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

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

The Open COVID Journal 29 Mar 2023 RESEARCH ARTICLE DOI: 10.2174/26669587-v3-230221-2022-30

Abstract

Background:

Antiviral drugs gained more importance due to SARS-COV-2 infection and many drugs are under investigation to end the pandemic. Molnupiravir is an investigational medicinal product being developed by Merck Sharp and Dohme in collaboration with Ridgeback for the treatment of COVID-19.

Objective:

A new, simple, and economical UV-spectrometric method was developed and validated for the estimation of Molnupiravir in a bulk and pharmaceutical dosage form.

Methods:

The maximum wavelength was found to be 236 nm. The developed method was validated according to ICH guidelines and found to be linear within the range of 10-50µg/ml with a correlation coefficient (R2) 0.9989.

Results:

The %RSD for precision, accuracy, LOD, LOQ, Ruggedness, and Robustness were within the range of acceptable limits as per ICH Q2 (R1). The accuracy of the method was determined at three concentration levels and found to be 99.87%, 99.53%, and 99.84%.

Conclusion:

All parameters obtained are within acceptable limits as per ICH guidelines. The molnupiravir was exposed to thermal, photolytic, hydrolytic, and oxidative stress conditions and its stability data was determined which will be useful for further formulation development.

Keywords: Molnupiravir, COVID-19, Spectrophotmetric, Stability, ICH, [Q2 (R1)].

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.

2.1. Preparation of Standard Stock Solution

The accurately weighed 10mg Molnupiravir was transferred in a volumetric flask containing 10ml methanol to prepare 1000 µg/ml, and from that 100 µg/ml was prepared as a working standard.

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).

Fig. (1). UV Spectra of 20 µg/ml Molnupiravir.

2.3. Method Validation

The new method was validated by following “Validation of Analytical procedures: Text and methodology Q2 (R1)”, guidelines given by the international conference on harmonization of technical requirements for registration of pharmaceuticals for human use. The objective of the analytical procedure will state different validation characteristics like linearity, precision, repeatability, intermediate precision, specificity, detection limit, quantitation limit, linearity, and range [15].

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.
Results of linearity range and absorbance.
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

Fig. (2). Calibration curve in the concentration range 10–50 µg/ml.

2.5. Precision

The inter-day and intra-day precision of the method was determined at three concentration levels 10, 20, and 30 µg/ml. Three replicates of each concentration were determined, and the % RSD of found concentration was determined. The results are given in Tables (2-5).

2.6. Repeatability

The method's repeatability was determined by analyzing six replicates within short time intervals. The results are given in Table 6.

2.7. Accuracy

The accuracy of the method was determined by the standard addition method at 80%, 100%, and 120% concentration levels. The samples were analyzed on a UV spectrophotometer and the concentration found was compared with the found concentration standards of the same concentration. The percent recovery was obtained in the range of 99.87 – 99.84%. The results are shown in Table 7.

2.8. Robustness

The robustness of the method was determined by small but deliberate changes in wavelength and the drug was analyzed. The results of %RSD are given in Table 8.

Table 2.
Results of intraday precision–morning.
Concentration (ppm) Absorbance Found Concentration % Found Concentration Mean % Found Concentration
- 0.55035 10.0 100 -
10 0.53998 9.815 98.15 98.796
- 0.54035 9.824 98.24 -
- 0.95947 19.709 98.545 -
20 0.94995 19.484 97.423 97.646
- 0.94613 19.394 96.972 -
- 1.36481 29.269 97.563 -
30 1.36951 29.379 97.93 98.001
- 1.37686 29.553 98.51 -
- - - Mean 98.147
- - - SD 0.588862
- - - %RSD 0.599975
Table 3.
Results of intraday precision –evening.
Concentration (ppm) Absorbance Found Concentration % Found Concentration Mean % Found Concentration
- 0.54293 9.885 98.85 -
10 0.53841 9.778 97.78 97.556
- 0.53101 9.604 96.04 -
- 0.94196 19.297 96.481 -
20 0.94996 19.484 97.424 96.911
- 0.94412 19.366 96.83 -
- 1.350 28.919 96.399 -
30 1.358 29.108 97.028 97.159
- 1.371 29.415 98.050 -
- - - Mean 97.208
- - - SD 0.325356
- - - %RSD 0.3347
Table 4.
Results of interday precision – day – I.
Concentration (ppm) Absorbance Found Concentration % Found Concentration Mean % Found Concentration
- 0.52774 9.5268 95.268 -
10 0.53680 9.7405 97.405 97.017
- 0.54093 9.8379 98.379 -
- 0.92998 19.0136 95.068 -
20 0.94948 19.4735 97.367 96.233
- 0.94012 19.2528 96.264 -
- 1.351 28.943 96.477 -
30 1.347 28.849 96.163 96.110
- 1.341 28.707 95.691 -
- - - Mean 96.453
- - - SD 0.492008
- - - %RSD 0.5101

2.9. Ruggedness

The ruggedness of the method was analyzed by two different analysts at three concentration levels. The results are given in Tables 9 and 10.


