Construction and application of highly sensitive spinel nanocrystalline zinc chromite decorated multiwalled carbon nanotube modified carbon paste electrode (ZnCr2O4@MWCNTs/CPE) for electrochemical determination of alogliptin benzoate in bulk and its dosage form: green chemistry assessment

A new sensor for alogliptin benzoate (ALG) estimation based on a simple and sensitive method was evolved on multiwalled-carbon-nanotube modified nanocrystalline zinc chromite carbon paste electrodes (ZnCr2O4@MWCNTs/CPEs). ALG electrochemical behavior was evaluated using a cyclic voltammetry (CV), square wave voltammetry (SWV) and chronoamperometry (CA). The new electrode materials were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), energy dispersive X-ray analysis (EDX) for elemental analysis and mapping, and X-ray diffraction (XRD) and the X-ray photoelectron spectroscopy (XPS) measurements. All these measurements exhibiting enhanced activity and high conductivity compared to the bare electrode without modification. The calibration curves obtained for ALG were in the ranges of 0.1–20 μmol L−1 with a quantification and detection limits of 0.09 and 0.03 μmol L−1, respectively. The prepared sensor showed a good sensitivity and selectivity with less over potential for ALG determination. Finally, the presented method was successfully applied as a simple, precise and selective electrochemical electrode for the estimation of ALG in its pharmaceutical dosage form.


Introduction
Diabetes is the ninth leading cause of mortality. 1 Diabetes has a detrimental effect on an individual's health and quality of life. Treatment of diabetes has become critical owing to its benecial effect on patient satisfaction and life quality. Diabetes occurs when the pancreas's beta cells are unable to release enough insulin to maintain a normal blood sugar level. There are two forms of diabetes: type 1 diabetes, which is considered an autoimmune illness, and type 2 diabetes, which is dened by inappropriate proglucagon gene expression. 2 There are many categories of oral antidiabetic drugs, such as sulfonylureas, meglitinides, biguanides, thiazolidines, a-glucosidase inhibitors, dipeptidyl peptidase (DPP-4) inhibitors, and sodiumglucose transport protein 2 inhibitors. 3 They act via a variety of mechanisms, including increasing insulin secretion, increasing muscle glucose uptake, increasing hepatic gluconeogenesis, increasing sensitivity to insulin, inhibiting polysaccharide reabsorption, inhibiting sucrose metabolism, decreasing glucagon release, increasing satiation, inhibiting glucose reabsorption, decreasing glucose production, and reversing insulin resistance. 3,4 Many considerations, such as comorbidities, cardiovascular assessment, and mortality, inuence the selection of an oral antidiabetic medication. 5 Alogliptin (ALG) is an oral antidiabetic that works by inhibiting the dipeptidyl peptidase enzyme, which is responsible for the degradation of glucagon like peptide 1 (GLP-1) and incretins glucose-dependent insulinotropic polypeptide (GIP). Inhibiting DPP 4 increases incretin levels, which has a benecial impact on glycemic control. Additionally, inhibiting GIP and GLP-1 stimulates insulin secretion. Additionally, GLP-1 suppresses the release of glucose-dependent glucagon, induces satiety, lowers food intake, and slows stomach emptying rate. 6 ALG may decrease inammatory responses by inhibiting the production of proinammatory cytokines by toll-like receptor 4 (TLR-4). 7 ALG is a (2-((6-((3R)-3-aminopiperidin-1-yl)-3-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl) benzonitrile, its chemical structure is shown in Fig. 1S. † 7 Its molecular weight is 339.4 g and soluble in dimethylsulfoxide; slightly soluble in ethanol; sparingly soluble in water, methanol; very slightly soluble in isopropyl acetate and octanol. 7 While ALG as benzoate salt is more soluble in water than alogliptin free base. 8 ALG has been determined spectrophotometric, [9][10][11] spectro-uorometric, 12,13 chromatographic, [14][15][16][17][18][19][20] and HPTLC. 21 While the literature review revealed no electrochemical method.
On a practical level, electrochemical methods provide a number of signicant benets include their high sensitivity, selectivity, reproducibility, portability, and economics, as well as their design exibility, disposable nature, rapid analysis, minimal sample requirement, ease of use, wide variety of electrodes, and speedy readout. [22][23][24][25][26] Electrochemical methods can be applied in the detection of exosomes from certain diseases, drug analysis, food analysis, the precipitation of organic layers on other metals, the corrosive effect of metals on stainless steel, soil cleaning from oil contamination, and the analysis of biological uids are all examples of electrochemistry being used in numerous sectors. 22,23,[25][26][27][28][29] Moreover, nanoparticles inclusion to electrodes enhance electrical conductivity, charge-transfer complex transfer ability, surface area, mass transport, long-term stability, multiple functionalization, amplication of signals, and catalytic activity all of which have a positive impact on the processing of the data collect. 22,[30][31][32] Cr/ZnO is an example of these nanoparticles and has a variety of applications, including the photolytic degradation of aniline, the removal of nitrogen oxide pollutants, the photocatalytic degradation of thymol blue, the strengthening of antibacterial activity, and the photodegradation of congo red under visible light. [33][34][35][36][37] Spinel-structured ZnCr 2 O 4 has a limited research studies in pharmaceutical eld.
The present study describes the development of the rst electrochemical technique for determining ALG in bulk and dosage form. Additionally, the inclusion of different nanoparticles as ZnCr 2 O 4 or ZnCr 2 O 4 /MWCNT nanoparticles during electrode fabrication allows marvelous electrocatalytic activity toward ALG, thus enhancing the sensitivity and voltammetric signal-to-noise ratios as well as the resulting response stability for CPE quickly and accurately. Finally, the produced novel nanoparticle electrode was assessed on an eco-scale for environmental friendliness.

