INTRODUCTION:

Umifenovir (UMI) (1-methyl-2-((phenylthio)methyl)-3-carbethoxy-4-((dimethylamino)methyl)-5-hydroxy-6-bromindole hydrochloride. Fig. 1, is an antiviral drug and indole derivative^1-2. UMI is marketed under the brand name Arbidol and is a broad-spectrum antiviral used to treat influenza in both China and Russia. UMI is a fusion inhibitor. It works by blocking the fusion of the virus and the target host cells. It also stimulates the immune system. UMI is effective against a variety of enveloped and nonenveloped viruses³. It has also been used to treat influenza virus A and B viruses and has recently been used to treat hepatitis C virus. UMI has also been shown to be effective in inhibiting COVID-19 infection by interfering with the release of SARS-CoV-2 from intracellular vesicles^4,5,6.

Figure 1: Chemical structure of UMI

According to a literature survey the drug has been estimated in biological fluids such as human urine, plasma, and rat plasma, Wang et al., identified the metabolites in human urine and studied the fragmentation pathways using HPLC coupled with ion trap mass spectrometry⁷. Liu X, et al., developed a method based on cloud-point extraction to determine arbidol in rat plasma by HPLC and UV detection⁸ and used the LC-ESI-MS method for arbidol determination in human plasma⁹ while Annampura et al., established a stability-indicating LC method for the determination of UMI in tablets¹⁰ and quantified arbidol by RP-HPLC with PDA detection¹¹. Surabhi et al. validated the stability-indicating method for the determination of UMI-remdesivir in the presence of its degradation products¹². Damle et al. developed a validation HPTLC method for the determination of arbidol from a pharmaceutical formulation¹³. As far as we know, there is no UV spectrophotometric method for the determination of UMI. Therefore, we believe that developing a UV method is worth exploring, and very few HPLC methods have been reported, to provide an alternative HPLC method that is simple, has high reproducibility, has a good retention time, has more percent recovery and utilizes very less time of analysis, i.e., 6 minutes to the existing methods for estimation of UMI in API and in tablet formulation along with validation of the methods as referred to in ICH guidelines¹⁴.

MATERIALS AND METHODS:

Pure sample:

UMI was procured from Mylan Hyderabad as a gift sample.

Formulation:

UMI is available as a tablet (Arpeflu; label claim: 50 mg; 100mg) and capsule (Arbidol; label claim: 100mg). Tablets were prepared in the laboratory as formulations were not available in India.

Chemicals and reagents:

Double distilled water was obtained from the Millipore unit. HPLC grade acetonitrile (ACN), methanol, orthophosphoric acid and triethylamine (TEA) were all purchased from Sisco Research Laboratories Pvt. Ltd. Mumbai, India.

Instrumentation:

A double-beam UV-1800 Shimadzu UV spectrophotometer with a pair of 10mm matched quartz cells was used. The HPLC system was a Shimadzu model no. DGU-20A5R, a Shimadzu ATX224 digital analytical balance and a PCi analytics ultrasonic bath.

Sample preparation for the UV method development:

Standard stock solution (100μg/ml):

5mg of precisely measured pure UMI was poured into a clean dry 50ml volumetric flask, methanol was used for dissolution, and the volume was made up to 50ml.

Standard working solution (10μg/ml):

1ml from the standard stock solution was transferred and dissolved in 10ml of methanol.

Sample solution for the UV assay method:

Ten tablets (containing 100mg of UMI) were precisely weighed and prepared in our laboratory, and the average weight of the tablets was determined. Tablets were reduced to a powdery consistency. The tablet powder equivalent to 5mg of UMI was weighed and further transferred into a 50ml volumetric flask. The content was dissolved in methanol and then sonicated for ten minutes. The volume was brought up to the mark (Conc.100μg/ml) and the solution was filtered with Whatman filter paper No. 41. From the clear sample stock solution, appropriate dilutions were made to obtain a concentration of 10µg/ml sample solution.

Sample preparation for the HPLC method development:

Standard stock solution (A1) (1000 μg/ml):

10mg of precisely measured pure UMI was poured into a clean dry 50ml volumetric flask, acetonitrile was used for dissolution and volume was made up to 50ml.

Standard stock solution (A2) (100μg/ml):

1ml of A1 stock solution was added to a 10ml volume flask, and the volume was made up to the mark with acetonitrile.

Standard working solution (A3) (10μg/ml):

1ml of A2 stock solution was added to a 10ml volume flask, and the volume was made up to the mark with acetonitrile.

