Method optimization
The Influence of pH and buffer type:
The influence of pH on 1500 ng EDTM was studied by DPV utilizing 0.04M BR-buffer at various pH values at bare carbon paste electrode and scan rate 100 mV/S as shown in Fig. 2,3. At BR-Buffer pH 2–8 the voltammogram shows broad peaks which have a shoulder. At pH 9 the anodic peak is slightly broad without a shoulder upon increasing pH the current increase till reach pH 10 which has a symmetrical and uniform anodic peak and starts to decrease gradually till pH 12. According to these observations pH 10 was chosen as the best pH to be utilized in our suggested method. The influence of solution pH on peak potentials of EDTM at bare CPE has also been studied an increase in pH of the solution caused a shift in the oxidative peak potential to the negative direction which indicates that the protonation/deprotonation process takes place during the charge transfer process of the EDTM at the surface of the electrode(26) as shown in Fig. 4. A linear relationship between peak potential and the solution pH was obtained and the linear equation and correlation were found to be E (V/S) = -0.0325 pH + 1.1692, R2 = 0.9967 according to Nernst Eq. (27) E = -(0.0592 y/n) pH + K, where y is the number of protons and n is the number of electrons. The slope value of our suggested method is -0.0473 which is very close to the Nernst slope indicating that the number of protons included in the oxidation reaction of EDTM is nearly equal to the number of included electrons.
As pH 10 was selected as the optimum pH for our method we tried borate buffer with different molarities of 0.02M, 0.1M, and 0.2 M besides the BR-buffer which was utilized to study their influence on the electrochemical behavior of our drug. 0.2 M borate buffer pH 10 was used as the optimum solution for EDTM oxidation due to its high peak current as observed in Fig. 5.
The influence of surfactants:
In the past few decades, the surfactant has a very large impact on electrochemistry. They affect electrons transfer on the surface of the electrode which controls the rate of the electrochemical reactions (28) and they also affect the solubility of organic molecules in an aqueous solution (29). So, we tried different types of surfactants such as anionic as sodium dodecyl sulfate (SDS), cationic as cetrimide, and non-ionic as tween 20 with a concentration of 3x 10− 4 M. Successive addition of different volumes (100–400µL) of different surfactants were added to 10 µg of EDTM in pH 10 borate buffer solution at bare CPE and scan rate 100 mV/s. Both tween 20 and cetrimide showed a remarkable decrease in peak current while SDS showed a remarkable increase in the peak current until reached the maximum effect by adding 300 µL of SDS as shown in Fig. 6. This effect is due to the adsorption of surfactant molecules on the electrode surface, which may be followed by the formation of micelle aggregates as the distance from the electrode surface increases (30).This increase the chances of EDTM accumulating on the surface of the electrode with improving the sensitivity.
The influence of carbon paste modifications:
In our study of the electrochemical behavior of EDTM, we used CPE due to its wide potential range, ease of preparation, and surface regeneration. We also can increase its sensitivity and selectivity by easy incorporation of different modifiers. Different types of modifications were tried as MWCNT, Gr, and metal oxide nanoparticles as (ZnO – Fe2O3) all these nanoparticles have many advantages of high surface area, stability, and biocompatibility. As shown in Fig. 7. the most sensitive peak with the highest current was obtained in the case of using iron oxide 5% Fe2O3 modification so it was selected as the optimum electrode modification for our suggested method.
The influence of scan rate:
The influence of scan rate on the current and potential of our anodic peak was investigated. Different potential sweep rates of 20–200 mV/s using 1500 ng EDTM in 0.1 M borate buffer pH 10 at 10% Fe2O3-CPE were tried. Upon Plotting the logarithm of anodic peak current (log I) against the logarithm of the scan rate ʋ a linear relationship was obtained as shown in Fig. 8. The slope of the equation was found to be 0.2289 which is less than 0.5 which indicates that the reaction of EDTM is a diffusion-controlled process at the electrode surface (31). Scan rate 100 mV/s showed a lower %RSD after 3 repetitions so it was used in the following measurements. Upon increasing the scan rate from (20–200) mv/s, there was a shift in peak potential to a more positive value, as shown in Fig. 9. which confirms the irreversibility of the reaction of EDTM (32) according to the equation E(V) = 0.0503 log ʋ + 0.7581 (R2 = 09925) the number of electrons involved in the electro-oxidation reaction can be calculated by applying Laviron equation for the irreversible reaction (33). EP = Ep + 2.303RT/αnF[logRTKp/αnF + logν] Where R is gas constant 8.314 J K/ mol, T is the temperature (298 k), α is the electron transfer coefficient, n is the number of electrons, F is Faraday constant (96485c /mol). The slope value is 0.0503, The number of electrons (n) involved in the reaction of EDTM was found to be 2.35 ≈ 2 electrons which matches with the suggested mechanism of EDTM as shown in Fig. 10.
