Electroanalytical determination of estriol hormone using a boron-doped diamond electrode

Talanta 80 (2010) 1999–2006

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Electroanalytical determination of estriol hormone using a boron-doped diamond electrode Keliana D. Santos, Otoniel C. Braga, Iolanda C. Vieira, Almir Spinelli ∗ Grupo de Estudos de Processos Eletroquímicos e Eletroanalíticos, Universidade Federal de Santa Catarina, Departamento de Química – CFM, 88040-900 Florianópolis, SC, Brazil

a r t i c l e

i n f o

Article history: Received 12 June 2009 Received in revised form 24 October 2009 Accepted 27 October 2009 Available online 1 November 2009 Keywords: Hormone Estriol Urine Pharmaceuticals Electroanalytical method Boron-doped diamond electrode

a b s t r a c t A boron-doped diamond (BDD) electrode was used for the electroanalytical determination of estriol hormone in a pharmaceutical product and a urine sample taken during pregnancy by square-wave voltammetry. The optimized experimental conditions were: (1) a supporting electrolyte solution of NaOH at a pH of 12.0, and (2) a frequency of 20 Hz, a pulse height of 30 mV and a scan increment of 2 mV (for the square-wave parameters). The analytical curve was linear in the concentration range of 2.0 × 10−7 to 2.0 × 10−5 mol L−1 (r = 0.9994), with a detection limit of 1.7 × 10−7 mol L−1 and quantification limit of 8.5 × 10−7 mol L−1 . Recoveries of estriol were in the range of 98.6–101.0%, for the pharmaceutical sample, and 100.2–103.4% for the urine sample, indicating no significant matrix interference effects on the analytical results. The accuracy of the electroanalytical methodology proposed was compared to that of the radioimmunoassay method. The values for the relative error between the proposed and standard methods were −7.29% for the determination of estriol in the commercial product and −4.98% in a urine sample taken during pregnancy. The results obtained suggest a reliable and interesting alternative method for electroanalytical determination of estriol in pharmaceutical products and urine samples taken during pregnancy using a boron-doped diamond electrode. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Estriol (1,3,5,(10)-estratriene-3,16␣,17␤-triol) (Fig. 1) is by far the most abundant estrogen present in pregnant mammals. Estriol is produced by placental syncytiotrophoblasts from dehydroepiandrosterone sulfate and its 16␣-derivatives produced by fetal adrenals and the liver, respectively. Since the production of estriol relies on fetal and placental reactions, estriol determinations in blood and urine can be used to monitor the fetus-placental unit and indicate fetal well being. Free estriol is hydrophobic but it is conjugated to hydrosoluble glucuronides and sulfates (70–80% as 16␣-glucuronides) by the maternal liver and excreted in the urine [1–2]. The conjugated forms of estriol in urine are quickly hydrolyzed after excretion, leaving estriol in the free form. Oral estriol tablets have been used with good results, primarily for the treatment of local urogenital complaints in postmenopausal women, for over 40 years. One of its product characteristics is that it does not stimulate the endometrium and can, therefore, be used uninterrupted without the cyclical addition of a progestogen to

∗ Corresponding author at: Universidade Federal de Santa Catarina (UFSC), Centro de Ciências Físicas e Matemáticas (CFM), Departamento de Química, Campus Universitário Trindade, 88040-900 Florianópolis, SC, Brazil. Tel.: +55 48 3721 6844; fax: +55 48 3721 6850. E-mail address: [email protected] (A. Spinelli). 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.10.058

protect the endometrium. Although all available data confirm the endometrial safety of oral estriol tablets, it is considered relevant to update the existing long-term data on this topic [3–4]. Estriol levels in urine, serum and amniotic fluid have been measured by HPLC using various detectors including UV [5,6], fluorescence [7,8] and electrochemical (EC) [9], and different methods such as GC–MS [10,11], micellar electrokinetic chromatography [12], enzyme immunoassay (EIA) [13–15], and radioimmunoassay (RIA) [16]. However, most of these methods require sample preparation steps such as extraction from biological fluids, enzymatic hydrolysis or acidic solvolysis and derivatization. These procedures are tedious and time-consuming, require high-cost equipment and sometimes adversely affect the analytical results. In the case of immunoassays, for example, it is necessary to obtain a specific antibody for this steroid. RIA is currently considered to be the screening method of choice for routine measurements of estriol. Although this method is sensitive and reliable, there are drawbacks associated with the use of radioisotopes, for example, the cost of scintillation fluids, radioactive waste disposal, and its use being restricted to institutes granted permission to handle radioisotopes. Electrochemical studies on alkyl phenols with estrogenic properties have been reported. Vega et al. [17] developed a methodology using a carbon nanotube-modified electrode for amperometric detection of phenolic estrogenic compounds, including estriol, by HPLC. The determination of estriol was obtained in the range of 1.0–100 ␮mol L−1 with a detection limit of 0.340 ␮mol L−1 . The

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K.D. Santos et al. / Talanta 80 (2010) 1999–2006

Fig. 1. Chemical structure of estriol.

