key: cord-0768716-vah1bgv1
authors: Reçber, Tuba; Timur, Selin Seda; Erdoğan, Sevilay; Yalçın, Fatma; Karabulut, Tutku Ceren; Neslihan Gürsoy, R.; Eroğlu, Hakan; Kır, Sedef; Nemutlu, Emirhan
title: A Stability Indicating RP-HPLC Method for Determination of the COVID-19 Drug Molnupiravir Applied Using Nanoformulations in Permeability Studies
date: 2022-02-26
journal: J Pharm Biomed Anal
DOI: 10.1016/j.jpba.2022.114693
sha: bd255031d0ec8098b4ced321a1cc5f9bc2c60357
doc_id: 768716
cord_uid: vah1bgv1

Antiviral drugs have gained much more attention in recent years due to SARS-CoV-2 infection and many drug candidates are currently under investigation in order to end pandemic. Molnupiravir, a prodrug of the synthetic nucleoside derivative N4-hydroxycytidine, is one of the promising candidates for SARS-CoV-2 treatment. In this study, a RP-HPLC method was developed for the determination of Molnupiravir and applied for in vitro permeability studies of self-emulsifying drug delivery system (SEDDS) formulations using Caco-2 cell line. Discovery® HS C18 Column (75 ×4.6 mm, 3 μm) was used at 30 °C. Isocratic elution was performed with ACN:Water (20:80 v/v) mixture. The flow rate was 0.5 mL/min and UV detection was at 240 nm. Molnupiravir eluted within 5 minutes. Molnupiravir was exposed to thermal, photolytic, hydrolytic, and oxidative stress conditions. Peak homogeneity data of Molnupiravir in the stressed samples peak obtained using photodiode array detector, in the stressed sample chromatograms, demonstrated the specificity of the method for their estimation in presence of degradants. The developed method was validated according to the ICH guidelines and found to be linear within the range 0.1 - 60.0 μg/mL. The method was simple, rapid, selective, sensitive, accurate, precise, robust and rugged. Thus, it was applied successfully for permeability quantitation of Molnupiravir in nanoformulations. The apparent permeability of Molnupiravir in SEDDS formulations, which have droplet size under 350 nm, was calculated as 3.20± 0.44 ×10(-6) cm/s.

Since the announcement of the pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in March 2020, approximately 300 million confirmed cases and over 5 million death has been reported globally by World Health Organization, and it has been still causing serious public health problems [1]. There have been over 9 billion COVID-19 vaccine doses administrated worldwide, and there still has been a seek for effective therapy options to control the pandemic. Here, the antiviral agents could be one of the treatments of coronavirus disease, especially for high-risk patient groups.

Molnupiravir (MLP, Figure 1 ) is an oral broad-spectrum antiviral agent originally designed for the treatment of Alphavirus infections and now has been used for the treatment of COVID-19 disease caused by the nasopharyngeal SARS-CoV-2 infectious virus [2] . An emergency use authorization (EUA) has been issued on December 2021 for MLP by the U.S. Food and Drug Administration (FDA) for the treatment of mild-to-moderate COVID-19 in adults with positive results of direct SARS-CoV-2 viral testing and who are at high risk for progression to severe COVID-19 and EMA has issued advice on the use of MLP in adults with the increased risk of developing severe COVID-19 on November 2021. MLP, a prodrug of the synthetic nucleoside derivative N4-hydroxycytidine, has been shown to be directly effective in reducing viral RNA and SARS-CoV-2 virus and is known to have favorable safety and tolerability profile. It shows its antiviral effect by adding transcription errors in viral RNA replication. In pharmacokinetic profiling studies, the oral bioavailability of MLP in ferrets and non-human primates was found to be sufficient, and it was also found to be effective in preventing viral transmission in animal studies. If ongoing clinical trials result as expected, MLP is considered to become an important tool to counter the effects of the COVID-19 pandemic [3] [4] [5] .

Pharmaceutical analyses are very important in the pharmaceutical industry because of the need for accurate and precise analytical methods to analyze drugs, impurities and drug metabolites in drug development processes. Permeability studies are one of the applications of pharmacokinetic studies and require the analysis of drug substances in the transport media to determine the J o u r n a l P r e -p r o o f permeability of targeted drugs [6] . According to the guidelines, permeability studies could be performed utilizing human pharmacokinetic studies. In addition to that validated and standardized in vitro systems capable of predicting the extent of drug absorption could also be applied [7, 8] . Among these in vitro models, Caco-2 cell monolayers are one of the most preferred cellular-based alternatives for drug transport evaluation due to the expression of a wide range of drug transporters [9] . However, variability of results generated in different laboratories due to inter-laboratory culturing differences, and the need for precise and validated analytical methods, especially for poorly water-soluble drug molecules, could be considered [9] [10] [11] .