Mobile phase preparation:

The mobile phase consisted of 60:40% v/v acetonitrile and 0.1% trimethylamine (with orthophosphoric acid pH adjusted to 2.7).

Sample preparation for HPLC assay:

Ten tablets (containing 100mg of UMI) were accurately weighed and prepared in our laboratory, and the average weight was determined. Tablets were reduced to a powdery consistency. The tablet powder equivalent to 10mg of UMI was weighed and further transferred into a 50ml volumetric flask. The content was dissolved in methanol and then sonicated for ten minutes. The volume was brought up to the mark with methanol (Conc.100μg/ml), and the solution was filtered with Whatman filter paper No. 41. From the clear sample stock solution, appropriate dilutions were made to obtain a concentration of 10µg/ml sample solution.

Validation of the Proposed Method:

System suitability:

It is considered under every validation parameter. Five replicate injections of 100 μg/ml drug standard solution were auto injected. Parameters including the tailing factor and theoretical plate counts were noted.

Linearity and Range:

It is the ability to obtain results that are proportional to the analyte concentration. From a standard stock solution of UV, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2ml aliquots were added to a series of 10 volumetric flasks, and the volume was brought to the mark by methanol, resulting in concentrations of 2, 4, 6, 8, 10 and 12µg/ml respectively. At 223nm their absorbance was measured against a blank. Similarly, 5-30µg/ml concentrations from the working standard solution of HPLC were prepared and auto injected with optimized chromatographic parameters.

Accuracy:

It is the assessment of the difference between the measured value and the real value. Accuracy was measured at 80%, 100%, and 120% by spiking a standard solution of UMI (0.8, 1.0, 1.2μg/ml) into the sample solution. The percentage recoveries were calculated.

Precision:

It is the degree of agreement under specified conditions between a set of measurements acquired from multiple samplings of homogeneous samples. This was demonstrated by intraday and interday variation studies. Six sample solutions were prepared, and %RSD was calculated.

Intraday and Interday variation:

The absorbance of the standard working solution (10 µg/ml) was recorded by UV at 0hours, 6hours, the 2^nd day and the 3^rdday. Similarly, for the HPLC method, a 10μg/ml concentration of six standard solutions of UMI was injected and chromatograms were recorded.

RESULTS AND DISCUSSION:

Determination of wavelength of maximum absorbance (λmax) for UMI:

The working standard solution of UMI (10μg/ml) was scanned in the range of 200-400nm in a 1.0cm matched quartz cell against ablank (methanol) and the spectrum was recorded. From the UV spectra, λmax was found to be 223nm, which was selected as the analytical wavelength for further analysis. The spectrum was recorded as shown in Fig. 2

Figure 2: UV spectrum of 10μg/ml UMI standard solution.

Preliminary optimization of the mobile phase and other chromatographic conditions:

To obtain the best possible chromatographic results, one or more parameters were adjusted in each trial. The trial runs are shown in Table 1.

Table 2: Optimized chromatographic parameters

Column	Shimatzu C₁₈ (250mm × 4.6mm i.d., 5µm particle size)
Mobile Phase	Acetonitrile:0.1% trimethylamine (60:40) (pH 2.7 adjusted with orthophosphoric acid) on isocratic mode
Flow rate	1 ml/min
Wavelength	223 nm
Injection volume	20µl
Column oven temperature	25 ˚c
Retention time	3.515 min
Total run time	6 min

A 20µL aliquot of solution was autoinjected into the HPLC system, and the chromatogram was recorded as shown in Fig. 3 and its 3D image in Fig. 4.

Figure 3: A model chromatogram showing the separation of UMI.

Study of system suitability:

After equilibrating the column with the mobile phase, the solution (conc. 10µg/ml) was auto-injected five times and the chromatograms were recorded. The results of the system suitability are reported in Table 3.

Fig. 4: 3D image of UMI chromatogram.

Table 3: Observations of system suitability parameters

Sr. No.	Weight of Std. drug taken (mg)	Peak area (mV)	Tailing factor	Theoretical plates
1	10.0	798006	1.291	3313
2		791841	1.293	3318
3		791959	1.265	3387
4		797809	1.312	3287
5		798511	1.267	3369
% RSD		0.88	0.92	0.95
Limit		NMT 2%	NMT 2	NLT 2000

Linearity and range:

The UV and HPLC methods confirmed linearity in the ranges of 2-12µg/ml and 5-30µg/ml, and the linearity equations were y = 0.064x+0.0065 and y = 7523x+ 63074 with correlation coefficients of 0.9995 and 0.9997 respectively. The results are tabulated in Table 4. The calibration plots for UV and HPLC are depicted in Figs. 5 and 6, respectively.

