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Electrochemistry of raloxifene on glassy carbon electrode and its determination in pharmaceutical formulations and human plasma
Bioelectrochemistry 88 (2012) 164–170
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Electrochemistry of raloxifene on glassy carbon electrode and its determination in pharmaceutical formulations and human plasma Akbar Bagheri ⁎, Hadi Hosseini Department of Chemistry, Faculty of Science, Shahid Beheshti University, Evin, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 6 August 2011 Received in revised form 22 March 2012 Accepted 26 March 2012 Available online 13 April 2012 Keywords: Raloxifene Voltammetry Glassy carbon electrode Pharmaceutical formulations Plasma samples
a b s t r a c t The electrochemical behavior of raloxifene (RLX) on the surface of a glassy carbon electrode (GCE) has been studied by cyclic voltammetry (CV). The CV studies were performed in various supporting electrolytes, wide range of potential scan rates, and pHs. The results showed an adsorption-controlled and quasi-reversible process for the electrochemical reaction of RLX, and a probable redox mechanism was suggested. Under the optimum conditions, differential pulse voltammetry (DPV) was applied for quantitative determination of the RLX in pharmaceutical formulations. The DPV measurements showed that the anodic peak current of the RLX was linear to its concentration in the range of 0.2–50.0 μM with a detection limit of 0.0750 μM, relative standard deviation (RSD %) below 3.0%, and a good sensitivity. The proposed method was successfully applied for determination of the RLX in pharmaceutical and human plasma samples with a good selectivity and suitable recovery. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Raloxifene (RLX) is a selective estrogen receptor modulator that belongs to the benzothiophene class of compounds [1]. The chemical designation is methanone [6-hydroxy-2-(4-hydroxyphenyl) benzo[b] thien-3-yl]-[4-[2-(1-piperidinyl) ethoxy] phenyl] hydrochloride. The RLX is currently used for the prevention of osteoporosis and also for risk reduction of breast cancer in postmenopausal women [2–5]. It is known that RLX is adsorbed rapidly after oral administration and has an absolute bioavailability of about 2.0%. The drug has a halflife of about 28.0 h and is eliminated primarily in the feces after hepatic glucuronidation [6]. Despite the numerous studies, mechanistic details of the RLX function in various tissues have not been understood, yet. The most common side effects of RLX include deep-vein thrombosis, hot flashes, and pulmonary embolism and leg cramps [6]. Drug analysis, an important branch of analytical chemistry, has an important role in drug quality control. Therefore, the development of a sensitive, simple, rapid, accurate, and reliable method for determination of active ingredient is of great importance and interest. Only few methods have been used for determination of RLX in pharmaceutical and biological systems, such as high-performance liquid chromatography (HPLC) coupled with various detectors [7–13], capillary electrophoresis [14], spectrophotometry [15], and Rayleigh scattering [16]. Among the HPLC methods, the HPLC coupled with mass detectors [7–9] have lower detection limits (b6.0 μg/L) compared to other HPLC methods
⁎ Corresponding author. Tel.: + 98 21 29903251; fax: + 98 21 22431661. E-mail address: [email protected] (A. Bagheri). 1567-5394/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2012.03.007
(>8.0 μg/L). Although HPLC has a good sensitivity for determination of RLX, the method has many disadvantages such as expensive equipments, time consuming and complex sample preparations. Electrochemical methods have proven to be very sensitive for quantitative determination of organic molecules, including drugs and related molecules in pharmaceutical formulations and biological fluids [17–29]. The advances in experimental electrochemical techniques in the field of drug analysis are due to simplicity, low-cost and relatively short analysis time compared to other techniques. Only one paper based on the use of electroanalytical methods is reported for determination of RLX [30]. In this paper, the multi-walled carbon nanotube modified glassy carbon electrode was used as a sensor for determination of RLX. The aim of the present work is to carry out a detailed study on the electrochemical behavior and the redox mechanism of RLX at the surface of a glassy carbon electrode (GCE). Moreover, a sensitive, rapid, simple, and accurate voltammetric method for direct determination of RLX in human plasma samples and pharmaceutical formulations using differential-pulse voltammetric (DPV) technique has been established, and the results were compared to developed methods that have been reported in the literatures [7–16]. 2. Experimental 2.1. Chemicals and reagents RLX (>99.0% purity) was purchased from Abureihan pharmaceutical company (Tehran, Iran). Due to low solubility of RLX in water, a stock solution of RLX (0.1 mM) was made in methanol, and more dilute solutions (1.0 × 10 − 3–1.0 × 10 − 8 M) were prepared by diluting
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with buffer solutions. Ascorbic acid and uric acid were purchased from Merck. The RLX tablets (60.0 mg) were purchased from a local pharmacy. Phosphate buffers from pH 2.0 to 10.0 (0.1 M) which were used for pH experiments and supporting electrolyte were prepared in double distilled water. Fresh human plasma samples were supplied by a local hospital (Taleghani hospital, Tehran, Iran). All other reagents used were of analytical grade, and all solutions were prepared with double distilled water.
centrifuged at 5000.0 rpm for 10.0 min. An amount of 2.0 mL of the supernatant was transferred to the voltammetric cell and diluted 10.0 times with 0.1 M phosphate buffer at pH 3.0 then the solution was analyzed according to the procedure for voltammetric analysis.