Characterization of the modified electrodes:
Surface area of the modified electrode:
The surface area of the bare CPE and the modified electrodes MWCNT, Gr, ZnO-NPs, and Fe2O3-NPs were investigated by the Randels-Sevcik Eq. (34):
Ip = 2.69 × 105 n3/2 ACD1/2 ν1/2 where Ip is peak current, n is the number of electrons involved in the redox reaction, A is the surface area of the electrode (cm2), C is the molar concentration of potassium Ferrocyanide (0.02 M), D is diffusion coefficient of electroactive species (7.6 × 10–6 cm2/s), ʋ is scan rate (V/s). by plotting peak current (Ip) against the square root of different scan rate. From the slope, the calculated surface area of the bare CPE is 0.095 cm2, MWCNT/CPE is 0.145 cm2, Gr-NPs/CPE is 0.032, ZnO-NPs/CPE is 0.113, and Fe2O3-NPs/CPE is 0.183. From these results, Fe2O3-NPs/CPE was found to have the largest surface area which increases its sensitivity for EDTM quantification in our method.
Morphology of the modified electrode:
The morphology of our modified electrode Fe2O3-NPs/CPE was investigated using the scanning electron microscopy (SEM) technique as shown in Fig. 11a the morphology of Fe2O3-NPs/CPE surface includes many groves and cavities which have small particle size increasing electroactive surface area more than other electrodes and these cavities can act as a selective adsorbent to the EDTM that enhance electrons transfer through the electrode surface. Energy dispersive X-ray spectroscopy (EDX) was utilized to confirm the content of Fe2O3-NPs/CPE with the main elements carbon (91.84%), oxygen (7.13%), and iron (1.02%) EDX spectrum shows a weak peak of oxygen due to the diffusion of air in the paste during preparation procedure as shown in Fig. 11b.
Method validation:
After optimization of all method factors our electrochemical method was validated in pure form and pharmaceutical dosage form according to ICH guidelines (35) using the DPV technique concerning linearity and range, precision, accuracy, Limit of detection, and limit of quantification.
Table 1
Validation data of the suggested method for EDTM quantification at Fe2O3-NPs/CPE Electrode in pure form.
Parameter | Pure form |
linearity (ng/ml) | 200–3500 |
Slop | 0.0025 |
Correlation coefficient (R2) | 0.9995 |
Intercept | 0.2587 |
SD of intercept | 0.084 |
SE | 0.023 |
% RSD | 1.48 |
LOD (ng/mL) | 60 |
LOQ (ng/mL) | 100 |
Recovery | 100.81 |
% E | 0.396 |
Linearity and range:
Under the optimum conditions, the anodic peak current was proportional to the concentrations of EDTM over the concentration range of 200 to 3500 ng/mL as shown in Fig. 12. The regression equation was calculated as shown
I = 0.0025C + 0.2587, R2 = 0.9995 where I is the anodic peak current and C is EDTM concentration in ng/mL
Limit of detection and limit of quantification:
The limit of detection (LOD) and limit of quantification (LOQ) can be estimated according to ICH guidelines by visual inspection method and were found to be 60 and 100 ng/mL for LOD and LOQ respectively as shown in Table 1.
Accuracy and Precision:
The accuracy of our suggested method was determined by the % recovery of the authentic drug and it was found to be 100.81% ±1.48 as presented in Table 1. High values of recoveries prove the accuracy of our suggested method. The precision of our suggested method was determined by measuring 3 various concentrations of EDTM 3 times per day (intraday precision) and on 3 successive days (inter-day precision) the result is presented in Table 2. Low values of % RSD represent the high precision of our suggested method.
Table 2
Accuracy and precision results of EDTM authentic powder utilizing the suggested method.
Parameters | Intra-day precision | Inter-day precision |
Conc (ng/mL) | 700 900 1500 | 700 900 1500 |
Recovery % | 101.45 100.64 101.10 100.19 100.06 101.90 101.10 99.39 100.84 | 100.93 99.66 101.91 100.70 101.12 101.37 101.10 101.44 100.30 |
Mean | 100.91 100.03 101.28 | 100.91 100 .74 101.19 |
±SD | 0.53 0.51 0.45 | 0.16 0.78 0.67 |
RSD% | 0.54 0.51 0.46 | 0.17 0.78 0.67 |
%E | 0.30 0.29 0.26 | 0.09 0.44 0.38 |
Selectivity:
The selectivity of our suggested method was proved by its ability to determine EDTM in its pharmaceutical dosage form with a high % recovery with no excipient interference. Our procedure could detect EDTM in more complex matrices such as human plasma and urine with no interference of some metals that could be present as Na+, Fe+ 2,, Ca+ 2, Mg+ 2, K+, and other compounds such as ascorbic acid, uric acid, glucose, sucrose, and lactose.
Robustness:
To evaluate the robustness of our method, minor changes in the experimental factors such as the borate buffer pH (10 ± 0.02) and concentration of SDS surfactants (3.0 ± 0.02 x 10− 4M) there was no significant change on the peak current.
