.4 1.8% (m-AgSAE), 100.3 3.5% (p-AgSAE) and 102.0 1.0% (HMDE) (n ¼ 10). There was no significant difference between the values gained by proposed voltammetric methods and the HPLC-UV recommended by the United States Pharmacopoeia regarding the mean values and standard deviations. Keywords: Azidothymidine, Zidovudine, Silver solid amalgam electrode, Differential pulse voltammetry, Pharmaceutical preparations, Antiviral agents, Voltammetry DOI: 10.1002/elan.200904660

Dedicated to Professor Karel Vytrˇas on the Occasion of His 65th Birthday

1. Introduction Nucleoside reverse transcriptase inhibitors (NRTI) were the first agents shown to be safe and effective for the treatment of patients infected with human immunodeficiency virus (HIV). Among them, 3’-azido-2’,3’-dideoxythymidine (AZT, zidovudine; Fig. 1) was the first compound to be officially approved, in 1987, and marketed (as Retrovir) for the treatment of AIDS, within 2 years after its in vitro activity against the infectivity and cytopathicity of HIV in cell culture had been demonstrated [1]. It is still widely used for antiretroviral (ARV) therapy, either alone or in combination with other ARV agents [2, 3]. Several analytical methods have been developed for the determination of AZT alone or simultaneously with other NRTI [4, 5], in plasma [6, 7], other biological fluids [8], or pharmaceutical preparations [9 – 14]. Methods based on liquid chromatography prevail, either coupled with ultra violet (UV) detection [5, 8, 10, 11, 14] or more recently coupled with tandem mass spectrometry (MS/MS) [6, 7]. Several immunoassays have been also described (e.g., radioimmunoassay [15] and amperometric immunoassays [12, 13]). Electroanalysis 2009, 21, No. 15, 1750 – 1757

Beside mentioned methods, micellar electrokinetic chromatography [16], high-performance thin-layer chromatography [17], and spectrophotometry and titrimetry [18] were used for AZT determination in pharmaceutical preparations. The electrochemical methods for AZT determination (as well as its biological action) rely on the presence of the reducible azido group. Basic mechanistic studies were

Fig. 1. Structural formula of AZT. 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Voltammetric Determination of Azidothymidine Using Silver Solid Amalgam Electrodes

Table 1. Electroanalytical methods using mercury electrodes for AZT determination. DPV: differential pulse voltammetry, DPP: differential pulse polarography, CV: cyclic voltammetry, SWV: square wave voltammetry, EVLS: elimination voltammetry with linear scan; PB: phosphate buffer; LLE: liquid-liquid extraction. Method

Conditions

Matrix

Detection limit (mmol L1)

Ref.

DPV

1 mol L1 KNO3, 1 mmol L1 EDTA, 0.1 mol L1 PB pH 7.2 1 mol L1 KNO3, 1 mmol L1 EDTA, 0.1 mol L1 PB pH 7.9 0.1 mol L1 PB pH 7.0

Buffer

0.0041

[23]

Blood

0.029

Buffer Serum, urine Buffer Urine Urine, plasma Buffer Urine, serum, blood Buffer þ ssDNA or BSA [a] Keratinocyte cells Buffer þ dsDNA, ssDNA, or ODNs [b]

0.2 5 0.005 0.5 0.5 0.001 1 0.250 not given not given

HPLC – off line DPV DPP CV

L1 L1 L1 L1

PB PB PB PB

pH 7.0 pH 8.0 pH 7.0 pH 8.0

LLE – CV SWV

0.1 mol 0.1 mol 0.1 mol 0.1 mol

SWV, EVLS

0.1 mol L1 PB pH 8.0

[22] [20]

[24]

[40]

[a] denaturated calf thymus ssDNA (c ¼ 10 mg L1) or bovine serum albumin (c ¼ 100 mg L1); [b] native calf thymus dsDNA, denaturated calf thymus ssDNA, synthetic oligodeoxynucleotides.