2.2. Apparatus
The effective surface area of the glassy carbon electrode was calculated by cyclic voltammetry method using 1.0 mM K3Fe (CN)6 as a probe at a different scan rate [19,31]. The peak-to-peak separation (ΔEp) was about 61.0 mV in the scan rate ranging from 10.0 to 100.0 mV/s which it was close to the expected value (59.0 mV) for a reversible one-electron process. For a reversible process, the following Randles–Sevcik formula can be used:
Voltammetric experiments were performed using a μAutolab Type III electrochemical system. A conventional three-electrode cell consisting of a glassy carbon working electrode (GCE) , a platinum wire counter electrode, and a saturated Ag/AgCl reference electrode was used for voltammetric experiments. A digital pH-meter (Ion Analyzer 827, Metrohm) with precision of ±0.001 was used for pH measurements. All experiments were carried out at room temperature (25.0 ± 1.0 °C). 2.3. Analytical procedure Before each experiment and transferring the GCE to the voltammetric cell, it was cleaned by polishing with 0.05 μm alumina slurry on a polishing cloth and rinsed thoroughly with double distilled water. After each polishing step, to get reproducible current-potential curves, cyclic voltammetry was performed at the scan rate of 100.0 mV/s between 0.0 and 1.6 V for 10.0 times in 0.1 M H2SO4 solution [19]. For cyclic voltammetry studies, 2.0 ml of 10.0 mM RLX stock solution was diluted with 18.0 mL of a 0.1 M buffer solution in voltammetric cell for obtaining 1.0 mM RLX solution, and then cyclic voltammograms were recorded at 100.0 mV/s scan rate. For determination of concentration ranges and preparation of the calibration curve, each RLX concentration was transferred into the voltammetric cell and deoxygenated by purging with pure nitrogen for 5.0 min then the DPV voltammograms were recorded. Operating conditions for DPV were: pulse amplitude, 50.0 mV; pulse width, 30.0 ms; scan rate, 30.0 mV/s. 2.4. Sample preparation 2.4.1. Procedure for pharmaceutical preparations Five tablets of RLX were weighed accurately and finely powdered and mixed. A quantity of the powder equivalent to the average weight of one tablet was transferred into a 100.0 mL calibrated flask and 50.0 mL of methanol was added. The content of the flask was sonicated for 15.0 min and then filled up to the volume with methanol. The solution was centrifuged for 10.0 min at 5000.0 rpm. Appropriate volumes of the clear supernatant were transferred into the volumetric cell and then diluted with 0.1 M phosphate buffer solution at pH 3.0 for proper concentrations. The solution was analyzed according to the procedure for voltammetric analysis and the quantity of RLX per tablet was determined using the calibration curve method. To study the accuracy and reproducibility of the proposed method, and to check the interference of expedients used in the pharmaceutical formulations, recovery experiments were carried out using the standard addition method by adding the known amounts of RLX to the pre-analyzed tablet formulations of RLX, and then the mixtures were analyzed by the proposed method. 2.4.2. Procedure for spiked human plasma preparations For analysis of plasma samples and recovery studies, a plasma sample (3.0 mL) was spiked with RLX at the desired concentration levels and then, was acidified with 100.0 μL hydrochloric acid (37.0%) to disturb the RLX protein binding. Then, 150.0 μL trichloroacetic acid (TCA) was added to denature the proteins. These processes eventually led to precipitation of proteins. Subsequently, the samples were
3. Results and discussion 3.1. Determination of surface area
1=2 3 3=2 1=2 1=2 Ip ¼ 0:4463 F =RT An D 0 C0 ν
ð1Þ
where Ip (A) refers to the peak current, F is Faraday's constant (96,485 C/mol), R is the universal gas constant (8.314 J/mol K), T is the absolute temperature (298.0 K), A (cm 2) is the surface area of the electrode, n is the number of electron transfer number, D0 (cm 2/s) is diffusion coefficient, C0 (mol/cm 3) is the concentration of K3Fe (CN)6, and υ (V/s) is the scan rate. For 1.0 mM K3Fe (CN)6 in the 0.1 M KCl electrolyte: n = 1.0 and D0 = 7.6 × 10− 6 cm2/s [19,31], then from the slope of Ip vs. υ1/2 plot, the effective surface area of the electrode can be calculated. In this experiment, the slope was 15.4 × 10− 6A/(V/s) 1/2 and the effective surface area of the glassy carbon electrode was found to be 0.0207 cm2. 3.2. Electrochemistry of RLX 3.2.1. Cyclic voltammetry In order to understand the electrochemical process occurring at the GCE, cyclic voltammetry was used. Fig. 1A shows the cyclic voltammetric response of RLX at GCE over the range of + 0.2 to +1.2 V at 100.0 mV/s scan rate and pH of 3.0. As shown in Fig. 1A, RLX gives a pair of redox peaks (p1, p2) with a well-defined oxidation peak at + 0.827 V and a weak reduction peak at + 0.720 V. The peakto-peak separation (ΔEp) was found to be 0.107 V, and the ratio of redox peak currents (Ipc/Ipa) was 0.13. The results indicated that the electrode response of RLX is a typical quasi-reversible electrode reaction. To further study the redox process, The number of electrons involved in the redox reaction was determined by electrolysis of a 0.1 mM RLX solution using controlled potential coulometry at +0.837 V (oxidation peak potential of RLX). The electrolysis progress was monitored using cyclic voltammetry and the total charge consumed by the electrolytic reaction (Qt) was estimated from Ipa vs. Q plot [32–36]. Then, the coulometric n was calculated using the equation, Qt = nFN, where F is Faraday's constant and N is the number of moles of the substrate. In our experiment, the number of the electrons involved in the redox process, n, was found to be 2.0. 3.2.2. Effect of supporting electrolyte The effect of supporting electrolytes on current–potential curves (peak height and the peak shape) was tested by a 0.1 M solution of various supporting electrolytes such as phosphate buffer, acetate buffer, sodium perchlorate–perchloric acid, and potassium chlorate– hydrochloric acid. The results showed that the phosphate buffer can give the best background and signal response. 3.2.3. Effect of pH The pH is an important factor in the electrochemical behavior of organic compounds because protons are always involved in the
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Fig. 1. (A): cyclic voltammogram of 1.0 mM RLX in 0.1 M phosphate buffer solution (pH 3.0) at GCE in the potential range from + 0.2 to + 1.2 V at a scan rate of 100.0 mV/s. (B): cyclic voltammograms of 1.0 mM RLX in 0.1 M phosphate buffer in the potential range from 0.0 to + 1.0 V at a scan rate of 100 mV/s under different pHs: (a) 2.0, (b) 3.0, (c) 4.0, (d) 6.0, (e) 8.0, and (f) 10.0.
electrochemical reactions and exert a significant impact on the reaction speed. Hence, for study of pH effect of phosphate buffer solution on voltammetric response of RLX, the cyclic voltammograms of 1.0 mM RLX were recorded from pH 2.0 to 10.0 at a scan rate of 100 mV/s (Fig. 1B). As Fig. 1B shows, the voltammetric response of RLX was strongly pH dependent. The oxidation peak current was suddenly increased from pH 2.0 to 3.0 and conversely decreased when the pH was gradually increased from pH 3.0 to 10.0 (Fig. 2A). According to the literature [37], although RLX is zwitterionic in chemical character and carries an ionic charge at all pH values, it is increasingly insoluble and relatively hydrophobic as pH increases to about 9.0. Moreover, the rate of RLX hydrolysis increases with an increase in pH. Therefore, the mentioned factors may be the reasons for decrease in current response of RLX along with an increase in electrolyte pH. Therefore, due to the high solubility and the high-current response of RLX, the pH of 3.0 was chosen for the subsequent analytical experiments. The relationship between the peak potentials and pH is shown in the Fig. 2B. As it can be seen, with increase in pH of the solution, both anodic and cathodic peak potentials linearly shift to the less positive values. The results indicate that the protons are participated in the oxidation of RLX: þ
−
ðRLXÞRed ⇌ðRLXÞOx þ mH þ ne
where m is the number of protons involved in the reaction. The Nernstian equation is given below: Ep ¼ EpðpH¼0:0Þ −ð2:303mRT=nFÞpH
ð2Þ
where Ep (pH = 0.0) is the peak potential at pH 0.0, R, T, and F have their usual meanings. The Ep–pH diagrams (Fig. 2B) are showing
that the dependence of Ep on pH on both the anodic and cathodic peaks can be expressed by the following relations: 2
Epa =V ¼ 1:029−0:06pH; R ¼ 0:984 2
Epc =V ¼ 0:907−0:058pH; R ¼ 0:998: The slope of Ep vs. pH corresponds to 60.0 mV/pH and 58.0 mV/pH for the anodic peak (p1) and cathodic peak (p2), respectively. According to Eq. (2), the results revealed that the number of protons in the process is equal to the number of the transferred electrons [38]. Hence, two electrons and two protons are involved in a redox process. Furthermore, RLX has three pKa values as 8.95, 9.83, and 10.91, which correspond to the nitrogen and hydroxyl groups, respectively [13,37]. In the pH ranges from 2.0 to 10.0, deviation from linearity is not observed in Ep-pH diagram (Fig. 2B) even at 8.95 (pKa value for nitrogen). These results suggested that the oxidation peak may be corresponding to the oxidation of hydroxyl groups in the RLX molecule. A proposed redox mechanism of RLX is described in Scheme 1. 3.2.4. Effect of scan rate In the electrochemical investigations, useful information involving the electrochemical reaction mechanisms usually can be found from the potential scan rate. Therefore, the electrochemical behavior of RLX at scan rate ranges from 50.0 to 300.0 mV/s was also investigated at pH 3.0 by cyclic voltammetry in 1.0 mM solution of RLX (Fig. 3A). By increasing the scan rate, the Epa and Epc shift to more positive and more negative values, respectively, and the peak-to-peak separation also increases. These results illustrated that the electron transfer was quasi-reversible.
