INTRODUCTION:

Nirmatrelvir¹ chemically known as(1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azobicyclo[3.1.0]hexane-2-carboxamide is an antiviral medication which acts as an orally active 3C-like protease inhibitor. Nirmatrelvir is a white to pale colored powder with practical solubility in methyl isobutyl ketone, 1-butanol and isopropyl acetate, sparingly soluble in anisole, n-proply acetate, n-butyl acetate and insoluble in heptane. It has Log P value 2.12 and pKa 7.1. Nirmatrelvir/ritonavir, sold under the brand name, Paxlovid, is a co-packaged medication used in treatment COVID-19.

Ritonavir², 1,3-thiazol-5-ylmethylN-[(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-diphenylhexan-2-yl]carbamate and sold under the brand name norvir, is an antiretroviral medication used along with other medications to treat HIV/AIDS. Ritonavir is white to light tan powder. Ritonavir is practically insoluble in water, freely soluble in methanol and ethanol, soluble in isopropanol. Ritonavir is a weakly basic drug with a pH-dependent solubility. It has Log P value 3.9 and pKa 6.27.

Several methods^2-6 have been reported to quantify nirmatrelvir and ritonavir as separate entities but only few methods were reported for determining both the drugs in combination samples and these procedures include thin layer chromatography⁷ (TLC), high performance liquid chromatography^8,9 (HPLC), and liquid chromatography- mass spectrometry^10,11(LC-MS/MS). To the best of our knowledge, no UPLC methodologies for the simultaneous determination of nirmatrelvir and ritonavir have been described. The current research work explains the develpoment and validation of RP-UPLC method for the simultaneous determination of nirmatrelvir and ritonavir in a copackaged tablet formulation as per ICH^12-14 recommendations.

Name

Molecular formula

Molecular weight

(g/mol.)

Nirmatrelvir

C₂₃H₃₂F₃N₅O₄

499.53

Name

Molecular formula

Molecular weight

(g/mol.)

Ritonavir

C₃₇H₄₈N₆O₅S₂

720.94

Table 1: Chemical structures of Nirmatrelvir and Ritonavir

MATERIALS AND METHODS

Chemicals and reagents

Acetonitrile, Triethyl amine (TEA, 0.1 % v/v) and water were supplied by Merk (India) Ltd.Worli, Mumbai, India (UPLC grade). The active pharmaceutical ingredients for nirmatrelvir and ritonavir that served as reference standards (99.9 % w/w) were provided by Zydus Cadila, Ahmedabad, India as gift samples. Marketed formulation, Paxlovid (Pfizer Limited) (labeled claim, nirmatrelvir-150 mg, ritonavir- 100 mg) was purchased from licensed pharmacy.

Instrumentation

For UPLC analysis, an agilent 1290 infinity II LC system, a system with a quaternary pump, and a photodiode array detector, semi micro balance (sartorius), pH meter (eutech), ultra sonicator (UCA 701, unichrome), constant temperature water bath (remi) and 0.45µ membrane filters were used. The data was collected and the peak purity was verified using the empower 2.0 program.

Method development

To optimize the chromatographic conditions, different ratios of TEA (0.1 % v/v) and acetonitrile as mobile phase with isocratic and gradient mode were tested. However the mobile phase composition was modified at each trial to enhance the resolution and also to achieve acceptable retention times. Finally TEA (0.1 % v/v) and acetonitrile with isocractic elution was selected because it resulted in a greater response of active ingredients. During the optimization of the method various stationary phases such as C₈, C₁₈ and amino, phenyl columns were tested. From these trials the peak shapes were relatively good with phenyl column of 100 x 2.1 mm, 1.7 µ with a PDA detector. The mobile phase flow rate was altered for effective separation.

Selection of wavelength

An attempt was made to improve the sensitivity of the method by scanning 25 µg/mL concentrated solutions prepared from dilution of selected drugs using several detection wavelengths ranging from 200 to 400 nm in PDA detector. For all the components tested, the wavelength of 267 nm proved to be the most sensitive.

Selection of chromatographic conditions

Initial trials were conducted by injecting the diluted standard solutions of analyte to get the optimized chromatographic conditions by changing various solvents and solvent compositions. The effective separation and consistent peak shapes were achieved by changing different columns and flow rate was adjusted to get good peak resolution and shape.

