Materials Science and Engineering C 52 (2015) 297–305
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Electrochemical determination of hydrochlorothiazide and folic acid in real samples using a modified graphene oxide sheet paste electrode Hadi Beitollahi a,⁎, Mozhdeh Hamzavi b, Masoud Torkzadeh-Mahani c a b c
Environment Department, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran Department of Chemistry, Graduate University of Advanced Technology, Kerman, Iran Biotechnology Department, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran
a r t i c l e
i n f o
Article history: Received 11 February 2014 Received in revised form 24 January 2015 Accepted 22 March 2015 Available online 24 March 2015 Keywords: Hydrochlorothiazide Folic acid Carbon paste electrode Graphene oxide sheets
a b s t r a c t A new ferrocene-derivative compound, 2-chlorobenzoyl ferrocene, was synthesized and used to construct a modified graphene oxide sheet paste electrode. The electrooxidation of hydrochlorothiazide at the surface of the modified electrode was studied. Under optimized conditions, the square wave voltammetric (SWV) peak current of hydrochlorothiazide increased linearly with hydrochlorothiazide concentration in the range of 5.0 × 10−8 to 2.0 × 10−4 M and a detection limit of 20.0 nM was obtained for hydrochlorothiazide. The diffusion coefficient and kinetic parameters (such as electron transfer coefficient and the heterogeneous rate constant) for hydrochlorothiazide oxidation were also determined. The prepared modified electrode exhibits a very good resolution between the voltammetric peaks of hydrochlorothiazide and folic acid which makes it suitable for the detection of hydrochlorothiazide in the presence of folic acid in real samples. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Thiazidic diuretics, such as hydrochlorothiazide, which is a prototype of thiazide drugs comprising of an important class of diuretics, increase the rate of urinary excretion of sodium and water by sodium reabsorption inhibition in the renal tubules. Hydrochlorothiazide is used for the treatment of edemas associated with the heart (congestive heart failure), the liver (hepatic cirrhosis) and the kidneys (nephrotic syndrome, chronic renal failure, and acute glomerulonephritis). It has also been used for all degrees of hypertension, being efficient as antihypertensive agents of the other classes [1]. Various analytical methods have been described for the determination of hydrochlorothiazide in pharmaceutical preparations, blood and plasma, including chemiluminescence [2], capillary zone electrophoresis [3], spectrophotometric [4], high performance liquid chromatography (HPLC) [5], derivative spectroscopy [6], thin-layer chromatography [7] and liquid chromatographic methods [8]. The main deficiency of such methods lies in the fact that their use requires time-consuming derivatization techniques or that they suffer from low detection limits. Hence, there have been increasing demands for new, fast, simple, convenient and sensitive methods for the determination of hydrochlorothiazide. Therefore, given the advantages of having a lower cost, providing faster response, simple instrumentation, high sensitivity, facile miniaturization, and low power requirement, numerous electrochemical methods for the determination of hydrochlorothiazide have been developed [9–13]. ⁎ Corresponding author. E-mail address: [email protected] (H. Beitollahi).
http://dx.doi.org/10.1016/j.msec.2015.03.031 0928-4931/© 2015 Elsevier B.V. All rights reserved.
Several chronic diseases (for example, gigantocytic anemia, leucopoenia, mentality devolution, psychosis, heart attack, and stroke), especially those concerned with malformation during pregnancy and carcinogenic processes, are related to the deficiency of folic acid [14] which is a water-soluble vitamin. Since folic acid is detected in biological fluids at very low concentrations, a highly specific and sensitive assay is called for. Available methods for this purpose are generally based on spectrophotometry [15], chromatography [16], fluorescence [17] or phosphorescence detection [18], together with bioassay [19]. Among the different methods, electrochemical methods are found to be very promising [20–24]. It has been discovered that when pregnant women consume hydrochlorothiazide due to high blood pressure, their body not only loses its necessary liquids and sodium but the level of folic acid in their body also decreases due to frequent urination. Therefore, as it is possible that a number of difficulties, such as congenital malformations and neural tube defects, may be caused by low levels of folic acid in a pregnant woman's body, they need to use 800 mg of folic acid, which is twice as much as the amount used by an ordinary person (400 mg). Hence, by the simultaneous determination of hydrochlorothiazide and folic acid, it is possible to identify the best type of treatment for each specific patient while decreasing the possible harm that is likely to occur as a result of side-effects caused by incorrect prescriptions [25]. A new kind of two-dimensional carbon material, graphene (G) has attracted increasing attention due to its unique properties including a high surface area, excellent electrical conductivity, quick electron mobility at room temperature, high mechanical strength, and ease of functionalization [26,27]. Graphene based electrochemical sensors
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for the determination of hydrochlorothiazide and folic acid in real samples. 2. Experimental 2.1. Apparatus and chemicals
Fig. 1. TEM image of synthesized graphene oxide sheets.
have been proven to possess excellent electrocatalytic ability and good performance [28–30]. Among all the carbon electrodes, the carbon paste electrode (CPE) is an appealing and widely used electrode material in the fields of electrochemistry, electroanalysis, etc. due to its attractive advantages, such as simple preparation, low-cost implementation, renewability, low background current, and wide potential window [31–35]. The preparation of CPE usually involves the dispersion of graphite powder in a hydrophobic binder to form a homogeneous paste, followed by filling a tube with the resulting paste [36,37]. Sometimes, the modifiers including various biomolecules, organic compounds and nanoparticles are also doped into the carbon paste, which makes CPE quite suitable for bioanalysis [38–44]. To the best of our knowledge, no study has been published so far reporting the simultaneous electrocatalytic determination of hydrochlorothiazide and folic acid using a modified graphene oxide sheet paste electrode. Thus, in this paper, the preparation and suitability of a 2chlorobenzoyl ferrocene (2CBF) modified graphene oxide sheet paste electrode (2CBFGPE) as a new electrode in the electrocatalysis and determination of hydrochlorothiazide in an aqueous buffer solution was initially described. Then the analytical performance of the modified electrode in quantification of hydrochlorothiazide in the presence of folic acid was also evaluated. Finally this new constructed electrochemical sensor was used
The electrochemical measurements were performed with an Autolab potentiostat/galvanostat (PGSTAT 302N, Eco Chemie, the Netherlands). The experimental conditions were controlled with the General Purpose Electrochemical System (GPES) software. A conventional three electrode cell was used at 25 ± 1 °C. An Ag/AgCl/KCl (3.0 M) electrode, a platinum wire, and 2CBFGPE were used as the reference, auxiliary and working electrodes, respectively. Finally a Metrohm 710 pH meter was used for pH measurements. All solutions were freshly prepared with double distilled water. Hydrochlorothiazide, folic acid and all of the other reagents were of analytical grade and were obtained from Merck (Darmstadt, Germany). The buffer solutions were prepared from orthophosphoric acid and its salts in the pH range of 2.0–11.0. 2.2. Synthesis of graphene oxide sheets Graphene oxide sheets were synthesized from natural graphite flakes based on the modified Hummers and Offeman method [45,46]. In a typical synthesis process, 1.0 g of pristine graphite flakes was immersed in 50 mL of formic acid, and then sonicated for 2 h at room temperature. These resulting graphite plates were washed with acetone, and then dried in an oven at 95 °C for 12 h. Then, 100 mL H2SO4 (95%) was added into a 500 mL flask, and cooled by immersion in an ice bath followed by stirring. About 1.0 g treated graphite powder and 0.5 g NaNO3 were added under vigorous stirring to avoid agglomeration. After the graphite powder was well dispersed, 3 g KMnO4 was added gradually under stirring and cooling so that the temperature of the mixture was maintained at below 10 °C. The mixture was stirred for 2 h and diluted with deionized double distilled water (in an ice bath). After that, 25 mL 15% H2O2 was slowly added to the mixture until the color of the mixture changed to brilliant yellow, indicating fully oxidized graphite. The as-obtained graphite oxide slurry was re-dispersed in deionized double distilled water and then exfoliated to generate graphene oxide
Fig. 2. FTIR of synthesized graphene oxide sheets.
