INTRODUCTION:

Analytical method development, regulated by the ICH Q14 standards, is the application of new or modified analytical procedures used for stability and allow testing of commercial pharmaceutical compounds and products. The chemical definition of nimatrelvir is (1R,2s,5s)-N-[(1s)-1-cyano-2 (3S)-2-oxopyrrolidin-3-yl] ethyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2 trifluoro acetyl) amino] butanoyl]-6,6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide.

The first part is Nimatrelvir, which is a combination of the peptidomimetic inhibitor of main protease and primary protease of SARS-CoV-2. By blocking main protease, the virus is unable to digest the precursors of polyproteins needed for viral replication. Nimatrelvir is efficacious against both major and omicron forms of SARS CoV-2. For Nirmatrelvir, plasma protein binding (PPB) is 69%. Human cytochrome CYP3A4 substrate Nimatrelvir has a negligible metabolic clearance when combined with ritonavir¹.A literature analysis indicates that the availability of methods employing UV-spectroscopic techniques to obtain Nirmatrelvir is limited. Many methods focus on estimating the levels of Ritonavir and Nirmatrelvir in pharmaceutical dose forms and bulk. The simultaneous measurement of Nirmatrelvir and Ritonavir using LC-MS/MS, HPLC-DAD, and HPTLC, respectively, is not widely available. Also, there are some methods that are developed by using highly concentrated buffers, acids, long run time, high temperature and a large number of solvents. Therefore, the developed methods are simple, economical and specified for Nirmatrelvir². Figure 1 and Table 1 display Nirmatrelvir's chemical structure and chemical profile, respectively.

Table 1. Drug profile of Nirmatrelvir

Attribute	Description
Name of drug	Nirmatrelvir
Functional category	Antiviral medication
Melting point	192.9℃
Molecular formula	C23H32F3N5O4
Molecular weight	499.535gm/mol
Chemical name	(1R,2s,5s)-N-[(1s)-1-cyano-2 (3S)-2-oxopyrrolidin-3-yl] ethyl]-3-[(2S)-3,3-dimethyl-2- [(2,2,2 trifluoro acetyl) amino] butanoyl]-6,6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxamide
Solubility	Methanol

Figure 1. Chemical structure of Nirmatrelvir

MATERIALS AND METHODS:

Materials

Chemicals:

The free sample of Nirmatrelvir was received from Cipla Pvt. Ltd. Analytical grade solvents were used in the analysis of the drug.

Instruments:

The UV spectrophotometer utilised was a Shimadzu UV-1800 which records the UV spectra of the drug, which is a double beam spectrophotometer. The materials were weighed by using an electronic analytical balance. The instrument used for the analysis of the drug by liquid chromatography was the Jasco Extrema LC System-4000, and the results were analysed through the ChromNav software.

Methods:

The drug's authenticity was confirmed via FTIR spectroscopy and melting point analysis, matching Nirmatrelvir’s known data.³.

Development of UV-visible spectrophotometric method:

The previous study indicated the development of analytical techniques, including UV-visible spectrophotometry and HPLC, to estimate Nirmatrelvir in bulk pharmaceuticals. However, as few UV spectrophotometric techniques were developed for medicinal formulations, this work aims to design and verify a UV spectrophotometer for its determination.

a) Selection of diluent: A 70:30 methanol-water mixture was used, ensuring good solubility and well-resolved peaks while minimizing methanol usage.

b) Preparation of standard stock solution: Weigh out 100mg of Nirmatrelvir and pour it into a 100ml volumetric flask to create the standard stock solution. To fully dissolve the medicine, add 20ml of diluent, shake for two minutes, or sonicate, and then dilute with diluent to the required volume. As necessary, more dilutions were prepared.

c) Determination of wavelength: A Shimadzu UV-1800 spectrophotometer scanned over 200–400nm to establish the optimal wavelength for detection⁴.

d) Method validation: UV-visible spectrophotometric validation followed ICH standards, for assessing linearity, accuracy, precision, LOD, and LOQ⁵.

