INTRODUCTION:

Nirmatrelvir is chemically (1R,2S,5S)-N-[(1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluoroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide.¹It is a SARS- COV-2 main protease inhibitor. Ritonavir is 1,3-thiazol-5-ylmethyl N-[(2S,3S,5S)-3-hydroxy-5- [(2S)-3-methyl-2-{[methyl({[2-(propan-2-yl)-1,3-thiazol-4-yl]methyl})carbamoyl]amino}butanamido]-1,6-diphenyl hexan-2-yl]carbamate.²

Ritonavir is an antiretroviral used together with other medicines for the treatment of the infection caused by the human immunodeficiency virus(HIV). To treat corona virus disease, a combination of Nirmatrelvir and Ritonavir is administered. It prevents the growth of virus that causes COVID-19.^3,4

Fig 1(a) Chemical Structure of Nirmatrelvir

Fig 1(b) Chemical Structure of Ritonavir

For bulk and formulation, a chromatographic method for the simultaneous determination of Nirmatrelvir and Ritonavir in a combination dosage form has to be developed because there are not many available. Numerous techniques were presented for the assessment of Ritonavir and Nirmatrelvir both alone and in combination with other antiviral medications.^5-12The developed method can be validated according to ICH guidelines and applied successfully to regular tablet quality control analysis since it is rapid, inexpensive, accurate, and precise.¹³

EXPERIMENTAL:

Agilent Eclipse XDB (250x4.6mm, particle size 5µ) HPLC was used for the method. Trials with several mobile phases were used to determine the optimal conditions for the separation of Ritonavir and Nirmatrelvir. It was discovered that the best mobile phase was acetonitrile: octane sulphonic acid buffer pH 2.5 (30:70 v/v) with a flow rate of 1.0mL/min. The PDA detection was at 287nm. The sample mixture and mobile phase were filtered using a membrane filter featuring a pore size of 0.45 µm. The mobile phase was examined at room temperature and degassed before to use using an ultrasonic bath sonicator.

MATERIALS AND METHODS:

Pharmaceutical grade working standards of Nirmatrelvir and Ritonavir was obtained from Hetero Pvt.Labs. All chemicals and reagents were of HPLC grade.

Preparation of standard solution of Nirmatrelvir:

About 150mg/100mL of Nirmatrelvir was weighed and transferred into 100mL volumetric flask and the sample is dissolved with few mL of diluent(1500µg/mL). From the prepared solution, 5mL was pipetted out into 10mL volumetric flask and made upto final mark using diluent(150µg/mL).

Preparation of standard Solution of Ritonavir:

About 100mg/100mL of Ritonavir was weighed and transferred into 100mL volumetric flask and the sample is dissolved with few mL of diluent(1000µg/mL). From the prepared solution 5mL was pipetted out into 10mL volumetric flask and made upto final mark using diluent(100µg/mL).

Preparation of sample solution:

Weigh equivalent amount of 273mg of NIT and 242mg of RIT was accurately measured and solubilized in a diluents to 100mL. A solution containing 150µg/mL of NIT and 100µg/mL of RIT was prepared by diluting 5mL of resulting solution by 50mL again. Potential particulate matter from the sample solution was removed by using 0.45µm Nylon Filters.

Method development:

The HPLC procedure was optimized with a view to develop a simultaneous assay method for Nirmatrelvir and Ritonavir. 10µl of the working standard solutions for each component were injected in HPLC. Different ratios of Acetonirile: Formic acid /OSA/OPA were tested.

Method validation:

The devised procedure was validated in compliance with ICH criteria. The following validation parameters were used such as system suitability, linearity, accuracy, precision, specificity, robustness, limit of detection and limit of quantification. The linearity of the method was determined in concentration range of 37.50 – 225.50 µg/mL for Nirmatrelvir and 25 – 150µg/mL for Ritonavir. It is recommended to create a set of samples where the analyte concentrations are within the procedure's claimed ranges. Test findings should be assessed using the proper statistical techniques if a linear relationship was found. A minimum of six within (25, 50, 75, 100, 125, 150%) should be used. The linearity was evaluated by plotting responses vs concentration and calculating the correlation of coefficient. By assessing the impact of minor but intentional changes in the chromatographic conditions, robustness was investigated. The conditions studied were flow rate (altered by ±0.2mL/min) and Acetonitrile ratio in mobile phase. The resolution between Nirmatrelvir and Ritonavir as well as the percentage of drugs assay were assessed using these chromatographic variations. Injecting the standard and sample solutions at regular intervals for up to 30 minutes helped to establish the stability of the analytical solutions. The responses of standard solution and sample solution were measured and the % difference of peak area were calculated. System suitability parameters with respect to retention time, peak area, plate count, tailing factor and resolution between Nirmatrelvir and Ritonavir peaks were defined.

The method's accuracy was investigated through recovery experiments. To the pre-analyzed drug sample (Nirmatrelvir and Ritonavir bulk and formulation) known amounts of Nirmatrelvir and Ritonavir raw materials corresponding to 50, 100 and 150% of label claim were prepared by standard addition method and determined by running chromatograms in optimized chromatographic conditions. The experiment was carried out in triplicate, and percentages of recovery and RSD were computed.

RESULTS AND DISCUSSION:

Method Optimization:

The HPLC procedure was optimized with a view to develop a suitable LC method for the estimation of Nirmatrelvir and Ritonavir in fixed dose combined dosage form. Initially, acetonitrile and octane sulphonic acid in various ratios were performed. It was determined that acetonitrile: ocatne sulphonic acid pH 2.5 (30:70 v/v) gave acceptable retention time.