Standard solution
To make a standard stock solution of ALG (1 Â 10 À3 mol L À1 ), we dissolved 46.15 mg of ALG in 70 ml of distilled water, stirring it for 15 minutes, and then ultrasonically dissolving the remaining water to make it 100 ml. The nal standard solution was prepared by transferring the necessary aliquots into a 10 ml volumetric ask.

Synthesis of ZnO, ZnCr 2 O 4 and ZnCr 2 O 4 /MWCNT nanoparticles
In order to synthesize ZnO nanoparticles, 10 mmole Zn(CH 3 -COO) 2 $2H 2 O dissolved in 50 ml water under stirring. 20 ml of 2 mol L À1 NaOH solution was slowly added dropwise and pH of the solution was maintained at 11 into the starting materials under vigorous stirring. The solution was transferred into Teon lined sealed stainless steel autoclaves and maintained at 180 C for 12 h under endogenous pressure. Aer completion the reaction, the formed white solid precipitate was washed with ethanol, ltered and dried in oven at 60 C. 38 ZnCr 2 O 4 nanoparticles were prepared by the same procedure in addition to 3 mmol of amount Cr(NO 3 ) 2 $4H 2 O was added to the starting solution. Aer completion the reaction, the formed grey black precipitate was washed with ethanol, ltered and dried in oven at 60 C. 39 ZnCr 2 O 4 /MWCNT nanoparticles was prepared as before under ultrasonication of ZnCr 2 O 4 to MWCNT in a ratio 1 : 4, then the grey black powder was dried under the same condition. 40

Working electrode construction
For the bare carbon electrode, 0.4 g graphite powder and 0.2 ml paraffin oil were mixed into a homogenous paste, then the electrode cavity was lled with formulated carbon paste, smoothing the electrode surface to give a shiny surface, and nally, dipping copper wires into the electrode to connect electricity. ZnO, ZnCr 2 O 4 and ZnCr 2 O 4 /MWCNT modied electrodes fabrication, blend 0.6 g graphite powder and different percent modier ranged from (2-12%) nanoparticles powder with 0.4 ml paraffin oil to create a homogenous paste, then pack the electrode cavity with the formulated carbon paste and smooth the electrode surface to get a shiny surface. Finally, through dipping copper wire, connect the electrode to the electrical supply.

