Isobutyric acid is short-chain fatty acid. In pharmaceutical analysis, quantifying isobutyric acid is essential for ensuring the purity and stability of pharmaceutical products. Molnupiravir, for instance, is an antiviral drug approved by the FDA to treat COVID-19. Isobutyric acid is a known impurity that may be present in Molnupiravir formulations due to the synthesis process or degradation over time. Isobutyric acid can irritate the lungs. Repeated exposure may cause bronchitis to develop with cough, phlegm, and/or shortness of breath. The current study is the first study for isobutyric acid determination used carbon paste electrodes. Tetradodecyl ammonium chloride surfactant (TDAC) used as ionophore in a quality-by-design approach to develop and optimize a carbon-paste sensor for isobutyric acid . Twenty-one sensors with different compositions were tested, and the results were computationally analyzed to generate prediction models. Using the prediction models, the function predicted with 96.7% desirability that a sensor containing 16.85% ion-exchanger, 0% graphene, and o-NPOE as a plasticizer would achieve a Nernstian slope of 57.035 mV/decade, an R-squared approaching unity, and a response time of 6.2 seconds. The sensor accurately measured isobutyric acid concentrations within a wide linear range (9.0×10-8 –1.0×10-2 mol L-1) with high sensitivity and selectivity. The sensor’s morphologya and compostion were examined using the scanning electron microscope and energy-dispersive X-ray analysis. Isobutyric acid was determined in different smamples like waste water, urine and plasma and spiked Molnupiravir standard samples. Molnupiravir thermal stability also was examined using proposed electrode. The results obtained from electrochemical method was compared with data obtained from HPLC method.
Article
Experimentally designed sensor for the real-time detection of Molnupiravir degradation: Optimization, Characterization, and Application"
https://doi.org/10.21203/rs.3.rs-5097456/v1
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Isobutyric acid
Molnupiravi
COVID-19
surfactant
ionophore
plasticizer
sensor
Isobutyric acid, also known as 2-methylpropanoic acid, is a carboxylic acid with the chemical formula CH₃CH(CH₃)COOH. It is a colorless liquid with a strong, unpleasant odor resembling sweaty socks or cheese. Isobutyric acid is classified as a short-chain fatty acid due to its relatively small carbon chain length. It is miscible in water and most organic solvents. It is commonly found in nature as a metabolite in various organisms, including bacteria, fungi, and mammals. The acid is produced during the metabolism of branched-chain amino acids such as valine and threonine, as well as through the bacterial fermentation of carbohydrates in the gastrointestinal tract of animals. Isobutyric acid finds applications in several industrial processes and products. It is a precursor in synthesizing various chemicals, including pharmaceuticals, flavoring agents, and perfumes. Additionally, it serves as a raw material in producing esters, commonly employed as solvents, plasticizers, and fragrance ingredients. Isobutyric acid derivatives also play a role in manufacturing polymers and resins. In biological systems, isobutyric acid participates in metabolic pathways crucial for energy production and cellular function. It is involved in the synthesis of acetyl-CoA, a key intermediate in the citric acid cycle, which plays a central role in the aerobic respiration of organisms. Furthermore, isobutyric acid regulates gene expression and signaling processes in certain physiological contexts [1–4].
