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Electrochemical behavior of progesterone at an ex situ bismuth film electrode
Electrochimica Acta 107 (2013) 542–548
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Electrochemical behavior of progesterone at an ex situ bismuth film electrode Camila Alves de Lima, 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 29 January 2013 Received in revised form 28 May 2013 Accepted 28 May 2013 Available online 18 June 2013 Keywords: Bismuth film electrode Progesterone Electroanalysis Pharmaceuticals
a b s t r a c t An ex situ bismuth film electrode (BiFE) was used for the electrochemical study of the hormone progesterone. Two peaks were observed by cyclic voltammetry, at −1.68 V and −1.47 V, respectively, associated with the reduction and oxidation reactions of progesterone in 0.1 mol L−1 Britton–Robinson solution at pH 12.0, in agreement with a quasireversible electrode process. In relation to the mechanism, an adsorption-controlled reaction rate with one electron involved in the electrochemical step was observed. Square-wave adsorptive stripping voltammetry was used for the analytical procedures after accumulation of the progesterone for 60 s at −1.0 V. A peak current at −1.63 V related to the reduction of progesterone was obtained, which increased linearly with the hormone concentration in the range of 0.40–7.90 mol L−1 (R2 = 0.9983). The limit of detection attained was 0.18 mol L−1 . The method was applied in the determination of progesterone in four pharmaceutical samples with satisfactory results being obtained compared with a spectrophotometric method. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction In the search for new and mercury-free electrode materials, different bismuth-based electrodes (BiEs) have been developed by the scientific community [1–6]. As demonstrated by different authors [7–11], they serve for the determination of both metals and organic compounds as pharmaceuticals [12–15], pesticides [16,17], herbicides [18–20] and more complex matrices [21]. Bismuth-film electrodes (BiFEs) have been widely used because (i) they are economically viable; (ii) they have high mechanical stability; (iii) bismuth can be deposited on various types of substrates; and (iv) the physico-chemical properties of bismuth are similar to those of mercury. In this study, we show the electroanalytical determination of the hormone progesterone (P4) at an ex situ bismuth film-plated electrode. Progesterone (P4) (pregn-4-ene-3,20-dione) (Fig. 1) belongs to a group of steroid hormones derived from cholesterol and it plays an important role in the stabilization and maintenance of pregnancy in mammals, acting in the synthetic route of various biologically active steroids including gluco-corticoids, mineral corticoids, androgens and estrogens [22]. An imbalance of P4 in the
∗ 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). 0013-4686/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.electacta.2013.05.141
human organism can cause infertility as well as malformations within the reproductive system [23,24]. In the pharmaceutical industry, P4 is the active principle of many drugs used in hormone replacement therapies for post-menopausal women [25–27]. Various chromatography-based methods have been reported for P4 determination in commercial formulations, water, urine, serum, human and bovine milk, and rat plasma. The techniques of HPLC and LC have been coupled with different detectors, including DAD [28], UV [22,29], MS [30,31], and DCE [32]. In addition, various other methods, such as time-resolved fluoroimmunoassay (TRFIA) [33], fluorescent immunoassay [34], and chemiluminescence enzyme immunoassay (CLEIA) [35], have also been employed for P4 determination. Nevertheless, most of these methods are timeconsuming and require expensive equipment, sample preparation steps, such as extraction from biological fluids and/or derivatization, which can adversely affect the analytical results. Some attempts have also been made to include voltammetric methods as alternatives for the detection and electrochemical study of several hormones. Progesterone and other sex hormones have been investigated using a static mercury drop electrode and adsorptive stripping voltammetry [36]. The cyclic voltammogram for P4 showed a well-defined reduction peak at −1.52 V in 0.005 mol L−1 NaOH solution. A limit of detection of 0.2 nmol L−1 was achieved with 15 min of preconcentration. Hu et al. [37] developed a linear sweep polarographic method for the determination of P4 based on the enhancement effect of surfactants. In alkaline media, P4 was reduced at a dropping mercury electrode and a limit of detection of 20.0 nmol L−1 was attained. The method
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was diluted to 50 mL with ethanol. An aliquot of 125 L was used to determine P4 concentration in sample C. Sample D: the label value was 100 mg of P4 by tablet. Ten tablets were weighed and macerated. The mean mass for one tablet was dissolved in 50 mL of ethanol. A new solution was prepared with 2.5 mL of the original solution and 20 mL of ethanol. An aliquot of 200 L was used to determine P4 concentration in sample D. Fig. 1. Chemical structure of progesterone.
was successfully applied for determinations of P4 in blood and pharmaceutical products. Also, Arévalo et al. [24] have reported the adsorption of P4 onto a glassy carbon electrode. Applying square wave voltammetry, the authors observed a reduction peak at −1.65 V in pH 8.0 buffer solution. The same researchers, in a different publication [38], reported the use of cyclic and square wave voltammetries to study the mechanism involved in the electrochemical reduction of P4 at a glassy carbon electrode in N(C4 H9 )4 PF6 + acetonitrile solution. A few papers [39–45] have reported the development of immunosensors for P4 detection in bovine serum and cow’s milk. In this context, the objective of this paper is to report the electrochemical behavior of progesterone at an ex situ bismuth film-plated electrode and the determination of this hormone in pharmaceutical formulations. The BiFE is a cost-effective, single-use, feasible, accurate and reliable sensor for many organic molecules; however, there are no other publications reporting the electrochemical behavior or the electroanalytical determination of hormones at the BiFE surface. Thus, the results of this study could stimulate and aid research in this field. 2. Experimental 2.1. Reagents, solutions and samples All reagents used in this study were of analytical grade and obtained from Sigma Aldrich. The solutions were prepared with water purified using a Milli-Q system manufactured by Millipore (Bedford, MA, USA). A solution of 2.0 mmol L−1 Bi(NO3 )3 was obtained by dissolving an appropriate mass of the bismuth salt in 1.0 mol L−1 HCl and employing it for the deposition of the bismuth film onto a glassy carbon substrate. Britton–Robinson and Ringer buffers and NaOH and LiOH solutions were tested as supporting electrolytes at pH 12.0. Stock solutions of each supporting electrolyte were prepared at concentration of 0.1 mol L−1 . Their pH was adjusted to 12.0 and they are referred in the text as, for example, 0.1 mol L−1 NaOH with pH of 12.0 or 0.1 mol L−1 Britton–Robinson buffer (pH 12.0). A stock solution of 20.0 mmol L−1 P4 dissolved in ethanol was prepared daily and less concentrated solutions were prepared by dilution. For the recovery experiments, the standard addition method was used, with a standard solution of P4 being added to the samples. Samples of progesterone gel, cream, hair lotion and tablets, designated as samples A, B, C and D, respectively, were obtained commercially. The solution for the determination of each sample was prepared as follows. Sample A: the labeled value was 450 mg of P4 for 50 g of gel. 5.0 g of gel were weighed and dissolved in 20 mL of ethanol. After filtration, the volume was completed to 20 mL with ethanol. 8.0 mL of this solution were diluted to 50 mL, also using ethanol. An aliquot of 75 L was used to determine the P4 concentration in sample A. Sample B: the label value was 750 mg of P4 for 50 g of cream. A mass of 11.5 g was weighed and dissolved in 50 mL of ethanol. 750 L were used to prepare a new solution with 50 mL of ethanol. An aliquot of 100 L was used to determine P4 concentration in sample B. Sample C: the labeled value was1000 mg of P4 for 50 mL of hair lotion. A volume of 630 L
