Synthesis of N-Hydroxycytidine and Dimethyl Dioxol Impurities, Method Development and Validation for Their Simultaneous Analysis in Molnupiravir

Abstract

WHO declared the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus as a global pandemic (coronavirus disease 2019 (COVID-19)) in March 2020. Molnupiravir (MPV) is an oral antiviral drug that received use authorization for mild to moderate COVID-19 treatment in adults. However, the global and national drug testing and research of MPV is still difficult due to lack of standards and validated procedures. Therefore, this work aims to synthesize the standard substance of two main molnupiravir impurities, N-hydroxycytidine (NHC) and dimethyl dioxol (DMDO), followed by development of a HPLC-photo diode array (PDA) for their simultaneous analysis. The procedure was validated in compliance with the international pharmaceutical analysis guideline (ICH), and employed to test these compounds in molnupiravir on the market. As a result, NHC and DMDO were successfully synthesized by a hydrolysis in an alkaline environment, and acetalization in an acid environment with very high yields of 83.76 and 73.51%, respectively, along with the purities of over 99%. NHC was detected below the allowable threshold whereas DMDO could not be detected in our samples. The findings show the high applicability of our synthesis and determination procedures in the large-scale production and quality control of impurities of commercial molnupiravir medicines.

Introduction

A severe acute respiratory syndrome caused by a novel coronavirus (SARS-CoV-2) was declared by WHO as a global coronavirus disease 2019 (COVID-19) pandemic on March 11, 2020.^1,2) The number of people infected with COVID-19 in Vietnam was the highest in Southeast Asia and the 13th highest in the world, with a total of over 11 million cases, of which 43000 deaths.¹⁾ The majority of cases aged between 21 and 30 years.³⁾ Therefore, besides vaccines, there was an urgent need for drug approval to ameliorate the pandemic burden and combat potential new variants. The evidence has proved the effectiveness of molnupiravir (MPV) in treatments against influenza A virus (IAV), Ebola virus, Venezuelan equine encephalitis virus (VEEV), SARS-CoV, and most recently SARS-CoV-2.^4,5) In the context of complicated epidemic progression, a pharmaceutical containing active molnupiravir was licensed in the name LAGEVRIO in November 2021 in the United Kingdom,⁶⁾ then approved for treating high-risk adults with mild to moderate COVID-19 by Food and Drug Administration (FDA) in December 2021.⁷⁾ On February 17, 2022, three MPV products (from Boston Pharmaceutical Vietnam, Mekophar Chemical Pharmaceutical, and Stellapharm) were eventually licensed for the domestic COVID-19 treatment by the Drug Administration of Vietnam.⁸⁾ The data show no significant side effects of MPV in phase 1, 2, and 3 clinical trials,^9,10) reduces the risk of hospitalization or death by 50% in patients at risk.^9,11–13) Thus, this pharmaceutical has drawn the attention of global researchers in developing cost-effective synthesis and drug testing procedures to ensure a sufficient global supply chain of COVID-19 medicines.

Molnupiravir is an antivirial active ingredient that was first introduced by a research group at Emory University in Atlanta, U.S.A. in 2003,^14,15) with the structure similar to a nucleoside developed from β-D-N4-hydroxycytidine.^16,17) MPV is a synthetic RNA-dependent RNA polymerase (RdRp) inhibitor,¹⁸⁾ which impairs SARS-CoV-2 replication by enhancing the frequency of viral RNA mutations. When the drug enters human cell, the active molnupiravir forms N-hydroxycytidine hydrate (NHC triphosphate), which can be replaced by cytidine triphosphate, or uridine triphosphate under the action of (RdRp) of SARS-CoV-2.^17,19) The active pharmaceutical of most drugs could consist of various unwanted compounds such as water, small electrophiles, peroxides, and metals, which will affect efficacy and safety of the final drug products. Although MPV is a new drug and the clinical studies are still in phase 3, its amount ingested into the body is very large, with a licensed dose of 1600 mg/d for five consecutive days,^20,21) which means an undetermined content of potential impurities would enter the patients. Hence, impurity profiling has become mandatory according to various regulatory authorities.

