Multiwall carbon nanotube-ionic liquid modified paste electrode as an efficient sensor for the determination of diazepam and oxazepam in real samples M.A. Zare*, M. Saber Tehrani, S. Waqif Husain, P. Aberoomand Azar Department of Chemistry, Science and Research Branch, Islamic Azad University, P.O. Box: 1477893855, Tehran, Iran E-mail:
[email protected] (Corresponding Author) Abstract A carbon-based electrode using multiwall carbon nanotube as a modifier and room temperature ionic liquid as a binder has been applied for the determination of diazepam (DZP) and oxazepam (OZP) in real samples including serum, urine and tablets. Square wave voltammetry as an appropriate electrochemical technique was applied to achieve improved limits of detection and higher sensitivities. The electrochemical studies were investigated under various experimental conditions such as pH, buffer concentration, ionic strength, deposition potential, deposition time and scan rate to achieve higher sensitivities. Linear concentration ranges for DZP and OZP were 0.02–0.76 mgL−1 and 0.05–1.90 mgL−1 with the detection limits of 4.1 µgL−1 and 5.8 µgL−1, respectively. The proposed method was successfully applied for the analysis of commercially available tablets as well as serum and urine samples and satisfactory results were obtained.
Keywords: Diazepam, oxazepam, ionic liquid, multiwall carbon nanotube, serum
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1. Introduction Benzodiazepines such as diazepam, oxazepam, nordiazepam, medazepam, temazepam etc. are a group of drugs with important clinical applications. They are widely prescribed as anxiolytic, hypnotic, anticonvulsant, myorelaxant, amnesic agents sleep regulator agents [1,2]. The major impact of these compounds operates widely in the brain, affecting emotional reactions, memory, thinking, muscle tone and coordination [3]. Diazepam (DZP) and oxazepam (OZP) are two representative benzodiazepines. DZP (7-chloro-1-methyl-5-phenyl-3H-1,4-benzodiazepin-2(1H)one) is mainly used to treat anxiety, insomnia, and symptoms; and OZP (7-chloro-1,3-dihydro-3hydroxy-5-phenyl-2H-1,4-benzod-iapin-2-one) is indicated for the acute management of aggressive or delirious patients. These applications gained popularity among medical professionals, increasing the number of pharmaceutical preparations in the market and consequently a need of development of different techniques for quality control. However, abuse of DZP can have serious consequences, even causing death when taken in overdose [4]. DZP as well as many 1,4-benzodiazepines is metabolized in the organisms to OZP [5]. As all hydroxylated metabolites coming from DZP, OZP is excreted in bile and urine in the form of glucuronide conjugates. These glucuronides are establishing enterohepatic circulation, which is one of the factors affecting more the duration of the effect of these pharmaceuticals. So, it is implied that the determination of OZP and DZP in biological samples is very important in patients’ health point of view. Because, the accurate control of these substances gives important information about their plasmatic concentrations in order to adequate the dose of these compounds to the needs of the patients. Various analytical methods including chromatography [6-8], spectrophotometry [2,9], spectrofluorimetry
[10,11],
enzymatic
and
immunoassay 2
methods
[12,13],
capillary
electrophoresis [14,15] and electrochemical techniques [5,16-20] have been reported for the determination of 1,4-benzodiazepines especially diazepam and its metabolites in biological samples. Electrochemical techniques have attracted special attempts for their simplicity, low cost and adaptability for in situ analysis. Few reports are anent the electrochemical measurements of DZP or OZP, in which they were investigated by a lead film electrode [21], sonogel-carbon electrode modified with bentonite [3], screen printed sensor [20], and carbon paste electrode modified by zeolite and bentonite [5]. It was also reported that the presence of various forms of CNT on the surface of modified electrodes cause a considerable improvement in their electrochemical responses toward many biologically important compounds [22-27]. So, the work on CNT-modified electrodes seems to be growing for biosensing purposes. Ionic liquid based carbon electrodes have attracted more attention for the determination of biologically important compounds and pharmaceuticals [28-31]. Among them, CNT-modified carbon ionic liquid electrodes due to the promoting electron transfer by CNT between the electroactive species and electrodes, have been proposed as appropriate electrodes for biological applications [32-34]. In this work, multiwall carbon nanotube (MWCNT)-modified electrode by using ionic liquid as binder is proposed for the fabrication of a biosensor for the determination of diazepam (DZP) and oxazepam (OZP) in real biological as well as pharmaceutical samples. To achieve higher sensitivities, square wave voltammetry (SWV) was used as an appropriate electrochemical technique. To the best of our knowledge, there is not any report on the determination of DZP and OZP by carbon ionic liquid electrode (CILE) modified with MWCNT. The modification method is rather simple and the reproducibility of the method is satisfied. The method was applied to the determination of DZP and OZP in real samples. 3
2. Experimental 2.1. Materials All chemicals were of analytical grade and were used as received without any further purification. Diazepam and oxazepam were purchased from Kimidarou Farma and Hakim Farma (Tehran, Iran). 1-Iodooctane and pyridine as reagents for the synthesis of ionic liquid, 1octylpyridinum hexafluorophosphate, paraffin oil and diethyl ether were obtained from Merck. The ionic liquid, 1-octylpyridinum iodide, was synthesized as described elsewhere [35]. 1Octylpyridinum hexafluorophosphate (OPFP) was prepared by anion exchange of 1octylpyridinum iodide with ammonium hexafluorophosphate (Fluka). Graphite powder (5-9 µm, >99.5%), multiwall carbon nanotube used in the electrodes preparation were from SigmaAldrich. All solutions were prepared using double distilled water. The buffer solutions of pH 7.0 and pH 4.0 were prepared by adding the appropriate volumes of 5.0 M of NaOH (Merck) in corresponding acid solution containing 0.5M phosphoric acid (Merck) and 0.8M acetic acid (Merck), respectively. The stock solutions of DZP (1.00 gL-1) and OZP (0.500 gL−1) were prepared by dissolving the substance in ethanol:H2O (3:2 v/v) and methanol:H2O (2:3 v/v), respectively, and diluting with solvent to the mark . All these solutions were maintained under refrigerated conditions in the absence of light. Pharmaceutical formulations including Valium® tablet formulated to contain 5 mg DZP per tablet, and OZP tablet formulated to contain 10 mg OZP per tablet were obtained from Kimidarou Farma (Tehran, Iran) and Hakim Farma (Tehran, Iran), respectively.
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2.2. Instrumentation All electrochemical measurements were performed on Electrochemical Work Station, model Autolab PGSTAT 302N (Nova, Netherlands). Voltammetric experiments were carried out in the buffered solutions of DZP and OZP, deoxygenated by purging the pure nitrogen (99.99%). During the experiments, nitrogen gas was passed over the surface of the test solutions in order to avoid entrance of oxygen into the solution. The Autolab software, NOVA 1.8 was used for waveform generation and data acquisition and elaboration. A three-electrode system, employing a platinum wire and Ag/AgCl (KCl 3.0M) were used as auxiliary and reference electrodes, respectively. Different working electrodes were used in this study including carbon ionic liquid electrode (CILE), carbon paste electrode (CPE) (1.8 mm in diameter) and glassy carbon electrode (GCE) (Metrohm, 3.0 mm inner diameter). SWV and cyclic voltammery (CV) were applied to carry out the electrochemical study of DZP and OZP. pH measurements were performed using WTW701 pH meter.
2.3. Preparation of the working electrodes Conventional CPE was prepared by hand-mixing of paraffin oil and graphite powder with a ratio of 70/30 graphite/paraffin oil (w/w). The paste was packed into the cavity of a Teflon tube (1.8 mm diameter). MWCNT/CILE was prepared by hand-mixing of the graphite powder, MWCNT and OPFP with a ratio of 35: 15: 50 (%w/w), respectively. A portion of the resulting paste was packed firmly into the cavity (1.8 mm i.d.) of a Teflon holder. The electrode was then heated either in an oven to a temperature higher than the melting point of OPFP (mp: 65 ºC) or by using a domestic hair drier for 2 min. It was then left to cool to room temperature [28]. The
5
electric contact was established via a copper wire. A new surface of CPE, CILE or MWCNT/CILE was obtained by smoothing the electrode onto a weighing paper.