2.2. Electrochemical apparatus Voltammetric measurements were carried out with an EG&G PARC model 263A potentiostat/galvanostat interfaced with a personal computer using the “SoftCorr II Model 252/352” software (from the same company) for data acquisition and analysis. Cyclic and square wave voltammograms were recorded using an electrochemical cell containing three electrodes: the BiFE as the working electrode, platinum wire as the auxiliary electrode and Ag/AgCl saturated with KCl as the reference electrode. All of the potentials measured are quoted versus the reference electrode. 2.3. Ex situ preparation of BiFE The surface of a glassy carbon electrode (GCE-A = 0.07 cm2 ) was employed as the substrate for the plating of the bismuth film. Before each deposition of the film, the glassy carbon electrode was handpolished using alumina slurry with a sequence of particle sizes of 0.50 and then 0.05 m, followed by washing and sonication for 3 min in purified water. After the insertion of the GCE into the electrochemical cell containing 2.0 mmol L−1 Bi(NO3 )3 in a 1.0 mol L−1 HCl solution, the electrolytic deposition was carried out at −0.3 V versus Ag/AgCl for 12 s while stirring the solution. A thin film was obtained, which was considered free of roughness and with the same area of the substrate. The BiFE was then transferred to another cell and the voltammetric measurements were taken in the presence of P4. Due to the adsorption of P4 onto the BiFE a new film had to be prepared after each voltammetric experiment. 2.4. Square wave adsorptive stripping voltammetry-SWAdSV Initial SWAdSV experiments were carried out with 6.0 mol L−1 P4. After the application of an accumulation step, square wave voltammograms were obtained from −1.4 to −1.8 V with the BiFE immersed in 0.1 mol L−1 Britton–Robinson buffer at pH 12.0 in the presence of P4. Thus, the dependence of the SWAdSV response on the parameters scan increment (Es), amplitude (a) and frequency (f) was analyzed in order to optimize their values for P4 determination. The ranges studied were: Es = 1–5 mV, a = 1–100 mV and f = 20–90 Hz. The optimized values, that is, Es = 2 mV, a = 50 mV and f = 70 Hz, were selected because they provided the best compromise between the sensitivity and the voltammogram profile. SWAdSV was therefore employed for the construction of the calibration curve and the determination of P4 in the four samples of pharmaceutical formulations. 2.5. Comparative method Based on the Brazilian Pharmacopoeia [46], ultraviolet absorption spectrophotometry (UV) was used as the comparative technique, aiming to evaluate the accuracy of the results provided by the BiFE for the detection of P4 in pharmaceuticals. The spectra were obtained on a Varian 50 Bio Ultraviolet Absorption Spectrophotometer at 240 nm, using a quartz cell with optical path length of 1 cm. Solutions and samples used in the comparative method had concentrations in the same range as those employed in the case of the BiFE.
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Fig. 3. Cyclic voltammograms for 40 mol L−1 P4 in 0.1 mol L−1 Britton–Robinson buffer (pH 12.0) obtained with the BiFE as a function of the potential scan rate (a–f = 50–300 mV s−1 ).
electrode under similar conditions [36]. The separation between the anodic and cathodic peaks is 210 mV, indicating the quasireversible nature of the electrochemical reaction. At pH 8.0 (Fig. 2A-b) a single reduction peak was observed at −1.48 V. No peaks were observed in solutions with pH lower than 8.0. The reduction peak potential (Epr ) shifted linearly to more negative potentials as the solution pH increased from 8.0 to 11.0, remaining almost constant at higher pH values (Fig. 2B). The slope for the linear portion of the Epr × pH plot was −66.7 mV dec−1 , which is consistent with the exchange of one electron and one proton, as expected for progesterone reduction [38]. The influence of the solution pH on the cathodic current (ipc ) is shown in Fig. 2C. It can be noted that at pH values below 12.0 the P4 reduction leads to a gradual decrease in the intensity of the current. The best combination of voltammetric profile and sensitivity was obtained at pH 12.0, which was chosen as the optimum pH for subsequent tests. 3.2. Electrochemical behavior of progesterone at the BiFE surface in Britton–Robinson buffer (pH 12.0)
Fig. 2. (A) Cyclic voltammograms, (B) reduction potential and (C) cathodic current as a function of solution pH for 40 mol L−1 P4 in 0.1 mol L−1 Britton–Robinson buffer obtained using a BiFE, v = 200 mV s−1 .
3. Results and discussion 3.1. Selection of the supporting electrolyte and solution pH The electrochemical reduction of 40 mol L−1 P4 at the BiFE was evaluated by cyclic voltammetry in Britton–Robinson buffer, Ringer’s solution, NaOH and LiOH, all at a concentration of 0.1 mol L−1 and pH 12.0. The best compromise between the detection potential, the peak current and the cyclic voltammogram profile was achieved using Britton–Robinson buffer as the supporting electrolyte, which was chosen for further experiments. In the cyclic voltammetric experiments, the potential was swept from −1.0 to −1.8 V and back to −1.0 V. As can be seen in Fig. 2A-a, at pH 12.0 the cyclic voltammogram of P4 exhibits a well-defined reduction peak at −1.68 V and a poorly defined oxidation peak at −1.47 V, in good agreement with results obtained at a mercury
The cyclic voltammograms shown in Fig. 3 depict the electrochemical behavior of 40 mol L−1 P4 in Britton–Robinson buffer solution (pH 12.0) at the BiFE surface as a function of the potential scan rate, considered over the range of 50–300 mV s−1 . As can be clearly observed, the Epr -value shifts to more negative potentials with an increase in the scan rate. The dependence of the Epr with log v is linear according to the equation Epr /V = −1.438 − 0.101 log v. From the slope of the plot, a value of ˛n = 0.585 was calculated, which provides n = 1.17 for ˛ = 0.5, where n is the stoichiometric number of the electrons involved in the electrode reaction and ˛ is the transfer coefficient. On the other hand, although the oxidation peak is perceptible at low scan rates it is best defined at 300 mV s−1 . According to the literature [38], even a quasireversible mechanism can be detected at very fast scan rates. Furthermore, the interdependence between the current function (−ipc v−1/2 ) and v indicates that a chemical reaction is coupled to the electrode process, i.e., the reaction follows an EC mechanism. Finally, the slope of the plot log ipc × log v was 0.9, that is, very close to 1.0, verifying the adsorption of P4 onto the BiFE. De Boer et al. [47] have studied the electrochemical properties of corticosteroids, including progesterone, in different supporting electrolytes and Arévalo et al. [38] investigated the electrochemical reduction of P4 in acetonitrile. In both cases, a dimer was proposed
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as the final product of the P4 reduction, although the media used in the studies were different, as well as the reduction patterns. To explain our results, the deduction proposed by De Boer et al. seems to be most appropriate. According to these authors [47 and references therein], in alkaline medium the dimerization takes place initially via a one-electron reaction with the reduction of the C-3 keto group on the A-ring of the P4 molecule and the formation of an unprotonated radical. The radical undergoes protonation and reacts with another unprotonated radical, resulting in a dimer. The overall reaction can be described as follows:
3.3. Adsorption of P4 at the BiFE surface As commented earlier, the plot of log ipc × log v with a slope of close to 1.0 indicates the adsorption of P4 onto the BiFE surface. This behavior was also identified by the successive cyclic voltammograms recorded at 200 mV s−1 for the reduction of 40 mol L−1
Fig. 4. (A) Cyclic and (B) square wave voltammograms for 40 mol L−1 P4 in 0.1 mol L−1 Britton–Robinson buffer (pH 12.0) obtained with the BiFE. (A-a) first cycle, (A-b) tenth cycle, (A) v = 200 mV s−1 , (B-a) without accumulation step, (B-b) Eacc = −1.0 V, tacc = 60 s, (B) Es = 4 mV, a = 60 mV, f = 50 Hz.