Literature reports several methods to deterime isobutyric acid, such as high-performance liquid chromatography (HPLC) [5–10], Spectroscopy [11–13], and gas-chromatography [14–18]. Ion-selective electrodes (ISEs) offer several advantages over other techniques. ISEs allow for direct measurement of specific ions in a sample without the need for sample preparation or complex procedures. This simplifies the analysis process and reduces the time required for analysis compared to techniques like HPLC, which often involve sample preparation and separation steps. ISEs provide real-time monitoring capabilities, allowing for continuous or frequent measurements of ion concentrations. This is particularly useful for applications requiring continuous monitoring of ion levels in processes or environments, such as environmental monitoring or industrial processes. Many ISEs are portable and relatively compact, making them suitable for fieldwork or on-site measurements. In contrast, other systems are typically larger and less portable, requiring a dedicated laboratory setting. ISEs are often more cost-effective than other systems in terms of initial equipment cost and ongoing operational expenses. They generally require simpler instrumentation and fewer consumables, resulting in lower overall costs and more straightforward analysis. So far literature has not yet reported an ISE-potentiometric method for the assay of isobutyric acid[19–20]
Carbon paste electrodes (CPEs) are widely used in electrochemical analysis due to their versatility, ease of preparation, and excellent electrochemical properties. These electrodes are composed of a mixture of graphite powder and a binder material, such as mineral oil or paraffin wax, packed into a non-conductive body, typically glass or plastic. CPEs offer several advantages over other types of electrodes, making them valuable tools in various research and analytical chemistry fields. One significant advantage of CPEs is their ability to be easily modified to tailor their electrochemical properties for specific applications. By incorporating various modifiers, such as nanoparticles, enzymes, or ionophores, into the carbon paste matrix, CPEs can be selectively tuned to detect specific analytes with high sensitivity and selectivity. This versatility makes CPEs suitable for a wide range of applications, including environmental monitoring, biomedical analysis, and food quality control [21, 22]. The experimental design (DoE) is an industrial and scientific research methodology to understand and optimizes different processes. DoE plays a pivotal role in efficiently and systematically evaluating the effects of different factors on sensor response. Unlike the traditional one-factor-at-a-time (OFAT) approach, which varies only one factor while keeping others constant, DoE allows for the simultaneous evaluation of multiple factors. This comprehensive approach not only minimizes the number of required experiments but also enhances the reliability and robustness of the results. By employing DoE, researchers can understand the factors influencing sensor response and develop predictive models that accurately forecast the sensor's performance[23].
The primary objective of our research is to develop a novel carbon paste ISE-potentiometric sensor tailored for the sensitive and selective detection of isobutyric acid. A custom experimental design guided the optimization process to optimize the carbon paste reipe for maximum sensor performance. We aim to achieve accurate and reliable quantification of isobutyric acid. The potentiometric response characteristics of the proposed electrode were evaluated and refined for a range of ions, alongside investigating the impact of variables such as the composition of carbon paste matrices, amount of ionophore, type of plasticizer, pH, response time and temperature. In pharmaceutical analysis, quantifying isobutyric acid is essential for ensuring the purity and stability of pharmaceutical products. Molnupiravir, for instance, is an antiviral drug approved by the FDA to treat COVID-19. Isobutyric acid is a known impurity that may be present in Molnupiravir formulations due to the synthesis process or degradation over time. Therefore, accurate determination of isobutyric acid levels in Molnupiravir drug samples is vital to guarantee the safety and efficacy of the medication. Also, isobutyric acid can be a metabolic byproduct, so biological samples, including urine and plasma, were examined. Environmental samples such as water, especially in areas with industrial activities, can contain isobutyric acid as waste, so industrial wastewater samples were examined by the proposed electrode.
Instrumentation and Chemicals
Graphite powder (synthetic 1–2 µm), graphene, and tetradodecyl ammonium chloride surfactant (TDAC) were sourced from Aldrich. Isobutyric acid was obtained from Noor Elsharq, while EVA Pharmaceuticals kindly donated Molnupiravir. Fluka provided o-Nitro phenyloctyl ether (o-NPOE). Tricresylphosphate (TCP), dibutyl phthalate (DBP), and dioctyl phthalate (DOP) were acquired from BDH. Interfering inorganic salts were obtained from El Nasr Company. Potential measurements were conducted using a digital Hanna pH/mV meter (model 8417). Ag-AgCl double-junction reference electrode (Metrohm 6.0222.1) was utilized along with various isobutyric acid ion-selective electrodes. pH measurements were carried out using a Jenway 3505 pH meter. The electrode surface was examined via scanning electron microscopy (Quanta FEG250) at the National Research Center, Egypt.