Method Validation:

For linearity, the working concentration ranges of Nirmatrelvir was 37-225µg/mL and Ritonavir was 25-150µg/mL. Correlation coefficient of Nirmatrelvir was 0.999 and that of Ritonavir was 0.999.

Table-1: Data of Linearity

Percentage level	Nirmatrelvir		Ritonavir
Percentage level	Conc (µg/mL)	Peak area	Conc (µg/mL)	Peak area
25%	37.50	638564	25.00	486526
50%	75.00	1257127	50.00	863053
75%	112.50	1815690	75.00	1239579
100%	150.00	2554255	100.00	1646107
125%	187.50	3192819	125.00	2022633
150%	225.50	3712383	150.00	2489159

Fig 2(a) Linearity graph of Nirmatrelvir

Fig 2(b) Linearity graph of Ritonavir

Precision was measured at the repeatability and intermediate precision levels. For repeatability, six portions of a sample solution of Nirmatrelvir and Ritonavir bulk and formulation (150mg/100mL and 100mg/100mL) were processed through the full analytical method and results were evaluated obtaining a % RSD values for NIR and RIT were 0.59 and 0.35.

Specificity was performed by injecting of the placebo to demonstrate the absence of interferences with the signals of Nirmatelvir and Ritonavir, as shown in Fig 3(a&b). On the other hand, the chromatogram of the solution of bulk and formulation with the two compounds showed clear, compact and well separated peaks of Nirmatelvir and Ritonavir. Therefore, the method was considered as specific.

Fig 3(a) Chromatogram of placebo

Fig 3(b) Chromatogram of sample

LOD, LOQ were determined based on standard deviation of Y- intercepts of the regression line. The standard deviation of Y- intercepts obtained from the replicate measurements (n=3), was substituted for standard deviation in the equation 3.3*SD/S and 10*SD/S respectively. SD is standard deviation and S is the mean of slope of the calibration curves. The limit of detection (LOD) and limit of quantification (LOQ) results are given in table no. 2. Precision of the method was done by 6 standard and sample injections and mean % assay and %RSD were calculated.

Table-2: Summary of Validation Parameters

Validation parameter		Nirmatrelvir	Ritonavir
Precision	Mean, % assay	99.7	100.4
Repeatability, N=6	%RSD	0.59	0.35
Coefficient of determination		0.999	0.999
Linearity		37.5 to 225.5	25 to 150
LOD (µg/mL)		0.45	0.3
LOQ (µg/mL)		1.5	1

Table-3: System suitability

Parameter	Nirmatrelvir	Ritonavir
RT (min)	2.312	4.238
Tailing factor	1.15	0.97
Theoretical plate count	12158	9628
Resolution	9.14

Six standard solution injections were used to assess the appropriateness of the system in accordance with ICH recommendations. Evaluation of analyte peak parameters provided high quality results (table no. 3). The results agree with those specified in USP and ICH guidelines, demonstrating that the chromatographic system is adequate and reliable.

Through recovery studies, the method's accuracy was determined. The different concentrations of solutions were injected and the recovery of the known amount of the added analyte was computed for each sample. The values of % recovery and % RSD indicating the method was found to be accurate, are listed in table no 4.

Table-4: Results of % Recovery at different levels

Drug Name	% Level	Recovered Amount (µg/Ml)	% Recovery	Mean % Recovery	Limit
Nirmatrelvir	50	74.9	99.9	99.4	100±2
	100	149.1	99.4
	150	222.9	99.1
Ritonavir	50	48.65	99.3	100.1
	100	99.58	99.6
	150	151.59	101.6

Table-5: Robustness study

Injection No.	Flow rate		M.P(org.phase)
Nirmatrelvir	Plus	Minus	Plus	Minus
1	2836528	2315618	2941157	2232789
2	2846485	2335221	2925699	2242601
3	2834147	2356583	2934583	2223154
Ritonavir
1	1868925	1554868	1959548	1359292
2	1854859	1534177	1961859	1347392
3	1861564	1549834	1951551	1361513

Robustness study was conducted by deliberate changes in flow rate and organic phase, revealed that there was no significant variation in %assay, retention time, tailing factor and resolution.

Table-6: Results of Assay

Drug	Peak Name	R.T	Area	Tailing	PC	% Assay
Nirmatrelvir	1	2.310	2556154	1.19	12175	100.1
Nirmatrelvir	2	2.308	2554423	1.21	12178	100.1
Ritonavir	1	4.213	1634571	0.97	96632	99.8
Ritonavir	2	4.215	1647890	0.95	9665	99.8

Stability studies of Nirmatelvir and Ritonavir in the analytical solution were studied within a period of 30 min and they were stable within that period. The values are presented in table no. 7

Table-7: % Degradation data

Type of degradation	Nirmatrelvir		Ritonavir
Type of degradation	Area	% Degradation	Area	% Degradation
Control	2552669	0	1644002	0
Acid	2209624	13.5	1455241	11.5
Alkali	2228027	12.8	1426147	13.3
Peroxide	2167129	15.1	1401625	14.8
Thermal	2443757	4.3	1491462	3.2
Photo	2497264	2.2	1595417	3

CONCLUSION:

A more recent, precise and focused RP-HPLC technique has been developed to measure Nirmatelvir and Ritonavir in bulk and formulation. ICH regulations were followed in the validation of the development procedure. Consequently, the method can be used for routine simultaneous estimation of Nirmatelvir and Ritonavir in bulk and formulation.

ACKNOWLEDGEMENT:

We wish to take this opportunity to express our deep sense of gratitude and profound indebtedness to School of Pharmacy, Anurag University, Hyderabad for providing necessary facilities to carry out the research work.

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Received on 20.02.2024 Revised on 07.06.2024

Accepted on 13.08.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):594-598.

DOI: 10.52711/0974-360X.2025.00088

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