Department of Pharmaceutical Analysis and Quality Assurance, Viswanadha Institute of Pharmaceutical Sciences, Visakhapatnam, A.P, India
A simple, rapid, and reliable visible spectrophotometric method has been developed for the estimation of Molnupiravir, an antiviral drug, using 1,10-phenanthroline as a chromogenic reagent. The method is based on the formation of a red-colored complex between the ferrous ions, generated in situ, and 1,10-phenanthroline, in the presence of Molnupiravir which acts as a reducing agent for ferric to ferrous ions. The resulting colored complex exhibits maximum absorbance at 510 nm. The reaction conditions were optimized, and the Beer-Lambert law was found to be obeyed in the concentration range of [insert range, e.g., 1–5 µg/mL]. The method has been validated according to ICH guidelines for parameters such as linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). . Linearity range was 1 - 5µg/ml with correlation coefficient (R2) of 0.999. LOD and LOQ were found to be 0.05831µg/ml and 0.17672µg/ml respectively. The percentage relative standard deviation for intraday precision was 1.2% and for interday precision was 0.71%.,The robustness were found to be 0.7% and ruggedness 1.0%. The proposed method was successfully applied to the estimation of Molnupiravir in bulk and pharmaceutical dosage forms without interference from common excipients. This method offers a cost-effective and efficient alternative for routine quality control analysis of Molnupiravir.
The most commonly employed reagents for the determinations are the following:
Oxidation Followed by Complexation
1,10-Phenanthroline Synonym for 1,10-phenanthroline is o-phenanthroline. It is extensively chromogenic reagent used for the colorimetric or visible spectrophotometric determination of a number of drugs and pharmaceutical substances. It is also known as ortho- phenanthroline (O- phenanthroline). It is constantly utilized in combination with a popular oxidant or oxidizing agent namely ferric chloride.
MECHANISM
The analysis of drug by using 1,10- phenanthroline involves two steps. Initially the solution of the drug under investigation is allowed to react with 1,10- phenanthroline and Ferric chloride (at elevated temperature). During this process, the drug gets oxidized and results in the generation of ferrous iron [i.e., ferric chloride is converted to ferrous chloride, i.e., (II)]. The ferrous now complexes with 1,10- phenanthroline to form an orange red colored complex. In this method, the reason of the addition of phosphoric acid is to stop the photochemical reduction. The following are the chemical reactions involved:
Drug+ FeCl3
Reduced form of Fe+3 + 1,10-Phenanthroline → (Fe+2-1,10-Phenanthroline) complex
MATERIALS AND METHODS
Chemicals and reagents: Molnupiravir (pure drug) was obtained as a gift sample from Hetero Labs. Ferric Chloride(fecl3) Grade (Thermo Fisher Scientific India Pvt.Ltd), 1,10 Phenanathroline, Grade (Thermo Fisher Scientific India Pvt.Ltd) were purchased from Qualigens Company, Methanol, Rankem(Laboratory Reagent) and All chemicals and reagents used were of analytical grade. Tablets containing drug Molnupiravir (Molnuvid (200mg) were procured from local market.
Instruments: Electronic Precision balance (Infra Instruments Pvt. Ltd., Chennai), PC Based Double Beam Spectrophotometer 2202 (Systronics, India).
Drug name: Molnupiravir
Structure:
Fig.3.1: Chemical structure of Molnupiravir
Iupac Name: [(2R,3S,4R,5R)-3,4-dihydroxy-5-[(4Z)-4-(hydroxyimino)-2-oxo-1,2,3,4 tetrahydropyrimidin-1-yl] oxolan-2-yl] methyl 2- methyl propanoate
Category: Anti -viral drug.
Molecular weight: 336.35 g/mol
Molecular formula: C13H19N3O7
Melting point: 156-157?
Dosage form: Capsules
Description: White solid
pH: 7.2
Solubility: Soluble in methanol.
Route of administration: Oral route.
Storage: Stored in a closed container at room temperature
Dose: Movfor(200mg)
Principle:
Molnupiravir by using 1,10-Phenanthroline involves two steps. Initially the solution of the drug Under investigation is allowed to react with 1,10-Phenanthroline and Ferric chloride (at elevated Temperature). During this process, the drug gets oxidized and results in the generation of ferrous Iron [i.e., ferric chloride converted to ferrous chloride. The ferrous now complexes with 1,10- Phenanthroline to form an orange red colored complex
Step:1
Step:2
Experimental Work
UV-Visible Spectrometry for Molnupiravir
Preparation of Solutions
Preparation of Stock solution: 10mg of Molnupiravir was taken in a 10ml volumetric flask, dissolved in 2ml of Methanol and made upto 10ml with Distilled water. (1mg/ml or 1000µg/ml)
Preparation of Standard solution: 1ml of stock solution was taken in a 10 ml volumetric flask, and made upto 10 ml with Distilled water (100µg/ml)
Preparation Of 0.5% 1,10-Phenanthroline: 500mg 0f 1,10-Phenanthroline was taken in a 100ml volumetric flask , dissolved in 2 ml of methanol and made upto 10ml with Distilled water.
