INTRODUCTION:

The objective is to create a completely novel, green,easy to understand, precise, cost-efficient method for determining the presence of molnupiravir using UV spectrophotometry and to validate it in accordance with ICH standards. An isopropylesterprodrug of N4-hydroxycytidine called as molnupiravir is marketed under the brand names Molnaflu and Lagevrio. The active 5 triphosphate form is generated in vivo after its metabolized and distributed into tissues. In non-human primates, this increases oral bioavailability¹. The accumulation of mutationsbrought on by the active medication integrating into the RNA virus genome is known as a viral error catastrophe. Airway epithelial cell proliferation of Cov-2 SARS other human and bat coronaviruses of humans and mice is inhibited by molnupiravir, according to current studies. It has molecular formula C₁₃H₁₉N₃O₇.

Molnupiravir is a crystalline solid and soluble in DMSO, Ethanol, Water, Methanol and the Molecular weight is 329.31g/mol. The melting point is 156-157^oC. It is having the purity > 98%. It was stored under the 20-25^oC.

Figure 1: Structure of Molnupiravir

Molnupiravir undergoes hydrolysis to N4-hydroxycytidine (NHC) in living tissue where it is phosphorylated to create the active 5-phosphate-3 form². This integration into the growing virions genomes promotes the accumulation of these mutations. Additionally, it has been demonstrated that a mouse mutant hepatitis virus that is resistant to remdesivir is more receptive to N4-hydroxycytidine². NHC, the active ingredient, is given orally in dosages of 800mg every 12 hours, and after the first dose, it reaches a Cmax of 2970 ng/ml, a Tmax of 1.5hours, and an AUC0-12h of 8360 hr ng/ml². The ribonucleoside triphosphate pharmacologically active form (NHC-TP) is produced by the phosphorylation of the prodrug molnupiravir, which is subsequently transported into cells. As N4-Hydroxycytidine, which is distributed throughout tissues, hydrolyzes molnupiravir. When N4-hydroxycytidine enters cells, it is phosphorylated to the 5-triphosphate form 1 and excreted in the urine as the active metabolite N4-hydroxycytidine, which is responsible for around 3% of an oral dosage of molnupiravir.

The^3-7review of the literature^8-15suggests that there aren't many ways to use Molnupiravir.

MATERIALS AND METHODS:

Materials:

Molnupiravir was acquired from Vkt Pharma Pvt. Ltd., Srikakulam, as a freebie sample. Methanol is used as a solvent for this method and this methanol was procured from laboratory.

Formulation Used:

From the nearby pharmacy, 200mg of Molupiravir in "Molnutor" capsules were acquired.

Preparationof Reagents:^16,17,18

Preparation of Stock 1 solution:

To yield a final concentration of 1000µg/ml, accurately weigh 100mg of molnupiravir in a 100mL volumetric flask and thoroughly dissolve in methanol.

Preparation of stock-2 solution:

To achieve a concentration of 100µg/ml, dilute 10ml of the above stock-1 solution with methanol in a volumetric flask of 100ml.

Preparation of sample solution:

The suggested technique was used to examine the marketed Molnupiravir capsules, MOLNUTOR (200mg). The equivalent of 100mg of molnupiravir from 10 capsules was carefully weighed, grounded, and added to a 100mL volumetric flask with methanol¹⁹. The volume was then violently shaken and sonicated for 15 mins bringing the volume to 100ml. Filtration of the solution was done using Whatmann filter paper #44. To obtain a final solution with a concentration of 100 µg/ml, the filtrate was appropriately diluted with methanol. After that, it was examined in the UV range of 200–400nm using aUV–VIS double beam spectrophotometer and blank as a methanol²⁰. The spectrum's 236nm wavelength was noted. From the linear regression equation, the concentration of drug was calculated.

Methodology:

Method development:

The drug's standard stock solution was made based on its solubility and physical characteristics, and their wave length maximum was found to be 236nm²¹. Several dilutions were made based on the drug's absorbance maxima, and formulation estimation was done.