3. Results and discussion Cyclic voltammetry (CV) was used to study the electrochemical behaviour of OZP and DZP on MWCN/CILE and square wave voltammetry (SWV) was used for the determination of these species. The instrumental parameters in SWV technique including frequency, square-wave potential amplitude and potential step height were optimized to get the best analytical performance for DZP and OZP. These are given in Supporting Information (Fig.’s S1 and S2). CV of 10.0 mgL-1 of DZP in 0.5 M of PBS solution (pH 7.0) and 10.0 mgL-1 of OZP in acetate buffer solution (pH 5.0) on MWCNT/CILE (15%, w/w) at the scan rate of 100 mVs-1 are given in Fig. 1. The cathodic peak due to the reduction of DZP at MWCNT/CILE appears at about -0.99 V. In comparison with that of CILE (Fig. 1a), CPE and GCE (Fig. S3), the cathodic peak potential of DZP on MWCNT/CILE is shifted to more positive potentials and the peak current is increased. The superiority of IL modified electrode can be attributed to its inherent electrocatalytic activity which is the consequence of substituting the traditional liquid paraffin with IL as it was reported previously [36,37] as well as electrocatalytic activity of WMCNT [32]. The peak potential of MWCNT/CILE for DZP reduction is more positive than that reported on screen printed sensor (-1.5 V) [20] and zeolite-modified CPE (-1.320V) [5]. As for DZP, the reduction peak potential of OZP on MWCNT/CILE (Fig. 1c) has also been shifted to more positive potentials compared to CILE (Fig. 1d), CPE and GCE (Fig. S3). The reduction peak for OZP on MWCNT/CILE is occurred in -0.92 V vs. Ag/AgCl.
6
3.1. The effect of pH and supporting electrolytes
The effect of pH on the reduction peak potentials of DZP and OZP was studied by linear
sweep voltammetry (LSV). It is observed that for both drugs (Fig.’s S4 and S5), as pH of the solution is increased, the reduction peak potentials (Ep) are shifted to more negative values, indicating the involvement of protons in the electrochemical reactions of both OZP and DZP on MWCNT/CILE. From the plots of Ep vs. pH of the solution, the slopes of -54.47 mV/pH and 55.1 mV/pH were achieved in the pH range of 3.0 to 9.0 for DZP and OZP, respectively (Fig. S6). It can be concluded that the electrochemical reduction of DZP and OZP is 2-electron/2proton process as reported previously [3,20]. The effect of pH values on the reduction peak currents (ip) of DZP and OZP on MWCNT/CILE was also studied. From the obtained results, the optimal pH values for DZP and OZP are pH 7.0 and pH 5.0, respectively. As an interesting result, OZP does not show any reduction peak at pH > 8.5. This observation i.e. electro-inactivity of OZP at high pH values has also been reported previously by Chaves et.al. [5]. After the addition of OZP in PBS buffer solution, pH 8.5, SWV of MWCNT/CILE immersed in solution did not show any reduction peak corresponding to the electrochemical reduction of OZP (Fig. 2). By the addition of DZP in solution, the reduction peak corresponding to DZP was appeared in -1.05 V. By the sequential addition of OZP in solution, the peak current and potential of DZP was not changed indicating that DZP can be selectively determined in the presence of OZP by adjusting the pH value of the solution to pH > 8.5. For investigating the effect of ionic strength on voltammograms of DZP and OZP, KNO3 was added into the solution up to 0.65 M. For DZP, the higher the added KNO3 concentration up to 0.45 M, the better the signal obtained i.e. the ip values increased with increase in ionic strength of
7
solution. For OZP, however, the peak current was not changed significantly by increase in ionic strength. So, due to the highest peak intensity of DZP in solution containing 0.45 M of KNO3, this optimized condition was chosen for the next studies.
