Abstract

A simple, precise, accurate method was developed for the estimation of molnupiravir by RP HPLC. Chromatographic conditions used are stationary phase Hemochrom C18 (4.6mm x 250mm x 5?m), mobile phase methanol for 5 minutes then 0.1% orthophosphoric acid and methanol in a ratio of 50:50 from 5 – 20 min. The flow rate was maintained at 1.0 ml/min, the detection wavelength was 235 nm, column temperature was set to 30. C and diluent were water and methanol (50:50) was finalized as an optimized method. System suitability parameters were studied by injecting the standard six times and the results were well under acceptance limits. Theretention time for molnupiravir was 10 min. The method was validated for linearity, accuracy, precision, specificity, the limit of quantification, detection, and robustness. The limit of detection and limit of quantificationwas found to be 0.5 ?g/ml and 1.5 ?g/ml respectively and recovery of molnupiravir from molnupiravir capsule was found 98.72 %. The proposed method was successfully applied for the quantitative determination of molnupiravir capsule formulation.

Introduction

I. INTRODUCTION

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a new coronavirus (CoV), which is the cause of the COVID- 19 pandemic, has grown to be a significant concern to public health. More than 115 millionindividuals will have contracted the disease by March 8th, 2021, and sadly, approximately 2.5 million will have perished [1]. Molnupiravir, an antiviral medication with two brand names (MK-4482 and EIDD-2801) that is usedto treat influenza, is one of the useful medications in this regard [2]. This prodrug, which is being developed by Merckfirm as a novel oral anti-viral medication for the treatment of COVID-19, has a nucleoside scaffold of N4-hydroxycytidine [3],[4]. It was initially created at Emory University with the assistance of the university's drug discovery company. Molnupiravir efficacy in preventing viral transmission and inhibiting SARS-CoV 22 was shown by testing the medication on various animal species [5],[6],[7],[8],[9]. This medication, which is based on an oral antiviral ribonucleoside analog, is known as a 5′-isobutyrate prodrug of the direct-acting antiviral ribonucleoside analog EIDD-1931 or - D N4-hydroxy cytidine. EIDD-1931 is produced in the plasma when molnupiravir cleaves[10],[11]. The active anti-viral drug, EIDD-1931 5′-triphosphate, is phosphorylated intracellularly by host kinases. Molnupiravir IUPAC name is ((2R,3S,4R,5R)Its chemical name is C13H19N3O7, and its molecular formula is4-(hydroxyimino)-2-oxo-3,4-dihydropyrimidin-1(2H)-yl)methyl isobutyrate. Molnupiravir has a 329.31 molecular weight [12]

A review of the literature found that molnupiravir pharmacokinetics studies were done in people. The effectiveness and safety of molnupiravir and numerous other drugs were also compared in controlled field research. Molnupiravir can be separated using a liquid chromatographic process that has been verified [13],[14],15].

Here, efforts were undertaken to establish a reverse phase chromatographic method (RP-HPLC) that was straightforward, precise, quick, accurate, and cost-effective for estimating molnupiravir in bulk and formulation.Additionally, techniques like uv-vis techniques are insufficient to calculate an exact amount of API. The study ofthis API similarly reveals insufficient resolution due to poor reproducibility [16],[17],[18]. Keeping this in mind the RP-HPLC method was developed and validated for quantification. Therefore, in the present investigation, a novel HPLC method was developed and validated for the quantification of this API and degradation studies under ICH guidelines [19].

???????II.  MATERIAL AND METHODS

A. Instrumentation

HPLC instrument used was of Shimadzu iprominence with PDA detector. The software used is Lc lab solutions. UV – VIS spectrophotometer PG instrument T 60 with special bandwidth of 2nm and 10 nm and matched quartz was used for measuring absorbance for molnupiravir [20].

???????B. Chemicals

The molnupiravir reference standard was obtained from the capsule of the brand Moluzen (Xenon pharma) of 200mg was obtained from Shuban Pharmaceutical Pvt Ltd, Bhiwandi (India), and also molnupiravir standard was supplied by Shuban Pharmaceutical Pvt Ltd, Bhiwandi (India).