HPLC is one of the widely used analytical techniques in pharmaceutical analysis, as it enables precise, accurate, fast, and low-cost analysis [12] . In the literature, there is an LC-MS/MS method for the determination of MLP and its metabolite in human plasma and saliva [13] .

However, there is no method reported for the quantification of MLP in pharmaceutical dosage forms. This study, it was aimed to develop a rapid, selective, accurate, precise, and robust RP-HPLC method for the determination of MLP in transport samples. The developed method was fully validated according to the ICH guidelines [14] . The stability of MLP was evaluated and also a forced degradation procedure was applied under stress conditions for selectivity analysis.

The application of the method to in vitro permeability studies was conducted using Caco-2 cell lines. After the characterization of Self-Emulsifying Drug Delivery Systems (SEDDS) containing MLP, the quantification of the drug was conducted in transport media and apparent permeability of MLP formulation was determined. 

J o u r n a l P r e -p r o o f MLP was purchased from Optimus Drugs, India. The analytical standard of MLP and HPLCgrade acetonitrile (ACN) were purchased from Merck-Sigma Aldrich (Darmstadt, Germany).

Milli-Q water wa o tained from a Barn tead anopure™ y tem from Thermo. a rafil ® M1944C, Labrafac TM Lipophile WL1349, Transcutol ® HP were kind gift from Kura Chemicals, Turkey. Tween 80 was obtained from Sigma-Aldrich, Germany. All the solutions were prepared in Milli-Q water.

HPLC analyses were performed on a Shimadzu UFLC system. The liquid chromatographic system was comprised of a solvent delivery system (Shimadzu LC-20AB) and a diode array detector (Shimadzu SPD-M20A). The autosampler (Shimadzu SIL-20AC) was used for sample injection and the column was kept in the oven (Shimadzu CTO-20AC).

Chromatographic separations were carried on a Discovery ® HS C18 Column (75 x 4.6 mm, 3 μm) via i ocratic elution of AC :water (20:80, v/v) mixture a mo ile pha e. The column temperature was set at 30 °C and the flow rate was 0.5 mL/min. The injection volume was 10 μ and 240 nm wa elected a the detection wavelength for the diode array detector. Peak identity was confirmed by retention time comparison.

Data acquisition and processing were done with the LabSolutions software (version 1.25, Shimadzu). The quantification of MLP in the samples was performed using calibration curves. Therefore, least squares regression was used to create the calibration curves by plotting MLP concentration to the peak areas of MLP. The statistical calculations were carried out using Microsoft Excel software.

The tandard tock olution of M P (1000 μg/m ) wa prepared in water. Calibration 

Self-Emulsifying Drug Delivery Systems (SEDDS) were prepared using ternary phase diagrams. All excipients selected in formulation studies were suitable for oral administration.

For the selection of the oil phase, the solubility of MLP was assessed using Labrafac TM lipophile W 1349 and Peceol™. For the preparation of SEDDS formulation , the oil phase;

Labrafac TM lipophile WL1349 surfactant mixture; Labrafil ® M1944C:Tween 80 (1:2), and J o u r n a l P r e -p r o o f cosolvent Transcutol ® HP was used. Briefly, the aforementioned excipients were weighed in screw-capped borosilicate vials and the mixture was heated above the melting point of the solid excipients and stirred with a mechanical stirrer. MLP was added to the formulations by simply mixing until a homogenous mixture was obtained. All the formulations were kept for 24 h at ambient temperature to reach equilibrium before transport studies and diluted with transport media prior to the application [15, 16] . The formulations were assessed in terms of organoleptic properties, as well as droplet size and zeta potential values were obtained. The characterization studies were conducted using Malvern Nanosizer ZS 2000 (United Kingdom).

Caco-2 cell line (passage number [15] [16] [17] [18] [19] [20] was used for the evaluation of permeability. Cells were seeded in 75 cm 2 flasks using high glucose DMEM containing 4mM L-Glutamine and sodium pyruvate, 10% FBS, 0.5% penicillin (10,000 units) and streptomycin sulfate (10,000 μg/m ). The cell were cultured at 37 °C in an environment containing 5% CO 2 .

After reaching confluency, Caco-2 cells were seeded into the inserts at a concentration of 12 × 10 4 cells/mL (ThinCert TM , 12 well, 1 μM pore ize, tran parent). Plate were incu ated at 37 °C containing 5% CO 2 . The culture medium was changed every other day, and transport studies were carried out after 21 days when the cell monolayer reached confluency.

The integrity of the cell monolayer was assessed by measuring the transepithelial electrical resistance (TEER). TEER values of Caco-2 cell monolayers were measured by Millicell-ERS epithelial voltammetry. The cell monolayers with an electrical re i tance of 500 Ω.cm 2 and above were used for experiments.