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Table 1 Cyclic voltammetric data obtained for constructed 2CBFGPE in 0.1 M PBS (pH 7.0) at 10 mV s−1. Epa (V)
a
0.655 ± 1.1b a b
Fig. 3. SEM image of GPE.
sheets by sonication for 2 h. Then, the solution was filtered and washed with diluted HCl solution to remove metal ions. Finally, the product was washed with deionized double distilled water until the solution became acid free, and dried under vacuum at 50 °C. A typical transmission electron microscopy (TEM) for synthesized graphene oxide sheets is shown in Fig. 1. Also FTIR of synthesized graphene oxide sheets is shown in Fig. 2. In the FT-IR spectrum of the graphene oxide sample, a broad band in the range of 2500–3650 cm−1 corresponds to the stretching and bending vibration of OH groups of water molecules adsorbed on graphene oxide. The peaks at 1699 and 1369 cm−1 are C_O stretching vibration peaks of carboxyl and carbonyl and also, the peak at 1631 cm−1 is attributed to the stretching vibration of aromatic C_C. The absorption peaks at 1236 and 1099 cm−1 correspond to the C\O stretching vibration of an epoxy group and C\OH of alcohol, respectively. The presence of these oxygen-containing groups reveals that the graphite has been oxidized. 2.3. Synthesis of 2-chlorobenzoyl ferrocene To a 100 mL round-bottomed flask under argon atmosphere, 1.86 g (10 mmol) of ferrocene, 1.75 g (10 mmol) of 2-chlorobenzoyl chloride and 20 mL of dichloromethane is added. The reaction mixture is cooled in an ice bath (0–5 °C), then 1.40 g (11 mmol) of anhydrous aluminum chloride was added in small portions at such a rate that the reaction
Fig. 4. CVs of 2CBFGPE (A) and CPE (B) in 0.1 M PBS (pH 7.0). In all cases scan rate is 100 mV s−1.
Epc (V)
E1/2 (V)
ΔEp (V)
Ipa (μA)
Ipc (μA)
0.540 ± 1.2
0.597 ± 1.2
0.115 ± 1.1
9.5 ± 1.4
5.7 ± 1.6
Versus Ag/AgCl/KCl (3.0 M) as reference electrode. All the ‘±’ values are RSD% (n = 5).
mixture remains below 5 °C. The resulting solution is stirred for 30 min at 0–5 °C and 2 h at room temperature. Then, the flask is placed in an ice bath again and 20 mL of water is added cautiously to give a two-phase mixture. After stirring for 30 min the aqueous layer is extracted with two 15 mL portions of dichloromethane. The combined dichloromethane extracts are washed once with water, twice with 10% sodium hydroxide solution, dried over magnesium sulfide and evaporated at reduced pressure. The crude residue is purified by recrystallization from heptane to afford (2-chlorobenzoyl)ferrocene in 95% yield. M.p. 99–100 °C, 1
H NMR (400 MHz, CDCl3): δ (ppm) = 4.30 (s, 5H), 4.62 (t, J = 1.6 Hz, 2H), 4.77 (t, J = 1.6 Hz, 2H), 7.36 (dt, J = 1.6, 7.6 Hz, 1H), 7.42 (dt, J = 2.0, 7.6 Hz, 1H), 7.47 (dd, J = 1.2, 8.0 Hz, 1H), 7.52 (1.6, 7.2 Hz, 1H).
IR (KBr) (νmax, cm− 1): 3080.8, 3052.5 (C\H aromatic), 1644.9 (C_O), 1442.7, 1292.8, 1031.5, 827.7. 2.4. Preparation of the electrode The 2CBFGPEs were prepared by dissolving 0.01 g 2CBF in 3 mL diethyl ether and then adding this to 0.89 g graphite powder and 0.1 g graphene oxide sheets with a mortar and pestle. Then, 0.7 mL of paraffin was added to the above mixture and mixed for 15 min until a uniformly wetted paste was obtained. The paste was then packed into the end of a glass tube. A copper wire inserted into the carbon paste provided the electrical contact. When necessary, a new surface was obtained by pushing an excess of the paste out of the tube and polishing with a weighing paper. For comparison, a 2CBF modified CPE electrode (2CBFCPE) without G, a G paste electrode (GPE) without 2CBF, and an unmodified CPE in the absence of 2CBF and G were also prepared in the same way. A typical scanning electron microscope (SEM) image for GPE is shown in Fig. 3.
Fig. 5. CVs of 2CBFCPE (A) and 2CBFGPE (B) in 0.1 M PBS (pH 7.0) containing 100.0 μM hydrochlorothiazide. (C) and (D) are CVs of CPE and GPE in 0.1 M PBS (pH 7.0) containing 100.0 μM hydrochlorothiazide and (E) is CV of CPE in 0.1 M PBS (pH 7.0) In all cases scan rate is 10 mV s−1.
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Fig. 6. CVs of 2CBFGPE in 0.1 M PBS (pH 7.0) containing 10.0 μM hydrochlorothiazide at various scan rates; numbers 1–5 correspond to 5, 10, 20, 30 and 40 mV s−1, respectively. Insets: variation of (A) anodic peak current vs. square root of scan rate; (B) normalized current (Ip/ν1/2) vs. ν and (C) anodic peak potential (Ep) vs. logarithm of scan rate.
Fig. 7. Chronoamperograms obtained at 2CBFGPE in 0.1 M PBS (pH 7.0) for different concentrations of hydrochlorothiazide. The numbers 1–6 correspond to 0.0, 0.1, 0.2, 0.4, 0.6 and 1.0 mM of hydrochlorothiazide. Insets: (A) plots of I vs. t−1/2 obtained from chronoamperograms 2–6. (B) Plot of the slope of the straight lines against hydrochlorothiazide concentration. (C) Dependence of Ic/Il on t1/2 derived from the data of chronoamperograms 1–6.
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Fig. 8. SWVs of 2CBFGPE in 0.1 M PBS (pH 7.0) containing different concentrations of hydrochlorothiazide. Numbers 1–15 correspond to 0.05, 0.35, 1.0, 2.5, 5.0, 7.5, 10.0, 25.0, 50.0, 75.0, 100.0, 125.0, 150.0, 175.0 and 200.0 μM of hydrochlorothiazide. Inset: plots of the electrocatalytic peak current as a function of hydrochlorothiazide concentration in the range of 0.05 to 200.0 μM.
2.5. Procedure for the preparation of real samples Five hydrochlorothiazide tablets (labeled 50 mg) were ground and a hydrochlorothiazide solution was prepared by dissolving 50 mg of the powder in 100 mL water by ultrasonication. Then, different volumes of the diluted solution were transferred into a 10 mL volumetric flask and were diluted to the mark with phosphate buffer solution (PBS) (pH 7.0). The hydrochlorothiazide content was analyzed by the proposed method using the standard addition method. Also, five folic acid tablets (labeled 1.0 mg) were ground and the tablet solution was prepared by dissolving 50 mg of the powder in 100 mL water by ultrasonication. Then, different volumes of the diluted solution were transferred into a 10 mL volumetric flask and were diluted to the mark with PBS (pH 7.0). The folic acid content was analyzed by the proposed method using the standard addition method. Urine samples of healthy people were stored in a refrigerator immediately after collection. Ten milliliters of the sample was centrifuged for 15 min at 2000 rpm. The supernatant was filtered out using a 0.45 μm filter. Then, a different volume of the solution was transferred into a 25 mL volumetric flask and diluted to the mark with PBS (pH 7.0). The diluted urine sample was spiked with different amounts of hydrochlorothiazide and folic acid. The serum sample of healthy people was prepared from a local laboratory. It was centrifuged and after filtering, diluted with PBS (pH 7.0) without any further treatment. The diluted serum sample was spiked with different amounts of hydrochlorothiazide and folic acid. 3. Results and discussion 3.1. Electrochemical behavior of 2CBFGPE 2CBFGPE was prepared and its electrochemical behaviors were studied using a cyclic voltammetry (CV) technique (Fig. 4). The experimental
results show well-defined and reproducible anodic and cathodic peaks related to a 2-chlorobenzoyl ferrocene/2-chlorobenzoyl ferricenium ion (Fc/Fc+) redox system, which show a quasireversible behavior in an aqueous medium. The electrode capability for the generation of a reproducible surface was examined by cyclic voltammetric data obtained in optimum solution at pH 7.0 from five separately prepared 2CBFGPEs (Table 1). The calculated relative standard deviation (RSD) for various parameters is accepted as the criteria for a satisfactory surface reproducibility (about 1–4%), which is virtually the same as that expected for the renewal or ordinary carbon paste surface. However we regenerated the surface of 2CBFGPE before each experiment based on our previous results. In addition, the long-term stability of 2CBFGPE was tested over a three-week time period. Once the CVs were recorded after the modified electrode was stored in atmosphere at an ambient temperature, the peak potential for hydrochlorothiazide oxidation was unchanged and the current signals showed less than 2.3% decrease relative to the initial response. The antifouling properties of the modified electrode toward hydrochlorothiazide and its oxidation products were investigated by recording the CVs of the modified electrode before and after use in the presence of hydrochlorothiazide. CVs were recorded in the presence of
Table 2 Comparison of the efficiency of some modified electrodes used in the determination of hydrochlorothiazide. Electrode
Method
Carbon paste Boron-doped diamond Boron-doped diamond Glassy carbon Carbon paste
Voltammetry Voltammetry
LOD (M) 3.7 × 10−4 1.2 × 10−6
LDR (M) 8.0 × 10−8–5.0 × 10−4 3.0 × 10−6–7.4 × 10−5
Ref. [10] [13]
Voltammetry 6.39 × 10−7 1.97 × 10−6–8.81 × 10−5 [49] Voltammetry Voltammetry
4.3 × 10−9 2.0 × 10−8
4.0 × 10−6–4.0 × 10−5 5.0 × 10−8–2.0 × 10−4
[50] This work
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Fig. 9. SWVs of 2CBFGPE in 0.1 M PBS (pH 7.0) containing 1000.0 μM folic acid and different concentrations of hydrochlorothiazide. From inner to outer: 50.0, 65.0, 90.0, 110.0, and 125.0 μM of hydrochlorothiazide. Inset: plot of the electrocatalytic peak current as a function of hydrochlorothiazide concentration.