Selection of solvent:

According to Indian Pharmacopeia guidelines, the solubility of Molnupiravir was assessed in a range of different solvents. Molnupiravir's solubility was tested in a variety of polar solvents. Methanol was chosen as a suitable solvent for the suggested approach from the solubility investigations.

Selection of λ _max:

To yield a 100µg/ml concentrations, the normal stock solution was further diluted using methanol²². Using methanol as a blank, the solution was scanned in the 200–400nm range. 236nm was chosen for examination from the UV spectrum.

Figure 2: UV absorption spectra of Methanol at 236nm

METHOD VALIDATION:

Linearity:

Molnupiravir (0.2-2.0ml of 100µg/ml) was put into a 10mL VF and diluted to the desired strength with methanol in this methanolic stock solution. At 236 nm, the absorbance of solutions of various concentrations was measured in comparison to a blank²³. From 2 to 20 µg/ml, the samples were found to be linear. Concentration versus absorbance was used to plot the calibration curve [Figure 3]. At the concentration range of 2-20µg/ml, the curve that was obtained was linear. [Table 1]

Precision:

Studies on intra-day and inter-day fluctuation provided evidence of the method's accuracy. In the interday study investigation, solutions with the same concentration (10 g/ml) were made analysed six times over 3 days, and absorbance was recorded [Table 2]. Three times a day morning, afternoon, and evening six separate solutions of the same concentration (10g/ml) were created for the intra-day and inter-day variation investigation²⁴. As well as being computed, the % RSD was also reported²⁵[Table 3,4].

Accuracy:

The accuracy of the procedure was tested by making solutions with varying conc. (80, 100, and 120%), in which the amount of the commercially available Molnupiravir was changed while the amount of the pure pharmaceuticals remained constant²⁶. The solutions were made in three replicas, and correctness was shown by % recovery, which was computed and published in [Table 5].

Robustness:

By analysing the sample with two distinct wavelengths and recording the corresponding absorbance, the robustness of the procedure was tested²⁷. [Table 6] listed the outcomes.

Ruggedness:

Analysing the sample with two separate analyzers and recording their distinct absorbances was done to test the robustness of the procedure²⁸. In [Table 7], the findings are shown.

RESULTS AND DISCUSSION:

Table 1: Results of Linearity

S. No.	Concentration (µg/ml)	Absorbance (nm)
1	2	0.0849
2	4	0.1854
3	6	0.2785
4	8	0.3568
5	10	0.4645
6	12	0.5549
7	14	0.6332
8	16	0.7358
9	18	0.8213
10	20	0.9023

Figure 3: Calibration Curve of Molnupiravir

Table 2: Precision Data

Concentration(µg/ml)	Injections	Absorbance(nm)
10(µg/ml)	1	0.4452
	2	0.4433
	3	0.4412
	4	0.4468
	5	0.4426
	6	0.4478
Statistical Analysis	Mean	0.444483
	SD	0.00231
	RSD	0.005244
	%RSD	0.52

Table 3: Study of Intraday Results

Concentration (µg/ml)	%RSD			Avg. %RSD
Concentration (µg/ml)	1	2	3	Avg. %RSD
10	0.5488	0.5457	0.532	0.542

Table 4: Study of Interday Results

Concentration(µg/ml)	%RSD			Avg. %RSD
Concentration(µg/ml)	1^stDay	2^ndDay	3^rdDay	Avg. %RSD
10	0.5243	0.5417	0.5134	0.5233

Table 5: Accuracy data

Accuracy	Concentration of standard (µg/ml)	Concentration of Marketed formulation (µg/ml)	Amount Found	% Recovery	Statistical Parameters
80%	12	9.6	9.50	98.9%	Mean= 99.1 SD=0.305 %RSD=0.307
	12	9.6	9.56	99.5%
	12	9.6	9.52	99.1%
100%	12	12	11.9	99.1%	Mean= 98.71 SD=0.401 %RSD=0.406
	12	12	11.8	98.3%
	12	12	11.85	98.75%
120%	12	14.4	14.3	99.3%	Mean= 98.93 SD=0.351 %RSD=0.354
	12	14.4	14.2	98.6%
	12	14.4	14.25	98.9%

