INTRODUCTION:

Molnupiravir is anti-viral drug (CAS No. 2349386-89-4) used for the treatment of mild to moderate COVID-19 adult patients¹. It is chemically [(2R, 3S, 4R, 5R)-3,4-dihydroxy-5-[4-(hydroxyamino)-2-oxopyrimidin-1-yl]oxolan-2-yl]methyl 2-methylpropanoate with molecular weight 329.31 g/mol and molecular formula C₁₃H₁₉N₃O₇ (Figure 1). It acts by interfering with the replication of the SARS-CoV-2 virus, specifically by inhibiting the viral RNA polymerase, an enzyme crucial for viral replication².

Figure 1: Chemical structure of Molnupiravir

Literature survey reveals that Molnupiravir can be estimated using spectrophotometry, HPLC, LC-MS, LC-MS/MS, LC-HRMS in pharmaceutical formulations as well as in biological samples.

Pritam Jain et al., have developed a spectrophotometric method³ in distilled water for the estimation of Molnupiravir from bulk and pharmaceutical formulation where the λ_max of Molnupiravir was found to be 235 nm and the linearity was shown over the concentration range 5-30 µg/ml. Aboras et al., have developed a stability indicating HPLC-DAD method⁴ for the estimation of Molnupiravir and its major metabolite, N4-hydroxycytidine, its main metabolite using Agilent HC-C18 analytical column and a mobile phase system consisting of deionized water and Acetonitrile (75:25, v/v) (Detection wavelength 236 nm) and the linearity was obeyed over the concentration range 0.1-100 μg/ml. Sravanthi et al., have developed a RP-HPLC method⁵ for the estimation of Molnupiravir in bulk and tablet dosage form using Symmetry ODS C18 column and mobile phase consisting of Methanol and Phosphate buffer (pH 4.2 adjusted with Orthophosphoric acid solution) (35:65, % v/v) in an isocratic mode (Detection wavelength 236 nm) and the linearity was obeyed over the concentration range 20-100 μg/ml. Tuba Reçber et al., have developed a stability indicating RP-HPLC method⁶ for determination of Molnupiravir in nano formulations (Isocratic mode) using mobile phase, Acetonitrile: water (20:80, v/v) with flow rate 0.5 ml/min with UV detection wavelength at 240 nm and the linearity was found to be 0.1-60.0 μg/ml. Ravi Kumar et al., have developed a stability indicating RP‐HPLC method⁷ for the determination of Molnupiravir using Waters 2695 HPLC instrument with Agilent Zorbax Eclipse C18 column with the mobile phase consisting of 30 mM Ammonium phosphate and Methanol (47:53, %v/v) with UV detection wavelength at 260 nm and the linearity was found to be 25 -150 μg/ml. Annadi et al., have developed a RP-HPLC method⁸ for the estimation of Molnupiravir bulk powder and pharmaceutical formulation using an Inertsil C₁₈ column with mobile phase mixture consisting of 20 mM phosphate buffer (pH 2.5) and Acetonitrile (80 : 20, v/v%) with flow rate 1.0 ml/min and the linearity was found to be 0.2-80 μg/ml. Hebatallah had developed a stability indicating LC-MS method⁹ for the determination of Molnupiravir and its major degradation product using Acquity UPLC H-Class system with a XevoTM triple-quadrupole tandem mass spectrometer with Acquity BEH C18 column with electrospray ionization interface using negative mode. For the RP-HPLC method, Kromasil Eternity Nouryon C18 column was used with 0.1 % phosphoric acid in ultra-pure water and Acetonitrile (85:15, v/v) as mobile phase with a flow rate of 1 ml/min (Detection wavelength 254 nm) with a run time of 10 minutes. For the LC-MS method, a mobile phase consisting of 0.1 % Formic acid and Acetonitrile with a flow rate of 0.2 ml/min and Acquity BEH C18 column were used on gradient mode of elution and the run time was 15 minutes. The linearity was found to be 10 -200 μg/ml and four green metric scales; National environmental metric index (NEMI), Analytical-ECO scale Green Analytical Procedure Index (GAPI) and Analytical GREEnness Metric Approach (AGREE) were used during the study. Komarov et al., have developed HPLC-ESI-MS/MS method¹⁰ for the quantification of major Molnupiravir metabolite (β-D-N4-hydroxycytidine) in human plasma using Shim-pack GWS C18 column (Gradient mode) using 0.1% aq. Formic acid solution with 0.08% Ammonia solution (eluent A, v/v) and 0.1% aq. Formic acid solution in methanol with 0.08% Ammonia solution mixed with acetonitrile in a 4:1 ratio (eluent B, v/v) as mobile phase. Amara et al., have developed a LC-MS/MS method¹¹ for the simultaneous quantification of Molnupiravir and its metabolite ss-d-N4-hydroxycytidine in human plasma and saliva using Atlantis C₁₈ column (Gradient mode) and mobile phase consisting of 1 mM Ammonium acetate in water (pH 4.3) and 1 mM Ammonium acetate in Acetonitrile and the linearity was found to be 2.5-5000 ng/ml for both human plasma and saliva. Jain et al., have studied LC and LC-HRMS methods¹² to study the stability behavior of Molnupiravir. RP-HPLC method for the estimation of Molnupiravir. The HPLC separation was achieved using Waters Xselect HSS T3 column using a mixture of Ammonium formate and Acetonitrile as mobile phase (Gradient mode) with flow rate 0.7 ml/min (Detection wavelength 272 nm). In the present study the authors have proposed a new stability indicating RP-UFLC method for the estimation of Molnupiravir and the method was validated as per ICH guidelines.