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P4 in 0.1 mol L−1 Britton–Robinson buffer at pH 12.0 (Fig. 4A). The first cycle (Fig. 4A-a) was compared to the tenth cycle (Fig. 4A-b) and it was apparent that the cathodic current sharply decreased. This means that the surface of the electrode was blocked by the adsorption of P4, preventing new molecules from reaching the surface. Based on this behavior, an accumulation step was performed before carrying out the voltammetric experiments. In this way, the variables accumulation potential – Eacc and time – tacc were optimized. The Eacc -values were varied in the range of −0.9 V to −1.4 V at constant tacc = 60 s, while the tacc -values ranged from 40 to 120 s at Eacc = −1.0 V. The ipc for P4 reduction increased for Eacc values of up to −1.0 V and then decreased. The ipc value increased with the tacc , indicating an increasing accumulation of P4 at the electrode surface over time. However, for electroanalytical purposes, a reasonably short time of 60 s was chosen. Therefore, to proceed with the studies, Eacc and tacc values of −1.0 V and 60 s, respectively, were selected. In order to verify that this procedure is appropriate for the electroanalytical determination of P4, the accumulation step was tested for square wave experiments. As can be seen in Fig. 4B, a more defined peak and a significant increase in the current for the reduction of P4 were obtained with the application of the accumulation step. Thus, square wave adsorptive stripping voltammetry (SWAdSV) was applied in further experiments. 3.4. Calibration curve for P4 obtained using BiFE-SWAdSV Fig. 5 shows the square wave voltammograms obtained under optimized conditions for P4 at the BiFE surface after successive additions of a standard solution of the sex hormone. A well-defined and sharp reduction peak is observed at −1.63 V, with the current increasing proportionally to the P4 concentration. The calibration curve displayed good linearity in the range of 0.40–7.90 mol L−1 P4 (R2 = 0.9983). The equation for the straight line can be expressed as i/mA = −0.0049 + 0.5691 [progesterone]/mol L−1 . The limits of detection (LD) and quantification (LQ) were calculated according to the equations: LD = 3Sb /B and LQ = 10Sb /B, where Sb is the standard deviation of the y-coordinate obtained from the line of best fit (linear coefficient) and B is the slope (angular coefficient) of this line. The LD calculated for P4 was 0.18 mol L−1 and the LQ was 0.61 mol L−1 . The repeatability of the signals produced by BiFE was verified using a 6.0 mol L−1 P4 solution under optimized conditions. The repeatability of the current peak was tested using two methodologies: intra-day, using ten different solutions prepared on the same day at a concentration of 6.0 mol L−1 , and inter-day,
Fig. 5. Square wave voltammograms for P4 in 0.1 mol L−1 Britton–Robinson buffer (pH 12.0) obtained with a BiFE under optimized conditions: (a) blank, (b) 0.4 mol L−1 , (c) 0.7 mol L−1 , (d) 1.0 mol L−1 , (e) 2.0 mol L−1 , (f) 3.0 mol L−1 , (g) 4.0 mol L−1 , (h) 5.0 mol L−1 , (i) 5.9 mol L−1 , (j) 6.9 mol L−1 , and (k) 7.9 mol L−1 .
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Table 1 Study of potential interferents in the determination of P4 in pharmaceutical products. Sample
Excipient
P4: interferent ratio (1:0) ipc (mA cm−2 )a
(1:0.01)
(1:0.1)
(1:1)
(1:10)
A
Parabens Phenoxyethanol Hydroxyethylcellulose
3.35
3.36
3.37
3.32
3.27
B
Cetostearyl alcohol mineral oil Lanolin alcohol Parabens Phenoxyethanol propylene glycol
3.31
3.33
3.35
3.34
3.32
C
Ethanol
3.33
3.33
3.35
3.34
3.36
D
Soya lecithin Peanut oil
3.34 3.32
3.34 3.33
3.36 3.33
3.34 3.33
2.10 3.32
a
n = 3.
over ten days using different solutions prepared at a concentration of 6.0 mol L−1 P4. The relative standard deviations (n = 10) were 1.7% and 1.5%, respectively. These results demonstrate the suitability of the BiFE for the determination of P4 by SWAdSV. 3.5. Study of potential interferents in the determination of P4 in pharmaceuticals The presence of excipients in pharmaceuticals can interfere with electroanalytical measurements. Therefore, a study on the possible interferents in the samples used for the progesterone determination was carried out. The excipients present in samples A–D analyzed in this study are shown in Table 1. The possible interferents were tested in the presence of 6.0 mol L−1 P4 in 0.1 mol L−1 Britton–Robinson buffer (pH 12.0) with the BiFE under optimized conditions by SWAdSV. The P4: interferent ratios studied were 1:0; 1:0.01; 1:0.1; 1:1 and 1:10. The results for the current density obtained for the P4 reduction in the absence and presence of interferents are also shown in Table 1. As can be noted from the results, the current for the P4 reduction remained almost constant, except in the presence of soya lecithin (sample D) for the ratio of 1:10 (P4: soya lecithin). The soya lecithin exhibits a reduction peak at −1.39 V, interfering by decreasing the current for the P4 reduction by around 37%. This means that P4 determination using BiFE-SWAdSV cannot be performed in the presence of soya lecithin when its concentration is ten times higher than that of P4. On the other hand, as can be observed, no other excipient contained in the samples interfered in the P4 determination.
with the slope of the calibration curve. The slopes of the standard addition plots were 0.639 mA L mol−1 cm−2 (R2 = 0.9921), 0.534 mA L mol−1 cm−2 (R2 = 0.9961), 0.499 mA L mol−1 cm−2 (R2 = 0.9900) and 0.634 mA L mol−1 cm−2 (R2 = 0.9855) for samples A, B, C and D, respectively. These values are very close to that of the calibration curve: 0.569 mA L mol−1 cm−2 (R2 = 0.9983). Thus, it can be concluded that there is no interference from the matrix components in the P4 determination. This behavior is in agreement with the results obtained in the study on potential interferents described in the preceding section.
3.6. Determination of P4 in pharmaceuticals using BiFE-SWAdSV and UV techniques The electroanalytical methodology described above using BiFESWAdSV and the comparative method were applied in the quantitative determination of P4 in four pharmaceutical formulations. The electroanalytical determination of P4 and recovery studies were carried out by adding standard solutions of the hormone to the four samples (A, B, C and D). The solutions prepared from the samples were diluted to a concentration included in the range used to obtain the calibration curve. Fig. 6A shows the square wave voltammograms obtained in the determination of P4 in sample A, and Fig. 6B depicts the resulting standard addition plot together with the calibration curve for the purposes of comparison. For all samples analyzed the voltammograms showed well-defined reduction peaks and the standard addition plots were linear. The selectivity, i.e., absence of interferences, was tested by comparing the slopes of the standard addition plots
Fig. 6. (A) Square wave voltammograms for: (a) blank, (b) 75 L of sample A, (c)–(e) 25 L and (f) 50 L of 0.5 mmol L−1 P4. (B) The standard addition plot for sample A and calibration curve.
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Table 2 Determination of P4 in commercial pharmaceutical products by BiFE-SWAdSV and UV techniques. Sample
A
Method
UV
BiFE
UV
BiFE
UV
BiFE
UV
BiFE
Label values (mg) Found values (mg)a RSD (%) Er1 (%)b Er2 (%)c tvalue d Fvalue e
450 446 1.03 −0.89 – −1.51 1.45
450 447 1.23 −0.67 0.22 −0.94 1.45
750 757 0.75 0.93 – 2.13 2.50
750 752 1.15 0.27 −0.66 0.38 2.50
1000 1023 1.37 2.30 – 2.84 3.23
1000 1033 2.44 3.30 0.97 2.27 3.23
100 102 1.50 2.00 – 2.13 1.00
100 101 1.51 1.00 −0.97 1.13 1.00
a b c d e
B
C
D
n = 3. Er1 = relative error between BiFE-SWAdSV or UV techniques and label values. Er2 = relative error between BiFE-SWAdSV and UV techniques. ttheoretical = 4.30. Ftheoretical = 19.