performed by means of cyclic and linear sweep voltammetry, differential pulse voltammetry (DPV), chronocoulommetry using static mercury drop electrode [19] and direct current, alternating current and differential pulse polarography at dropping mercury electrode [20]. The results are rather ambiguous, a two electron [20 – 22] or four electron [19] reduction of AZT was proposed in alkaline media. Electroanalytical methods published so far for the determination of AZT are summarized in Table 1. The optimized pulse methods in basic buffers resulted in the lowest detection limits of units of nmol L1 [23, 24]. Higher values were reported for direct determination of AZT in various biological matrices without any purification steps [24]. All these studies were performed using mercury electrodes enabling easy renewal of electrode surface. As the proposal on mercury ban in European Union [25] and other countries should be respected in scientific and commercial sphere, intensive effort for development of new electrode materials and their implementation in practice is necessary [26 – 29]. In the framework of this trend, electrodes based on amalgam materials were re-introduced in electroanalytical chemistry in the year 2000 [30, 31]. They gained deserved popularity especially for analysis of reducible organic compounds [32 – 34] and heavy metals [35, 36] in various matrices thanks to following favorable electrochemical properties: Relatively high hydrogen overvoltage and thus potential window comparable to that of mercury electrodes, low and stable background current and simple electrochemical regeneration of electrode surface. The solid amalgam electrodes (SAE) prepared by our group feature a variety of metals used for amalgam preparation (e.g., silver, copper, gold). They can be used either as mercuryfree electrodes after polishing of solid amalgam disk (pMeSAE) or after modification of their surfaces by mercury film (MF-MeSAE) or mercury meniscus (m-MeSAE). 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Among all the metals used, silver SAE (AgSAE) feature electrochemical properties comparable with mercury electrodes for analytes missing specific interactions (e.g., complexation with metal cations) with metals of the amalgam [35, 37]. Nevertheless, the development of new solid electrode materials is always a certain compromise between achieving the desired properties and maintaining the reproducibility on a necessary level. The equivalency of results achieved using m-AgSAE and HMDE, or by application of other analytical methods (e.g. AAS) was demonstrated on analysis of heavy metals (Cd, Cu and Pb) contents in four different reference materials of plant origin [36]. Similar equivalency in case of organic compounds (phenylglyoxylic acid) was confirmed as well [38]. In this work, we report on the comparison of hanging mercury drop electrode (HMDE) and amalgam electrodes (m-AgSAE, p-AgSAE) for the determination of AZT in standard chemical form using DPV regarding linearity, sensitivity and repeatability. Further, for the first time an electroanalytical method for quantitation of AZT in commercially available pharmaceutical preparations using these electrodes is proposed and compared with the HPLC-UV recommended by the United States Pharmacopoeia (USP) [39].

2. Experimental 2.1. Reagents The 1 103 mol L1 and 5 103 mol L1 stock solutions of AZT (Merck, 99.9%) were prepared by dissolving 26.73 mg and 133.65 mg of the substance in 100 mL of deionized water (Milli-Qplus system, Millipore, USA). More diluted solutions

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were prepared by exact dilution of the stock solutions with deionized water or with mobile phase. All the solutions were stored in the dark. Britton – Robinson (BR) buffers were prepared in a usual way, i.e. by mixing a solution of 0.04 mol L1 in phosphoric acid, 0.04 mol L1 in acetic acid and 0.04 mol L1 in boric acid with an appropriate amount of 0.2 mol L1 sodium hydroxide solution. Following chemicals of analytical grade purity were used: Acetic acid, phosphoric acid, boric acid, natrium borate (all Lach-Ner, Neratovice, CZ). Methanol (Merck, Prague, CZ) of gradient grade purity was used for mobile phase preparation.

in the solution of 0.2 mol L1 KCl by applying 2.2 V for 300 s while stirring the solution. The computer program for regeneration of the amalgam electrode surfaces prior each measurement was inserted into the software for individual measuring methods implemented under Polar Pro 5.1. It consisted of 50 electrochemical potential jumps between 1800 mVand a potential about 50 – 100 mV more negative than the potential of the anodic dissolution of the electrode material for 0.3 s of each directly in the analyzed solutions. The other solid amalgam electrodes based on different metals were made as described in [35]. A HMDE (EcoTrend Plus, Prague, CZ) of an area of 0.72 mm2 obtained by opening the valve for 0.1 s was used.