Development of RP-HPLC method:

a) Optimization of Mobile Phase:

The ideal composition for a well-resolved, sharp, and symmetric peak was optimized by varying the Acetonitrile: Water ratio (50:50, 40:60, 55:45, 60:40, 70:30) and adjusting pH. Separation was achieved using isocratic elution with a 55:45v/v acetonitrile: water mixture at pH 7.0.

b) Optimization of flow rate:

Analysis time and separation efficiency are impacted by the flow rate of the mobile phase. A steady 1 ml/min flow ensured well-resolved peaks with an optimal retention time, balancing flow rate and column size.

c) Optimization of chromatographic conditions:

Several mobile phase trials were conducted in order to determine the optimal composition for generating a well-resolved, sharp, and symmetric peak. A 0.45 µ membrane filter was used to filter the mixture, which was then degassed before use. Acetonitrile and water (55:45 v/v) were used to achieve separation in isocratic elution mode. Ten minutes of run time, a 10 µl injection volume, and a 1 ml/min flow rate were employed. For isocratic elution, an Inertsil ODS 3V (250x4.6mm, 5μ) column was used at 25°C. The optimized chromatographic conditions for the analysis of drug are shown in Figure 2.

Figure 2. Chromatogram of standard

d) Validation of Method:

The process was validated in accordance with ICH criteria, taking into consideration a number of factors such as robustness, linearity, range, accuracy, precision, LOD, and LOQ⁶.

Forced degradation studies:

A forced degradation study of Nirmatrelvir was carried out in accordance with ICH recommendations under acid, base, peroxide, thermal, and photolytic conditions to evaluate API stability and the specificity of stability-indicating techniques. All experiments were carried out in complete darkness to avoid light-induced effects.

a) Acid-induced degradation: Ten milligrammes of Nirmatrelvir were dissolved in five millilitres of diluent, and then one millilitre of 1N HCl was added. The mixture then refluxed for a day at room temperature. After 24 hours, the acid was neutralised using 1N NaOH, and the volume was completed to 10 ml with diluent.

b) Base-induced degradation: Ten milligrammes of Nirmatrelvir were dissolved in five millilitres of diluent, and then one millilitre of 1N NaOH was added. The mixture then refluxed for a day at room temperature. After 24 hours, the base was neutralised using 1N HCl, and the volume was completed to 10 ml with diluent.

c) Hydrogen peroxide-induced degradation:

One millilitre of 3% hydrogen peroxide was added after ten milligrammes of Nirmatrelvir had been dissolved in five millilitres of diluent. Ten millilitres of the solution were prepared and allowed to sit at room temperature.

d) Photolytic degradation: In a UV cabinet, nirmatrelvir was exposed to visible UV light for six hours. To achieve the necessary concentration, the degraded samples were diluted with diluent once they had cooled to room temperature.

e) Thermal degradation: For six hours, nirmatrelvir was placed in oven at 80°C. To get the required concentration, 10mg of the material was weighed and diluted in diluent after cooling^7-17.

RESULTS:

UV-visible spectrophotometry:

The development of an innovative straightforward UV-visible spectroscopic technique turned out to be successful in bulk Nirmatrelvir assessment. The UV-visible spectrum of Nirmatrelvir is depicted in Figure 3.

Table 2: Results of linearity By Spectroscopy

Concentration (µg/ml)	Absorbance
10	0.102
20	0.275
30	0.449
40	0.638
50	0.829
60	0.994
Regression coefficient (r²)	0.9996
Equation	y = 0.018x - 0.0833

Figure 3. UV spectrum of Nirmatrelvir

a) Linearity:

To confirm the method's linearity, a range of dilutions, from 10 to 60 µg/ml, were made and their absorbance examined. Figure 4 illustrates the plotting of the calibration curve between concentration values on the x- and absorbance values on the y-axes. Table 2 outlines the results. The correlation coefficient for the linear curve obtained for standard preparations of Nirmatrelvir was found to be 0.9996 and the overlay spectrum of Nirmatrelvir is shown in Figure.

b) Precision:

Reanalysing the same sample allowed us to assess the reliability of the UV-visible spectrophotometric approach. evaluating the same sample on the same day revealed that the intra-day precision which was determined by evaluating the same sample across a day showed acceptable variability, or 0.24%. There was a relative deviation from the baseline of 0.25% for the inter-day precision, which is less than 2%. Table 3 and Table 4 show the precision results.