???????C. Chromatographic conditions

Glasswares in each procedure were soaked overnight in a hot air oven. Hemochrom C18 (4.6mm x 250mm x 5 μm) was used at

30. C. Gradient elution was performed with 0-5 min (100% of 0.1% OPA), and 5-20 min (0.1% OPA: methanol in ratio of 50:50).

???????D. Preparation of standard solutions

Accurately weigh 50 mg std molnupiravir in a 50 ml volumetric flask and sonicated for 10 minutes. Flask was made up to the mark by water ( stock sample solution ). Take 1ml from stock , add in 100 ml flask. Add some amount of diluent ( water and methanol in the ratio of 50:50 ). Sonicated it for 10 minutes. Flask was made up tothe mark by diluent.

???????E. Preparation of sample solution

Ten capsules of ( Moluzen, 200 mg) have been weighed and mixed with the powder. Accurately weighed tablet powder containing 60 mg of molnupiravir was transferred to a 50 ml calibrated flask and dissolved in deionized water. The content was shaken for 30 min. The volume was completed with deionized water to get the concentration of 1000 μg mL-1 ( stock sample solution). Then further sample solution was made to get 10 μg/mLsolution by adding 1 ml from stock in a 100 ml flask and then diluting with diluent in the ratio of 50:50 (water: methanol). The final solution was filtered using a Whatman filter paper (No. 42) [21].

III. METHOD VALIDATION

The proposed method was validated as per ICH Q2 (R1) guidelines for specificity, linearity, range, accuracy, precision, and robustness.

???????A. Specificity

Specificity, also known as selectivity, is the ability of an analytical method to measure the analyte of interest accurately in the presence of potential interfering substances. It ensures that the method can distinguish and quantifythe target analyte without being affected by other components in the sample matrix. To assess specificityforced Degradation Studies is carried out Subject the analyte to various stress conditions (e.g., heat, light, acid, base) to assess if the method can accurately detect and quantify degradation products.

???????B. Linearity

Linearity refers to the ability of the analytical method to produce results that are directly proportional to the concentration of the analyte in the sample over a specific range. A linear relationship implies that the method's response is consistent and predictable across a range of concentrations.

???????C. Range

In High-Performance Liquid Chromatography (HPLC) method validation, the term "range" refers to the concentration interval over which the analytical method is expected to provide accurate and precise results. Determining the appropriate range for your HPLC method is crucial for ensuring that the method can effectively quantify the analyte of interest within the desired concentration levels.

???????D. Accuracy

To assess the accuracy of the proposed method, recovery studies were carried out at three different levels i.e. 50 %, 75%, 100%, and 150%. To the pre-analyzed sample solution, a known amount of standard drug solution wasadded at three different levels, absorbance was recorded. The % recovery was calculated by using the formula

% recovery= A- B / C

Where A = total amount of drug estimated, B= amount of drug found on pre analyzed basis, C= amount of puredrug added

???????E. Precision

Precision assessment in HPLC method validation provides insights into the method's ability to generate consistentand reliable results, which is crucial for obtaining accurate data in analytical work. It helps ensure that the methodis suitable for its intended purpose and that the variability in results is within acceptable limits.

???????F. Robustness

Small deliberate changes in a method like flow rate, mobile phase ratio, and temperature are made but there wereno recognized changes in the result, and are within the range as per ICH guidelines [22],[23],[24].

???????IV.  RESULTS

???????A. Selection of detection wavelength

The UV spectra of molnupiravir in methanol in the region between 200 to 400 nm are shown in Fig 2. It shows that at 235 nm molnuiravir has maximum absorbance. Hence max of molnupiravir in the mobile phase was selected as the optimum detection wavelength for quantification of molnupiravir.