General procedure
An adequate volume of ALG standard solution was added to the voltammetric cell, which had been previously prelled with 25 ml of PBS buffer, pH 3.0 to obtain a nal concentration of 1.00 Â 10 À4 mmol L À1 . Then, for a set period of time, each prepared electrode is exposed to a known voltage. Aer the specied time interval, the potential and stirring were suspended for 5.0 seconds. Each concentration sample was treated identically. Finally, square wave voltammograms were recorded at the same optimal condition in CV in concentration range 0.1-20 mmol L À1 with a sweep rate of 100 mV s À1 , pulse amplitude of 20 mV and frequency of 20 Hz.

Application to pharmaceutical formulation
To calculate inhiglip® 12.5 mg, smash twenty tablets containing 12.5 mg ALG. The crushed tablets were weighed, and the weight equivalent to one tablet was transferred to a 100 ml volumetric ask, to which 70 ml of distilled water was added for dissolution. Aer vigorously shaking for ten minutes, the mixture was sonicated for 15 minutes. Distilled water was utilized to ll the volumetric ask to the mark. The linear regression equation used to determine the concentration of ALG per tablet aer centrifugation of the prepared solution at 5000 rpm for 20 minutes at a temperature of 20 .    Fig. 1E and F which indicates the presence of multi-layered compact graphite, conrming the observations suggests that the synthetic graphite is of high quality with some surface modication owing to nanoparticles incorporation. 41,42 Fig. 2A-C shows the TEM images of ZnO, ZnCr 2 O 4 and ZnCr 2 O 4 /MWCNT, which suggests ZnO nanoparticle formation with an average size ranged 5-10 nm, Fig. 2A. Interestingly, chromium nanoparticles' dispersion on ZnO surface with spinel structure is evident from the TEM image as shown in Fig. 2B, where particle size is about (25-35 nm) Fig. 3. Moreover, ZnCr 2 O 4 /MWCNT reveals close chemical composition with low intensity and higher carbon percentage related to MCNTs addition, Fig. 3.  X-ray diffraction of studied nanoparticles reveal that, no characteristic peaks were detected other than ZnO and all the diffraction peaks acceptable with the reference JCPDS no. 36 44 and 45) and disappearance of some peaks for ZnO NPs which conrms formation of two phase. Moreover, the small shiing and broadening of the diffraction peaks increases which conrms the substitution of smaller chromium atoms on zinc sites surface. 46 The full width at half maximum of the peaks for ZnCr 2 O 4 NPs increase due to a segregation of chromium atoms to the particle. Upon incorporation of MCNTs with ZnCr 2 O 4 , the most diffraction peaks for ZnO NPs and ZnCr 2 O 4 were disappeared and appearance of broad peak at 25.12 (corresponding to (002) crystal plane), which assure the high percent ratio of MCNTs to ZnCr 2 O 4 spinel nanocrystalline particles, Fig. 5.

