Zinc(II) complexation strategy for ultra‐sensitive fluorimetric estimation of molnupiravir: Applications and greenness evaluation

The endemicity of the pandemic coronavirus disease 2019 (COVID‐19) infection proved to be transitional only. Spikes are forming again in 2023, and high expectations are returning for reinfections and viral mutations. Molnupiravir (MOL) has been approved as an oral antiviral drug for the treatment of the COVID‐19 causative virion. Therefore, the development of an ultrasensitive, instantaneous, and cost‐effective method for the quantification of MOL in real plasma samples and formulated dosage form are mandatory. The proposed approach is based on the synthesis of a MOL metal‐chelation product. MOL as a ligand was chelated with 1.0 mM zinc(II) in an acetate buffer (pH 5.3). After illumination at 340 nm, the intensity of the MOL fluorescence measured at 386 nm was increased by about 10‐fold. The linearity range was found to be from 60.0 to 800.0 ng mL−1 with limit of quantitation (LOQ) of 28.6 ng mL−1. Two methods were utilized for measuring the greenness of the proposed method (Green Analytical Procedure Index [GAPI] and analytical greenness metric [AGREE] methods), with results equal to 0.8. The binding stoichiometry of MOL with the zinc(II) ion was found to be 2:1. All the experimental parameters were optimized and validated using International Conference on Harmonization (ICH) and United States Food and Drug Administration (US‐FDA) recommendations. Furthermore, the fluorescent probes were successfully utilized in real human plasma with high percentages of recovery (95.6%–97.1%) without any matrix interferences. The mechanism of fluorescent complex formation was confirmed using 1H NMR in the presence and absence of Zn(II). The method was further utilized for testing content uniformity of MOL in its marketed capsule dosage forms.


