Hydrochlorothiazide (HCTZ), 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide, is a drug widely used around the world for hypertension treatment, either alone or in combination with other anti-hypertensive drugs. HCTZ acts on the kidneys inhibiting sodium and chloride ions reabsorption into nephron-contoured tubules, and also preventing water reabsorption, which results in a decrease in blood pressure. Moreover, HCTZ is also used in the treatment of renal tubular acidosis, diabetes insipidus, edema, and the prevention of kidney stones [1
The determination of HCTZ in different matrices is currently carried out by means of different analytical techniques. Prominent among them is the high performance liquid chromatography (HPLC) with UV-VIS detection [3
], which is the analytical technique recommended by the United States Pharmacopeia [6
]. Although HPLC/tandem MS [7
] or capillary electrophoresis [8
] are also considered. Methods for the individual determination of HCTZ are also described. Thus, Youssef [10
] describes the use of an optical sensor for the fluorimetric determination of this compound. In [11
] chemiluminescence was considered. Nevertheless, these techniques have some disadvantages, such as high initial investment (equipment), the need for sample pre-treatments, time-consuming procedures, required expertise, and the high cost of consumables. In this sense, electroanalytical methods play a fundamental role and are stated as a very notable alternative for the determination of this anti-hypertensive drug [13
]. In particular, voltammetric techniques provide excellent detection and quantification limits, high sensitivity and selectivity, with relatively low economic cost. Taking into account that the performance of voltammetry is strongly influenced by the working electrode used, the design of this electrode is an area of major concern.
In the last decade, electrode modification through the immobilization of different species on the electrode surface has caused great interest in the development of new electrochemical sensors for the detection and quantification of different analytes in solution [19
]. Thus, an essential aspect in the design of these new sensors is the molecule immobilization procedure. In this sense, one of the widely used electrode modification approaches is the electropolymerization, where through consecutive voltammetry sweeps it is possible to generate a polymer layer on the electrode surface, which also enables to study the charge transfer kinetics [22
]. However, another suitable strategy for molecule immobilization that has aroused interest in recent years is based on aryl diazonioum salt monolayers anchored on the electrode surface [23
]. This approach allows the incorporation of a wide range of functional groups to the electrode surface [26
] and leads to the development of a recognition device with high repeatability, reproducibility, and stability in the measurements reported [27
Glutamic acid is one of the 20 most common amino acids that can be easily immobilized on the electrode surface, linked through an amino bond between α-amino and β-carboxylic acid groups [32
]. In the literature there are many works based on the application of electrochemical sensors modified with glutamic acid by electropolymerization for the determination of different analytes, including caffeic acid [34
], hydrazine [35
], ascorbic acid [36
], and hydrochlorotiazide [38
] among others, which provide good detection and quantification limits. Nevertheless, from the best of our knowledge studies on the application of glutamic acid modified electrodes via electrografting have not yet been attempted.
Regardless of the modification approach, it can be applied to different types of carbon surfaces such as graphite, glassy carbon, diamond, carbon nanomaterials and screen-printed carbon ink, among others. In this sense, in the last years, screen-printed carbon electrodes (SPCE) have generated great interest as a support for electrode modification. The screen-printing technology allows the mass production of reproducible, disposable, and relatively economical devices that usually include a three-electrode configuration printed on the same strip. Other important characteristics of these screen-printed electrodes (SPEs) are related with their miniaturized size and their capability to be connected to portable instrumentation, which makes them especially suitable for on-site analysis [39
In this work, both electropolymerization and electrografting modification approaches have been considered for the first time in the development of a glutamic acid modified electrode using a screen-printed carbon electrode as a support. Glutamic acid screen-printed carbon electrodes modified by both approaches electropolymerization (SPCE/PGA) and electrochemical grafting (SPCE/EGA) will be compared in terms of their electrochemical characterization and their analytical performance in the determination of hydrochlorothiazide. Moreover, the applicability of SPCE/EGA as a better sensor will be tested through its determination in a commercial anti-hypertensive drug.
