Introduction

Up to today, metallic nanoparticles have attracted significant scientific interest because their distinct optical, catalytic, magnetic, electronic, and biological properties are applicable in diverse areas. Their excellent catalytic properties and large surface area offer new perspectives in electrocatalysis and electroanalysis [1, 2]. Furthermore, they increase the redox reversibility on electrode surfaces and improve the reproducibility, sensitivity, and detection limit of electrodes/sensors [3, 4]. AgNPs have exceptional physical, electronic, and chemical properties among several metallic nanoparticles devoted to electrochemical applications. Some reports focus on synthesizing, characterizing, and using AgNPs in electroanalytical applications [5]. For example, SPCE modified with AgNPs indicated excellent selectivity for detecting dopamine (DA) with a low LOD and high sensitivity [6]. Patel et al. developed an AgNP-based electrochemical detection method for Shiga toxin 1 detection [7]. Palisoc et al. reported graphene paste electrodes modified with AgNPs as effective sensors for heavy metals such as lead, cadmium, and copper [8].

The synthesis of AgNPs often involves chemical reagents to reduce silver ions and stabilize the nanoparticles. These reagents can be toxic and present potential health risks. Green chemistry has emerged as a promising AgNP synthetic approach to address this issue. During the last decade, biological systems, including plants, algae, bacteria, yeast, and fungi, have been employed to transform inorganic metal ions into metal nanoparticles through the reducing capabilities of their components [9]. Green synthesis is a promising and environmentally friendly approach to producing AgNPs with unique properties. The synthesis of AgNPs using plants and their extracts has attracted much attention since they are readily available in large quantities and utilize non-toxic phytochemicals. Ashraf et al. demonstrated that AgNPs synthesized from aloe vera leaf extract have potential anti-glycating ability, which may enable their use as therapeutics in treating diabetes-related complications [10]. Kumar et al. synthesized AgNPs using the sacha inchi leaf extract, which exhibited a significant antioxidant capability [11].

Hibiscus (Hibiscus rosa-sinensis) is a medicinal plant used effectively in native medicines against hypertension, pyrexia, and liver disorders. Previous studies have revealed that the leaves contain flavonoids, glycosides, tannins, saponins, and terpenoids, while the petals contain anthocyanins, quercetin, and kaempferol [12]. Red petals have more anthocyanins, while white petals have slightly higher tannins. Philip et al. reported silver and gold nanoparticles with triangular, hexagonal, dodecahedral, and spherical shapes after the bioreduction of Ag and Au ions by Hibiscus rosa-sinensis leaf extract [13]. Lu et al. performed a green synthesis of AgNPs using Hibiscus rosa-sinensis leaf aqueous extract to treat human liver cancer. The synthesized nanoparticles exhibited a notable anti-liver cancer activity against common liver cancer cell lines, attributable to their antioxidant activities [14]. As another fascinating application, Kumar et al. developed a sustainable and environment-friendly electrochromic device using raw hibiscus flower extract [15].

This work explores the electroanalytical application of AgNPs synthesized using Hibiscus rosa-sinensis flower extract to determine N-acetyl-para-aminophenol (acetaminophen, APAP) in pharmaceutical formulations. APAP is one of the most widely used analgesic and antipyretic medications worldwide and an emerging environmental contaminant [16]. Its traditional analysis methods include spectrophotometry [17], HPLC [18], and capillary zone electrophoresis [19], among others. Electroanalytical methods are reliable alternatives since APAP is an electroactive species that can be easily electrooxidized. Using electroanalysis in quantitative analysis has remarkable advantages, such as low-cost equipment, low reagent consumption, simple pre-treatment procedure, excellent sensitivity, and suitable selectivity [20]. Furthermore, they offer the possibility of acquiring information about the mechanism involved in the electrochemical reactions. The electrode surface modification draws attention because of its highly enhanced sensitivities. Several modified electrodes have been developed for APAP determination, from the simplest to the most complex and laborious procedure [21,22,23].

This paper also describes the electrochemical behavior of APAP and its oxidation mechanism.