method was successfully applied to determine estriol in tap water. At the present time, there are no other publications reporting the electrochemical determination or behavior of estriol. Some articles have described the use of chromatographic methods with amperometric detectors to investigate hormones like bisphenol A [18,19], or to develop biosensors for the determination of the hormones 17␤-estradiol and estrone [20–24]. In the present study a boron-doped diamond (BDD) electrode was used to develop an electrochemical methodology for determination of estriol. Diamond is an electrical insulator, but conducts heat even more effectively than copper and can withstand very high electric fields. With these physical properties, diamond is an attractive material for electronic applications, particularly when it is rendered conductive through the introduction of charge carriers [25]. Boron has one less electron than carbon and because of its small atomic radius it is relatively easily incorporated into diamond. BDD electrodes have been extensively studied in recent years, in relation of their fundamental electrochemical properties [25] and also in terms of their applications [25,26]. BDD electrodes have high stability and hardness, inert surfaces and very low capacitive background currents. Thus, they have been extensively used in electrosynthesis, electroanalysis, electrochemical combustion, and as electrocatalyst supports [25–27]. Recent reports in the literature have shown that several bio-molecules can be satisfactorily determined using BDD electrodes. Souza et al. [28] quantified sulfonamides in pharmaceutical products and obtained an analytical curve in the concentration range of 8.01 × 10−6 to 1.19 × 10−4 mol L−1 . Lourenc¸ão et al. [29] developed an electrochemical method for the single or simultaneous determination of paracetamol and caffeine; the calibration curves for the simultaneous determination showed an excellent linear response, ranging from 5.0 × 10−7 to 8.3 × 10−5 mol L−1 for both compounds. A methodology to determine perfloxacin in pharmaceuticals and serum presented a linear range for concentrations between 2.0 × 10−6 and 2.0 × 10−4 mol L−1 [30]. Many other bio-molecules, including pentachlorophenol, 4-nitrophenol, lincomycin, purine and pyrimidine, dopamine and ascorbic acid, aspartame and cyclamate [31–36], have been determined using BDD electrodes and in all cases the methodologies presented good selectivity, repeatability, reproducibility, precision and accuracy. However, BDD electrodes have not yet been used for the electroanalytical determination of hormones. Thus, the objective of this study is to develop a cost-effective, single-use, feasible, accurate, and reliable method to detect estriol in a complex matrix (urine) and in a drug sample using a boron-doped diamond electrode by square-wave voltammetry. 2. Experimental 2.1. Chemicals and solutions All reagents used in this study were of analytical grade purchased from Sigma. The solutions were prepared with water

purified using a Milli-Q system manufactured by Millipore (Bedford, MA, USA). Sodium hydroxide (pH 12.0) and five buffer solutions were tested as supporting electrolytes: Britton–Robinson (pH 2.0–12.0), acetate (pH 4.5), ammonium (pH 9.4) and Ringer’s solution (pH 12.0). All of these solutions had a concentration of 5.0 × 10−3 mol L−1 . A solution of 0.1 mol L−1 NaOH was used to adjust the pH of the 5.0 × 10−3 mol L−1 NaOH supporting electrolyte to 12.0. Progesterone, urea, uric acid, albumin and creatinine were evaluated as potential interferents for estriol determination in urine. The excipients present in the pharmaceutical preparation used in this study (Ovestrion® ) are amido, lactose, magnesium stearate and amilopectine. They were tested as potential interferents for estriol determination in the commercial product. Stock solutions of 1.4 × 10−2 mol L−1 estriol dissolved in methanol were prepared daily and less concentrated solutions were prepared by dilution. For the recovery studies, standard solutions of the hormone were added to samples of urine and tablets of a commercial product (Ovestrion® ). The urine sample was obtained from a woman in the third trimester of pregnancy and no pretreatment was necessary to determine its estriol levels. Commercially available estriol contained 1 mg of estriol. Ten tablets of the medicament were finely macerated in a mortar with a pestle. The powder was weighed in the quantity necessary to prepare the sample solutions, diluted using methanol and mixed for 5 min. The non-dissolved excipients were left to settle and a sample was then withdrawn from the clear supernatant liquor. 2.2. Cell and electrodes Linear scan, cyclic and square-wave voltammograms were recorded using a 25-mL three-electrode electrochemical cell. The electrochemical detection of estriol was initially tested on five electrodes: copper, platinum, gold, glassy carbon and boron-doped diamond. The electrodes (with the exception of the BDD electrode) were hand-polished before each experiment with 0.05 ␮m alumina paste and ultrasonically rinsed in deionized water. The BDD working electrode (A = 0.23 cm2 ) employed for the development of the electroanalytical methodology was a BDD film with around 8000 ppm of boron. A pretreatment procedure was initially applied to the BDD electrode to achieve reproducible results. The procedure consisted of the polarization at −3.0 V of the BDD electrode in 0.5 mol L−1 H2 SO4 for 3 min in a separate cell, as recommended in the literature [27]. Afterwards, the electrode was transferred to the measuring cell containing the supporting electrolyte and estriol, and polarized under stirring at −3.0 V for 30 s between each run. Following this method, excellent electrode response and reproducibility were systematically observed for estriol determination. Platinum wire and Ag/AgCl electrodes were used as auxiliary and reference electrodes, respectively. All potentials were measured versus this reference electrode. 2.3. Instruments Voltammetric measurements and surface pretreatment of the BDD electrode were performed on a Voltalab PGZ 100 potentiostat/galvanostat interfaced with a computer using the Voltamaster 4.0 software. Linear scan and cyclic voltammmetry were employed for preliminary studies on the electrochemical behavior of the estriol compound. Square-wave voltammetry was used for the development of the electroanalytical methodology and estriol determination in urine and in a commercial drug sample. The accuracy of the proposed method was compared to the standard radioimmunoassay method [37].

