Method Development and Validation of Stability Indicating RP-HPLC Assay for The Determination of Molnupiravir in Capsules Formulation

Molnupiravir, [(2R,3S,4R,5R)-3,4-dihydroxy-5-[4-(hydroxyamino)-2-oxopyrimidin-1-yl]oxolan-2-yl]methyl 2-methylpropanoate ^[1]. Molecular Formula is C13H19N3O7 ^[2]. The FDA has approved the drug molnupiravir for use in treating COVID-19 in cases of emergency needs. It should be used by adults aged 18 and older who have previously tested positive for the coranavirus, have mild to severe signs, lasting not and over 5 days, and have not been admitted to the hospital ^[11]. There have also been numerous reviews written on the pathogenesis, diagnosis, and management of COVID-19 ^[2,3,4]. An antiviral medication called molnupiravir stops the replication of several RNA viruses ^[5,9,10]. It is utilised to treat COVID-19 in people with SARS-CoV-2 infection ^[6].  Molnupiravir is an EIDD-1931 isopropyl prodrug (Figure 1). N-Hydroxycytidine (NHC) and N4-Hydroxycytidine are two additional names for EIDD-1931 ^[7]. Literature survey found that there is no assay method of molnupiravir was developed. This prompted us to develop simple, accurate, rapid, economical and sensitive assay method for determination of molnupiravir.

Figure 1 Structure of Molnupiravir

MATERIALS AND METHODS:

Reagents and chemicals:

IPC provided 200 mg of Molnupiravir I.P. in working standard and commercial pharmaceutical capsules as a gift sample. We procured from Finar Limited acetonitrile HPLC grade, glacial acetic acid HPLC grade, and spectroscopy grade. Merck Specialities Private Limited provided the ammonium acetate used in the Mili Q water system. Mili-Q water of BIO-AGE water purification system, PVDF membrane filters of 0.45 μm was used for the filtration of mobile phase and 0.45μm PVDF syringe filter for sample preparation.

Instrumentation:

The chromatographic separation was performed on a Thermo Fisher Scientific, Dionex Ultimate 3000 equipped with an integrated degasser, column oven, autosampler, and UV detector. To compile and process the data, Chromeleon console 7.2 software was used. Chromatographic separation and quantification were carried out at 8ºC temperature using a YMC C18 150 x 4.6 mm, 3 μm column.

Chromatographic condition:

The mixture of 0.1% formic acid and acetonitrile in 98:2 v/v and 100% ACN were used as mobile phase with gradient elution. For optimal separation, YMC C18 150 × 4.6 mm, 3 μm column was used. For a total of 16 minutes, the mobile phase was injected into the column at a flow rate of 1.0 ml/min. Injection volume was 5 µL, the detection of effluent was performed at 260 nm, and at a given temp.,the separation was performed.

0.1% formic acid preparation:

1 ml of formic acid was taken with the help of pipette in 1000 ml of milli-Q water. Mixed well and filtered it through a 0.45 µm micropore membrane filter and degassed it.

Mobile phase-A:

Took 0.1% Formic acid: Acetonitrile in a ratio of 98:2, mixed well, then degassed it by sonicator. The solution which has prepared was used as mobile phase.

Mobile phase-B:

100% degassed Acetonitrile.

Preparation of pH 4.75 - 0.02M acetate buffer:

Weighed and transferred about 1.54 g of ammonium acetate into a clean container, then added about 1000 ml of water and mixed to dissolve. Adjusted the pH to 4.75 ± 0.05 with glacial acetic acid.

Preparation of diluent:

Mixed 900 ml of pH 4.75 - 0.02M Acetate Buffer with 100 ml of Methanol and degassed.

