INTRODUCTION:

Molnupiravir, is chemically, isopropylester prodrug of N4-hydroxycytidine. Molecular formula as C₁₃H₁₉N₃O₇(molecular weight 329.31 g/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 trialled, but unsuccessfully, 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 Monupiravir 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 catastrophe⁷. 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 methods and miscellaneous¹³ 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

Experimental:

Instrumentation:

An potentiometric titrator was used (Lab- India-Auto titrator) for assay method development and validation. A Shimadzu analytical balance with 0.01mg was used.

Reagents and chemical:

Reference standard of molnupiravir was obtained from reputed firm with certificate of analysis.

Potassium hydrogen phthalate, perchloric acid, mercury acetate and glacial acetic acid of A. R. grade were used.

General procedure:

Standardization of 0.1 N perchloric acid:

About 0.350mg of potassium hydrogen phthalate (previously powdered lightly, dried at 120^oC for 2 hours) was weighed accurately into clean and dry titration jar. It was dissolved in 50ml of glacial acetic acid. It was titrated with 0.1N perchloric acid by using auto titrator. Blank determination was performed out for necessary correction. The titration was performed in duplicate.

One ml of 0.1N HClO₄ is equivalent to 0.2042g. of potassium hydrogen phthalate (C₈H₅KO₄)

Normality of perchloric acid = W

--------------------

B.R. x 0.2042

Where W is weight of potassium hydrogen phthalate in g.

B.R. is burette reading in ml.

Quantitative determination of Molnupiravir:

About 0.200g. of molnupiravir test sample was weighted accurately into a clean and dried titration jar. It was dissolved in 60ml. of anhydrous glacial acetic acid. It was heated to dissolve molnupiravir.

It was titrated with 0.1N perchloric acid potentiometrically.

Blank determination was also carried out for necessary correction.

One ml of 1N perchloric acid is equivalent to 0.0164g. of molnupiravir.

% (Percentage) Molnupiravir on the dried basis was calculated as below.

B.R. x N x 0.0164 x 100 x dilution factor

% Assay = _____________________________

Where B.R. is burette reading in ml at the potentiometric end point.

N is actual normality of 0.1N perchloric acid.

W is weight of the sample taken in g.

RESULT AND DISCUSSION:

Determination of Molnupiravir:

The objective of this work was to determine accurately the content of molnupiravir. The assay of molnupiravir (on the dried basis) of various batches of test sample was analyzed using the above method. It was in the range of 99.33 to 100.84%.

Analytical method validation:

The method precision was checked after analyzing six different preparations of homogeneous test sample of molnupiravir. The %RSD of results obtained was found to be 0.7634. It confirms good precision of the method. The results are presented in table 1.

Table no. 1: Method of precision

Sr. No	Weight of molnupiravir	Burette reading in ml	Normality of perchloric acid	% assay
1	0.02	1.8	0.1007	99.33048
2	0.0202	1.81	0.1007	98.89338
3	0.0199	1.79	0.1007	99.27502
4	0.0197	1.8	0.1007	100.8431
5	0.0202	1.81	0.1007	98.89338
6	0.0201	1.79	0.1007	98.28721
			Mean	99.25377
			Std. Deviation	0.863295
			RSD	0.869786

Linearity:

For the establishment of method linearity, five different weights of molnupiravir test samples corresponding to 20%, 40%, 60%, 80%, 100% and 120% of the about weight (0.500g.) were taken and analyzed for % (percentage) of molnupiravir content. The results are in table 2.

Table no.2: Linearity

Sr. No.	Weight of Molnupiravir in gm	Burette reading	Normality of Perchloric acid	% assay
1	0.01	0.9	0.1007	99.38486
2	0.02	1.81	0.1007	99.937
3	0.03	2.69	0.1007	99.01677
4	0.04	3.61	0.1007	99.66093
5	0.05	4.49	0.1007	99.164
	0.06	5.4	0.1007	99.38486
			Std. Deviation	0.372119
			RSD	0.374422

The potentiometric titration was conducted once at each level. Linearity curve Figure no .1 was drawn by plotting test sample weight in gram on x axis and titre values on y axis.

Fig. no.1: Linearity curve

The values of correlation coefficient, slope and intercept are given in table 3.

Table No.3: Regression values

Parameter	Values
Slope	90.536
Intercept	0.0143
Coefficient of co-relation	0.9999

Accuracy and recovery:

Accuracy was determined at five different levels i.e., 10 %, 20%, 30%, 40% and 50% of the nominal concentration. (0.100 g.) The titration was conducted in triplicate at each level and the titre value was recorded. The tire value obtained in linearity study was considered as true value during the calculation of percentage (%) recovery. The percentage recovery is calculated using following equation.

Titre value

Percentage recovery = --------------------------- x 100

True titre value

The percentage range recovery of Molnupiravir was in 99.05 to 99.81 %. It confirms the accuracy of the proposed method. (Table 4).

Table No. 4: accuracy and precision

Level No.	Weight of Molnupiravir added	Weight of Molnupiravir found	% assay	Mean % assay
1	0.01	0.009902	99.02234	99.4254
	0.0101	0.010012	99.13127
	0.01	0.010012	100.1226
2	0.02	0.019804	99.02234	99.0769
	0.0202	0.020025	99.13127
	0.0201	0.019914	99.07708
3	0.03	0.029707	99.02234	98.81593
	0.0301	0.029817	99.05889
	0.0302	0.029707	98.36656
4	0.04	0.039719	99.2974	99.05041
	0.0402	0.039609	98.52969
	0.0401	0.039829	99.32415
5	0.05	0.049621	99.24239	99.11762
	0.0501	0.049621	99.0443
	0.0502	0.049731	99.06617

Ruggedness:

The ruggedness of the method is defined as degree of reproducibility of results obtained by analysis of molnupiravir sample under variety of normal test conditions such as different laboratories, different analysts and different lots of reagents. Quantitative determination of molnupiravir was conducted potentiometrically on one laboratory. It was again tested in another laboratory using different instrument by different analyst. The assays obtained in two different laboratories were well in agreement. It proved ruggedness of the proposed method.

CONCLUSION:

The proposed method of non-aqueous potentiometric titration was found to be precise, accurate and rugged. The values of percentage recovery and standard deviation showed sensitivity. The method was completely validated. It showed satisfactory data for all the parameters of validation. Hence it can be applied for routine quality control application.

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12. Rele Rajan V. Tiwatane Prathamesh P. Determination of Molnupiravir by Extractive Colorimetric Method from Pharmaceutical Dosage Form, Asian J. Research Chem. 15(4);256-258: 2022; DOI: 10.52711/0974-4150.2022.00046

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Received on 03.08.2022 Modified on 29.08.2022

Asian J. Research Chem. 2022; 15(6):439-442.

DOI: 10.52711/0974-4150.2022.00077