K.D. Santos et al. / Talanta 80 (2010) 1999–2006

3. Results and discussion 3.1. Electrode selection In order to compare the electrochemical oxidation of estriol, copper, platinum, gold, glassy carbon and BDD were tested as catalyst electrodes. In this study, linear scan voltammetric measurements were carried out from 0.0 to +0.63 V at 50 mV s−1 using 2.0 × 10−5 mol L−1 estriol in NaOH (pH 12.0) solution. Under these experimental conditions, estriol was electrochemically inactive on copper, platinum and gold electrodes, since no oxidation current was observed. On the other hand, as can be seen in Fig. 2, the voltammograms obtained with the glassy carbon (a and c) and BDD (b and d) electrodes without (a and b) and with (c and d) 2.0 × 10−5 mol L−1 estriol solution show that the electrochemical oxidation of estriol produced a good, sensitive and well-defined anodic current peak. However, the current at the BDD electrode was almost 10 times higher than at the glassy carbon electrode. Similar behavior has been systematically observed by other authors for this type of electrode and for other bio-molecules [26,30]. The good performance of BDD electrodes as electrochemical catalysts is due to properties such as low sensitivity to dissolved oxygen and low background currents. The capacitive background current, for example, is ten times lower than that at glassy carbon electrodes [38], making BDD electrodes highly sensitive toward the oxidation/reduction of organic molecules. This study confirms, therefore, that the BDD electrode also offers excellent electrocatalytic properties for the oxidation of hormones, in particular estriol, which is here under study for the first time using this type of electrode surface. Accordingly, the BDD electrode was selected to develop the methodology for the electrochemical determination of estriol.

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voltammograms obtained for 5.0 × 10−5 mol L−1 estriol in different supporting electrolytes. In all voltammograms, just one oxidation peak was observed when the potential was swept from 0.0 to +1.2 V and back to 0.0 V at a scan rate of 100 mV s−1 . As can be seen, estriol oxidized at a high potential of 0.90 V in Britton–Robinson (pH 2.0) solution (Fig. 3A(a)), and a well-defined oxidation peak and analytical signal, i.e. the anodic current density, were observed. In acetate (pH 4.5) buffer (Fig. 3A(b)), the estriol oxidation was observed at 0.75 V with a small current being produced. A very poorly defined current peak was generated at 0.64 V in Britton–Robinson (pH 7.0) buffer (Fig. 3A(c)). The estriol oxidation in ammonium (pH 9.4) (Fig. 3A(d)) and Ringer’s solution (pH 12.0) (Fig. 3A(e)) buffers was observed at almost the same potential of 0.45 V. In spite of the well-defined peaks, the analytical signals were also poor. In Britton–Robinson (pH 12.0) solution, a peak with low current and ill-defined peak was observed at 0.40 V (Fig. 3A(f)). The most well-defined oxidation peak and the highest anodic current density were obtained in NaOH (pH 12.0) solution (Fig. 3A(g)). Under this experimental condition the hormone estriol oxidized at 0.38 V, the lowest oxidation potential observed. The relatively high peak current found for estriol oxidation in solutions of pH 12.0 suggests that the electrochemical reaction follows a different mechanism to that in solutions of lower pH and also with a different number of electrons involved. As can be seen in Fig. 3A, the cyclic voltammograms shifted to less positive potentials as the solution pH increased. As a consequence, the water oxidation reaction was anticipated and a narrower cyclic voltammogram was

3.2. Selection of supporting electrolyte The choice of the supporting electrolyte is an important stage in electroanalytical studies because its composition and pH affect the properties of the solution as well as the electrode–solution interface, modifying the thermodynamics and kinetics of the charge transfer process and the adsorption at the electrode surface [39]. In this study, solutions with different compositions and pH values were tested as the supporting electrolyte to investigate the estriol oxidation at a BDD electrode. Therefore, the buffers Britton–Robinson (pH 2.0–12.0), acetate (pH 4.5), ammonium (pH 9.4) and Ringer’s solution (pH 12.0), and the NaOH (pH 12.0) solution, were evaluated. All of these solutions had a concentration of 5.0 × 10−3 mol L−1 . Fig. 3A shows the cyclic

Fig. 2. Linear scan voltammograms for (a) glassy carbon and (b) BDD electrodes in NaOH (pH 12.0) solution and (c and d) in the presence of 2.0 × 10−5 mol L−1 estriol, v = 50 mV s−1 .

Fig. 3. (A) Cyclic voltammograms for 5.0 × 10−5 mol L−1 estriol at a BDD electrode immerse in (a) Britton–Robinson (pH 2.0), (b) acetate (pH 4.5), (c) Britton–Robinson (pH 7.0), (d) ammonium (pH 9.4), (e) Ringer’s (pH 12.0), (f) Britton–Robinson (pH 12.0), and (g) NaOH (pH 12.0) solutions, v = 100 mV s−1 . (B) Influence of Britton–Robinson solution pH on the estriol oxidation peak potential.