Fig. 2. (A): variation of the anodic peak current with pH. (B): plots of variation of peak potential with pH at a scan rate of 100.0 mV/s: (a) Epa vs. pH and (b) Epc vs. pH.
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constant ks (1/s) can be determined from the intercept of ΔEp vs. log ν plot (Eq. (4)). Under different scan rates of 100.0, 150.0, 200.0, and 250.0 mV/s, the potential shifts (ΔEp) were 0.107, 0.117, 0.126, and 0.133, respectively and the ks was calculated to be 0.282 1/s. An estimate of the surface coverage of the electrode was made approximately by adopting the method used by Sharp et al [41]. According to the method, the peak current is related to the surface concentration of electroactive species, Г, by the following equation: 2 2 Ipa ¼ αn F ГνA =2:718RT
Scheme 1. A proposed redox mechanism of RLX at glassy carbon electrode.
Moreover, when the scan rate was increased, a linear relationship between the peak current and the scan rate in the range of 50.0–300.0 mV/s was found for both anodic and cathodic peak currents (Fig. 3B), which suggests an adsorption behavior [39]. The equations related to peak current and the scan rate can be represented as Ipa (μA) = 43.74 ν (V/s) + 1.66, R2 = 0.997 and Ipc (μA)= −5.817 ν (V/s) − 0.222, R2 = 0.997. A good linearity between logarithm of the anodic peak current and logarithm of scan rate with the slope of 0.958 was also found (Fig. 3C), which it is close to the theoretical value of 1.0 for the adsorption-controlled electrode process [39]. The linear equation obtained was: log Ipa (μA) = 0.958 log ν (V/s) + 1.55, R 2 = 0.995. According to the Laviron's equation [40], for an adsorptioncontrolled and quasi-reversible interfacial reaction, the relationship between Epa and scan rate is defined by the following equations: Epa ¼ K þ ½2:33RT=ð1−α Þ F log ν
ð3Þ
log ks ¼ α logð1−α Þ þ ð1−α Þ log α−logðRT=F ν Þ−ð1−α Þ αFΔEp =2:3RT
ð5Þ
Where n represents the number of electrons involved in reaction, A is the effective surface area (0.0207 cm 2) of the GCE, α is the transfer coefficient (0.57), Г (mol/cm 2) is the surface coverage, and other symbols have their usual meanings. The surface concentration of electroactive species (Г) on GCE was calculated to be 4.7 × 10 − 10 mol/cm 2 from the slop of Ip vs. υ plot (Fig. 3B). 3.2.5. Accumulation time Because the electrochemical reaction of RLX is an adsorptiondriven process, accumulation can improve the pre-concentration of RLX on the electrode surface, and obviously improve sensitivity. Hence, the influence of accumulation time (tacc) on peak current was investigated by DPV in 0.1 M phosphate buffer (pH 3.0) containing 1.0 μM RLX. (Fig. 4). As it can be seen, the peak current was reached the maximum value after immersing GCE into solution after 70.0 s. Thus, the 70.0 s was chosen as the best accumulation time for the subsequent analytical experiments. 4. Method validation
ð4Þ 4.1. Calibration curve and detection limit where α is the transfer coefficient, ν (V/s) is the scan rate, and K is a constant value. From the slope of Epa vs. log ν plot (Fig. 3D), The value of α was found to be 0.57. In addition, the value of apparent rate
Differential-pulse voltammetry (DPV) is one of the most sensitive electrochemical detection methods. Therefore, DPV was applied for
Fig. 3. (A): cyclic voltammograms of 1.0 mM RLX in 0.1 M phosphate buffer solution (pH 3.0) in the potential ranges from + 0.2 to + 1.2 V at a different scan rates: (a → f) 50.0, 100.0, 150.0, 200.0, 250.0 and 300.0 mV/s. (B): plot of anodic (a) and cathodic (b) peak currents vs. potential scan rate. (C): plot of the logarithm of anodic peak current vs. logarithm of scan rate. (D): plot of the variation of anodic peak potential with logarithm of the scan rate.
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A. Bagheri, H. Hosseini / Bioelectrochemistry 88 (2012) 164–170 Table 1 Regression data of the calibration lines for determination of RLX by DPV. DPV Measured potential (V) Linearity range (μM) Number of point Slope (μA/μA) Intercept (μA) SE of slope SE of intercept Correlation coefficient LOD LOQ
Fig. 4. Effect of accumulation time (tacc) on DPV peak current of 1.0 μM RLX in 0.1 M phosphate buffer solution (pH 3.0).
determination of RLX. Under the optimized experimental conditions (phosphate buffer, pH 3.0; accumulation time, 70.0 s), the DPV curves were obtained using different concentrations of RLX (Fig. 5A). As shown in Fig. 5A, the height of the DPV peaks (Ipa) increases with increase in concentration of RLX. The calibration curve of the peak current vs. concentration was obtained using data from these measurements (inset Fig. 5A). According to the obtained results, linear calibration graphs were obtained for RLX with the linear dynamic range of 0.2–50 μM (0.096–23.9 ppm). The linear regression equations were Ipa (μA) = 0.782 + 0.198CRLX (μM), with a correlation coefficient of 0.998 and a sensitivity of 0.198 μA/μM. A limit of detection (LOD) of 0.075 μM (0.036 ppm) and limit of quantification (LOQ) of 0.25 μM (0.12 ppm) were calculated according to the 3 sb/m and 10 sb/m criterions, respectively, where m is the slope value of the calibration curve and sb is the calculated standard deviation for the peak currents of the blank (five runs). Regression and necessary validation data of the calibration plots are summarized in Table 1 according to the standard procedures [42–45]. The correlation coefficient greater than 0.99 and the low values of standard error of the slope and intercept confirmed the precision of the proposed methods.
