Journal Pre-proofs

Batch injection analysis with electrochemical detection for the simultaneous

determination of the diuretics furosemide and hydrochlorothiazide in synthetic
urine and pharmaceutical samples

Eduardo Fonseca Silva, Auro Atsushi Tanaka, Ridvan Nunes Fernandes,

Rodrigo Alejandro Abarza Munoz, Iranaldo Santos da Silva

PII: S0026-265X(20)30540-3
DOI: https://doi.org/10.1016/j.microc.2020.105027
Reference: MICROC 105027

To appear in: Microchemical Journal

Received Date: 20 February 2020

Revised Date: 11 May 2020
Accepted Date: 12 May 2020

Please cite this article as: E. Fonseca Silva, A. Atsushi Tanaka, R. Nunes Fernandes, R. Alejandro Abarza
Munoz, I. Santos da Silva, Batch injection analysis with electrochemical detection for the simultaneous
determination of the diuretics furosemide and hydrochlorothiazide in synthetic urine and pharmaceutical samples,
Microchemical Journal (2020), doi: https://doi.org/10.1016/j.microc.2020.105027

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 Original Article

Batch injection analysis with electrochemical detection for the

simultaneous determination of the diuretics furosemide and

hydrochlorothiazide in synthetic urine and pharmaceutical samples

Eduardo Fonseca Silva 1, Auro Atsushi Tanaka 2,3, Ridvan Nunes Fernandes 2, Rodrigo

Alejandro Abarza Munoz 3,4 *, Iranaldo Santos da Silva 1**

1 Departamento de Tecnologia Química, Centro de Ciências Exatas e Tecnologia, Universidade

Federal do Maranhão, CEP 65080-805, São Luís, MA, Brasil

2 Departamento de Química, Centro de Ciências Exatas e Tecnologia, Universidade Federal do

Maranhão, CEP 65080-805, São Luís, MA, Brasil

3 Instituto Nacional de Ciência e Tecnologia de Bioanalítica, Caixa Postal 6154, CEP 13083-

970, Campinas, SP, Brasil.

4 Instituto de Química, Universidade Federal de Uberlândia, 38400-902, Uberlândia, Minas

Gerais, Brazil

Corresponding authors:

* munoz@ufu.br ORCID: 0000-0001-8230-5825

** iranaldo.ss@ufma.br ORCID: 0000-0001-6216-9141

Abstract

This works shows the combination of batch-injection analysis with multiple-pulse

amperometric detection (BIA-MPA) for the determination of the diuretics furosemide

(FRD) and hydrochlorothiazide (HCT) in biological and pharmaceutical samples. Under

optimized electrolyte conditions (0.04 mol L−1 Britton-Robinson buffer solution at pH

4.0) FRD was oxidized at +1.10 V and both compounds were oxidized at +1.30 V. The

MPA detection involves the continuous application of two potential pulses as a function

of time to determine FRD at the first potential and HCT at the second after subtracting

the FRD current obtained at the second potential. A correction factor was needed because

the current of FRD at both potentials was different. A boron-doped diamond working

electrode was selected for the simultaneous determination as its surface is less susceptive

to fouling. The compounds exhibited a linear response range of 2 to 100 μmol L−1 for

HCT (r = 0.9990) and 2 to 300 μmol L−1 for FRD (r = 0.9993), satisfactory repeatability

(RSD lower than 5% for n = 30), detection limits of 0.65 μmol L−1 for FRD and 0.63

μmol L−1 for HCT, and analytical frequency of 130 injections per hour. The proposed

method was applied for the analysis of synthetic urine samples spiked with both

compounds and proper recovery values were obtained (95-110%). Moreover, the

proposed method was compared to UV-Vis spectrophotometry for the analysis of

pharmaceutical formulations and they were found to be statistically similar at the 95%

confidence level.

Keywords: Furosemide; Hydrochlorothiazide; Boron-doped diamond; Flow analysis;

Anti-doping.
 1. Introduction

Furosemide (FRD) [5-(aminosulfonyl)-4-chloro-2-[(furanylmethyl)amino

benzoic acid] and hydrochlorothiazide (HCT) [6-chloro-3,4-dihydro-2H-1,2,4-

benzothiadiazine-7-sulfonamide-1,1 dioxide] are substances belonging to the classes of

loop and thiazide diuretics, respectively. These are groups of substances used to adjust

the composition and volume of bodily fluids in numerous diseases [1]. From the

pharmacological standpoint, these drugs increase urinary volume and the urinary

excretion of sodium through direct action on the kidneys [2,3].

