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11 Carbon Black Composite Electrode for Flow Injection

Analytical Methods Accepted Manuscript

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12 Amperometric determination of Hydrochlorothiazide

13 Received 00th January 20xx,
14 Accepted 00th January 20xx Francyelle Calegaria, Paulo Roberto de Oliveiraa, Luiz Humberto Marcolino Juniora, Márcio F.
15 Bergaminia†
DOI: 10.1039/x0xx00000x
16
This work describes a composite electrode based on carbon black (CB) and poly(ethylene co-vinyl)acetate (EVA) for
17 www.rsc.org/
development of a robust, low cost and time-saving method for the amperometric determination of hydrochlorothiazide
18
(HCTZ) in a pharmaceutical formulation and synthetic human urine samples. This composite electrode exhibits favourable
19
features such as mechanical resistance, low cost and simple preparation The proposed electrode was used as an
20 amperometric detector in a flow injection analysis (FIA) based on HCTZ oxidation. The best amperometric responses were
21 found employing a composite electrode made with 75% (w/w) of carbon black (CB) and 25% (w/w) of poly(ethylene co-
22 vinyl)acetate (EVA), sample volume of 100 µL, flow rate of 5.3 mL min-1 and potential of detection of +900 mV vs.
23 Ag/AgCl/3.0 mol L-1. Under optimized conditions, it was possible to achieve an analytical frequency of 128 injections h-1,
24 linear dynamic range (LDR) of 10 to 200 µmol L-1, limit of quantification (LOQ) of 4.4 µmol L-1 and a limit of detection (LOD)
25 of 1.3 µmol L-1 of HCTZ. The proposed method was successfully applied for determination of HCTZ in commercial tablets
26 and synthetic human urine samples without any chemical pretreatment. Composite electrode based on EVA and carbon
27 black is feasible and totally adaptable to portable devices for HCTZ quantification.
28
29 electroanalytical applications7. On this matter, the present
Introduction work aims to contribute with the scientific advance in
30
electroanalytical systems using new composite electrode
31 Electrochemical sensors can be constructed with different
materials, since very expensive ones, as diamond1 and noble coupled to a flow injection analysis (FIA) system for
32
metal nanoparticles2, 3, or even with really cheap ones, as amperometric determination of Hydrochlorothiazide (HCTZ) in
33 human urine and pharmaceutical samples.
carbon black4, 5. Several approaches can be used for
34 HCTZ belongs to thiazide class of diuretics, which are widely
construction of electrochemical sensors including the
35 used as therapeutic agents for the treatment of arterial
preparation of composite electrodes. This strategy is based on
36 use of at least two components in order to yield a new hypertension and other disorders that are result from the
37 material. For electrodes preparation, a conductive material (eg retention of body fluids8, 9. HCTZ is among the most extensively
38 graphite powder) and a binder (mineral oil or polymers) are prescribed thiazide in Brazilian clinical practice10, being the
39 combined to produce a conductive composite. One material first choice for the treatment of mild and moderate
40 that deserves attention is Ethylene-Vinyl Acetate (EVA). EVA hypertension and one of the cheapest antihypertensive drugs
41 was discovered in 1930 and has interesting features, such as available11. After oral drug administration the absorption of
42 excellent flexibility, malleability and easy processing6. These HCTZ rapidly occurs by the gastro-intestinal tract and around
43 properties allow its application in different areas, from 80% is eliminated unmetabolized from the body via urine8.
44 electrical and thermal coatings and adhesives, as well as its use One patient which makes use of chronic use of this drug can
45 in the construction of electrochemical sensors. Recently, a new present a minimum value of 5.0 mg L-1 of HCTZ in urine in the
unchanged form12. Therefore, the monitoring of this drug is
46 composite electrode constructed only with carbon black (CB)
(electric conductor) and EVA was reported7. This composite possible not only in quality control by pharmaceutical industry,
47
electrode exhibits favourable features such as mechanical but also in urine samples from patients who are treated
48
resistance, low cost, simple preparation, easy to modify and accordingly.
49 Besides of pharmaceutical control, HCTZ is present in the list of
also quite versatile due to many possibilities for
50 prohibited substances of World Anti-Doping Agency’s (WADA).
51 This list is used as an international reference for identifying
52 a. a Laboratório de SensoresEletroquímicos (LabSensE), Departamento de Química,
substances that are not allowed in sports practices and,
Universidade Federal do Paraná (UFPR), CEP 81.531-980, Curitiba-PR, Brazil.
53 b. † e-mail: bergamini@ufpr.br therefore, are frequently monitored in athletes body fluids
54 c. Electronic Supplementary Information (ESI) available: [details of any
(blood plasma, saliva, and urine)13.
supplementary information available should be included here]. See
55 DOI: 10.1039/x0xx00000x
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EVA/CB composite electrode as the working electrode. For (methodology recommended by the Brazilian
3 comparative studies others solid carbon-based electrodes Pharmacopedia)21.
4 were used as working electrode: Glassy carbon electrode (GCE)
5 with diameter of 3 mm by Metrohm, Screen printed carbon
6 electrode (SPCE), provided by Prof. Craig E. Banks from 3. Results and Discussion
7 Manchester Metropolitan University19, Epoxy resin and 3.1. Electrochemical behavior of hydrochlorothiazide (HCTZ)
8 graphite composite electrode (GEE)20. The SPCE (3 mm The first step addressed for the development of the FIA
9 diameter working electrode) were fabricated with a stencil procedure for the determination of HCTZ in pharmaceutical
10 designs using a microDEK 1760RS screen-printing machine formulations and synthetic urine samples was the investigation
11 (DEK,Weymouth, UK). A metallic thread is placed on the of the electrochemical behavior of the drug (Fig. 2A). Cyclic