Table 3: Intra-day precision by UV Spectroscopy

Concentration (µg/ml)	Sample absorbance	Calculated concentration (µg/ml)	S. D	% RSD
60	0.968	59.1	0.14	0.24%
60	0.971	59.2
60	0.972	59.3
60	0.975	59.5
60	0.973	59.4
60	0.970	59.2
Mean	0.971	59.28

Table 4: Inter-day precision by UV Spectroscopy

Concentration (µg/ml)	Sample absorbance	Calculated concentration (µg/ml)	S. D	% RSD
60	0.967	59.1	0.15	0.25%
60	0.966	59.0
60	0.968	59.4
60	0.972	59.3
60	0.970	59.2
60	0.971	59.3
Mean	0.969	59.21

c) Accuracy:

Analyte concentrations were introduced into different matrix types in recovery experiments to determine the accuracy of the UV-visible spectrophotometric technique. The average recovery values ranged from 98% to 102%, indicating a satisfactory level of accuracy. The accuracy results are displayed in table 5.

Table 5: Nirmatrelvir accuracy results by UV

Level (%)	Sample conc (µg/ml)	Standard conc (µg/ml)	Total conc (µg/ml)	Absorbance	Calc. conc. (µg/ml)	Mean	% Recovery	S. D	% RSD
80%	11	8.8	19.8	0.361	19.6	19.7	99.4 %	0.07	0.35
	11	8.8	19.8	0.363	19.8
	11	8.8	19.8	0.364	19.8
100%	11	11	22	0.397	22.03	22.05	100.2 %	0.02	0.09
	11	11	22	0.397	22.03
	11	11	22	0.398	22.09
120%	11	13.2	24.2	0.432	24.3	24.3	100.4 %	0.05	0.2
	11	13.2	24.2	0.435	24.5
	11	13.2	24.2	0.433	24.3

d) Detection limit and Quantification limit:

The limits of detection (LOD) and quantitation (LOQ) were established using the slope of the regression and the standard deviation of the observations. The respective findings are 2.4 µg/ml and 7.3 µg/ml.

RP-HPLC

a) Linearity:

Constructing a curve for calibration using standard solutions at known concentrations enabled us to evaluate the linearity of the HPLC technique. In concentration range between 10 to 60µg/ml, Nirmatrelvir exhibited a good correlation coefficient (R² = 0.9997) for HPLC. There was no significant difference observed in the standard curve. Table 6 and Figures 6 and 7 reveal how linear the method is. This indicates that the target analyte can be precisely quantified using the HPLC technique across a broad concentration range.

Figure 4. Calibration plot of Nirmatrelvir by RP-HPLC

Table 6: Linearity results of Nirmatrelvir by RP-HPLC

Concentration	Peak Area
10	932346
20	1793159
30	2731463
40	3530488
50	4442130
60	5321608

Figure 5. Linearity chromatograms of Nirmatrelvir by RP-HPLC

b) Precision:

Analysing the same sample repeatedly allowed for the evaluation of the HPLC technique's precision. After analysing many aliquots of the same sample on the same day, it was found that the intra-day precision had a relative standard deviation (RSD) of less than 2%. As determined by testing the same sample over multiple days, the inter-day precision also showed acceptable variation, with an RSD < 2%. Tables 7 and 8 display the precision results. These findings demonstrate the method's great precision and reproducibility, confirming accurate and consistent observations.