Table 2 shows the results obtained in the determination of P4 in four samples independently analyzed by BiFE-SWAdSV and UV techniques as well as the statistical data. Firstly, it can be observed that the values obtained using the two techniques are very close to the label values for the pharmaceutical products. The relative standard deviation (RSD) for the mean of three determinations of P4 in the four samples was between 1.15% and 2.44% for the measurements with BiFE-SWAdSV and between 0.75% and 1.50% using the UV technique. The relative errors in relation to the label values for BiFE-SWAdSV and the UV technique were <3.30% and <2.30%, respectively, while the relative difference between the values obtained with the two techniques was <1.0%. To examine whether the data provided by a certain technique is precise, accurate or superior to another technique, we can compare the results obtained by the proposed technique with those available in other data sets. Two of the most widely used tests for the comparison of results are the t- and F-tests. The t-test was used to compare the results obtained by the two techniques with the label values (accepted as the true values). At the 95% confidence level all values calculated for tvalue were lower than those for ttheoretical (4.30), indicating no significant differences between the data obtained using the two techniques and the true (label) values. The discrepancy between the data provided by BiFE-SWAdSV and UV techniques was verified by applying the F-test. The values for Fvalue obtained in the determination of P4 in pharmaceutical samples were lower than those for Ftheoretical (19) at the 95% confidence level, indicating that there is no significant difference in the precision provided by the two techniques. 3.7. Recovery experiments Recovery essays were also carried out to investigate the accuracy of the data furnished by BiFE-SWAdSV in the determination of P4 in pharmaceutical formulations. Four additions of P4 standard solutions yielded recoveries between 104% and 115% for sample A and 90% and 110% for sample D. For the samples B and C, five additions of P4 standard solutions yielded recoveries between 94% and 101% for sample B and 97% and 115% for sample C. These data verify that the matrix components do not interfere in the determination of P4. Moreover, these results corroborate the accuracy of the data obtained with BiFE-SWAdSV. 4. Conclusions The data presented allow concluding that the BiFE associated with cyclic voltammetry can provide reliable information on the electrochemical behavior of the sex hormone progesterone. In addition, when associated with square wave voltammetry, the BiFE also exhibited the capacity to furnish data comparable to those
obtained with the UV technique in the determination of progesterone in varied and complex matrices such as gel, cream, lotion and tablets. Furthermore, the sample preparation is very simple and rapid, necessitating only dilution in ethanol or water. The excellent performance of the BiFE in the determination of progesterone was demonstrated by the parameters of the calibration curve, recovery experiments and the similarity of the data with those provided by the comparative technique. Acknowledgements The authors wish to thank the Brazilian government agencies CAPES (Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for scholarships and financial support. References [1] J. Wang, J. Lu, S.B. Hoˇcevar, P.A.M. Farias, B. Ogorevc, Bismuth-coated carbon electrodes for anodic stripping voltammetry, Analytical Chemistry 72 (2000) 3218. [2] E.A. Hutton, B. Ogorevc, S.B. Hoˇcevar, F. Weldon, M.R. Smyth, J. Wang, An introduction to bismuth electrode for use in cathodic electrochemical detection, Electrochemistry Communications 3 (2001) 707. [3] R. Pauliukaité, R. Metelka, I. Svancara, A. Królicka, A. Bobrowski, K. Vytˇras, E. Norkus, K. Kalcher, Carbon paste electrodes modified with Bi2 O3 as sensors for the determination of Cd and Pb, Analytical and Bioanalytical Chemistry. 374 (2002) 1155. [4] R. Pauliukaité, S.B. Hoˇcevar, B. Ogorevc, J. Wang, Characterization and applications of a bismuth bulk electrode, Electroanalysis 16 (2004) 719. [5] S.B. Hoˇcevar, I. Svancara, K. Vytˇras, B. Ogorevc, Novel electrode for electrochemical stripping analysis based on carbon paste modified with bismuth powder, Electrochimica Acta 51 (2005) 706. ˜ [6] M.T. Castaneda, B. Pérez, M. Pumera, M. del Valle, A. Merkoc¸i, S. Alegret, Sensitive stripping voltammetry of heavy metals by using a composite sensor based on a built-in bismuth precursor, Analyst 130 (2005) 971. [7] H. Sopha, S.B. Hocevar, B. Pihlar, B. Ogorevc, Bismuth film electrode for stripping voltammetric measurement of sildenafil citrate, Electrochimica Acta 60 (2012) 274. [8] K. Asadpour-Zeynali, P. Najafi-Marandi, Bismuth modified disposable pencillead electrode for simultaneous determination of 2-nitrophenol and 4-nitrophenol by net analyte signal standard addition method, Electroanalysis 23 (2011) 2241. [9] V. Guzsvany, Z. Papp, J. Zbiljic, O. Vajdle, M. Rodic, Bismuth modified carbonbased electrodes for the determination of selected neonicotinoid insecticides, Molecules 16 (2011) 4451. [10] I. Svancara, C. Prior, S.B. Hoˇcevar, J. Wang, A decade with bismuth-based electrodes in electroanalysis, Electroanalysis 22 (2010) 1405. [11] C. Kokkinos, A. Economou, Stripping analysis at bismuth-based electrodes, Current Analytical Chemistry 4 (2008) 183. [12] G.L. Kreft, O.C. de Braga, A. Spinelli, Analytical electrochemistry of vitamin B12 on a bismuth-film electrode surface, Electrochimica Acta 83 (2012) 125. [13] I. Campestrini, O.C. de Braga, I.C. Vieira, A. Spinelli, Application of bismuth-film electrode for cathodic electroanalytical determination of sulfadiazine, Electrochimica Acta 55 (2010) 4975. [14] B. Nigovic, B. Simunic, S. Hocevar, Voltammetric measurements of aminosalicylate drugs using bismuth film electrode, Electrochimica Acta 54 (2009) 5678.
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[15] J.A. Rodriguez, E. Barrado, Y. Castrillejo, J.R. Santos, J. Lima, Validation of a tubular bismuth film amperometric detector: determination of diclofenac sodium by multisyringe flow injection analysis, Journal of Pharmaceutical and Biomedical Analysis 45 (2007) 47. [16] D. Du, X. Ye, J. Zhang, D. Liu, Cathodic electrochemical analysis of methyl parathion at bismuth-film-modified glassy carbon electrode, Electrochimica Acta 53 (2008) 4478. [17] A.S. Arribas, E. Bermejo, M. Chicharro, A. Zapardiel, Voltammetric detection of the herbicide metamitron at a bismuth film electrode in nondeaerated solution, Electroanalysis 18 (2006) 2331. [18] L.C.S. de Figueiredo-Filho, D.C. Azzi, B.C. Janegitz, T.B. Guerreiro, O. FatibelloFilho, Determination of atrazine in natural water samples by differential pulse adsorptive stripping voltammetry using a bismuth film electrode, Electroanalysis 24 (2012) 303. [19] L.C.S. de Figueiredo-Filho, V.B. dos Santos, B.C. Janegitz, T.B. Guerreiro, O. Fatibello-Filho, R.C. Faria, L.H. Marcolino-Junior, Differential pulse voltammetric determination of paraquat using a bismuth-film electrode, Electroanalysis 22 (2010) 1260. [20] M. Moreno, E. Bermejo, M. Chicharro, A. Zapardiel, A.S. Arribas, Cathodic electrochemical determination of herbicides in acid media using a bismuth film electrode, Electroanalysis 21 (2009) 415. [21] M. Frena, I. Campestrini, O.C. de Braga, A. Spinelli, In situ bismuth-film electrode for square-wave anodic stripping voltammetric determination of tin in biodiesel, Electrochimica Acta 56 (2011) 4678. [22] V. Pucci, F. Bugamelli, R. Mandrioli, B. Luppi, M.A. Raggi, Determination of progesterone in commercial formulations and in non-conventional micellar systems, Journal of Pharmaceutical and Biomedical Analysis 30 (2003) 1549. [23] M.S. Christian, R.L. Brent, P. Calda, Embryo–fetal toxicity signals for 17␣hydroxyprogesterone caproate in high-risk pregnancies: a review of the non-clinical literature for embryo–fetal toxicity with progestins, Journal of Maternal–Fetal and Neonatal Medicine 20 (2007) 89. [24] F.J. Arévalo, P.G. Molina, M.A. Zón, H. Fernández, Studies about the adsorption of progesterone (P4) at glassy carbon electrodes in aqueous buffer solution by square wave voltammetry, Journal of Electroanalytical Chemistry 629 (2009) 133. [25] D.T. Baird, A.F. Glasier, Drug therapy: hormonal contraception, The New England Journal of Medicine 328 (1993) 1543. [26] R. Sitruk-Ware, Progestogens in hormonal replacement therapy: new molecules, risks, and benefits, Menopause 9 (2002) 6. [27] P.E. Belchetz, Drug Therapy, Hormonal treatment of postmenopausal women, The New England Journal of Medicine 330 (1994) 1062. [28] C. Almeida, J.M.F. Nogueira, Determination of steroid sex hormones in water and urine matrices by stir bar sorptive extraction and liquid chromatography with diode array detection, Journal of Pharmaceutical and Biomedical Analysis 41 (2006) 1303. [29] L. Wang, Y.-Q. Cai, B. He, C.-G. Yuan, D.-Z. Shen, J. Shao, G.-B. Jiang, Determination of estrogens in water by HPLC–UV using cloud point extraction, Talanta 70 (2006) 47. [30] P. Keski-Rahkonen, K. Huhtinen, M. Poutanen, S. Auriola, Fast and sensitive liquid chromatography–mass spectrometry assay for seven androgenic and progestagenic steroids in human serum, Journal of Steroid Biochemistry and Molecular Biology 127 (2011) 396.