0.7 0.2–50 12.0 0.198 0.782 0.0047 0.104 0.998 0.075 0.25
The repeatability and reproducibility of method were examined by RSD% and by the difference between theoretical and measured concentrations. Intra-day precision (repeatability) and accuracy of the proposed method were evaluated by assaying freshly prepared solutions at three different concentrations. Inter-day precision (reproducibility) and accuracy of the proposed method were evaluated by assaying freshly prepared solutions for 4 different days. The obtained results are shown in Table 2. The results reveal that there is no significant difference between the experiments which were carried out within-day and between days. Thus, the results confirmed the excellent reproducibility, repeatability, accuracy, and precision of the proposed method for determination of RLX. The detection limit obtained by proposed method is better than HPLC coupled with coulometric and UV detector [13] and it is close to some other developed methods [12,14]. Although the detection limits of HPLC coupled with mass detectors [7–9] are better compared to the proposed method, these methods need expensive equipments, are time-consuming, and require sample pretreatment, which make them unsatisfactory for routine or cost-effective screening. 4.2. Tablet analysis and recovery test The developed method was applied for determination of RLX in their dosages forms. Hence, the amount of RLX in tablets was determined by recording DPVs under the optimized experimental conditions. The results of proposed method were compared to the published methods [13,14], and are presented in Table 3. It was found that the obtained quantitative results were in agreement with the reported values. According to Table 3, the recovery tests were carried out by adding known amounts of pure drug to various pre-analyzed formulations. The mean percentage recovery showed no significant excipient interference which clearly demonstrates the
Fig. 5. (A): differential pulse voltammograms of RLX at GCE in 0.1 M phosphate buffer solution (pH 3.0) at 70 s accumulation time. RLX concentrations (from a to h): 0.7, 3.0, 10.0, 15.0, 20.0, 30.0, 40.0 and 50.0 μM. Inset: corresponding linear calibration curves of the anodic peak currents vs. RLX concentration. (B): differential-pulse voltammograms of the plasma sample spiked with different RLX concentration: (a) 0.3, (b) 1.5 and (c) 3.0 μM.
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Table 2 Intra- and inter-dye analysis of RLX (n = 4). Intra-day Inter-day RLX concentration Recovery% Accuracy Precision Recovery% Accuracy Precision (μM) RE % RSD % RE % RSD % 15 10 5
− 1.5 1.2 − 1.9
98.5 101.2 98.1
2.6 1.2 2.1
− 3.1 2.5 − 2.1
97.7 102.5 97.9
2.9 1.7 3.1
Table 3 Comparative studies for raloxifene in tablet by proposed and literature methods and mean recoveries in spiked tablet. HPLC with UV detector [13] Claimed dose (mg) Found (mg)a RSD% tvalue Bias% Recovery: added amount (mg) Recovery: found (mg) Recovery (%)c RSD% of recovery Bias% a b c
HPLC with Coulometric detector [13]
60.0 59.48 2.8
60.0 60.3 3.1
20.0
20.0
19.95 99.75 0.273
Capillary electrophoresis [14]
Present method
60.0 59.3 2.7
60.0 59.64 2.3 0.64b 0.6 20.0
20.06 100.3 0.297
19.97 99.85 0.253 0.15
Each value is the mean of six experiments. t-theoretical :2.57. Recovery value is the mean of six experiments.
accuracy and selectivity of the method. Thus, the procedure was able to determine RLX in the presence of excipients. 4.3. Interference studies Some inorganic ions and organic compounds may coexist with drugs in biological samples (such as plasma or urine) and tablet formulations, and these compounds can affect the voltammetric response of the drugs. Thus, in order to evaluate the specificity of the proposed method, the influence of potentially interfering substances such as Mg 2+, Ca 2+, Cu 2+, Zn 2+, Fe 3+, Na +, K +,SO42 −,Cl −, nitrate, glucose, fructose, sucrose, talc, citric acid, dopamine, ascorbic acid (AA), and uric acid (UA) on the determination of RLX were investigated by using DPV. Some results are presented in Table 4. Furthermore, Fig. 6 shows the voltammetric response of RLX in the presence of AA and UA which are the most important electroactive molecules which coexisting with RLX in a biological system. The results indicated that no interference was observed on the signals of RLX in the presence of interfering substances with concentration of 100.0 fold interfering substance to drug with a deviation below
Table 4 Influence of potential interferents on the voltammetric response of 10 μM raloxifene. Interferent 3+
Fe Cl− Glucose Fructose Talc Sucrose Ascorbic acid Uric acid
Concentration (mM)
Signal change (%)a
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
− 1.20 + 1.49 − 2.15 + 1.33 + 1.71 − 1.97 − 2.32 − 1.5
a The difference between the peak currents in the absence and presence of interferents.
Fig. 6. Differential pulse voltammograms of 10.0 μm RLX at GCE in the absence (…..) and the presence (—) of 1.0 mM AA and 1.0 mM UA in 0.1 M phosphate buffer solution (pH 3.0).
5.0%. It was observed that the method can be safely applied for determination of RLX in biological samples. 4.4. Determination of RLX in plasma samples The determination of RLX in plasma was also a challenging task because of its very low concentration; on average, its maximal plasma concentration (cmax) reaches only 1.36 (μg/L)/(mg/kg) or 1.36 ppm [8,46]. In order to evaluate a voltammetric method, the proposed method was applied to determine RLX in plasma samples, duo to low LOD (0.036 ppm) and LOQ (0.12 ppm). According to Section 2.4.2, the recovery studies were carried out by spiking pure drug free plasma with known amounts of RLX (standard addition method), and the calibration plot was used for determination of spiked RLX in human plasma. DPVs for determination of RLX in human plasma are shown in Fig. 5B. The peak currents of DPVs were linearly increased by adding higher concentrations of RLX to plasma samples. The results for detection of RLX in three samples are presented in Table 5. The recoveries were determined in the range of 98.4%–101.9% with RSD (%) below 3.0%. The results illustrated that the proposed method is accurate enough for practical applications. 5. Conclusion The electrochemical behavior of RLX at GCE surface was studied by CV. Obtained results from the CV measurements suggested that electrode reaction process is quasi-reversible and adsorption-controlled and two electrons and two protons are involved in the process and consequently, a probable redox mechanism was proposed. The proposed differential-pulse voltammetry has been validated, and it was successfully applied for quantitative determination of RLX in tablet and human plasma samples. The effects of potential interfering substances were studied, and it was found that the proposed method is free from interferences. Comparison of the proposed method with
Table 5 Determination of raloxifene in plasma samples. Plasma
Spiked (μM)
Found (μM)a
Recovery(%) + RSD%
Bias%
Sample 1.0 Sample 2.0 Sample 3.0
0.3 1.5 3.0
0.303 1.476 3.056
101 ± 1.56 98.4 ± 1.93 101.9 ± 2.37
− 1.0 + 1.6 − 1.87
a
Average of six determinations.
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HPLC [13], offers the advantages of simplicity, low cost, and short analysis time, and it can be preferred to HPLC method for the analysis of RLX. References [1] A. Smith, P.E. Heckelman, J.R. Obenchain, J.A.R. Gallipeau, M.A.D. Arecca, S. Budavari, The Merck Index, 13th Ed Whitehouse Station, 2001. [2] J.Y. Reginster, J.P. Devogelaer, Raloxifene reduces fractures in postmenopausal women with osteoporosis, Clin. Orthop. Relat. Res. 443 (2006) 48–54. [3] P.D. Delmas, K.E. Ensrud, J.D. Adachi, K.D. Harper, S. Sarkar, C. Gennari, J.Y. Reginster, H.A. Pols, R.R. Recker, S.T. Harris, W. Wu, H.K. Genant, D.M. Black, R. Eastell, Efficacy of raloxifene on vertebral fracture risk reduction in postmenopausal women with osteoporosis: four-year results from a randomized clinical trial, J. Clin. Endoc. Meta. 87 (2002) 3609–3617. [4] E. Barrett-Connor, L. Mosca, P. Collins, et al., Effects of raloxifene on cardiovascular events and breast cancer in postmenopausal women, N. Engl. J. 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Akbar Bagheri received his PhD in 1976 in Analytical Chemistry at Howard University (United States). Now, he is an associate professor in Chemistry Department of Shahid Beheshti University (Tehran, Iran). His research fields of interest include electrochemical sensors, novel electrode material and electroanalytical chemistry.