At the end of 21 days, the medium in the apical and basolateral sections was collected and formulation containing 0.8 mg/mL MLP was applied using HBSS transport medium (without phenol red) containing 10 mM HEPES to the apical compartment. The samples were kept in the water bath at 37 °C for 2 hours with shaking at 60 rpm. After 2 hours, the media in the basolateral section was collected and stored at -20 °C for analysis ( Figure 1 ). The cell integrity was confirmed by measuring the electrical resistance of the cell monolayers after 2 hours of incubation.

Thermal degradation J o u r n a l P r e -p r o o f 100 μ of M P tandard tock olution wa diluted to 1000 μ with water. Then the solution was kept at 80 °C for 2 hours. The solution was cooled to room temperature, and then it was diluted with mo ile pha e to 20 μg/m and injected into the RP-HPLC system.

100 μ of M P tandard tock olution wa diluted to 1000 μ y adding 3% H 2 O 2 . Then the solution was kept at 40 °C for 15 minutes. The solution was boiled at 100 °C for 15 minutes and then cooled to room temperature. The solutions were diluted with mobile phase to 20 μg/m and injected into the RP-HPLC system.

100 μ of M P tandard tock olution wa diluted to 1000 μ y adding 0.1 M HC , or 0.1 M NaOH. Then the solution was kept at 40 °C for 2 hours. These solutions were cooled at room temperature after they were neutralized with a suitable amount of HCI or NaOH. The olution were diluted with mo ile pha e to 20 μg/m and injected into the RP-HPLC system. MLP was exposed to different concentrations of NaOH solutions (0.01 and 0.1 M) at different temperatures and times (i-40 °C for 2 hours, ii-40 °C for 15 minutes, iii-room temperature for 15 minutes, iv-room temperature for 2 minutes).

100 μ of M P tandard tock olution wa diluted to 1000 μ with water. The olution wa kept under UV light (254 nm) combined with tungsten lamp for 24 hours at room temperature. The olution wa then diluted with mo ile pha e to 20 μg/m and injected into the RP-HPLC system.

The RP-HPLC method was validated for selectivity, linearity, sensitivity, matrix effect, carryover, precision, accuracy, robustness, and ruggedness following the ICH guidelines [17] .

The selectivity of the RP-HPLC method was investigated by comparing chromatograms obtained for blank transport medium and MLP spiked transport medium at LOQ concentration (0.10 μg/m ). Moreover, matrix effect were te ted for tran port medium and 

The linearity studies were carried out by analyzing calibration solutions containing different concentrations of MLP (0.1-60.0 μg/m ) u ing the propo ed RP-HPLC method. The calibration plots were created by plotting the peak area of MLP against the concentrations with least-squares linear regression analysis.

The sensitivity of the developed method was evaluated with limit of detection (LOD) and

limit of quantitation (LOQ) values based on signal-to-noise ratio at 3:1 and 10:1, respectively.

Intra-day and inter-day precision and accuracy were estimated by analyzing three replicates containing M P at four different concentration level ( OQ, 1.0, 10.0, and 50.0 μg/m ) on the same day and on six consecutive days, respectively. Relative standard deviation (RSD %)

for precision and relative error (RE) for accuracy was calculated at each concentration level.

In addition, the accuracy of the developed method was also examined with recovery studies.

The robustness of analytical methods is checked by examining the effect of small deliberate changes in experimental conditions on the analysis results. In this study, an experimental design was applied with the simultaneous change of factors to determine the robustness of the RP-HPLC method. Small changes were made in ACN percentage of the mobile phase (19-21%), flow rate (0.495-0.505 mL/min) and column temperature (29-31°C). The results were compared statistically.

The ruggedness of the developed method was achieved by analyzing the MLP standard solution (20.0 µg/mL) by two different analysts (Analyst 1 and Analyst 2) under optimum conditions. The results were compared statistically.

The ta ility of the method wa inve tigated u ing 20 μg/ml tandard olution of MLP for different conditions such as short-term (room temperature and +4° C for 24 h), autosampler [17] , and it has been determined that the system is suitable for the analysis of MLP.

The RP-HPLC method was validated for selectivity, linearity, sensitivity, matrix effect, carryover, precision, accuracy, robustness, and ruggedness following the ICH guidelines [17] .

The chromatograms obtained in the selectivity study are given in Figure 3 . No distractive peaks were detected at the analyte retention time. This indicates that the developed method is selective for the analysis of MLP from transport samples. Moreover, forced degradation studies were performed to detect its degradation products. For this purpose, different stress conditions such as high temperature, oxidative degradation, acid-base hydrolysis, and irradiation with UV light were applied. The results obtained are presented in Table 1 and 

The recovery was found to be 98.8% when exposed to high temperatures of the MLP standard solution. The recovery obtained as a result of oxidative degradation was found to be 93.7%.

