A UiO-66-NH2 MOF/PAMAM Dendrimer Nanocomposite for Electrochemical Detection of Tramadol in the Presence of Acetaminophen in Pharmaceutical Formulations

In this work, we prepared a novel electrochemical sensor for the detection of tramadol based on a UiO-66-NH2 metal–organic framework (UiO-66-NH2 MOF)/third-generation poly(amidoamine) dendrimer (G3-PAMAM dendrimer) nanocomposite drop-cast onto a glassy carbon electrode (GCE) surface. After the synthesis of the nanocomposite, the functionalization of the UiO-66-NH2 MOF by G3-PAMAM was confirmed by various techniques including X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS), field emission-scanning electron microscopy (FE-SEM), and Fourier transform infrared (FT-IR) spectroscopy. The UiO-66-NH2 MOF/PAMAM-modified GCE exhibited commendable electrocatalytic performance toward the tramadol oxidation owing to the integration of the UiO-66-NH2 MOF with the PAMAM dendrimer. According to differential pulse voltammetry (DPV), it was possible to detect tramadol under optimized circumstances in a broad concentration range (0.5 μM–500.0 μM) and a narrow limit of detection (0.2 μM). In addition, the stability, repeatability, and reproducibility of the presented UiO-66-NH2 MOF/PAMAM/GCE sensor were also studied. The sensor also possessed an acceptable catalytic behavior for the tramadol determination in the co-existence of acetaminophen, with the separated oxidation potential of ΔE = 410 mV. Finally, the UiO-66-NH2 MOF/PAMAM-modified GCE exhibited satisfactory practical ability in pharmaceutical formulations (tramadol tablets and acetaminophen tablets).


Introduction
Acetaminophen (N-acetyl-p-aminophenol), also referred to as paracetamol, is an analgesic and antipyretic medication as well as a key ingredient in some flu and cold treatments. Acetaminophen is functionally similar to aspirin, and is therefore considered a suitable alternative for aspirin-sensitive people. Combining acetaminophen with non-steroidal anti-inflammatory drugs (NSAIDs) and opioids is reportedly an effective strategy for treating pain such as post-operative or cancer pain [1][2][3]. The metabolism of acetaminophen occurs mainly in the liver, resulting in the production of toxic metabolites. Acetaminophen overdose is associated with accumulated toxic metabolites leading to the development of nephrotoxicity, hepatotoxicity, and sometimes kidney failure. Other side effects have been reported to be liver problems, pancreatitis, and skin rashes. Such bottlenecks may be due to high doses, chronic abuse, or concurrent abuse with alcohol or other agents [4][5][6].
Tramadol, (1RS, 2RS)-2-[(dimethylamine)-methyl]-1-(3-methoxy phenyl)-cyclohexanol hydrochloride), is a centrally serving synthetic analgesic with poor affinity for µ-opioid receptors. It is prescribed to manage moderate-to-severe pain and has monoaminergic performance to impede the reabsorption of serotonin and noradrenaline. Tramadol alone or in combination with NSAIDs can be prescribed for people with chronic pain, spinal The current work was conducted to produce and characterize the UiO-66-NH 2 /G3-PAMAM nanocomposite, which was then anchored on a GCE to achieve an electrocatalytic and voltammetric tramadol sensor. The sensor based on the UiO-66-NH 2 MOF/PAMAM nanocomposite demonstrated a high degree of catalysis toward tramadol, with a narrow LOD and a wide linear range. Additionally, the sensor was tested in terms of catalytic behavior for the tramadol determination in the co-existence of acetaminophen. The ability of the modified electrode for sensor applications was examined in real specimens of acetaminophen tablets and tramadol tablets.
The novelty of this research lies in the combination of the UiO-66-NH 2 MOF and the PAMAM dendrimer as a sensing platform, which has enabled the detection of tramadol in the presence of acetaminophen.

Instruments and Reagents
An Autolab PGSTAT302N electrochemical device was employed to conduct electrochemical determinations. The solutions' pHs were adjusted by a pH-meter (Metrohm 710). The deionized water used in each experiment was also taken from a Millipore Direct-Q ® 8 UV (ultra-violet) (Millipore, Darmstadt, Germany).
Field-emission scanning electron microscopy (MIRA3TESCAN-XMU) was utilized for morphological studies. Energy-dispersive X-ray spectroscopy (EDS)-FE-SEM (MIRA3TESCAN-XMU, Tescan, Czech Republic) was also applied for elemental analysis. XRD patterns were recorded to obtain the data on structure using a Panalytical X'Pert Pro X-ray diffractometer (Etten Leur, The Netherlands) via Cu/Kα radiation (λ = 1.5418 nm). The FT-IR spectra were also obtained through a Bruker Tensor II spectrometer (Mannheim, Germany).
The precursors for the synthesis of the UiO-66-NH 2 /G3-PAMAM nanocomposite, tramadol, acetaminophen, and other chemicals were also of analytical grade and were used upon delivery without any additional purification. It is noted that they were obtained from Merck and Sigma-Aldrich chemical companies.