| INTRODUCTION
Coronavirus infectious disease (COVID-19) is a pandemic infection that emerged in the late 2019. COVID-19 causes an acute respiratory syndrome with about 3.6 million cases around the world that lead to high mortality rates. [1] Patients over 65 years old were the most vulnerable, with fatality rates of 6.27% for hospitalized patients and 3.40% for nonhospitalized patients. [2,3] This pandemic showed high prevalence, even during the strong restrictive measures that were effected in areas characterized by small population density in geographically isolated islands. [4] Although the produced vaccinations performed an essential impact in reducing COVID-19's transmission, the use of a directly acting antiviral drug was a necessity during acute infections. [5] Molnupiravir (MOL, Figure 1) is an isopropyl ester prodrug of N 4 -hydroxycytidine. It is hydrolyzed in vivo and then distributes to different body tissues, where it is converted to the active 5'-triphosphate form. The active form binds to the genome of RNA viruses, causing viral error catastrophe, or a build-up of mutations. [6,7] MOL inhibits the replication of human and bat coronaviruses, including SARS-CoV-2, in mice and human airway epithelial cells, according to recent research. It was also discovered that a remdesivir-resistant mutant mouse hepatitis virus exhibits increased sensitivity to N 4 -hydroxycytidine. [6,7] Literature review showed only a few methods that have been reported for MOL determination using UV-spectroscopy, [8] liquid chromatography mass spectroscopy method (LC-MS/MS), [9] high performance liquid chromatography with UV spectroscopy detection (HPLC-UV), [8,10,11] Spectrofluorimetry, [6] high performance thin layer chromatography (HPTLC) [7] and electrochemical analysis. [12] Some of the published methodologies suffered, in a way or another, from some limitations and/or drawbacks. The lack of sensitivity, need for expensive instrumentations, and consumption of large amounts of organic solvents, which affected the environment and increased environmental pollution are representative samples of those limitations. [9][10][11] Compared with those techniques, spectrofluorimetric methods proved to be simple, sensitive, time-saving, to have wide availability at low cost for the estimation of the drug in different matrices. [13,14] Therefore, the development of a spectrofluorimetric method that is capable for providing sensitive determination for trace amounts of MOL in different matrices, in simple procedure, while saving the analysis time would have a wide applicability. [13,14] Additionally, the proposed method would be totally green due to the absence of any required organic solvents with low-energy spectrofluorometric equipment. [9][10][11] Moreover, the metal-enhanced fluorescence is a promising approach used to improve the fluorescence intensity of certain analytes using different metals such as silver nanoparticles. [13] Furthermore, metal-enhanced approach has gained significant uses due to their extraordinary characters, including large surface areas and tuneable effect making it an attractive tool for various applications, including fluorescence analysis, imaging, electroanalysis, imaging, and biomedicine. [15][16][17] Therefore, the proposed research studies the practical implementation of Zn(II) metal for the detection of MOL in pharmaceutical formulations and human plasma samples as a novel and inexpensive method based on spectrofluorimetric technique. All optimization processes concerning the analytical methodology and the validation measurements were successfully conducted. In addition, a confirmation of the complexation process between MOL and Zn(II) was estimated using 1 H-NMR spectrometry. Additionally, since the evaluation of analytical methodologies aids in the reduction of pollution caused by such operations, two greenness assessment approaches (GAPI and analytical greenness metric [AGREE]) were presented for evaluating the ecological implications of the presented analytical methodology.
F I G U R E 1 Suggested reaction mechanism between MOL and Zinc(II). MOL, Molnupiravir. disease. [7] MOL has a very weak native fluorescence. Therefore, the development of an ultra-sensitive, instantaneous, and cost-effective method for its quantification in real plasma and formulated dosage forms was necessary.
The proposed method was based on metal-chelation with zinc(II) in acetate buffer, pH 5.3 to form a fluorescent product as in Figure 1 The fluorescent product was measured at 386 nm after excitation at 340 nm ( Figure 2a). The binding mode of MOL and Zn(II) was confirmed by Job's plot [18][19][20] and was found to be in the ratio of 2:1, As depicted in Supporting Information: Figure S1 Figure S2).
It was found that the value of K for zinc is equal to 2.5 × 10 5 M −1 .
The standard reaction-free energy change (ΔG°) was determined using the following equation: [22] G R T K Δ°= -2 : 303 log , where R is the gas constant 8.  Therefore, 150.0 µM of Zn(II) was chosen for the proposed procedure as shown in Figure 2b.
As shown in Figure 2c, the pH effect on the reaction of MOL with Zn 2+ was studied in the range from 4.5 to 6.1. It was observed that the maximum fluorescence intensity was achieved using acetate buffer at pH 5.3 ± 0.2. Besides, the volume of buffer was studied from 0.25 to 2.00 mL, the maximum relative fluorent intensity (RFI) was obtained using 1.00 ± 0.25 mL (Supporting Information: Figure S3). Different metals such as Cu 2+ , Ca 2+ , Mg 2+ , Ba 2+ , and Co 2+ were utilized for comparison with Zn(II) results. It was found maximum RFI was obtained using Zn(II) as seen in Figure 2d.
The reaction time was studied at different intervals from 0.0 to 60.0 min, it was observed the reaction spontaneously occurred. In addition, the time stability was studied to 60.0 min, and it was found that the reaction was stable for 60 min using distilled water as summarized in Supporting Information: Figure S4 and the results represented in Supporting Information: Figure S5 ensured the utility of distilled water and ethanol produces the same fluorescence intensity, water was chosen as optimum diluting solvents when compared with other solvents since it is the greenest as well as cheapest one. [23] The mechanism of the reaction represented in Figure 1

| Reaction validation
The reaction was validated using the International Conference on

Harmonization (ICH) andUnited States Food and Drug Administration
(US-FDA) standards. [24,25] As shown in Table 1, the given study is ultra-sensitive, with low detection (LOD) and quantitation (LOQ)  The robustness of the studied methodology was carried out via inducing minor changes in the optimum analytical parameters. As shown from the data presented in Supporting Information: Table ST5, no significant changes were observed after such minor modifications which proved the method's robustness.