hydrochlorothiazide after having cycled the potential 20 times at a scan rate of 10 mV s−1. The peak potentials were unchanged and the currents decreased by less than 2.4%. Therefore, at the surface of 2CBFGPE, not only the sensitivity increased, but also, the fouling effect of the analyte and its oxidation product also decreased. 3.2. Electrocatalytic oxidation of hydrochlorothiazide at 2CBFGPE The electrochemical behavior of hydrochlorothiazide is dependent on the pH value of the aqueous solution, whereas the electrochemical
properties of an Fc/Fc+ redox couple are independent of pH [22,24,33, 43]. Therefore, pH optimization of the solution seems to be necessary in order to obtain the electrocatalytic oxidation of hydrochlorothiazide. Thus the electrochemical behavior of hydrochlorothiazide was studied in different pH values (2.0 b pH b 11.0) at the surface of 2CBFGPE by CV. It was found that the electrocatalytic oxidation of hydrochlorothiazide at the surface of 2CBFGPE was more favored under neutral conditions than in acidic or basic medium. This appears as a gradual growth in the anodic peak current and a simultaneous decrease in the cathodic peak current in the CVs of 2CBFGPE. Thus, pH 7.0 was chosen as the optimum pH for electrocatalysis of hydrochlorothiazide oxidation at the surface of 2CBFGPE. Fig. 5 depicts the CV responses for the electrochemical oxidation of 100.0 μM hydrochlorothiazide at unmodified CPE (curve C), GPE (curve D), 2CBFCPE (curve A) and 2CBFGPE (curve B). As can be seen, while the peak potential for hydrochlorothiazide oxidation at the GPE, and unmodified CPE are 910 and 970 mV, respectively, the corresponding potential at 2CBFGPE and 2CBFCPE is ~ 655 mV. These results indicate that the peak potential for hydrochlorothiazide oxidation at 2CBFGPE and 2CBFCPE shifts by ~ 255 and 315 mV toward negative values compared to GPE and unmodified CPE, respectively. However, 2CBFGPE shows a much higher anodic peak current for the oxidation of hydrochlorothiazide compared to 2CBFCPE, indicating that the combination of G and the mediator (2CBF) has significantly improved the performance of the electrode toward hydrochlorothiazide oxidation. 2CBFGPE, in 0.1 M PBS (pH 7.0) and without hydrochlorothiazide in solution (Fig. 4 curve A), exhibited a well-behaved redox reaction and with addition of 100.0 μM hydrochlorothiazide, increased the anodic peak current (Fig. 5 curve B), indicating a strong electrocatalytic effect [47]. The effect of potential scan rate on the electrocatalytic oxidation of hydrochlorothiazide at 2CBFGPE was investigated by CV (Fig. 6). As can be observed in Fig. 6, the oxidation peak potential shifted to more positive potentials with an increasing scan rate, confirming the kinetic limitation in the electrochemical reaction. Also, a plot of peak height (Ip) vs. the square root of scan rate (ν1/2) was found to be linear in the
Fig. 10. SWVs of 2CBFGPE in 0.1 M PBS (pH 7.0) containing different concentrations of hydrochlorothiazide and folic acid in μM, from inner to outer: 15.0 + 100.0, 25.0 + 150.0, 40.0 + 250.0, 50.0 + 500.0, 90.0 + 750.0, 115.0 + 1500.0 and 160.0 + 2000.0 respectively. Insets: (A) plots of Ip vs. hydrochlorothiazide concentration and (B) plot of Ip vs. folic acid concentrations.
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Table 3 The application of 2CBFGPE for simultaneous determination of hydrochlorothiazide and folic acid in their tablet samples (n = 5). Sample
Spiked (μM)
Hydrochlorothiazide tablet
Folic cid tablet
Found (μM)
Recovery (%)
Folic acid
Hydrochlorothiazide
Folic acid
Hydrochlorothiazide
Folic acid
Hydrochlorothiazide
Folic acid
0 10.0 20.0 30.0 40.0 0 15.0 25.0 35.0 45.0
0 100.0 125.0 150.0 175.0 0 20.0 40.0 60.0 80.0
12.0 22.3 31.2 41.6 53.3 ND 15.2 24.6 35.9 44.6
ND 98.1 127.1 154.1 173.7 100.0 118.3 141.1 159.1 182.3
– 101.4 97.5 99.0 102.5 – 101.3 98.4 102.6 99.1
– 98.1 101.7 102.7 99.3 – 98.6 100.8 99.4 101.3
3.3 2.1 2.9 1.7 3.3 – 3.4 1.7 2.9 2.5
– 3.2 2.1 2.3 2.4 2.9 2.7 3.1 2.1 1.9
range of 5–40 mV s−1, suggesting that, at sufficient overpotential, the process is diffusion rather than surface controlled (Fig. 6A). A plot of the scan rate-normalized current (Ip/ν1/2) vs. scan rate (Fig. 6B) exhibits the characteristic shape typical of an electrocatalytic (EC') process [47]. The Tafel slope (b) can be obtained from the slope of Ep vs. log ν using Eq. (1): [47] Ep ¼ b=2 log ν þ constant
ð1Þ
The Tafel slope was found to be 112.0 mV (Fig. 6C), which indicates that a one-electron transfer process is the rate limiting step assuming that a transfer coefficient (α) is about 0.47. 3.3. Chronoamperometric measurements Chronoamperometric measurements of hydrochlorothiazide at 2CBFGPE were carried out by setting the working electrode potential at 0.75 V for the various concentrations of hydrochlorothiazide in 0.1 M PBS (pH 7.0) (Fig. 7). For an electroactive material (hydrochlorothiazide in this case) with a diffusion coefficient of D, the current observed for the electrochemical reaction at the mass transport limited condition is described by the Cottrell equation [47]. Experimental plots of I vs. t−1/2 were employed, with the best fits for different concentrations of hydrochlorothiazide (Fig. 7A). The slopes of the resulting straight lines were then plotted vs. hydrochlorothiazide concentration (Fig. 7B). From the resulting slope and Cottrell equation the mean value of the D was found to be 1.1 × 10−5 cm2/s which is comparable with values reported by other research groups (1.1 × 10−5 cm2/s [10] and 1.75 × 10−5 cm2/s [12]). Chronoamperometry can also be employed to evaluate the catalytic rate constant, k, for the reaction between hydrochlorothiazide and 2CBFGPE according to the method described by Galus [48]: 1=2
IC =IL ¼ γ
h
π
1=2
i 1=2 1=2 þ exp ð−γÞ=γ erf γ
ð2Þ
where IC is the catalytic current of hydrochlorothiazide at 2CBFGPE, IL is the limited current in the absence of hydrochlorothiazide and γ = kCbt is the argument of the error function (Cb is the bulk concentration of hydrochlorothiazide). In cases where γ exceeds the value of 2, the error function is almost equal to 1 and therefore, the above equation can be shortened to: IC = IL ¼ π
1=2 1=2
γ
¼ π
1=2
RSD (%)
Hydrochlorothiazide
1=2
ðkCb tÞ
ð3Þ
where t is the time elapsed. The above equation can be used to calculate the rate constant, k, of the catalytic process from the slope of IC/IL vs. t1/2 at a given hydrochlorothiazide concentration. From the values of the slopes (Fig. 7C), the average value of k was found to be 9.1 × 102 M−1 s−1 which is comparable with values reported by other research groups (6.38 × 103 M−1 s−1 [10] and 3.38 × 102 M−1 s−1 [12]).