Optimised chromatographic conditions

Chromatographic separations of the selected drugs were perormed on Agilent 1290 Infinity II LC System, phenyl (100 x 2.1mm, 1.7 µ) column with a mobile phase consisting of acetonitrile : TEA (0.1 % v/v) in the ratio of 30:70 v/v, pumped at a flow rate of 0.5 mL/min. and detection wavelength of 267 nm was set at room temperature, the injection volume of 5 µL and 5 min. run time for effective simultaneous separation of selected drugs were set.

Table 2: Optimized chromatographic conditions

Column		Phenyl (100 x 2.1 mm, 1.7 µ)
Column temperature		25 °C
Mobile phase		Acetonitrile : TEA (0.1 % v/v) (30 : 70)
Flow rate		0.5 mL/min.
Injection volume		5 µL
Elution mode		Isocratic
Wave length		267 nm
Retention Time (Rt)	Nirmatrelvir	1.262 min.
	Ritonavir	1.873 min
Run time		5 min.

Preparation of TEA (0.1 % v/v)

1 mL of TEA (0.1 % v/v) was transferred into a 1000 mL volumetric flask, made up to the volume with HPLC water, filtered through a 0.45µ membrane filter and degassed.

Diluent: Mobile phase

Preparation of the standard stock solution

Nirmatrelvir (150 mg) and ritonavir (100 mg) were accurately weighed and transferred into a 100 mL volumetric flask, sonicated for 20 min. and made up the volume with diluent. It was filtered through a 0.45 μ membrane filter.

Preparation of the sample solution

20 tablets from each sample of nirmatrelvir and ritonavir were accurately weighed and triturated to get a fine powder. An equivalent to average weight nearly 273 mg of nirmatrelvir sample and 242 mg ritonavir was transferred into a 100 mL volumetric flask, sonicated for 20 min. and made up the volume with diluent. The solution was then filtered through 0.45 μ membrane filter.

Assay of Formulation

5 mL of standard stock solution and sample stock solutions were transferred separately into 50 mL volumetric flasks, diluted and made up to final volume with diluent, the resulting solutions were sonicated for about 20 min. and filtered through 0.45 μ membrane filter.

Method validation

Validation of the optimized method parameters includes linearity, system suitability, accuracy, precision, robustness, limit of detection and limit of quantification according to ICH guidelines.

System suitability

The system suitability test was carried out by injecting six replicate injections of a standard solution containing 150 µg/mL of nirmatrelvir and 100 µg/mL of ritonavir into the UPLC system. The system suitability parameters were evaluated from chromatograms obtained, calculated the % RSD of retention times, tailing factor, theoretical plates and peak areas as below:

Table 3: System suitability

Parameter	Nirmatrelvir	Ritonavir
Retention time (min.)	1.262	1.873
Peak area	2085128	1385301
Theoretical plates	7138	5856
Tailing factor	1.06	1.15
Resolution	-	3.33
% RSD	0.295	0.339

Specificity

In this test method placebo, sample and standard solutions were analyzed individually to examine the interference. The below figures (Fig. 1a to 1c) shows that the active ingredients were well separated from blank and their excipients and there was no interference of placebo with the principal peak. Hence the method was found to be specific.

Fig. 1a: Chromatogram of blank

Fig. 1b: Chromatogram of standard

Fig. 1c: Chromatogram of sample

Linearity

The linearity of the developed method was determined by preparing the aliquots of standard drug solutions with concentrations of 37.5-225 µg/mL of nirmatrelvir and 25-150 µg/mL of ritonavir from the standard stock solution. The linearity parameters like regression value, slope, y-intercept were evaluated by plotting the calibration curves (Fig. 2) taking concentration in µg/mL on x-axis, and peak area on y-axis for linearity concentrations. The correlation coefficients achieved were greater than 0.9990.

Table 4: Linearity data

S. No	Conc. (µg/mL)	Nirmatrelvir area count	Conc. (µg/mL)	Ritonavir area count
1	37.50	587392	25.00	368414
2	75.00	1073025	50.00	679383
3	112.50	1521134	75.00	1039478
4	150.00	2084502	100.00	1381917
5	187.50	2560044	125.00	1710677
6	225.00	3043429	150.00	2029838
R²		0.99956		0.99982
Slope		13416.26		13537.96
Intercept		43460.71		14610.93

Fig. 2: Calibration plots of (A) Nirmatrelvir (B) Ritonavir

Accuracy

In this method, accuracy was conducted in triplicate by analyzing active pharma ingredient sample solution at three different concentration levels of 50 %, 100 % and 150 % of each at a specified limit. Percentage recoveries were measured and found to be within the limit. The accuracy and reliability of the developed method were established. The results are given in table 5.