Results and discussion
Scherrer equation applied for the crystallite size determination on the highest peak (101) using the following formula where, l ¼ 1.540Å the wavelength of incident X-ray, D the crystallite size, K ¼ 0.94 the shape factor b the full width at half maximum (FWHM ¼ 3.911) and q symbolizes for the diffraction angle. ZnO and ZnCr 2 O 4 crystallite sizes have been predicted to be nearly 10 and 35 nm, respectively.
The obtained results indicate that ZnO, ZnCr 2 O 4 and ZnCr 2 O 4 /MWCNT samples exhibit a good structural characteristic observed in SEM, TEM, EDX and XRD analysis. Fig. 6 shows a full survey spectrum of the ZnCr 2 O 4 /MCNTs nanocrystalline. Characteristic peaks for Zn, Cr, O and C elements can be observed in the obtained curve. XPS spectra examination of nanoparticles spectrum shows presence of a main peak Cr(III) species with value of binding energy equal 576.98 and 586.4 eV Fig. 2S. † One can generally deduce the oxidation state of chromium and we nd oxidation states which correspond to Cr 2 O 3 . 47 In the survey of spectrum for Zn 2p, the strong resolution Zn 2p spectrum is presented in Fig. 3S, † in which two strong peaks at 1022.07 and 1044.40 eV can be clearly seen, corresponding to the binding energy of Zn 2p 3/2 and Zn 2p 1/2 respectively, indicating the presence of Zn 2+ in the ZnCr 2 O 4 structure. From the O 1s spectrum Fig. 6, it can be seen that the spectrum can be tted to gauss peak at 529.52 eV is attributed to the lattice oxygen from the ZnCr 2 O 4 nanoparticles 48 XPS results in Fig. 6 with the high resolution of C 1s and O 1s explain the deconvoluted peaks of MWCNTs where the  where I pa symbolize for the anodic peak current, A 0 symbolize for the surface area of the electrode, n symbolize for electrons number contributed in the oxidation reaction C 0 symbolize for K 4 Fe(CN) 6 solution concentration, D o symbolize for the diffusion coefficient, and y refers to scan rate (V s À1 ). The obtained slope of the relation between peak current response and square root of scan rate (y 1/2 ) was used for determination of electroactive area. 50 Accordingly, the signal from ZnCr 2 O 4 /MWCNT was superior to that from the other modied electrodes ZnO/CPE, ZnCr 2 O 4 / CPE and bare CPE due to the fact that the inclusion of the nanoparticle increased the electrochemical signal and the effective surface area of ZnCr 2 O 4 /MWCNT electrode was 0.281 compared to 0.195 and 0.146, 0.052 for ZnCr 2 O 4 , ZnO and plain CPE electrodes, respectively. The higher surface area implies the existence of more active sites, which improves the electrostatic interaction between ALG and ZnCr 2 O 4 /MWCNT surface. When these four unique electrode responses were compared to a solution that contains (1.0 Â 10 À4 mol L À1 ) ALG at a pH of 3 and a scan rate of 100 mV s À1 , it was determined that ZnCr 2 O 4 / CPE had a greater intensity than plain CPE, ZnO where the highest current response and less positive overpotential shi at ZnCr 2 O 4 /MNCT/CPE which related to the capability of ZnCr 2 O 4 NPs to penetrate through the spacing between MCNTs and graphite layers of CPE and owing to less surface fouling ability and the higher unique surface/volume MCNTs ratio, Fig. 7.
These results suggest that inclusion of the electrode with ZnCr 2 O 4 /MWCNT enhances its surface area, increasing the number of active sites accessible for interaction with ALG and thereby improving the electrode's conductivity and sensitivity to ALG. On further investigation, it was found that 10% ZnCr 2 O 4 / MWCNT NPs was excellent for achieving the appropriate conductivity and sensitivity, as demonstrated by SWV, whereas larger concentrations of ZnCr 2 O 4 /MWCNT were found to be undesirable Fig. 4S, † leading in a loss to active sites and electrode surface blocking.

Optimization of the experimental conditions
3.3.1. pH effect. First parameter is the effect of pH. The impact of pH on the potential of 10% ZnCr 2 O 4 /MWCNT/CPE is rst measured in several solutions with the same ALG content (1 Â 10 À4 mol L À1 ), but with varied pH values ranging from 2 to 8. Anodic peak shi owing to ALG electron involvement in the electro-oxidation process was discovered as shown in Fig. 8. The ratio of protons to electrons in this reaction is 1 : 1, and the slope of the reaction is 0.052, which is quite near to the Nernstian theoretical value (0.059). As a result, the reaction is pH-dependent.