According to WHO (2022), molnupiravir has three main impurities: N-hydroxycytidine, dimethyl dioxol and acetyl impurity²²⁾ (Fig. 1). Hydrolysis is a common reaction for the ester type of pharmaceuticals, and molnupiravir is not an exception. N-Hydroxycytidine (NHC, impurity A), a process-related and degradation-related impurity, has the highest likelihood of appearing in MPV,^23–25) because the drug can be produced from cytidine through NHC²⁶⁾ and molnupiravir is degraded to N-hydroxycytidine in all harsh conditions.²³⁾ Dimethyl dioxol (DMDO, impurity B) is a process impurity in the raw material, since it is the final intermediate substance in synthesis from uridine.^23,27,28) Acetyl impurity (impurity C) is also a process impurity,²¹⁾ but it has not been used as an intermediate in the production process of molnupiravir.

Fig. 1. The Mutual Relationship between MPV with NHC and DMDO Impurities

The raw materials may contain a small amount of acetic anhydride produced from the conjugation with isobutyric anhydride, and acetyl ester may appear during the reaction. Nevertheless, the possibility of acetyl impurity in MPV is very low since acetic anhydride is the only impurity of the material. Hence, impurity C was not investigated in our study. The presence of impurities, even in a trace amount can affect the efficacy and safety of the active drug. The most crucial factors of drug testing are impurity profiles, especially of novel medication.²⁹⁾ In addition, techniques and procedures must also always be taken into account when employed.³⁰⁾ To meet the limit requirements of impurities, a convenient HPLC procedure with high accuracy and specificity was established to determine the impurities A and B. There are no comprehensive instructions on the analysis of molnupiravir-related impurities in the raw materials as well as finished products in the pharmacopeias. Our study is the first to establish procedures for quantitation of these impurities, especially in the finished MPV products.

On the other hand, pharmaceutical manufacturers are facing challenges with MPV impurity testing, because the standard of its impurities is very rare and expensive. The national production and quality control of molnupiravir pharmaceuticals are facing difficulties since no standard substances of NHC and DMDO are produced in Vietnam.³¹⁾ The import of materials is not a sustainable solution owing to the unstable supply source, product unavailability and degradation risks, customs procedures, long shipping time and very high cost. Previous studies focused on researching synthesis of molnupiravir rather than its impurities.³²⁾ To the best of the author’s knowledge, this work is the first to develop a simple synthetic process for establishing the impurity standard from molnupiravir, which helps manufacturers save manufacturing and testing costs of molnupiravir pharmaceuticals.

For all urgent reasons, the main goals of our work are (1) synthesize the standards of N-hydroxycytidine and dimethyl dioxol impurities, (2) develop and validate a procedure for simultaneous analysis of these impurities, (3) apply the procedure to determine these impurities in commercial MPV pharmaceuticals. The results provide not only cost-effective synthetic and validated procedures of impurity analysis in the drug quality control of COVID-19 pharmaceutical, but also purified NHC and DMDO impurities which are utilized as the standard materials for synthesis and analysis of molnupiravir in pharmaceutical factories.

Experimental

Chemicals

All chemicals with the highest purity for pharmaceutical research and manufacture were purchased from Fischer (U.S.A.), Merck (Germany), VWR Chemical (France): sodium hydroxide, triethylamine, concentrated ammonia and sulfuric acid, acetone, ethanol absolute, methanol, n-hexane, ethyl acetate, chloroform, dichloromethane, glacial acetic acid, acetonitrile.

