Introduction
Coronaviruses are single-stranded, positive-sense, and enveloped RNA viruses that can cause zoonosis and are named for the appearance of a solar corona under an electron microscope. The third coronavirus, SARS-CoV-2, which is causing the global pandemic of coronavirus disease 2019 (COVID-19), was initially identified in December 2019 in China, alongside the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV).1
Molnupiravir is an antiviral medication candidate for the treatment of COVID-19 patients that is now in phase III trials.2 Molnupiravir is an isopropyl ester prodrug that is converted into an active nucleoside analogue -D-N4-hydroxycytidine (NHC) or EIDD-1931 by host esterases in the plasma.3 NHC exhibits antiviral activity against a variety of positive-and negative-sense RNA viruses. Recent research has centred on the development of molnupiravir for the treatment of influenza and coronavirus infections, respectively.4 Molnupiravir is a pyrimidine ribonucleoside analogue having a chemical name of ((2R, 3S, 4R, 5R)-3,4-dihydroxy-5-(4-(hydroxyamino)-2-oxopyrimidin-1-(2H)-yl)-tetrahydrofuran-2-yl) methyl isobutyrate.5 The literature survey revealed that two methods of analysis for Molnupiravir have been reported, which included LC-MS/MS for the quantification of β-D-N4-hydroxycytidine in human plasma and β-D-N4-hydroxycytidine-triphosphate in peripheral blood mononuclear cell lysates; 6 and novel LC-MS/MS method for the simultaneous quantification of Molnupiravir and its metabolite β -d-N4-hydroxycytidine in human plasma and saliva.7 Accordingly, the objective of this study was to develop and validate the first order derivative method for the estimation of Molnupiravir in bulk and pharmaceutical formulation as per ICH guidelines.8
Table 1
Linearity study of molnupiravir
Table 2
Recovery studies (Method A)
|
Drug |
Initial Amount (µg/ml) |
Amount Added (µg/ml) |
Amount Recovered (µg/ml) |
% Recovery |
% RSD |
|
|
MPV |
15 |
12 |
11.99 |
99.97 |
0.23 |
|
|
15 |
15 |
14.90 |
99.34 |
0.23 |
||
|
15 |
18 |
17.84 |
99.15 |
0.17 |
||
Table 3
Recovery studies (Method B)
|
Drug |
Initial Amount (µg/ml) |
Amount Added (µg/ml) |
Amount Recovered (µg/ml) |
% Recovery |
% RSD |
|
MPV |
15 |
12 |
11.94 |
99.50 |
0.14 |
|
15 |
15 |
14.93 |
99.56 |
0.13 |
|
|
15 |
18 |
17.93 |
98.85 |
0.37 |
Table 4
Precision studies
Table 5
Repeatability studies
|
|
|
Method A |
Method B |
||
|
Component |
Amount taken (µg/ml) (n=6) |
Amount found* (%) ± SD |
%R.S.D. |
Amount found*(%) |
%R.S.D. |
|
Molnupiravir |
15 |
99.98 ± 0.10 |
0.1062 |
99.96 ± 0.39 |
0.3971 |
Table 6
Ruggedness studies (Method A)
|
Component |
Amount taken (µg/ml) (n=3) |
Amount Found (%) * |
|
|
Analyst I ±S.D. |
Analyst II ±S.D. |
||
|
Molnupiravir |
15 |
99.23 ± 0.82 |
98.92 ± 0.95 |
Table 7
Ruggedness studies (Method B)
|
Component |
Amount taken (µg/ml) (n=3) |
Amount Found (%) * |
|
|
Analyst I ±S.D. |
Analyst II ±S.D. |
||
|
Molnupiravir |
15 |
99.27 ± 0.65 |
98.89 ± 0.81 |
Table 8
Analysis of molnupiravir in bulk
Table 9
Analysis of formulation of molnupiravir capsule
Materials and Methods
Materials
Molnupiravir was bought from Alain Pharmaceuticals, Hyderabad, Telangana. As the solubility of drug Molnupiravir is freely soluble in water, a distilled water is used as a solvent.
Preparation of standard stock solution
Accurately weighed 10 mg of Molnupiravir was transferred to 100 ml volumetric flask and the volume was adjusted by distilled water. The volume was adjusted with the same up to the mark to give final strength of 100 µg/ml.
Selection of wavelength for analysis of Molnupiravir
Appropriate volume 1 ml of standard stock solution of Molnupiravir was transferred into 10 ml volumetric flask, diluted to mark with distilled water to give concentration of 10µg/ml. The resulting solution was scanned in UV range (200 nm — 400 nm). In spectrum Molnupiravir showed absorbance maximum at 235 nm (Figure 2).