Scan rate effect.
For a solution containing 1 Â 10 À4 mol L À1 of ALG, the ZnCr 2 O 4 /MWCNT electrode's peak current (I) was measured using a scanning rate of 20 to 180 mV s À1 for ALG, Fig. 5S. † Different scan rates reveal the oxidation behavior of ALG diffusion on the ZnCr 2 O 4 /MWCNT electrode, as shown in Fig. 5S. † The validity of the diffusion process was shown by plotting peak current oxidation versus the square root of scan frequency, as illustrated in Fig. 5S (inset A). † The equation is I pa ¼ 3.2265y 1/2 + 1.1483 (2) Moreover, on plotting the logarithm of peak current response vs. logarithm of the scan rate, the relation was linear, Fig. 5S ( The slope value was 0.55 which conrm that the mechanism of electroactive transport is diffusion technique. Upon plotting log(i) versus potential Fig. 5S (inset C), † a value can be determined from the Tafel plot. 51 The experimentally slope value was 7.69 is compatible with the value of 0.46 for transfer coefficient.
The transfer coefficient value (a n ) for ALG can be calculated by using the following equation 52 where E p and E p/2 were measured in mV and a n was calculated to be 0.981, suggesting two electrons for ALG that was in agreement with the recommended electro-oxidation mechanisms of the cited drug as shown in Scheme 1.

Chronoamperometric studies
Chronoamperometry investigations were used to determine the diffusion coefficient of ALG. The study employs four different concentrations of ALG, and choronoamperogrammes were obtained for a ZnCr 2 O 4 /MWCNT/CPE over a xed potential (pH ¼ 3), as shown in the Fig. 6S (inset A). † Further analysis of the obtained choronoamperogramms revealed a linear relationship between current obtained and t À1/2 for the all studied concentrations, as illustrated in Fig. 6S (inset B). † To calculate the diffusion coefficient, the obtained slopes were plotted against the corresponding concentration of ALG, as illustrated in Fig. 6S (inset C) † and the diffusion coefficient was calculated to be 1.18 Â 10 À5 using the Cottrell equation. 53

Determination of ALG in pure form
The working potential range 0-1.4 V utilized for ALG electro oxidation on the ZnCr 2 O 4 /MWCNT/CPE in. However, ALG showed an oxidation peak at 0.34 V potential at ZnCr 2 O 4 / MWCNT/CPE by square voltammetry as described in Fig. 9. The calibration curve of ALG versus peak current was linear. The linearity of the method was found to be 0.1-20 mmol L À1 and statistical parameters are described in Table 1.

Linearity, limit of detection (LOD) and limit of quantitation (LOQ)
The linearity, the coefficient of determination and percent relative standard deviation (% RSD) of ALG were determined utilizing the new ZnCr 2 O 4 /MWCNT/CPE as reported in Table 1.
For each concentration, the average of three replicates was calculated; the linear regression parameters for the approach are provided in Table 1. According to ICH recommendations, the limit of detection (LOD) and limit of quantitation (LOQ) are the lowest concentrations that can be detected and quantied, as stated in Table 1.
LOD ¼ 3.3 Â SD/slope and LOQ ¼ 10 Â SD/slope where, SD ¼ standard deviation of the intercept for constructed calibration curve. b ¼ slope of constructed calibration curve.

Accuracy
As a consequence of the new ZnCr 2 O 4 /MWCNT/CPE fabrication, accuracy should be assessed through measuring % recovery of ALG concentrations, the nal result should be close to the theoretical value that was previously recorded. The novel ZnCr 2 O 4 /MWCNT electrode's accuracy was tested by comparing the new suggested technique for determining drug concentration with the real concentration of ALG. Table 1 shows the average percentage of recovery as calculated.