Stock isobutyric acid solution (1.0×10− 1 mol L− 1) was prepared by dissolving isobutyric acid in double distilled water. Other diluted solutions (1.0×10− 2 -1.0×10− 10 mol L− 1) were prepared by serial dilution from the stock isobutyric acid solution. Stock molnupiravir standard solution (1.0×10− 2 mol L− 1) was prepared using methanol as a solvent, subsequent dilutions were performed using double distilled water to prepare the aquoes molnupiravir standard solution 1.0×10− 3 mol L− 1.
Modified carbon paste electrode (MCPE) preparation
The Design Expert® Version 10 was used to generate a custom design of 21 sensors (Table 1). The carbon paste electrodes were prepared by thoroughly mixing TDAC as an ion-excahnger with graphene, plasticizer (TCP and o-NPOE), and graphite as shown in Table 1. Intimate homogenization was achieved through careful mixing using an agate pestle and mortar, followed by intensive pressing with the pestle. The resulting paste was then packed into the electrode body, and the carbon paste was smoothed onto a wet filter paper until it appeared shiny. The electrode assembly comprised a Teflon holder measuring 12 cm in length, featuring a hole at one end specifically designed to accommodate the carbon paste. This aperture had dimensions of 7 mm in diameter and 3.5 mm in depth. To ensure electrical connectivity, a stainless-steel rod was inserted through the center of the holder. This rod boasted adjustability, facilitated by a screw mechanism, enabling vertical movement to compress the paste and rejuvenate the electrode surface as required. Surplus paste was extruded and wiped clean for surface renewal, resulting in a lustrous surface, subsequently polished on a sheet of paper to confirm its freshness.
Tabe 1. The recipes and responses of the 21 studied sensors according to the custom experimental design.
| Sensor No. | A:%IE | B:%Graphene | C:PS Type | Graphene (mg) | Graphite (mg) | Ion-Exchanger (mg) | PS (mg) | PS type | NS (mV decade− 1) | pLOQ (M) | R-Squared | RT (s) |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 11 | 0 | TCP | 0 | 250 | 11 | 114.3 | TCP | 55.3 | 6 | 0.999 | 5 |
| 2 | 2 | 1 | TCP | 250 | 0 | 2 | 114.3 | TCP | 50.5 | 4 | 0.998 | 12 |
| 3 | 20 | 1 | TCP | 250 | 0 | 20 | 114.3 | TCP | 49.6 | 5 | 0.995 | 9 |
| 4 | 11 | 50 | TCP | 125 | 125 | 11 | 114.3 | TCP | 56.4 | 5 | 0.999 | 7 |
| 5 | 11 | 1 | o-NPOE | 250 | 0 | 11 | 104.0 | o-NPOE | 44.5 | 4 | 0.955 | 14 |
| 6 | 20 | 0 | o-NPOE | 0 | 250 | 20 | 104.0 | o-NPOE | 56.3 | 7 | 0.999 | 6 |
| 7 | 11 | 50 | o-NPOE | 125 | 125 | 11 | 104.0 | o-NPOE | 53.4 | 5 | 0.999 | 5 |
| 8 | 2 | 0 | TCP | 0 | 250 | 2 | 114.3 | TCP | 58.3 | 6 | 0.999 | 5 |
| 9 | 20 | 0 | TCP | 0 | 250 | 20 | 114.3 | TCP | 56.4 | 7 | 0.999 | 5 |
| 10 | 11 | 1 | o-NPOE | 250 | 0 | 11 | 104.0 | o-NPOE | 52.4 | 4 | 0.989 | 8 |
| 11 | 11 | 50 | o-NPOE | 125 | 125 | 11 | 104.0 | o-NPOE | 50.5 | 5 | 0.988 | 15 |
| 12 | 11 | 50 | TCP | 125 | 125 | 11 | 114.3 | TCP | 49.4 | 4 | 0.998 | 13 |
| 13 | 20 | 1 | o-NPOE | 250 | 0 | 20 | 104.0 | o-NPOE | 55.3 | 5 | 0.999 | 5 |
| 14 | 2 | 50 | o-NPOE | 125 | 125 | 2 | 104.0 | o-NPOE | 50.4 | 5 | 0.988 | 15 |
