New UV Spectrophotometric Method for the Estimation of Molnupiravir used in the treatment of COVID-19

1. INTRODUCTION

The Molnupiravir, N-Hydroxy-5¹-0-isobutyryl-3, 4-dihydrocytidine (C2R, 3S, 4R, 5R)-3, 4-dihydroxy-5-((4Z)-4- (hydroxyimino)-2-oxo-3, 4-dihydropyrimidin-1(2H)-yl) oxolan-2-yl) methyl 2-methyl propanoate, was approved by UKS medicines and health product regulatory agency on 04 November 2021 and on 23 December 2021 was granted emergency use of authorization by FDA developed by Merck Sharp and Dohme in collaboration with Ridge back for treatment of COVID-19 [1]. An oral bioavailable isopropyl ester prodrug of the ribonucleoside analog -d-N 4-Hydroxycytidine (NHC, also known as EIDD-1931), molnupiravir (MPV), also known as EIDD-2801/MK-4482, has antiviral activity against several RNA viruses. With its active metabolite NHC showing broad-spectrum antiviral efficacy against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, pharmacokinetic (PK) profiling revealed that MPV is orally bioavailable in ferrets and nonhuman primates. Additionally, it demonstrated improved effectiveness against CoV with mutations conferring resistance to the nucleoside analog inhibitor, Remdesivir. In mice models of SARS-CoV or MERS-CoV infection, prophylactic and therapeutic MPV treatment markedly improved pulmonary function, decreased viral titer, and prevented body weight loss [2]. An immunocompromised mouse model's response to molnupiravir treatment revealed suppression of SARS-CoV-2 replication in vivo, supporting the drug's potential for the treatment of COVID-19. Data from a phase 2a clinical trial presented at the 2021 Conference on Retroviruses and Opportunistic Infections (CROI) demonstrated that, in comparison to the placebo, treatment with molnupiravir significantly decreased the time to the negativity of infectious virus from nasopharyngeal swabs in COVID-19 participants [3].

Molnupriavir was recently approved for the treatment of COVID-19, there are no spectrophotometric methods available for this drug [2-14]. Therefore the new spectrophotometric method with forced degradation was developed and validated in bulk as well as in formulation for Molnupiravir.

2. MATERIALS AND METHODS

The Molnupiravir standard was provided by Swapnroop Research Pvt. Ltd., India. The Molnupiravir tablet containing 200 mg of Molnupiravir and the inactive ingredient used in the drug matrix was obtained from the market under the brand name “Molflu 200”. The analytical grade methanol was obtained from Merck life Science Pvt. Ltd. (Mumbai). The UV visible double beam spectrometer with matched quartz cells (1 cm) of Shimadzu with model no. 1800 was used for analysis.

2.2. Selection of Wavelength

The standard solution of Molnupiravir was scanned on a UV spectrophotometer between 200 nm to 400 nm on spectrum mode using diluents as blank. The Molnupiravir shows λmax at 236 nm (Fig. 1).

2.4. Linearity

Five points calibration curve was obtained in a concentration range from 10– 50 µg/ml for Molnupiravir. The response of the drug was found to be linear in concentration range and the linear regression equation was y = 0.042357x+0.123828 with a correlation coefficient of 0.9989. The results are shown in Table 1 and Fig. (2).

Table 1.

Concentration (ppm)	Absorbance
10	0.55148
20	0.96286
30	1.38023
40	1.85464
50	2.22342
y-intercept	0.12382
Slope	0.042357

2.11. Force Degradation Study

In the view of determining stability indicating data of the drug, the forced degradation was carried out by thermal, oxidative, acid, alkali, and photolytic degradation. The details of the procedure of forced degradation are given in the below sections [16-20].

2.11.1. Thermal Degradation

The 0.5 ml of MLP standard stock solution was diluted to 4.5 ml of water, and then the solution was kept at 40^oC for 2 hours. The solution was cooled to room temperature, and then it was diluted with a suitable solvent to 25 µg/ml and then absorbance was taken on UV spectrophotometry (Fig. 3).

2.11.2. Oxidative Degradation

The 0.5 ml of MLP standard stock solution was diluted to 4.5 ml of 3% H₂O₂. Then the solution was kept at 40^oC for 15 minutes. The solution was boiled at 100^oC for 15 minutes and then cooled to room temperature. The solution was diluted with diluent to 25 µg/ml and then absorbance was taken on UV spectrophotometry (Fig. 4).

2.11.3. Acid and Alkali Hydrolysis

The 0.5 ml of MLP standard stock solution was diluted to 4.5 ml of 0.1M HCI or 0.1M NaOH. The solution was kept at 40^oC for 2 hours. The solution was cooled at room temperature after they were neutralized with a suitable amount of HCI or NaOH. Then the solution was diluted with a suitable diluent to 25 µg/ml and then absorbance was taken on UV spectrophotometry (Figs. 5 and 6).

2.11.4. Photolytic Degradation

The 0.5 ml of MLP standard stock solution was diluted to 4.5 ml with water. The solution was kept under UV light combined with a tungsten lamp for 24 hours at room temperature diluent to 25µg/ml and then absorbance was taken on UV spectrophotometry (Fig. 7 and Table 11).

Table 11.

Degradation Parameter	Absorbance of Unstressed Sample (236nm)	Absorbance of Stressed Sample (236nm)	% Degradation
Thermal Degradation	0.26532	0.00679	2.559%
Oxidative Degradation	0.33719	0.16051	47.602%
Acidic Degradation	0.03770	0.02690	71.352%
Basic Degradation	0.16367	0.04633	28.306%
Photolytic Degradation	0.13666	0.6917	50.614%

Note: The % degradation was obtained by using the following formu Table (la (Table 11), % Degradation=Stressed sample/Unstressed sample×100.

3. RESULTS AND DISCUSSION

The correlation coefficient of linearity shows that the method was linear over the range of 10-50 µg/ml. The %RSD for inter-day and intra-day precision and repeatability was found to be within the acceptable limit as per ICH Q₂ (R₁) guidelines. The accuracy of the method was determined at 80,100,120% levels and the results obtained were within the range of acceptable limit as per ICH Q₂ (R₁) guidelines. The %RSD for ruggedness and robustness were within the acceptable range as per ICH Q₂ (R₁) guidelines. The sensitivity of the method was determined by obtaining LOD and LOQ and their values obtained were within the acceptable limit as per ICH Q₂ (R₁) guidelines. The forced degradation behaviour of Molnupiravir was tried to determine and percent degradation in thermal, oxidative, acidic, basic, and photolytic degradation were reported, which are helpful for determining the stability indicating the method by using hyphenated techniques.

CONCLUSION

Since there is no method available for the determination of Molnupiravir by UV spectrophotometry, therefore in the present study, a new, simple, economical UV spectrophotometric method for determination of Molnupiravir in bulk and dosage form was developed and validated for the first time. The developed method was linear, accurate, precise, robust, rugged and having stability indicating characteristics that is capable of determining Molnupiravir in the presence of degradation products. The present analytical method was validated as per ICH Q₂ (R₁) guidelines and it meets specific acceptance criteria.

REFERENCES