Molnupiravir, is chemically, isopropyl ester pro-drug of [N4-hydroxycytidine]. Molecular formula as C₁₃H₁₉N₃O₇and molecular weight 329.31g/mol. Molnupiravir is an antiviral medication that inhibits the replication of certain RNA viruses It is used to treat Covid 19 in those infected by SARS-CoV-2. There is a growing list of both novel and repurposed antiviral being tested for the treatment of COVID-19. The number of antiviral were tested for Covid 19 treatment. The drugs like lopinavir/ritonavir and darunavir/cobicistat have been trialed but they were found to be unsuitable for COVID-19^1,2, but early in the pandemic, a number of antiretroviral drugs were investigated.

Among these, protease inhibitors lopinavir/ritonavir and also darunavir/cobicistat have been trialed, but unsuccessful in patients with COVID-19^2,3. The drug like Remdesivir which was used for the treatment of Hepatitis C and Ebola, it is approved by the FDA for treatment of COVID-19 infection.⁴ Molnupiravir is an orally bio-available isopropyl ester pro-drug of the ribonucleoside which is analogue β-d-N 4-Hydroxycytidine.

It exhibits antiviral activity against number of RNA viruses. Pharmacokinetic profiling indicated that molnupiravir is orally bio-available in ferrets and nonhuman primates⁵, with its active metabolite. It gave broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs. It also showed increased potency against a CoV bearing resistance mutations to the nucleoside analogue inhibitor Remdesivir. Prophylactic and therapeutic administration of molnupiravir, in mice models of infection with SARS-CoV or MERS-CoV, showed significantly improved pulmonary function and reduced virus titre and body weight loss⁶. Molnupiravir is unstable in plasma and is rapidly converted to β-d-N 4-Hydroxycytidine is much more stable in plasma (t½>6.5h). Once β-d-N 4-Hydroxycytidine is absorbed into animal plasma it is widely distributed in tissues where rapid conversion to the active β-d-N 4-Hydroxycytidine -5’-triphosphate occurs. The primary mechanism of action of Molnupiravir is inhibition of viral RNA replication by incorporation of the β-d-N 4-Hydroxycytidine mono-phosphate metabolite into the viral RNA genome resulting in induction of viral error catastrophe ⁷. In addition, the active metabolite, β-d-N 4-Hydroxycytidine -5’-triphosphate, may act directly as a chain terminator to prevent replication.

A literature reveals the analytical methods for these compounds shows limited results. Plasma levels of individual drugs have been validated using liquid chromatography/tandem mass spectrometry (LC-MS/MS)⁸, in animal models⁹, but the validation details have not been fully available. A LC-MS method¹⁰, HPLC¹¹ Colorimetric¹², non aqueous titration¹³ and miscellaneous^14,methods were reported in literature. Molnupiravir is not officially available in any pharmacopoeia. Simple, rapid and reliable colorimetric method is developed for the determination of molnupiravir. This method can be used for the routine analysis. In the proposed methods optimization and validation of this method are reported.

Structure of Molnupiravir:

MATERIALS AND METHODS:

A Shimadzu -160 A double beam UV-Visible recording spectrophotometer with pair of 10mmmatched quartz cell was used to measure absorbance of solutions. A Shimadzu analytical balance was used.

Bromophenol blue, solochrome dark blue, bromocresol green, hydrochloric acid, potassium hydrogen phthalate and chloroform of A.R. grade were used in the study.

Preparation of standard solution and reagents:

Stock solution of molnupiravir (100μg/ml) was prepared in ethanol. From this stock solution working standard (10μg/ml) was prepared by diluting 10ml stock solution to 100ml with ethanol. A 0.6% w/v solution of bromophenol blue, solochrome dark blue and bromocresol green were prepared in distilled water respectively.

Potassium hydrogen phthalate buffer solution of pH 4.01was prepared in distilled water. Dilute hydrochloric acid was used to adjust desired pH of buffer solution.

EXPERIMENTAL:

Method 1(with bromophenol blue):

Into a series of separating funnels appropriate amount of the working standard drug solutions were pipetted out. To each funnel 1.0ml of buffer (pH= 4.2) and 4ml of 0.6 %w/v bromophenol blue were added. 10ml of chloroform was added to each funnel. The solutions were shaken for thorough mixing of the two phases and were allowed to stand for clear separation of the layers. The absorbance values of the chloroform layers were measured against their respective reagent blank at the wavelength of the maximum absorbance (λ max 420 nm).

Method 2(with solochrome dark blue):

Into a series of separating funnels appropriate amount of the working standard drug solutions were pipetted out. To each funnel 4.0ml of buffer (pH = 1.10) and 3.2ml of 0.25% w/v solochrome dark blue were added. 10ml of chloroform was added to each funnel. The solutions were shaken for thorough mixing of the two phases and were allowed to stand for clear separation of the layers. The absorbance values of the chloroform layers were measured against their respective reagent blank at the wavelength of the maximum absorbance (λ max=495 nm).

Method 3(with bromocresol green):

Into a series of separating funnels appropriate amount of the working standard drug solutions were pipetted out. To each funnel 1.5ml of buffer (pH= 3.7) and 4. 2ml of 0.02% w/v bromocresol green was added. 10ml of chloroform was added to each funnel. The solutions were shaken for thorough mixing of the two phases and were allowed to stand for clear separation of the layers. The absorbance values of the chloroform layers were measured against their respective reagent blank at the wavelength of the maximum absorbance (λ max =430 nm).