Experimental

Materials and reagents

All the reagents were of analytical grade and used without further purification. Silver nitrate (AgNO3), potassium chloride (KCl), potassium ferrocyanide (K2[Fe(CN)6]), N-acetyl-para-aminophenol (APAP), ascorbic acid (AA), and caffeine (CA) were purchased from Sigma-Aldrich, Darmstadt, Germany. Hibiscus rosa-sinensis dried flowers were acquired from a local market in Porto Alegre, Brazil. Paracetamol tablets and oral powder sachets were purchased from local pharmacies in Porto Alegre, Brazil. The Britton-Robinson (B-R) buffer solution consisted of a mixture of acetic acid (CH3COOH, Sigma-Aldrich, Darmstadt, Germany), phosphoric acid (H3PO4, Sigma-Aldrich, Germany), and boric acid (H3BO3, Sigma-Aldrich, Darmstadt, Germany) at a concentration of 0.04 mol L−1. The pH was adjusted with a 1.0 mol L−1 sodium hydroxide (Merck, Darmstadt, Germany) solution when necessary.

Instrumentations

The UV–Vis absorbance measurements were performed on a spectrophotometer model UV-1800 from Shimadzu, Kyoto, Japan. The spectra were recorded in triplicate, in the wavelength range from 800 to 350 nm, using a quartz cuvette with a 1 cm optical path length. The X-ray diffraction (XRD) patterns were collected on a Siemens D5000 diffractometer (Siemens, Berlin, Germany) using a Cu Kα1 (0.15406 nm) voltage of 40 kV and a current intensity of 30 mA. The scanning range (2q) was 10–90°, with a step size of 0.05° and a counting time per step of 1 s. To capture the scanning electron microscope (SEM) images of the modified electrode, an Inspect F50-FEI scanning electron microscope (FEI Company, Eindhoven, The Netherlands) equipped with a field emission electron gun (FEG) accelerated with a voltage of 30 kV was used. DLS measurements were performed on a Brookhaven Instruments (BI-200 goniometer, BI-9000 at digital correlator) with a He–Ne laser (λ = 632.8 nm) as the light source. In the experimental setup, the photomultiplier tube (PMT) is on the scattering plane at a 90° angle, with the laser lie and the scatter volume. Electrochemical measurements were performed using a PGSTAT-128N potentiostat/galvanostat (Metrohm, Herisau, Switzerland) controlled by NOVA 2.1.6 software. The electrochemical cell consisted of a disposable and commercial screen-printed carbon electrode (SPCE, 110) produced by Metrohm. The conventional three-electrode configuration comprises a carbon disk shape with a 4 mm diameter as the working electrode, silver as the pseudo-reference, and a carbon counter electrode printed on a ceramic substrate (3.4 × 1.0 × 0.05 cm, L × W × H). Stock and buffer solutions were prepared with ultrapure water with a resistivity of 18.2 MΩ cm at 25 °C obtained by a Millipore Milli-Q purification system (Bedford, MA, USA). The solutions’ pH was verified and adjusted using a pH meter from MS Tecnopon Instrument (Mpa 210).

Synthesis of AgNPs

The flower extract was obtained by macerating 4 g of small pieces of dried flowers in 20 mL of ethanol for 48 h. The mixture was filtered and saved for AgNP biosynthesis. Subsequently, 50 µL of 0.05 mol L−1 AgNO3 solution was added to 20 mL of the extract, previously diluted with ultrapure water to a final concentration of 0.01 g mL−1. A yellow color emerged after a few minutes of reaction and became darker and more stable after 24 h. The AgNP suspension was stored in the refrigerator at 4–8 °C. A more concentrated suspension was prepared to promote the precipitation of AgNPs and obtain the solid powder for XRD analysis.

Electrochemical measurements

To produce SPCE/AgNPs, 15.0 µL of the AgNPs suspension was dropped onto the working electrode surface, 5 µL at a time, and allowed to dry overnight at room temperature. The electrochemical response of the electrode before and after modification was evaluated by recording cyclic voltammograms of 1.0 × 10−3 mol L−1 ferrocyanide solution in 0.1 mol L−1 KCl supporting electrolyte. CV and DPV techniques were employed to perform the electrochemical behavior and quantitative analysis of APAP in a B-R buffer solution at different scan rates and pH values.