| 15 | 2 | 0 | o-NPOE | 0 | 250 | 2 | 104.0 | o-NPOE | 59.3 | 7 | 0.999 | 5 |
| 16 | 20 | 1 | TCP | 250 | 0 | 20 | 114.3 | TCP | 51.3 | 5 | 0.999 | 15 |
| 17 | 20 | 50 | o-NPOE | 125 | 125 | 20 | 104.0 | o-NPOE | 50.00 | 6 | 0.998 | 14 |
| 18 | 20 | 0 | TCP | 0 | 250 | 20 | 114.3 | TCP | 58.40 | 7 | 0.999 | 6 |
| 19 | 2 | 0 | TCP | 0 | 250 | 2 | 114.3 | TCP | 59.00 | 7 | 0.999 | 5 |
| 20 | 2 | 1 | TCP | 250 | 0 | 2 | 114.3 | TCP | 55.40 | 5 | 0.999 | 7 |
| 21 | 11 | 0 | o-NPOE | 0 | 250 | 11 | 104.0 | o-NPOE | 59.40 | 7 | 0.999 | 4 |
| %IE = percent of the ion-exchanger, PS = plasticizer, RT = Response time | ||||||||||||
Potentiometric measurement
All potentiometric measurements were conducted using the following cell assembly: Ag | AgCl | saturated KCl || sample solution | sensor (MCPE). Electromotive force (EMF) was plotted against the negative logarithm of isobutyric acid concentration. Calibration of the prepared sensors was established by immersing the carbon paste electrode, in conjunction with the Ag/AgCl reference electrode, in a 25 mL beaker containing 10.0 mL aliquots of isobutyric acid solutions ranging from 1.0×10–10 to 1.0×10–1 mol L–1. The limit of quantification (LOQ) and detection (LOD) were determined using the formulas:
LOD = 3SD/S
LOQ = 10SD/S
Where (SD) represents the standard deviation of the intercept of the response of the blank, and (S) denotes the calibration curve slope. The limit of detection (LOD) was assessed to identify the lowest concentration of isobutyric acid that could be reliably detected [24].
Selectivity
The sensors' ability to accurately and precisely measure a target analyte in the presence of background noise or interfering chemicals is referred to as selectivity. In other word, it refers to the ability to identify and react exclusively to the target analyte while disregarding or downplaying the impact of the accompanying elements. High selectivity is necessary to ensure the accuracy and reliability of potentiometric measurements in various contexts, including industrial quality control, pharmaceutical analysis, and environmental monitoring. In this study, the separate solution method (SSM) and the fixed interfering method (FIM) were adopted to determine the selectivity coefficients of the proposed electrodes toward many anionic species. In the separate solution method, the proposed electrode is exposed to a solution containing only isobutyric acid without interference from other substances. The selectivity coefficient (\(\:{K}_{A,B}^{pot}\)) can be determined by measuring the potential response of the proposed electrode to solutions containing 1.0x10− 2 and 1.0x10− 7 mol L–1 of the isobutyric acid and interfering ions separately, and the selectivity coefficients were determined using the following equation [25]:
\(\:{logK}_{A,B}^{SSM}\) = \(\:\frac{{E}_{B}-{E}_{A}}{S}+(\)1-\(\:\frac{{Z}_{A}}{{Z}_{B}}){\:loga}_{A}\)
where \(\:{Z}_{A}\) and \(\:{Z}_{B}\) are the charge of isobutyric acid interfering ions, respectively. \(\:{E}_{A}\) and \(\:{E}_{B}\) are the measured potential of isobutyric acid and interfering ions, respectively; S is the Nernstian slope; and \(\:{\:a}_{A}\) is the activities of the isobutyric acid.