Estimation from tablets:

Twenty tablets of labelled claim 200mg of molnupiravir were weighed accurately. Average weight of each tablet was determined. Tablets were crushed into fine powder. An accurately weighed quantity of powder equivalent to 10mg of molnupiravir was transferred into a beaker and it was shaken with 50ml of ethanol and filtered. The filtrate and the washing were collected in a 100.0 ml volumetric flask. This filtrate and the washing were diluted up to the mark with ethanol to obtain final concentration as 100μg/ml. This solution was further diluted to give 10μg/ml. Such solution was used for methods (1-3) respectively.

Table 1: Values of results of optical and regression of drug

Parameter	Bromophenol blue	Solochrome dark blue	Bromocresol green
Detection Wavelength (nm)	420	495	430
Beer Law Limits (µg/ml)	1-12	2-14	1-10
Correlation coefficient(r²)	0.9999	0.9999	0.9999
Regression equation (y=b+ac)
Slope (a)	0.0251	0.0301	0.0404
Intercept (b)	0.0002	0.0009	0.00005

Appropriate aliquots of drug solution were taken and the individual assay procedures were followed for the estimation of drug contents in tablets. The concentration of the drug in the tablets was calculated using calibration curve. The recovery experiment was carried out by standard addition method. Results of analysis are given in table 1.

RESULTS:

The extractive spectrophotometric methods are popular due to their sensitivity in assay of the drug and hence ion pair extractive spectrophotometric methods have gain considerable attention for quantitative determination of many pharmaceutical preparations. These proposed methods are extractive spectrophotometric methods for the determination of molnupiravir by using chloroform as solvent from its formulations i.e. tablets. The colour ion pair complexes formed are very stable. The working conditions of these methods were established by varying one parameter at time and keeping the other parameters fixed by observing the effect produced on the absorbance of the colour species. The various parameters involved for maximum colour development for these methods were optimized. The proposed methods were validated statistically and by recovery studies. The molar absorptivity show the sensitivity of methods while the precision was confirmed by %RSD (relative standard deviation). The optical characteristics such as absorption maxima (nm), molar absorptivity (l -mole-1 cm-1), co-relation coefficient (r) were calculated and are also summarized. Assay results of recovery studies are given in table 2 (A, B, C).

Table no 2:A (Bromophenol blue)

Amount of Sample Added in (µg/ml)	Amount of Standard Added in (µg/ml)	Total amount recovered	Percentage recovery (%)	Standard deviation	Percentage of relative standard deviation (C.O.V.)
1	0	1.002857	100.2857	0.004451	0.443811
2	1	2.031429	101.5714	0.021594	1.062979
3	2	3.001143	100.0381	0.005014	0.167079
4	3	4.074286	101.8571	0.053807	1.320659
				Mean= 0.021217	Mean= 0.748632

Table no 2:B (Solochrome dark blue)

Amount of Sample Added in (µg/ml)	Amount of Standard Added in (µg/ml)	Total amount recovered	Percentage recovery (%)	Standard deviation	Percentage of relative standard deviation (C.O.V.)
2	0	2.009368	100.4684	0.036482	1.81559
2	2	3.976581	99.41452	0.036472	0.917418
2	4	5.967213	99.45355	0.046368	0.77704
2	6	7.953162	99.41452	0.037177	0.467447
				Mean=0.039127	Mean= 0.994374

Table no 2:C (Bromocresol green)

Amount of Sample Added in (µg/ml)	Amount of Standard Added in (µg/ml)	Total amount recovered	Percentage recovery (%)	Standard deviation	Percentage of relative standard deviation (C.O.V.)
2	0	2.007055	100.3527	0.027474	1.368872
2	2	4.003527	100.0882	0.038854	0.970497
2	4	5.975309	99.58848	0.031876	0.533469
2	6	7.978836	99.73545	0.038854	0.486965
				Mean= 0.034265	Mean= 0.839951

Results are in good in agreement with labelled value.

DISCUSSION:

The percent recovery obtained indicates noninterference from the common excipients used in the formulation. The reproducibility, repeatability and accuracy of these methods were found to be good, which is evidenced by low standard deviation. The proposed methods are simple, sensitive, accurate, precise and reproducible. They are directly applied to drug to form chromogen. Hence they can be successfully applied for the routine estimation of molnupiravir, in bulk and pharmaceutical dosage form even at very low concentration and determination of stability of drug in formulation such as tablets. The strong recommendation is made here for the proposed methods for determination of molnupiravir from its formulation.

ACKNOWLEDGMENTS:

Authors express sincere thanks to the Principal, of D.G. Ruparel College, for providing necessary facility for research work.

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13. Rele Rajan V., Tiwatane Prathamesh P. A Validated Potentiometric Titration Method for Quantitative Determination of Favipiravir from Pharmaceutical Preparation, Asian journal of research in chemistry 15(1): 2022; 49-51.

14. Rele Rajan V., Tiwatane Prathamesh P.Simple Extractive spectrophotometric method for determination of favipiravir from pharmaceutical formulation Asian Journal of Research Chem. 2022; 15(4):2022; 299-302. DOI: 10.52711/0974-4150.2022.00053.

Received on 04.01.2023 Modified on 28.01.2023

Asian J. Research Chem. 2023; 16(2):155-158.

DOI: 10.52711/0974-4150.2023.00025