2.2. Apparatus Voltammetric measurements were carried out using computer controlled Eco-Tribo-Polarograph with Polar Pro software, version 5.1 for Windows 95/98/Me/2000/XP (both Eco-Trend Plus, Prague, CZ) in combination with a three electrode arrangement with a platinum wire auxiliary electrode and silver/silver chloride (1 mol L1 KCl) reference electrode (both Monokrystaly, Turnov, CZ), to which all the potential values are referred. HPLC system consisted of a high-pressure pump LaChrom-7100, UV spectrophotometric detector L-7400, autosampler L-7200 with interface D-7000 (all MerckHitachi), and software HPLC System Manager v. 4.0 (Hitachi) working under WinNT (Microsoft Corp.). The mobile phase was degassed by ultrasonication using PS 02000A ultrasonic bath (Powersonic, USA) followed by passing helium for 5 min. The spectrophotometric detector was set at the wavelength of 265 nm. The measurements were carried out at laboratory temperature. The pH measurements were carried out by pH meter Jenway 4330 (Jenway, UK) with combined glass electrode (Ag/AgCl/3 mol L1 KCl). The pH meter was calibrated with standard pH buffers (Sevac, Prague, CZ).

2.3. Procedures

2.3.2. Differential Pulse Voltammetry For DPV measurements, the pulse amplitude of 50 mV, pulse duration of 0.1 s, sampling time of 0.02 s beginning 0.08 s after the onset of the pulse and interval between pulses of 0.1 s was applied. The scan rate of 20 mV s1 was used. The general procedure to obtain voltammograms was as follows: A required amount of the stock solution of AZTwas placed in a 10 mL volumetric flask, an appropriate volume of deionized water to total volume of 1 mL was added and the system was diluted to volume with a BR buffer of the required pH or other supporting electrolyte to 10 mL. Oxygen was removed from the measured solutions by purging with nitrogen for five minutes. Peak heights were measured from linear baseline (tangent to the curve joining beginning and end of the given peak). All calibration curves were measured in triplicate. The statistical parameters (e.g., linear dynamic range (LDR), slope, intercept, standard deviation (s), relative standard deviation (sr), confidence interval (L1,2), t-test, F-test) were calculated using statistic software ADSTAT ver. 2.0 (Trilobyte, CZ) [41]. Limit of detection (LD) and limit of quantitation (LQ) were determined using the standard deviation sc of the mean of the peak heights (a ¼ 0.05) obtained for eleven voltammograms of lowest measurable concentration and the slope b of the analytical curve related by LD ¼ 3 sc/b and LQ ¼ 10 sc/b [42].

2.3.1. Preparation and Pretreatment of Working Electrodes A polished silver solid amalgam electrode (p-AgSAE) with the disk diameter of 0.55 mm was prepared as described in [37]. It was first ground on a soft emery paper and then polished using polishing kit (Electrochemical Detectors, Turnov, CZ) consisting of polishing polyurethane stocks, Al2O3 suspension (particle size 1.1 mm), and soft polishing Al2O3 powder (particle size 0.3 mm). The mercury meniscus modified AgSAE (m-AgSAE) was prepared by immersion of the p-AgSAE into liquid mercury for 15 s (while swirling the flask with mercury). At the daily used m-AgSAE, the meniscus was renewed once a week by mechanical removal of the old meniscus and redipping the electrode in liquid mercury. Before starting measurements with p-AgSAE and m-AgSAE, after every pause longer than 1 h or in the case of passivation of electrode surface, the electrode was activated Electroanalysis 2009, 21, No. 15, 1750 – 1757

2.3.3. Quantitative Determination of AZT in Capsules The pharmaceutical preparations analyzed by DPV and HPLC were capsules Zidovudina with declared AZT content 100 mg (Laborato´rio Farmaceˆutico do Estado de Pernambuco (LAFEPE), Recife, Brazil). The content of ten capsules was weighed and homogenized in an agar mortar. The average weight of one capsule content was 189.0 3.2 mg. For DP voltammetric quantitation using HMDE, mAgSAE, and p-AgSAE, an aliquot of the powder mass from capsule content corresponding to 10 mg of AZT (ca. 19 mg) was accurately weighed (n ¼ 10), transferred to 50 mL volumetric flask and made up to the mark with 0.05 mol L1 borate buffer, pH 9.3. After 3 min of sonication followed by centrifugation, 1.00 mL of the supernatant was diluted to