The fixed interfering method keeps the concentration of interfering ions constant while varying the concentration of isobutyric acid (from 1.0×10− 9 to 1.0×10− 2 mol L− 1). Prepare a series of solutions containing different concentrations of the isobutric acid and add the same concentration of the interfering ion to each concentration of the isobutric acid. Measure the potential using the proposed electrode in each solution containing the constant concentration of the interfering ion and varying concentrations of the isobutyric acid. Ensure the electrode reaches equilibrium in each solution before recording the potential. Plot the measured potentials against the logarithm of the isobutyric acid concentration. The selectivity coefficient (\(\:{K}_{A,B}^{FIM}\)) can be calculated from the slope of the linear plot using the following equation [25]:
$$\:{K}_{A,B}^{FIM}\:=\frac{{a}_{A}}{{{(a}_{B})}^{\raisebox{1ex}{${Z}_{A}$}\!\left/\:\!\raisebox{-1ex}{${Z}_{B}$}\right.}}$$
Where \(\:{a}_{A}\) is the isobutyric acid activity at the detection limit and \(\:{a}_{B}\) is the interfering ion activity in the background and\(\:\:{Z}_{A}\) and \(\:{Z}_{B}\) are the charge of the interfering ion and isobutyric acid, respectively. A selectivity coefficient (\(\:{K}_{A,B}^{FIM}\)) less than 1 indicates low interference from the interfering ion, while a value significantly higher than 1 indicates higher interference.
Determination of isobutyric acid in different samples
Wastewater samples
A volume of 10 mL industrial wastewater samples was accurately transferred into a 25 mL beaker and the content was analyzed through potentiometric calibration using the proposed sensing electrode and the reported HPLC method [6]. Spiked wastewater samples were similarly analyzed to assess the accuracy of the proposed method.
Urine and plasma samples
A volume of 100 µL isobutyric acid standard solution (1×10− 3 mol L− 1) was separately mixed with 900 µL urine and plasma samples, the solutions were diluted to 10 mL using double distilled water. The diluted solutions were analyzed using the developed sensor and the reported HPLC method [6] to determine their isobutyric acid.
Assessment of isobutyric acid in spiked Molnupiravir standard
One mL of stock Molnuporavir standard solution 1×10− 2 mol L− 1 was mixed with 0.1 mL of isobutyric acid standard solution 1×10− 4 mol L− 1 and the volume was completed to 10 mL using double distilled water. The solution was analyzed for isobutyric acid content using the developed sensor and the reported HPLC method [6].
Assessment of molnupiravir thermal stability
A pure molnupiravir standard powder was incubated at 50°C for one week. Samples of the powder were collected and analyzed prior to incubation and subsequently on a daily basis for seven consecutive days. Each powder sample was utilized to prepare an aqueous molnupiravir standard solution at a concentration of 1×10− 3 mol L− 1. The prepared solutions were then analyzed using the developed sensor to quantify their isobutyric acid content.
An ion-selective electrode potentiometric sensor's performance is dependent on the composition of the sensor and the experimental setup. A custom design guided the sensor optimization study through 21 experimentally tested sensors to reach the targeted electroanalytical performance. The design quantitatively evaluated the effects of the studied factors on the measured responses and predicted the optimum sensor recipe through limited number of sensors.