3.4. Calibration plot and limit of detection The electrocatalytic peak current of hydrochlorothiazide oxidation at the surface of 2CBFGPE can be used for the determination of hydrochlorothiazide in solution. Therefore, square wave voltammetry (SWV) experiments were performed using a modified electrode in 0.1 M PBS (pH 7.0) containing various concentrations of hydrochlorothiazide (Fig. 8). The plot of peak current vs. hydrochlorothiazide concentration consisted of a linear segment with slope of 0.135 μA/μM−1 in the concentration range of 5.0 × 10−8–2.0 × 10−4 M. The detection limit (3σ) of hydrochlorothiazide was found to be 20.0 nM. This value is comparable with values reported by other research groups for the determination of hydrochlorothiazide (see Table 2).
3.5. Simultaneous determination of hydrochlorothiazide and folic acid To our knowledge, no study has used the modified graphene oxide sheet electrode and specially modified graphene oxide sheet paste electrode for simultaneous determination of hydrochlorothiazide and folic acid and this is the first report for simultaneous determination of hydrochlorothiazide and folic acid using the modified graphene oxide paste electrode. The determination of hydrochlorothiazide and folic acid in mixtures was performed at 2CBFGPE using SWV. The concentration of hydrochlorothiazide was varied, while keeping the folic acid concentration constant. The results are shown in Fig. 9. When the concentration of folic acid is kept constant at 1000.0 μM, the peak current of hydrochlorothiazide is proportional to its concentration. No changes in the peak current and potential of folic acid can be found. Also, determination of two compounds was performed by simultaneously changing the concentrations of hydrochlorothiazide and folic acid, and recording the SWVs (Fig. 10). The voltammetric results showed well-defined anodic peaks at potentials of 600 and 820 mV, corresponding to the oxidation of hydrochlorothiazide and folic acid, respectively, indicating that simultaneous determination of these compounds is feasible at 2CBFGPE as shown in Fig. 10. The sensitivity of the modified electrode toward the oxidation of hydrochlorothiazide was found to be 0.134 μA/μM−1. This is very close to the value obtained in the absence of folic acid (0.135 μA/μM−1, see Section 3.4), indicating that the oxidation processes of these compounds
Table 4 Comparison of the total values of hydrochlorothiazide and folic acid of some pharmaceutical preparations obtained using 2CBFGPE with declared values in the label of the samples (n = 5). Samples
Declared value
Found value
RSD%
Hydrochlorothiazide tablet (mg per tablet) Folic acid tablet (mg per tablet)
50.00 1.00
50.10 1.05
2.7 2.5
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Table 5 The application of 2CBFGPE for simultaneous determination of hydrochlorothiazide and folic acid in human blood serum and urine samples (n = 5). Sample
Human blood serum
Urine
Spiked (μM)
Found (μM)
Recovery (%)
RSD (%)
Hydrochlorothiazide
Folic acid
Hydrochlorothiazide
Folic acid
Hydrochlorothiazide
Folic acid
Hydrochlorothiazide
Folic acid
10.0 20.0 30.0 40.0 17.5 27.5 37.5 47.5
120.0 140.0 160.0 180.0 115.0 135.0 155.0 175.0
10.3 19.6 30.2 39.6 17.2 27.7 37.1 49.2
118.1 141.9 159.2 175.3 117.1 133.2 160.1 172.1
103.0 98.0 100.7 99.0 98.3 100.7 98.9 103.6
98.4 101.3 99.5 97.4 101.8 98.7 103.3 98.3
2.1 3.2 1.7 2.9 2.6 2.2 1.9 3.5
1.7 2.9 2.8 3.3 3.1 2.8 2.7 1.8
at 2CBFGPE are independent and therefore, simultaneous determination of their mixtures is possible without significant interferences.
3.6. Interference studies The influence of various substances as compounds potentially interfering with the determination of hydrochlorothiazide was studied under optimum conditions. The potentially interfering substances were chosen from the group of substances commonly found with hydrochlorothiazide in pharmaceuticals and/or in biological fluids. The tolerance limit was defined as the maximum concentration of the interfering substance that caused an error of less than ±5% in the determination of hydrochlorothiazide. According to the results, neither a 600-fold +2 excess of Mg2+, Al3+, NH+ , Fe+3, F−, SO2− and S2−, nor a 4004 , Fe 4 fold excess of L-lysine, glucose, NADH, acetaminophen, uric acid, L-asparagine, L-serine, L-threonine, L-proline, L-histidine, L-glycine, L-tryptophan, L-phenylalanine, lactose, saccharose, fructose, benzoic acid, methanol, ethanol, urea, and caffeine, did not show interference in the determination of hydrochlorothiazide.
3.7. Real sample analysis 3.7.1. Determination of hydrochlorothiazide and folic acid in pharmaceutical preparations In order to evaluate the analytical applicability of the proposed method, it was also applied to the determination of hydrochlorothiazide and folic acid in their tablets. The results are listed in Table 3. The reliability of the proposed modified electrode was also evaluated by comparing the obtained results with those declared in the label of pharmaceutical preparations (Table 4). The results in Table 3 show that the relative standard derivations (RSD) and the recovery rates of the spiked samples are acceptable. Also, the data in Table 4 indicate that the results obtained by utilizing 2CBFGPE are in good agreement with those declared in the label of the preparations. Thus, the modified electrode can be efficiently used for individual or simultaneous determination of hydrochlorothiazide and folic in pharmaceutical preparations.
3.7.2. Determination of hydrochlorothiazide and folic in human blood serum and urine samples In order to evaluate the analytical applicability of the proposed method, it was additionally applied for the determination of hydrochlorothiazide and folic acid in human blood serum and urine samples. The results for determination of the two species in real samples are given in Table 5. Satisfactory recovery of the experimental results was found for hydrochlorothiazide and folic acid. The reproducibility of the method was demonstrated by the mean relative standard deviation (RSD).