Table 7: Intra-day precision results by RP-HPLC

Concentration (µg/ml)	Peak area	Calculated concentration (µg/ml)	S. D	% RSD
50	4654661	51.4	0.49	0.95%
50	4612629	51.2
50	4594098	51.2
50	4651871	51.5
50	4502026	51.7
50	4537406	51.1
Mean	4592115	51.1

Table 8: Inter-day precision results by RP-HPLC

Concentration (µg/ml)	Peak area	Calculated concentration (µg/ml)	S. D	% RSD
50	4423630	49.8	0.089	0.17%
50	4495797	50.6
50	4527594	50.5
50	4496697	50.6
50	4497897	50.6
50	4498783	50.6
Mean	4490066	50.45

c) Accuracy:

In recovery tests, analyte concentrations are injected into different matrices to assess the HPLC technique's accuracy. The average recovery values ranged from 98% to 102%, indicating a satisfactory level of accuracy. These findings imply that matrix interferences do not considerably impact the HPLC method, which can yield accurate and dependable results for a wide range of sample types. Table 9 presents the findings.

Table 9: Results of Nirmatrelvir accuracy (n=3±SD) by RP-HPLC

Level (%)	Sample conc (µg/ml)	Standard conc (µg/ml)	Total conc (µg/ml)	Peak area	Mean peak area	Cal. conc (µg/ml)	Mean conc (µg/ml)	% Recovery	Mean % recovery	S. D	% RSD
	10	8	18	1645854	1645855	18.1	18.1	101.6	101.6	0.07	0.38
80%	10	8	18	1645857		18.1		101.6
	10	8	18	1645855		18.1		101.6
	10	10	20	1825896	1825540	20.1	20.1	101.8	101.7	0.03	0.14
100%	10	10	20	1825459		20.1		101.7
	10	10	20	1825265		20.1		101.4
	10	12	22	1987562	1976605	22.0	21.8	100.2	99.3	0.08	0.36
120%	10	12	22	1954799		21.6		97.1
	10	12	22	1987456		22.02		100.2

Table 10: Robustness evaluation by RP-HPLC

Parameters	Set Value	Factors	1	2	3	Mean	S. D	%RSD
Flow rate 1 ±0.2 ml/min	0.80ml/min	Area	136042	135621	137421	136361.3	941.53	0.69
		RT	4.65	4.68	4.74	4.69	0.045	0.97
		NTP	4360	4364	4378	4367.3	9.4	0.21
	1.2 ml/min	Area	1268661	1268451	1268551	1268554	105.03	0.08
		RT	4.41	4.45	4.3	4.38	0.077	1.77
		NTP	4550	4580	4695	4608.3	76.5	1.66
Temperature 25 ± 5 ⁰C	20 ⁰C	Area	158562	158624	162241	159809	2106.40	1.3
		RT	4.531	4.53	4.53	4.53	0.0005	0.01
		NTP	4895	4954	4975	4941.3	41.47	0.83
	30 ⁰C	Area	1230599	1268661	1269561	1256274	22239.47	1.77
		RT	4.5	4.52	4.52	4.51	0.01	0.25
		NTP	4968	4855	4995	4939.3	74.2	1.5
Wavelength 220 ± 5 nm	215 nm	Area	1761916	1761504	1761981	1761800	258.68	0.01
		RT	4.532	4.53	4.53	4.53	0.001	0.02
		NTP	4358	4381	4396	4378.3	19.13	0.43
	225 nm	Area	1382039	1389045	1396587	1389224	7275.64	0.52
		RT	4.4	4.4	4.4	4.4	0.004	0.09
		NTP	4621	4657	4698	4658.6	38.52	0.8

Table 11: Forced degradation of Nirmatrelvir by RP-HPLC

Sr No.	Stress condition	Time (h)	% degradation	Retention time of degradation product
1.	Acid induced degradation	24 hours at dark room temperature	6.15 %	2.83 and 4.05 min
2.	Base induced degradation	24 hours at dark room temperature	5.8 %	2.49 and 4.04 min
3.	Hydrogen peroxide induced degradation	1 hour at dark room temperature	4.6 %	2.12 and 3.45 min
4.	Photolytic degradation	6 hours in UV cabinet	1.23%	4.0 min
5.	Thermal degradation	6 hours at 80℃	2%	Did not exhibit an additional peak

Forced degradation studies of Nirmatrelvir:

Forced degradation studies using HPLC revealed that Nirmatrelvir degrades under acidic and basic conditions but remains stable under thermal, photolytic, and oxidative conditions. These findings, illustrated in Table 11, aid in determining suitable storage conditions.