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12 female connector. Then, a sheet of circular copper is welded. voltammograms were obtained in the absence and in the
13 Subsequently, the PVC tube (6 mm diameter) is placed around presence of a sufficient amount of HCTZ to obtain a significant
14 the connector. The prepared composite (epoxy resin + process for the visualization and evaluation of the
15 graphite) is introduced into the virtual cavity this tube. performance of the proposed electrode (1.0 mmol L-1 of HCTZ
For pH measurements, a Metrohm pH-meter with a combined
16 in 0.04 mol L-1 BR buffer solution at 50 mV s-1). One of the
pH reference electrode was used.
17 advantages of the composite electrode proposed is its high
FIA experiments were carried out using a flow cell with an
18 effective volume of 95 L. The working electrode (EVA/CB
mechanical resistance promoted by the presence of EVA. Thus,
19 electrode), reference electrode (Ag/AgCl/KCl, 3.0 mol L-1) and
other carbon-based electrodes with similar feature were used
20 in order to compare the voltammetric profile of HCTZ (Fig. 2B).
the platinum auxiliary electrode were introduced through
21 holes in its cover. The electrochemical flow cell was inserted in 30 In absence of HCTZ
22 a one-channel flow injection analysis system7. The system was
In the presence de HCTZ

23 assembled with a peristaltic pump (Ismatec, model IPS12, 20

24

I/ A
Switzerland) and a manual homemade injector of
25 polyurethane. The manifold connections were made with 10
26 tygon tubing (0.5 mm i.d.). Britton-Robinson (BR) buffer
27 solution (0.04 mol LR pH 7.5) was used as the carrier solution. 0
A
28 The analytical path was 30 cm long and the entire flow -0.3 0.0 0.3 0.6 0.9 1.2
29 injection system was kept at room temperature. E / V (vs. Ag/AgCl/KCl, 3.0 mol L )
-1