FRD is one of the most potent and effective diuretic medications available. It

exerts its action on the loop of Henle, which is the portion of the nephron that regulates

the balance of water and sodium in the organism, inducing the loss of sodium chloride

and water [4]. However, its diuretic properties have recently made FRD a useful

medication for the treatment of hypertension and the retention of liquids (edema) in

patients with congestive heart failure, liver disease and kidney dysfunction, such as

nephrotic syndrome, by inhibiting the co-transport of sodium, potassium and chlorides,

and inducing the loss of calcium, magnesium, bicarbonate and potassium ions, thereby

causing hypocalcemia [5]. HCT is a thiazide diuretic that is widely used in the treatment

of diseases such as systemic arterial hypertension, renal edema, liver disease, heart

disease and nephrogenic diabetes insipidus due to its therapeutic efficacy and low cost

[6]. It is quite useful when taken orally, as it is well absorbed by the gastrointestinal tract

and excreted mainly through urine. Among short-acting drugs, such as HCT, the

maximum effect is seen in four to six hours, lasting eight to 12 hours. The main side

effects of HCT result from its action in the kidneys, such as the reduction of plasma

potassium, which could induce ventricular arrhythmia [7]. Other undesirable effects are
metabolic alkalosis, an increase in plasma levels of uric acid and hyperglycemia,

underscoring the need to use this drug with caution [5].

Neither of these diuretics require a prescription. This easy access can pose

serious health risks. Their use in sports is prohibited because these drugs mask substances

in anti-doping exams [8] by favoring the increase in the elimination of electrolytes

(especially sodium and chloride ions) and water along with other substances, such as

anabolic steroids. Several methods are found in the current literature for the determination

of drugs containing FRD and HCT, such as chromatography [9], spectrophotometry [10]

and voltammetry [11–15]. As both FRD and HCT are electrochemically active, batch

injection analysis (BIA) with multiple pulse amperometry (MPA) is an attractive

detection method. To the best of our knowledge, there is no electroanalytical method

described in the literature for the simultaneous determination of these two analytes.

BIA is an alternative tool to flow-injection analysis (FIA) with some

advantages including a simpler apparatus (injector and cell) in comparison to FIA (valves,

pump, tubing and cell) that makes BIA portable [16–19]. BIA‒MPA has been

successfully explored to execute simultaneous determinations using only a working

electrode, a reference electrode, and a counter electrode [20–23]. For such, a combination

of potential pulses is chosen such that only one analyte is detected in one of the pulses

and the rest are detected in the other pulses. In analyses involving two substances, a

simple correction factor is used to obtain the relative current of the second analyte [24,25].

This method also enables the application of a third or fourth potential pulse to avoid

contamination or promote the constant electrochemical cleaning of the working electrode

[24,26].

In the present study, we present a novel, simple, fast, low-cost,

electroanalytical method for the simultaneous determination of FRD and HCT in drugs
and biological samples (synthetic urine) using BIA‒MPA with a boron-doped diamond

(BDD) working electrode. The method is considered versatile as it enables the

simultaneous quantification of FRD and HCT in urine as well as the quality control of

commercial-available pharmaceutical products containing either FRC or HCT (the

mixture of both compounds is not found in commercial products). The method was

evaluated using analytical performance parameters, such as linearity, repeatability, limit

of detection, and limit of quantification, and the results were compared to those obtained

using UV‒Vis spectrophotometry.

2. Experimental Part

2.1. Reagents and samples

All aqueous solutions were prepared with highly pure deionized water

(resistivity no less than 18 MΩ cm) obtained in a Millipore Direct 8 water purification

system. All reagents used in the experiment were of analytical purity. Boric acid,

potassium chloride and sodium hydroxide were obtained from ISOFAR. Glacial acetic

acid and sulfuric acid 95-97% were obtained from EMSURE. FRD and HCT were

obtained from GALENA QUÍMICA E FARMACÊUTICA Ltda, Campinas, SP, Brazil.

Britton-Robinson buffer solution was used as the supporting electrolyte in the

experiments. Samples and stock solutions of the standards were prepared daily.

Pharmaceutical formulations containing either FRD or HCT were obtained from local

pharmacies. Twenty pills of the same sample were weighed and ground using a porcelain

mortar and pestle until complete homogenization. Next, an adequate mass of each sample

was diluted in the supporting electrolyte to obtain solutions with concentrations in the

linear range and on the calibration curve of the method. For the initial study of the

electrochemical behavior, stock solutions of FRD and HCT were prepared in an alkaline
medium (0.1 mol L⁻1 NaOH) at a concentration of 4.1 × 10−3 mol L−1. The Britton-

Robinson (BR) buffer solution was prepared from a mixture of acetic acid, boric acid and

phosphoric acid (all at 0.04 mol L⁻1) and 0.1 mol L⁻1 KCl. The pH of each solution was

adjusted with NaOH 3.0 mol L⁻1 to obtain buffer solutions with different pH values.