3.2. The effect of amount of MWCNT on the electrochemical behavior of DZP and OZP on MWCNT/CILE
The influence of the amounts of MWCNT contained in MWCNT/CILE modified electrode on the SWV responses of DZP and OZP were evaluated. Four CILE-modified electrodes containing different amounts of MWCNT (0, 5, 15 and 25 % w/w) were prepared and examined for their SWV signals in solutions containing DZP and OZP under the optimized conditions. By the addition of MWCNT up to 15% to the CILE matrix, the peak current was increased and then decreased beyond 15% MWCNT. The observed increase in the cathodic peak current, using MWCNT/CILE, represents the electrocatalytic activity of MWCNT and the larger microscopic area of the modified electrode. However, at higher amounts of MWCNT, capacitive background current was increased, caused to worsen the signal to noise ratio in the electrochemical measurements. Maximum peak currents for both DZP and OZP were obtained in 15% MWCNT contained in MWCNT-modified CILE. Therefore, 15% MWCNT was selected as the optimum amount for the preparation of the MWCNT/CILE modified sensor.
3.3 Study of the potential sweep rate effects Cyclic voltammetry for DZP and OZP was carried out at different scan rates. Results indicated that there is a linear relationship between the peak current (ip) of DZP and the scan rate (ν) in the range of 0.05 to 1.0 Vs-1 (Fig. S7). The peak current increased linearly with the scan rate (ν) and 8
the corresponding linear equation is I (µA) = 13.24 ν+ 0.41 with a correlation coefficient (R2) of 0.994. This means that the electrochemical process of DZP at the MWCNT/CILE is an adsorption-controlled mechanism. In addition, with increase in the scan rate, Ep has a slight shift toward more negative potentials, indicating slow kinetic reaction corresponding to quasireversible reactions. The effect of scan rate on voltammograms of OZP was also studied. The results indicated a linear relationship between the reduction peak current of OZP and the square root of the scan rate (ν1/2) in the ν range of 0.05 Vs-1 to 5.0 Vs-1 (Fig. S8). The regression equation was I (µA) = 17.78 ν1/2 – 3.853, with R2 = 0.996. This indicates the diffusion-controlled process of electrochemical reduction of OZP on MWCNT/CILE.
3.4. Effect of accumulation potential and time As described in previous section, the linear ip- ν relation for DZP indicates the adsorptioncontrolled mechanism of electrochemical DZP reduction. So, the accumulation of DZP and OZP on proposed electrode was investigated. The accumulation potential as well as accumulation time is an effective factor which affects the response sensitivity. The effect of accumulation potential on the peak current of DZP and OZP was examined over the range of +0.4 V to -0.8 V keeping the accumulation time to 60 s (Fig. S9). Clearly, peak currents for DPZ and OZP increased from +0.4 V to a maximum value at -0.3 V (vs. Ag/AgCl), then decreased at more negative potentials. So, -0.3 V was chosen as optimized accumulation potential for the further studies. The effect of accumulation time was also evaluated up to 240 s at the accumulation potential of -0.3 V (Fig. S10). It was observed that for DZP, the peak current increased with increase in the accumulation time up to 75 s, indicating the adsorption of DZP on MWCNT/CILE. These results confirm the adsorption-controlled mechanism of DZP reduction. So, the optimized accumulation time for 9
DZP was selected as 75s. However, for OZP, accumulation time up to 240 s did not have any significant effect on the response, confirming the diffusion-controlled process of OZP reduction on MWCNT/CILE.