Table 4: Calibration curve data of the UV and HPLC methods

UV method		HPLC method
Conc. (µg/mL)	Absorbance	Conc. (µg/mL)	Area
2	0.138	5	424472
4	0.266	10	837809
6	0.385	15	1183954
8	0.513	20	1570907
10	0.644	25	1944492
12	0.782	30	2316201
r²	0.9995	r²	0.9997

Figure5: The calibration curve of UMI for UV

Figure 6: The calibration curve of UMI for HPLC

Accuracy:

The percentage recovery of UMI at different spiked levels (i.e., 80%, 100%, 120%) was ascertained to be in the range of 98.50 – 101.6% and 99.97– 102% for UV and HPLC respectively. The % RSD was less than 2 as shown in Table 5. This means that there is no interference of the excipients when determining umifenovir. Therefore, the method is accurate. Tables 13 and 14, show the percentage recovery results that are statistically compared for the F test and the T test respectively.

Table 5: Results of Accuracy (Recovery) Studies

Sr. No.	Conc.(%)	Tablet (μg/ml)	Pure drug (μg/ml)	Total amount (μg/ml)	UV Method			HPLC Method
Sr. No.	Conc.(%)	Tablet (μg/ml)	Pure drug (μg/ml)	Total amount (μg/ml)	Actual amount found (μg/mL)	% Recovery	% RSD	Actual amount found (μg/mL)	% Recovery	% RSD
1	80	10	8	18	17.98	99.85	0.44	18.09	101.2	0.60
2	80	10	8	18	18.05	100.71		18.06	100.75
3	80	10	8	18	18.10	100.12		18.0	100
4	100	10	10	20	19.85	98.50	1.59	20.1	101	0.60
5	100	10	10	20	20.16	101.60		20.2	102
6	100	10	10	20	19.94	99.42		20.09	100.9
7	120	10	12	22	22.08	100.60	0.27	21.99	99.97	0.19
8	120	10	12	22	21.95	100.09		22.04	100.33
9	120	10	12	22	22.02	100.19		22.0	100

%RSD = percentage relative standard deviation

Precision:

It was studied by analyzing homogenous samples intraday and interday. The results are shown in Table. 6. The %RSD for UMI of the standard solution was less than 2, which significantly assures the precision of the proposed method.

Table 6: Intraday andInterday Precision Result

UV Method
Concentration (10 μg/ml)		Intraday Study (n=6)		Interday Study (n=6)
		0 Hour	6 Hours	2^nd Day		3^rd Day
*Mean Absorbance		0.685	0.637	0.566		0.536
Standard deviation		0.006	0.0054	0.0047		0.0045
% RSD		0.88	0.85	0.83		0.84
HPLC Method
Concentration (10 μg/ml)	Intraday Study (n=6)				Interday Study (n=6)
	0 Hour		6 Hours		2^nd Day	3^rd Day
*Mean Area	794781		784506		772668	764276
Standard deviation	7104.50		6048.82		6698.09	6281.27
% RSD	0.89		0.77		0.87	0.82

n = number of measurements, %RSD = percentage relative standard deviation, *Mean of six observations

Ruggedness:

It was determined by performing the same procedure by two different analysts and two different UV spectrophotometers were also used for the study. The results are reported in Tables 7 and 8.

Table 7: Ruggedness Study Result (Analysis Performed by Different Analysts)

UV Method
Conc. (10 μg/ml)	Analyst 1	Analyst 2
*Mean Absorbance	0.653	0.662
Standard deviation	0.0060	0.0059
% RSD	0.92	0.89
HPLC Method
Conc. (10 μg/ml)	Analyst 1	Analyst 2
*Mean Area	794455	794788
Standard deviation	7576.24	7101.41
% RSD	0.95	0.85

RSD= Relative standard deviation, *Mean of six observations

Table 8: Ruggedness Study (Analysis Performed on Different Instruments)

Conc. (10 μg/ml)	Instrument Model & Make
Conc. (10 μg/ml)	UV-1800 Shimadzu UV Spectrometer	Perkin Elmer UV-Visible Spectrometer
*Mean Absorbance	0.653	0.638
Standard deviation	0.0064	0.0059
% RSD	0.98	0.92

RSD = Relative standard deviation,*Mean of six observations

Robustness:

In the proposed UV method, to validate the robustness parameter, slight variation was employed in wavelength (±2nm), and HPLC robustness was assessed by introducing small, intentional variations in the chromatographic conditions which comprised the percent of acetonitrile in the mobile phase (±5%), the flow rate of mobile (±0.2ml/min) and pH (±1). The data are represented in Tables 9 and 10. The %RSD was less than 2, which significantly represents the robustness of the method.