MATERIALS AND METHODS:

Molnupiravir API (> 99.9 purity) was procured from Natco Pharma Ltd as gift sample. Molnupiravir is available as capsules with different brand names Molnumize 200 (Torrent Pharmaceuticals), Molnunat 200 (Natco Pharma Ltd), Molflu (Dr. Reddy’s Laboratories Ltd) (Label claim: 200 mg) etc and Mobiflue 200 as tablets. HPLC grade Acetonitrile was procured from Merck (India) and all other chemicals glacial acetic acid, sodium hydroxide, hydrochloric acid and hydrogen peroxide (30% w/v) were purchased from Merck (India). HPLC grade water was obtained from Millipore system.

Preparation of stock and standard solutions

25 mg Molnupiravir was accurately weighed, transferred and dissolved in Acetonitrile in a 25 ml volumetric flask and made up to volume and a series of 2-100 µg/ml solutions were prepared on dilution with the mobile phase and filtered through 0.42 µ membrane filter before injecting in to the UFLC system.

Instrumentation and Chromatographic conditions

Shimadzu Model CBM-20A/20 Alite UFLC system with PDA detector and Zorbox C₁₈ column were used for the chromatographic study. Mobile phase mixture consisting of 0.1% Acetic acid and Acetonitrile in the ratio 35: 65, v/v with a flow rate 0.8 ml/min was chosen for the chromatographic elution of Molnupiravir (Detection wavelength 245 nm). The injection volume was 20 µl and the total run time was 10 minutes.

Method validation¹³

Linearity, Precision Accuracy Robustness

A series of 2-100 µg/ml of Molnupiravir solutions were prepared from the stock and working standard solutions using the diluent and each of these solutions were injected thrice into the UFLC system and the chromatograms were observed. The area under curve or the peak area of each of the solutions injected was noted at its retention time and the mean peak area was calculated. A calibration curve was drawn by plotting the concentration of Molnupiravir solutions on the x-axis and the corresponding mean peak area values on the y-axis. The LOD and LOQ were calculated from the signal to noise ratio (S/N). The LOD is 3.3 times the signal to noise ratio and that of LOQ is 10 times the signal to noise ratio. Precision of the method was evaluated intra-day and inter-day precision studies. Three different concentration solutions (10, 20 and 40 µg/ml) of Molnupiravir were prepared within the linearity range on the same day (intra-day precision) and on three consecutive days (inter-day precision) and the chromatographic study was performed. The mean peak area (n=3) and thereby the % RSD was calculated. Accuracy of the method was measured by spiking the drug formulation (20 µg/ml) solution (50, 100, 150%) with a known concentration of standard drug (n=3) where the final concentrations were found to be 30, 40 and 50 µg/ml. The mean peak area was calculated from the chromatograms obtained and finally the % RSD was calculated from the linear regression equation. The robustness of the method was performed using Molnupiravir drug solution (20 μg/ml) proved by incorporating a very small changes in the optimized chromatographic conditions such as mobile phase composition (± 5%; 30:70 and 40:60), flow rate (± 0.1 ml; 0.7 and 0.9 ml/min) and detection wavelength (± 5 nm; 240 and 250 nm).