Sensor optimization
The present study aims to enhance the sensor's performance while minimizing the time and cost involved in the experimental process. Initially, TDAC was chosen as the ion-exchanger, and o-NPOE and TCP were selected as plasticizers. Subsequently, an experimental design approach was employed to assess the influence of three variables on the sensor's performance: the percentage of the ion-exchanger (TDAC), the percentage of graphene, and the type of plasticizer used. A custom experimental design suggested fabricating 21 sensors with varying compositions. The performance of these sensors was evaluated by measuring parameters such as slope, quantification limit, linearity (R-squared), and response time. These response parameters helped in assessing the sensor's performance as the studied factors were varied in the sensor composition. Furthermore, the DoE approach deduced predictive models to forecast the sensor's response under different conditions.
A one-way ANOVA statistical analysis demonstrated the model's ability to predict the studied responses, showing a non-significant lack of fit. The analysis identified the graphene percentage as the primary factor influencing sensor performance, highlighting the importance of carefully controlling the graphene content in the carbon paste to achieve optimal performance. However, the model indicated that the interactions between the studied factors were not significant (Table 2).
Table 2. One-way ANOVA statistical analysis of the four studied parameters.
To optimize sensor performance, the desirability function was adjusted to target the best possible Nernstian slope, maximum pLOQ, maximum R-squared, and minimal response time. Using the prediction models, the function predicted with 96.7% desirability that a sensor containing 16.85% ion-exchanger, 0% graphene, and o-NPOE as a plasticizer would achieve a Nernstian slope of 57.035 mV/decade, a pLOQ of 7.049, an R-squared approaching unity, and a response time of 6.2 seconds, Fig. 1.
Surface Characterization
SEM was employed to investigate the morphology of the proposed electrode, as illustrated in Fig. 2. Prior to immersion in isobutyric acid solution, the electrode surface appeared homogeneous, permeable, and displayed cracks that potentially facilitated the diffusion of isobutyric acid ions, as depicted in Fig. 2a. Subsequent to soaking, there was noticeable filling of the cavities between the carbon grains and the surface becomes rough with white spots due to isobutyric acid ions, Fig. 2b. The mechanism was further supported by EDX analysis, which confirmed the presence of isobutyric acid ions. Figure 3 provides quantitative data regarding the surface composition of carbon paste both a) before and b) after soaking in the isobutryic acid solution, illustrating the changes induced by the immersion process.
Response time and lifetime
Response time the time it takes for the electrode potential to stabilize after being immersed in a sample solution or after a change in the sample composition. In other words, it's the time required for the electrode to reach equilibrium and provide a consistent and accurate potential measurement [26]. The dynamic response time of the sensors below study was investigated for the concentration vary from 9.0×10− 8 to 1.0×10− 2 mol L− 1for the proposed electrode. The proposed sensor has a relatively short response time of 7s for the proposed electrodes as shown in Fig. 4, indicating that the selected plasticizer and paste composition performed optimally and that the calibration time did not exceed a few minutes.
The lifetime of a carbon paste electrode refers to the duration over which the electrode maintains its functionality and performance within acceptable parameters for its intended application. It is a measure of the electrode's stability and durability. The lifetime of the proposed electrode was determined by measuring the slope under optimum conditions for an extended period. The proposed electrode showed high stability over long time reached up to 90 days without any significant change in slope as shown in Fig. 5. After 90 days the slope started to decline. During usage, the electrode surface can accumulate contaminants from the sample matrix or from the environment, leading to fouling. This fouling can decrease the effective surface area of the electrode, reduce its sensitivity, and ultimately shorten its lifetime. Continuous usage of the electrode can result in loss of material from the electrode surface, changes in surface morphology, and degradation of electrode performance over time.
Effect of pH
Studying the effect of pH on electrode response in potentiometric determination involves systematically varying the pH of the solution while measuring the potential of the proposed electrode. Over the pH range of 1.0–12.0, the variation of the potential with the pH of the isobutyric acid solutions (1.0×10− 3 and 1.0×10− 4 mol L− 1) was investigated. Measure the potential of the electrode in each solution with varying pH levels. Ensure that the electrode has reached equilibrium in each solution before recording the potential. As shown in Fig. 6, the potential readings were plotted against the pH values. The results indicated that the electrodes were independent on pH changes in the range of 3.50–7.50 for the proposed electrode. At pH lower than 3.5, the electrode is sensitive toward H+and the potential readings were increased gradually. At pH higher than 7.50 the while the potential readings decrease due to the precipitation of sodium isobutyrate.