Synthetic urine samples were prepared to contain the majority of constituents

of a real sample, following the procedure described by Laube et al. [27]. For the proposed

method, the determination of both compounds in synthetic urine was performed using the

addition and recovery process: 200 µL of the synthetic urine was added to 5 mL of

solution containing only the BR buffer 0.04 mol L⁻1, pH 4, and to each standard solution

at three different concentrations and the percentage of recovery was calculated. Spiking

of the synthetic urine with FRD and HCT was performed before sample dilution in the

supporting electrolyte.

2.2. Instruments

The electrochemical measurements were performed using three electrodes in

the BIA cell: a BDD working electrode, an Ag/AgCl (KCl 3 mol L−1) reference electrode

and a platinum auxiliary or counter electrode – all coupled to the 302 N

potentiostat/galvanostat connected to a microcomputer and controlled using the GPES

software, version 4.9.007. Before the experiments, cleaning and electrochemical

activation of the BDD electrode were performed by applying a current of +0.01 A for

1000 s using the BR buffer solution 0.04 mol L⁻1 pH 2.0 (anodic activation), followed by

the application of −0.01 A for 1000 s in H2SO4 solution 0.1 mol L⁻1 (cathodic activation).

These pretreatments were similar to those employed in previously published studies [28].

The electrochemical (anodic and cathodic) pretreatments were only performed on new

electrodes or when a very accentuated reduction of the analytical signal occurred. It was
also necessary to perform the pretreatments (anodic and cathodic) again when the

electrode was not used for several days. Otherwise, only the cathodic treatment was

required once at the beginning of each workday.

The electrochemical cell used in the BIA system was fabricated with a fused

deposition modeling 3D‒printer as described by Cardoso et al. [29] This cell consisted of

a cylindrical recipient with a maximum capacity of 100 mL made from the filament of

the copolymer acrylonitrile butadiene styrene. The upper portion (lid) had three orifices

for the positioning of the reference electrode, auxiliary electrode and electronic

micropipette (EDP3-Plus) so that the tip of the micropipette was firmly inserted into the

orifice over the surface of the working electrode. To ensure sensitive and, reproducible

analytical signals, a fixed distance of 2 mm between the tip of the micropipette and

electrode was used throughout the entire study. The BDD electrode was positioned in the

lower portion of the cell with the aid of a rubber O‒ring and pressed by a steel plate to

enable electrical contact. The lower portion was fixed to the nesting by screw pins made

from the same material as the cell. The useful area of the BDD electrode was defined as

the geometric area of the O‒ring (0.126 cm2). The stirring of the solution was possible by

the use of a magnetic stirrer positioned in the center of the cell.

2.3. UV-Vis spectrophotometric analysis

The results obtained using the proposed method were compared to those

obtained using the reference method described in the Brazilian pharmacopeia [30]. The

pharmaceutical samples were analyzed by UV‒Vis spectrophotometry, with a 1 cm quartz

cuvette, using 0.1 mol L−1 NaOH as the solvent. Readings were performed at 271 nm for

FRD and 273 nm for HCT. The procedure consisted of the measure of the background
signal for both analytes individually, followed by the reading of the standard solution and,

finally, the pharmaceutical samples.

3. Results and Discussion

The electrochemical behavior of FRD and HCT (both at a concentration of

1 mmol L−1) was initially investigated using cyclic voltammetry in BR buffer solution pH

10 at a concentration of 0.04 mol L−1 on different working electrodes. These initial studies

were conducted to evaluate different materials for the working electrode with the aim of

the simultaneous detection of FRD and HCT. The overlap of oxidation peaks occurred

with both compounds when the gold electrode was used, and the compounds were easily

adsorbed to the surface of the glassy carbon electrode (data not shown). Therefore, these

electrodes presented significant limitations to the simultaneous determination of FRD and

HCT. In contrast, the BDD electrode achieved adequate separation of the voltammetric

signals for the two compounds, with a low background current and weak molecular

adsorption of the species to the surface of the electrode. Figure 1A displays the cyclic

voltammograms for FRD and HCT on the BDD electrode in 0.04 mol L−1 BR buffer pH

10. The voltammetric responses revealed the occurrence of oxidation in the potential

region of +1.13 V and +1.25 V for FRD and HCT, respectively. However, no reduction

processes were observed for either species, suggesting that both processes are irreversible

under these conditions. According to the literature [28,31], the electro-oxidation of FRD

involves the transfer of two electrons and two voltammetric peaks may be observed,

depending on the pH of the solution. In the present study, only the first oxidation peak

was used, corresponding to the oxidation of the sulfonyl amine group. For HCT, the

mechanism of electrochemical oxidation on BDD electrodes involves the transfer of two

electrons and two protons [32].