30 GCE
2.4. Flow injection analysis
31 300 SPCE
EVA/CB
32
-2

Flow injection experiments were carried out using the EVA/CB GEE
j / A cm

200
33 composite electrode as an amperometric detector. All
34 experimental parameters (flow rate, volume of injection and 100
35 potential applied) were studied by analyzing the sensor
response to HCTZ oxidation process in 0.04 mol LR BR buffer 0
36 B
solution, pH 7.5. Parameters such as detection potential, flow
37 -0.3 0.0 0.3 0.6 0.9 1.2
rate and volume of injection were evaluated and chosen based
38 E / V (vs. Ag/AgCl/KCl, 3.0 mol L )
-1

on sensibility of sensor response verified for successive

39
injections of 1.0 x 10-4, 2.5 x 10-4 and 5.0 x 10-4 mol L-1 HCTZ in
40 0.04 mol L-1 BR buffer solution (pH 7.5). Optimized analytical
41 curves were obtained by injecting 100 µL of HCTZ into the
42 carrier solution, at a flow rate of 5.3 mL min-1. The
43 C
amperometric signal was monitored at +900 mV vs.
44 Ag/AgCl/3.0 mol L-1. The proposed method was applied for the
45 determination of HCTZ in human urine spiked samples using Fig.2: Cyclic voltammograms obtained (A) in the absense and
46 the calibration curve method in three levels of concentration: presence of 1.0×10-3 mol L-1 of HCTZ for EVA/CB; (B) in the presence
47 S1- 10 µmol L-1, S2 - 50 µmol L-1 and S3 - 100 µmol L-1. The of 1.0×10-3 mol L-1 of HCTZ for different carbon-based solid
48 synthetic urine samples were prepared by dilution in 0.040 electrodes. Conditions: 0.04 mol L-1 BR buffer solution in pH 7.5 and
49 mol L-1 BR buffer with pH 7.5 starting from synthetic urine scan rate of 50 mV s-1; (C) suggested reaction for HCTZ oxidation.
50 stock solution fortified with 0.020 mol L-1 HCTZ. The results
were evaluated from the relation between the added and Faradaic processes are not observed in CV recorded in the
51
recovery values of analyte concentration. Similarly, the absence of HCTZ showing that electrode composite EVA/CB is
52 not electroactive under these experimental conditions. On the
percentage content of HCTZ in the pharmaceutical formulation
53 other hand, in the presence of HCTZ a well-defined irreversible
also was determined by the standard addition method and
54 peak at 850 mV can be observed, which corresponds to the
compared with that given by B;R' spectroscopy
55 oxidation of HCTZ. Anodic reaction is an irreversible process
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probably caused by a minimal dispersion of the analytical
3 solution 27. Thus, a volume of injection of 100 µL was selected A e B
3.0 3.0
4 for furthers studies.
5 Flow rate of the carrier solution is a hydrodynamic parameter

I/ A
6

I/ A
that plays a key role since it changes the efficiency of mass 2.0 d d 2.0
7 transport of the analyte from bulk solution to EVA/CB
8 electrode surface. The influence on the peak current was
9 evaluated for flow rate values in the range of 1.7 to 8.0 mL c c 1.0
1.0
10 min-1 using potential of detection of 900 mV and sample
11 volume of 100 µL (Fig.4 C). Sensitivity increased continuously b b

Analytical Methods Accepted Manuscript

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12 up to 2.9 mL min-1 and remains practically constant for higher

0.0 0.0 Decreasing

13 flow rate values. Increasing the flow rate promotes a

0 200 400 600 800 0 50 100 150 200
14 decreasing in the diffusion layer formed closer to the electrode
t/s CHCTZ / mol L
-1