Preparation Of 0.3% Fecl3 : 300mg of Fecl3 was taken in a 100ml volumetric flask , dissolved in 2 ml of methanol and made upto 10ml with Distilled water.
Method Development
Determination Of Λ Max of Molnupiravir
Preparation of solution: 1ml of standard drug solution (100µg/ml) was taken in a 10ml volumetric flask, 1ml of ferric chloride and 1,10-phenanthroline reagent (0.5%) was added. It was heated for 15mins in a water bath for colour development and was allowed to cool. Then it was made up to 10ml with distilled water.
Preparation of blank solution: 1ml of ferric chloride and 1,10-phenanthroline was taken in 10ml of volumetric flask, then it was heated for 15mins. Then it was allowed to cool and made upto 10ml with distilled water.
Fig: λmax of Molnupiravir using 1,10-Phenanthroline
|
S. No |
Wavelength(nm) |
Absorbance |
|
1 |
400 |
0.284 |
|
2 |
405 |
0.293 |
|
3 |
410 |
0.305 |
|
4 |
415 |
0.312 |
|
5 |
420 |
0.329 |
|
6 |
425 |
0.341 |
|
7 |
430 |
0.353 |
|
8 |
435 |
0.384 |
|
9 |
440 |
0.402 |
|
10 |
445 |
0.419 |
|
11 |
450 |
0.443 |
|
12 |
455 |
0.465 |
|
13 |
460 |
0.478 |
|
14 |
465 |
0.504 |
|
15 |
470 |
0.526 |
|
16 |
475 |
0.537 |
|
17 |
480 |
0.564 |
|
18 |
485 |
0.583 |
|
19 |
490 |
0.607 |
|
20 |
495 |
0.635 |
|
21 |
500 |
0.672 |
|
22 |
505 |
0.724 |
|
23 |
510 |
0.748 |
|
24 |
515 |
0.735 |
|
25 |
520 |
0.712 |
|
26 |
525 |
0.698 |
|
27 |
530 |
0.683 |
|
28 |
535 |
0.492 |
|
29 |
540 |
0.398 |
|
30 |
545 |
0.378 |
|
31 |
550 |
0.238 |
|
32 |
555 |
0.202 |
|
33 |
560 |
0.158 |
Absorption Spectrum of Molnupiravir
Result: The absorption maximum (λmax) of Molnupiravir was found to be 510 nm
Optimization Of (0.3%) Fecl3 Solution
|
S. No |
Contents in flask |
Absorbance |
|
|
Blank |
Sample |
||
|
|
0.5 ml of FeCl3 +1ml of 1,10-PTL then heat it for 15min and made up to 10ml with distilled water |
1ml working standard solution + 0.5ml of FeCl3 +1ml of 1,10- PTL then heat it for 15 min and made up to 10ml with distilled water |
0.122 |
|
|
1 ml of FeCl3 +1ml of 1,10- PTL then heat it for 15 min and made up to 10ml with distilled water |
1ml working standard solution + 1ml of FeCl3 +1ml of 1,10- PTL then heat it for 15 min and made up to 10ml with distilled water |
0.259 |
|
|
1.5 ml of FeCl3 +1ml of 1,10- PTL then heat it for 15 min and made up to 10ml with distilled water |
1ml working standard solution + 1.5ml of FeCl3 +1ml of 1,10- PTL then heat it for 15 min and made up to 10ml with distilled water |
0.135 |
|
|
2 ml of FeCl3 +1ml of 1,10- PTL of then heat it for 15 min and made up to 10ml with distilled water |
1ml working standard solution+ 2ml of FeCl3 +1ml of 1,10- PTL then heat it for 15 min and made up to 10ml with distilled water |
0.142 |
|
|
2.5 ml of FeCl3 +1ml of 1,10- PTL then heat it for 15 min and made up to 10ml with distilled water |
1ml working standard solution+ 2.5ml of FeCl3 +1ml of 1,10- PTL then heat it for 15 min and made up to 10ml with distilled water |
0.156 |
Result: The optimium volume of 0.3% Fecl3 was found to be 1.0.ml
Optimization Of 0.5% 1,10- Phenanthroline Solution
|
S. No |
Contents in flask |
Absorbance |
|
|
Blank |
Sample |
||
|
|
1ml of FeCl3+0.5ml of 1,10-PTL then heat it for 15 min and made up to 10ml with distilled water |
1ml stock solution + 1 ml of FeCl3+0.5ml of 1,10- PTL then heat it for 15 min and made up to 10ml with distilled water |
0.129 |
|
|
1ml of FeCl3+1ml of 1,10-PTL then heat it for 15 min and made up to 10ml with distilled water |