[31] Á. Tölgyesi, Z. Verebey, V.K. Sharma, L. Kovacsics, J. Fekete, Simultaneous determination of corticosteroids, androgens, and progesterone in river water by liquid chromatography–tandem mass spectrometry, Chemosphere 78 (2010) 972. [32] R. Wehner, A. Handke, Simple and rapid method for the determination of progesterone in rat plasma by gas–liquid chromatography with electron-capture detection, Journal of Chromatography A 177 (1979) 237. [33] Y. Oku, T. Osawa, T.-I. Hirata, N. Kon, S. Akasaka, W.S. Senosy, T. Takahashi, Y. Izaike, Validation of a direct time-resolved fluoroimmunoassay for progesterone in milk from dairy and beef cows, Veterinary Journal 190 (2011) 244. [34] L. Trapiella-Alfonso, J.M. Costa-Fernández, R. Pereiro, A. Sanz-Medel, Development of a quantum dot-based fluorescent immunoassay for progesterone determination in bovine milk, Biosensors & Bioelectronics 26 (2011) 4753. [35] S.-Q. Ren, W. Xu, B.-J. Tang, G.-M. Hu, Z.-J. Li, G.-N. Chen, J.-M. Lin, Micro-plate chemiluminescence enzyme immunoassay for clinical determination of progesterone in human serum, Chinese Journal of Analytical Chemistry 36 (2008) 729. [36] J. Wang, P.A.M. Farias, J.S. Mahmoud, Adsorptive stripping voltammetry of sex hormones at the static mercury drop electrode, Analytica Chimica Acta 171 (1985) 195. [37] S. Hu, Y. Yan, Z. Zhao, Determination of progesterone based on the enhancement effect of surfactants in linear sweep polarography, Analytica Chimica Acta 248 (1991) 103. [38] F.J. Arévalo, P.G. Molina, M.A. Zón, H. Fernández, Novel studies about the electrochemical reduction of progesterone (P4) in acetonitrile at glassy carbon electrodes, Journal of Electroanalytical Chemistry 619–620 (2008) 46. [39] M.J. Monerris, F.J. Arévalo, H. Fernández, M.A. Zon, P.G. Molina, Integrated electrochemical immunosensor with gold nanoparticles for the determination of progesterone, Sensors and Actuators B: Chemical 166–167 (2012) 586. [40] F.J. Arévalo, G.A. Messina, P.G. Molina, M.A. Zon, J. Raba, H. Fernández, Determination of progesterone (P4) from bovine serum samples using a microfluidic immunosensor system, Talanta 80 (2010) 1986. [41] R.M. Pemberton, J.P. Hart, P. Stoddard, J.A. Foulkes, A comparison of 1-naphthyl phosphate and 4 aminophenyl phosphate as enzyme substrates for use with a screen-printed amperometric immunosensor for progesterone in cows’ milk, Biosensors & Bioelectronics 14 (1999) 495. [42] R.W. Claycomb, M.J. Delwiche, C.J. Munro, R.H. BonDurant, Rapid enzyme immunoassay for measurement of bovine progesterone, Biosensors & Bioelectronics 13 (1998) 1165. [43] R.W. Claycomb, M.J. Delwiche, Biosensor for on-line measurement of bovine progesterone during milking, Biosensors & Bioelectronics 13 (1998) 1173. [44] S. Cosnier, Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films. A review, Biosensors & Bioelectronics 14 (1999) 443. [45] S. Storri, T. Santoni, M. Minunni, M. Mascini, Surface modifications for the development of piezoimmunosensors, Biosensors & Bioelectronics 13 (1998) 347–357. [46] Farmacopeia Brasileira, Agência Nacional de Vigilância Sanitária e Fundac¸ão Oswaldo Cruz, v. 2, p. 1225–1226, Brasília, 2010. [47] H.S. De Boer, J. Den Hartigh, H.H.J.L. Ploegmakers, W.J. Van Oort, Polarographic analysis for corticosteroids. Part 1. The electroanalytical properties of corticosteroids, Analytica Chimica Acta 102 (1978) 141.