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obtained since the initial potential was the same for all experiments. The influence of the pH on the oxidation peak of 5.0 × 10−5 mol L−1 estriol in Britton–Robinson solutions of pH 2.0–12.0 is shown in Fig. 3B. The increase in pH shifted the estriol oxidation potential to less positive values. This is a typical behavior for the oxidation of phenols and was also observed by Vega et al. [17] for the oxidation of phenolic estrogenic compounds. The intersection of the two straight lines observed in Fig. 3B matches the pKa of the phenolic group present in the estriol chemical structure. The value found (10.0) is very close to 10.3–10.4, the value reported in the literature [40]. As demonstrated, a good compromise between low detection potential and high peak current was achieved using NaOH (pH 12.0) solution as the supporting electrolyte, which was chosen for further experiments and development of the methodology. 3.3. Electrochemical behavior of estriol at BDD electrode in NaOH (pH 12.0) solution The cyclic voltammograms shown in Fig. 4 show the electrochemical behavior of 2.0 × 10−5 mol L−1 estriol in NaOH (pH 12.0) solution at a BDD electrode. The influence of the potential scan rate on the estriol oxidation was studied over the range of 10–200 mV s−1 . The potential was swept from 0.0 to +0.7 V and back to 0.0 V. The cyclic voltammograms are characteristic of an electrochemically irreversible reaction showing just one oxidation peak potential at 0.365 V for a scan rate of 20 mV s−1 . The peak potential shifted to more positive values as the scan rate increased, also in agreement with an irreversible electrochemical behavior. Furthermore, a linear j × v1/2 plot was obtained indicating a diffusion-controlled process of the oxidation reaction. The log j × log v plot showing a slope of 0.64 (r = 0.999) confirmed this finding with a small contribution from the adsorption process. Finally, interdependence between the current function (jv−1/2 ) and the potential scan rate indicated that the chemical reaction was coupled to the electrode process, i.e. the reaction followed an EC mechanism. The data collected on the influence of the solution pH and potential scan rate on the electrochemical behavior of estriol at the BDD electrode allow some speculations regarding its oxidation mechanism. The mechanism for the electrochemical oxidation of estriol seems to follow the typical oxidation of phenolic compounds having just one hydroxyl group in their structure [41–43]. Additionally, the cyclic voltammograms of estriol (Fig. 4) are similar to that of ␤-estradiol [22], a hormone with similar chemical structure.

Fig. 4. Cyclic voltammograms for 2.0 × 10−5 mol L−1 estriol at a BDD electrode immerse in NaOH (pH 12.0) solution, v (a) 10 mV s−1 , (b) 20 mV s−1 , (c) 30 mV s−1 , (d) 40 mV s−1 , (e) 50 mV s−1 , (f) 60 mV s−1 , (g) 70 mV s−1 , (h) 80 mV s−1 , (i) 90 mV s−1 , (j) 100 mV s−1 , (k) 150 mV s−1 and (l) 200 mV s−1 .

The electrochemical behavior of both hormones shows one oxidation peak and characteristics of an electrochemically irreversible charge transfer. Hence, we propose that the electrochemical oxidation of estriol forms a phenoxonium ion, the electrochemical step of the EC mechanism, in a two-electron reaction process. Such an oxidation reaction is diffusion-controlled, as indicated by the data obtained at different scan rates. The phenoxonium ion can undergo homogeneous chemical reactions in the sequence, the chemical step of the EC mechanism. The reactions are fast, dependent on the solution pH and irreversible in alkaline media. The resulting products vary according to the rearrangements in the oxidized molecule. In acetonitrile medium, for example, the final identified product was the ketone derivative of ␤-estradiol [22]. The stability of estriol in NaOH (pH 12.0) solution was initially studied over 6 h by cyclic voltammetry. The cyclic voltammograms obtained over time were reproducible indicating that the estriol solution was stable. The same behavior was observed for a solution studied over 10 days. These experiments indicated that the determination of estriol can be carried out in NaOH (pH 12.0) solution without modifying the composition of the solution containing the analyte. 3.4. Optimization of square-wave voltammetry parameters Square-wave voltammetry is a pulse technique that offers the advantages of fast speed and good sensitivity for reversible as well as irreversible reactions. The excitation signal is obtained applying a series of forward and reverse pulses superimposed onto a constantheight potential staircase to the working electrode. The current is sampled twice in each wave cycle: once at the end of the forward pulse and once at the end of the reverse pulse. The signal is given by the difference between these two currents. As a consequence, an important step in the development of the electroanalytical methodology is the optimization of the parameters that can influence the voltammetric response. Hence, the dependence of the SWV responses on parameters such as frequency (f), pulse height (PH) and scan increment (Es ) was analyzed in order to optimize the experimental set-up for estriol determination. The SWV parameter optimization was carried out in solutions of 2.0 × 10−5 mol L−1 estriol in NaOH (pH 12.0) solution as the supporting electrolyte. The ranges studied were 5–100 Hz for frequency, 10–50 mV for pulse height and 1–5 mV for scan increment. The frequency is one of the most important parameters of SWV since it determines the intensity of the signals and, consequently, the sensitivity of the method. The anodic current density increased with the increase in the frequency at constant PH and Es values, as shown in Fig. 5A. At higher frequencies, a broadening and a distortion in the voltammograms were observed (data not shown). An excellent compromise between voltammetric profile and sensitivity was obtained at a frequency of 20 Hz. As a consequence, the frequency of 20 Hz was chosen for use in subsequent experiments. For totally irreversible redox systems, analytical sensitivity in the SWV is greatly influenced by variations in the pulse height [44]. It is observed in Fig. 5B that the current density values increased linearly up to 30 mV and for higher values remained almost constant. Thus, a pulse height of 30 mV was selected for further experiments. The scan rate in SWV is the result of the product of the f and Es . Therefore, a higher Es can increase the analytical signal and, thus, improve the sensitivity of the method [44]. However, with higher scan increments a broadening of the peak can occur, and thus the resolution of the voltammograms can be affected. Consequently, this is a parameter which also has to be analyzed. In Fig. 5C, it can be seen that the anodic current density remained almost constant for Es values higher than 2 mV and therefore this Es value was selected. The optimized values were also used to validate the methodology