 INSERT FIGURE 1

The analysis of the hydrogen ion concentration revealed that the potential

oxidation peaks of both compounds varied with the change in the pH of the BR buffer

solution at a concentration of 0.04 mol L⁻1 (Figure 1B). However, the difference in the

potential variation of FRD and HCT was 110 mV between oxidation peaks for both

species at pH 4, unlike what occurred at other pH values. Therefore, this value was chosen

for the subsequent analyses.

After the choice of pH 4.0 for the BR buffer solution at a concentration of

0.04 mol L⁻1 used as the supporting electrolyte, tests were performed to determine the

electrochemical behavior of FRD and HCT under hydrodynamic conditions using the

BIA system with amperometric detection. For such, solutions containing 50 µmol L⁻1 of

FRD and 50 µmol L⁻1 of HCT were injected (in triplicate) into the BIA cell filled with

BR buffer solution 0.04 mol L−1 pH 4. Figure 2 illustrates the results of the application of

13 potential pulses of 100 ms each on the BDD working electrode. For the simultaneous

determination of two compounds by the amperometric detection technique [33], the

electrochemical behavior of each must present a difference of at least 100 mV, in which

one compound may present the redox process and the other may not. Analyzing Figure 2,

we see that only FRD was oxidized at E = +1.10 V, whereas the signal of HCT oxidation

was negligible at this potential. In contrast, both compounds were oxidized at E =

+1.30 V. Thus, the combination of potential pulses was selected such that only the

oxidation of FRD occurred at +1.10 V and the oxidation of both FRD and HCT occurred

at +1.30 V, thereby obtaining good separation.

 INSERT FIGURE 2

Based on these data, a potential pulse application waveform was proposed

and optimized (+1.10 V/100 ms and +1.30 V/100 ms) with the aim of the simultaneous

determination of the two compounds by BIA-MPA. As long as the application of two

potential pulses (100 ms each) is performed, two amperograms are obtained, one

registered at +1.10 V and the other one at +1.30 V. The MAP detection enables the

application of two or more potential pulses and current recording at all applied pulses,

which is not possible in all software destinated pulsed amperometric detection. As seen

in the amperograms presented in Figure 3, only FRD is oxidized at +1.10 V. At this

potential, the current detected for HCT is minimal and can, therefore, be disregarded.

However, this also indicates that there is no chemical interaction between HCT and FRD

or with oxidation products. On the other hand, both HCT and FRD were oxidized at

+1.30 V.

INSERT FIGURE 3

Another phenomenon observed was the fact that the oxidation of FRD did not

generate the same current magnitude at both potential pulses (7.7 µA at +1.10 V and 29.3 µA

at +1.30 V), which impedes the use of a simple subtraction between the currents detected

at the two potential pulses (I+1.30 V – I+1.10 V) to have access to the oxidation current related

only to the oxidation of HCT. This problem was circumvented with the use of a correction

factor (CF), which was obtained by the ratio (division) between the oxidation current of

FRD obtained at +1.30 V and the current of FRD at +1.10 V through the injection of the

solution containing only FRD [28]. Thus, when a solution containing the two compounds

is injected into the BIA‒MPA system, the oxidation current of HCT can be obtained by
the subtraction between the current detected at +1.30 V and that detected at +1.10 V

multiplied by the CF using Equations 1 and 2, which were used in the calculations for the

obtainment of the currents related to the oxidation of HCT and the CF, respectively.

CF = IFRD 1.30V / IFRD 1.10V Eq 1

IHCT = I 1.30V – (CF × I 1.10V) Eq 2

With the two amperograms acquired at +1.10 and +1.30 V and the equations

above, we can selectively detect the oxidation currents of HCT and FRD when present in

the same solution.

For the optimization of the method, we evaluated the influence of the

electronic micropipette dispensing rate and volume on the BIA-MPA response. For such,

the dispensing rate ranged from 19 µL s−1 to 143 µL s⁻1 and an increase in the oxidation

currents was found with the increase in the dispensing rate. This effect is relating to the

increase in the mass transport rate on the surface of the BDD electrode. Evaluating the

intensity of the amperometric signal and the smallest relative standard deviation (RSD),

the best response for both compounds at both potentials was achieved with a dispensing

rate of 100 µL s⁻1. Moreover, a dispensing volume of 120 µL led to a higher peak current

with the least standard deviation, thereby ensuring better stability of the system.