Instruments and Equipment

a water bath Memmert WNB22, rotary evaporator Buchi R110S, ultrasonic bath Elma T840 DH, vacuum drying cabinet Memmert VO400, HPLC–PDA Water Alliance 2695 XE–InertSustain C8 (250 × 4.6 mm; 5 µm), InertSustain C18 (150 × 4.6 mm; 5 µm), InertSustain C18 (250 × 4.6 mm; 5 µm), Shim-pack XR^® C18 (250 × 4.6 mm; 5 µm), UV-Vis Spectrophotometer Hitachi U-3900H, Bruker Alpha II IR spectroscopy, UPLC I-Class System ESI-QTOF source, and NMR Bruker Advance II.

Synthesis, Evaluation and Standard Establishment of Impurities

NHC and DMDO was synthesized from molnupiravir batch No. MPRV210501 with humidity 0.16% and purity 99.5% (Zhejiang Hongyuan Pharmaceutical Co., Ltd., China). NHC was synthesized based on the degradation of MPV in alkaline agents.³³⁾ DMDO was synthesized by the acetalization of MPV with acetone in the presence of sulfuric acid at different reaction times, adjusted to an alkaline environment with TEA,³⁴⁾ and purified through liquid-liquid extraction.

The purity was tested by TLC and HPLC-PDA with the configuration in Tables 1 and 2. The procedure was carried out three times to determine the yield, and validated in accordance with ICH Q2A.³⁵⁾ The properties of synthesized impurities including solubility, melting point, humidity, structure and purity were evaluated according to ICH Q3A (R2).³⁶⁾ The uniformity, assigned values–declared values were according to ISO 13528: 2015³⁷⁾ and uncertainty of measurement were determined by following EURACHEM guidelines^38,39) in three laboratories certified in GLP and ISO/IEC 17025.

Table 1. Mobile Phases in TLC and HPLC-PDA

TLC mobile phases
TLC	NHC		MP 1: Acetone–water–glacial acetic acid (14 : 3 : 0.3)
			MP 2: Ethyl acetate–methanol–glacial acetic acid (18 : 8 : 0.3)
			MP 3: Chloroform–ethanol–glacial acetic acid (16 : 7 : 0.3)
	DMDO		MP 1: Ethyl acetate–methanol–glacial acetic acid (18 : 8 : 0.3)
			MP 2: Dichloromethane–acetone–glacial acetic acid (16 : 8 : 0.3)
			MP 3: n-Hexane–acetone–glacial acetic acid (14 : 7 : 0.3)
HPLC mobile phases
		Time (min)	Mobile phase 1		Mobile phase 2
			Methanol (%)	Water (%)	Acetonitrile (%)	Water (%)
HPLC-PDA	NHC	0	1	99	1	99
		60	99	1	99	1
	DMDO	0	5	95	5	95
		60	95	5	95	5

Table 2. Chromatographic Conditions of the Purity Determination

	N-Hydroxycytidine (NHC)	Dimethyl dioxol (DMDO)
Chromatographic column	InertSustain C18 (250 × 4.6 mm; 5 µm)	InertSustain C18 (150 × 4.6 mm; 5 µm)
Flow rate	1.0 mL/min	1.0 mL/min
Detector	236 nm	236 nm
Injection volumn	20 µL	10 µL
Column temperature	30 °C	30 °C
Gradient	Mobile phase 2	Mobile phase 2
Diluent solvent	Water	Acetonitrile–water (4 : 6)
Sample	100 µg/mL in diluent solvent	300 µg/mL in diluent solvent
Extreme conditions:
0.1 N NaOH	24 h	5 min
0.1 N HCl	24 h	1 h
Water	24 h	24 h
3% H2O2	24 h	24 h
Thermal degradation	100 °C for 6 h	80 °C for 6 h
Photo degradation (UV)	12 h	12 h

Method Development, Validation and Application in Analyzing Impurities in Commercial Molnupiravir Pharmaceutical

Diluent solvent was a mixture of acetonitrile–water (1 : 1). The sample solution was prepared by mixing impurity standards of 1.8 µg/mL NHC and DMDO with a 1000 µg/mL MPV standard in the diluent solvent, then filtered through a 0.45 µm PTFE filter.