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Electrochemical behavior of progesterone at an ex situ bismuth film electrode Camila Alves de Lima, 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
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Article history: Received 29 January 2013 Received in revised form 28 May 2013 Accepted 28 May 2013 Available online 18 June 2013 Keywords: Bismuth film electrode Progesterone Electroanalysis Pharmaceuticals
a b s t r a c t An ex situ bismuth film electrode (BiFE) was used for the electrochemical study of the hormone progesterone. Two peaks were observed by cyclic voltammetry, at −1.68 V and −1.47 V, respectively, associated with the reduction and oxidation reactions of progesterone in 0.1 mol L−1 Britton–Robinson solution at pH 12.0, in agreement with a quasireversible electrode process. In relation to the mechanism, an adsorption-controlled reaction rate with one electron involved in the electrochemical step was observed. Square-wave adsorptive stripping voltammetry was used for the analytical procedures after accumulation of the progesterone for 60 s at −1.0 V. A peak current at −1.63 V related to the reduction of progesterone was obtained, which increased linearly with the hormone concentration in the range of 0.40–7.90 mol L−1 (R2 = 0.9983). The limit of detection attained was 0.18 mol L−1 . The method was applied in the determination of progesterone in four pharmaceutical samples with satisfactory results being obtained compared with a spectrophotometric method. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction In the search for new and mercury-free electrode materials, different bismuth-based electrodes (BiEs) have been developed by the scientific community [1–6]. As demonstrated by different authors [7–11], they serve for the determination of both metals and organic compounds as pharmaceuticals [12–15], pesticides [16,17], herbicides [18–20] and more complex matrices [21]. Bismuth-film electrodes (BiFEs) have been widely used because (i) they are economically viable; (ii) they have high mechanical stability; (iii) bismuth can be deposited on various types of substrates; and (iv) the physico-chemical properties of bismuth are similar to those of mercury. In this study, we show the electroanalytical determination of the hormone progesterone (P4) at an ex situ bismuth film-plated electrode. Progesterone (P4) (pregn-4-ene-3,20-dione) (Fig. 1) belongs to a group of steroid hormones derived from cholesterol and it plays an important role in the stabilization and maintenance of pregnancy in mammals, acting in the synthetic route of various biologically active steroids including gluco-corticoids, mineral corticoids, androgens and estrogens [22]. An imbalance of P4 in the
∗ 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). 0013-4686/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.electacta.2013.05.141
human organism can cause infertility as well as malformations within the reproductive system [23,24]. In the pharmaceutical industry, P4 is the active principle of many drugs used in hormone replacement therapies for post-menopausal women [25–27]. Various chromatography-based methods have been reported for P4 determination in commercial formulations, water, urine, serum, human and bovine milk, and rat plasma. The techniques of HPLC and LC have been coupled with different detectors, including DAD [28], UV [22,29], MS [30,31], and DCE [32]. In addition, various other methods, such as time-resolved fluoroimmunoassay (TRFIA) [33], fluorescent immunoassay [34], and chemiluminescence enzyme immunoassay (CLEIA) [35], have also been employed for P4 determination. Nevertheless, most of these methods are timeconsuming and require expensive equipment, sample preparation steps, such as extraction from biological fluids and/or derivatization, which can adversely affect the analytical results. Some attempts have also been made to include voltammetric methods as alternatives for the detection and electrochemical study of several hormones. Progesterone and other sex hormones have been investigated using a static mercury drop electrode and adsorptive stripping voltammetry [36]. The cyclic voltammogram for P4 showed a well-defined reduction peak at −1.52 V in 0.005 mol L−1 NaOH solution. A limit of detection of 0.2 nmol L−1 was achieved with 15 min of preconcentration. Hu et al. [37] developed a linear sweep polarographic method for the determination of P4 based on the enhancement effect of surfactants. In alkaline media, P4 was reduced at a dropping mercury electrode and a limit of detection of 20.0 nmol L−1 was attained. The method
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was diluted to 50 mL with ethanol. An aliquot of 125 L was used to determine P4 concentration in sample C. Sample D: the label value was 100 mg of P4 by tablet. Ten tablets were weighed and macerated. The mean mass for one tablet was dissolved in 50 mL of ethanol. A new solution was prepared with 2.5 mL of the original solution and 20 mL of ethanol. An aliquot of 200 L was used to determine P4 concentration in sample D. Fig. 1. Chemical structure of progesterone.
was successfully applied for determinations of P4 in blood and pharmaceutical products. Also, Arévalo et al. [24] have reported the adsorption of P4 onto a glassy carbon electrode. Applying square wave voltammetry, the authors observed a reduction peak at −1.65 V in pH 8.0 buffer solution. The same researchers, in a different publication [38], reported the use of cyclic and square wave voltammetries to study the mechanism involved in the electrochemical reduction of P4 at a glassy carbon electrode in N(C4 H9 )4 PF6 + acetonitrile solution. A few papers [39–45] have reported the development of immunosensors for P4 detection in bovine serum and cow’s milk. In this context, the objective of this paper is to report the electrochemical behavior of progesterone at an ex situ bismuth film-plated electrode and the determination of this hormone in pharmaceutical formulations. The BiFE is a cost-effective, single-use, feasible, accurate and reliable sensor for many organic molecules; however, there are no other publications reporting the electrochemical behavior or the electroanalytical determination of hormones at the BiFE surface. Thus, the results of this study could stimulate and aid research in this field. 2. Experimental 2.1. Reagents, solutions and samples All reagents used in this study were of analytical grade and obtained from Sigma Aldrich. The solutions were prepared with water purified using a Milli-Q system manufactured by Millipore (Bedford, MA, USA). A solution of 2.0 mmol L−1 Bi(NO3 )3 was obtained by dissolving an appropriate mass of the bismuth salt in 1.0 mol L−1 HCl and employing it for the deposition of the bismuth film onto a glassy carbon substrate. Britton–Robinson and Ringer buffers and NaOH and LiOH solutions were tested as supporting electrolytes at pH 12.0. Stock solutions of each supporting electrolyte were prepared at concentration of 0.1 mol L−1 . Their pH was adjusted to 12.0 and they are referred in the text as, for example, 0.1 mol L−1 NaOH with pH of 12.0 or 0.1 mol L−1 Britton–Robinson buffer (pH 12.0). A stock solution of 20.0 mmol L−1 P4 dissolved in ethanol was prepared daily and less concentrated solutions were prepared by dilution. For the recovery experiments, the standard addition method was used, with a standard solution of P4 being added to the samples. Samples of progesterone gel, cream, hair lotion and tablets, designated as samples A, B, C and D, respectively, were obtained commercially. The solution for the determination of each sample was prepared as follows. Sample A: the labeled value was 450 mg of P4 for 50 g of gel. 5.0 g of gel were weighed and dissolved in 20 mL of ethanol. After filtration, the volume was completed to 20 mL with ethanol. 8.0 mL of this solution were diluted to 50 mL, also using ethanol. An aliquot of 75 L was used to determine the P4 concentration in sample A. Sample B: the label value was 750 mg of P4 for 50 g of cream. A mass of 11.5 g was weighed and dissolved in 50 mL of ethanol. 750 L were used to prepare a new solution with 50 mL of ethanol. An aliquot of 100 L was used to determine P4 concentration in sample B. Sample C: the labeled value was1000 mg of P4 for 50 mL of hair lotion. A volume of 630 L
2.2. Electrochemical apparatus Voltammetric measurements were carried out with an EG&G PARC model 263A potentiostat/galvanostat interfaced with a personal computer using the “SoftCorr II Model 252/352” software (from the same company) for data acquisition and analysis. Cyclic and square wave voltammograms were recorded using an electrochemical cell containing three electrodes: the BiFE as the working electrode, platinum wire as the auxiliary electrode and Ag/AgCl saturated with KCl as the reference electrode. All of the potentials measured are quoted versus the reference electrode. 2.3. Ex situ preparation of BiFE The surface of a glassy carbon electrode (GCE-A = 0.07 cm2 ) was employed as the substrate for the plating of the bismuth film. Before each deposition of the film, the glassy carbon electrode was handpolished using alumina slurry with a sequence of particle sizes of 0.50 and then 0.05 m, followed by washing and sonication for 3 min in purified water. After the insertion of the GCE into the electrochemical cell containing 2.0 mmol L−1 Bi(NO3 )3 in a 1.0 mol L−1 HCl solution, the electrolytic deposition was carried out at −0.3 V versus Ag/AgCl for 12 s while stirring the solution. A thin film was obtained, which was considered free of roughness and with the same area of the substrate. The BiFE was then transferred to another cell and the voltammetric measurements were taken in the presence of P4. Due to the adsorption of P4 onto the BiFE a new film had to be prepared after each voltammetric experiment. 2.4. Square wave adsorptive stripping voltammetry-SWAdSV Initial SWAdSV experiments were carried out with 6.0 mol L−1 P4. After the application of an accumulation step, square wave voltammograms were obtained from −1.4 to −1.8 V with the BiFE immersed in 0.1 mol L−1 Britton–Robinson buffer at pH 12.0 in the presence of P4. Thus, the dependence of the SWAdSV response on the parameters scan increment (Es), amplitude (a) and frequency (f) was analyzed in order to optimize their values for P4 determination. The ranges studied were: Es = 1–5 mV, a = 1–100 mV and f = 20–90 Hz. The optimized values, that is, Es = 2 mV, a = 50 mV and f = 70 Hz, were selected because they provided the best compromise between the sensitivity and the voltammogram profile. SWAdSV was therefore employed for the construction of the calibration curve and the determination of P4 in the four samples of pharmaceutical formulations. 2.5. Comparative method Based on the Brazilian Pharmacopoeia [46], ultraviolet absorption spectrophotometry (UV) was used as the comparative technique, aiming to evaluate the accuracy of the results provided by the BiFE for the detection of P4 in pharmaceuticals. The spectra were obtained on a Varian 50 Bio Ultraviolet Absorption Spectrophotometer at 240 nm, using a quartz cell with optical path length of 1 cm. Solutions and samples used in the comparative method had concentrations in the same range as those employed in the case of the BiFE.