Therefore, this volume was chosen for subsequent experiments. The influence of stirring

in the BIA cell was also evaluated and was found to accelerate the removal of the

electroactive material from the electrode/solution interface, with the current returning to

baseline more quickly. This increases the analytic frequency of the system and the

analytes and their oxidation products are quickly removed from the surface of the working

electrode, which reduces the possibility of the contamination of the electrode [34].

Although the simultaneous determination of FRD and HCT could be performed without

stirring in the BIA cell, we opted for using the BIA‒MPA system with stirring at 150 rpm
to diminish the dispersion of the analytical signals and increase the analytical frequency,

achieving more stable, more reproducible analytical signals.

Satisfactory repeatability was found for both compounds, with a low RSD

value. The RSD values were lower than 5% and no memory effect was found between

successive injections, even when alternating solutions of different concentrations (data

not shown). To confirm the possibility of adsorption to the surface of the BDD working

electrode at the highest concentration, we performed successive injections (n = 30) of a

mixture containing FRD and HCT, as shown in Figure 4. In this study, the RSD (n = 30)

was 1.53% at E = +1.30 V and 1.39% at E = +1.10 V for injections of a mixture containing

FRD and HCT at a concentration of 100 µmol L−1. Considering the results of peak

currents, the proposed BIA‒MPA system presented proper repeatability and stability with

the previously optimized variables, with low RSDs and without adsorption to the electrode

surface between successive injections of FRD and HCT at a concentration of 100 µmol

L−1, achieving an analytical frequency of around 130 injections per hour.

INSERT FIGURE 4

For the simultaneous determination of FRD and HCT employing MPA, the

CF needs to remain constant along with the calibration range used to identify the oxidation

current of HCT. Thus, it is necessary to perform studies for the identification of the

concentration range in which there is a linear relationship between the current and

concentration. Based on the results, an FRD concentration of 20 µmol L−1 was used for

the CF, as this concentration exhibited a linear trend with the lowest standard deviation

compared to the other concentrations.

Figure 5 illustrates the results related to the linear range, in which we used an

FRD concentration of 20 µmol L⁻1 for the CF and a mixture of increasing concentrations
of the FRD and HCT standards. Linear regression was only used in the region where the

correlation between the concentration and detected oxidation current was linear.

FRD: I/µA = 0.076 [FRD, µmol L−1] + 0.27 R = 0.9993

HCT: I/µA = 0.13 [HCT, µmol L−1] + 5.9 R = 0.9990

INSERT FIGURE 5

The standard solutions injected in increasing order of concentration had the

same response, with low deviations in the values of the slopes of the curves and excellent

linearity in the concentration range studied, which demonstrates that the memory effect

phenomenon or electrode contamination was prevented. For concentrations of HCT

higher than 100 µmol L−1, the current response did not increase in the same intensity

probably due to adsorption of HCT or its oxidation product on the electrode surface. The

oxidation occurs at the aromatic ring of HCT [24], which probably leads to a greater

interaction between HCT species and the electrode surface (π-π interaction).

Nevertheless, the obtained linear concentration range is wider than the one obtained in a

previous work for HCT [24].

The analytical characteristics of the BIA‒MPA method for the simultaneous

determination of FRD and HCT are displayed in Table 1. Considering the analysis

pharmaceutical tablets (40 mg of FRD and 25-50 mg of HCT), the values of limit of

quantification (LQ) are within the required concentration range for the quality control of

such samples. The values possibly found in urine samples can be estimated by considering

the recommended daily dosage of FRD and HCT (80 and 100 mg, respectively) and that

these compounds are largely excreted unchanged (around 90% for FRD and 70% for

HCT) [35,36]. The expected value of FRD and HCT in urine after the consumption of the
recommended daily dosage is 48 mg L−1 of FRD and 47 mg L−1 of HCT (equivalent to

145 and 157 µmol L−1, respectively). Considering the 26-fold dilution of urine samples

before analysis and the LQ values listed in Table 1, the LQ values for the analysis of urine

samples can be estimated as 51 and 50 µmol L−1. Therefore, these LQ values indicate that

the proposed method is suitable to quantify both FRD and HCT in urine samples of a

volunteer that consumed the recommended daily dosage.

Table 1 Analytical performance of the proposed BIA‒AMP method to the FRD and HCT

Parameters compounds

FRD HCT

Linear range, µmol L⁻1 2 – 300 2 – 100

Sensitivity (a), µA/µmol L−1 0.076 0.13
R 0.9993 0.9990
Intercept, µA 0.27 5.9
LD, µmol L⁻1 0.65 0.63
LQ, µmol L⁻1 1.97 1.92

SD 0.015 0.025

R = Correlation coefficient; LD = Limit of detection (3.3*SD/a); LQ = Limit of quantification

(10*SD/a); SD = blank standard deviation; Intercept = Linear Coefficient.