Optimization of the Sample Preparation

Sonicate extraction with different solvent systems (water, acetonitrile, methanol, mixture of water and acetonitrile/methanol) at different ratios was performed on simulated samples.

Optimization of the Analytical Procedure

Chromatographic conditions were optimized by investigating wavelengths (200 to 400 nm), stationary phase (column C8, C18), mobile phases.

Validation of the Analytical Procedure

According to ICH and AOAC guidelines,^35,36,39) the experiments were carried out at normal and stress conditions (Table 2) in which the impurities were assumed to be degraded to identify the specificity in conditions of with and without decomposition products. The standard curve of impurities concentrations (0.09–0.36–0.90–1.44–1.80–2.52–2.88–3.60 µg/mL) was built to calculate the linearity, LOD and LOQ (S/N is more than 3 and 10, respectively, peak area RSD at LOQ ≤5.3%.). Impurity-spiked samples prepared at 1.8 µg/mL of each impurity and measured repeatedly intra-day and inter-day to evaluate the precision (RSD % accepted is ≤3.7%). They were injected at different times to evaluate the stability. The accuracy was assessed based on the recovery range (95.0–105.0%) of repeated measurements (RSD ≤3.7%) of impurity-spiked samples at 1.08–1.80–2.52 µg/mL. The impurity-spiked samples 1.8 µg/mL were measured at different chromatographic columns and conditions (±10% flow rate) to investigate the robustness of the method. The validated procedure was applied to analyze NHC and DMDO in commercial molnupiravir pharmaceuticals.

Results and Discussion

Synthesis and Characterization of Synthesized NHC and DMDO

Based on the experimental results, the use of triethylamine created an unidentified by-product, while the the procedure with NaOH required many time-consuming steps to remove the excess solution. Hence, an ammonia solution was proposed for the synthesis of NHC (Supplementary Fig. S1). The decomposition of MPV is due to the cleavage of its ester group to form a carboxylic acid and RO− ions, which are protonated to NHC and a by-product called isobutyrate salt⁴⁰⁾ (Supplementary Fig. S2). The spectral analysis of synthesized NHC (C₉H₁₃N₃O₆) UV λ_max (H₂O) nm: 236, 271; IR (KBr) cm⁻¹: 3371.17, 2991.02, 1692.95, 1670.74, 1620.75, 1242.99; MS m/z 260,08699 (M⁺)^41,42); ¹H-NMR (CD₃OD, 400 MHz): 3.70 (1H, d), 3.78 (1H, d), 3.95 (1H, q), 4.14 (2H, m), 5.61 (1H, d), 5.83 (1H, d), 7.13 (1H, d)⁴³⁾ (Supplementary Fig. S3); ¹³C-NMR (CD₃OD, 100 MHz): 61.44 (C₁₂), 70.32 (C₈), 73.26 (C₉), 84.70 (C₁₀), 88.67 (C₅), 97.98 (C₇), 130.87 (C₆), 145.01 (C₄), 150.37 (C₂)⁴³⁾ (Supplementary Fig. S4). Isobutyrate salt has a characteristic odor and high solubility in cold water, thereby being removed through the crystallization and washing with cold water. Besides, our product has a high hygroscopicity, especially under high humid conditions. Thus, the product should be dried in vacuum and stored in a sealed jar. In previous studies, NHC was synthesized from cytidine in continuous stirring conditions for 48 h at 40 °C.²⁶⁾ The product obtained a chromatographic purity of about 98%, with an overall yield of only 50%.²⁶⁾ Other synthesis with the purification took up to 24 h, but the obtained product had an overall yield of up to 80%.²³⁾ Our impurity synthesis from MPV achieved a very great efficiency of about 84%. The product chromatographic purity was up to 99.4%, suitable for application in impurity analysis. More importantly, the synthesis and purification times were reduced to 7 h, which makes the procedure highly applicable in establishing impurity standards in laboratories and pharmaceutical manufacturers.