2. Materials and Methods
L-glutamic acid (≥ 99%), 4-aminobenzoic acid (ABA), N-hydroxysulfosuccinimide (sulfo-NHS), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), potassium dihydrogen phosphate, sodium monophosphate, ethanol and sodium nitrite were provided from Sigma-Aldrich (St. Louis, MO, USA). Potassium ferrocyanide K4[Fe(CN)6]·3H2O, hydrochloride acid, 2-(N-morpholino)-ethanesulfonic acid (MES) and sodium hydroxide were supplied by Merck (Darmstadt, Germany). Potassium ferricyanide K3[Fe(CN)6] was purchased from Panreac (Barcelona, Spain). All reagents were of analytical grade. Hydrochlorothiazide (Pure) was provided by Laboratorio Chile® (Lab Chile®, Santiago, Chile). Commercial capsules of HCTZ (Hidroronol) from ITF-Labomed® (Santiago, Chile, capsules declared 50 mg hydrochlorothiazide per tablet) were commercially obtained.
Deionized and ultrapure water (Milli-Q plus 185 system, Millipore, Billerica, MA, USA) was used in all experiments.
Cyclic voltammetric (CV) and differential pulse voltammetric (DPV) measurements were performed using an Autolab System PGSTAT12 (Eco Chemie BV, Utrecht, The Netherlands) attached to a Metrohm 663 VA Stand (Metrohm, Herisau, Switzerland). The acquisition and treatment of data were carried out by means of a personal computer with GPES software, version 4.9 (Eco Chemie).
A traditional electrochemical cell based on a three-electrode system was used in all the experiments: (i) an Ag/AgCl in saturated KCl (Ag/AgCl/KClsat, CH-Instruments, Austin, TX, USA) electrode was used in aqueous media as a reference electrode; (ii) a platinum wire (CH-Instruments, USA) was used as a counter electrode; and (iii) the working electrode was a screen-printed carbon electrode modified with L-glutamic acid via both electropolymerization (SPCE/PGA) and electrografting (SPCE/EGA) procedures. SPCE/PGA and SPCE/EGA were prepared using a commercial screen-printed carbon disk electrode of 4 mm of diameter (reference DRP-110, DS SPE) supplied by DropSens (Oviedo, Spain). SPEs were connected to the Autolab System by means of a flexible cable (reference CAC, DropSens).
For pH measurements, a Crison micro pH 2000 pH-meter was used, and all electrochemical measurements were carried out in a glass cell at room temperature (20 °C) without oxygen removal.
2.3.1. Preparation of Modified SPCEs by Electropolymerization with L-Glutamic Acid (SPCE/PGA)
Before starting the preparation of the L-glutamic acid modified screen-printed carbon electrode, the main parameters affecting the electropolymerization approach were optimized. Thus, both the number of voltammetric cycles and the scan rate applied between −0.2 to +2.8 V vs. Ag/AgCl/KClsat
were studied. The optimization was performed considering the oxidation current peak response of 100.0 µmol L−1
HCTZ in 0.01 mol L−1
HCl by DPV. Figure 1
shows the results of a two-factor central composite design (9 experiments) for the screening of a wide range of cycles (1–60) and scan rates (1–200 mV s−1
). The fitting of a quadratic second order polynomial model allowed us to draw a rough estimate of the response surface (the mesh in Figure 1
), which exhibits the highest peak currents in the region corresponding to a small number of voltammetric cycles (between 0 and 10) and high scan rates (between 150 and 200 mV s−1
). A new set of measurements carried out inside this restricted area (not shown) produced the optimum response for the combination of five cycles and 180 mV s−1
. Therefore, according to these results, the unmodified SPCEs were immersed in L-glutamic acid 0.02 mol L−1
prepared in hydrochloric acid 0.04 mol L−1
solution, and five voltammetric cycles were applied between −0.2 and +2.8 V with a scan rate of 180 mV s−1
. The obtained SPCE/PGA was rinsed with ultrapure water and dried at room temperature.
2.3.2. Preparation of Modified SPCEs by Electrografting with L-Glutamic Acid (SPCE/EGA)
SPCE/EGA electrodes were prepared according to a two-step procedure previously described in the literature [29
] with minor changes.