Precision (repeatability, intermediate precision)
Three distinct ALG concentrations were evaluated on one day and three consecutive days to determine the repeatability and intermediate precision (inter-day and intra-day) of the novel ZnCr 2 O 4 /MWCNT electrode method. The novel ZnCr 2 O 4 / MWCNT electrode method ndings were analyzed and found to have a good degree of precision, enabling it to be utilized for quality control of ALG as shown in Table 1.

Robustness, reusability, reproducibility and stability
Robustness is a term that relates to an analytical technique's ability to stay unaffected by little changes in the procedure's conditions. 54 The used technique robustness was established by noticing peak current response to purposefully minor changes in method parameters. Among the circumstances tested were the equilibrium period (10 s AE 0.2 s) and pH variation (3 AE 0.1). The peak current of ALG remained unaffected by these minor alterations that occurred throughout the experimental method, indicating the suggested approach's robustness. The sensor's reusability was determined by comparing the voltammetric response of a previously used new ZnCr 2 O 4 /MWCNT/CPE to that of the newly synthesised sensor (more than ten times). The fact that both sensors generated a similar response demonstrates the sensor's reusability. The reproducibility of the ZnCr 2 O 4 /MWCNT/CPE was evaluated by fabricating ve sensors under similar conditions and all sensors produced almost similar response with a % RSD of 1.87, showing that the sensor is highly reproducible. The constructed sensor's stability was tested over a 30 day period by measuring the resultant current gathered using the provided technique. The stability of the constructed sensor is investigated through this period. According to previous data, the newly synthesized ZnCr 2 O 4 /MWCNT/CPE exhibits a high degree of reliability.

Analytical applications of commercial tablets
An investigation was conducted into the effectiveness of a novel ZnCr 2 O 4 /MWCNT/CPE electrode for the analysis of commercial tablets containing ALG. To accurately measure ALG in pharmaceutical products using a novel ZnCr 2 O 4 /MWCNT/CPE electrode sensor, the SWV method was applied. Standard addition technique was applied and satisfactory results were obtained. The results displayed provided successful application with good recovery as presented in Table 2.

Statistical comparison
Statistical analysis of the gained results of estimation of ALG in its dosage form was applied and compared to those of a reported HPLC method. 15 F-and t-values were then estimated and compared as in Table 3. The recovery percentage obtained for the results of the pharmaceutical dosage form for the proposed sensors and the reported HPLC method were statistically   Our work compared using one way-ANOVA at a 95% condence interval (P ¼ 0.05). No statistically signicant difference between the proposed electrodes assay and the reported HPLC method from the results obtained from ANOVA test, as shown in Table 4 to conrm the precise assessment of studied drug in the bulk and its pharmaceutical formulation and assuring the validity of applying the adopted method. Moreover, the proposed method was more applicable than the published methods as the proposed method is more sensitive than the reported methods (Table 5).

Assessment of greenness of the analytical methods greenness
According to the eco-scale approach, the new fabricated electrode was assessed for its greenness by calculating penalty points from the newly established processes, including chemicals, instruments, and trash. 55,56 The approach was determined by the result of deducting these penalty points from 100; a high result implies that the method is more environmentally friendly. The penalty points in these new approaches were 12, which resulted in a total score of 88, as shown in Table 6, indicating that this newly created electrode is regarded as environmentally favorable.

Conclusion
The proposed electrochemical method was applied successfully to determine ALG in its pure and dosage forms (inhiglip). ZnCr 2 O 4 /MWCNT electrode increased the process of electron transfer as well as improved the electrode active surface area mainly due to the ability of ZnCr 2 O 4 NPs to penetrate through the spacing between graphite layers of CPE and MCNTs. The higher molecular-scale wires resemblance and less surface fouling ability of MCNTs with unique high surface/volume ratio and their high porosity character of ZnCr 2 O 4 nanocrystalline exhibits very sensitive results with low detection limits and higher stability with no risk of interference from excipients and its co-formulation drug were acquired.