K.D. Santos et al. / Talanta 80 (2010) 1999–2006

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Fig. 6. Square-wave voltammograms for estriol using NaOH (pH 12.0) solution at a BDD electrode: (a) blank; (b) 2.0 × 10−7 mol L−1 ; (c) 8.0 × 10−7 mol L−1 ; (d) 2.0 × 10−6 mol L−1 ; (e) 4.0 × 10−6 mol L−1 ; (f) 6.0 × 10−6 mol L−1 ; (g) 8.0 × 10−6 mol L−1 ; (h) 1.0 × 10−5 mol L−1 ; (i) 1.2 × 10−5 mol L−1 ; (j) (k) 1.6 × 10−5 mol L−1 ; (l) 1.8 × 10−5 mol L−1 ; (m) 1.4 × 10−5 mol L−1 ; 2.0 × 10−5 mol L−1 (f = 20 Hz; PH = 30 V; Es = 2.0 mV). Inset: the analytical curve.

at the peak potential. Fig. 6 shows the SWV voltammograms for estriol obtained after successive additions of the respective standard solution and the inset depicts the corresponding analytical curve. For the construction of the analytical curve, the currents shown in the square-wave voltammograms were subtracted from the background current. Table 1 shows some of the validation parameters for the proposed method for estriol determination. As can be seen, a well-defined irreversible oxidation peak was obtained, with the current density increasing proportionally to the estriol concentration. The analytical curve shows a linear response in the range of 2.0 × 10−7 to 2.0 × 10−5 mol L−1 . The equation for the straight line can be expressed according to j/␮A cm−2 = 1.02 × 105 [estriol]/mol L−1 − 0.0044. From this plot, the detection limit (DL) was calculated according to the equation: DL = 2 Sb/B, where Sb is the standard deviation of the y-coordinate from the line of best fit (linear coefficient) and B the slope (angular coefficient) of this line. The calculated DL for estriol was 1.7 × 10−7 mol L−1 . Additionally, the quantification limit (QL) was calculated using the equation: QL = 10 Sb/B. The calculated QL for estriol was 8.5 × 10−7 mol L−1 . The repeatability for five measurements of the current peak for solutions of 5.0 × 10−7 mol L−1 estriol, under optimized conditions, was very good, with a relative standard deviation of 0.64%. The reproducibility of the Table 1 Validation parameters for electroanalytical determination of estriol. Fig. 5. Dependence of the anodic current density on (A) frequency at PH = 40 mV and Es = 2 mV, (B) pulse height at f = 60 Hz and Es = 2 mV and (C) scan increment at PH = 40 mV and f = 60 Hz obtained by square-wave voltammetry for 2.0 × 10−5 mol L−1 estriol at a BDD electrode immerse in NaOH (pH 12.0) solution.

proposed as well as for estriol determination in a pharmaceutical product and in urine. 3.5. Analytical curve and validation parameters of the method proposed for estriol determination The previously optimized SWV parameters were employed to record the analytical curve for estriol in NaOH (pH 12.0) solution as the supporting electrolyte using the BDD electrode. The square-wave voltammograms were collected from 0.0 to 0.7 V and the resultant anodic current density was registered

Parameter

Value

Peak potential (V) Linear range (mol L−1 ) Correlation coefficient Slope (␮A L mol−1 cm−2 ) (×105 ) Standard deviation of slope (␮A L mol−1 cm−2 ) (×102 ) Intercept (␮A cm−2 ) Standard deviation of intercept (␮A cm−2 ) Detection limit (mol L−1 ) Quantification limit (mol L−1 ) Repeatability of peak current (%)a , b Reproducibility of peak current (interday) (%)a , b Reproducibility of peak current (intraday) (%)a , b Repeatability of peak potential (%)a , b Reproducibility of peak potential (interday) (%)a , b Reproducibility of peak potential (intraday) (%)a , b

0.38 2.0 × 10−7 to 2.0 × 10−5 0.9994 1.02 7.73 −0.0044 0.0088 1.7 × 10−7 8.5 × 10−7 0.64 0.98 0.78 1.05 1.58 1.23

a b

Relative standard deviation. n = 5.