Results and discussion

Synthesis and characterization of AgNPs

After mixing the silver nitrate and extract solutions, a prompt visible color change from light yellow to brownish yellow was observed, indicating the formation of AgNPs. The bioactive compounds in the extract promote the reduction of silver ions to metallic silver, and a further crystallization process is beginning. The colloidal suspension remains stable for weeks when appropriately stored under refrigeration, which prevents evaporation and agglomeration. Figure 1A shows the UV–Vis spectrum with a maximum absorption band at 421 nm. It is well-known that this band reflects the nanoparticle size and aggregate state since AgNPs absorb the visible part of light due to the surface plasmon resonance (SPR). The particle size of 62 nm was estimated using Mie’s theory and the Palik database [24]. This value is very close to the 60 nm found by dynamic light scattering (DLS) analysis (Fig. S1).

Fig. 1
figure 1

A UV–Vis spectrum obtained for AgNPs (solid red line) and the extract solution (black dashed line). B XRD pattern of AgNPs

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The XRD analysis shown in Fig. 1B confirms the crystalline nature of the green-synthetized AgNPs. The peaks observed at 2θ values of 38.17, 44.17, 64.54, 77.46, and 81.41° correspond to (111), (200), (220), (311), and (211) reflection planes of a face-centered cubic (FCC) crystal structure of metallic silver (JCPDS 04–0783). The extra peaks at 32.31 and 46.38° were indexed as planes (111) and (200) of silver oxide (Ag2O) (JCPDS 76–1393). The unassigned peak (*) at 27.88° may be associated with a phytochemical precipitated product. In this sense, Nikam et al. find the same peak in eco-friendly syntheses of AgNPs [25]. The crystallite size was calculated using the Debye–Scherrer Eq. (1).

$$D\;(\text{nm})= \frac{k\lambda }{\beta \text{cos}\theta }$$
(1)

where the dimensionless factor K is assigned a value of 0.94, λ is the wavelength of the copper alpha-line (0.15406 nm), and β is the full width at half maximum (FWHM) of a diffraction intensity peak. The crystallite size from seven main intensity peaks is 14 ± 2 nm. The reference intensity ratio (RIR) using X-ray diffraction (XRD) is a convenient method for phase quantification [26]. The phase percentage of Ag and Ag2O was found to be 98% and 2%, respectively.

SPCE/AgNP characterization

The SEM images of the modified electrode surface shown in Fig. 2A confirm a uniform distribution of granulated AgNPs.

Fig. 2
figure 2

A SEM images of the SPCE/AgNP surface. B Cyclic voltammograms for the SPCE (black line) and SPCE/AgNPs (red line) in the presence of 1.0 × 10−3 mol L−1 K4[Fe(CN)6] solubilized in 0.1 mol L−1 KCl at a scan rate of 50 mV s.−1

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Cyclic voltammograms of the ferrocyanide/ferricyanide redox couple offer valuable information about the electrochemical response of the electrode before and after its modification. As shown in Fig. 2B, it is noticeable that the presence of AgNPs barely affected the current intensity but significantly improved the reversibility of the redox system. The variation of peak current as a function of the square root of the scan rate gave a linear relationship as predicted by the Randles–Sevcik equation for a diffusion-controlled process, as shown in Figs. S2 and S3. From these slopes, the electroactive surface areas were calculated to be 0.077 and 0.091 cm2 for the unmodified and modified electrode, respectively, which represents an increase of 18%.

On the other hand, the modified electrode’s response was surprisingly more sensitive to APAP oxidation than the bare electrode, and a potential shift was also evident, which indicates a catalytic effect of AgNPs (Fig. 3A).

Fig. 3
figure 3

A Differential pulse voltammograms of 2.5 × 10−5 mol L−1 APAP in B-R buffer solution at pH 5.0 at SPCE (black line) and SPCE/AgNPs (red line). B Cyclic voltammograms of 3.0 × 10−4 mol L−1 APAP in B-R buffer solution (pH 3.0–9.0) at a scan rate of 50 mV s−1. The inset shows the variation of the peak potential and peak current as a function of the pH

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Electrooxidation of APAP

The electrochemical properties of APAP at SPCE/AgNPs were investigated using CV in a B-R buffer solution with pH values from 3 to 9. Figure 3B exhibits an anodic peak in the forward scan and a cathodic peak in the reverse scan. According to the literature, APAP exhibits a quasi-reversible two-electron process due to its transformation to N-acetyl-p-benzoquinone-imine (NAPQI) and vice versa, as shown in Eq. (2).