Table 6: Robustness Data

Concentration (µg/ml)	Absorbance(nm)
10(µg/ml)	Change in wavelength
	235	236	237
	0.4173	0.4294	0.4625
	0.4138	0.4285	0.4638
	0.4130	0.4275	0.4610
	0.4175	0.4298	0.4625
	0.4132	0.4273	0.4624
	0.4129	0.4265	0.4636
%RSD	0.47	0.275	0.198

Table 7: Results of Ruggedness

Concentration (µg/ml)	Absorbance(nm)
	Analyst 1		Analyst 2
10	0.4296		0.4213
10	0.4292		0.4225
10	0.4275		0.4203
10	0.4287		0.4236
10	0.4236		0.4221
10	0.4258		0.4217
	Mean	0.4274	Mean	0.4219
	%RSD	0.49	%RSD	0.41

Table 8: LOD and LOQ

LOD(µg/ml)	LOQ(µg/ml)
0.1516	0.4595

ASSAY OF MOLNUPIRAVIR:

For the analysis of 10 capsules of Molnupiravir were weighed and coarsely pulverised. An precise weighed quantity of powder equivalent to 100mg quantity of molnupiravir was taken in a 100 mL volumetric flask. Few mL of methanol was added and sonicated for fifthteen minutes and then filtered through Whatmann filter paper (#44) and volume was adjusted to solvent. From that 10 ml taken into another 100ml volumetric flask to that methanol is added upto the mark. The percentage assay was found to be 99%.

Table 9: Assay results of Molnupiravir

Brand Name	Drug name	Label Claimed (mg)	Amount found (mg)	% Recovery
MOLNUTOR	Molnupiravir	200mg	198mg	99%

DISCUSSION:

In accordance with ICH principles, the procedure was created and verified. Precision, linearity, robustness, accuracy, ruggedness, LOQ, LOD and specificity were all used to verify the approach. Regression analysis ensures that Beer's law is followed across the concentration range of 2–20(μg/ml). y=0.0457x–0.0031, where x and y have a correlation value of r2=0.999. The precision findings at each level show %RSD<2, which denotes the level's accuracy. Recovery experiments that were used to evaluate the method's accuracy revealed a range of 98–102%. The approach is more sensitive, as evidenced by the robustness and ruggedness results. The fact that there was no interference from the excipients used in the dosage form suggests that the procedure is precise. Analysis of the Molnupiravir content in tablet dosage form revealed that it was nearly identical to the quantity listed on the label. All of the parameter's %RSD values fell within the allowable range <2%.

CONCLUSION:

The medicine molnupiravir is used to both treat and prevent viral infections. Currently, several pharmaceutical companies are marketing it. The suggested analytical techniques for estimating molnupiravir are straightforward, dependable, quick, sensitive, and accurate. In the treatment of viral infections, molnupiravir is used in a variety of applications. The FDA is in favour of using prescription drugs outside of their label, provided that the use is backed by reliable medical data. The use of molnupiravir must yet be improved in many areas, according to researchers and healthcare professionals. But there exist a number of signs that have solid proof behind them. The potential for molnupiravir to advance global health exists. The validation test outcomes were deemed adequate.

CONFLICT OF INTEREST:

Concerning this investigation, the researchers don't have any conflicts of interest.

ACKNOWLEDGMENTS:

We appreciate the free Molnupiravir sample provided by Vkt Pharma Pvt. Ltd. in Srikakulam as well as the cooperation and encouragement received from the administration of Vignan Institute of Pharmaceutical Technology in providing the required resources for the research project.

REFERENCES:

1. Toots M, Yoon JJ, Cox RM, Hart M, Sticher ZM, Makhsous N, Plesker R, Barrena AH, Reddy PG, Mitchell DG, Shean RC. Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia. Science Translational Medicine. 2019; Oct 23; 11(515). DOI: 10.1126/scitranslmed.aax586.