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K.D. Santos et al. / Talanta 80 (2010) 1999–2006 Table 2 Estriol determination in commercial pharmaceutical product and in urine sample taken during pregnancy. Method

Labeled values (mg) Found (mg)a Found (mol L−1 ) a R.S.D. (%) Er1 (%)b Er2 (%)c tvalue d Fvalue e a b c d e

Pharmaceutical product

Urine

Radioimmunoassay

BDD electrode

Radioimmunoassay

BDD electrode

1.00 1.03 – 6.26 2.91 – 0.72 0.032

1.00 0.96 – 1.20 −4.17 −7.29 5.5 0.032

– – 8.43 × 10−5 1.37 – – – 1.00

– – 8.03 × 10−5 1.44 – −4.98 – 1.00

n = 3. Er1 = relative error between radioimmunoassay or electrochemical methods and labeled values. Er2 = relative error between electrochemical method and radioimmunoassay method. ttheoretical = 5.99. Ftheoretical = 19.

current peak was tested using two methodologies: interday, over 5 days using different solutions prepared in the concentration of 5.0 × 10−7 mol L−1 and intraday, using five different solutions prepared on the same day and using the same concentration of estriol (5.0 × 10−7 mol L−1 ). The relative standard deviations (n = 5) were 0.98 and 0.78%, respectively. Good data were also obtained for the repeatability and reproducibility of peak potential, as can be seen in Table 1. These results demonstrate that the measurement of the current density and peak potential, as well as of the method proposed, show excellent accuracy, precision, reproducibility, repeatability and sensitivity for estriol determination.

the real matrix. The acceptable range of recovery for the analysis of residues is generally between 80 and 120% [45]. As demonstrated in Table 3, the estriol recovery ranged from 98.6 to 101.0%. Furthermore, the slope of 1.09 × 105 ␮A L mol−1 cm−2 (r = 0.9982) obtained from the curve shown in Fig. 7A, is very close to that obtained from the analytical curve (Table 1), which indicates the excellent selectivity of the method proposed. The results shown in Fig. 7A were converted in order to obtain the same units for the slope of both

3.6. Estriol determination, recovery and potential interferent studies on a commercial pharmaceutical product The electroanalytical methodology described above and the standard radioimmunoassay method for estriol determination were applied in pharmaceutical dosage for the hormone quantification. The electroanalytical determination is shown in Fig. 7A. The mean results for three determinations (n = 3) of both techniques were very close to the declared value of 1.00 mg; the confidence limits were calculated for a significance level of 0.05. Table 2 summarizes the results obtained. The standard method resulted in a higher value for estriol determination than the value labeled on the commercial pharmaceutical product, while the electroanalytical method resulted in lower values. The relative error between the standard method and the labeled value was 2.91%, while for the method proposed it was −4.17%. The relative error between the method proposed and the radioimmunoassay was −7.29%. The performance of the two methods (proposed and standard) was verified using the Student’s t- and F-tests. The t-test was carried out to check the validity of the data obtained using the standard and proposed methods. At the 95% confidence level both results for tvalue calculated were lower than the result for ttheoretical (5.99), indicating no significant differences between the data obtained using the two methods and the true (labeled) values. The precision of the proposed method was compared to that of the standard method through statistical examination of the values obtained from F-tests, also at the 95% confidence level. The Fvalue was 0.032, which is lower than the Ftheoretical (19), indicating excellent performance of the electroanalytical method when compared to the standard method. Taking into consideration that other components of the matrix of the pharmaceutical dosage may interfere with the analysis or accurate quantification of the analyte, potential effects from matrix components must be investigated. If no adequate placebo can be prepared, a known amount of drug substance can also be added to an authentic batch of a drug product (standard addition). In this case, recovery experiments are performed in the presence of

Fig. 7. Electroanalytical determination and recovery studies for estriol in (A) a pharmaceutical product and (B) in urine samples taken during pregnancy using a BDD electrode.

K.D. Santos et al. / Talanta 80 (2010) 1999–2006 Table 3 Recovery studies for estriol in commercial pharmaceutical drug and in urine sample taken during pregnancy. Analytes

Addeda

Found

Recovery (%)

Pharmaceutical (10−7 mol L−1 )

6.00 7.00 8.00 9.00 10.00

5.97 6.98 7.89 8.97 10.10

99.5 99.7 98.6 99.7 101.0

2.00 3.00 4.00 5.00 6.00

2.07 3.05 4.01 5.08 6.05

103.4 101.6 100.2 101.6 100.8

Urine (10−7 mol L−1 )

a

n = 3.

curves. Finally, amido, lactose, magnesium stearate and amilopectine (the excipients present in the pharmaceutical preparation used in this study) were also tested as potential interferents. The studies showed that all of them are insoluble in methanol, the solvent used to prepare the solutions. The experiments carried out in the presence of these potential interferents did not change significantly the results described above. These results showed that the proposed method could be applied with great success to assays of estriol in tablet form without any significant matrix interference effects. 3.7. Study of potential interferents for estriol determination in urine It is well known that urine is a complex matrix and the determination of its components is not an easy task. Urine is composed essentially of water, salts, proteins, principally albumin and creatinine, and uric acid. During pregnancy, it is common for glucose and progesterone concentrations to increase. Most of these compounds could hinder the electrochemical determination of estriol, therefore, studies on interferents were carried out to identify any such influence. The major components of urine, progesterone, urea, uric acid, creatinine and albumin were tested as interferents in a 5.0 × 10−7 mol L−1 estriol solution. The estriol:interferent ratios studied were 1:0.01, 1:0.1, 1:1, 1:10 and 1:100. The results for the determination of the anodic current density obtained from the voltammograms recorded for estriol oxidation in the absence and in the presence of the potential interferents are listed in Table 4. As can be seen, the current density generated by the estriol oxidation was almost the same in the absence and presence of the potential interferents. This signifies that progesterone, urea, uric acid, albumin and creatinine did not significantly interfere in the analytical signal produced by the electrochemical oxidation of estriol. Thus, we can extrapolate this observation to a real urine sample and consider that the components of the urine will not significantly interfere in the determination of estriol in urine samples taken during pregnancy.