Hadi Hosseini received his MS degree in 2009 from Shahid Beheshti University (Tehran, Iran). Now, he is a PhD student in Chemistry Department of Shahid Beheshti University (Tehran, Iran). His research interests focus on electrode materials and electroanalytical chemistry.
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Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem
Electrochemistry of raloxifene on glassy carbon electrode and its determination in pharmaceutical formulations and human plasma Akbar Bagheri ⁎, Hadi Hosseini Department of Chemistry, Faculty of Science, Shahid Beheshti University, Evin, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 6 August 2011 Received in revised form 22 March 2012 Accepted 26 March 2012 Available online 13 April 2012 Keywords: Raloxifene Voltammetry Glassy carbon electrode Pharmaceutical formulations Plasma samples
a b s t r a c t The electrochemical behavior of raloxifene (RLX) on the surface of a glassy carbon electrode (GCE) has been studied by cyclic voltammetry (CV). The CV studies were performed in various supporting electrolytes, wide range of potential scan rates, and pHs. The results showed an adsorption-controlled and quasi-reversible process for the electrochemical reaction of RLX, and a probable redox mechanism was suggested. Under the optimum conditions, differential pulse voltammetry (DPV) was applied for quantitative determination of the RLX in pharmaceutical formulations. The DPV measurements showed that the anodic peak current of the RLX was linear to its concentration in the range of 0.2–50.0 μM with a detection limit of 0.0750 μM, relative standard deviation (RSD %) below 3.0%, and a good sensitivity. The proposed method was successfully applied for determination of the RLX in pharmaceutical and human plasma samples with a good selectivity and suitable recovery. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Raloxifene (RLX) is a selective estrogen receptor modulator that belongs to the benzothiophene class of compounds [1]. The chemical designation is methanone [6-hydroxy-2-(4-hydroxyphenyl) benzo[b] thien-3-yl]-[4-[2-(1-piperidinyl) ethoxy] phenyl] hydrochloride. The RLX is currently used for the prevention of osteoporosis and also for risk reduction of breast cancer in postmenopausal women [2–5]. It is known that RLX is adsorbed rapidly after oral administration and has an absolute bioavailability of about 2.0%. The drug has a halflife of about 28.0 h and is eliminated primarily in the feces after hepatic glucuronidation [6]. Despite the numerous studies, mechanistic details of the RLX function in various tissues have not been understood, yet. The most common side effects of RLX include deep-vein thrombosis, hot flashes, and pulmonary embolism and leg cramps [6]. Drug analysis, an important branch of analytical chemistry, has an important role in drug quality control. Therefore, the development of a sensitive, simple, rapid, accurate, and reliable method for determination of active ingredient is of great importance and interest. Only few methods have been used for determination of RLX in pharmaceutical and biological systems, such as high-performance liquid chromatography (HPLC) coupled with various detectors [7–13], capillary electrophoresis [14], spectrophotometry [15], and Rayleigh scattering [16]. Among the HPLC methods, the HPLC coupled with mass detectors [7–9] have lower detection limits (b6.0 μg/L) compared to other HPLC methods
⁎ Corresponding author. Tel.: + 98 21 29903251; fax: + 98 21 22431661. E-mail address: [email protected] (A. Bagheri). 1567-5394/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2012.03.007
(>8.0 μg/L). Although HPLC has a good sensitivity for determination of RLX, the method has many disadvantages such as expensive equipments, time consuming and complex sample preparations. Electrochemical methods have proven to be very sensitive for quantitative determination of organic molecules, including drugs and related molecules in pharmaceutical formulations and biological fluids [17–29]. The advances in experimental electrochemical techniques in the field of drug analysis are due to simplicity, low-cost and relatively short analysis time compared to other techniques. Only one paper based on the use of electroanalytical methods is reported for determination of RLX [30]. In this paper, the multi-walled carbon nanotube modified glassy carbon electrode was used as a sensor for determination of RLX. The aim of the present work is to carry out a detailed study on the electrochemical behavior and the redox mechanism of RLX at the surface of a glassy carbon electrode (GCE). Moreover, a sensitive, rapid, simple, and accurate voltammetric method for direct determination of RLX in human plasma samples and pharmaceutical formulations using differential-pulse voltammetric (DPV) technique has been established, and the results were compared to developed methods that have been reported in the literatures [7–16]. 2. Experimental 2.1. Chemicals and reagents RLX (>99.0% purity) was purchased from Abureihan pharmaceutical company (Tehran, Iran). Due to low solubility of RLX in water, a stock solution of RLX (0.1 mM) was made in methanol, and more dilute solutions (1.0 × 10 − 3–1.0 × 10 − 8 M) were prepared by diluting
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with buffer solutions. Ascorbic acid and uric acid were purchased from Merck. The RLX tablets (60.0 mg) were purchased from a local pharmacy. Phosphate buffers from pH 2.0 to 10.0 (0.1 M) which were used for pH experiments and supporting electrolyte were prepared in double distilled water. Fresh human plasma samples were supplied by a local hospital (Taleghani hospital, Tehran, Iran). All other reagents used were of analytical grade, and all solutions were prepared with double distilled water.
centrifuged at 5000.0 rpm for 10.0 min. An amount of 2.0 mL of the supernatant was transferred to the voltammetric cell and diluted 10.0 times with 0.1 M phosphate buffer at pH 3.0 then the solution was analyzed according to the procedure for voltammetric analysis.