Effect of temperature
The thermal stability of modified isobutyric acid sensor was investigated by generating calibration graphs across a range of solution temperatures (10, 25, 40, 50, and 60°C). Standard cell potentials (Eocell) were then determined from this graph, with the intercept at [isobutyrate (I) ions] = 0 serving as reference points. Plotting these potentials against (t − 25) yielded straight-line plots in accordance with Antropov's equation [27]. Consequently, the slope of this line revealed the isothermal coefficient of the electrode is 5.22×10− 3 V °C− 1. This coefficients affirm the high thermal stability of the proposed electrode within the experimental temperature range. Additionally, the electrode demonstrated consistent Nernstian behavior across the tested temperatures. Analysis of Fig. 7 indicated that the proposed electrode remained stable up to 50°C. High temperatures can alter the kinetics and thermodynamics of electrochemical reactions occurring at the electrode surface. This can affect the sensitivity of the electrode by changing the rate of electron transfer or the equilibrium potential.
Selectivity
The performance of the proposed electrodes was further assessed in the presence of various foreign substances. Potentiometric selectivity coefficients (\(\:{K}_{A,B}^{pot}\)) were determined according to IUPAC guidelines using separate solutions (SSM) and fixed interfering (FIM) methods. Analysis of the results, as depicted in Table 3, revealed no substantial interference from inorganic cations. This underscores the high selectivity of the proposed electrodes towards isobutyrate (I).
|
Interfering compound |
\(\:{logK}_{A,B}^{SSM}\) |
\(\:{logK}_{A,B}^{FIM}\) |
|---|---|---|
|
Cl− |
-1.3342 |
-3.4432 |
|
NO3− |
-5.3321 |
-7.4321 |
|
SO₄²⁻ |
-2.4342 |
-1.3323 |
|
PO₄³⁻ |
-2.7878 |
-3.4432 |
|
Cr2O7 − 2 |
-3.2987 |
-4.5543 |
|
CH₃COO− |
-1.9854 |
-1.5543 |
|
CO₃²⁻ |
-3.9677 |
-1.8656 |
|
C₂O₄²⁻ |
-6.7696 |
-1.5643 |
|
C4H4O6 − 2 |
-3.4432 |
-6.4432 |
|
C6H5O7 − 3 |
-5.3322 |
-6.5587 |
|
Molnupiravir |
-6.8923 |
-6.5576 |
Application
The proposed electrode show a relatively higher selectivity toward isobutryic acid compared to other common interfering ions in the sample matrix, Table 3. The later enabled for a variety of applications in different sampling matrices including industrial wastewater samples, urine, plasma, and molnupiravir standard powder. The results were compared to those obtained using the reported HPLC method[6]. Student's t-test and variance ratio F-test showed that the developed method successfully compares to the reported HPLC method in terms of accuracy and precision, Table 4.
Isobutyric acid is a known impurity that may be present in molnupiravir formulations due to the synthesis process or degradation over time due to bad storage conditions. After storage at different temperatures, the sensor assessed the isobutyric acid content of 1.0×10− 3 mol L− 1 molnupiravir solution stored at relatively high temperature. Molnupiravir expressed a relatively high thermal stability and barely no degradation along the storage period at 50°C storage temperarure.