Figure 6: Chromatogram of Nirmatrelvir and its acid degradation products

Figure 7: Chromatogram of Nirmatrelvir and the products of its base degradation

Figure 8: Chromatogram of Nirmatrelvir and the products of its oxidative degradation

Figure 9: Chromatogram of Nirmatrelvir and product of its photolytic degradation

Figure 10: Chromatogram of Nirmatrelvir and product of its thermal degradation

DISCUSSION:

Nirmatrelvir's technique development and validation utilising RP-HPLC and UV-visible spectrophotometry was completed satisfactorily. The ICH criteria were followed when executing each validation parameter. For UV- vis spectrophotometry, the method was developed by determining the appropriate wavelength. The results received from the execution of all validation parameters fall within the specified range. In order to obtain suitable chromatographic conditions for the medication, multiple trials were conducted before developing the RP-HPLC method. The results obtained after performing validation parameters were found to be satisfactory. The force degradation studies were performed under various stress conditions to determine the characteristics of the drug. The degradation studies were carried out under acidic, basic, oxidative, photolytic and thermal conditions. The information obtained from the force degradation study was helpful to determine the intrinsic stability of the drug as well as it will help to maintaining the storage conditions for the drug.

CONCLUSION:

In accordance with ICH guidelines, an innovative, straightforward, accurate, and reliable UV-spectroscopic and RP-HPLC method has been designed and verified. In a literature survey, it was found that the development of a method for Nirmatrelvir was done by using gradient mode and buffers in higher concentrations. But in the present study, the method was developed by using isocratic elution mode, and the mobile phase was free from the buffers. The new approach can extract the drug from its acidic, basic, and oxidative products of degradation and can give valuable insights into the possible degradation products that may arise during drug storage. Hence, understanding chemical behaviour may be applied to enhance the quality of medicinal products, thereby facilitating pharmaceutical development in areas including formulation development, manufacturing, and packaging. The method has been successfully applied to carry out both accelerated and long-term stability studies of Nirmatrelvir. The study indirectly emphasizes the benefit of using a stress testing technique to identify the stability of the drug.

CONFLICT OF INTEREST:

There are no conflicts of interest pertaining to this inquiry for the authors.

REFERENCES:

1. Rai DK, Yurgelonis I, McMonagle P, Rothan HA, Hao L, Gribenko A, Titova E, Kreiswirth B, White KM, Zhu Y, Anderson AS. Nirmatrelvir, an orally active Mpro inhibitor, is a potent inhibitor of SARS-CoV-2 Variants of Concern. BioRxiv. 2022; Jan 19: 2022-01. doi: https://doi.org/10.1101/2022.01.17.476644.

2. Ilayaraja P, Manivannan M, Parthiban P. Novel stability indicating HPLC method for the quantification of Nirmatrelvir in bulk drugs. Microchemical Journal. 2024; 196: 109707. doi: https://doi.org/10.1016/j.microc.2023.109707.

3. Reyes VM, Martínez O, Hernández GF. National center for biotechnology information. Plant Breeding. Universidad Autonoma Agraria Antonio Narro, Calzada Antonio Narro. 1923.

4. Sisubalan AP, Manickam C, Ramaswamy V, Natesan S. analytical method development and validation for simultaneous estimation of nirmatrelvir and ritonavir in pharmaceutical dosage form by RP-HPLC. 2024. doi: 10.20959/wjpr20249-32174.

5. European Medicines Agency. "ICH Guideline Q2 (R2) on Validation of Analytical Procedures." (2022).

6. Alegete P, Byreddy S. Development of a novel quality by design–enabled stability‐indicating HPLC method and its validation for the quantification of nirmatrelvir in bulk and pharmaceutical dosage forms. Biomedical Chromatography. 2024; 38(3): 5812. doi: 10.1002/bmc.5812.