4. Conclusions In this paper a carbon paste electrode modified with graphene oxide sheets and a ferrocene derivative was fabricated and used for the investigation of the electrochemical behaviors of hydrochlorothiazide. The electrochemical behavior of hydrochlorothiazide at the surface of 2CBFGPE showed that hydrochlorothiazide electrooxidation is catalyzed at pH 7.0. It has been found that under an optimum condition (pH 7.0), the oxidation of hydrochlorothiazide at the surface of such an electrode occurs at a potential of about 315 mV less positive than that of an unmodified carbon paste electrode. The modified electrode successfully resolves the overlapped voltammetric peaks of hydrochlorothiazide and folic acid (approximately 220 mV), so that the modified electrode displays high selectivity in the SWV measurement of hydrochlorothiazide and folic acid in their mixture solutions. Finally, the modified electrode was also examined as a selective, simple, and precise new electrochemical sensor for the determination of hydrochlorothiazide and folic acid in real samples. References [1] A.O. Youssef, J. Fluoresc. 22 (2012) 827. [2] J. Ouyang, W.R.G. Baeyens, J. Delanghe, G. Van der Weken, A.C. Calokerinos, Talanta 46 (1998) 961. [3] M.R. Balesteros, A.F. Faria, M.A.L. Oliveira, J. Braz. Chem. Soc. 18 (2007) 554. [4] R.C. Mashru, V.B. Sutariya, A. Ythakker, J. Ars Pharm. 47 (2006) 375. [5] L.R. Bhat, R.K. Godge, A.T. Vora, M.C. Dam Le, J. Liq. Chromatogr. Relat. Technol. 30 (2007) 3059. [6] I.F. Al-Momani, Turk. J. Chem. 3 (2005) 17. [7] B. Shaikh, N. Rummel, J. Agric. Food Chem. 46 (1998) 1039. [8] L. Li, J. Sun, P. Yang, Z. He, Anal. Lett. 39 (2006) 2797. [9] S. Tajik, M.A. Taher, H. Beitollahi, J. Electroanal. Chem. 704 (2013) 137. [10] H. Karimi-Maleh, A.A. Ensafi, H.R. Ensafi, J. Braz. Chem. Soc. 20 (2009) 880. [11] O.A. Razak, J. Pharm. Biomed. Anal. 34 (2004) 433. [12] H. Beitollahi, F. Ghorbani, Ionics 19 (2013) 1673. [13] M.C. Gardenal Santos, C.R. Teixeira Tarley, L.H. Dall'Antonia, E.R. Sartori, Sens. Actuators B 188 (2013) 263. [14] S. Wei, F. Zhao, Z. Xu, B. Zeng, Microchim. Acta 152 (2006) 285. [15] G.R. Rao, G. Kanjilal, K.R. Mohan, Analyst 103 (1978) 993. [16] R.H.F. Chenug, P.D. Morrison, D.M. Small, P.J. Marriott, J. Chromatogr. A 1213 (2008) 93. [17] E.J.M. Konings, J. AOAC Int. 82 (1999) 119. [18] R.M.A. Von Wandruzska, R.J. Hurtubise, Anal. Chim. Acta 93 (1977) 331. [19] A. Lermo, S. Fabiano, S. Hernández, R. Galve, M.P. Marco, S. Alegret, M.I. Pividori, Biosens. Bioelectron. 24 (2009) 2057. [20] H. Beitollahi, I. Sheikhshoaie, J. Electroanal. Chem. 661 (2011) 336. [21] L. Mirmoghtadaie, A.A. Ensafi, M. Kadivar, P. Norouzi, Mater. Sci. Eng. C 331 (2013) 1753. [22] H. Beitollahi, I. Sheikhshoaie, Anal. Methods 3 (2011) 1810. [23] M. Arvand, M. Dehsaraei, Mater. Sci. Eng. C 33 (2013) 3474. [24] H. Beitollahi, I. Sheikhshoaie, Electrochim. Acta 56 (2011) 10259. [25] http://www.wikipedia.org. [26] J. Tang, L. Niu, J. Liu, Y. Wang, Z. Huang, S. Xie, L. Huang, Q. Xu, Y. Wang, L.A. Belfiore, Mater. Sci. Eng. C 34 (2014) 186. [27] A. Inaba, K. Yoo, Y. Takei, K. Matsumoto, I. Shimoyama, Sens. Actuators B 195 (2014) 15. [28] B.C. Janegitz, F.A. dos Santos, R.C. Faria, V. Zucolotto, Mater. Sci. Eng. C 37 (2014) 14. [29] S.P. Zhang, B. Liu, C.Y. Li, W. Chen, Z.J. Yao, D.T. Yao, R.B. Yu, H.O. Song, Chin. Chem. Lett. 25 (2014) 355.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Electrochemical determination of hydrochlorothiazide and folic acid in real samples using a modified graphene oxide sheet paste electrode Hadi Beitollahi a,⁎, Mozhdeh Hamzavi b, Masoud Torkzadeh-Mahani c a b c
Environment Department, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran Department of Chemistry, Graduate University of Advanced Technology, Kerman, Iran Biotechnology Department, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran
a r t i c l e
i n f o
Article history: Received 11 February 2014 Received in revised form 24 January 2015 Accepted 22 March 2015 Available online 24 March 2015 Keywords: Hydrochlorothiazide Folic acid Carbon paste electrode Graphene oxide sheets
a b s t r a c t A new ferrocene-derivative compound, 2-chlorobenzoyl ferrocene, was synthesized and used to construct a modified graphene oxide sheet paste electrode. The electrooxidation of hydrochlorothiazide at the surface of the modified electrode was studied. Under optimized conditions, the square wave voltammetric (SWV) peak current of hydrochlorothiazide increased linearly with hydrochlorothiazide concentration in the range of 5.0 × 10−8 to 2.0 × 10−4 M and a detection limit of 20.0 nM was obtained for hydrochlorothiazide. The diffusion coefficient and kinetic parameters (such as electron transfer coefficient and the heterogeneous rate constant) for hydrochlorothiazide oxidation were also determined. The prepared modified electrode exhibits a very good resolution between the voltammetric peaks of hydrochlorothiazide and folic acid which makes it suitable for the detection of hydrochlorothiazide in the presence of folic acid in real samples. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Thiazidic diuretics, such as hydrochlorothiazide, which is a prototype of thiazide drugs comprising of an important class of diuretics, increase the rate of urinary excretion of sodium and water by sodium reabsorption inhibition in the renal tubules. Hydrochlorothiazide is used for the treatment of edemas associated with the heart (congestive heart failure), the liver (hepatic cirrhosis) and the kidneys (nephrotic syndrome, chronic renal failure, and acute glomerulonephritis). It has also been used for all degrees of hypertension, being efficient as antihypertensive agents of the other classes [1]. Various analytical methods have been described for the determination of hydrochlorothiazide in pharmaceutical preparations, blood and plasma, including chemiluminescence [2], capillary zone electrophoresis [3], spectrophotometric [4], high performance liquid chromatography (HPLC) [5], derivative spectroscopy [6], thin-layer chromatography [7] and liquid chromatographic methods [8]. The main deficiency of such methods lies in the fact that their use requires time-consuming derivatization techniques or that they suffer from low detection limits. Hence, there have been increasing demands for new, fast, simple, convenient and sensitive methods for the determination of hydrochlorothiazide. Therefore, given the advantages of having a lower cost, providing faster response, simple instrumentation, high sensitivity, facile miniaturization, and low power requirement, numerous electrochemical methods for the determination of hydrochlorothiazide have been developed [9–13]. ⁎ Corresponding author. E-mail address: [email protected] (H. Beitollahi).
http://dx.doi.org/10.1016/j.msec.2015.03.031 0928-4931/© 2015 Elsevier B.V. All rights reserved.
Several chronic diseases (for example, gigantocytic anemia, leucopoenia, mentality devolution, psychosis, heart attack, and stroke), especially those concerned with malformation during pregnancy and carcinogenic processes, are related to the deficiency of folic acid [14] which is a water-soluble vitamin. Since folic acid is detected in biological fluids at very low concentrations, a highly specific and sensitive assay is called for. Available methods for this purpose are generally based on spectrophotometry [15], chromatography [16], fluorescence [17] or phosphorescence detection [18], together with bioassay [19]. Among the different methods, electrochemical methods are found to be very promising [20–24]. It has been discovered that when pregnant women consume hydrochlorothiazide due to high blood pressure, their body not only loses its necessary liquids and sodium but the level of folic acid in their body also decreases due to frequent urination. Therefore, as it is possible that a number of difficulties, such as congenital malformations and neural tube defects, may be caused by low levels of folic acid in a pregnant woman's body, they need to use 800 mg of folic acid, which is twice as much as the amount used by an ordinary person (400 mg). Hence, by the simultaneous determination of hydrochlorothiazide and folic acid, it is possible to identify the best type of treatment for each specific patient while decreasing the possible harm that is likely to occur as a result of side-effects caused by incorrect prescriptions [25]. A new kind of two-dimensional carbon material, graphene (G) has attracted increasing attention due to its unique properties including a high surface area, excellent electrical conductivity, quick electron mobility at room temperature, high mechanical strength, and ease of functionalization [26,27]. Graphene based electrochemical sensors
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for the determination of hydrochlorothiazide and folic acid in real samples. 2. Experimental 2.1. Apparatus and chemicals
Fig. 1. TEM image of synthesized graphene oxide sheets.