MPV was acetonized for 3 h and neutralized with trimethylamine, followed by an extraction with ethyl acetate and water. The nucleophilic addition reaction of acetone is more sterically hindered than other aldehydes, which was improved by the catalysis of concentrated sulfuric acid (Supplementary Fig. S5). Ethyl acetate is easily saturated with 3% water³⁶⁾ thereby, the obtained layer was filtered through anhydrous sodium sulfate to remove unwanted water (Supplementary Fig. S6). The spectral analysis of synthesized DMDO (C₁₆H₂₃N₃O₇) UV λ_max (methanol) nm: 234.5, 275; IR (KBr) cm⁻¹: 3271.26, 1663.10, 2990.76, 1376.28, 1749.05, 1281.56, 1692.07, 1246.79; MS m/z 370,16054 (M⁺)^39,42); ¹H-NMR (CD₃OD, 400 MHz): 1.15–1.17 (6H, d), 1.34 (3H, s), 1.53 (3H, s), 2.60 (1H, sep), 4.22 (1H, q), 4.26 (2H, d), 4.78–4.81 (1H, d), 4.97–5.00 (1H, d), 5.57 (1H, d), 5.69 (1H, d), 6.85 (1H, d).²²⁾ (Supplementary Fig. S7); ¹³C-NMR (CD₃OD, 100 MHz): 17.90–17.92 (C_24,25), 24.14–26.10 (C_20,21), 33.67 (C₂₃), 63.85 (C₁₂), 81.18 (C₉), 83.88–83.92 (C_8,10), 92.85 (C₅), 98.06 (C₇), 114.05 (C₁₉), 132.52 (C₆), 144.82 (C₂), 149.74 (C₄), 176.94 (C₂₂).²²⁾ (Supplementary Fig. S8). Similar to NHC, DMDO is an intermediate in the synthesis of molnupiravir.^23,27) In patent number U.S. 20200276219A1, DMDO was synthesized from uridine through 4 steps. Besides, it could be produced from cytidine through 3 steps as described by Gopalsamuth et al. These processes use common input materials (cytidine and uridine), however, there are too many intermediate steps and reaction agents such as 1,2,4-triazole, N,N-diethylethanamine, phosphoryl trichloride, etc. are less common.^23,27) Therefore, the procedure is likely to be less applicable in testing laboratories as well as pharmaceutical manufacturers. In our study, reaction agents such as acetone, concentrated sulfuric acid, trimethylamine and ethyl acetate are available. The reaction and purification took place in only 4 h, which is feasible to be applied to establish standards. The yields of process were so high (73.51%).

The reaction and generation of by-products were monitored by TLC, mobile phase 2 and the reaction screening results are described in Table 3. The purity tests gave “Pass” results and over 99% from the calculation (Fig. 2). Their 2D and 3D chromatograms were presented in Supplementary Fig. S9. The results of TLC with three mobile phases showed only one single spot on the chromatogram. The HPLC-PDA method with two mobile phases also gave a single principal peak at 236 nm. The developed procedure for identifying the purity of NHC and DMDO compounds was validated according to Q2A,³⁷⁾ and the results were in Supplementary Table S1. The chromatogram of the degradation samples showed the degradation impurity peaks and these peaks were completely separated from the NHC peak and DMDO peak. The validation results also show a wide linear range, a high value of correlation coefficient, acceptable accuracy. The RSD% of the retention time and peak area were less than 2.0%, the resolution was >2.0, the tailing factor of these peaks were in the range of 0.8–1.5, the number of theoretical plates was high.