Preparation of reference solution:

Weighed accurately and transferred 50 mg of molnupiravir working standard into a volumetric flask i.e 25 mL. Added about 20 ml of diluent, sonicated for 5 minutes to dissolve drug, made volume up to 25 mL with diluent, and mixed well. 5 ml of above solution was pipetted into a 20 ml volumetric flask, made volume with diluent, and mixed well. And filtered the final reference solution through a 0.45 µm PVDF syringe filter. First 3 ml of the filtrate was discarded and subsequent filter was used for analysis.

Preparation of Test solution:

Transferred the content of 20 capsules into a mortar and crushed with pestle. Then weighed accurately and transferred the capsules content equivalent to 250 mg of Molnupiravir into a dry 500 ml volumetric flask. Added about 400 ml of diluent and stirred at 500 RPM for about 15 minutes. Then, sonicated for approximately 15 minutes with occasionally shaking, diluted it to volume, and thoroughly mixed. Then centrifuged at 5000 rounds per minute (RPM) for about 10 min. Filtered above portion of solution through a 0.45 µm PVDF syringe filter. First 3 ml of the filtrate was discarded and subsequent filter was used for analysis.

Selection of the detection wavelength:

Solution of molnupiravir working standard (500.0 µg/ml) was scanned, and the UV spectrophotometer was used to observe the spectra between 200 and 400 nm.

Final method description:

The final method for analysing molnupiravir was provided in light of the results of the optimization of chromatographic conditions.

System Suitability:

The drug at a concentration of 500.0 μg/mL was used to check the system suitability. The majority of the characteristics, including peak area, theoretical plate, tailing factor were evaluated using USP standards.

Accuracy:

Recovery studies were done utilising the conventional addition method to verify method reliability and accuracy. The accuracy studies involved introducing molnupiravir drug sample in triplicate at a level of 50% to 150%. Nine measurements were made at three different concentration levels, providing data that covered the whole concentration range.

Robustness:

Changes in column temperature, wavelength and flow rate were used to calculate robustness, and the %RSD was obtained. Robustness may be evaluated using minor changes to the given parameter, and the method's resistivity was also developed. Peak area, retention time, and %RSD could all differ significantly as a result of the parameter change.

Specificity

Specificity in an assay means that the signal is from the target substance alone, unaffected by excipients, degradation products, or contamination. Chromatograms of the standard solution and sample solution should show comparable retention times following each injection. The blank and placebo chromatogram should show no signal and there should be no interference from blank and placebo at the RT of analyte peak. Also in forced degradation study no interference observed at the retention time of analyte peak. The process is specific as a result.

Precision:

In intraday precision (Repeatability), 2 injections of blank and 5 injections of molnupiravir working standard were injected in chromatographic system. Freshly prepared molnupiravir 6 sample solution than 6 injections of 5 μL of sample were injected in the chromatographic system. In interday precision (Ruggedness), On a different day and using a different system, a different analyst ran an interday precision test. Five injections of molnupiravir working standard were injected. Freshly prepared 6 sample solutions then 6 injections of 5 μL of samples were ran in new HPLC system.

Filter compatibility:

It is frequently necessary to filter samples to stop undissolved drug components from entering the analytical sample system and further dissolving. It was investigated whether 0.45 µm nylon and 0.45 µm PVDF filters may coexist. For each centrifuged and filtered sample, the assay value %difference was calculated after filtering the sample solution. The %difference in assay value between filtered and centrifuged samples was calculated ^[8].

Solution stability:

Sample and standard solutions were prepared, and solution stability was evaluated by analysis performed at various time intervals. Studies on solution stability were carried out for 48 hours, and the %RSD of the measured area. The solution was administered at regular intervals for 48 hours.

Linearity:

It was carried out by making various dilutions of the drug solution. Many molnupiravir concentrations were developed for linearity in the 20–150 μg/mL range. A calibration curve was created by plotting the peak area v/s drug concentration for each concentration level using 5 µL of the solution.