2. 67209 - Food and Drug Administration (FDA). Fact sheet for healthcare providers: emergency use authorization for molnupiravir. Retrieved February 1, 2023 Available on the World Wide Web.

3. Annadi AM, El Zahar NM, Abdel-Sattar NE, Mohamed EH, Mahmoud SA, Attia MS. Development and validation of molnupiravir assessment in bulk powder and pharmaceutical formulation by the RP-HPLC-UV method. RSC Advances. 2022; 12(53): 34512-9. DOI: 10.1039/D2RA05066H

4. Reçber T, Timur SS, Kablan SE, Yalçın F, Karabulut TC, Gürsoy RN, Eroglu H, Kır S, Nemutlu E. A stability indicating RP-HPLC method for determination of the COVID-19 drug molnupiravir applied using nanoformulations in permeability studies. Journal of Pharmaceutical and Biomedical Analysis. 2022; May 30; 214: 114693. https://doi.org/10.1016/j.jpba.2022.114693

5. Abdelazim AH, Abourehab MA, Abd Elhalim LM, Almrasy AA, Ramzy S. Green adherent spectrophotometric determination of molnupiravir based on computational calculations; application to a recently FDA-approved pharmaceutical dosage form. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2023; Jan 15; 285: 121911. https://doi.org/10.1016/j.saa.2022.121911

6. Bindu M, Gandla K, Vemireddy S, Samuel S, Praharsha Y. A validated stability indicating RP-HPLC method for the determination of molnupiravir in pharmaceutical dosage form. World Journal of Advanced Research and Reviews. 2022; 15(1): 580-90. DOI: 10.30574/wjarr.2022.15.1.0720.

7. Amara A, Penchala SD, Else L, Hale C, FitzGerald R, Walker L, Lyons R, Fletcher T, Khoo S. The development and validation of a novel LC-MS/MS method for the simultaneous quantification of Molnupiravir and its metabolite ß-d-N4-hydroxycytidine in human plasma and saliva. Journal of Pharmaceutical and Biomedical Analysis. 2021 Nov 30; 206: 114356. https://doi.org/10.1016/j.jpba.2021.114356

8. Mohamed AR, Abolmagd E, Nour IM, Badrawy M, Hasan MA. Earth-friendly-assessed silver-nanoparticles spectrophotometric method for rapid and sensitive analysis of Molnupiravir, an FDA-approved candidate for COVID-19: application on pharmaceutical formulation and dissolution test. BMC Chemistry. 2023; Mar 10; 17(1): 13.10.1186/s13065-023-00933-2

9. Saraya RE, Deeb SE, Salman BI, Ibrahim AE. Highly sensitive high‐performance thin‐layer chromatography method for the simultaneous determination of molnupiravir, favipiravir, and ritonavir in pure forms and Pharmaceutical formulations. Journal of Separation Science. 2022; Jul; 45(14): 2582-90. https://doi.org/10.1002/jssc.202200178

10. Reddy KT, Haque MA. Develop and validate a highly sensitive method for the estimation of Molnupiravir in rat plasma by high-performance liquid chromatography-tandem mass spectroscopy and its application to pharmacokinetic studies. Journal of Pharmaceutical Negative Results. 2022; Sep 21: 28-34. DOI: https://doi.org/10.47750/pnr.2022.13.S01.04

11. Mahmoud NE, Youssef AO, Attia MS. Spectrophotometric Method Development and Validation for the Determination of Molnupiravir in Bulk Powder and Pharmaceutical Formulation. Egyptian Journal of Chemistry. 2023 Oct 1;66(10):423-9.DOI: 10.21608/ejchem.2023.135533.5970

12. Kumar GR, Babu BS, Rao RR, Vardhan VM, Abbaraju VD. Method development and validation of a specific stability indicating RP-HPLC method for molnupiravir API. Journal of Pharmaceutical Research International. 2021 Dec 27;33(60B):3026-https://doi.org/10.9734/jpri/2021/v33i60B34973

13. Sravanthi G, Gandla KS, Repudi L. New analytical method development and validation for estimation of molnupiravir in bulk and tablet dosage form by RP-HPLC method. Cellular, Molecular and Biomedical Reports. 2023 Sep 1;3(3):130-6.https://doi.org/10.55705/cmbr.2023.375093.1087.