Diazonium Salt Electrografting
The aryl diazonium salt was obtained in-situ by adding 2 mmol L−1
of sodium nitrite to a cooled acidic solution (1 mol L−1
aqueous HCl) of 73 mmol L−1
4-aminobenzoic acid. The resulting solution was stirred for 30 min in an ice bath before the electrochemical grafting process [42
] was performed. Then, the SPCE was immersed in 20 mL of the diazonium salt solution and 15 CV cycles between 0 V and −1 V at scan rate of 0.2 V s−1
were performed. Finally, the functionalized SPCE were carefully rinsed with Milli-Q water and methanol to remove any physisorbed compounds on the electrode surface.
Covalent Immobilization of L-Glutamic Acid via Carbodiimide Coupling
Carboxyl groups generated during the electrografting process on SPCE surface were activated by dropping 10 μL of 26 mmol L−1 N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and 35 mmol L−1 of N-hydroxysulfosuccinimide (sulfo-NHS) in 100 mmol L−1 MES buffer (pH 4.5) onto the electrode surface for about one hour, then the electrodes were rinsed with Milli-Q water and dried at room temperature. The activated carboxyl groups reacted overnight at 4 °C with amine groups of L-glutamic acid by placing 10 µL of 2.9 mg/100 μL of L-glutamic acid solution prepared in 90:10 of 0.1 mol L−1 MES buffer (pH 4.5) and ethanol, respectively.
2.3.3. Voltammetric Measurements
For differential pulse voltammetric measurements of hydrochlorothiazide using both SPCE/PGA and SPCE/EGA, the experimental conditions were a pulse amplitude of 5 mV, a pulse width of 0.0050 s, a scan rate of 5 mV s−1 scanning the potential from 0.70 to 1.60 V vs. Ag/AgCl/KClsat.
Linear calibration plots were obtained by increasing HCTZ concentration in hydrochloric acid 0.01 mol L−1
solution, according to previous studies [38
The assay of HCTZ tablets was performed according to the Razak protocol [44
]: three tablets were crushed and homogenized, and the amount equivalent to one tablet was dissolved in 50 mL 0.02 mol L−1
NaOH and sonicated for 10 min. Then, a volume of the sample in 0.01 mol L−1
hydrochloric acid solution was placed in the cell and the scan was recorded. Calibration was performed by the standard addition method: four aliquots of HCTZ standard solutions were further added and the respective curves were recorded.
In both linear calibration plots and analysis of the tablet samples, to improve the repeatability of both electrodes SPCE/PGA and SPCE/EGA, a conditioning step was performed before each measurement by applying a conditioning potential (Econd) of 0.7 V for 30 s in the same measuring solution. In all cases, peak currents were calculated considering the background current.
The developed sensors for the determination of HCTZ are the first approach on glutamic acid-based screen-printed electrodes. Two different modification approaches, electropolymerization and electrochemical grafting, were considered for the immobilization of the glutamic acid on the SPCE surface. Thus, in this work the analytical performance of both SPCE/PGA and SPCE/EGA were compared, concluding that the SPCE/EGA has an enhanced chemical and mechanical stability with respect to SPCE/PGA and also performs much better for HCTZ determination. In comparison with the unique existing glutamic acid-based electrode for HCTZ determination, the repeatability, the reproducibility, as well as the calibration data obtained in this study for both developed sensors are much better than those achieved by the preceding glutamic acid glassy carbon electrode modified via electropolymerization [38
]. Moreover, the developed sensors present all the addition advantages provided by the use of low-cost commercially available SPCE as a support which, unlike glassy carbon substrate, does not require polishing of the surface of the carbon screen-printed prior to glutamic acid immobilization.
The applicability of SPCE/EGA, as the best-developed sensor, for the determination of HCTZ by DPV was demonstrated using a commercial anti-hypertensive drug with a good trueness and a high reproducibility inferred by the relative error (%) and the RSD (%), respectively.
Thus, the above presented results suggest that the SPCE/EGA can be very appropriate for the determination of HCTZ at μmol L−1 levels in drug samples. It could be also applicable to biological samples like plasma or urine, but this would require a careful study about the possible interferences that could be present in such complex media.