(2)

Figure S4 shows the variation of the peak current ratio (ipc/ipa) as a function of pH, which reflects the chemical stability of the reaction product on the electrode surface. As can be seen, the ipc/ipa is less than one, demonstrating the instability of NAPQI. According to Nematollahi et al., in an acidic media (pH < 4), NAPQI will decompose by hydrolysis, and in an alkaline media (pH > 9), it will undergo hydroxylation [27]. On the other hand, a dimerization process of NAPQI may occur at the interval of 5 to 9. Therefore, relative stability is expected at pHs 5 and 9.

The peak potentials shifted to negative potentials as pH increased, confirming that protons participated in the oxidation reaction. Equation (3) represents the relationship between the peak potential and pH value.

$$Epa={E}_{(\text{pH}=0)}-\frac{2.303 \;mRT}{2F}\text{ pH}$$
(3)

where m is the number of protons involved in the reaction; R, T, and F have their usual meanings. From the linear relationship, a slope of 0.028 V/pH was obtained which indicates that one proton is involved in the electrooxidation of APAP.

The linear plot of potential versus pH fitted with the equation E(V) = 0.442 − 0.028 pH (R2 = 0.984). The slope of 0.028 pH V−1 was almost half the theoretical value of 0.059, indicating that the number of protons (H+) and electrons (e−) has a stoichiometric ratio of 1:2. Nagles et al. reported similar findings that they attributed to an alternated mechanism, shown in Fig. S5. In this case, the modified electrode favors the protonation of APAP below its pKa value (pKa = 9.5), resulting in a protonated NAPQI that undergoes a subsequent chemical reaction producing p-benzoquinone [28].

Regarding the influence of the pH on the current intensity, differential pulse voltammograms exhibited higher current intensities at pH 3–5, as shown in Fig. S6, offering greater sensitivity to the analytical method. Therefore, the pH chosen for future studies was 5.

The effect of the scan rate reveals essential information about the reaction kinetics of the electrochemical processes. Figure 4 shows the cyclic voltammograms of APAP in B-R buffer solution at pH 5.01, with the scan rates varying from 10 to 500 mV s−1. As can be observed, the increase in scan rate shifted the anodic and cathodic peaks towards more positive and negative potentials, respectively. Such behavior is characteristic of a quasi-reversible redox process [29].

Fig. 4
figure 4

Cyclic voltammograms of 3.0 × 10−4 mol L−1 APAP in B-R buffer solution at pH 5.0 at scan rates from 10 to 500 mV s−1. The inset shows the correlation between the peak current and the square root of the scan rate

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In the inset, the plot of the peak current as a function of the square root of the scan rate displayed a linear relationship, which indicates that the kinetics of the redox processes of APAP on the SPCE/AgNPs is a diffusion-controlled process, as predicted by the Randles–Sevcik equation at 25 °C [29]:

$$ip=2.69\times {10}^{5}{n}^{3/2}A{D}^{1/2}C{v}^{1/2}$$
(4)

where ip is the peak current (A), n is the number of electrons, A is the electrode surface area (cm2), D is the diffusion coefficient (cm2 s−1), ѵ is the scan rate (V s−1), and C is the bulk concentration (mol cm−3).

Furthermore, Fig. S7 shows the linear correlation between log ip and log v with an angular coefficient of 0.525 for the anodic peak of APAP close to the theoretical value of 0.5, demonstrating that the oxidation process is governed mainly by diffusion.