Table 3. The Reaction Screening Results

Reaction screening NHC				Reaction screening DMDO
Alkaline	Temp.	Time	Result	Acetone	Conc. H2SO4	Time	Result
TEA	60 °C	4 h	Unidentified by-product	3 mL	0.1 mL	5 h	A lot of MPV remaining
NaOH 0.5 N	60 °C	4 h	A single point	3 mL	0.2 mL	5 h	MPV traces
NH3	60 °C	4 h	A single point	3 mL	0.3 mL	5 h	MPV traces
NH3	60 °C	2 h	MPV traces	3 mL	0.2 mL	1 h	A lot of MPV remaining
NH3	80 °C	1 h	MPV traces	3 mL	0.2 mL	3 h	MPV traces
NH3	80 °C	2 h	A single point

Fig. 2. Purity Test Results of Synthesized NHC and DMDO in Two Mobile Phase Systems

Mobile phase 1 (pictures marked A); mobile phase 2 (pictures marked B).

Reference Standard Establishment

Two synthesized impurities were fully evaluated and finally described in Table 4. The final products were stored in tightly sealed containers in the dark at 2–8 °C, packaged into 100 vials (5 mg of each compound per vial) and ready to submit to the authority for approval. The results were determined by three laboratories certified in GLP, ISO/IEC 17025 and calculted based on the ISO 13528 : 2015³⁷⁾ EURACHEM guidelines.^38,39) They are being used as reference standards in the Institute of Drug Quality Control Ho Chi Minh City, Vietnam.

Table 4. Summary of Parameters of the Synthesized N-Hydroxycytidine (NHC) and Dimethyl Dioxol (DMDO) Impurities

Method Development and Validation for MPV Impurity Analysis

Validation of the analytical procedure according to ICH and AOAC guidelines.^37,41,44) Diluent solvent was a mixture of acetonitrile–water (1 : 1). The sample solution was prepared by mixing impurity standards of 1.8 µg/mL NHC and DMDO with a 1000 µg/mL MPV standard in the diluent solvent, then filtered through a 0.45 µm PTFE filter.

Samples diluted in water, methanol, respectively and their mixture (1 : 1) did not produce a pure peak of NHC. However, samples prepared in acetonitrile and its mixture with water (1 : 1) gave high-area NHC and DMDO peaks, tailing factors from 0.8 to 1.5. NHC and DMDO were not detected in the second extraction. Thus, water-acetonitrile (1 : 1) was chosen due to low consumption of organic solvent. The wavelength of 236 nm was chosen due to the highest and stable absorbance of both impurities. C18 column (25 cm) was selected based on the highest resolution time, 6 min for NHC and 31 min for DMDO. The gradient with a higher ratio of acetonitrile and water (Gradient 2, Supplementary Table S2) was selected to receive a high tailing ratio and stable peaks in a short chromatography time.

System Suitability

RSD (%) of NHC and DMDO peaks were 1.02 and 1.09%, respectively, less than 2.0%, the tailing factor was in the range of 0.8–1.5, and resolution (Rs) was >1.5 (Table 5). Therefore, the procedure met requirements of the system suitability.

Table 5. The Validation Results of Procedure for Simultaneous Analysis of N-Hydroxycytidine and Dimethyl Dioxol Impurities