3.5. Interference studies The important problem under the optimum conditions for the determination of DZP and OZP are the interference of several substances which are mostly companion drugs in biologicalfluids or pharmaceuticals. So, a dozen was investigated for their potential interferences (Table 1). Furthermore, the influence of some inorganic ions such as Zn2+, Ni2+, Fe2+, Fe3+ and Cu2+ were also studied. The tolerance limit was defined as the molar ratio of the additive/DZP or additive/OZP that caused an error less than 5% for the determination of 0.9 mgL-1 (3.16 µM) of DZP and 0.5 mgL-1 (1.74 µM) of OZP. Despite their interference effects, ascorbic acid and uric acid are not present at significant levels in the urine and tablet samples [38]. The normal level of ascorbic acid in blood is under 0.1 mM [39] and for uric acid in blood and urine is about 200-430 µM [40] and 1.48-4.43 mM [41], respectively, and the normal level of dopamine in blood and urine is less than 1-100 µM and 65-400 micrograms per 24 hours, respectively [42]. It should be noted that the reported concentration of dopamine, uric acid and ascorbic acid in Table 1 is higher than their respective level in blood serums and urine except for determination of DZP and OZP in the presence of dopamine, uric acid and ascorbic acid. As the serum was diluted ten times, the concentration of dopamine, uric acid and ascorbic acid for determination of DZP and OZP were also lower than their tolerance level. 10
3.6. Analytical figures of merits The analytical performance of the proposed sensor was examined using SWV and the results
have been shown in Fig. 3 and Fig. 4. The slopes of calibration curves for DZP and OZP were calculated by a linear regression method in the linear concentration ranges of 0.020 to 0.76 mgL1
for DZP and 0.050 to1.90 mgL-1 for OZP using their relevant reduction peaks. The detection
limits, calculated as 3σ, were 3.52 μg L-1 and 5.8 μg L-1 for DZP and OZP, respectively. The relative standard deviations (%RSD) were obtained to be 2.4% and 2.6% for ten repetitive determinations of DZP (0.5 mgL-1) and OZP (0.5 mgL-1), respectively. The electrochemical responses of MWCNT/CILE in terms of linear range and detection limits were compared to the other modified electrodes reported in the literature (Table 2).
3.7. Determination of DZP and OZP in real samples Because the benzodiazepines have important risks to be subject of an excessive or abusive utilization, especially by drug addicts, and their effects on the central nervous system, the modified electrode we applied for the determination of DZP and OZP (the major metabolite of DZP in human body) in real samples including serum, urine and pharmaceutical by the proposed method.
3.7.1. Determination of DZP and OZP in pharmaceutical and serum samples Three tablets of DZP and OZP were weighed separately and ground and their solutions were prepared. Then, their SWV voltammograms were taken under the optimized conditions (Table 11
3). The amounts of both drugs were found to be in satisfactory agreement with the declared amounts of 10 and 5 mg per tablet for DZP and OZP, respectively. The proposed method was also applied for the analysis of DZP and OZP in human serum samples using the standard addition method. Before analysis, the serum samples were prepared and pre-treated as reported previously [43]. 1.0 mL of serum sample was diluted 10 times with 0.5 M PBS (pH 7.0). The results of real sample analysis are presented in Table 3. Recovery of the results was not affected significantly, and consequently, the described method is accurate for the assay of DZP and OZP in serum samples. As reported, the therapeutic concentration range for most drugs is approximately from 0.1 to 2.0 mgL-1, while toxic effects can occur when their concentrations in plasma exceed 3.0 to 5.0 mgL-1 [44].
3.7.2. Determination of OZP in urine samples Urine samples were diluted before analysis without pre-treatment as reported previously [29]. A portion of 5.0 mL of urine sample and 2.5 mL of KNO3 (1.79 M) were added to 2.5 mL of acetate buffer solution, pH 5.0. Then, adequate volumes of OZP stock solution (100.0 mgL-1) was spiked into the buffered sample solution (as detailed in Table 3) and SW voltammograms were worked under the optimized conditions. The obtained results demonstrated the ability of MWCNT/CILE for the voltammetric determination of OZP in urine samples with good recoveries of the spiked OZP and reproducibility (Table 3).
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Conclusions MWCNT/CILE was used successfully for the determination of DZP and OZP in tablets, urine and serum samples. As the benzodiazepines have important risks to be subject of an excessive or abusive utilization, we successfully used the proposed electrode for the determination of OZP (the major metabolite of DZP in human body) in human urine real samples. The advantage of the proposed composite electrode is its ability in detection of DZP in the presence of OZP with good sensitivity and without the need for separation of the two compounds prior to electrochemical measurements. As stated, OZP did not have any response in pH > 8.5. Therefore, in these conditions, DZP could be determined selectively in the presence of OZP.
Acknowledgements The authors gratefully acknowledge the Science and Research Branch of Islamic Azad University.