| Applications of the proposed method in human plasma
In a bio-analytical validation study, six concentrations were used   waste is generated daily from a conventional high performance liquid chromatography (HPLC) system, [26] therefore, the greenness assessment became a must-do evaluation. Since the proposed method is suitable for application in determining MOL in pharmaceutical dosage forms as well as plasma samples, the method is to be compared with two previously reported methodologies [8,9] which have the same applications to evaluate its ecological ranking.
The Green Analytical Procedure Index (GAPI) was introduced in 2018. [27] GAPI presents 15 pictograms, each representing a step within the main 5 pentagrams, each corresponding to an analytical T A B L E 1 Analytical parameters for estimation of MOL using the proposed complexation method. process. The color code applied in GAPI is indicated by red, yellow, and green colors. The red and green colors indicate the highest and lowest ecological impacts, respectively. As shown in Table 2 acetonitrile (ACN) for plasma protein precipitation. [28] As shown in the AGREE [29] is another assessment tool that has been recently introduced on the color code based on GAPI. The main difference from GAPI is that it was based on the 12 green analytical chemistry (GAC) principles. [28,30] AGREE shows a clock-shaped pictogram, in which the perimeter is divided into 12 sections, each corresponding to a GAC principle. The center of the pictogram shows a numerical value estimating the ecological impact, where the closer to 1, the better impact. As shown in Table 2 Proposed method Reported method [8] Reported method [9] Technique Spectrofluorimetry HPLC-UV LC-MS/MS  low-energy spectrofluorometric equipment, its higher throughput, and simple sample preparation procedures without the need for derivatizing agents account for the better environmentally friendly behavior of the proposed methodology.

| Comparison study between the proposed method and other reported methods
The proposed method was compared with three previously reported methodologies. [7,8,10] As shown (Table 3), the working range of the proposed methodology is much lower than the reported methods with lower detection and quantification limits.
The linear range and LOQ refer to the proposed method's higher sensitivity and reliability than other reported methods. The proposed technique also does not require expensive instrumentation such as those needed in HPLC techniques. [8,10] Moreover, the thorough output of the spectrofluorimetric analysis is much improved than in chromatographic techniques which require time for elution of the studied analytes either HPLC or HPTLC. [7,8,10] Besides, the proposed method was successfully applied for the estimation of COVID-19 drug in real human plasma without matrix interference.
Thus, an innovative, simple tool with high through output was provided for the selective determination of trace amounts of MOL in matrices including pharmaceutical products and human plasma compared with other reported method. [7,8,10]

| CONCLUSION
A novel ultra-sensitive fluorimetric method was developed and validated for the estimation of MOL using Zn(II) complexation.
The method was evaluated for the mechanism of complexation and the results were confirmed using 1H-NMR. The proposed method was applied in the determination of MOL in biological samples as well as marketed formulations. The method provides a low limit of quantitation with high percentages of recovery from plasma sample analysis.

| Plotting calibration curve
The working solutions were prepared within a concentration range of Abbreviations: HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography.

| Estimation in pharmaceutical product and content uniformity tests
Ten capsules of Molcovir ® capsules (each labeled to contain 200.0 mg per capsule) were weighed and then their contents were mixed. The average weight of each capsule was calculated and an amount equivalent to 10.0 mg of MOL was dissolved into 50.0 mL ultra-pure water with sonication for 3 min, followed by filtration to remove undissolved excipients. The final volume was completed to 100.0 mL with ultrapure water to get a solution concentration of 100.0 µg mL −1 .
For content uniformity testing, the content of each capsule of Molcovir ® was individually weighed and then an amount equivalent to 10.0 mg MOL was dissolved into 50.0 mL ultra-pure water with sonication for 3 min followed by filtration to remove any nondissolved excipients then volume completed to 100.0 mL with ultrapure water to get the concentration of 100.0 µg mL −1 . Then the analytical procedure was followed.

| Analysis of MOL in spiked human plasma samples
The plasma samples were analyzed and collected following the

| Stoichiometry mechanism
The stoichiometry of the reaction was investigated using MOL and Zn(II) at equimolar concentrations (