15 surface and consequently a more efficient mass transport of

the analyte. A flow rate of 5.3 mL min-1 provided the best Fig.5: (A) Transient current signals obtained (n=3) for HCTZ
16
response and it was used for further studies since it combines solutions: (a) 1.0 x 10-5, (b) 2.0 x 10-5, (c) 5.0 x 10-5, (d) 1.0 x 10-4 and
17
low reagent consumption, more efficient mass transport, good (e) 2.0 x 10-4; (B) Correlation curve for analytical determination of
18 reproducibility (according to standard deviation) and high HCTZ (Increasing direct slope = 16.0 µA L mmol-1 and Decreasing
19 analytical frequency (128 injections h-1). direct slope = 16.3 µA L mmol-1). Conditions: 0.04 mol L-1 BR buffer
20 Under optimized experimental conditions, amperometric solution (pH 7.5), applied potential of 900 mV, sample volume of
21 measurements were carried out in 0.04 mol L-1 BR buffer 100 µL and flow rate of 5.3 mL min-1.
22 solution (pH 7.5) using different HCTZ concentrations (Fig.5 A).
23 Analytical curves (Fig.5 B) were recorded for concentrations
Table 1. Analytical performance of several electrochemical
24 ranging from 10 to 200 mol L-1 (R2 = 0.9854) showing a methodologies employed for HCTZ determination.
25 sensitivity of A L mmol-1 (slopes average), limit of
26 detection (LOD) and limit of quantification (LOQ) of 1.3 mol L- Detection/Electrode F LOD LDR Ref.
(System)
27 1 and 4.4 mol L-1, respectively. Thus, based on the presented

EVA/CB electrode 128 1.3 10 to 200 *

28 performance, the proposed methodology seems to be clearly
(FIA)
29 feasible in the quantification of HCTZ in urine samples for
BDD (BIA) 130 1.9 5.3 to 26.3 28
30 doping control (cut-off concentration of 16.7 mol L-1)12 as 28
BDD (CE-C) 90 10 105 to 527
well as in pharmaceutical formulations. In addition, no
31 BDD (FIA) 89 0.2 0.4 to 8.0 29
cleaning or pretreatment of the EVA/CB composite electrode
32 CL (FIA) 200 0.15 0.33 to 130 30
surface was necessary between successive injections and any
33 BDD (BIA) 100 0.27 10 to 30 31
memory effect was verified, since the slopes of increasing and
34 decreasing HCTZ concentration presented practically the same LDR: Linear Dynamic Range (µmol L-1);
LOD: Limit of Detection
35 magnitude. RSD for the transient signal of 3.34 % was found
(µmol L-1); F: Analytical Frequency (h-1); BDD - Boron-Doped
36 Diamond; BIA - Bath injection analysis; CE-C - Capillary
for successive injections (n= 10) of HCTZ (50.0 µmol L-1) which Electrophoresis with Capacitively Coupled Contactless Conductivity
37 was considered a satisfactory repeatability, confirming that Detection; CL – Chemiluminescence; * Proposed method.
38 under flow conditions there is no blocking of electrode
39 surface. In addition, EVA/CB electrode has shown a stable and
40 reproducible response with a capacity to perform 128 3.3. Application of composite electrode for amperometric
41 injections per hour. Figures of merits found using the proposed detection of HCTZ
42 methodology are very attractive in the determination of HCTZ The proposed methodology using the EVA/CB composite
43 and they were comparable to other electrochemical strategies electrode as an amperometric detector for the determination
44 described in literature (Table 1). of HCTZ was tested by analyzing synthetic human urine spiked
45 samples and a pharmaceutical formulation. Fig.6 shows the
46 transient signals obtained for standard (a-e) and spiked urine
47 samples (S1-S3) solutions. Recoveries varying from 98.0 to
48 113% (n = 3) of HCTZ were obtained and statistical calculations
49 showed good concordance (at 95% confidence level) between
50 the added and recovery value. This is relevant point, since the
evaluated HCTZ concentrations agrees with the minimum
51
average drug concentration available in the non-metabolized
52
form present in urine samples of patients in chronic
53
treatment32.
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ARTICLE Journal Name
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Conflicts of interest
e e
4 2.0 There are no conflicts to declare.
5
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7
1.5
d Acknowledgements
I/ A

S3
8 d We gratefully acknowledge financial support from Brazilian
9 1.0
c agencies Fundação Araucária, CAPES and CNPQ.
c
10 S2
11 0.5 b b a

Analytical Methods Accepted Manuscript

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12 S1 References
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