The accuracy of the method was evaluated by comparing the results obtained

using the BIA-MPA method to those obtained using the reference method [30]. For such,

the pharmaceutical formulations were also analyzed using UV‒Vis spectrophotometry.

Table 2 displays the results of the analyses of these samples and respective standard

deviations (n = 3) using both the proposed method and the reference method.
Table 2 Comparison of the labeled, BIA‒AMP, and UV‒Vis results in the simultaneous

determination of FRD and HCT

S Labeled FRD HCT

FRD HCT BIA‒AMP UV‒Vis RD1% BIA‒AMP UV‒Vis RD2%

A 40 mg 25 mg 39.4 ± 0.8 39.5 ± 0.5 − 0.3 24.8 ± 0.6 25.1 ± 0.4 − 1.2
B 40 mg 50 mg 38.9 ± 0.8 40.1 ± 0.4 − 3.0 51.3 ± 0.5 49.1 ± 0.3 + 4.5
C 40 mg 25 mg 39.5 ± 0.7 41.3 ± 0.4 − 4.0 25.2 ± 0.5 24.3 ± 0.3 + 3.7
D 40 mg 50 mg 39.3 ± 0.8 39.2 ± 0.4 + 0.3 50.3 ± 0.4 48.9 ± 0.3 + 2.9

S = samples; FRD = Furosemide; HCT = Hydrochlorothiazide; UV-Vis = Spectrophotometry UV-Vis; E1 and

E2 =[(BIA‒AMP value − UV‒Vis value) / (UV‒Vis value) ] × 100

The results obtained using the BIA‒MPA method are in agreement with those

obtained using UV‒Vis spectrophotometry. The Student's t‒test for paired samples was

used to compare the values obtained using the two methods. The results were similar

(tcalculated = 0.61 < tcritical = 2.78), with no significant differences at the 95% confidence

level. Moreover, the relative errors found comparing the results obtained with two

methods to the value declared on the label were within the acceptable range.

The accuracy of the proposed method was also evaluated using the addition

and recovery test. Based on the results, the standard solutions injected in increasing order

of simultaneous concentration of FRD and HCT presented excellent responses, with the

recovery rate ranging from 92% to 113% for HCT and 92% to 107% for FRD.

In the interference study, HCT was quantified using FRD as the interferent.

For such, the HCT concentration remained constant (5 µmol L⁻1) in all standard solutions

and the FRD concentration was varied to determine its percentage of interference in the

determination of HCT. Based on the results, FRD at proportions of 1:1 to 1:3 did not

significantly interfere in the determination of HCT, as demonstrated by the RSD% less

than 5%. At an FRD concentration above 20 µmol L⁻1 (1:4 proportion or more), a

significant increase in the RSD% occurred, demonstrating that FRD exerted considerable
interference in the determination of HCT under this condition. Therefore, working with

this proportion or higher should be avoided. In the quantification of FRD using HCT as

the interferent, the concentration of FRD was maintained constant (5 µmol L⁻1) in all

standard solutions and the concentration of HCT was varied to investigate its interference

in the determination of FRD. Based on the results, HCT at proportions of 1:1 to 1:4 did

not significantly interfere in the determination of FED, as demonstrated by the RSD%

less than 5%. At an HCT concentration above 40 µmol L⁻1, an increase was found in the

percentage of interference of HCT in the determination of FRD. Therefore, working with

this proportion or higher should be avoided.

In the synthetic urine samples, the method achieved recovery rates ranging

from 104% to 110% for HCT and 95% to 100% for FRD (Table 3). The added amount

presented in Table 3 corresponds to the final concentration after sample dilution.

Therefore, considering the 26-fold dilution, the concentration of HCT and FRD in the

synthetic urine samples ranged from 520 to 780 µmol L−1. Considering the LQ values

listed in Table 2 There are no upper limits for both compounds in urine samples

established by World Anti-Doping Agency (WADA) as any concentration of both

compounds in urine samples of athletes are not allowed [37]. Therefore, simultaneous

determination using a BDD electrode is viable. This is a sensitive, stable, and

reproducible analysis that is easy to execute without the interference of the compounds

on the sample matrix. Thus, the proposed method can be satisfactorily applied for the

simultaneous determination of FRD and HCT in samples of synthetic urine. Moreover,

we can affirm that all components present in the synthetic urine (several inorganic ions

and urea) did not interfere in the response of FRD and HCT. However, if other

electroactive species are present in the analyzed samples, e.g. ascorbic acid, and if they

oxidize at potentials covering the range of detection of FRD and HCT and even less
positive potentials than +1.10 V, these electroactive species will interfere in this analysis.

If such interfering species are present, they need to be removed from the samples. In the

case of ascorbic acid, it would be possible to use ascorbate oxidase to remove it from the

samples.