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Fig. 3. Cyclic voltammograms for 40 mol L−1 P4 in 0.1 mol L−1 Britton–Robinson buffer (pH 12.0) obtained with the BiFE as a function of the potential scan rate (a–f = 50–300 mV s−1 ).
electrode under similar conditions [36]. The separation between the anodic and cathodic peaks is 210 mV, indicating the quasireversible nature of the electrochemical reaction. At pH 8.0 (Fig. 2A-b) a single reduction peak was observed at −1.48 V. No peaks were observed in solutions with pH lower than 8.0. The reduction peak potential (Epr ) shifted linearly to more negative potentials as the solution pH increased from 8.0 to 11.0, remaining almost constant at higher pH values (Fig. 2B). The slope for the linear portion of the Epr × pH plot was −66.7 mV dec−1 , which is consistent with the exchange of one electron and one proton, as expected for progesterone reduction [38]. The influence of the solution pH on the cathodic current (ipc ) is shown in Fig. 2C. It can be noted that at pH values below 12.0 the P4 reduction leads to a gradual decrease in the intensity of the current. The best combination of voltammetric profile and sensitivity was obtained at pH 12.0, which was chosen as the optimum pH for subsequent tests. 3.2. Electrochemical behavior of progesterone at the BiFE surface in Britton–Robinson buffer (pH 12.0)
Fig. 2. (A) Cyclic voltammograms, (B) reduction potential and (C) cathodic current as a function of solution pH for 40 mol L−1 P4 in 0.1 mol L−1 Britton–Robinson buffer obtained using a BiFE, v = 200 mV s−1 .
3. Results and discussion 3.1. Selection of the supporting electrolyte and solution pH The electrochemical reduction of 40 mol L−1 P4 at the BiFE was evaluated by cyclic voltammetry in Britton–Robinson buffer, Ringer’s solution, NaOH and LiOH, all at a concentration of 0.1 mol L−1 and pH 12.0. The best compromise between the detection potential, the peak current and the cyclic voltammogram profile was achieved using Britton–Robinson buffer as the supporting electrolyte, which was chosen for further experiments. In the cyclic voltammetric experiments, the potential was swept from −1.0 to −1.8 V and back to −1.0 V. As can be seen in Fig. 2A-a, at pH 12.0 the cyclic voltammogram of P4 exhibits a well-defined reduction peak at −1.68 V and a poorly defined oxidation peak at −1.47 V, in good agreement with results obtained at a mercury
The cyclic voltammograms shown in Fig. 3 depict the electrochemical behavior of 40 mol L−1 P4 in Britton–Robinson buffer solution (pH 12.0) at the BiFE surface as a function of the potential scan rate, considered over the range of 50–300 mV s−1 . As can be clearly observed, the Epr -value shifts to more negative potentials with an increase in the scan rate. The dependence of the Epr with log v is linear according to the equation Epr /V = −1.438 − 0.101 log v. From the slope of the plot, a value of ˛n = 0.585 was calculated, which provides n = 1.17 for ˛ = 0.5, where n is the stoichiometric number of the electrons involved in the electrode reaction and ˛ is the transfer coefficient. On the other hand, although the oxidation peak is perceptible at low scan rates it is best defined at 300 mV s−1 . According to the literature [38], even a quasireversible mechanism can be detected at very fast scan rates. Furthermore, the interdependence between the current function (−ipc v−1/2 ) and v indicates that a chemical reaction is coupled to the electrode process, i.e., the reaction follows an EC mechanism. Finally, the slope of the plot log ipc × log v was 0.9, that is, very close to 1.0, verifying the adsorption of P4 onto the BiFE. De Boer et al. [47] have studied the electrochemical properties of corticosteroids, including progesterone, in different supporting electrolytes and Arévalo et al. [38] investigated the electrochemical reduction of P4 in acetonitrile. In both cases, a dimer was proposed
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as the final product of the P4 reduction, although the media used in the studies were different, as well as the reduction patterns. To explain our results, the deduction proposed by De Boer et al. seems to be most appropriate. According to these authors [47 and references therein], in alkaline medium the dimerization takes place initially via a one-electron reaction with the reduction of the C-3 keto group on the A-ring of the P4 molecule and the formation of an unprotonated radical. The radical undergoes protonation and reacts with another unprotonated radical, resulting in a dimer. The overall reaction can be described as follows:
3.3. Adsorption of P4 at the BiFE surface As commented earlier, the plot of log ipc × log v with a slope of close to 1.0 indicates the adsorption of P4 onto the BiFE surface. This behavior was also identified by the successive cyclic voltammograms recorded at 200 mV s−1 for the reduction of 40 mol L−1
Fig. 4. (A) Cyclic and (B) square wave voltammograms for 40 mol L−1 P4 in 0.1 mol L−1 Britton–Robinson buffer (pH 12.0) obtained with the BiFE. (A-a) first cycle, (A-b) tenth cycle, (A) v = 200 mV s−1 , (B-a) without accumulation step, (B-b) Eacc = −1.0 V, tacc = 60 s, (B) Es = 4 mV, a = 60 mV, f = 50 Hz.
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P4 in 0.1 mol L−1 Britton–Robinson buffer at pH 12.0 (Fig. 4A). The first cycle (Fig. 4A-a) was compared to the tenth cycle (Fig. 4A-b) and it was apparent that the cathodic current sharply decreased. This means that the surface of the electrode was blocked by the adsorption of P4, preventing new molecules from reaching the surface. Based on this behavior, an accumulation step was performed before carrying out the voltammetric experiments. In this way, the variables accumulation potential – Eacc and time – tacc were optimized. The Eacc -values were varied in the range of −0.9 V to −1.4 V at constant tacc = 60 s, while the tacc -values ranged from 40 to 120 s at Eacc = −1.0 V. The ipc for P4 reduction increased for Eacc values of up to −1.0 V and then decreased. The ipc value increased with the tacc , indicating an increasing accumulation of P4 at the electrode surface over time. However, for electroanalytical purposes, a reasonably short time of 60 s was chosen. Therefore, to proceed with the studies, Eacc and tacc values of −1.0 V and 60 s, respectively, were selected. In order to verify that this procedure is appropriate for the electroanalytical determination of P4, the accumulation step was tested for square wave experiments. As can be seen in Fig. 4B, a more defined peak and a significant increase in the current for the reduction of P4 were obtained with the application of the accumulation step. Thus, square wave adsorptive stripping voltammetry (SWAdSV) was applied in further experiments. 3.4. Calibration curve for P4 obtained using BiFE-SWAdSV Fig. 5 shows the square wave voltammograms obtained under optimized conditions for P4 at the BiFE surface after successive additions of a standard solution of the sex hormone. A well-defined and sharp reduction peak is observed at −1.63 V, with the current increasing proportionally to the P4 concentration. The calibration curve displayed good linearity in the range of 0.40–7.90 mol L−1 P4 (R2 = 0.9983). The equation for the straight line can be expressed as i/mA = −0.0049 + 0.5691 [progesterone]/mol L−1 . The limits of detection (LD) and quantification (LQ) were calculated according to the equations: LD = 3Sb /B and LQ = 10Sb /B, where Sb is the standard deviation of the y-coordinate obtained from the line of best fit (linear coefficient) and B is the slope (angular coefficient) of this line. The LD calculated for P4 was 0.18 mol L−1 and the LQ was 0.61 mol L−1 . The repeatability of the signals produced by BiFE was verified using a 6.0 mol L−1 P4 solution under optimized conditions. The repeatability of the current peak was tested using two methodologies: intra-day, using ten different solutions prepared on the same day at a concentration of 6.0 mol L−1 , and inter-day,
Fig. 5. Square wave voltammograms for P4 in 0.1 mol L−1 Britton–Robinson buffer (pH 12.0) obtained with a BiFE under optimized conditions: (a) blank, (b) 0.4 mol L−1 , (c) 0.7 mol L−1 , (d) 1.0 mol L−1 , (e) 2.0 mol L−1 , (f) 3.0 mol L−1 , (g) 4.0 mol L−1 , (h) 5.0 mol L−1 , (i) 5.9 mol L−1 , (j) 6.9 mol L−1 , and (k) 7.9 mol L−1 .