Table 5.
Results of interday precision day – II.
Concentration (ppm) Absorbance Found Concentration % Found Concentration Mean % Found Concentration
- 0.54003 9.8107 98.167 -
10 0.53235 9.6356 96.356 95.218
- 0.52292 9.4132 94.132 -
- 0.94837 19.4474 97.237 -
20 0.94008 19.2518 96.259 96.176
- 0.92963 19.0066 95.033 -
- 1.349 28.896 96.320 -
30 1.341 28.849 96.163 96.032
- 1.340 28.683 95.613 -
- - - Mean 95.8086
- - - SD 0.09755
- - - %RSD 0.1018
Table 6.
Results of repeatability.
Concentration (ppm) Absorbance Found Concentration % Found Concentration
30 1.38147 29.6914 98.971
30 1.37120 29.4490 98.163
30 1.36880 29.3923 97.974
30 1.36754 29.3626 97.875
30 1.35810 29.1397 97.132
30 1.35609 29.0922 96.974
- - Mean 97.843
- - SD 0.72849
- - %RSD 0.7445
Table 7.
Results of accuracy.
%
Recovery level
Stock Solution of dosage form API added Concentration in ppm Absorbance Found Concentration Conc. found in Std. API % Recovery Mean
80% 1 ml 0.8 ml 18 ppm 0.881 17.85 17.86 99.97% -
- 1 ml 0.8 ml 18 ppm 0.870 17.60 17.65 99.73% 99.87%
- 1 ml 0.8 ml 18 ppm 0.850 17.13 17.14 99.91% -
100% 1 ml 1 ml 20 ppm 0.943 19.33 19.34 99.92% -
- 1 ml 1 ml 20 ppm 0.947 19.43 19.49 99.68% 99.53%
- 1 ml 1 ml 20 ppm 0.940 19.25 19.45 98.99% -
120% 1 ml 1.2 ml 22 ppm 1.041 21.65 21.74 99.57% -
- 1 ml 1.2 ml 22 ppm 1.037 21.55 21.78 98.93% 99.84%
- 1 ml 1.2 ml 22 ppm 1.048 21.80 21.58 101.03% -
Table 8.
Results of robustness (change in wavelength)
Conc.
µg/ml
Absorbance Found Concentration % Found Concentration Mean
R1 R2 R3 R1 R2 R3 R1 R2 R3
30 ppm (λ=236) 1.361 1.351 1.359 29.208 28.972 29.160 97.36 96.57 97.20 97.04
30 ppm (λ=237) 1.357 1.348 1.350 29.113 28.901 28.948 97.04 96.33 96.49 96.62
30 ppm (λ=238) 1.350 1.345 1.349 28.948 28.830 28.924 96.49 96.47 96.47 96.35
- - - - - - - Mean 96.671
- - - - - - - SD 0.347707
- - - - - - - %RSD 0.359680
Table 9.
Results of ruggedness analyst–I.
Concentration (ppm) Absorbance Found Concentration % Found Concentration Mean % Found Concentration
- 0.55035 10.0 100 -
10 0.53998 9.815 98.15 98.796
- 0.54035 9.824 98.24 -
- 0.95947 19.709 98.545 -
20 0.94995 19.484 97.423 97.646
- 0.94613 19.394 96.972 -
- 1.36481 29.269 97.563 -
30 1.36951 29.379 97.93 98.001
- 1.37686 29.553 98.51 -
- - - Mean 98.147
- - - SD 0.588862
- - - %RSD 0.599975
Table 10.
Results of ruggedness analyst–II.
Concentration (ppm) Absorbance Found Concentration % Found Concentration Mean % Found Concentration
- 0.54011 9.827 98.27 -
10 0.55003 10.0 100 98.68
- 0.53797 9.777 97.77 -
- 0.95747 19.681 98.405 -
20 0.94981 19.500 97.500 97.61
- 0.94514 19.390 96.950 -
- 1.3671 29.352 97.84 -
30 1.3602 29.189 97.29 97.77
- 1.37155 29.457 98.19 -
- - - Mean 98.02
- - - SD 0.577148
- - - %RSD 0.588806

2.10. Limit of Detection and Limit of Quantitation

2.10.1. LOD:

It is the lowest amount of analyte in the sample that can be detected. LOD can be calculated from the following formula.

2.10.2. LOQ:

It is the lowest amount of analyte in a sample that can be quantitatively determined. LOQ can be calculated from the following formula:

The LOD and LOQ were found to be 7.59 µg/ml and 23.01 µg/ml.

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 40oC 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 40oC for 15 minutes. The solution was boiled at 100oC 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).

Fig. (3). Comparative results of stressed to unstressed samples for thermal degradation.
Fig. (4). Comparative results of stressed to unstressed samples for Oxidative degradation.

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 40oC 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).

Fig. (5). Comparative results of stressed to unstressed samples for Acid degradation.
Fig. (6). Comparative results of stressed to unstressed samples for Base degradation.
Fig. (7). Comparative results of stressed to unstressed samples for photolytic degradation.
Table 11.
Results of forced degradation.
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.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

No humans/ animals were used for studies that are the basis of this research.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

The data and supportive information are available within the article.

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

The authors are very much thankful to the Management and Principal, Dr.M.J.Chavan, of Amrutvahini College of Pharmacy, Sangamner for providing necessary facilities to carryout this work.

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