Table 3 Recovery values for the analysis of synthetic urine spiked with HCT and FRD

[HCT], µmol L−1 [FRD], µmol L−1

samples Added Found % Rec Added Found % Rec
A + 20 20 22 110 20 19 95
A + 25 25 26 104 25 24 96
A + 30 30 32 110 30 30 100

Table 4 presents a comparison of the proposed methods with other

electroanalytical methods reported in the literature for the determination of diuretics in

urine samples. The proposed BIA‒MPA method presents higher sample throughput

(analytical frequency) compared with all previous methods, keeping comparable

detectability, sensitivity and precision. Although other methods did not report sample

throughput, it is possible to estimate in around 60 h−1, considering a voltammetric scan

of around 1 min. However, all previous works used the standard addition method while

the proposed method made use of an external calibration curve, hence the number of

sample analysis that can be performed using BIA‒MPA is much higher than a simple

comparison of the time for the obtaining of simple response. Therefore, the proposed

method can be successfully applied for routine analysis of biological samples.

Table 4 Comparative study on the determination of FRD or HCT in synthetic and human urine using other electroanalytical methods

Analyte Technique Ep (V)* Refence Electrode Working Supp. Electrolyte LD (µM) LQ (µM) AF Urine Type Ref.

Electrode

FRD SWV 1.02 Ag/AgCl (KCl 3M) CPE PB, pH 5 4.5×10−3 1.4×10−2 - Human [3]

FRD SWV 1.08 Ag/AgCl (KCl 3M) BPPG H2SO4, pH 1 0.47 - - Synthetic [4]

HCT CV ⁓0.45 Ag/AgCl (KCl Sat) CPE‒FCD BRB, pH 9 0.037 - - Human [12]

HCT DPV 1.04 Ag/AgCl (KCl 3M) GCE BR, pH 3.3 16.8 47 - Human [13]

FRD CV 0.98 Ag/AgCl (KCl Sat) GC‒MWG PB, pH 6.8 0.10 0.61 - Human [31]

HCT SWV 1.1 Ag/AgCl (KCl 3M) BDD BRB, pH 5 0.16 - - Synthetic [38]

HCT SWV 1.3 Ag/AgCl (KCl 3M) BDD BRB, pH 3 2.0 - - Synthetic [39]

FRD SWV - Ag/AgCl ECG‒P AcB, pH 5.3 2.7 9.06 - Synthetic [40]

HCT CV 1.2 Ag/AgCl GCE BRB, pH 3 4.3×10−3 - - Human [41]

FRD SWV 1.3 Ag/AgCl (KCl Sat) NPs/CS/PGE BRB, pH 3 3.9×10−3 0.011 - Human [42]

FRD BIA-MPA 1.0 Ag/AgCl (KCl 3M) BDD BRB, pH 4 0.65 1.97 130 Synthetic This work

HCT BIA-MPA 1.3 Ag/AgCl (KCl 3M) BDD BRB, pH 4 0.63 1.92 130 Synthetic This work

AF: Analytical Frequency; SWV: Square wave voltammetry; BPPG: Basal-Plane Pyrolytic Graphite; CV: Cyclic voltammetry; CPE-FCD: Carbon paste electrode
modified with ferrocenodicarboxylic acid; DPV = Differential pulse voltammetry; GCE = Glassy carbon electrode; GC-MWG = Glassy carbon electrode modified
with graphene oxide; ECG‒P = Graphite-paraffin composite electrode; BDD = Boron-doped diamond electrode; CPE = Carbon paste electrode; NPs/CS/PGE =
MnO2 nanoparticles/chitosan-modified pencil graphite electrode; PB = Phosphate buffer; BRB = Britton-Robinson buffer; AcB = Acetate buffer.
 Although the BDD surface is less susceptive to fouling effects, its use in a

flow system such as BIA under stirring facilitates the electrode cleaning and reduce the

possible adsorption of analytes or their oxidation products on electrode surface. This is

another unique advantage of using BIA systems for routine high-throughput analysis

combined with BDD electrodes [43].

Conclusion

In this paper, we present a fast, precise method with low limits of detection

and quantification for the simultaneous determination of FRD and HCT using the BIA-

MPA system with a BBD working electrode. After the optimization of the experimental

variables, the validation tests revealed excellent results, such as a high analytical

frequency, low limits of detection and quantification, excellent correlation coefficients (r

> 0.99), small standard deviations and satisfactory accuracy for both compounds studied.