2.2. Apparatus
The effective surface area of the glassy carbon electrode was calculated by cyclic voltammetry method using 1.0 mM K3Fe (CN)6 as a probe at a different scan rate [19,31]. The peak-to-peak separation (ΔEp) was about 61.0 mV in the scan rate ranging from 10.0 to 100.0 mV/s which it was close to the expected value (59.0 mV) for a reversible one-electron process. For a reversible process, the following Randles–Sevcik formula can be used:
Voltammetric experiments were performed using a μAutolab Type III electrochemical system. A conventional three-electrode cell consisting of a glassy carbon working electrode (GCE) , a platinum wire counter electrode, and a saturated Ag/AgCl reference electrode was used for voltammetric experiments. A digital pH-meter (Ion Analyzer 827, Metrohm) with precision of ±0.001 was used for pH measurements. All experiments were carried out at room temperature (25.0 ± 1.0 °C). 2.3. Analytical procedure Before each experiment and transferring the GCE to the voltammetric cell, it was cleaned by polishing with 0.05 μm alumina slurry on a polishing cloth and rinsed thoroughly with double distilled water. After each polishing step, to get reproducible current-potential curves, cyclic voltammetry was performed at the scan rate of 100.0 mV/s between 0.0 and 1.6 V for 10.0 times in 0.1 M H2SO4 solution [19]. For cyclic voltammetry studies, 2.0 ml of 10.0 mM RLX stock solution was diluted with 18.0 mL of a 0.1 M buffer solution in voltammetric cell for obtaining 1.0 mM RLX solution, and then cyclic voltammograms were recorded at 100.0 mV/s scan rate. For determination of concentration ranges and preparation of the calibration curve, each RLX concentration was transferred into the voltammetric cell and deoxygenated by purging with pure nitrogen for 5.0 min then the DPV voltammograms were recorded. Operating conditions for DPV were: pulse amplitude, 50.0 mV; pulse width, 30.0 ms; scan rate, 30.0 mV/s. 2.4. Sample preparation 2.4.1. Procedure for pharmaceutical preparations Five tablets of RLX were weighed accurately and finely powdered and mixed. A quantity of the powder equivalent to the average weight of one tablet was transferred into a 100.0 mL calibrated flask and 50.0 mL of methanol was added. The content of the flask was sonicated for 15.0 min and then filled up to the volume with methanol. The solution was centrifuged for 10.0 min at 5000.0 rpm. Appropriate volumes of the clear supernatant were transferred into the volumetric cell and then diluted with 0.1 M phosphate buffer solution at pH 3.0 for proper concentrations. The solution was analyzed according to the procedure for voltammetric analysis and the quantity of RLX per tablet was determined using the calibration curve method. To study the accuracy and reproducibility of the proposed method, and to check the interference of expedients used in the pharmaceutical formulations, recovery experiments were carried out using the standard addition method by adding the known amounts of RLX to the pre-analyzed tablet formulations of RLX, and then the mixtures were analyzed by the proposed method. 2.4.2. Procedure for spiked human plasma preparations For analysis of plasma samples and recovery studies, a plasma sample (3.0 mL) was spiked with RLX at the desired concentration levels and then, was acidified with 100.0 μL hydrochloric acid (37.0%) to disturb the RLX protein binding. Then, 150.0 μL trichloroacetic acid (TCA) was added to denature the proteins. These processes eventually led to precipitation of proteins. Subsequently, the samples were
3. Results and discussion 3.1. Determination of surface area
1=2 3 3=2 1=2 1=2 Ip ¼ 0:4463 F =RT An D 0 C0 ν
ð1Þ
where Ip (A) refers to the peak current, F is Faraday's constant (96,485 C/mol), R is the universal gas constant (8.314 J/mol K), T is the absolute temperature (298.0 K), A (cm 2) is the surface area of the electrode, n is the number of electron transfer number, D0 (cm 2/s) is diffusion coefficient, C0 (mol/cm 3) is the concentration of K3Fe (CN)6, and υ (V/s) is the scan rate. For 1.0 mM K3Fe (CN)6 in the 0.1 M KCl electrolyte: n = 1.0 and D0 = 7.6 × 10− 6 cm2/s [19,31], then from the slope of Ip vs. υ1/2 plot, the effective surface area of the electrode can be calculated. In this experiment, the slope was 15.4 × 10− 6A/(V/s) 1/2 and the effective surface area of the glassy carbon electrode was found to be 0.0207 cm2. 3.2. Electrochemistry of RLX 3.2.1. Cyclic voltammetry In order to understand the electrochemical process occurring at the GCE, cyclic voltammetry was used. Fig. 1A shows the cyclic voltammetric response of RLX at GCE over the range of + 0.2 to +1.2 V at 100.0 mV/s scan rate and pH of 3.0. As shown in Fig. 1A, RLX gives a pair of redox peaks (p1, p2) with a well-defined oxidation peak at + 0.827 V and a weak reduction peak at + 0.720 V. The peakto-peak separation (ΔEp) was found to be 0.107 V, and the ratio of redox peak currents (Ipc/Ipa) was 0.13. The results indicated that the electrode response of RLX is a typical quasi-reversible electrode reaction. To further study the redox process, The number of electrons involved in the redox reaction was determined by electrolysis of a 0.1 mM RLX solution using controlled potential coulometry at +0.837 V (oxidation peak potential of RLX). The electrolysis progress was monitored using cyclic voltammetry and the total charge consumed by the electrolytic reaction (Qt) was estimated from Ipa vs. Q plot [32–36]. Then, the coulometric n was calculated using the equation, Qt = nFN, where F is Faraday's constant and N is the number of moles of the substrate. In our experiment, the number of the electrons involved in the redox process, n, was found to be 2.0. 3.2.2. Effect of supporting electrolyte The effect of supporting electrolytes on current–potential curves (peak height and the peak shape) was tested by a 0.1 M solution of various supporting electrolytes such as phosphate buffer, acetate buffer, sodium perchlorate–perchloric acid, and potassium chlorate– hydrochloric acid. The results showed that the phosphate buffer can give the best background and signal response. 3.2.3. Effect of pH The pH is an important factor in the electrochemical behavior of organic compounds because protons are always involved in the
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Fig. 1. (A): cyclic voltammogram of 1.0 mM RLX in 0.1 M phosphate buffer solution (pH 3.0) at GCE in the potential range from + 0.2 to + 1.2 V at a scan rate of 100.0 mV/s. (B): cyclic voltammograms of 1.0 mM RLX in 0.1 M phosphate buffer in the potential range from 0.0 to + 1.0 V at a scan rate of 100 mV/s under different pHs: (a) 2.0, (b) 3.0, (c) 4.0, (d) 6.0, (e) 8.0, and (f) 10.0.
electrochemical reactions and exert a significant impact on the reaction speed. Hence, for study of pH effect of phosphate buffer solution on voltammetric response of RLX, the cyclic voltammograms of 1.0 mM RLX were recorded from pH 2.0 to 10.0 at a scan rate of 100 mV/s (Fig. 1B). As Fig. 1B shows, the voltammetric response of RLX was strongly pH dependent. The oxidation peak current was suddenly increased from pH 2.0 to 3.0 and conversely decreased when the pH was gradually increased from pH 3.0 to 10.0 (Fig. 2A). According to the literature [37], although RLX is zwitterionic in chemical character and carries an ionic charge at all pH values, it is increasingly insoluble and relatively hydrophobic as pH increases to about 9.0. Moreover, the rate of RLX hydrolysis increases with an increase in pH. Therefore, the mentioned factors may be the reasons for decrease in current response of RLX along with an increase in electrolyte pH. Therefore, due to the high solubility and the high-current response of RLX, the pH of 3.0 was chosen for the subsequent analytical experiments. The relationship between the peak potentials and pH is shown in the Fig. 2B. As it can be seen, with increase in pH of the solution, both anodic and cathodic peak potentials linearly shift to the less positive values. The results indicate that the protons are participated in the oxidation of RLX: þ
−
ðRLXÞRed ⇌ðRLXÞOx þ mH þ ne
where m is the number of protons involved in the reaction. The Nernstian equation is given below: Ep ¼ EpðpH¼0:0Þ −ð2:303mRT=nFÞpH
ð2Þ
where Ep (pH = 0.0) is the peak potential at pH 0.0, R, T, and F have their usual meanings. The Ep–pH diagrams (Fig. 2B) are showing
that the dependence of Ep on pH on both the anodic and cathodic peaks can be expressed by the following relations: 2
Epa =V ¼ 1:029−0:06pH; R ¼ 0:984 2
Epc =V ¼ 0:907−0:058pH; R ¼ 0:998: The slope of Ep vs. pH corresponds to 60.0 mV/pH and 58.0 mV/pH for the anodic peak (p1) and cathodic peak (p2), respectively. According to Eq. (2), the results revealed that the number of protons in the process is equal to the number of the transferred electrons [38]. Hence, two electrons and two protons are involved in a redox process. Furthermore, RLX has three pKa values as 8.95, 9.83, and 10.91, which correspond to the nitrogen and hydroxyl groups, respectively [13,37]. In the pH ranges from 2.0 to 10.0, deviation from linearity is not observed in Ep-pH diagram (Fig. 2B) even at 8.95 (pKa value for nitrogen). These results suggested that the oxidation peak may be corresponding to the oxidation of hydroxyl groups in the RLX molecule. A proposed redox mechanism of RLX is described in Scheme 1. 3.2.4. Effect of scan rate In the electrochemical investigations, useful information involving the electrochemical reaction mechanisms usually can be found from the potential scan rate. Therefore, the electrochemical behavior of RLX at scan rate ranges from 50.0 to 300.0 mV/s was also investigated at pH 3.0 by cyclic voltammetry in 1.0 mM solution of RLX (Fig. 3A). By increasing the scan rate, the Epa and Epc shift to more positive and more negative values, respectively, and the peak-to-peak separation also increases. These results illustrated that the electron transfer was quasi-reversible.