Table 4 Study of potential interferents for determination of estriol in urine. Estriol:interferent ratio

Progesterone Urea Uric acid Creatinine Albumin a

n = 3.

(1:0)

(1:0.01)

0.15 0.15 0.15 0.14 0.15

Anodic current density (␮A cm−2 )a 0.15 0.16 0.15 0.16 0.15 0.13 0.16 0.15 0.16 0.15 0.16 0.15 0.14 0.13 0.14 0.14 0.15 0.16 0.16 0.16

(1:0.1)

(1:1)

(1:10)

(1:100) 0.16 0.16 0.14 0.15 0.17

2005

3.8. Estriol determination and recovery studies in urine sample taken during pregnancy Without any sample preparation, extraction, filtration or evaporation step other than an adequate dilution of the sample, the electroanalytical methodology developed was successfully applied to the determination of estriol in a urine sample taken during pregnancy (Fig. 7B). Table 2 summarizes the results obtained. As can be seen, the value found for estriol determination in urine (8.03 × 10−5 mol L−1 , n = 3) was lower than that of the comparative radioimmunoassay method (8.43 × 10−5 mol L−1 ). Both values found were in good agreement with the values published in the pertinent literature [4]. The relative error for the mean of three determinations was −4.98% considering the electroanalytical and radioimmunoassay methods. The precision of the proposed method was compared to the radioimmunoassay method through statistical examination of the values obtained from the F-test, considering a 95% confidence level (Table 2). The value for Fvalue (1.0) was lower than that for Ftheoretical (19.0), indicating excellent performance of the electroanalytical method using the BDD electrode when compared to the radioimmunoassay method. Recovery experiments were performed in the presence of the real matrix in order to check the matrix interference effects. As demonstrated in Table 3, the recovery ranged from 100.2 to 103.4% for estriol, indicating that the composition of the matrix does not significantly interfere in the analytical response of the method proposed. This behavior, i.e. the selectivity of the proposed method, was confirmed by comparing the slopes of the analytical curve (Table 1) and that obtained in Fig. 7B (1.04 × 105 ␮A L mol−1 cm−2 , r = 0.9991), which were very close. The linearity and slope of the analytical curve and that obtained by the standard addition method, the repeatability and reproducibility of the data and the recovery results obtained for estriol determination using the optimized parameters for SWV and a suitable surface pretreatment for the BDD electrode indicated that the proposed electroanalytical methodology can be successfully applied in control processes and estriol determinations in urine samples taken during pregnancy. The direct determination of estriol in urine here reported is comparable to the existing radioimmunoassay methodology, but with clear advantages, as no preliminary separation steps or pretreatment of the matrix are required. 4. Conclusions According to the results obtained in this study, it can be concluded that the electroanalytical methodology proposed using a BDD electrode and the SWV technique presented a concentration range and detection limit compatible with the conventional techniques of urine analysis for estriol determination and can also be applied to routine analysis for quality control in the pharmaceutical industry. Another positive aspect observed was the total absence of interferences in the determination of this hormone in a complex sample, in this case urine. The determination of estriol in a urine sample taken during pregnancy was successfully achieved without sample pretreatment. Therefore, this confers credibility and feasibility to the methodology proposed for the determination of estriol in urine samples taken during pregnancy. In addition, due to the simplicity of the preparation procedures, fast routine determinations can also be achieved in the direct electrochemical analysis of estriol and other hormones in pharmaceutical preparations. Acknowledgements The authors wish to thank CAPES and CNPq for scholarships and financial support. Prof. Orlando Fatibello-Filho and Prof. Romeu