Table 9: Robustness Study for UV

Conc. (10 μg/ml)	At 221 nm	At 225 nm
*Mean Absorbance	0.687	0.658
Standard deviation	0.0062	0.0059
% RSD	0.90	0.89

*Mean of six observations, RSD = Relative standard deviation

Table 10: Robustness Study for HPLC

Parameters	*Mean Peak Area (mV)	Standard deviation	% RSD
ACN: TEA (55:45)	798005	7144.50	0.86
ACN: TEA (65:35)	799733	9424.45	0.95
Flow rate (0.8ml/min)	791844	7576.23	0.99
Flow rate (1.2ml/min)	792364	7860.87	0.98
pH (2.6)	789065	7112.30	0.99
pH (2.8)	792011	7781.20	0.95

Sensitivity:

The LOD and LOQ values computed from the stated formulae are reported in Table 11.

Table 11: Sensitivity result

Sensitivity	UV Method	HPLC Method
LOD (μg/ml)	0.58	0.29
LOQ (μg/ml)	1.75	0.88

Assay procedure of UMI by UV and HPLC methods:

A standard working solution of UMI (conc. 10µg/ml) and sample solution (conc. 10µg/ml) were used, and the absorbance of both solutions was measured at 223nm. Similarly, both solutions were autoinjected into HPLC. The content of UMI present in the laboratory prepared tablet was evaluated by UV and HPLC. The obtained results of the % label claim by UV and HPLC are shown in Table 12. The % contents of UMI obtained by UV and HPLC were statistically compared for the F test and T test, as shown in Tables 13 and 14, respectively.

Table 12: Results of Analysis of Tablet Formulation

Methods	Sample	Label Claim (100mg/tablet)	% Label claim estimated (% content)	Mean	% RSD
UV	Laboratory prepared Tablet	100	99.24	99.54	0.23
			99.57
			99.45
			99.51
			99.93
			99.53
HPLC	Laboratory prepared Tablet	100	99.97	99.90	0.83
			98.79
			100.87
			99.16
			100.77
			99.85

%RSD = percentage relative standard deviation

Statistical comparison between % contents and % recovery study of assay samples by UV and HPLC methods:

F Test:

Table 13: Observations and results of the Ftest for % contents and % recovery study

Parameter	Variance		F Value	P (F<=f) one tail	F critical one tail
Parameter	UV Method	HPLC Method	F Value	P (F<=f) one tail	F critical one tail
% Content	0.0504	0.6970	13.8274	0.0059	5.0503
% Recovery	0.7453	0.4650	0.6239	0.2599	0.2909

From the above F test results, it could be inferred that the % content and % recovery F values were higher than the F-critical values; hence, the null hypothesis is rejected.

T Test:

Table 14: Observations and results of T test for % contents and % recovery study

Parameter	t Stat	P(T<=t) one tail	t Critical one tail	P(T<=t) two-tail	t Critical two-tail
%Content	1.0954	0.1616	2.0150	0.3233	2.5706
%Recovery	1.6823	0.06551	1.8595	0.1310	2.3060

From the above T test results, it could be inferred that the P value is higher than the alpha value (P = 0.05). Hence the null hypothesis is accepted, i.e., there is a statistically significant difference between the mean % content and % recovery determined by UV and HPLC.

CONCLUSION:

The proposed UV and HPLC methods were found to be simple, precise, fast, robust and rugged. The % RSD was less than 2%. Our studies revealed a recovery percentage ranging from 98 to 102%, indicating that the developed methods were accurate. The method did not interfere with excipients while determining umifenovir. Its chromatographic run time of 6 minutes allows the analysis of samples in a short period. The simple UV method could be implemented in laboratories that lack high-tech analytical instruments, which are complicated, expensive and time consuming. Based on the statistical tests, the F test and the T test, it can be concluded that the HPLC method is better than the UV method for drug determination. Therefore, the developed UV method and HPLC method can be used for routine quality control of UMI in bulk and tablet formulation.

ACKNOWLEDGMENT:

The authors are grateful to Mylan Labs Ltd. (India) for providing the gift samples of UMI and to colleague Pranita Kanojiya for helping in the preparation of tablets in the laboratory.