Criteria	N-Hydroxycytidine	Dimethyl dioxol
System suitability test
Area (μV × second)	141427 ± 1.02	149226 ± 4.2
Tailing factor	1.35	1.17
Resolution	—	57.58
Regression equation	y = 76522x	y = 73491x
Range (µg/mL)	0.089–2.505 µg/mL	0.095–2.656 µg/mL
Correlation coefficient (R)	0.9999	0.9999
LOD (n = 2)	0.0089% (0.089 µg/mL)	0.0095% (0.095 µg/mL)
LOQ (n = 6)	0.0267% (0.267 µg/mL)	0.0285% (0.285 µg/mL)
RSD%	4.65%	2.59%
Precision (in 2 d, n = 6) at conc 0.18%, ± RSD%	0.18 ± 0.86%	0.18 ± 1.01%
	0.18 ± 0.89%	0.18 ± 0.93%
Accuracy: Recovery (%) ± RSD% (n = 3) at 3 conc.:
1.08 µg/mL (0.108%)	103.06 ± 0.51%	99.02 ± 0.59%
1.80 µg/mL (0.18%)	102.99 ± 0.64%	100.40 ± 0.99%
2.52 µg/mL (0.252%)	100.96 ± 0.14%	98.41 ± 0.76%
Robustness: + 10% flow rate
Area (μV × s) ± RSD%	137939 ± 0.44%	137766 ± 0.48%
Tailing factor	1.27	1.32
Resolution	—	43.4
Robustness: − 10% flow rate
Area (μV × s) ± RSD%	136795 ± 0.41%	136452 ± 0.32%
Tailing factor	1.4	1.48
Resolution	—	40.9
Change column
Area (μV × s) ± RSD%	142936 ± 0.86%	140996 ± 0.88%
Tailing factor	1.40	1.46
Resolution	—	48.6

Robustness

The system suitability still met the requirements when changing 10% flow rate and column with equivalent parameters (Table 5).

Specificity

The tests were performed on placebo and degraded MPV samples (in strong alkaline, strong acid, oxidizing agent, high temperature and UV). On the chromatogram of the placebo, there were no peaks of NHC and DMDO. In the harsh conditions, molnupiravir was degraded into only NHC, therefore no peaks of DMDO were observed in the chromatograms (Figs. 3d–i). The content of NHC (Imp A) increased in the order of degradation by HCl (Fig. 3e), NaOH (Fig. 3d) and H₂O₂ solution (Fig. 3f). The temperature, light and humidity did not cause significant differences in the decomposition of MPV to NHC (Figs. 3g–i). The peaks of impurities are clearly separated from the adjacent peaks including molnupiravir peak (Rs ≥1.5) in all the chromatograms (Fig. 3). Thus, the procedure met requirements of the specificity.

Fig. 3. Specificity Test Results of Simultaneous Quantitative Determination of NHC (Imp A) and DMDO (Imp B) in Molnupiravir (Molnu)

(a) Placebo; (b) NHC standard 1.8 mg/mL; (c) DMDO standard 1.8 mg/mL; Impurities in MPV samples degraded in (d) NaOH 0.01 N for 5 min; (e) HCl 0.01 N for 2 h; (f) H₂O₂ 3% for 8 h; (g) by heat at 80 °C for 12 h; (h) by sunlight for 12 h; (i) in humid environment for 12 h; (j) stability test in diluent solvent for 24 h; Impurities in commercial MPV medicines including product A (k) and product B (l).

Stability

They were injected at different times to evaluate the stability. RSD values of the percentage content of impurities was less than 2%. This means, the difference in the percentage content in samples spiked with impurity standards did not change statistically significantly during 24-hour storage at 25 °C (Fig. 3j).

Linearity, LOD and LOQ

The linearity was established base on impurities concentrations 0.09–0.36–0.90–1.44–1.80–2.52–2.88–3.60 µg/mL. The R-value >0.999 proves the linear correlation between the impurity concentration and peak area. The low LOD and LOQ values indicate high sensitivity of the method (Table 5).

Precision

Impurity-spiked samples prepared at 1.8 µg/mL of each impurity. The RSD values of NHC and DMDO analyzed in intra-day and inter-day were all less than 3.7% (Table 5). Thus, the procedure met requirements of precision.

Accuracy

The recoveries at 3 concentrations of impurities varied in a strictly allowable range (95.0–105.0%) with RSD ≤3.7% (Table 5). Thus, the procedure met requirements of accuracy.

Conclusion

The simultaneous analytical procedure for N-hydroxycytidine and dimethyl dioxol impurities was proved to achieve the accuracy and precision, specificity and sensitivity.