2006

K.D. Santos et al. / Talanta 80 (2010) 1999–2006

Costa Rocha-Filho (UFSCar) are gratefully acknowledged for providing the BDD electrode. References [1] U. Goebelsmann, R.B. Jaffe, Acta Endocrinol. 66 (1971) 679. [2] Y. Yaron, M. Cherry, R.L. Kramer, J.E. O’Brien, M. Hallak, M.P. Johnson, M.I. Evans, Am. J. Obstet. Gynecol. 181 (1999) 968. [3] L.A. Bradley, J.A. Canick, G.E. Palomaki, J.E. Haddow, Am. J. Obstet. Gynecol. 176 (1997) 531. [4] M.A. Reyes-Romero, Med. Hypotheses 56 (2001) 107. [5] M.J.L. Alda, S. Díaz-Cruz, D. Barceló, J. Chromatogr. A 1000 (2003) 503. [6] V. Ingrand, G. Herry, J. Beausse, M. Roubin, J. Chromatogr. A 1020 (2003) 99. [7] R.W. Reis Filho, J.C. Araújo, E.M. Vieira, Quim. Nova 29 (2006) 817. [8] X. Huang, D. Yuan, B. Huang, Talanta 75 (2008) 172. [9] M. Kuster, M.J.L. Alda, M.D. Hernando, M. Petrovic, J. Martín-Alonso, D. Barceló, J. Hydrol. 358 (2008) 112. [10] N. Tagawa, H. Tsuruta, A. Fujinami, Y. Kobayashi, J. Chromatogr. B 723 (1999) 39. ˜ [11] A. Penalver, E. Pocurull, F. Borrull, R.M. Marcé, J. Chromatogr. A 964 (2002) 153. [12] Y.K.K. Koh, T.Y. Chiu, A. Boobis, E. Cartmell, J.N. Lester, M.D. Scrimshaw, J. Chromatogr. A 1173 (2007) 81. [13] S. Impens, K. Wasch, M. Cornelis, H.F. Brabander, J. Chromatogr. A 970 (2002) 235. [14] P. Marchand, B. Bizec, C. Gade, F. Monteau, F. André, J. Chromatogr. A 867 (2000) 219. [15] M.R. Fuh, S.Y. Huang, T.Y. Lin, Talanta 64 (2004) 408. [16] D. Wu, J. Lin, Z. Li, X. Wang, X. Ying, Fenxi Huaxue 35 (2007) 1241. ˜ ˜ J.M. Pingarrón, Talanta [17] D. Vega, L. Agüí, A. González-Cortés, P. Yánez-Sede no, 71 (2007) 1031. [18] M.A. Méndez, M.F. Suárez, M.T. Cortés, J. Electroanal. Chem. 590 (2006) 181. [19] G.V. Korshin, J. Kim, L. Gan, Water Res. 40 (2006) 1070. [20] G. Volpe, G. Fares, F. Quadri, R. Draisci, G. Ferretti, C. Marchiafava, D. Moscone, G. Palleschi, Anal. Chim. Acta 572 (2006) 11. [21] J. Tschmelak, G. Proll, G. Gauglitz, Talanta 65 (2005) 313. [22] M.M. Ngundi, O.A. Sadik, T. Yamaguchi, S. Suye, Electrochem. Commun. 5 (2003) 61.

[23] K.H.R. Baronian, S. Gurazada, Biosens. Bioelectron. 22 (2007) 2493. [24] T. Hahn, K. Tag, K. Riedel, S. Uhlig, K. Baronian, G. Gellissen, G. Kunze, Biosens. Bioelectron. 21 (2006) 2078. [25] F.B. Liu, J.D. Wang, B. Liu, X.M. Li, D.R. Chen, Diam. Relat. Mater. 16 (2007) 454. [26] L. Codognoto, S.A.S. Machado, L.A. Avaca, Diam. Relat. Mater. 11 (2002) 1670. [27] G.R. Salazar-Banda, L.S. Andrade, P.A.P. Nascente, P.S. Pizani, R.C. Rocha-Filho, L.A. Avaca, Electrochim. Acta 51 (2006) 4612. [28] C.D. Souza, O.C. Braga, I.C. Vieira, A. Spinelli, Sens. Actuators B 135 (2008) 66. [29] B.C. Lourenc¸ão, R.A. Medeiros, R.C. Rocha-Filho, L.H. Mazo, O. Fatibello-Filho, Talanta 78 (2009) 748. [30] B. Uslu, B.D. Topal, S.A. Ozkan, Talanta 74 (2008) 1191. [31] L. Codognoto, V.G. Zuin, D. Souza, J.H. Yariwake, S.A.S. Machado, L.A. Avaca, Microchem. J. 77 (2004) 177. [32] V.A. Pedrosa, L. Codognoto, S.A.S. Machado, L.A. Avaca, J. Electroanal. Chem. 573 (2004) 11. [33] K. Boonsong, S. Chuanuwatanakul, N. Wangfuengkanagul, O. Chailapakul, Sens. Actuators B 108 (2005) 627. [34] T.A. Ivandini, K. Honda, T.N. Rao, A. Fujishima, Y. Einaga, Talanta 71 (2007) 648. [35] D.A. Tryk, H. Tachibana, H. Inoue, A. Fujishima, Diam. Relat. Mater. 16 (2007) 881. [36] R.A. Medeiros, A.E. Carvalho, R.C. Rocha-Filho, O. Fatibello-Filho, Talanta 76 (2008) 685. [37] E. Diczfaluzy, S. Mancuso, in: A. Kloper, E. Diczfalusy (Eds.), Foetus and Placenta, Blackwell Scientific Publications, Oxford, 1969, pp. 191–248. [38] I. Duo, P.-A. Michaud, W. Haenni, A. Perret, C. Comninellis, Electrochem. SolidState Lett. 3 (2000) 325. [39] S.M.L. Agostinho, R.F.V. Villamil, A. Agostinho Neto, H. Aranha, Quim. Nova 27 (2004) 813. [40] J.B. Quintana, J. Carpinteiro, I. Rodriguez, R.A. Lorenzo, A.M. Carro, R. Cela, J. Chromatogr. A 1024 (2004) 177. [41] D. Galato, K. Ckless, M.F. Susin, C. Giacomelli, R.M. Ribeiro-do-Valle, A. Spinelli, Redox Rep. 6 (2001) 243. [42] C. Giacomelli, K. Ckless, D. Galato, F.S. Miranda, A. Spinelli, J. Braz. Chem. Soc. 13 (2002) 332. [43] C. Giacomelli, F.S. Miranda, N.S. Gonc¸alves, A. Spinelli, Redox Rep. 9 (2004) 263. [44] D. Souza, S.A.S. Machado, L.A. Avaca, Quim. Nova 26 (2003) 81. [45] M. Ribani, L.F.C. Melo, Quim. Nova 27 (2004) 7.