Analytical method validation

SWV and DPV were employed to develop the analytical procedure for APAP determination. They are well-known for exhibiting high sensitivity, low background current, and low detection limits. Figures 5 and 6 show the voltammetric responses at different concentrations of APAP and the corresponding calibration plot for DPV and SWV, respectively. Table 1 summarizes the analytical parameters associated with each analytical curve. As can be inferred from these parameters, the same linear concentration range was observed for both techniques, although the SWV sensitivity was 2.7 times higher than the DPV sensitivity, as well as the detection limits (LOD) and quantification limit (LOQ). The values of LOD and LOQ were calculated using the relation LOD = 3 s/m and LOQ = 10 s/m, where “s” is the standard deviation of five blank measurements and “m” is the slope of the analytical curve. The precision of SPCE/AgNPs, regarding the repeatability of the APAP signal, was evaluated by recording ten voltammograms at 0.54% and 0.35% and were the relative standard deviation (RSD) obtained for DPV and SWV, respectively, indicating excellent precision. SPCE/AgNPs remained stable and reusable for at least 2 months without special storage conditions. They were cleaned with ultrapure water, allowed to dry, and kept at room temperature. Figure S8 shows the voltammetric response after 2 months of use, showing that the signal maintained 99% of the original intensity.

Fig. 5
figure 5

Differential pulse voltammograms after successive addition of APAP, from 4.9 × 10−7 to 1.0 × 10−4 mol L.−1. The insets show the corresponding calibration plot; error bars indicate the standard deviation for N = 3

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Fig. 6
figure 6

Square wave voltammograms after successive addition of APAP, from 4.9 × 10−7 to 1.0 × 10−4 mol L.−1. The insets show the corresponding calibration plot; error bars indicate the standard deviation for N = 3

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Table 1 Analytical parameters obtained from SWV and DPV
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For comparison, Table 2 presents the LOD and linear concentration range of electroanalytical methods using different electrodes and techniques for APAP determination. As can be seen, SPCE/AgNPs showed better or similar analytical performance than the previously reported ones.

Table 2 Comparison of different modified electrodes for the determination of APAP
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APAP is commercially available alone or combined with other compounds such as ascorbic acid (AA) and caffeine (CA). The possible interferences of AA and CA in the voltammetric determination of APAP were evaluated. Figure S9 shows the differential pulse voltammograms of APAP alone and in the presence of AA and CA at a ratio concentration of 1:0.5 and 1:1. As can be observed, the peak potential shifted towards positive potentials when AA was added, but the current intensity remained unchanged. The interaction between the analytes and the electrode surface could be responsible for the potential shift since the electrolyte solution consisted of BR buffer.

Real sample analysis

SPCE/AgNPs were used to quantify APAP content in two different pharmaceutical samples: 750 mg tablets and 500 mg oral sachets. Ten APAP tablets were weighed and ground into a fine powder using a pestle and mortar. A quantity of this powder was weighed, transferred to a 10 mL volumetric flask, and adjusted with ultrapure water to obtain a stock solution at a concentration of 1.0 × 10−2 mol L−1. Recovery tests were carried out by spiking APAP in the sample and determined by DPV and SWV using the standard addition method. Figures S10 and S11 show the excellent linearity obtained for techniques, which indicates a null matrix effect, thus guaranteeing the reliability of the proposed sensor. The tablet analysis obtained satisfactory results of 109.3% and 102.0% by DPV and SWV, respectively. The content of APAP in the oral sachets was assessed in the same manner, dissolving the powder in ultrapure water for a concentration of 1.0 × 10−2 mol L−1. Figures S12 and S13 show the voltammograms and the corresponding analytical curves. The oral sachet samples also obtained satisfactory results of 106.2% and 100.5% for DPV and SWV, respectively. The SWV values fulfilled the accuracy requirement within 98–102%, suggesting that the proposed method is reliable for routine quality control analysis of APAP in pharmaceutical preparations.

Conclusions

An environmentally friendly electrochemical sensor platform based on AgNPs synthesized by Hibiscus rosa-sinensis flower extract is proposed. The synthesized AgNPs had an average diameter of 60 nm, calculated from the UV–Vis and DLS spectrum; XRD analysis confirmed their crystallinity. By incorporating AgNPs into an SPCE, a sensitive and selective voltammetric sensor for APAP was developed. APAP undergoes a quasi-reversible pH-dependent redox process controlled by diffusion. APAP was successfully determined in tablets and oral sachets using the standard addition method. The principal advantage of the proposed sensor is that it can be directly applied to the analysis of pharmaceutical forms without extensive sample preparation since there was no observed interference from excipients. Another advantage is that the preparation method is fast and simple and follows a green chemistry approach.