Table 5 : Results of Accuracy

Level (%)	Nirmatrelvir
Level (%)	Conc. recovered (µg/mL)	Recovery (%), % RSD
50	74.87	99.8, 1.57
100	150.49	100.3, 0.37
150	225.34	100.2, 0.61

Level (%)	Ritonavir
Level (%)	Conc. recovered (µg/mL)	Recovery (%), % RSD
50	49.60	99.3, 0.32
100	100.09	100.1, 0.59
150	148.90	99.3, 0.67

Precision

In method precision study, six different samples (nirmatrelvir (150 µg/mL) and ritonavir (100 µg/mL)) were prepared and injected into the UPLC system.

Intraday precision:

Six replicates of a sample solution containing nirmatrelvir (150 µg/mL) and ritonavir (100 µg/mL) were analysed on the same day. Peak areas were calculated, which were used to calculate mean, SD and % RSD values. Method Precision results are given below in table 6.

Inter-day precision (Intermediate precision):

In this six replicates of a sample solution containing nirmatrelvir (150 µg/mL) and ritonavir (100 µg/mL) were analysed on different days. Peak areas were calculated which were used to calculate mean, SD and % RSD values. The present method was found to be precise as the RSD values were less than 2 %. Intermediate Precision results were given below in table 6.

Table 6: Results of Precision study

Parameter	Nirmatrelvir	Ritonavir
Parameter	Assay ± SD, % RSD	Assay ± SD, % RSD
Method precision	99.5 ± 0.627 , 0.63	99.7 ± 0.634 , 0.64
Intermediate Precision (Day-1)	99.5 ± 0.519 , 0.52	100.2 ± 0.591 , 0.59
Intermediate Precision (Day-2)	99.8 ± 0.624 , 0.85	100.7 ± 0.563 , 0.64

LOD and LOQ:

The LOD and LOQ concentrations for nirmatrelvir and ritonavir were calculated from the signal to noise (s/n) ratio.

Table 7: LOD and LOQ

Nirmatrelvir				Ritonavir
LOD		LOQ		LOD		LOQ
Conc.	s/n	Conc.	s/n	Conc.	s/n	Conc.	s/n
0.45 µg/mL	3	1.5 µg/mL	10	0.3 µg/mL	3	1 µg/mL	10

Robustness

The conditions of the experiment were designed to test the robustness of established system by altering the chromatographic conditions such as flow rate and mobile phase organic percentage in all these varied conditions. Robustness results for nirmatrelvir and ritonavir were found to be within the limit and results are given in Table 8.

Table 8: Robustness data

Parameter	Nirmatrelvir		Ritonavir
Parameter	Assay ± SD	% RSD	Assay ± SD	% RSD
Flow rate (0.45 mL/min.)	100.2 ± 1.054	1.05	100.5 ± 0.907	0.9
Flow rate (0.55 mL/min.)	99.7 ± 0.802	0.8	100.5 ± 1.258	1.25
Mobile phase (27:73)	99.9 ± 0.929	0.93	100.5 ± 0.917	0.91
Mobile phase (33:67)	100.9 ± 1.153	1.14	100.1 ± 1.050	1.05

Degradation studies

The nirmatrelvir and ritonavir sample was subjected to various forced degradation conditions to effect partial degradation of the drug. Studies of forced degradation were carried out to find out the suitability of the method in stability studies and also to establish the specificity in the presence of degradants. In addition, the studies provide details about the conditions during which the drug is unstable, in order that the measures are often taken during formulation to avoid potential instabilities.

Acid degradation:

1 mL of the sample stock solution was transferred to a volumetric flask (10 mL), added 1 mL of 1N HCl and left it for 15 min. This solution was then neutralized with 1N NaOH and made up to the mark. This solution was filtered using syringe filter and injected into the UPLC system. The obtained chromatogram is shown in Fig.3a.

Alkali degradation:

1 mL of sample stock solution was transferred to a volumetric flask (10 mL), added 1 mL of 1N NaOH and left it for 15 min. This solution was then neutralized with 1 mL of 1N HCl and made up to the mark with the diluent. This solution was filtered using syringe filter and injected into the UPLC system. The obtained chromatogram is shown in Fig.3b.

Hydrolysis degradation:

1mL of sample stock solution was transferred to a volumetric flask (10 mL), added 1mL of HPLC water and made up to the mark with diluent. The solution was filtered using syringe filter and injected into the UPLC system. The obtained chromatogram is shown in Fig.3c.