Synthesis of the UiO-66-NH 2 MOF/G3-PAMAM Nanocomposite
The previous report, with slight modification, was followed to construct the UiO-66-NH 2 MOF [62]. Thus, ZrCl 4 , 2-aminoterephthalic acid, and glacial acetic acid (0.2 mmol, 0.2 mmol, and 5 mL, respectively) were poured into dimethylformamide (20 mL, DMF) while ultra-sonicating for 45 min, followed by their placement in a 50 mL Teflon-lined autoclave and subsequently in the oven at 120 • C for 48 h. After cooling down, a centrifugation was conducted to separate the precipitate, followed by rinsing thoroughly with DMF/methanol and then vacuum-drying at 70 • C for 12h to obtain the UiO-66-NH 2 MOF.

Preparation of GCE Modified with the UiO-66-NH 2 MOF/G3-PAMAM Nanocomposite
A facile drop-casting protocol was followed to modify the surface of GCE with the UiO-66-NH 2 MOF/G3-PAMAM nanocomposite. Thus, a 1 mg/mL suspension of prepared nanocomposite underwent a 20-min sonication, and then 4 µL of the suspension was poured on the GCE surface in a dropwise manner, followed by drying under room conditions to achieve the UiO-66-NH 2 MOF/PAMAM/GCE.

Preparation of Pharmaceutical Formulations
Five tablets of the tramadol (labeled value of tramadol = 100 mg per tablet) and acetaminophen (labeled value of acetaminophen = 325 mg per tablet) purchased from a local pharmacy in Kerman (Iran) were completely powdered in a mortar and pestle. Then, an accurately weighed amount of the homogenized tramadol and acetaminophen powders was transferred into 100 mL 0.1 mol/L PBS (pH 7.0). For better dissolution, the solutions inside the flasks were sonicated (20 min). After that, the resulting samples were filtered. Finally, a specific volume of the prepared samples was transferred into volumetric flasks and diluted with 0.1 M PBS (pH = 7.0). The diluted solutions were then put into the electrochemical cell for DPV analysis.

Results and Discussion
3.1. Characterization of the UiO-66-NH 2 MOF/PAMAM Nanocomposite FT-IR spectroscopy authenticated that the PAMAM cross-linked on the UiO-66-NH 2 MOF ( Figure 1). The characteristic peaks of the FT-IR spectrum prepared from the UiO-66-NH 2 MOF at 1577 cm −1 and 1657 cm −1 were attributed to the stretching vibration of C=C on the benzene ring and the vibration of coordinated carboxylate moieties, respectively [64]. The bonds at 1384 cm −1 and 1259 cm −1 were attributed to the stretching vibrations of C-N related to 2-aminoterephthalic acid. The characteristic peaks at 3366 cm −1 and 3462 cm −1 were attributed to symmetric and asymmetric stretching vibrations of N-H related to the primary amine group, respectively. The characteristic peaks at 768 cm −1 and 663 cm −1 were attributed to the vibration of Zr-O on the UiO-66-NH 2 [65]. In the FT-IR spectrum of the UiO-66-NH 2 MOF/PAMAM nanocomposite, the observed absorption peak at 1657 cm −1 of UiO-66-NH 2 shifted to 1625 cm −1 following PAMAM immobilization, authenticating the formation of imine bands (C=N) from a Schiff base reaction between the carbonyl groups, belonging to glutaraldehyde, and the amine group, belonging to the UiO-66-NH 2 MOF and the PAMAM dendrimer.
Five tablets of the tramadol (labeled value of tramadol = 100 mg per tab aminophen (labeled value of acetaminophen = 325 mg per tablet) purchased pharmacy in Kerman (Iran) were completely powdered in a mortar and pe accurately weighed amount of the homogenized tramadol and acetaminop was transferred into 100 mL 0.1 mol/L PBS (pH 7.0). For better dissolution, inside the flasks were sonicated (20 min). After that, the resulting samples Finally, a specific volume of the prepared samples was transferred into volu and diluted with 0.1 M PBS (pH = 7.0). The diluted solutions were then put trochemical cell for DPV analysis.