Analysis of NHC and DMDO Impurities in Commercial Molnupiravir Pharmaceuticals

The synthesized impurities were used to develop the procedure to analyze these impurities in two molnupiravir products—Product A (Batch No. 010921RD, mfg. date: 05/09/2021), and Product B (Batch No. 010821, mfg. date: 10/08/2021, exp. date: 24 months). As a result, N-hydroxycytidine was detected in products A (Fig. 3k) and B (Fig. 3l) with the content of 0.09 and 0.08%, respectively, whereas dimethyl dioxol could not be detected.

Currently, the current version of Vietnam Pharmacopoeia and reference Pharmacopoeia do not have detailed information for the raw material of molnupiravir as well as preparations containing molnupiravir. According to the WHO’s documents, the allowable limit of N-hydroxycytidine in raw materials is no more than 0.5% and in finished products is no more than 3.0%.²²⁾ However, according to ICH Q3B guidelines on the allowable limit of impurities in new drugs, with a maximum daily dose of molnupiravir of 1600 mg, N-hydroxycytidine has an allowable limit of no more than 0.1875%.⁴⁴⁾ Regarding dimethyl dioxol, WHO suggested that the content of dimethyl dioxol in raw materials and in finished products are lower than 0.15 and 0.2%, respectively.²²⁾ Besides, according to ICH Q3B guidelines, impurity B must be lower than 0.1875% if the maximum dose of molnupiravir is 1600 mg per day.⁴⁴⁾ Compared with both WHO and ICH regulations, the pharmaceuticals met the strict requirements about allowable limits of both N-hydroxycytidine and dimethyl dioxol impurities. Hence, these products are considered safe in terms of the composition of these two impurities.

NHC is a strong polar compound, in contrast to the DMDO, which is a weak polar compound. It is crucial to choose the chromatographic column and mobile phase to ensure a balanced retention time of NHC and DMDO in the simultaneous analysis of these compounds. The research results show that reversed-phase C18 column and the gradient program with a mixture of acetonitrile and water can elute two impurities well. In some previous publications, a mobile phase of phosphoric acid or phosphate buffer was used.^21,45,46) However, this study used a polar solvent-acetonitrile and purified water to ensure the longevity of the chromatographic column and reduce the risk of corrosion in HPLC-PDA system. The parameters show that this procedure has a great significance when applied at large-scale finished product factories.

Conclusion

N-Hydroxycytidine (impurity A) and dimethyl dioxol (impurity B) were successfully synthesized from molnupiravir with concentrated ammonia and acetone in sulfuric acid, respectively. These substances obtained chromatographic purity 99.4 and 99.8%, respectively and average overall yields of 83.76 and 73.51%, respectively. The simultaneous quantification of NHC and DMDO by HPLC-PDA was developed, validated according to ICH guidelines and applied to analyze molnupiravir pharmaceuticals on the market. The results showed that the contents of these impurities met the allowable limit for the circulation on the market. The synthetic and analytical procedures of N-hydroxycytidine and dimethyl dioxol developed and validated in this study were proved as simple, stable and easily applicable in any laboratory of pharmaceutical companies. The success of this work helps not only reduce difficulties in testing finished products of antiviral molnupiravir, but also contributes to building the standard substance bank in Vietnam’s drug testing system. Finally, the application of these research findings into routine service and pharmaceutical industry could reduce the total costs, which helps patients have access to the high-quality medicines throughout Vietnam and in the world. This work will continue to build a comprehensive impurity database of molnupiravir: (1) apply the validated procedures on a larger scale to determine impurities in molnupiravir medicines; (2) synthesize other impurities of molnupiravir; (3) propose quality levels for degradation-related impurities and total impurities.

Acknowledgments

The whole work was carried out in the laboratory of Boston Vietnam Pharmaceutical Joint Stock Company. The authors would like to thank Boston Vietnam Pharmaceutical Joint Stock Company for providing chemicals and facilities for the research.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials including the mechanism, diagrams of NHC and DMDO synthesis, the NMR spectra, 2D and 3D chromatograms of purified NHC and DMDO in the purity test.

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