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Figure captions
Fig. 1. Cyclic voltammograms of 10.0 mgL-1 DZP in PBS pH 7.0 on (a) MWCNT/CILE, and (b) CILE. Cyclic voltammograms of 10.0 mgL-1 OZP in acetate buffer solution, pH 5.0 on (c) MWCNT/CILE, and (d) CILE. The scan rates were 100 mVs-1. Fig. 2. SWV voltammograms of MWCNT/CILE immersed in (a) 0.5 M PBS, pH 8.5, and in PBS, pH 8.5 containing (b) 1.0 mgL-1 OZP, (c) 2.0 mgL-1 OZP, (d) 2.0 mgL-1 OZP and 1.0 mgL1
DZP, and (e) 1.0 mgL-1 DZP.
Fig. 3. SW voltammograms of MWCNT/CILE in PBS, pH 7.0 containing different concentrations of DZP: (a) 0.0, (b) 0.02, (c) 0.03, (d) 0.04, (e) 0.05, (f) 0.06, (g) 0.07, (h) 0.09, (i) 0.11, (j) 0.14, (k) 0.18, (l) 0.24, (m) 0.27, (n) 0.30, (o) 0.34, (p) 0.37, (q) 0.40, (r) 0.43, (s) 0.48, (t) 0.52, (u) 0.56, (v) 0.60, (w) 0.64, (x) 0.68, (y) 0.72 and (z) 0.76 mgL-1. Inset: Calibration plot of DZP. Fig. 4. SW voltammograms of MWCNT/CILE in acetate buffer, pH 5.0 containing different concentrations of OZP: (a) 0.0, (b) 0.05, (c) 0.15, (d) 0.25, (e) 0.35, (f) 0.45, (g) 0.70, (h) 0.95, (i) 1.15, (j) 1.40, (k) 1.65 and (l) 1.90 mgL-1. Inset: Calibration plot of OZP.
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Figure 1
1.0 -1.0
I (µA)
-3.0
b
-5.0
d a
-7.0
c -9.0 -11.0 -1.2
-1.0
-0.8 E, V (vs. Ag/AgCl)
18
-0.6
Figure 2 d
2.0
e
a
I (µA)
1.5
1.0
c
b
a b c d e
0.5
0.0 -1.10
-1.08
-1.06
-1.04 -1.02 E, V (vs. Ag/AgCl)
19
-1.00
-0.98
Figure 3 20.0
15.0
20.0
I (µA)
I (µA)
z
a
10.0
10.0
5.0
y = 23.808x - 0.0786 R² = 0.9981
0.0 0
0.2
0.4
0.6
DZP Conc. (mgL-1)
0.8
-0.1 -1.1
-1.0
-0.9
-0.8 -0.7 E, V (vs. Ag/AgCl)
20
-0.6
-0.5
Figure 4
5.0 5.0 4.0 I (µA)
4.0
I (µA)
3.0 l
3.0 y = 2.3074x + 0.0697 R² = 0.9988
2.0 1.0
2.0
0.0
a
0.0
0.5
1.0 OZP Conc.
1.5
2.0
(mgL-1)
1.0
0.0 -1.00
-0.90
-0.80 -0.70 E, V (vs. Ag/AgCl)
21
-0.60
-0.50
Table 1. Interference study for the determination of 0.9 mgL-1 of DZP and 0.5 mgL-1 of OZP under the optimized conditions Tolerance limits
Tolerance limits
([substance]/[DZP]
([substance]/[OZP])
1000
1000
Lactose
65
800
Uric acid
75
600
80
120
100
100
Species Ascorbic acid, fructose, Glucose, sucrose, starch
Dopamine +
+
2+
2+
2+
2+
K , Na , Mg , Zn , Mn , Ni 2+
3+
a
Fe , Fe
-
100
Cu2+
-b
30
a
Ferric and ferrous ions are precipitated in the presence of DZP.
b
Cupric ion is precipitated in the presence of DZP.