Characterization of the UiO-66-NH2 MOF/PAMAM Nanocomposite
FT-IR spectroscopy authenticated that the PAMAM cross-linked on the MOF (Figure 1). The characteristic peaks of the FT-IR spectrum prepared f 66-NH2 MOF at 1577 cm −1 and 1657 cm −1 were attributed to the stretching vib on the benzene ring and the vibration of coordinated carboxylate moieties [64]. The bonds at 1384 cm −1 and 1259 cm −1 were attributed to the stretching C-N related to 2-aminoterephthalic acid. The characteristic peaks at 3366 cm −1 were attributed to symmetric and asymmetric stretching vibrations of N the primary amine group, respectively. The characteristic peaks at 768 cm −1 were attributed to the vibration of Zr-O on the UiO-66-NH2 [65]. In the FT-IR the UiO-66-NH2 MOF/PAMAM nanocomposite, the observed absorption cm −1 of UiO-66-NH2 shifted to 1625 cm −1 following PAMAM immobilization ing the formation of imine bands (C=N) from a Schiff base reaction between groups, belonging to glutaraldehyde, and the amine group, belonging to the MOF and the PAMAM dendrimer.   Figure 2a shows the XRD pattern verifying crystallinity of the as-produced UiO-66-NH 2 MOF. As seen, the characteristic peaks generated from the UiO-66-NH 2 MOF were in line with earlier reports [60,66]. The characteristic peaks of the UiO-66-NH 2 MOF/PAMAM nanocomposite were the same as those of the UiO-66-NH 2 MOF (Figure 2b), which means that this sample did not change in crystal structure following modification with PAMAM. Figure 2a shows the XRD pattern verifying crystallinity of the as-produced UiO-66-NH2 MOF. As seen, the characteristic peaks generated from the UiO-66-NH2 MOF were in line with earlier reports [60,66]. The characteristic peaks of the UiO-66-NH2 MOF/PA-MAM nanocomposite were the same as those of the UiO-66-NH2 MOF (Figure 2b), which means that this sample did not change in crystal structure following modification with PAMAM.  The UiO-66-NH2 MOF had a particle size of ~275 nm. When PAMAM was loaded onto the UiO-66-NH2 MOF, the octahedral edges and corners of the UiO-66-NH2 MOF were destroyed and its surface was coated with the PAMAM dendrimer (Figure 3c,d).
The EDS was recruited to carry out the elemental analyses ( Figure 4). As seen, the main peaks in spectrum 4a exhibited the elements Zr, C, N, and O, which indicates the chemical purity of the as-constructed UiO-66-NH2 MOF. Figure Figure 3 illustrates the FE-SEM images prepared from the UiO-66-NH 2 MOF and the UiO-66-NH 2 MOF/PAMAM nanocomposite for morphological studies. As seen (Figure 3a,b), the UiO-66-NH 2 sample displayed octahedral morphology with proper crystallinity. The UiO-66-NH 2 MOF had a particle size of~275 nm. When PAMAM was loaded onto the UiO-66-NH 2 MOF, the octahedral edges and corners of the UiO-66-NH 2 MOF were destroyed and its surface was coated with the PAMAM dendrimer (Figure 3c,d).
The EDS was recruited to carry out the elemental analyses ( Figure 4). As seen, the main peaks in spectrum 4a exhibited the elements Zr, C, N, and O, which indicates the chemical purity of the as-constructed UiO-66-NH 2 MOF. Figure

Investigating the Effect of the UiO-66-NH2 MOF/PAMAM Nanocomposite on the Electro chemical Behavior of Tramadol
The tramadol electro-oxidation has an association with electron and proton e change. Thus, the effect of pH on the electrochemical response of the tramadol on the UiO

The Scan Rate Effect on the Oxidation of Tramadol on the UiO-66-NH 2 MOF/PAMAM-Modified GCE
The electrochemical response of tramadol on the UiO-66-NH 2 MOF/PAMAM-modified GCE was evaluated by the linear sweep voltammetry (LSV) method. Figure 6 shows the LSVs acquired for tramadol (70.0 µM) on the UiO-66-NH 2 MOF/PAMAM-modified GCE in PBS (pH = 7.0, 0.1 M) at variable scan rates. The LSVs show an increase in the oxidation peak currents with enhanced applied scan rates. Figure 6 (inset) depicts the anodic peak current (Ipa) of tramadol, representing a linear relationship with the scan rate square root (υ 1/2 ), Ipa = 1.1535υ 1/2 − 0.8797 (R 2 = 0.9985). Hence, the electrochemical behavior of tramadol follows a diffusion-controlled process.