Fig. 2. (A): variation of the anodic peak current with pH. (B): plots of variation of peak potential with pH at a scan rate of 100.0 mV/s: (a) Epa vs. pH and (b) Epc vs. pH.
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constant ks (1/s) can be determined from the intercept of ΔEp vs. log ν plot (Eq. (4)). Under different scan rates of 100.0, 150.0, 200.0, and 250.0 mV/s, the potential shifts (ΔEp) were 0.107, 0.117, 0.126, and 0.133, respectively and the ks was calculated to be 0.282 1/s. An estimate of the surface coverage of the electrode was made approximately by adopting the method used by Sharp et al [41]. According to the method, the peak current is related to the surface concentration of electroactive species, Г, by the following equation: 2 2 Ipa ¼ αn F ГνA =2:718RT
Scheme 1. A proposed redox mechanism of RLX at glassy carbon electrode.
Moreover, when the scan rate was increased, a linear relationship between the peak current and the scan rate in the range of 50.0–300.0 mV/s was found for both anodic and cathodic peak currents (Fig. 3B), which suggests an adsorption behavior [39]. The equations related to peak current and the scan rate can be represented as Ipa (μA) = 43.74 ν (V/s) + 1.66, R2 = 0.997 and Ipc (μA)= −5.817 ν (V/s) − 0.222, R2 = 0.997. A good linearity between logarithm of the anodic peak current and logarithm of scan rate with the slope of 0.958 was also found (Fig. 3C), which it is close to the theoretical value of 1.0 for the adsorption-controlled electrode process [39]. The linear equation obtained was: log Ipa (μA) = 0.958 log ν (V/s) + 1.55, R 2 = 0.995. According to the Laviron's equation [40], for an adsorptioncontrolled and quasi-reversible interfacial reaction, the relationship between Epa and scan rate is defined by the following equations: Epa ¼ K þ ½2:33RT=ð1−α Þ F log ν
ð3Þ
log ks ¼ α logð1−α Þ þ ð1−α Þ log α−logðRT=F ν Þ−ð1−α Þ αFΔEp =2:3RT
ð5Þ
Where n represents the number of electrons involved in reaction, A is the effective surface area (0.0207 cm 2) of the GCE, α is the transfer coefficient (0.57), Г (mol/cm 2) is the surface coverage, and other symbols have their usual meanings. The surface concentration of electroactive species (Г) on GCE was calculated to be 4.7 × 10 − 10 mol/cm 2 from the slop of Ip vs. υ plot (Fig. 3B). 3.2.5. Accumulation time Because the electrochemical reaction of RLX is an adsorptiondriven process, accumulation can improve the pre-concentration of RLX on the electrode surface, and obviously improve sensitivity. Hence, the influence of accumulation time (tacc) on peak current was investigated by DPV in 0.1 M phosphate buffer (pH 3.0) containing 1.0 μM RLX. (Fig. 4). As it can be seen, the peak current was reached the maximum value after immersing GCE into solution after 70.0 s. Thus, the 70.0 s was chosen as the best accumulation time for the subsequent analytical experiments. 4. Method validation
ð4Þ 4.1. Calibration curve and detection limit where α is the transfer coefficient, ν (V/s) is the scan rate, and K is a constant value. From the slope of Epa vs. log ν plot (Fig. 3D), The value of α was found to be 0.57. In addition, the value of apparent rate
Differential-pulse voltammetry (DPV) is one of the most sensitive electrochemical detection methods. Therefore, DPV was applied for
Fig. 3. (A): cyclic voltammograms of 1.0 mM RLX in 0.1 M phosphate buffer solution (pH 3.0) in the potential ranges from + 0.2 to + 1.2 V at a different scan rates: (a → f) 50.0, 100.0, 150.0, 200.0, 250.0 and 300.0 mV/s. (B): plot of anodic (a) and cathodic (b) peak currents vs. potential scan rate. (C): plot of the logarithm of anodic peak current vs. logarithm of scan rate. (D): plot of the variation of anodic peak potential with logarithm of the scan rate.
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A. Bagheri, H. Hosseini / Bioelectrochemistry 88 (2012) 164–170 Table 1 Regression data of the calibration lines for determination of RLX by DPV. DPV Measured potential (V) Linearity range (μM) Number of point Slope (μA/μA) Intercept (μA) SE of slope SE of intercept Correlation coefficient LOD LOQ
Fig. 4. Effect of accumulation time (tacc) on DPV peak current of 1.0 μM RLX in 0.1 M phosphate buffer solution (pH 3.0).
determination of RLX. Under the optimized experimental conditions (phosphate buffer, pH 3.0; accumulation time, 70.0 s), the DPV curves were obtained using different concentrations of RLX (Fig. 5A). As shown in Fig. 5A, the height of the DPV peaks (Ipa) increases with increase in concentration of RLX. The calibration curve of the peak current vs. concentration was obtained using data from these measurements (inset Fig. 5A). According to the obtained results, linear calibration graphs were obtained for RLX with the linear dynamic range of 0.2–50 μM (0.096–23.9 ppm). The linear regression equations were Ipa (μA) = 0.782 + 0.198CRLX (μM), with a correlation coefficient of 0.998 and a sensitivity of 0.198 μA/μM. A limit of detection (LOD) of 0.075 μM (0.036 ppm) and limit of quantification (LOQ) of 0.25 μM (0.12 ppm) were calculated according to the 3 sb/m and 10 sb/m criterions, respectively, where m is the slope value of the calibration curve and sb is the calculated standard deviation for the peak currents of the blank (five runs). Regression and necessary validation data of the calibration plots are summarized in Table 1 according to the standard procedures [42–45]. The correlation coefficient greater than 0.99 and the low values of standard error of the slope and intercept confirmed the precision of the proposed methods.