have been proven to possess excellent electrocatalytic ability and good performance [28–30]. Among all the carbon electrodes, the carbon paste electrode (CPE) is an appealing and widely used electrode material in the fields of electrochemistry, electroanalysis, etc. due to its attractive advantages, such as simple preparation, low-cost implementation, renewability, low background current, and wide potential window [31–35]. The preparation of CPE usually involves the dispersion of graphite powder in a hydrophobic binder to form a homogeneous paste, followed by filling a tube with the resulting paste [36,37]. Sometimes, the modifiers including various biomolecules, organic compounds and nanoparticles are also doped into the carbon paste, which makes CPE quite suitable for bioanalysis [38–44]. To the best of our knowledge, no study has been published so far reporting the simultaneous electrocatalytic determination of hydrochlorothiazide and folic acid using a modified graphene oxide sheet paste electrode. Thus, in this paper, the preparation and suitability of a 2chlorobenzoyl ferrocene (2CBF) modified graphene oxide sheet paste electrode (2CBFGPE) as a new electrode in the electrocatalysis and determination of hydrochlorothiazide in an aqueous buffer solution was initially described. Then the analytical performance of the modified electrode in quantification of hydrochlorothiazide in the presence of folic acid was also evaluated. Finally this new constructed electrochemical sensor was used
The electrochemical measurements were performed with an Autolab potentiostat/galvanostat (PGSTAT 302N, Eco Chemie, the Netherlands). The experimental conditions were controlled with the General Purpose Electrochemical System (GPES) software. A conventional three electrode cell was used at 25 ± 1 °C. An Ag/AgCl/KCl (3.0 M) electrode, a platinum wire, and 2CBFGPE were used as the reference, auxiliary and working electrodes, respectively. Finally a Metrohm 710 pH meter was used for pH measurements. All solutions were freshly prepared with double distilled water. Hydrochlorothiazide, folic acid and all of the other reagents were of analytical grade and were obtained from Merck (Darmstadt, Germany). The buffer solutions were prepared from orthophosphoric acid and its salts in the pH range of 2.0–11.0. 2.2. Synthesis of graphene oxide sheets Graphene oxide sheets were synthesized from natural graphite flakes based on the modified Hummers and Offeman method [45,46]. In a typical synthesis process, 1.0 g of pristine graphite flakes was immersed in 50 mL of formic acid, and then sonicated for 2 h at room temperature. These resulting graphite plates were washed with acetone, and then dried in an oven at 95 °C for 12 h. Then, 100 mL H2SO4 (95%) was added into a 500 mL flask, and cooled by immersion in an ice bath followed by stirring. About 1.0 g treated graphite powder and 0.5 g NaNO3 were added under vigorous stirring to avoid agglomeration. After the graphite powder was well dispersed, 3 g KMnO4 was added gradually under stirring and cooling so that the temperature of the mixture was maintained at below 10 °C. The mixture was stirred for 2 h and diluted with deionized double distilled water (in an ice bath). After that, 25 mL 15% H2O2 was slowly added to the mixture until the color of the mixture changed to brilliant yellow, indicating fully oxidized graphite. The as-obtained graphite oxide slurry was re-dispersed in deionized double distilled water and then exfoliated to generate graphene oxide
Fig. 2. FTIR of synthesized graphene oxide sheets.
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Table 1 Cyclic voltammetric data obtained for constructed 2CBFGPE in 0.1 M PBS (pH 7.0) at 10 mV s−1. Epa (V)
a
0.655 ± 1.1b a b
Fig. 3. SEM image of GPE.
sheets by sonication for 2 h. Then, the solution was filtered and washed with diluted HCl solution to remove metal ions. Finally, the product was washed with deionized double distilled water until the solution became acid free, and dried under vacuum at 50 °C. A typical transmission electron microscopy (TEM) for synthesized graphene oxide sheets is shown in Fig. 1. Also FTIR of synthesized graphene oxide sheets is shown in Fig. 2. In the FT-IR spectrum of the graphene oxide sample, a broad band in the range of 2500–3650 cm−1 corresponds to the stretching and bending vibration of OH groups of water molecules adsorbed on graphene oxide. The peaks at 1699 and 1369 cm−1 are C_O stretching vibration peaks of carboxyl and carbonyl and also, the peak at 1631 cm−1 is attributed to the stretching vibration of aromatic C_C. The absorption peaks at 1236 and 1099 cm−1 correspond to the C\O stretching vibration of an epoxy group and C\OH of alcohol, respectively. The presence of these oxygen-containing groups reveals that the graphite has been oxidized. 2.3. Synthesis of 2-chlorobenzoyl ferrocene To a 100 mL round-bottomed flask under argon atmosphere, 1.86 g (10 mmol) of ferrocene, 1.75 g (10 mmol) of 2-chlorobenzoyl chloride and 20 mL of dichloromethane is added. The reaction mixture is cooled in an ice bath (0–5 °C), then 1.40 g (11 mmol) of anhydrous aluminum chloride was added in small portions at such a rate that the reaction
Fig. 4. CVs of 2CBFGPE (A) and CPE (B) in 0.1 M PBS (pH 7.0). In all cases scan rate is 100 mV s−1.
Epc (V)
E1/2 (V)
ΔEp (V)
Ipa (μA)
Ipc (μA)
0.540 ± 1.2
0.597 ± 1.2
0.115 ± 1.1
9.5 ± 1.4
5.7 ± 1.6
Versus Ag/AgCl/KCl (3.0 M) as reference electrode. All the ‘±’ values are RSD% (n = 5).
mixture remains below 5 °C. The resulting solution is stirred for 30 min at 0–5 °C and 2 h at room temperature. Then, the flask is placed in an ice bath again and 20 mL of water is added cautiously to give a two-phase mixture. After stirring for 30 min the aqueous layer is extracted with two 15 mL portions of dichloromethane. The combined dichloromethane extracts are washed once with water, twice with 10% sodium hydroxide solution, dried over magnesium sulfide and evaporated at reduced pressure. The crude residue is purified by recrystallization from heptane to afford (2-chlorobenzoyl)ferrocene in 95% yield. M.p. 99–100 °C, 1
H NMR (400 MHz, CDCl3): δ (ppm) = 4.30 (s, 5H), 4.62 (t, J = 1.6 Hz, 2H), 4.77 (t, J = 1.6 Hz, 2H), 7.36 (dt, J = 1.6, 7.6 Hz, 1H), 7.42 (dt, J = 2.0, 7.6 Hz, 1H), 7.47 (dd, J = 1.2, 8.0 Hz, 1H), 7.52 (1.6, 7.2 Hz, 1H).
IR (KBr) (νmax, cm− 1): 3080.8, 3052.5 (C\H aromatic), 1644.9 (C_O), 1442.7, 1292.8, 1031.5, 827.7. 2.4. Preparation of the electrode The 2CBFGPEs were prepared by dissolving 0.01 g 2CBF in 3 mL diethyl ether and then adding this to 0.89 g graphite powder and 0.1 g graphene oxide sheets with a mortar and pestle. Then, 0.7 mL of paraffin was added to the above mixture and mixed for 15 min until a uniformly wetted paste was obtained. The paste was then packed into the end of a glass tube. A copper wire inserted into the carbon paste provided the electrical contact. When necessary, a new surface was obtained by pushing an excess of the paste out of the tube and polishing with a weighing paper. For comparison, a 2CBF modified CPE electrode (2CBFCPE) without G, a G paste electrode (GPE) without 2CBF, and an unmodified CPE in the absence of 2CBF and G were also prepared in the same way. A typical scanning electron microscope (SEM) image for GPE is shown in Fig. 3.
Fig. 5. CVs of 2CBFCPE (A) and 2CBFGPE (B) in 0.1 M PBS (pH 7.0) containing 100.0 μM hydrochlorothiazide. (C) and (D) are CVs of CPE and GPE in 0.1 M PBS (pH 7.0) containing 100.0 μM hydrochlorothiazide and (E) is CV of CPE in 0.1 M PBS (pH 7.0) In all cases scan rate is 10 mV s−1.
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Fig. 6. CVs of 2CBFGPE in 0.1 M PBS (pH 7.0) containing 10.0 μM hydrochlorothiazide at various scan rates; numbers 1–5 correspond to 5, 10, 20, 30 and 40 mV s−1, respectively. Insets: variation of (A) anodic peak current vs. square root of scan rate; (B) normalized current (Ip/ν1/2) vs. ν and (C) anodic peak potential (Ep) vs. logarithm of scan rate.
Fig. 7. Chronoamperograms obtained at 2CBFGPE in 0.1 M PBS (pH 7.0) for different concentrations of hydrochlorothiazide. The numbers 1–6 correspond to 0.0, 0.1, 0.2, 0.4, 0.6 and 1.0 mM of hydrochlorothiazide. Insets: (A) plots of I vs. t−1/2 obtained from chronoamperograms 2–6. (B) Plot of the slope of the straight lines against hydrochlorothiazide concentration. (C) Dependence of Ic/Il on t1/2 derived from the data of chronoamperograms 1–6.
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Fig. 8. SWVs of 2CBFGPE in 0.1 M PBS (pH 7.0) containing different concentrations of hydrochlorothiazide. Numbers 1–15 correspond to 0.05, 0.35, 1.0, 2.5, 5.0, 7.5, 10.0, 25.0, 50.0, 75.0, 100.0, 125.0, 150.0, 175.0 and 200.0 μM of hydrochlorothiazide. Inset: plots of the electrocatalytic peak current as a function of hydrochlorothiazide concentration in the range of 0.05 to 200.0 μM.