Chronoamperometric Determinations
The chronoamperometry was used to explore the tramadol oxidation on the UiO NH2 MOF/PAMAM-modified GCE surface (Figure 7). Chronoamperometric determ tions for variable tramadol levels on the modified electrode were carried out at the wo ing electrode potential of 825 mV. The diffusion coefficient (D) was measured for trama in aqueous solution in accordance with the Cottrell equation: Herein, Cb stands for the concentration, D for the diffusion coefficient, and A for electrode area. Figure 7A shows the plots of I against t −1/2 for variable tramadol cont Figure 7B indicates the slopes from the straight lines plotted against tramadol levels.

Chronoamperometric Determinations
The chronoamperometry was used to explore the tramadol oxidation on the UiO-66-NH 2 MOF/PAMAM-modified GCE surface (Figure 7). Chronoamperometric determinations for variable tramadol levels on the modified electrode were carried out at the working electrode potential of 825 mV. The diffusion coefficient (D) was measured for tramadol in aqueous solution in accordance with the Cottrell equation: Herein, C b stands for the concentration, D for the diffusion coefficient, and A for the electrode area. Figure 7A shows the plots of I against t −1/2 for variable tramadol content. Figure 7B indicates the slopes from the straight lines plotted against tramadol levels. The D value was computed to be 7.9 × 10 −5 cm 2 /s according to the slope of obtained plots and also the Cottrell equation. The D value in this work is comparable with the results reported in the literature (1.05 × 10 −5 cm 2 /s [67], 9.2 × 10 −6 cm 2 /s [68], 2.39 × 10 −5 cm 2 /s [69].

Quantitative Determination of Tramadol by the DPV Method
The quantitative determination of tramadol was performed using the DPV meth Figure 8 illustrates the DPVs acquired for the UiO-66-NH2 MOF/PAMAM-modified G in the exposure to variable tramadol levels. An elevation in the concentration of trama obviously resulted in an increase in the Ipa of tramadol. The calibration curve for vari tramadol levels revealed a linear dynamic range as broad as 0.5 μM to 500.0 μM, with equation of Ipa = 0.0881Ctramadol + 0.7839 (R 2 = 0.9997) (Figure 8, Inset). The sensitivity LOD were calculated to be 0.0881 μM/μA and 0.2 μM for the UiO-66-NH2 MOF/PAMA modified GCE in sensing tramadol, respectively. A comparison of tramadol detection ing various sensors is presented in Table 1.

Quantitative Determination of Tramadol by the DPV Method
The quantitative determination of tramadol was performed using the DPV method. Figure 8 illustrates the DPVs acquired for the UiO-66-NH 2 MOF/PAMAM-modified GCE in the exposure to variable tramadol levels. An elevation in the concentration of tramadol obviously resulted in an increase in the Ipa of tramadol. The calibration curve for variable tramadol levels revealed a linear dynamic range as broad as 0.5 µM to 500.0 µM, with the equation of Ipa = 0.0881C tramadol + 0.7839 (R 2 = 0.9997) (Figure 8, Inset). The sensitivity and LOD were calculated to be 0.0881 µM/µA and 0.2 µM for the UiO-66-NH 2 MOF/PAMAMmodified GCE in sensing tramadol, respectively. A comparison of tramadol detection using various sensors is presented in Table 1.

Quantitative Determination of Tramadol in the Presence of Acetaminophen
The current work aimed to fabricate a modified electrode capable of distinguish the tramadol and acetaminophen at the same time. The analytical tests were perform by varying the contents of tramadol and acetaminophen at the UiO-66-NH2 MOF/P MAM-modified GCE as the working electrode in PBS (pH 7.0, 0.1 M). The DPVs w acquired for the UiO-66-NH2 MOF/PAMAM-modified GCE at variable levels of trama and acetaminophen (Figure 9). Separate oxidation signals appeared at the potential about 780 mV and 370 mV, which correspond to the oxidation of tramadol and acetami phen, respectively. The sensitivity of the modified electrode relative to tramadol in absence (0.0881 μA/μM, Figure 8) and presence (0.0879 μA/μM, Figure 9B) of acetami phen was very similar, suggesting independent oxidation of tramadol and acetaminop on the UiO-66-NH2 MOF/PAMAM-modified GCE, and also the feasibility of simultane determinations of the two analytes with no interference.