0.7 0.2–50 12.0 0.198 0.782 0.0047 0.104 0.998 0.075 0.25
The repeatability and reproducibility of method were examined by RSD% and by the difference between theoretical and measured concentrations. Intra-day precision (repeatability) and accuracy of the proposed method were evaluated by assaying freshly prepared solutions at three different concentrations. Inter-day precision (reproducibility) and accuracy of the proposed method were evaluated by assaying freshly prepared solutions for 4 different days. The obtained results are shown in Table 2. The results reveal that there is no significant difference between the experiments which were carried out within-day and between days. Thus, the results confirmed the excellent reproducibility, repeatability, accuracy, and precision of the proposed method for determination of RLX. The detection limit obtained by proposed method is better than HPLC coupled with coulometric and UV detector [13] and it is close to some other developed methods [12,14]. Although the detection limits of HPLC coupled with mass detectors [7–9] are better compared to the proposed method, these methods need expensive equipments, are time-consuming, and require sample pretreatment, which make them unsatisfactory for routine or cost-effective screening. 4.2. Tablet analysis and recovery test The developed method was applied for determination of RLX in their dosages forms. Hence, the amount of RLX in tablets was determined by recording DPVs under the optimized experimental conditions. The results of proposed method were compared to the published methods [13,14], and are presented in Table 3. It was found that the obtained quantitative results were in agreement with the reported values. According to Table 3, the recovery tests were carried out by adding known amounts of pure drug to various pre-analyzed formulations. The mean percentage recovery showed no significant excipient interference which clearly demonstrates the
Fig. 5. (A): differential pulse voltammograms of RLX at GCE in 0.1 M phosphate buffer solution (pH 3.0) at 70 s accumulation time. RLX concentrations (from a to h): 0.7, 3.0, 10.0, 15.0, 20.0, 30.0, 40.0 and 50.0 μM. Inset: corresponding linear calibration curves of the anodic peak currents vs. RLX concentration. (B): differential-pulse voltammograms of the plasma sample spiked with different RLX concentration: (a) 0.3, (b) 1.5 and (c) 3.0 μM.
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Table 2 Intra- and inter-dye analysis of RLX (n = 4). Intra-day Inter-day RLX concentration Recovery% Accuracy Precision Recovery% Accuracy Precision (μM) RE % RSD % RE % RSD % 15 10 5
− 1.5 1.2 − 1.9
98.5 101.2 98.1
2.6 1.2 2.1
− 3.1 2.5 − 2.1
97.7 102.5 97.9
2.9 1.7 3.1
Table 3 Comparative studies for raloxifene in tablet by proposed and literature methods and mean recoveries in spiked tablet. HPLC with UV detector [13] Claimed dose (mg) Found (mg)a RSD% tvalue Bias% Recovery: added amount (mg) Recovery: found (mg) Recovery (%)c RSD% of recovery Bias% a b c
HPLC with Coulometric detector [13]
60.0 59.48 2.8
60.0 60.3 3.1
20.0
20.0
19.95 99.75 0.273
Capillary electrophoresis [14]
Present method
60.0 59.3 2.7
60.0 59.64 2.3 0.64b 0.6 20.0
20.06 100.3 0.297
19.97 99.85 0.253 0.15
Each value is the mean of six experiments. t-theoretical :2.57. Recovery value is the mean of six experiments.
accuracy and selectivity of the method. Thus, the procedure was able to determine RLX in the presence of excipients. 4.3. Interference studies Some inorganic ions and organic compounds may coexist with drugs in biological samples (such as plasma or urine) and tablet formulations, and these compounds can affect the voltammetric response of the drugs. Thus, in order to evaluate the specificity of the proposed method, the influence of potentially interfering substances such as Mg 2+, Ca 2+, Cu 2+, Zn 2+, Fe 3+, Na +, K +,SO42 −,Cl −, nitrate, glucose, fructose, sucrose, talc, citric acid, dopamine, ascorbic acid (AA), and uric acid (UA) on the determination of RLX were investigated by using DPV. Some results are presented in Table 4. Furthermore, Fig. 6 shows the voltammetric response of RLX in the presence of AA and UA which are the most important electroactive molecules which coexisting with RLX in a biological system. The results indicated that no interference was observed on the signals of RLX in the presence of interfering substances with concentration of 100.0 fold interfering substance to drug with a deviation below
Table 4 Influence of potential interferents on the voltammetric response of 10 μM raloxifene. Interferent 3+
Fe Cl− Glucose Fructose Talc Sucrose Ascorbic acid Uric acid
Concentration (mM)
Signal change (%)a
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
− 1.20 + 1.49 − 2.15 + 1.33 + 1.71 − 1.97 − 2.32 − 1.5
a The difference between the peak currents in the absence and presence of interferents.
Fig. 6. Differential pulse voltammograms of 10.0 μm RLX at GCE in the absence (…..) and the presence (—) of 1.0 mM AA and 1.0 mM UA in 0.1 M phosphate buffer solution (pH 3.0).
5.0%. It was observed that the method can be safely applied for determination of RLX in biological samples. 4.4. Determination of RLX in plasma samples The determination of RLX in plasma was also a challenging task because of its very low concentration; on average, its maximal plasma concentration (cmax) reaches only 1.36 (μg/L)/(mg/kg) or 1.36 ppm [8,46]. In order to evaluate a voltammetric method, the proposed method was applied to determine RLX in plasma samples, duo to low LOD (0.036 ppm) and LOQ (0.12 ppm). According to Section 2.4.2, the recovery studies were carried out by spiking pure drug free plasma with known amounts of RLX (standard addition method), and the calibration plot was used for determination of spiked RLX in human plasma. DPVs for determination of RLX in human plasma are shown in Fig. 5B. The peak currents of DPVs were linearly increased by adding higher concentrations of RLX to plasma samples. The results for detection of RLX in three samples are presented in Table 5. The recoveries were determined in the range of 98.4%–101.9% with RSD (%) below 3.0%. The results illustrated that the proposed method is accurate enough for practical applications. 5. Conclusion The electrochemical behavior of RLX at GCE surface was studied by CV. Obtained results from the CV measurements suggested that electrode reaction process is quasi-reversible and adsorption-controlled and two electrons and two protons are involved in the process and consequently, a probable redox mechanism was proposed. The proposed differential-pulse voltammetry has been validated, and it was successfully applied for quantitative determination of RLX in tablet and human plasma samples. The effects of potential interfering substances were studied, and it was found that the proposed method is free from interferences. Comparison of the proposed method with
Table 5 Determination of raloxifene in plasma samples. Plasma
Spiked (μM)
Found (μM)a
Recovery(%) + RSD%
Bias%
Sample 1.0 Sample 2.0 Sample 3.0
0.3 1.5 3.0
0.303 1.476 3.056
101 ± 1.56 98.4 ± 1.93 101.9 ± 2.37
− 1.0 + 1.6 − 1.87
a
Average of six determinations.
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Akbar Bagheri received his PhD in 1976 in Analytical Chemistry at Howard University (United States). Now, he is an associate professor in Chemistry Department of Shahid Beheshti University (Tehran, Iran). His research fields of interest include electrochemical sensors, novel electrode material and electroanalytical chemistry.
Hadi Hosseini received his MS degree in 2009 from Shahid Beheshti University (Tehran, Iran). Now, he is a PhD student in Chemistry Department of Shahid Beheshti University (Tehran, Iran). His research interests focus on electrode materials and electroanalytical chemistry.