Peroxide degradation:

1 mL of sample stock solution was transferred to a volumetric flask (10 mL). 1 mL of 30 % (v/v) hydrogen peroxide solution was added, made up to 10 mL with diluent, filtered using syringe filter and injected into the UPLC system. The obtained chromatogram is shown in Fig.3d.

Thermal degradation:

500 mg of nirmatrelvir sample and 500 mg of ritonavir were exposed in hot air oven at 105 °C for 6 hrs and the exposed sample was analysed. Equivalent weight of 273 mg of nirmatrelvir sample and 242 mg of ritonavir sample was transferred into a 100 mL volumetric flask, 70 mL diluent was added, sonicated to dissove and diluted to volume with diluent and mixed well. Further diluted 5 mL to 50 mL with diluent and the resultant solution was injected into the UPLC system. The obtained chromatogram is shown in Fig.3e.

Photo Degradation:

500 mg of nirmatrelvir and 500 mg of ritonavir samples were placed in photo stability chamber at 72 Lux hours for 6 hrs. Equivalent weight of 273 mg of nirmatrelvir sample and 242 mg of ritonavir sample was transferred into volumetric flask (100 mL). Added 70 mL diluent, sonicated to dissove and diluted to volume with diluent and mixed well. Further diluted 5 mL to 50 mL with diluent. The above solution was the injected into UPLC system. The obtained chromatogram is shown in Fig.3f.

Fig. 3a: Chromatogram of acid degradation

Fig. 3b: Chromatogram of alkali degradation

Fig. 3c: Chromatogram of hydrolysis degradation

Fig.3d: Chromatogram of peroxide degradation

Fig.3e: Chromatogram of thermal degradation

Fig.3f: Chromatogram of photo degradation

DISCUSSION:

The aim in developing the UPLC method is to achieve simultaneous separation and estimation of two drugs in tablet dosage under common conditions that are applicable for routine quality control , research and development of these drugs in ordinary laboratories. This work was intended to develop a precise, less time consuming, economical and a rapid method in reverse-phase UPLC separtion combined with PDA detection for simultaneous estimation in bulk samples and in dosage formulations. The developed method was rapid for simultaneous estimation of nirmatrelvir and ritonavir for elution of drugs with retention time (Rt) at 1.262 min., 1.873 min. Moreover , LOD and LOQ values indicated that the method was sensitive, % RSD values indicated that the method was precise, accurate and robust. The forced degradation studies performed indicated that both the drugs are highly senstive to acid, alkali and peroxide degaradations (> 13 %) while they degraded moderately in thermal stress conditions (> 5 %). Both the drugs showed minimal degradation (< 5 %) under hydrolysis and photo degradations.

CONCLUSION:

Till date no UPLC method related to simultaneous estimation of nirmatrlvir and ritonavir have been reported. Hence attempts were made towards the development of a method which separates the compounds with good resolution and less retention times by changing the mobile phase composition. Results indicated that the developed and optimized method for simultaneous estimation of nirmatrelvir and ritonavir in a copackaged tablet dosage form is rapid, simple, specific, accurate, robust and precise in nature with stability indicating capability. It would be easy to implement this method for the routine use in quality control laboratories as well as for the pharmaceutical formulations of nirmatrelvir and ritonavir.This method can also be extended to hyphenated techniques related to the bioanalytical methodologies associated with determination of nirmatrelvir and ritonavir in biological samples.

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Received on 03.07.2023 Modified on 06.08.2023

Research J. Pharm. and Tech 2023; 16(9):4370-4376.

DOI: 10.52711/0974-360X.2023.00715

Degradation condition	Nirmatrelvir				Ritonavir
Degradation condition	Drug recovered (% )	Drug decomposed (%)	Theoretical plates	Tailing factor	Durg recovered (% )	Drug decomposed (%)	Theoretical plates	Tailing factor
Control	99.9	-	7143	.05	99.9	-	5824	1.19
Acid degradation	85.6	14.3	7169	1.01	84.5	15.4	5809	1.12
Alkali degradation	86.8	13.1	7112	1.09	86.4	13.5	5875	1.13
Peroxide degradation	84.0	15.9	7138	1.10	83.4	16.5	5822	1.17
Thermal degradation	90.9	9.0	7152	1.03	92.8	7.1	5886	1.18
Hydrolysis degradation	95.8	4.1	7172	1.02	96.4	3.5	5827	1.16
Photo degradation	97.1	2.8	7101	1.08	98.1	1.8	5813	1.13