14. Camlik G, Beyazaslan F, Kara E, Ulker D, Albayrak I, Degim IT. A validated high-pressure liquid chromatography (HPLC) method for Molnupiravir. Medical Research Archives. 2022 Sep 20;10(9).DOI: https://doi.org/10.18103/mra.v10i9.3127.

15. Suresh CV, Mamatha T, Santhosh Illendula DK. An analytical new RP-HPLC method for the quantitative determination of molnupiravir in bulk and tablet dosage form. International Journal of Advanced Research in Medical & Pharmaceutical Sciences. 2023;8(1).

16. Rele Rajan V., Tiwatane Prathamesh P. Determination of Molnupiravir by Extractive Colorimetric Method from Pharmaceutical Dosage Form. Asian Journal of Research in Chemistry. 2022; 15(4): 256-8.

17. Rele Rajan V., Tiwatane Prathamesh P.. A Validated Potentiometric Titration Method for Quantitative Determination of Molnupiravir from Pharmaceutical Preparation. Asian Journal of Research in Chemistry. 2022; 15(6):439-2.

18. Rele Rajan V., Tiwatane Prathamesh P. Simple Extractive Spectrophotometric Method for Determination of Molnupiravir from Pharmaceutical Formulation. Asian Journal of Research in Chemistry. 2023; 16(2):155-8.

19. Validation of Compendial Procedures 1225>, the United States Pharmacopeia, 32th Rev., and the National Formulary, 27th Rev., Rockville, MD: The United States Pharmacopeial Convention Inc. 2009; I: 735.

20. Validation of Analytical Procedures: Text and Methodology Q2(R1), ICH Harmonized Tripartite guidelines, International Conference on Harmonization of Technical Requirements for Registration Of Pharmaceuticals For Human Use. 2005; 10.

21. Guideline on bioanalytical method validation, European Medicines Agency, London, UK, 2011; I: 8.

22. Validation of Analytical Procedures SC III F, British Pharmacopeia, British Pharmacopeia Commission. 2013.

23. Validation of Analytical Procedures: Text and Methodology Q2(R1), ICH Harmonized Tripartite guidelines, International Conference on Harmonization of Technical Requirements for Registration Of Pharmaceuticals For Human Use. 2005; 5.

24. Validation of Compendial Procedures 1225>, The United States Pharmacopeia, 32th Rev., and The National Formulary, 27th Rev., Rockville, MD: The United States Pharmacopeial Convention Inc. 2009; I: 737.

25. Validation of Analytical Procedures: Text and Methodology Q2(R1), ICH Harmonized Tripartite guidelines, International Conference on Harmonization of Technical Requirements for Registration Of Pharmaceuticals For Human Use. 2005; 9.

26. Validation of Compendial Procedures 1225>, The United States Pharmacopeia, 32th Rev., and The National Formulary, 27th Rev., Rockville, MD: The United States Pharmacopeial Convention Inc. 2009; I: 738.

27. Sahoo CK, Sudhakar M, Ramana DV, Satyanarayana K, Panda KC. Validation of analytical procedures-A review. Asian Journal of Pharmaceutical Analysis. 2018; 8(2): 109-16.

28. Shingote V, Mankar SD, Dighe SB. A review article on analytical methods development and validation. Research Journal of Science and Technology. 2022; 14(1): 77-83.

Received on 15.07.2023 Modified on 28.02.2024

Research J. Pharm. and Tech 2024; 17(11):5210-5214.

DOI: 10.52711/0974-360X.2024.00797