Assay of Molnupiravir capsules

Molnupiravir capsules are available in which Molnupiravir is 200 mg per capsule as label claim. Twenty capsules of Molnupiravir of different brands were procured accurately and the contents were weighed and powder equivalent to 25 mg of Molnupiravir was transferred in to two different 25 ml volumetric flasks and HPLC grade Acetonitrile was added, sonicated and filtered. The extracted solution i.e. the filtrate was then diluted with the mobile phase and 20 µl of each of these two different branded solutions was injected in to the system (n=3) and the average peak area was calculated from the resultant chromatograms. The assay was performed and the amount of Molnupiravir was calculated with the help of calibration curve.

Forced degradation studies¹⁴

Forced degradation studies were performed to determine the stability of Molnupiravir towards stress conditions such as acidic hydrolysis, basic hydrolysis, oxidation and thermal degradation. The specificity of the method can be known from the stability studies and therefore Molnupiravir was exposed to the following stress conditions and the stability was studied.

For acidic degradation studies Molnupiravir was treated with 1 ml of 0.1N hydrochloric acid solution, heated in a thermostat for about 30 minutes at 80ºC, cooled and then neutralized with 0.1N sodium hydroxide and diluted with the mobile phase and the resultant mixture was filtered through membrane filter and 20 µl of this solution was injected in to the UFLC system and the peak area of the chromatogram was noted and the percentage of degradation was calculated from the linear regression equation.

For alkaline degradation studies Molnupiravir was treated with 1 ml of 0.1N sodium hydroxide solution, heated in a thermostat for about 30 minutes at 80ºC, cooled and then neutralized with 0.1N hydrochloric acid and diluted with the mobile phase and the resultant mixture was filtered through membrane filter and 20 µl of this solution was injected in to the UFLC system and the peak area of the chromatogram was noted and the percentage of degradation was calculated from the linear regression equation.

For oxidative degradation studies Molnupiravir was treated with 1 ml of 30% hydrogen peroxide solution, heated in a thermostat for about 30 minutes at 80ºC, cooled and then diluted with the mobile phase and the resultant mixture was filtered through membrane filter and 20 µl of this solution was injected in to the UFLC system and the peak area of the chromatogram was noted and the percentage of degradation was calculated from the linear regression equation.

For thermal degradation studies Molnupiravir was heated in a thermostat for about 30 minutes at 80ºC, cooled and then diluted with the mobile phase and the resultant mixture was filtered through membrane filter and 20 µl of this solution was injected in to the UFLC system and the peak area of the chromatogram was noted and the percentage of degradation was calculated from the linear regression equation.

RESULTS AND DISCUSSION:

The authors have proposed a new stability indicating RP-UFLC method for the estimation of Molnupiravir capsules in the present study using ion pair chromatography technique and the method was validated as per ICH guidelines.

Shimadzu Model CBM-20A/20 Alite UFLC system with PDA detector and Zorbox C₁₈ column were used for the chromatographic study. Mobile phase mixture consisting of 0.1% Acetic acid: Acetonitrile (35:65, v/v) with a flow rate 0.8 ml/min was chosen for the chromatographic elution of Molnupiravir (Detection wavelength 245 nm). The run time was 10 minutes and the injection volume was 20 µl. The present RP-UFLC method was compared with the previously published methods and some of the important observations were highlighted in Table 1.