When exposed to 0.01 M HCI solution at 40 °C for 2 hours, it was observed that 81.8% recovery was obtained as a result of degradation (Table 1 and Figure 2 ).

It was determined that MLP was fully depredated when exposed to 0.1 M NaOH solution under different temperatures and times (i-40 °C for 2 hours, ii-40 °C for 15 minutes, iiiroom temperature for 15 minutes, iv-room temperature for 2 minutes). It was observed that almost all of MLP was degraded under these conditions and therefore the degradation study was continued with 0.01 N NaOH solution. Finally, when exposed to 0.01 M NaOH solution at room temperature for 2 minutes, it was observed that 65.2% recovery was obtained as a result of degradation. The recovery obtained as a result of photolytic degradation was found to be 84.0%. Our method was able to separate completely the degradation products from the intact MLP. The results obtained for MLP demonstrate the stability-indicating capability of the developed method. Therefore, the selectivity of the developed method has also been proven in the presence of degradation products (Figure 2 ). The mass of major degradation products of MLP in different forced degradation conditions have been identified using highresolution mass spectrometry ( Supplementary Figures 3 and 4) . 

The linearity of the calibration curves was determined over the concentration range of 0. 

The OD wa 0.05 μg/m and the OQ wa elected in thi tudy wa 0.10 μg/m (Supplementary Figure 2) . The developed method was highly sensitive for estimating MLP in transport samples.

The results of precision and accuracy are summarized in Table 2 . The low RSD and RE values showed that the method was precise and accurate. In addition, the accuracy of the developed method was also examined with recovery studies. To evaluate the recovery of the method, the slope values of the curves of MLP (1.0-50 μg/m ) prepared in ample lank matrix and water were compared and the recovery of the method was found as 100.63%. 

A nine-run fractional factor design with three experiments under optimized conditions was applied to determine whether a small deviation in experimental conditions produced a statistically significant variation in the obtained responses ( Table 3 ). The results of the analysis were statistically compared with ANOVA test and p values of regression coefficient and regression equation were calculated (Table 4 ). There was no statistical difference between the re ult (p≥0.05). Therefore, we can say that small changes do not have a statistically significant effect on the peak area ratio and the developed method is robust.

J o u r n a l P r e -p r o o f 

J o u r n a l P r e -p r o o f SEDDS formulations containing MLP was characterized and the results were summarized in Table 5 . The droplet size under 350 nm was obtained for transport studies. The zeta potential of formulations was found to be close to zero as expected due to the use of nonionic surfactants.

At the end of 21 days of culture, the formulation containing 0.8 mg/mL MLP was applied using HBSS transport medium (without phenol red) containing 10 mM HEPES to the apical compartment and RP-HPLC analysis was performed using samples from the basolateral compartment. After the RP-HPLC analysis, the apparent permeability coefficient (P app ) of the applied formulation was calculated as 3.20± 0.44 x 10 -6 cm/s. (n=4) [18] . Although these findings indicate the low permeability compared to Metoprolol tartrate, which is used as a reference for permeability classification [19] , the effect of the formulation on the permeability of MLP should be assessed by comparing the permeability of MLP in solution and formulation with further evaluation [19] [20] [21] . J o u r n a l P r e -p r o o f

In the present study, a simple, fast, and stable RP-HPLC method for the determination of MLP in transport medium was developed and validated for the first time. The developed method was selective, linear, sensitive, accurate, precise, robust, rugged, and could be used in permeability studies. The results obtained from stress testing show the method is stabilityindicating and capable of determining MLP in presence of its degradation products, which indicates the selectivity of the method. In addition, the fact that the mobile phase used does not contain any buffer solutions or ion-pairing agents allows for a simple, fast, and low-cost analysis, which makes the developed method advantageous. Considering all these features, it is understood that the developed HPLC method is more applicable for the routine analysis of MLP compared to the LC-MS/MS method proposed by Amara et al. [13] . Moreover, the availability of the regular HPLC systems compared to LC-MS systems makes our study much more applicable for routine analysis.

Although cell culture-based models offer numerous advantages to determine the permeability across the intestinal barriers, there are still disadvantages due to the biologic nature of the cell culture models including culturing conditions, culture media complexity, as well as solubility properties of the drug molecule. Thus, the use of precise analytical tools is crucial for reproducible results. The researchers working on permeability studies could easily apply the proposed RP-HPLC method on their investigations. The developed RP-HPLC method can be easily adapted for the analysis of MLP in different matrices due to all these advantages.

The authors state no conflict of interest

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J o u r n a l P r e -p r o o f

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.