7. Imam MS, Batubara AS, Gamal M, Abdelazim AH, Almrasy AA, Ramzy S. Adjusted green HPLC determination of nirmatrelvir and ritonavir in the new FDA approved co-packaged pharmaceutical dosage using supported computational calculations. Scientific reports. 2023; 13(1): 137. doi: https://doi.org/10.1038/s41598-022-26944-y.

8. Sutar SV, Yeligar VC, Patil SS. Stability Indicating Forced Degradation Studies. Research Journal of Pharmacy and Technology. 2019; 12(1): 429-33. doi. http://dx.doi.org/10.5958/0974-360X.2019.00078.7.

9. Sonia K, Nappinnai M, Panneerselvam P. Stability indicating method development and Validation of Telmisartan and Ramipril by UV Spectrophotometry. Research Journal of Pharmacy and Technology. 2018; 11(7): 3087-90. doi: 10.5958/0974-360X.2018.00567.

10. Naga Venkata Indira Devi Jajula, A. Krishnamanjari Pawar. Development and Validation of Stability Indicating Method for Simultaneous Estimation of Paritaprevir, Ombitasvir, and Ritonavir in Tablet Dosage Form. Research Journal of Pharmacy and Technology 2023; 16(5): 2306-0. doi: 10.52711/0974-360X.2023.00379

11. M. R. Ghante, R. B. Tangade, S. D. Sawant, P. D. Kulkarni , V. K. Bhusari. Development and Validation of stability indicating RP-HPLC method for the estimation of Ertugliflozin by forced degradation studies. Research Journal of Pharmacy and Technology. 2022; 15(11): 4945-9. doi: 10.52711/0974-360X.2022.00831

12. Thoddi P, Gautam GK, BH C. Stability indicating validated RP-HPLC method for Simultaneous estimation of Elbasvir and Grazopravir in Bulk and Pharmaceutical Dosage form. Research Journal of Pharmacy and Technology. 2020; 13(2): 950-5. doi: 10.5958/0974-360X.2020.00179.1

13. Manasa M, Aanandhi VM. Stability indicating method development and validation of semaglutide by RP-HPLC in pharmaceutical substance and pharmaceutical product. Research Journal Of Pharmacy And Technology. 2021; 14(3): 1385-9. doi: 10.5958/0974-360X.2021.00247.X

14. Gopal Prasad Agrawal. Validated Stability Indicating Method for Determination of Indapamide in Pharmaceutical Formulation. Research Journal of Pharmacy and Technology. 2021; 14(6): 3347-2. doi: 10.52711/0974-360X.2021.00582

15. Patel K, Verriboina SK, Vasantharaju SG. Stability indicating assay method development and validation of simultaneous estimation of chlorzoxazone, diclofenac sodium and paracetamol in bulk drug and tablet by RP-HPLC. Research Journal of Pharmacy and Technology. 2021; 14(9): 5024-8. doi: 10.52711/0974-360X.2021.00876

16. Kishore Konam, Somasekhar Reddy Kanala. A Stability Indicating Method Development and Validation for The Simultaneous Estimation of Emtricitabine, Tenofovir Alafenamide and Doultegravir in Pharmaceutical Formulation by Ultra Performance Liquid Chromatography. Research Journal of Pharmacy and Technology. 2021; 14(11): 6017-4. doi: 10.52711/0974-360X.2021.01046

17. A. Indira, N. Y. Sreedhar, D. Balakrishna. A Stability Indicating Method Development of Lopinavir and Rotinavir in Combined Tablet Dosage Forms by RP-HPLC. Research Journal of Pharmacy and Technology. 2022; 15(2): 661-4. doi: 10.52711/0974-360X.2022.00109

Received on 04.12.2024 Revised on 21.02.2025

Accepted on 30.04.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):439-445.

DOI: 10.52711/0974-360X.2026.00064

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