Moreover, the accuracy of the proposed method was tested in comparison to both

reference method and recovery tests, with no need for the use of cleaning and conditioning

pulses on the surface of the BDD electrode. The results were concordant with those

achieved with the official method described in the Brazilian pharmacopeia at the 95%

confidence level, demonstrating adequate accuracy with relative errors not higher than

±5%. Furthermore, the proposed method presented satisfactory recovery with no

significant interference when applied to pharmaceutical samples, with the exclusion of

organic solvents in the preparation of FRD and HCT. Thus, the method is fast, simple and

inexpensive. In the biological sample (synthetic urine), the BIA-MPA method was

entirely satisfactory, as demonstrated by the recovery rates of 104% to 110% for HCT

and 95% to 100% for FRD.

Conflicts of Interest

The authors declare that there are no conflicts of interest

Acknowledgments

The authors are grateful for the financial support provided by the Brazilian agencies

CNPq (205220/2018-5, 307172-2017-0 and 465389/2014-7 - INCTBio) and CAPES

(Finance Code 001). We also thank Rafael M. Cardoso for designing Scheme 1 and

Graphical abstract.

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Figure Captions

Scheme 1. View of the BIA cell (orthogonal cut of the cell for clear observation): (1)

electronic micropipette; (2) tip with solution (sample or standard); (3) Pt conter electrode;

(4) Ag/AgCl/KClsat reference electrode; (5) rubber O-ring; (6) BDD working electrode;

(7) steel plate for electric contact; the BDD working electrode is placed exactly below the

tip (3 mm distant); (8) screw pins to join the body and the lower portion of the BIA cell

and to fix the BDD electrode between the steel plate and the lower portion; (9) upper

portion; (10) body; (11) lower portion.

Fig. 1 (A) Cyclic voltammograms obtained using the BDD electrode in 0.04 mol L⁻1

Britton-Robinson buffer pH 10.0 before (blank as dotted line) and after the addition of 1

mmol L⁻1 of FRD (dashed line) and HCT (solid line). (B) Influence of pH on the values
of oxidation peak potentials of FRD and HCT. Scan rate: 50 mV s⁻1; potential step: 5.0

mV

Fig. 2 Hydrodynamic voltammograms for the study of FRD and HCT both at 50 μmol

L−1, using the BDD work electrode in 0.04 mol L−1 Britton-Robinson buffer solution (pH

4.0). Injection volume: 100 μL, dispensing rate: 67 μL s−1

Fig. 3 Amperogram obtained in the BIA-MPA system for injections (n = 3) of solutions

containing HCT (50 μmol L−1) and FRD (50 μmol L−1) and a mixture of HCT + FRD (50

μmol L−1 + 50 μmol L−1) in 0.04 mol L−1 BR buffer solution (pH 4.0). Sample

volume: 100 μL, dispensing rate: 67 μL s−1. MPA detection involves the sequential

application of +1.10 V for 100 ms and +1.30 V for 100 ms continuosuly.

Fig. 4 A) Amperograms obtained by BIA-MPA after continuous injections (n = 30) of a

mixture of HCT + FRD (100 μmol L−1 + 100 μmol L−1) in 0.04 mol L−1 BR buffer

solution (pH 4.0). Sample volume: 100 μL, dispensing rate: 67 μL s−1. Other conditions

similar to Fig. 3. B) Plot of peak current values obtained after each injection in A) obtained

at +1.10 and +1.30 V.

Fig. 5 A) Amperograms obtained by BIA-MPA after injections (n = 3) of solutions

containing

20 μmol L−1 FRD, eleven standard solutions containing increasing concentrations (a-k)

of both
FRD and HCT (a-2 μmol L−1, b-10 μmol L−1, c-20 μmol L−1, d-50 μmol L−1, e-100 μmol

L−1, f-

150 μmol L−1, g-200 μmol L−1, h250 μmol L−1, i-300 μmol L−1, j-350 μmol L−1 and k-400

μmol

L−1). B) Linear range to FRD and HCT. Other conditions similar to Fig. 3.

Sample CRediT author statement

Eduardo Fonseca Silva: Methodology, Data curation, Writing – Original
Draft. Auro Atsushi Tanaka: Resources, Project administration, Funding
acquisition. Ridvan Nunes Fernandes: Investigation, Writing – Review &
Editing. Rodrigo Alejandro Abarza Munoz: Writing – Review & Editing,
Visualization. Iranaldo Santos Silva: Writing- Reviewing and Editing,
Validation, Supervision.

Highlights

1 – Fast determination of furosemide and hydrochlorothiazide in tablet and urine samples.

2 – Multiple-pulse amperometric detection on a boron-doped diamond electrode.

3 – No electrode fouling and high precision due to fast flow of batch-injection analysis.

4 – Proper recovery values for the analysis of spiked urine after simple sample dilution.

5 – High sample throughput and submicromolar detection limits.