RESULT AND DISCUSSION:

Detection of Wavelength:

Molnupiravir standard solution (500 μg/mL) was scanned and spectrum was observed by UV spectrophotometer at 200-400 nm. Results suggest that 500 μg/mL standard solution had λ_max at 233.65 nm for new method development. Molnupiravir's UV-spectra are shown in figure 2.

Figure 2 Molnupiravir spectra

To resolve the molnupiravir peak, several mobile phase mixtures were attempted. The optimum mobile phases containing 0.1 per cent formic acid: ACN (98: 2) v/v and acetonitrile (100%) were selected because it could resolve the peak of molnupiravir. Using UV detection at 260 nm and area of peak at 1 ml/min flow rate, the determination was gained. A reprensented chromatogram of HPLC achieved during determination of molnupiravir is shown in Fig.3.

Figure 3 Chromatogram of Molnupiravir

The proposed technique was validated for criteria including system suitability, specificity, solution stability, precision, accuracy, forced degradation, filter compatibility, linearity, robustness, etc. in accordance with ICH requirements.

1. System suitability:

The system suitability parameters that were optimised in order to produce and construct system suitability conditions included the RT, theoretical plate, and tailing factor. The results of system suitability's theoretical plates, a tailing factor, a relative standard deviation of 5 duplicate injections, and a retention time were 234265, 0.95, 0.92%, and 8.84 minutes, respectively.

2. Accuracy:

The closeness between the results produced by an analytical procedure and the sample's actual value is known as accuracy. The conventional addition method is used to calculate it, and it is given as recovery (%). Samples were spiked at levels of 50, 100, and 150% of the standard. (%)Recovery and (%)RSD were calculated for molnupiravir and obtained as 98-102% and NMT 2% respectively.

3. Robustness:

Predetermined variations were done under test conditions to assess the robustness. We modified the column temperature ±5, the flow rate ±0.1 and the wavelength ±2. The %RSD was obtained and the limit of %RSD was not more than 2%. Results are showed in table 1.

Blank, solution of working standard, solution of test sample, and placebo solution ran into the HPLC. The blank & placebo did not interfere at the retention time of molnupiravir peak. According to peak purity, the molnupiravir peak was uniform and did not co-elute with any other peaks at its retention time.

5. Precision:

The inter precision & intra precision derived per cent RSD values were 1.75 and 0.38 respectively. The precision study's result reported in per cent RSD and meet the standard allowed by the ICH guidelines while showing strong repeatability, indicate that the developed method has excellent precision. The results are given in table 2.

A study was conducted on the compatibility of 0.45 µm nylon and 0.45 µm PVDF filters. The assay value variation was determined and summarised after filtering and analysing the sample solutions. Based on the analysis, it was determined that filtration by both filter were suitable. The result is given in table 3.

The stock solution's stability was assessed of working standard and  test sample for a specified time period of up to 48 hours at a given temperature. The fact that the percent difference values of assay at various interval of time was found NMT two of the initial time interval solution after the solution had been maintained at the specified temperature for 48 hours indicated that the solutions were stable. The result is given in table 4 and table 5.

8. Linearity and range:

The calibration curve demonstrated good linearity for molnupiravir API in the 20–150% range with R² (correlation coefficient) of 0.999. As shown in table 6, a typical calibration curve for molnupiravir has the regression equation y = 28301.2920x - 36333.7804. In figure 4, graph of linearity is shown.

Figure 4 calibration Curve for HPLC method

Forced degradation:

Sample was exposed to 1 ml of 1N HCl (hydrochloric acid) and 1 ml of 1N NaOH (sodium hydroxide) for 1 hours, oxidizing agent (1 ml of 30% H₂O₂) for 4 hours, thermal degradation at 105?C for 48 hours and for photolytic degradation, keeping sample in UV chamber for 7 days. Sample solutions were prepared of degraded samples and then analysed with compared to control sample. The drug was degraded in acid but it was within the limit. The drug was degraded significantly in base condition. Results are summarized in table 7.