2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


10 mL in volumetric flask by the same buffer, achieving theoretical concentration 7.48 105 mol L1 for AZT. The solution was transferred in the voltammetric cell and deoxygenated by nitrogen for 5 min. DP voltammogram was recorded for this sample. Exact concentration of AZT was determined applying two standard additions of 200 mL from the AZT standard stock solution (c ¼ 5 103 mol L1), and plotting resulting analytical curve. All measurements were performed in triplicate with initial potential 600 mV and end potential 1600 mV. For the HPLC quantitation, the method prescribed by the United States Pharmacopoeia (USP) [39] was modified using a Lichrospher 100 (RP-18, 250 4 mm, 5 mm, Merck, Darmstadt, Germany) column and UV detection at 265 nm. Mobile phase was composed by a mixture of methanol:water (20 : 80, v/v) at a flow rate of 1.2 mL min1. The standard solution of AZT was prepared by dissolution of 10 mg of AZT in 100 mL of methanol achieving final concentration of 3.74 104 mol L1. The samples from capsules were independently prepared to achieve theoretically the same final concentration: An aliquot of powder mass from capsules containing ca. 50 mg of AZT was accurately weighed and transferred to 50 mL volumetric flask with methanol:water (25 : 75, v/v) mixture. The samples (n ¼ 10) were sonicated for 20 minutes and after centrifuged. 10 mL of the supernatant was diluted in 100 mL volumetric flask to volume by methanol:water (25 : 75, v/v) mixture to get the final concentration. 10 mL of each sample and standard solution of AZT were injected, the samples were filtered using MN GF 3 filters (Macherey Nagel, Dren, Germany) prior injection.

3. Results and Discussion 3.1. Voltammetric Determination of AZT at Amalgam Electrodes and HMDE The electrochemical reduction of AZT was investigated at solid amalgam electrodes with surface modified by mercury meniscus (m-AgSAE, m-CuSAE, m-BiAgSAE, m-AuSAE) or in polished form (p-AgSAE, p-CdAgSAE) and compared with electroreduction at HMDE. Cyclic voltammograms recorded at these electrodes in alkaline media indicate irreversible one-step reduction as previously reported for HMDE [19, 24]. With a view to develop an analytical method for the AZT determination in drug dosage form, DPV was selected as common electroanalytical method with preferred sensitivity and selectivity in pharmaceutical analysis [43]. Obtained DP voltammograms compared to that obtained at HMDE are depicted at Figure 2. The easy reduction is obvious at both AgSAE with peak potentials at ca. 1050 mV, which is comparable to HMDE. The reduction of AZT at other types of SAE requires more negative potential with the maximum of 1300 mV for m-AuSAE. Nearly the same peak potential at m-AgSAE and HMDE was reported also for other organic compounds and inorganic ions (e.g., Cu(II), Pb(II), 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Fig. 2. DP voltammograms of AZT (c ¼ 1 104 mol L1) at HMDE and seven different amalgam electrodes (m-CuSAE, mAgSAE, p-AgSAE, m-BiAgSAE, m-CdAgSAE, p-CdAgSAE, and m-AuSAE) in 0.05 mol L1 borate buffer (pH 9.3).

Cd(II) and Zn(II)) in the absence of specific interactions between the analyte and silver from silver solid amalgam [34, 35, 37]. The complexation of AZT with any of the amalgam forming metals was not reported, only the detection of thymidine itself in complex with Hg(II) by cathodic stripping voltammetry was published [44]. Further experiments were performed with m-AgSAE and p-AgSAE as the most commonly used SAE for the determination of organic compounds [26]. The variability of DP voltammograms of AZTat m-AgSAE in the pH range of 2.0 – 12.0 in BR buffer is depicted at Figure 3. No clear reduction peaks were obtained for pH 2.0 – 4.0, at moderate acidic to neutral media (pH 4.5 – 7.0) significant increase of current between 800 mV and 1100 mV with no clear maximum is observable (Fig. 3A). The appearance of the reduction signal at pH 4.5 may be related to the pKa value 4.8 of azido group protonation reported in [20]. In that study, bellow pH value 4.8 the single polarographic wave at 1020 mV (vs. Ag/AgCl) was reported. This relatively negative reduction potential is already at the onset of hydrogen evolution at m-AgSAE and thus the reduction signals are only insinuated. The well-defined and regular reduction peaks were obtained in basic solutions (Fig. 3B) with peak potential shifting to more negative values according to the equation: Ep [mV] ¼ 31.0 pH 748 (R ¼ 0.9874). The slope of this shift is very close to the slope 30.7 mV pH1 reported for DPV at HMDE in the pH region 6.0 – 10.8 and the theoretical value of 30.0 mV pH1 for a reduction with a 1 : 2 proton to electron stoichiometry [19]. Presumably, the mechanism of the reduction in basic media follows that reported for mercury electrodes for AZT and other organic azides according to Scheme 1 [20]: The peak height maximum was obtained at pH 9.0, then a decrease was observable with increasing pH of the supporting electrolyte to pH 12.0 (Fig. 3B). All electrochemical methods reported so far for the AZT determination were based on reduction in neutral or basic buffers, most frequently 0.1 mol L1 phosphate buffer pH 7.0 – 8.0, as obvious from Table 1. For our further experiments, the