Table 1: Comparison of previously published methods with the present methods

Mobile phase(v/v) / Reagent	λ (nm)	Linearity (µg/ml)	Comment	Reference
Distilled water	235	5-30	Spectrophotometry	3
Water: Acetonitrile (75:25)	236	0.1-100	RP-HPLC (Metabolite)	4
Methanol and Phosphate buffer (pH 4.2 adjusted with Orthophosphoric acid solution) (35:65)	236	20-100	RP-HPLC	5
Water: Acetonitrile (20:80) (Nano formulations)	240	0.1-60.0	RP-HPLC	6
Ammonium phosphate monobasic: Methanol (47:53)	260	25 -150	RP-HPLC	7
20 mM phosphate buffer (pH 2.5) and Acetonitrile (80: 20)		0.2-80	RP-HPLC	8
0.1% aq. Phosphoric acid: Acetonitrile (85:15) 0.1 % Formic acid and Acetonitrile (Gradient mode)	254 -	10-200 -	RP-HPLC & LC-MS (Green metric scales)	9
0.1% aq. Formic acid solution with 0.08% Ammonia solution: 0.1% aq. Formic acid solution in Methanol with 0.08% Ammonia solution mixed with Acetonitrile (4:1)	-	-	HPLC-ESI-MS/MS ((Human plasma) (Gradient mode)	10
1 mM Ammonium acetate in water (pH 4.3): 1 mM Ammonium acetate in Acetonitrile (Gradient mode)	-	-	LC-MS/MS (Human plasma and saliva)	11
Ammonium formate: Acetonitrile	272	-	LC and LC-HRMS	12
0.1% Acetic acid: Acetonitrile (35:65) (Stability indicating)	245	2-100	RP-UFLC	Present method

Method validation

Molnupiravir obeys Beer-Lambert’s law over the concentration range of 2-100 µg/ml (% RSD 0.19-0.54) (Table 2). The representative chromatograms of Molnupiravir API and the blank were shown in Figure 2A and Figure 2B. The LOD and LOQ were found to be 0.4574 mg/ml and 1.548 mg/ml respectively. The method was linear over the concentration range 2.0-100 mg/ml with linear regression equation, y = 29137x + 3455.8 (R² = 0.9999) and the calibration curve was shown in Figure 3.

The % RSD in precision studies was found to be 0.0407-0.0545 (Intraday) (Table 3) and 0.0379-0.2483 (Inter-day) (Table 4) in precision studies which is less than 2.0 indicating that the method is precise. The % recovery in accuracy studies was found to be 99.66-99.77% (Table 5) and % RSD was (0.26-0.81) less than 2% indicating that the method is accurate. The % RSD in robustness study was also found to be 0.38-1.02 which is less than 2% indicating that the method is robust (Table 6).

Table 2: Linearity study of Molnupiravir

Conc. (µg/ml)	*Mean peak area	% RSD
0	0	0.21
2	58732	0.33
5	146939	0.42
10	293821	0.19
20	587429	0.27
40	1175023	0.54
50	1470431	0.46
80	2349912	0.37
100	2897188	0.31

*Mean of three replicates

Table 4: Inter day precision study of Molnupiravir

Conc. (µg/ ml)	*Mean peak area			*Mean ± SD (% RSD)
Conc. (µg/ ml)	Day 1	Day 2	Day 3	*Mean ± SD (% RSD)
10	293821	295221	294157	294399.6667 ± 730.8662 (0.2483)
20	587429	587158	586987	587191.3333 ± 222.8774 (0.0379)
40	1175023	1171127	1174578	1173576 ± 2132.5354 (0.1817)

*Mean of three replicates

Table 5: Accuracy study of Molnupiravir

Spiked drug Conc. (μg/ml)	Drug Formulation (μg/ml)	Total drug Conc. (μg/ml)	*Drug recovered (μg/ml) ± SD (% RSD)	% Recovery
20 (50%)	20 20 20	10 10 10	29.93 ± 0.0778 (0.26)	99.77
40 (100%)	20 20 20	20 20 20	39.89 ± 0.2553 (0.64)	99.73
60 (150%)	20 20 20	30 30 30	49.83 ± 0.0.4036 (0.81)	99.66

*Mean of three replicates

Table 6: Robustness study of Molnupiravir (20 μg/ml)