|
Sample |
Statistical parameters |
Proposed Electrode |
HPLC |
|---|---|---|---|
|
Industrial wastewater |
Mean recovery (%) |
99.56 |
98.30 |
|
n |
5 |
5 |
|
|
RSD (%) |
0.80 |
1.01 |
|
|
Urine |
Mean recovery (%) |
99.48 |
97.78 |
|
n |
5 |
5 |
|
|
RSD (%) |
0.65 |
0.95 |
|
|
Plasma |
Mean recovery (%) |
99.67 |
98.3 |
|
n |
5 |
5 |
|
|
RSD (%) |
0.55 |
0.76 |
|
|
Molnupiravir solution |
Mean recovery (%) |
98.55 |
97.56 |
|
n |
5 |
5 |
|
|
RSD (%) |
0.87 |
1.04 |
F-test = 0.08–0.88 and t-test = 1.30–1.90 for n = 5. 95% confidence limit: tabulated t value = 2.571 and tabulated F value = 5.05.
Method Validation
On the same day as well as three separate days, five replication experiments were carried out with industrial wastewater samples, urine, plasma, and isobutyric acid standard in molnupiravir solution. The results were acquired and listed in Table 5 verified the reproducibility, and repeatability of the developed potentiometric sensor. By figuring out the lowest concentration of isobutyric acid that can be easily identified, the limit of detection (LOD) was assessed. The sensors' high sensitivity was revealed by the LOD value of 3.33×10− 8 mol L− 1. The lowest concentration that can be detected in accordance with ICH Q2 (R1) recommendations (ICH, 25) is the limit of quantification (LOQ). Below this value, the calibration range was nonlinear, and the value was found to be 9.0×10− 7. Table 6 listed the response characteristics of the suggested electrode.
Table 5. Evaluation of intra- and inter-day precision and accuracy of the modified electrodes in pure isobutyric acid solution and different samples.
|
Sample |
Inter-day |
Intra-day |
||
|---|---|---|---|---|
|
Recovery (%) |
RSD |
Recovery (%) |
RSD |
|
|
Isobutyric acid standard solution |
99.55 |
0.32 |
99.44 |
0.78 |
|
Industrial wastewater |
98.56 |
0.81 |
99.86 |
0.54 |
|
Urine |
99.08 |
0.85 |
99.43 |
0.34 |
|
Plasma |
99.44 |
0.53 |
99.98 |
0.46 |
|
Molnupiravir solution |
99.77 |
0.56 |
98.67 |
0.49 |
|
Parameters |
Proposed Electrode |
|---|---|
|
Slop (mV decade− 1) |
57.3 |
|
Correlation coefficient (r) |
0.9999 |
|
Concentration rang (mol L− 1) |
9.0×10− 8-1.0×10− 2 |
|
Working pH range |
3.50–7.50 |
|
Response time (S) |
7 |
|
Isothermal coefficient (V °C− 1) |
5.22×10− 3 |
|
Detection limit (mol L− 1) |
9.0×10− 8 |
|
Quantification limit (mol L− 1) |
2.99×10− 7 |
|
RSD% |
0.55–0.80 |
|
Recovery % |
98.56–99.98 |
The current work optimised an ion-selective potentiometric sensor using a quality-by-design methodology. The procedure used a qualitative method to choose the best plasticiser and ion-exchanger, then a quantitative method to adjust the sensor recipe's ratios. The refined sensor made it possible to measure isobutyric acid in its pure form as well as samples of urine, plasma, industrial wastewater, and Molnupiravir solution. The suggested electrodes have excellent sensitivity, selectivity, and ease of application with a very low detection limit. The results were statistically equivalent to what the HPLC produced. The proposed method is incredibly simple and inexpensive compared to HPLC.
Author Contribution
Safa S. EL-Sanafery, Ahmed Sayed Saad and Gehad G. Mohamed conceived of the presented idea. Safa S. EL-Sanafery developed the theory and performed the computations.All authors verified the analytical methods and supervised the findings of this work. All authors discussed the results and contributed to the final manuscript.
Data Availability
To access article research data, you can make a request by sending an e-mail to [email protected]. It will be sent to you within a short time.
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No competing interests reported.