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Scheme 1.

Fig. 3. DP voltammograms of AZT (c ¼ 1 104 mol L1) at m-AgSAE in BR buffer. The number at particular voltammogram corresponds to given pH of BR buffer A) pH 2.0 – 7.0; B) pH 8.0 – 12.0.

universal BR buffer pH 9.0 was replaced by 0.05 mol L1 borate buffer (pH 9.3). The problematic feature in AZT electroanalysis is electrode fouling. Its course for m-AgSAE is depicted at Figure 4A. A significant peak height drop of ca. 60% is observable reaching a constant current value after 60 scans, when no activation is applied between measurements. The electrode surface gets passivated even after immersing of activated electrode into the solution containing AZT and products of its electroreduction. The composition of the

polymeric passivating layer remains unclear, as mentioned frequently for other amine-including electrode reactions. Several approaches were tested to overcome the fouling including application of constant potential between scans, cycling or stepwise repeated insertion of short potential pulses around the cathodic and anodic onset of potential window. All these methods prevented electrode surface fouling when potentials more negative than that of hydrogen evolution were included. The mechanical effect of formation of hydrogen micro bubbles or reductive ability of

Fig. 4. A) DP voltammograms of AZT (c ¼ 1 104 mol L1) at m-AgSAE in BR buffer, pH 9.0. Sixty consecutive scans without any regeneration of m-AgSAE between measurements. B) The peak heights Ip of DP voltammograms of AZT (c ¼ 1 104 mol L1) recorded in BR buffer, pH 9.0 during the period t, when given constant potential Ec was imposed at the electrode between individual DPV scans. Electroanalysis 2009, 21, No. 15, 1750 – 1757



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atomic hydrogen may play role. Figure 4B represents the changes of peak heights when constant potentials Ec between DPV scans of AZT were imposed at the electrode. After the first scan, the fast passivation of electrode surface proceeds with comparable rate when potentials Ec from 200 mV to 1400 mV are applied. At more negative values the hydrogen evolution starts to prevent the passivation. For Ec ¼ 1700 mV relative standard deviation of 4.52% for peak height of 1 104 mol L1 AZT in BR buffer pH 9.0 was achieved for 10 measurements in 5 min intervals. Nevertheless, from the practical point of view, the electrochemical regeneration of electrode surface applicable prior to each measurement is desirable. The process of automatic regeneration of SAE in analyzed solution inset in the program of the used computer-controlled instrument consists of the application of several hundreds of polarizing cycles, representing the switching the working potential from E1 to E2, each for tens of ms, so that the overall time for regeneration does not exceed 30 s. Usually the values of E1 and E2 are selected 50 100 mV more positive than the potential of electrolysis of hydrogen or base electrolyte and more negative than the dissolution of the electrode material. Under these conditions, the removal of the adsorbed species occurs, eventual oxides of mercury or silver are reduced and accumulation of most of the metals present in the analyzed solution is prevented. Reproducible response at AgSAE was obtained for many organic analytes using this type of regeneration [32, 34, 45 – 47]. In the case of AZT, E1 more negative than hydrogen evolution must be applied to prevent fouling. Using 50 polarization cycles between the potentials E1 ¼ 1800 mVand E2 ¼ 200 mV for 0.3 s each resulting in overall regeneration time of 30 s, excellent repeatabilities for peak heights 1.7% were achieved at mAgSAE and p-AgSAE. This is comparable with HMDE as obvious from Table 2 and confirms that the choice of reasonable electrochemical pretreatment enables determination of analytes with fouling-related problems even at high concentrations. In Table 2, the parameters of calibration dependencies in 0.05 mol L1 borate buffer, pH 9.3 are further listed. Figure 5 shows the set of DPV curves of (2 – 10) 105 mol L1