1ml stock solution+ 1 ml of FeCl3 +1ml of 1,10- PTL then heat it for 15 min and made up to 10ml with distilled water |
0.156 |
|
|
1 ml of FeCl3 +1.5ml of 1,10- PTL then heat it for 15 min and made up to 10ml with distilled water |
1ml stock solution+1ml of FeCl3 +1.5ml of 1,10- PTL then heat it for 15 min and made up to 10ml with distilled water |
0.228 |
|
|
1ml of FeCl3 +2ml of 1,10- PTL of then heat it for 15 min and made up to 10ml with distilled water |
1ml stock solution+1ml of FeCl3 +2ml of 1,10- PTL then heat it for 15 min and made up to 10ml with distilled water |
0.285 |
|
|
1ml of FeCl3 +2.5ml of 1,10-PTL then heat it for 15 min and made up to 10ml with distilled water |
1ml stock solutio+1ml of FeCl3+2.5ml of 1,10-PTL then heat it for 15min and made up to 10ml with distilled water |
0.240 |
Result: The optimum volume of 0.5% 1,10 Phenanthroline was found to be 2.0.ml
Method Validation
Linearity
Preparation of linearity solutions:
1mcg/ml Solution: 0.1ml of standard solution was taken, 1ml of fecl3(0.3%) and 2ml of 1,10-PTL (0.5%) was added. It was heated for 15 mins in a water bath for colour development and it was allowed to cool. Then it was made upto 10ml with distilled water.
2mcg/ml Solution: 0.2ml of standard solution was taken, 1ml of fecl3(0.3%) and 2ml of 1,10- PTL (0.5%) was added. It was heated for 15 mins in a water bath for colour development and it was allowed to cool. Then it was made upto 10ml with distilled water.
3mcg/ml Solution: 0.3ml of standard solution was taken, 1ml of fecl3(0.3%) and 2ml of 1,10- PTL (0.5%) was added. It was heated for 15 mins in a water bath for colour development and it was allowed to cool. Then it was made upto 10ml with distilled water.
4mcg/ml Solution: 0.4ml of standard solution was taken, 1ml of fecl3(0.3%) and 2ml of 1,10-PTL (0.5%) was added. It was heated for 15 mins in a water bath for colour development and it was allowed to cool. Then it was made upto 10ml with distilled water.
5mcg/ml Solution: 0.5ml of standard solution was taken, 1ml of fecl3(0.3%) and 2ml of 1,10-PTL (0.5%) was added. It was heated for 15 mins in a water bath for colour development and it was allowed to cool. Then it was made upto 10ml with distilled water.
Preparation of blank solution: 1ml of fecl3(0.3%) and 2ml of 1,10- PTL (0.5%) was added and then heated for 15 mins. Then it was allowed to cool and made upto 10ml with distilled water.
The absorbances of the solutions were observed.
Linearity data of Molnupiravir
|
Concentration(µg/ml) |
Absorbance |
|
1.0 |
0.174 |
|
2.0 |
0.359 |
|
3.0 |
0.541 |
|
4.0 |
0.732 |
|
5.0 |
0.909 |
A calibration curve or standard curve was plotted by taking concentration (mcg/ml) on X-axis and absorbance on Y-axis. Linearity equation (y=mx+c) and correlation coefficient (R2) was determined from the calibration curve.
Result: The linearity range- 1-5µg/ml
Correlation coefficient (R2) value obtained was 0.999
Precision
Preparation of 3mcg/ml solution: 0.3ml of standard solution was taken, 1ml of fecl3(0.3%) and 2ml of 1,10- PTL (0.5%) was added. It was heated for 15 mins in a water bath for colour development and it was allowed to cool. Then it was made upto 10ml with distilled water.
Preparation of Blank: 1ml of fecl3(0.3%) and 2ml of 1,10- PTL (0.5%) was added and then heated for 15 mins. Then it was allowed to cool and made upto 10ml with distilled water.