REFERENCES:

1. Balzarini J, Ruchko EA, Zakharova EK, Kameneva IY, Nawrozkij MB. Structural analogs of umifenovir.

2. Blaising J, Polyak SJ, Pécheur EI. Arbidol as a broad-spectrum antiviral: An update. Vol. 107, Antiviral Research. Elsevier B.V.; 2014. p. 84–94.http://dx.doi.org/10.1016/j.antiviral.2014.04.006.

3. Deng P, Zhong D, Yu K, Zhang Y, Wang T, Chen X. Pharmacokinetics, metabolism, and excretion of the antiviral drug arbidol in humans. Antimicrob Agents Chemother. 2013; 57(4): 1743–55.https://doi:10.1128/AAC.02282-12

4. Nojomi M, Yassin Z, Keyvani H, Makiani MJ, Roham M, Laali A, et al. Effect of Arbidol (Umifenovir) on COVID-19: a randomized controlled trial. BMC Infect Dis. 2020; 20(1): 1–10. https://doi.org/10.1186/s12879-020-05698-w

5. Wright ZVF, Wu NC, Kadam RU, Wilson IA, Wolan DW. Structure-based optimization and synthesis of antiviral drug Arbidol analogues with significantly improved affinity to influenza hemagglutinin. Bioorganic Med Chem Lett. 2017; 27(16): 3744–8.https://doi:10.1016/j.bmcl.2017.06.074.

6. Yuya Wang, Xiaoyan Chen QL and DZ. Metabolite identification of arbidol in human urine by the study of CID fragmentation pathways using HPLC coupled with ion trap mass spectrometry. J mass Spectrom. 2008;43:1099–1109.https://doi: 10.1002/jms.1394

7. XiaoLiuXiao-HuiChenYuan-YuanZhangWen-TaoLiuKai-ShunBi. Determination of arbidol in rat plasma by HPLC–UV using cloud-point extraction. J Chromatogr B. 2007; 856(1–2): 273–7. https://doi:10.1016/j.jchromb.2007.06.009

8. Liu X, Huang YW, Li J, Li XB, Bi KS, Chen XH. Determination of arbidol in human plasma by LC-ESI-MS. J Pharm Biomed Anal. 2007; 43(1):371–5.https://doi:10.1016/j.jpba.2006.06.039

9. Annapurna MM. New stability indicating ultrafast liquid chromatographic method for the determination of umifenovir in tablets. International Journal of Green Pharmacy. 2018; 11(9): 3851–6. https://doi.org/10.22377/ijgp.v12i01.1619

10. Narendra A, Annapurna MM. Quantification of arbidol by RP-HPLC with photo diode array detection. Asian J Pharm. 2018;12(2):S553–7. https://doi.org/10.22377/ajp.v12i02.2391

11. Surabhi SR. Validated Stability Indicating Method for Determination of Umifenovir- Remdesivir in Presence of Its Degradation Products. Int J Dev Res. 2021; 11(model 2695): 46227–32.https://doi.org/10.37118/ijdr.21666.04.2021

12. Damle MC, Phadtare SA, Raskar HD, Gadge KG, Mehendre R, Bothara KG. A Validated HPTLC Method for Determination of Arbidol from Pharmaceutical Formulation. Vol. 2, International Journal of ChemTech Research CODEN( USA).

13. National Center for Biotechnology Information. PubChem Compound Summary for CID 131411, Arbidol. https://pubchem.ncbi.nlm.nih.gov/compound/Arbidol. Accessed May 6, 2023.

14. ICH Validation of Analytical Procedures: Text and Methodology Q2 (R1), International Conference on Harmonization; 2005.

Received on 19.07.2023 Modified on 05.10.2023

Research J. Pharm. and Tech 2024; 17(3):1308-1313.

DOI: 10.52711/0974-360X.2024.00205

Mobile Phase	Flow rate (ml/min)	pH	Injection Vol. (µl)	Retention time (min)	Comment
ACN: Methanol (50:50)	0.6	-	10	-	Peak did not appear
ACN: Methanol (70:30)	0.6	-	10	-	Peak did not appear
ACN: TEA (1%) (50:50)	0.6	3.2	10	9.712	Peak show Tailing
ACN: TEA(1%) (55:45)	0.8	3.2	10	8.501	Peak show Tailing
ACN: TEA (1%) (55:45)	0.8	3.0	10	4.666	Peak show Tailing
ACN: TEA (1%) (60:40)	1.0	3.0	20	3.650	Peak show Tailing
ACN: TEA (1%) (60:40)	1.0	2.7	20	3.515	Sharp peak appear with no tailing