22
Table 2. Electrochemical detection of DZP and OZP reported at various electrodes
Sonogel-carbon
DZP
Linear range (mgL−1) 0.028 - 0.256
Carbon paste electrode (CPE) Carbon paste electrode (CPE)
DZP OZP
0.25 - 3.0 0.025 - 1.0
0.021 0.012
5 5
Screen printed carbon electrode
DZP
7.1 - 285
1.89
20
DZP
0.29 - 71.20
0.71
45
DZP OZP
0.02 - 0.76 0.05 - 1.90
4.1 5.8
This work This work
Electrode
Solid contact ion selective electrode MWCNT/CILE MWCNT/CILE
Analyte
23
LOD (µgL−1) 4.0
Ref. 3
Spiked (mgL-1)
Detected (mgL-1)
Recovery (%)
DZP
Tablet 1 Tablet 2 Tablet 3 Serum 1 Serum 2 Serum 3 Serum 4
0.20 0.40 0.60 0.30 0.50 0.70 0.90
0.18 0.39 0.61 0.29 0.51 0.72 0.89
90.0 97.5 101.6 96.6 102.0 102.8 98.8
OZP
Table 3. Determination of DZP and OZP in tablet, serum and urine samples using MWCNT/CILE.
Tablet 1 Tablet 2 Tablet 3 Serum 1 Serum 2 Serum 3 Serum 4 Urine 1 Urine 2 Urine 3 Urine 4
0.30 0.60 1.20 0.40 1.00 1.40 1.80 0.40 0.90 1.30 1.70
0.27 0.58 1.22 0.36 0.98 1.40 1.81 0.37 0.89 1.29 1.71
90.0 96.6 98.8 101.6 98.0 100.0 100.5 92.5 98.8 99.2 100.6
Sample
24
Supporting Information (SI)
Multiwall carbon nanotube-ionic liquid modified paste electrode as an efficient sensor for the determination of diazepam and oxazepam in real samples M.A. Zare*, M. Saber Tehrani, S. Waqif Husain, P. Aberoomand Azar Department of Chemistry, Science and Research Branch, Islamic Azad University, P.O. Box: 1477893855, Tehran, Iran
Optimization of the instrumental parameters in square-wave voltammetry (SWV) for the determination of DZP and OZP The peak currents in SWV depend on various instrumental parameters including frequency, potential amplitude and potential step height. Therefore, the effect of these parameters on the peak potentials and currents of DZP and OZP was studied. Although the SWV peak currents of DZP and OZP were affected by these instrumental parameters, they had a little effect on their peak potentials. The optimizing experiments for frequency and square wave amplitude are shown in Fig. S1 and Fig. S2. According to the experimental results, 120 Hz and 150 Hz were obtained as optimum frequency in the determination of DZP and OZP, respectively. For both drugs, square wave potential amplitude of 20 mV gave the best signal. The potential step height was found to have little effect on the peak current. Hence, 5mV was chosen as the increment of potential.
25
Figure S1
12.0
1.6
I (µA, DZP)
1.2 8.0
1.0 DZP
6.0
I (µA, OZP)
1.4
10.0
0.8
OZP
0.6
4.0
0.4 2.0
0.2
0.0
0.0 0
50
100 150 Frequncy (HZ)
200
250
Fig. S1. Effect of frequency on peak currents of 0.5 mgL-1 DZP and 0.75 mgL-1 OZP. The accumulation potential was -0.3V and pH of the solutions were 7.0 for DZP and 5.0 for OZP.
26
Figure S2
8.0
1.6 DZP
1.2
4.0
0.8
2.0
0.4
0.0 0.0
20.0
40.0 Amplitude (mV)
60.0
I (µA, OZP)
I (µA, DZP)
OZP
6.0
0.0 80.0
Fig. S2. The influence of applied potential amplitude (mV) on the peak currents of 0.5 mgL-1 DZP and 0.75 mgL-1 OZP. The experimental conditions were as Fig. S1.
27
Figure S3
0.5
I (µA)
-0.5
-1.5
-2.5
a b
c d
-3.5 -1.2
-1.0
-0.8
-0.6
E, V (vs. Ag/AgCl)
Fig. S3. Cyclic voltammograms of 10.0 mgL-1 DZP in PBS pH 7.0 on (a) CPE and (b) GCE. Cyclic voltammograms of 10.0 mgL-1 OZP in acetate buffer solution (pH 5.0) on (a) CPE and (d) GCE. The scan rates were100 mVs-1.