Quantitative Determination of Tramadol in the Presence of Acetaminophen
The current work aimed to fabricate a modified electrode capable of distinguishing the tramadol and acetaminophen at the same time. The analytical tests were performed by varying the contents of tramadol and acetaminophen at the UiO-66-NH 2 MOF/PAMAMmodified GCE as the working electrode in PBS (pH 7.0, 0.1 M). The DPVs were acquired for the UiO-66-NH 2 MOF/PAMAM-modified GCE at variable levels of tramadol and acetaminophen ( Figure 9). Separate oxidation signals appeared at the potentials of about 780 mV and 370 mV, which correspond to the oxidation of tramadol and acetaminophen, respectively. The sensitivity of the modified electrode relative to tramadol in the absence (0.0881 µA/µM, Figure 8) and presence (0.0879 µA/µM, Figure 9B) of acetaminophen was very similar, suggesting independent oxidation of tramadol and acetaminophen on the UiO-66-NH 2 MOF/PAMAM-modified GCE, and also the feasibility of simultaneous determinations of the two analytes with no interference.  The reproducibility of the prepared sensor was also evaluated by preparing five modified electrodes (UiO-66-NH2 MOF/PAMAM-modified GCE) using the same fabrication procedure. The RSD value for the peak currents obtained for these electrodes in a buffer solution (0.1 M, PBS) containing 60.0 μM of tramadol was calculated to be 2.8%, which revealed a very good reproducibility of the electrode preparation procedure. The repeatability of the response of the modified electrode (UiO-66-NH 2 MOF/ PAMAM-modified GCE) was estimated by performing the electrochemical experiment repeatedly (five measurements) with the same UiO-66-NH 2 MOF/PAMAM-modified GCE sensor in a buffer solution (0.1 M, PBS) containing 60.0 µM of tramadol. The relative standard deviation (RSD) based on five replicates was found to be 4.1%, which indicated that the UiO-66-NH 2 MOF/PAMAM-modified GCE has good repeatability.
The reproducibility of the prepared sensor was also evaluated by preparing five modified electrodes (UiO-66-NH 2 MOF/PAMAM-modified GCE) using the same fabrication procedure. The RSD value for the peak currents obtained for these electrodes in a buffer solution (0.1 M, PBS) containing 60.0 µM of tramadol was calculated to be 2.8%, which revealed a very good reproducibility of the electrode preparation procedure.

The Selectivity of the UiO-66-NH 2 MOF-PAMAM/GCE for the Detection of Tramadol
To evaluate the selectivity of the UiO-66-NH 2 MOF-PAMAM/GCE for tramadol, an investigation into the influence of potential interfering substances was performed under the optimized conditions. The DPV responses upon addition of interfering substances into 0.1 M PBS (pH 7.0) containing 50.0 µM tramadol were recorded. The obtained results revealed no significant changes in the current of tramadol in the presence of the interfering substances (1000-fold excess of Na + , Mg 2+ , Ca 2+ , NH 4 + , SO 4 2− , 500-fold excess starch, fructose, glucose, lactose, sucrose, L-lysine, L-serine, 100-fold excess dopamine, uric acid, epinephrine, and norepinephrine). However, ascorbic acid showed serious interference in the tramadol determination in equal concentration. The developed method can be used successfully for the determination of tramadol and acetaminophen in pharmaceutical formulations (tramadol tablets and acetaminophen tablets specimens). By using the standard addition method, this study was accomplished and the results of the analysis are shown in Table 2. The appreciable recovery rates (96.7-103.5%) confirmed the capability of the UiO-66-NH 2 MOF/PAMAM-modified GCE as a voltammetric sensor for the analysis of these two drugs in pharmaceutical formulations.

Conclusions
An attempt was made to produce a tramadol sensor through the modification of GCE surface with a UiO-66-NH 2 MOF/G3-PAMAM dendrimer. The sensor (a UiO-66-NH 2 MOF/PAMAM-modified GCE) exhibited commendable catalytic performance toward the tramadol oxidation. There was a linear relationship between the DPV response of the modified electrode and the tramadol contents (0.5 µM-500.0 µM). The LOD was calculated at 0.2 µM. The sensor also possessed an acceptable catalytic behavior for the tramadol determination in the co-existence of acetaminophen. In addition, it was found that the UiO-66-NH 2 MOF/PAMAM-modified GCE demonstrated good stability, repeatability, and reproducibility toward the detection of the analgesic drug, tramadol. The ability of the modified electrode for sensor applications was confirmed in specimens of acetaminophen tablets and tramadol tablets, with acceptable recovery rates.