Intraday precision: 6 determinations of 0.3mcg/ml solution were done 3 times in a day and observed for absorbance.
Intraday precision data of Molnupiravir
|
S.NO |
Morning |
Afternoon |
Evening |
|
|
0.545 |
0.539 |
0.537 |
|
|
0.540 |
0.538 |
0.536 |
|
|
0.542 |
0.540 |
0.539 |
|
|
0.544 |
0.543 |
0.542 |
|
|
0.539 |
0.538 |
0.537 |
|
|
0.546 |
0.544 |
0.543 |
|
% RSD |
1.1% |
1.1% |
1.4% |
Result: %RSD for intraday precision was found to be 1.2%
Interday precision: 6 determinations of 0.3mcg/ml solution were done 3 consecutive days and observed for absorbance.
|
S.NO |
Day– 1 |
Day– 2 |
Day-3 |
|
|
0.550 |
0.546 |
0.544 |
|
|
0.546 |
0.543 |
0.538 |
|
|
0.543 |
0.540 |
0.536 |
|
|
0.548 |
0.544 |
0.543 |
|
|
0.541 |
0.539 |
0.537 |
|
|
0.540 |
0.538 |
0.537 |
Result: %RSD for interday precision was found to be 0.71%.
Robustness: 6 determinations of 0.3mcg/ml solution were done and observed for absorbance at different wavelength (nm) with respect to optimized wavelength.
Robustness data of Molnupiravir
|
508nm |
510nm |
512nm |
|
0.542 |
0.544 |
0.545 |
|
0.534 |
0.539 |
0.540 |
|
0.540 |
0.538 |
0.538 |
|
0.535 |
0.543 |
0.544 |
|
0.533 |
0.536 |
0.537 |
|
0.531 |
0.545 |
0.546 |
Result: %RSD for wavelength of 508nm was found to be 0.7%
%RSD for wavelength of 510nm was found to be 0.7%
%RSD for wavelength of 512nm was found to be 1.1%
Ruggedness: 6 determinations of 0.3mcg/ml solution was done by two different analysts and observed for absorbances.
Ruggedness data of Molnupiravir
|
Analyst– 1 |
Analyst-2 |
|
0.544 |
0.543 |
|
0.545 |
0.544 |
|
0.537 |
0.536 |
|
0.546 |
0.545 |
|
0.538 |
0.538 |
|
0.540 |
0.539 |
Result: %RSD for analyst-1 was found to be 1.1%
%RSDforanalyst-2 was found to be 0.9%
Limit of Detection:
LOD=3.3σ/S
σ=standard deviation of intercept S = slope
σ=standard error of intercept* √N
SD intercept= 0.003232
Slope = 0.182886
LOD =3.3 ×SD intercept / Slope
=3.3×0.003232÷0.182886
=0.05831
Limit of Quantification:
LOQ=10σ/S
σ=standard deviation of intercept S = slope
σ=standard error of intercept*√N
SD intercept= 0.003232
Slope = 0.182886
LOQ =10×SDintercept/ Slope
=10 ×0.003232÷0.182886
=0.17672
RESULTS AND DISCUSSION
|
Parameters |
Molnupiravir |
|
Wavelength (nm) |
510nm |
|
Linearity range (μg/ml) |
1-5μg/ml |
|
Regression equation Slope (m) Intercept(c) |
Y= 0.182x-0.004 0.182 0.004 |
|
Correlation coefficient |
0.999 |
|
Precision (%RSD) 1. Intra day 2.Inter day |
1.2% 0.9% |
|
Robustness (%RSD)
|
0.7% 0.7% 1.1% |
|
Ruggedness (%RSD)
|
1.1% 0.9% |
|
Limit of detection(µg/ml) |
0.05831 |
|
Limit of quantification (µg/ml) |
0.17672 |
CONCLUSION:
A simple, sensitive, linear, precise and robust Visible Spectrophotometric method was developed for the determination of Molnupiravir using 1,10phenantroline reagent. The developed method was validated as per ICH guidelines. The % RSD for precision. robustness and ruggedness were found to be within the limits. The developed method has lower range of linearity which indicates that the method is more sensitive even at lower concentrations. Therefore, the developed method can be recommended for routine analysis of Molnupiravir.
REFERENCE
B. Rama Madhuri*, Dr. P. V. Madhavi Latha, M. Swapna, Visible Spectrophotometric Method for the Estimation of Molnupiravir Using 1,10 Phenanthroline Reagent, Int. J. Sci. R. Tech., 2025, 2 (11), 101-110. https://doi.org/10.5281/zenodo.17530391
10.5281/zenodo.17530391