Application of the suggested method
Application of the suggested method in pharmaceutical dosage form:
Our suggested method was efficiently utilized to determine EDTM in the pharmaceutical dosage form with high recoveries and low % RSD. There were no significant differences between the results of the reported and the suggested method that is proved the high accuracy and precision of our suggested method as shown in Table 3. The reported method used a mobile phase consisting of 0.1M K2HPO4: Methanol (65:35, v/v) at a flow rate of 1.0 ml/min at Hypersil BDS C18 column detected by a photodiode array set at 245 nm.
Table 3
Quantitative determination of EDTM in pharmaceutical dosage form by the standard addition method.
Taken (ng/ml) | Added (ng/ml) | Mean of found (ng/ml) | Recovery (%) * | Reference HPLC method (13) |
200 | 100 | 300.5 | 99.83 | 99.81 |
| 200 | 400.08 | 99.98 | 99.76 |
| 300 | 502.9 | 99.42 | 99.36 |
Mean* | | 401.16 | 99.75 | 99.64 |
Variance | | | 0.15 | 0.08 |
SD | | | 0.29 | 0.25 |
Student-t-test ** (4.3) | | | 0.41 | |
P | | | 0.39 | |
F-test (161.44) ** | | | 1.94 | |
*Each result is an average of 3 determinations |
** the tabulated F and t values at p = 0.05 |
Comparative analysis:
As shown in Table 4. We compared our proposed method with recently reported methods the results proved that our suggested method is more sensitive, has a wider linearity range, is rapid, and is economic compared to the HPLC technique which needs a large number of expensive organic solvents and is time-consuming.
Table 4
Comparison between the proposed method and the recently reported methods.
Method | Linear range | LOD | References |
Spectrophotometric | 5–25 µg/mL | 0.654 µg/mL | (36) |
HPLC-UV | 2–10 µg/mL | 0.225 µg/mL | (12) |
Potentiometric | 3.288-548.056 µg/mL | 1.858 µg/mL | (23) |
Our suggested method | 200–3500 ng/mL | 60 ng/mL | |
Green assessment of the suggested method:
As the analytical community has become more aware of the harmful effects of hazardous chemicals on the environment and health, efforts and trials are being made to minimize these effects. Herein green analytical chemistry was introduced to eliminate or reduce hazardous chemicals and energy consumption from analytical methods in order to improve environmental friendliness while maintaining method performance(37). Many assessment techniques were developed to assess the greenness of the analytical method based on solvents, wastes, consumption of energy, and solvents used per study. Because each tool has a distinct assessment protocol with advantages and disadvantages so, we used two assessment techniques in this work: the analytical eco-scale assessment (ESA) (38) and the newest green assessment tool called the complex green analytical procedure index (Complex-GAPI).
Analytical eco-scale assessment (ESA):
ESA has the advantage of providing a quantitative evaluation of the analytical methods by taking into account all of the reagents used, rather than just the most hazardous ones, as other matrices do(38, 39). It is determined by subtracting penalty points from a total eco scale score of 100, where more than 75 represents excellent greenness, more than 50 represents acceptable greenness, and less than 50 represents inadequate greenness.
The calculations based on the signal word, the pentagrams of the reagents that is listed on safety data sheet of each reagent, and the amounts of each reagent used. Table 5. shows the results for the suggested method. Our method recieved 83 penalty points, which demonstrated its excellent greenness. ESA has several drawbacks including insufficient information about the causes of the analytical procedure’s environmental impact and no data on the structure of the hazards is founded so we used another tool Complex-GAPI. Our method is considered to be an excellent green analysis.
Table 5
Green assessment of our suggested method by ESA
| Eco-scale parameter | Penalty points |
Reagents | Boric acid Graphite Paraffin oil Methanol Sodium hydroxide | 2 1 2 6 2 |
Instrument and waste | Voltameter pH meter vortex mixer centrifuge occupational hazard waste (10- 100mL, degradation) | 0 0 0 0 0 4 |
∑Penalty | | 17 |
Total score | | 83 |
Complex green analytical procedure index (Complex-GAPI):
Complex-GAPI is the most recent advanced assessment or technique of green chemistry. It is the final form of the GAPI technique. It represents a comprehension evaluation of the whole analytical process from sample collection to final analysis including storage, preservation, transportation, and sample preparation. The scale depends on using 5 pentagrams with the additional hexagonal part is the pre-analysis step. As shown in Fig. 13. the green color represents an eco-friendly step yellow color represents a medium environmental impact and the red color represents a hazardous environmental impact of this step(40). There is no need for a purification step, which corresponds to the white region in the hexagonal shape. This technique has a lot of advantages as the software is available that will facilitate the use of such a technique, it is friendly use, simple and includes all the parameters that characterize the analytical protocol as well as the pre-analysis procedure (reagents, techniques, and conditions).