Validation of the method
The method was validated in terms of linearity, accuracy, precision, and ruggedness.
Linearity study
Different aliquots of Molnupiravir in range 0.5-3 ml were transferred into series of 10 ml volumetric flasks and the volume was made up to the mark with distilled water to get concentrations 5, 10, 15, 20, 25 and 30 mg/ml, respectively. The solutions were scanned on spectrophotometer in the UV range 200 - 400 nm. The spectrum was recorded at 235 nm. The calibration plot was constructed as Absorbance versus concentration (Figure 3).
Accuracy
To the pre-analyzed sample solutions, a known amount of standard stock solution was added at different levels i.e. 80%, 100% and 120 %. The solutions were reanalyzed by proposed method.
Precision
Precision of the method was studied as intra-day and inter-day variations. Intra-day precision was determined by analyzing the 10, 15 and 20 µg/ml of Molnupiravir solutions for three times in the same day. Inter-day precision was determined by analyzing the 10, 15 and 20 µg/ml of Molnupiravir solutions daily for three days over the period of week.
Sensitivity
The sensitivity of measurements of Molnupiravir by the use of the proposed method was estimated in terms of the Limit of Quanfication (LOQ) and Limit of Detection (LOD). The LOQ and LOD were calculated using equation LOD = 3.3 x N/B and LOQ = 10 x N/B, where, ‘N’ is standard deviation of the peak areas of the drugs (n = 3), taken as a measure of noise, and ‘B’ is the slope of the corresponding calibration curve.
Determination of molnupiravir bulk
Accurately weighed 10 mg of Molnupiravir was transferred into 100 ml volumetric flask containing distilled water and volume was made up to the mark using same. Appropriate volume 1.5 ml of this solution was transferred to 10 ml volumetric flask and volume was adjusted to mark using distilled water. The resulting solution was scanned on spectrophotometer in the UV range 200 – 400 nm. The concentrations of the drug were calculated from linear regression equations.
Application of proposed method for pharmaceutical formulation
About 10 Molnupiravir in house capsules were taken and average quantity of the drug was determined. A quantity of powdered drug equivalent to 10 mg was transferred into a 100 ml volumetric flask containing distilled water and the volume was adjusted upto the mark using same solvent. From this 1.5 ml was taken and transferred to 10 ml volumetric flask and volume was made up to the mark with distilled water to give 15 μg/ml concentration. It was scanned on spectrophotometer in the UV range 200 – 400 nm. The spectrum was recorded at 235 nm. The concentrations of the drug were calculated from linear regression equation.
Results and Discussion
Method validation
The proposed method was validated as per ICH guidelines. The solutions of the drugs were prepared as per the earlier adopted procedure given in the experiment.
Linearity studies
The linear regression data for the calibration curves showed good linear relationship over the concentration range 5-30 µg/ml for Molnupiravir. Linear regression equation was found to be Y = 0.041 X + 0.017 (r2 = 0.999). The result is expressed in Table 1.
Accuracy
The solutions were reanalyzed by proposed method; results of recovery studies are reported in Table 2 which showed that the % amount found was between 98.54% to 99.98% with %R.S.D. >2.
Precision
The precision of the developed method was expressed in terms of % relative standard deviation (% RSD). These result shows reproducibility of the assay. The % R.S.D. values found to be less than 2, so that indicate this method precise for the determination of both the drugs in formulation (Table 3).
Sensitivity
The linearity equation was found to be Y = 0.009 X + 0.164. The LOQ and LOD for Molnupiravir were found to be 3.3 μg and 1.1 μg, respectively.
Repeatability
Repeatability was determined by analyzing 15 µg/ml concentration of Molnupiravir solution for six times and the % amount found was between 99.33% to 101% of Method A and 99.64% to 100.9% of Method B with % R.S.D. less than 2 (Table 4).
Ruggedness
Peak area was measured for same concentration solutions, six times. The results are in the acceptable range for both the drugs. The results are given in Table 5. The result showed that the % R.S.D. was less than 2%
Determination of molnupiravir in bulk
The concentrations of the drug were calculated from linear regression equations. The % amount found was between 99.17 % to 100.43 % (Table 6).
Application of proposed method for pharmaceutical formulation
The spectrum was recorded at 235 nm. The concentrations of the drug were calculated from linear regression equation. The % amount found was between 99.85% to 100.1% of Method A and 99.68% to 100.2% for Method B with % RSD less than 2%. (Table 7).
Conclusion
This UV-spectrophotometric technique is quite simple, accurate, precise, reproducible and sensitive. The UV method has been developed for quantification of Molnupiravir in capsule formulation. The validation procedure confirms that this is an appropriate method for their quantification in the bulk material and formulation. It is also used in routine quality control of the raw materials as well as formulations containing this entire compound.