Parameter	Condition	*Mean peak area ± SD (% RSD)
Flow rate (± 0.1 ml/min)	0.7	581537 ± 5931.68 (1.02)
	0.8
	0.9
Detection wavelength (± 5 nm)	250	587594 ± 2232.86 (0.38)
	245
	240
Mobile phase composition 0.1% Acetic acid: Acetonitrile (± 2 %, v/v)	30:70	587391 ± 2995.69 (0.51)
	35:65
	40:60

*Mean of three replicates

Assay of Molnupiravir capsules

The assay was performed for the Molnupiravir capsules of two different brands obtained from the local pharmacy store and the proposed chromatographic method was applied after extracting the active ingredient from the tablet dosage forms. The amount of Molnupiravir present in the capsule dosage forms was calculated by substituting the mean peak area in the linear regression equation obtained from the calibration curve and it was found that Molnupiravir was present as 99.46-99.64 (Table 7) and it was also observed that no excipients of the tablet formulations have interfered with the Molnupiravir drug peak. The typical chromatogram obtained for the tablet formulation against the placebo was shown in Figure 2C.

Table 7: Assay of Molnupiravir capsules

S. No	Brand name	Label claim (mg)	*Observed amount (%w/w)	% Recovery*
1	Brand I	200	198.91	99.46
2	Brand II	200	199.27	99.64

*Mean of three replicates

Forced degradation studies of Molnupiravir

During the chromatographic study Molnupiravir has been eluted as a sharp peak at 2.133 min and the system suitability parameters such as theoretical plates 2587 (> 2000) and tailing factor 1.114 (< 1.5) were within the acceptable criteria. During the acidic degradation studies Molnupiravir was eluted at 2.212 mins and has shown less than 20% degradation (19.01 %) with theoretical plates 2345 (> 2000) and tailing factor 1.437 respectively.

During the oxidative degradation study Molnupiravir was eluted at 2.164 mins with less than 2.0% degradation and the theoretical plates were found to be 2429 (> 2000) and tailing factor 1.041 indicating that Molnupiravir is highly resistant towards oxidation. During the alkaline degradation studies Molnupiravir was eluted at 2.186 min and has shown about 20.19 % degradation with theoretical plates 2814 and tailing factor 1.293. During thermal degradation Molnupiravir has shown less than 1.0 % degradation and the system suitability parameters such as theoretical plates (2473) and the tailing factor (1.046) were within the acceptable criteria indicating that Molnupiravir is highly resistant towards thermal degradation. The respective chromatograms obtained during the forced degradation studies of Molnupiravir were shown in Figure 4 and the other details were shown in Table 8.

Table 8: Forced degradation studies of Molnupiravir

Stress Conditions	Rt (min)	*Drug recovered (%)	*Drug decomposed (%)	Theoretical Plates	Tailing factor
Standard Drug	2.133	100	-	2587	1.114
Acidic degradation	2.212	80.99	19.01	2345	1.437
Oxidative degradation	2.164	98.59	1.41	2429	1.041
Alkaline degradation	2.186	79.81	20.19	2814	1.293
Thermal degradation	2.164	99.76	0.24	2473	1.046

*Mean of three replicates

Acidic degradation

Oxidative degradation

Alkaline degradation

Thermal degradation

Figure 5: Chromatograms of Molnupiravir during forced degradation studies

CONCLUSIONS:

A new stability indicating RP-UFLC method has been developed for the determination of Molnupiravir and validated as per ICH guidelines. The method is specific and no degradants were interfering with Molnupiravir peak and there is no interference of excipients used in the capsule formulation. Molnupiravir is more sensitive towards acidic and alkaline degradations and the proposed method is simple precise, accurate and robust and can be applied for the pharmaceutical formulations successfully.

ACKNOWLEDGEMENT:

The authors are grateful to Natco Pharmac Ltd (India) for providing the gift samples of Molnupiravir. The authors reported no conflict of interest.

REFERENCES:

1. Singh AK, Singh A, Singh R, Misra A. Molnupiravir in COVID-19: A systematic review of literature. Diabetes and Metabolic Syndrome. 2021; 15 (6): 102329.