2.5. Procedure for the preparation of real samples Five hydrochlorothiazide tablets (labeled 50 mg) were ground and a hydrochlorothiazide solution was prepared by dissolving 50 mg of the powder in 100 mL water by ultrasonication. Then, different volumes of the diluted solution were transferred into a 10 mL volumetric flask and were diluted to the mark with phosphate buffer solution (PBS) (pH 7.0). The hydrochlorothiazide content was analyzed by the proposed method using the standard addition method. Also, five folic acid tablets (labeled 1.0 mg) were ground and the tablet solution was prepared by dissolving 50 mg of the powder in 100 mL water by ultrasonication. Then, different volumes of the diluted solution were transferred into a 10 mL volumetric flask and were diluted to the mark with PBS (pH 7.0). The folic acid content was analyzed by the proposed method using the standard addition method. Urine samples of healthy people were stored in a refrigerator immediately after collection. Ten milliliters of the sample was centrifuged for 15 min at 2000 rpm. The supernatant was filtered out using a 0.45 μm filter. Then, a different volume of the solution was transferred into a 25 mL volumetric flask and diluted to the mark with PBS (pH 7.0). The diluted urine sample was spiked with different amounts of hydrochlorothiazide and folic acid. The serum sample of healthy people was prepared from a local laboratory. It was centrifuged and after filtering, diluted with PBS (pH 7.0) without any further treatment. The diluted serum sample was spiked with different amounts of hydrochlorothiazide and folic acid. 3. Results and discussion 3.1. Electrochemical behavior of 2CBFGPE 2CBFGPE was prepared and its electrochemical behaviors were studied using a cyclic voltammetry (CV) technique (Fig. 4). The experimental
results show well-defined and reproducible anodic and cathodic peaks related to a 2-chlorobenzoyl ferrocene/2-chlorobenzoyl ferricenium ion (Fc/Fc+) redox system, which show a quasireversible behavior in an aqueous medium. The electrode capability for the generation of a reproducible surface was examined by cyclic voltammetric data obtained in optimum solution at pH 7.0 from five separately prepared 2CBFGPEs (Table 1). The calculated relative standard deviation (RSD) for various parameters is accepted as the criteria for a satisfactory surface reproducibility (about 1–4%), which is virtually the same as that expected for the renewal or ordinary carbon paste surface. However we regenerated the surface of 2CBFGPE before each experiment based on our previous results. In addition, the long-term stability of 2CBFGPE was tested over a three-week time period. Once the CVs were recorded after the modified electrode was stored in atmosphere at an ambient temperature, the peak potential for hydrochlorothiazide oxidation was unchanged and the current signals showed less than 2.3% decrease relative to the initial response. The antifouling properties of the modified electrode toward hydrochlorothiazide and its oxidation products were investigated by recording the CVs of the modified electrode before and after use in the presence of hydrochlorothiazide. CVs were recorded in the presence of
Table 2 Comparison of the efficiency of some modified electrodes used in the determination of hydrochlorothiazide. Electrode
Method
Carbon paste Boron-doped diamond Boron-doped diamond Glassy carbon Carbon paste
Voltammetry Voltammetry
LOD (M) 3.7 × 10−4 1.2 × 10−6
LDR (M) 8.0 × 10−8–5.0 × 10−4 3.0 × 10−6–7.4 × 10−5
Ref. [10] [13]
Voltammetry 6.39 × 10−7 1.97 × 10−6–8.81 × 10−5 [49] Voltammetry Voltammetry
4.3 × 10−9 2.0 × 10−8
4.0 × 10−6–4.0 × 10−5 5.0 × 10−8–2.0 × 10−4
[50] This work
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Fig. 9. SWVs of 2CBFGPE in 0.1 M PBS (pH 7.0) containing 1000.0 μM folic acid and different concentrations of hydrochlorothiazide. From inner to outer: 50.0, 65.0, 90.0, 110.0, and 125.0 μM of hydrochlorothiazide. Inset: plot of the electrocatalytic peak current as a function of hydrochlorothiazide concentration.
hydrochlorothiazide after having cycled the potential 20 times at a scan rate of 10 mV s−1. The peak potentials were unchanged and the currents decreased by less than 2.4%. Therefore, at the surface of 2CBFGPE, not only the sensitivity increased, but also, the fouling effect of the analyte and its oxidation product also decreased. 3.2. Electrocatalytic oxidation of hydrochlorothiazide at 2CBFGPE The electrochemical behavior of hydrochlorothiazide is dependent on the pH value of the aqueous solution, whereas the electrochemical
properties of an Fc/Fc+ redox couple are independent of pH [22,24,33, 43]. Therefore, pH optimization of the solution seems to be necessary in order to obtain the electrocatalytic oxidation of hydrochlorothiazide. Thus the electrochemical behavior of hydrochlorothiazide was studied in different pH values (2.0 b pH b 11.0) at the surface of 2CBFGPE by CV. It was found that the electrocatalytic oxidation of hydrochlorothiazide at the surface of 2CBFGPE was more favored under neutral conditions than in acidic or basic medium. This appears as a gradual growth in the anodic peak current and a simultaneous decrease in the cathodic peak current in the CVs of 2CBFGPE. Thus, pH 7.0 was chosen as the optimum pH for electrocatalysis of hydrochlorothiazide oxidation at the surface of 2CBFGPE. Fig. 5 depicts the CV responses for the electrochemical oxidation of 100.0 μM hydrochlorothiazide at unmodified CPE (curve C), GPE (curve D), 2CBFCPE (curve A) and 2CBFGPE (curve B). As can be seen, while the peak potential for hydrochlorothiazide oxidation at the GPE, and unmodified CPE are 910 and 970 mV, respectively, the corresponding potential at 2CBFGPE and 2CBFCPE is ~ 655 mV. These results indicate that the peak potential for hydrochlorothiazide oxidation at 2CBFGPE and 2CBFCPE shifts by ~ 255 and 315 mV toward negative values compared to GPE and unmodified CPE, respectively. However, 2CBFGPE shows a much higher anodic peak current for the oxidation of hydrochlorothiazide compared to 2CBFCPE, indicating that the combination of G and the mediator (2CBF) has significantly improved the performance of the electrode toward hydrochlorothiazide oxidation. 2CBFGPE, in 0.1 M PBS (pH 7.0) and without hydrochlorothiazide in solution (Fig. 4 curve A), exhibited a well-behaved redox reaction and with addition of 100.0 μM hydrochlorothiazide, increased the anodic peak current (Fig. 5 curve B), indicating a strong electrocatalytic effect [47]. The effect of potential scan rate on the electrocatalytic oxidation of hydrochlorothiazide at 2CBFGPE was investigated by CV (Fig. 6). As can be observed in Fig. 6, the oxidation peak potential shifted to more positive potentials with an increasing scan rate, confirming the kinetic limitation in the electrochemical reaction. Also, a plot of peak height (Ip) vs. the square root of scan rate (ν1/2) was found to be linear in the
Fig. 10. SWVs of 2CBFGPE in 0.1 M PBS (pH 7.0) containing different concentrations of hydrochlorothiazide and folic acid in μM, from inner to outer: 15.0 + 100.0, 25.0 + 150.0, 40.0 + 250.0, 50.0 + 500.0, 90.0 + 750.0, 115.0 + 1500.0 and 160.0 + 2000.0 respectively. Insets: (A) plots of Ip vs. hydrochlorothiazide concentration and (B) plot of Ip vs. folic acid concentrations.