B. Optimized chromatographic conditions

1. Column: Hemochrom C18 (4.6mm x 250mm x 5μm)
2. Mobile phase: 0-5 min (100% of 0.1 % OPA), 5-20 min (OPA and methanol in ratio of 50:50)(iii)
3. Flow rate: 1.0ml/min
4. Detector: PDA detector
5. Temperature: 30. C
6. Injection volume: 10μL [25]

???????C. Robustness, limit of detection, and limit of quantification

Robustness was done bychanging parameters like mobile phase ratio, and flow rate. LOD and LOQ are calculatedby the S/N ratio.

???????D. System suitability

The Standard solution of molnupiravir working standard was prepared as per procedure and was injected 5 timesinto the HPLC system. The system suitability parameters were evaluated from a standard chromatogram obtainedby calculating % RSD of retention time, tailing factor, theoretical plates, and peak areas from the above replicateinjection are within range and results were shown in Table 1.

V. FORCED DEGRADATION STUDIES

Degradation studies were performed with formulation and degraded samples were injected. The assay of injectedsamples was calculated and all the samples were passed the limit of degradation. Degradation sample preparation are as given below.

A. Acid degradation studies

To the 1ml of stock sample solution, 1ml of 0.1 N HCL was added separately in a 100 ml flask. The solution waskept at 60. C for 4 hrs. for the HPLC study, the resultant solution was diluted with diluent to obtain 10 μg/ml, and10 μl was injected into the system after filtering through a syringe filter, and a chromatogram was recorded to assess the stability of the sample.

???????B. Alkali degradation studies

To the 1ml of stock sample solution, 1ml of 0.01 N NaOH was added separately in a 100 ml flask. The solution was kept at room temperature for 2 mins. For the HPLC study, the resultant solution was diluted with diluent to obtain 10 μg/ml, and 10 μl was injected into the system after filtering through a syringe filter, and chromatogramswere recorded to assess the stability of the sample.

???????C. Oxidative degradation studies

To the 1ml of stock sample solution, 1ml of 5 % H2O2 was added separately in a 100 ml flask. The solution waskept at 60. C for 4 hrs. For the HPLC study, the resultant solution was diluted with diluent to obtain 10 μg/ml, and10 μl was injected into the system after filtering through a syringe filter, and a chromatogram was recorded to assess the stability of the sample.

???????D. Water degradation studies

To the 1ml of stock sample solution, 1ml of water was added separately in a 100 ml flask. The solution was keptat 60. C for 4 hrs. For the HPLC study, the resultant solution was diluted with diluent to obtain 10 μg/ml, and 10 μl was injected into the system after filtering through a syringe filter, and a chromatogram was recorded to assessthe stability of the sample [29].

VII. ACKNOWLEDGMENT

The author is thankful to the Director of Gelnova Lab Pvt Ltd, Turbhe and Gahlot Institute of Pharmacy, Koparkhairane to provide the necessary facilities to carry out our research work and constant support to our entireresearch work. Special thanks to Dr. Ratnakar Mehendre, Dr. Sonali Mehendre, Dr. Smita Nayak, and Dr. V.H. Bhaskar for valuable guidance and support.

Conclusion

Chromatographic conditions used are stationary phase Hemochrom C18 ( 4.6mm x 250mm x 5?m ), mobile phase for 0-5 min 100% orthophosphoric acid, for 5- 20 min 0.1% orthrophosphoric acid and methanol in ratio of 50:50 and flow rate were maintained at 1.0 ml/min, detection wavelength was 235 nm, column temperature was set to 30. C and diluent methanol and water in a ratio of 50:50 were finalized as an optimized method. Systemsuitability parameters were studied by injecting the standard six times and the results were all under the acceptancecriteria. A linearity studywas carried out between 10–200, R2 value was found to be 0.9998. precision was foundto be 0.312 for the method and 251 for interday precision. LOD and LOQ were found to be 0.5 and 1.5 respectively. By using the above method assay of the marketed formulation was carried out. Degradation studies of molnupiravir were done, in all conditions purity threshold was more than the purity angle and within the acceptable range.

References

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Copyright

Copyright © 2023 Omkar Deshmukh, Sonali Mehendre, Smita Nayak. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.