Fig. 5. Selected DP voltammograms of AZT at m-AgSAE in 0.05 mol L1 borate buffer (pH 9.3). c(AZT) ¼ 0 mol L1 (1), 2 105 mol L1 (2), 4 105 mol L1 (3), 6 105 mol L1 (4), 8 105 mol L1 (5), 1 104 mol L1 (6). Electrode surface regeneration by 50 polarization cycles between the potentials E1 ¼ 1800 mV and E2 ¼ 200 mV (0.3 s each) before each scan.

AZT at m-AgSAE. The selected concentration range illustrates that the sensitivity of the electrode meets the requirements on the determination of higher concentrations common in pharmaceutical analysis. The analogous voltammetric curves were registered in the case of p-AgSAE and HMDE as well. The resulting peak height vs. concentration dependencies exhibited a linear course over three and four orders of magnitude for both AgSAE and HMDE, respectively. In the case of p-AgSAE, the sensitivity was found to be one third less than that in the case of m-AgSAE, also the LD and LQ (both in the 107 mol L1 concentration range) favor the latter electrode. This preference of m-AgSAE is common for organic analytes [26, 32], exemptions were reported for catalytic Brdicka-type processes [48] at pAgSAE. Nevertheless, in our study p-AgSAE provided completely satisfactory results as well and thus it should be preferred at conditions under which the presence of the liquid mercury (even in microvolumes) is undesirable [49]. The correlation coefficients R 0.9983 confirm very good linearity of calibrations dependencies.

Table 2. Analytical figures of merit for DP voltammetric determination of AZT at HMDE, m-AgSAE, and p-AgSAE in 0.05 mol L1 borate buffer, pH 9.3.

Peak potential (mV ) Peak height (nA ) [a, b] Repeatability (%) [a, c] Repeatability (%) [c, d] Linear dynamic range (mmol L1) Slope (nA mol1 L ) Intercept (nA ) Correlation coefficient Limit of detection (mmol L1) Limit of quantitation (mmol L1)

HMDE

m-AgSAE

p-AgSAE

1043 868.0 2.3 0.3 7.8 0.03 – 1900 7.62 106 6.7 0.9992 0.007 0.024

1050 207.9 3.5 1.7 9.7 0.4 – 1500 1.76 106 8.5 0.9983 0.12 0.41

1060 157.2 0.5 0.3 11.2 0.6 – 1500 1.24 106 þ 4.4 0.9994 0.20 0.67

[a] c( AZT ) ¼ 1.07 104 mol L1; uncertainty as [b] standard deviation or [c] relative standard deviation for peak heights, evaluated from eleven consecutive measurements at a ¼ 0.05; [d] concentration corresponding to the lowest concentration of the linear dynamic range. 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim



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3.2. Analysis of AZT in Pharmaceutical Dosage Form With optimized DP voltammetric parameters, an electroanalytical methodology was developed for the determination of AZT in pharmaceutical preparations – capsules Zidovudina containing 100 mg of AZT according the manufacturer. Firstly, the recovery of the DP voltammetric method for addition of known amounts of AZT in the presence of the capsule matrix was tested. Typically, it contains microcrystalline cellulose, lactose monohydrate, sodium starch glycolate or magnesium stearate and thus it is sparingly soluble in water. The solubility of AZT in water is sufficient (20.1 mg mL1 at 258) [50]. Real samples were prepared by dissolution of 10 mg of capsule content in 100 mL of 0.05 mol L1 borate buffer, pH 9.3. Known amounts of AZT (i.e., 200 mL of AZT standard stock solution (c ¼ 5 103 mol L1)) were added to 10 mL of this solution. The recoveries (R) for added AZT were calculated using the formula: R% ¼ 100 cAZT,found/cAZT,added, where the found value refers to the concentration obtained by extrapolation of the peak height of added AZT in the corresponding calibration dependence of AZT in buffer. A recovery of 97.8% 1.8%, 101.1% 2.2%, and 96.8% 2.9% (n ¼ 5) was found for HMDE, m-AgSAE and p-AgSAE. No interferences from the excipients were observable at the DP voltammograms. The content of AZT in capsules Zidovudina was determined by DPV using HMDE, m-AgSAE, p-AgSAE using the standard addition method described in working procedures (Chapter 2.3.3), and HPLC-UV recommended by USP [39]. Obtained mean values of AZT mass in capsules, standard deviations s and relative standard deviations sr, and confidence interval L1,2 are listed in Table 3. These results were compared with the value declared by manufacturer (100 mg). The following statistical methods were used: Twosample F-test for variances; two sample t-test for equal variances; two sample t-test for equal means. First of all, the normality of the distributions of processed groups of results was tested (e.g., by calculation of skew and excess): All groups of results exhibited normal statistical distribution. Two-sample F-test for variances was calculated for each pair of results in the first. If H0 hypothesis (Hypothesis H0: Variance of the first group of results is equal to the variance of the second group) was accepted on the basis of F-test results, the two sample t-test for equal variances for the result comparison was used. If the mentioned hypothesis was rejected, two sample t-test for equal means (unequal variances) was applied. We can conclude that the results are equal. Level of significance a ¼ 0.05 was accepted as limiting for the decision of equality. Further, one-factor ANOVA test was applied for conformation of results equivalency as well [36, 41]. It is possible to use this test to judge the influence of some factor on the equity of achieved results, but its importance for explanation is relatively low, because it does not give us the answer, which concrete group of results differs from others. It can be concluded that no influencing factor was proved in all Electroanalysis 2009, 21, No. 15, 1750 – 1757

Table 3. Recoveries of AZT samples (n ¼ 10) in commercial pharmaceutical preparations (capsules Zidovudina, declared content of AZT 100 mg) analyzed by differential pulse voltammetry at HMDE, m-AgSAE and p-AgSAE, and HPLC-UV.

DPV/HMDE DPV/m-AgSAE DPV/p-AgSAE HPLC-UV

mAZT (mg)

s (mg)

sr (%)

L1,2 (mg)

102.0 101.4 100.3 103.1

1.0 1.8 3.5 0.9

0.98 1.78 3.49 0.87

0.7 1.2 2.3 0.6

groups of results (a ¼ 0.05). This finding is in correspondence with statistical calculations obtained using t-tests. Thus, it can be concluded that results obtained for DPVat m-AgSAE, p-AgSAE, HMDE, and HPLC-UV are equal and the AZT content in capsules lie within the tolerance allowed by the USP protocol, which is 90 – 110% of the analytical standard target.

4. Conclusions For the first time, we have demonstrated the utility and advantages of solid electrodes for the analysis of AZT. AZT passivates easily the electrode surface during electrochemical reduction, and thus it was so far determined at mercury electrodes enabling easy renewal of the electrode surface. Reproducible and stable responses were obtained using silver solid amalgam electrodes (m-AgSAE and p-AgSAE) without electrode fouling when fast regeneration program preceding each measurement was implemented into the executive measuring program. The limits of quantitation using DPVat m-AgSAE and p-AgSAE are around 0.5 mmol L1. This is more than one order of magnitude higher than using HMDE (LQ ¼ 0.024 mmol L1), nevertheless, still attractive for determination of AZT in pharmaceutical preparations and in biological matrices, especially after a pre-concentration step using solid phase or liquid-liquid extraction. The applicability of the method was demonstrated on quantitation of AZT in capsules Zidovudina. Equivalent results regarding the mean values and standard deviations were obtained using DPVat m-AgSAE, p-AgSAE, HMDE, and HPLC-UV prescribed by USP. Easy sample preparation in a short time, inexpensive aqueous solvents and equipment prefer the electrochemical methods for quality control and in routine analysis of AZT in pharmaceutical preparations. Thus, it can be concluded that the silver solid amalgam electrodes fulfill the requirements on reliable, fast, sensitive, and relatively inexpensive determination of AZT and the proposed method represents a useful contribution to the analysis of pharmaceutical matrices.

5. Acknowledgements The research was supported by the Ministry of Education, Youth and Sports of the Czech Republic (projects LC 06035




and MSM 0021620857), Grant Agency of the Czech Republic (project GA CR 203/07/1195), and the Grant Agency of the Academy of Sciences (project GAAV IAA400400806). JCM thanks to the National Counsel of Technological and Scientific Development (CNPq) for support.

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