2. Kabinger F., Stiller C., Schmitzová J., Dienemann C., Kokic G., Hillen H.S., Höbartner C., Cramer P. Mechanism of Molnupiravir-induced SARS-CoV-2 mutagenesis. Nat. Struct. Mol. Biol. 2021; 28: 740-746.

3. Pritam Jain, Manali Bhamare, Sanjay Surana. Quantitative estimation of Molnupiravir by UV- spectrophotometric method. International Journal of Pharmaceutical Chemistry and Analysis. 2022; 9(1): 35-39.

4. Aboras, S.I., Megahed, A.A., El-Yazbi, Fawzy and Hadir M Maher. White targeted chromatographic screening method of Molnupiravir and its metabolite with degradation kinetics characterization and in-silico toxicity. Scientific Reports. 2023; 13: 17919.

5. Gandu Sravanthi, Kumara Swamy Gandla and Lalitha Repudi. New analytical method development and validation for estimation of Molnupiravir in bulk and tablet dosage form by RP-HPLC method. Cellular, Molecular and Biological Reports 2023; 3(3): 130-136.

6. Tuba Reçber , Selin Seda Timur , Sevilay Erdoğan Kablan , Fatma Yalçın , Tutku Ceren Karabulut , R Neslihan Gürsoy , Hakan Eroğlu , Sedef Kır , Emirhan Nemutlu. A stability indicating RP-HPLC method for determination of the COVID-19 drug Molnupiravir applied using nano formulations in permeability studies. J Pharm Biomed Anal. 2022; 214: 114693.

7. Ravi Kumar G, Suman Babua B, Rama Rao R, Megha Vardhan, V and VDN Kumar Abbaraju. Method development and validation of a specific stability indicating RP-HPLC method for Molnupiravir API. Journal of Pharmaceutical Research and Innovation. 2021; 33: 3026-3035.

8. Annadi AM, El Zahar NM, El-Din A Abdel-Sattar N, 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 Adv 2022; 12(53): 34512-34519.

9. Hebatallah A. Wagdy. A newly developed and validated environmentally friendly RP-HPLC stability indicating method for the COVID-19 antiviral Molnupiravir: Application to degradation kinetics structure suggestion using LC-MS and the effect on viable cells of the major degradation products. Microchemical Journal, 199; 2024: 109980.

10. Komarov T, Karnakova P, Archakova O, Shchelgacheva D, Bagaeva N, Popova M, Karpova P, Zaslavskaya K, Bely P, Shohin I. Development and validation of a high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) method for quantification of major molnupiravir metabolite (β-D-N4-hydroxycytidine) in human plasma. Biomedicines. 2023; 11(9): 2356.

11. Alieu Amara , Sujan Dilly Penchala, Laura Else, Colin Hale, Richard FitzGerald, Lauren Walker, Rebecca Lyons, Tom Fletcher and Saye Khoo. 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. J. Pharm. Biomed. Anal. 2021; 206: 114356.

12. Jain S, Giri S, Sharma N and Shah RP. LC and LC-HRMS studies on stability behavior of Molnupiravir an anti-COVID 19 drug. J. Liq. Chromatogr. Relat. Technol. 2022; 44(15): 1-10.

13. ICH, Validation of analytical procedures: Text and methodology Q2 (R1). International Conference on Harmonization. Geneva; 2005.

14. ICH, Stability Testing of New Drug Substances and Products Q1A (R2). International Conference on Harmonization. Geneva; 2003.

Received on 23.11.2024 Revised on 19.01.2025

Accepted on 27.03.2025 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1680-1687.

DOI: 10.52711/0974-360X.2025.00241

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Conc. (µg/ml)	*Mean peak area	Statistical Analysis
Conc. (µg/ml)	*Mean peak area	*Mean peak area ± SD (% RSD)
10	293821	293954.67 ± 119.7094 (0.0407)
10	294052
10	293991
20	587429	587736.33 ± 269.0006 (0.0458)
20	587851
20	587929
40	1175023	1174606.33 ± 640.3509 (0.0545)
40	1174927
40	1173869

GITAM School of Pharmacy, GITAM University, Visakhapatnam -530045, India.