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Table 1 Study of potential interferents in the determination of P4 in pharmaceutical products. Sample
Excipient
P4: interferent ratio (1:0) ipc (mA cm−2 )a
(1:0.01)
(1:0.1)
(1:1)
(1:10)
A
Parabens Phenoxyethanol Hydroxyethylcellulose
3.35
3.36
3.37
3.32
3.27
B
Cetostearyl alcohol mineral oil Lanolin alcohol Parabens Phenoxyethanol propylene glycol
3.31
3.33
3.35
3.34
3.32
C
Ethanol
3.33
3.33
3.35
3.34
3.36
D
Soya lecithin Peanut oil
3.34 3.32
3.34 3.33
3.36 3.33
3.34 3.33
2.10 3.32
a
n = 3.
over ten days using different solutions prepared at a concentration of 6.0 mol L−1 P4. The relative standard deviations (n = 10) were 1.7% and 1.5%, respectively. These results demonstrate the suitability of the BiFE for the determination of P4 by SWAdSV. 3.5. Study of potential interferents in the determination of P4 in pharmaceuticals The presence of excipients in pharmaceuticals can interfere with electroanalytical measurements. Therefore, a study on the possible interferents in the samples used for the progesterone determination was carried out. The excipients present in samples A–D analyzed in this study are shown in Table 1. The possible interferents were tested in the presence of 6.0 mol L−1 P4 in 0.1 mol L−1 Britton–Robinson buffer (pH 12.0) with the BiFE under optimized conditions by SWAdSV. The P4: interferent ratios studied were 1:0; 1:0.01; 1:0.1; 1:1 and 1:10. The results for the current density obtained for the P4 reduction in the absence and presence of interferents are also shown in Table 1. As can be noted from the results, the current for the P4 reduction remained almost constant, except in the presence of soya lecithin (sample D) for the ratio of 1:10 (P4: soya lecithin). The soya lecithin exhibits a reduction peak at −1.39 V, interfering by decreasing the current for the P4 reduction by around 37%. This means that P4 determination using BiFE-SWAdSV cannot be performed in the presence of soya lecithin when its concentration is ten times higher than that of P4. On the other hand, as can be observed, no other excipient contained in the samples interfered in the P4 determination.
with the slope of the calibration curve. The slopes of the standard addition plots were 0.639 mA L mol−1 cm−2 (R2 = 0.9921), 0.534 mA L mol−1 cm−2 (R2 = 0.9961), 0.499 mA L mol−1 cm−2 (R2 = 0.9900) and 0.634 mA L mol−1 cm−2 (R2 = 0.9855) for samples A, B, C and D, respectively. These values are very close to that of the calibration curve: 0.569 mA L mol−1 cm−2 (R2 = 0.9983). Thus, it can be concluded that there is no interference from the matrix components in the P4 determination. This behavior is in agreement with the results obtained in the study on potential interferents described in the preceding section.
3.6. Determination of P4 in pharmaceuticals using BiFE-SWAdSV and UV techniques The electroanalytical methodology described above using BiFESWAdSV and the comparative method were applied in the quantitative determination of P4 in four pharmaceutical formulations. The electroanalytical determination of P4 and recovery studies were carried out by adding standard solutions of the hormone to the four samples (A, B, C and D). The solutions prepared from the samples were diluted to a concentration included in the range used to obtain the calibration curve. Fig. 6A shows the square wave voltammograms obtained in the determination of P4 in sample A, and Fig. 6B depicts the resulting standard addition plot together with the calibration curve for the purposes of comparison. For all samples analyzed the voltammograms showed well-defined reduction peaks and the standard addition plots were linear. The selectivity, i.e., absence of interferences, was tested by comparing the slopes of the standard addition plots
Fig. 6. (A) Square wave voltammograms for: (a) blank, (b) 75 L of sample A, (c)–(e) 25 L and (f) 50 L of 0.5 mmol L−1 P4. (B) The standard addition plot for sample A and calibration curve.
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Table 2 Determination of P4 in commercial pharmaceutical products by BiFE-SWAdSV and UV techniques. Sample
A
Method
UV
BiFE
UV
BiFE
UV
BiFE
UV
BiFE
Label values (mg) Found values (mg)a RSD (%) Er1 (%)b Er2 (%)c tvalue d Fvalue e
450 446 1.03 −0.89 – −1.51 1.45
450 447 1.23 −0.67 0.22 −0.94 1.45
750 757 0.75 0.93 – 2.13 2.50
750 752 1.15 0.27 −0.66 0.38 2.50
1000 1023 1.37 2.30 – 2.84 3.23
1000 1033 2.44 3.30 0.97 2.27 3.23
100 102 1.50 2.00 – 2.13 1.00
100 101 1.51 1.00 −0.97 1.13 1.00
a b c d e
B
C
D
n = 3. Er1 = relative error between BiFE-SWAdSV or UV techniques and label values. Er2 = relative error between BiFE-SWAdSV and UV techniques. ttheoretical = 4.30. Ftheoretical = 19.
Table 2 shows the results obtained in the determination of P4 in four samples independently analyzed by BiFE-SWAdSV and UV techniques as well as the statistical data. Firstly, it can be observed that the values obtained using the two techniques are very close to the label values for the pharmaceutical products. The relative standard deviation (RSD) for the mean of three determinations of P4 in the four samples was between 1.15% and 2.44% for the measurements with BiFE-SWAdSV and between 0.75% and 1.50% using the UV technique. The relative errors in relation to the label values for BiFE-SWAdSV and the UV technique were <3.30% and <2.30%, respectively, while the relative difference between the values obtained with the two techniques was <1.0%. To examine whether the data provided by a certain technique is precise, accurate or superior to another technique, we can compare the results obtained by the proposed technique with those available in other data sets. Two of the most widely used tests for the comparison of results are the t- and F-tests. The t-test was used to compare the results obtained by the two techniques with the label values (accepted as the true values). At the 95% confidence level all values calculated for tvalue were lower than those for ttheoretical (4.30), indicating no significant differences between the data obtained using the two techniques and the true (label) values. The discrepancy between the data provided by BiFE-SWAdSV and UV techniques was verified by applying the F-test. The values for Fvalue obtained in the determination of P4 in pharmaceutical samples were lower than those for Ftheoretical (19) at the 95% confidence level, indicating that there is no significant difference in the precision provided by the two techniques. 3.7. Recovery experiments Recovery essays were also carried out to investigate the accuracy of the data furnished by BiFE-SWAdSV in the determination of P4 in pharmaceutical formulations. Four additions of P4 standard solutions yielded recoveries between 104% and 115% for sample A and 90% and 110% for sample D. For the samples B and C, five additions of P4 standard solutions yielded recoveries between 94% and 101% for sample B and 97% and 115% for sample C. These data verify that the matrix components do not interfere in the determination of P4. Moreover, these results corroborate the accuracy of the data obtained with BiFE-SWAdSV. 4. Conclusions The data presented allow concluding that the BiFE associated with cyclic voltammetry can provide reliable information on the electrochemical behavior of the sex hormone progesterone. In addition, when associated with square wave voltammetry, the BiFE also exhibited the capacity to furnish data comparable to those
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