H. Beitollahi et al. / Materials Science and Engineering C 52 (2015) 297–305
303
Table 3 The application of 2CBFGPE for simultaneous determination of hydrochlorothiazide and folic acid in their tablet samples (n = 5). Sample
Spiked (μM)
Hydrochlorothiazide tablet
Folic cid tablet
Found (μM)
Recovery (%)
Folic acid
Hydrochlorothiazide
Folic acid
Hydrochlorothiazide
Folic acid
Hydrochlorothiazide
Folic acid
0 10.0 20.0 30.0 40.0 0 15.0 25.0 35.0 45.0
0 100.0 125.0 150.0 175.0 0 20.0 40.0 60.0 80.0
12.0 22.3 31.2 41.6 53.3 ND 15.2 24.6 35.9 44.6
ND 98.1 127.1 154.1 173.7 100.0 118.3 141.1 159.1 182.3
– 101.4 97.5 99.0 102.5 – 101.3 98.4 102.6 99.1
– 98.1 101.7 102.7 99.3 – 98.6 100.8 99.4 101.3
3.3 2.1 2.9 1.7 3.3 – 3.4 1.7 2.9 2.5
– 3.2 2.1 2.3 2.4 2.9 2.7 3.1 2.1 1.9
range of 5–40 mV s−1, suggesting that, at sufficient overpotential, the process is diffusion rather than surface controlled (Fig. 6A). A plot of the scan rate-normalized current (Ip/ν1/2) vs. scan rate (Fig. 6B) exhibits the characteristic shape typical of an electrocatalytic (EC') process [47]. The Tafel slope (b) can be obtained from the slope of Ep vs. log ν using Eq. (1): [47] Ep ¼ b=2 log ν þ constant
ð1Þ
The Tafel slope was found to be 112.0 mV (Fig. 6C), which indicates that a one-electron transfer process is the rate limiting step assuming that a transfer coefficient (α) is about 0.47. 3.3. Chronoamperometric measurements Chronoamperometric measurements of hydrochlorothiazide at 2CBFGPE were carried out by setting the working electrode potential at 0.75 V for the various concentrations of hydrochlorothiazide in 0.1 M PBS (pH 7.0) (Fig. 7). For an electroactive material (hydrochlorothiazide in this case) with a diffusion coefficient of D, the current observed for the electrochemical reaction at the mass transport limited condition is described by the Cottrell equation [47]. Experimental plots of I vs. t−1/2 were employed, with the best fits for different concentrations of hydrochlorothiazide (Fig. 7A). The slopes of the resulting straight lines were then plotted vs. hydrochlorothiazide concentration (Fig. 7B). From the resulting slope and Cottrell equation the mean value of the D was found to be 1.1 × 10−5 cm2/s which is comparable with values reported by other research groups (1.1 × 10−5 cm2/s [10] and 1.75 × 10−5 cm2/s [12]). Chronoamperometry can also be employed to evaluate the catalytic rate constant, k, for the reaction between hydrochlorothiazide and 2CBFGPE according to the method described by Galus [48]: 1=2
IC =IL ¼ γ
h
π
1=2
i 1=2 1=2 þ exp ð−γÞ=γ erf γ
ð2Þ
where IC is the catalytic current of hydrochlorothiazide at 2CBFGPE, IL is the limited current in the absence of hydrochlorothiazide and γ = kCbt is the argument of the error function (Cb is the bulk concentration of hydrochlorothiazide). In cases where γ exceeds the value of 2, the error function is almost equal to 1 and therefore, the above equation can be shortened to: IC = IL ¼ π
1=2 1=2
γ
¼ π
1=2
RSD (%)
Hydrochlorothiazide
1=2
ðkCb tÞ
ð3Þ
where t is the time elapsed. The above equation can be used to calculate the rate constant, k, of the catalytic process from the slope of IC/IL vs. t1/2 at a given hydrochlorothiazide concentration. From the values of the slopes (Fig. 7C), the average value of k was found to be 9.1 × 102 M−1 s−1 which is comparable with values reported by other research groups (6.38 × 103 M−1 s−1 [10] and 3.38 × 102 M−1 s−1 [12]).
3.4. Calibration plot and limit of detection The electrocatalytic peak current of hydrochlorothiazide oxidation at the surface of 2CBFGPE can be used for the determination of hydrochlorothiazide in solution. Therefore, square wave voltammetry (SWV) experiments were performed using a modified electrode in 0.1 M PBS (pH 7.0) containing various concentrations of hydrochlorothiazide (Fig. 8). The plot of peak current vs. hydrochlorothiazide concentration consisted of a linear segment with slope of 0.135 μA/μM−1 in the concentration range of 5.0 × 10−8–2.0 × 10−4 M. The detection limit (3σ) of hydrochlorothiazide was found to be 20.0 nM. This value is comparable with values reported by other research groups for the determination of hydrochlorothiazide (see Table 2).
3.5. Simultaneous determination of hydrochlorothiazide and folic acid To our knowledge, no study has used the modified graphene oxide sheet electrode and specially modified graphene oxide sheet paste electrode for simultaneous determination of hydrochlorothiazide and folic acid and this is the first report for simultaneous determination of hydrochlorothiazide and folic acid using the modified graphene oxide paste electrode. The determination of hydrochlorothiazide and folic acid in mixtures was performed at 2CBFGPE using SWV. The concentration of hydrochlorothiazide was varied, while keeping the folic acid concentration constant. The results are shown in Fig. 9. When the concentration of folic acid is kept constant at 1000.0 μM, the peak current of hydrochlorothiazide is proportional to its concentration. No changes in the peak current and potential of folic acid can be found. Also, determination of two compounds was performed by simultaneously changing the concentrations of hydrochlorothiazide and folic acid, and recording the SWVs (Fig. 10). The voltammetric results showed well-defined anodic peaks at potentials of 600 and 820 mV, corresponding to the oxidation of hydrochlorothiazide and folic acid, respectively, indicating that simultaneous determination of these compounds is feasible at 2CBFGPE as shown in Fig. 10. The sensitivity of the modified electrode toward the oxidation of hydrochlorothiazide was found to be 0.134 μA/μM−1. This is very close to the value obtained in the absence of folic acid (0.135 μA/μM−1, see Section 3.4), indicating that the oxidation processes of these compounds
Table 4 Comparison of the total values of hydrochlorothiazide and folic acid of some pharmaceutical preparations obtained using 2CBFGPE with declared values in the label of the samples (n = 5). Samples
Declared value
Found value
RSD%
Hydrochlorothiazide tablet (mg per tablet) Folic acid tablet (mg per tablet)
50.00 1.00
50.10 1.05
2.7 2.5
304
H. Beitollahi et al. / Materials Science and Engineering C 52 (2015) 297–305
Table 5 The application of 2CBFGPE for simultaneous determination of hydrochlorothiazide and folic acid in human blood serum and urine samples (n = 5). Sample
Human blood serum
Urine
Spiked (μM)
Found (μM)
Recovery (%)
RSD (%)
Hydrochlorothiazide
Folic acid
Hydrochlorothiazide
Folic acid
Hydrochlorothiazide
Folic acid
Hydrochlorothiazide
Folic acid
10.0 20.0 30.0 40.0 17.5 27.5 37.5 47.5
120.0 140.0 160.0 180.0 115.0 135.0 155.0 175.0
10.3 19.6 30.2 39.6 17.2 27.7 37.1 49.2
118.1 141.9 159.2 175.3 117.1 133.2 160.1 172.1
103.0 98.0 100.7 99.0 98.3 100.7 98.9 103.6
98.4 101.3 99.5 97.4 101.8 98.7 103.3 98.3
2.1 3.2 1.7 2.9 2.6 2.2 1.9 3.5
1.7 2.9 2.8 3.3 3.1 2.8 2.7 1.8
at 2CBFGPE are independent and therefore, simultaneous determination of their mixtures is possible without significant interferences.
3.6. Interference studies The influence of various substances as compounds potentially interfering with the determination of hydrochlorothiazide was studied under optimum conditions. The potentially interfering substances were chosen from the group of substances commonly found with hydrochlorothiazide in pharmaceuticals and/or in biological fluids. The tolerance limit was defined as the maximum concentration of the interfering substance that caused an error of less than ±5% in the determination of hydrochlorothiazide. According to the results, neither a 600-fold +2 excess of Mg2+, Al3+, NH+ , Fe+3, F−, SO2− and S2−, nor a 4004 , Fe 4 fold excess of L-lysine, glucose, NADH, acetaminophen, uric acid, L-asparagine, L-serine, L-threonine, L-proline, L-histidine, L-glycine, L-tryptophan, L-phenylalanine, lactose, saccharose, fructose, benzoic acid, methanol, ethanol, urea, and caffeine, did not show interference in the determination of hydrochlorothiazide.
3.7. Real sample analysis 3.7.1. Determination of hydrochlorothiazide and folic acid in pharmaceutical preparations In order to evaluate the analytical applicability of the proposed method, it was also applied to the determination of hydrochlorothiazide and folic acid in their tablets. The results are listed in Table 3. The reliability of the proposed modified electrode was also evaluated by comparing the obtained results with those declared in the label of pharmaceutical preparations (Table 4). The results in Table 3 show that the relative standard derivations (RSD) and the recovery rates of the spiked samples are acceptable. Also, the data in Table 4 indicate that the results obtained by utilizing 2CBFGPE are in good agreement with those declared in the label of the preparations. Thus, the modified electrode can be efficiently used for individual or simultaneous determination of hydrochlorothiazide and folic in pharmaceutical preparations.
3.7.2. Determination of hydrochlorothiazide and folic in human blood serum and urine samples In order to evaluate the analytical applicability of the proposed method, it was additionally applied for the determination of hydrochlorothiazide and folic acid in human blood serum and urine samples. The results for determination of the two species in real samples are given in Table 5. Satisfactory recovery of the experimental results was found for hydrochlorothiazide and folic acid. The reproducibility of the method was demonstrated by the mean relative standard deviation (RSD).
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