28
Figure S4
-2.0
-4.0
I (µA)
-6.0
-8.0 pH=3
-10.0
pH=8 pH=7 pH=9
pH=4
pH=6 pH=5
-12.0 -1.2
-1.1
-1
-0.9 -0.8 E, V (vs. Ag/AgCl)
-0.7
-0.6
Fig. S4. Linear sweep voltammograms of 10 mgL-1 DZP at the MWCNT/CILE; pH from right to left: 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0. The scan rates were100 mVs-1.
29
Figure S5
-2.0
I (µA)
-4.0 -6.0 -8.0
pH 8.0 pH 7.0 pH 6.0 pH 4.0
-10.0
pH 3.0
pH 5.0
-12.0 -1.2
-1.1
-1.0
-0.9 -0.8 E, V (vs. Ag/AgCl)
-0.7
-0.6
Fig. S5. Linear sweep voltammograms of 10 mgL-1 OZP at the MWCNT/CILE; pH from right to left: 3.0, 4.0, 5.0, 6.0, 7.0, 8.0. The scan rates were100 mVs-1.
30
Figure S6
-800
Ep, mV (vs. Ag/AgCl)
y = -54.469x - 636.69 R² = 0.9983 -900 DZP y = -55.086x - 722.1 R² = 0.9984
-1000
OZP
-1100
-1200 2.0
3.0
4.0
5.0
6.0 pH
7.0
Fig. S6. Plot of Ep vs. pH for DZP and OZP on MWCNT/CILE.
31
8.0
9.0
10.0
Figure S7
-5.0
20.0
-35.0
a -50.0
y = 13.246x + 0.4104 R² = 0.994
I (µA)
I (µA)
-20.0
10.0
i 0.0 0.0
0.4
0.8
1.2
Scan rate (V/s)
-65.0 -1.2
-1.0
-0.8 E, V ( vs. Ag/AgCl)
-0.6
-0.4
Fig. S7. Cyclic voltammograms of 10.0 mgL-1 DZP in 0.5 M PBS, pH 7.0 on MWCNT/CILE at different scan rates of (a) 0.05, (b) 0.1, (c) 0.2, (d) 0.3, (e) 0.4, (f) 0.5, (g) 0.6 and (h) 0.8 and (i) 1.0 Vs-1. The inset shows the corresponding calibration plots of cathodic current as a function of scan rate.
32
Figure S8
-65.0
40.0
y = 17.777x - 3.8637 R² = 0.9965
I(µA)
I (µA)
-15.0
20.0
a
-115.0
o
0.0
0.0
1.3 Scan Rate0.5 (V0.5s-0.5)
2.5
-165.0
-1.2
-1.0
-0.8 E, V ( vs. Ag/AgCl)
-0.6
-0.4
Fig. S8. Cyclic voltammograms of 10.0 mgL-1 OZP in acetate buffer solution, pH 5.0 on MWCNT/CILE at different scan rates of (a) 0.05, (b) 0.1, (c) 0.2, (d) 0.3, (e) 0.4, (f) 0.5, (g) 0.6, (h) 0.7, (i) 0.8, (j) 0.9, (k) 1.0, (l) 1.5, (m) 2.0, (n) 3.0 and (o) 5.0 Vs-1. The inset shows the corresponding calibration plots of cathodic current as a function of square root of scan rate.
33
8.0
1.4
7.0
1.2
6.0
1.0
5.0
DZP
4.0
0.8
OZP
0.6
3.0
0.4
2.0
0.2
1.0 0.0 -0.85
I (µA, OZP)
I (µA, DZP)
Figure S9
0.0 -0.65
-0.45
-0.25 -0.05 E, V (vs. Ag/AgCl)
0.15
0.35
Fig. S9. The influence of accumulation potential on the peak currents of 0.5 mgL-1 DZP and 0.75 mgL-1 OZP. The pH value of the solutions was 7.0 for DZP and 5.0 for OZP. The accumulation time was 75s for both species.
34
Figure S10
7.0 6.0
I (µA)
5.0 4.0 3.0 OZP
2.0
DZP
1.0 0.0 0
50
100 150 Accumulation time (s)
200
250
Fig. S10. The effect of accumulation time for 0.5 mgL-1 DZP and 0.75 mg L-1OZP. The pH value of the solutions was 7.0 for DZP and 5.0 for OZP. The accumulation potential was -0.3V for both species.
35
