INTRODUCTION:

Nirmatrelvir is an antiviral drug used in the treatment of corona virus disease. Paxlovid, which is a combination of NRT and Ritonavir, is used under emergency use authorization for COVID-19 treatment^1,2.

The 3CL^PRO, also known as the main protease or non-structural protein 5, is responsible for cleaving polyproteins 1a and 1ab. The chemical structure was depicted in Figure 1. These polyproteins contain the 3CL^PRO itself, a papain-like (PL) cysteine protease, and 14 other non-structural proteins. Deprived of the activity of the 3CLPR^O, non-structural proteins (including proteases) cannot be released to perform their functions, inhibiting viral replication. The mean volume of distribution of NRT when given with Ritonavir is 104.7 liters.^3,4,5NRT, when given with Ritonavir, is 69% protein-bound in plasma. It is a substrate of CYP3A4, but undergoes minimal metabolism when administered alongside Ritonavir. The major route of NRT elimination is via renal elimination, due in part to its co-administration with Ritonavir, which inhibits its metabolism. The half-life of NRT when administered orally with Ritonavir is 6.05 hours.^6,7,8,9

MATERIALS AND METHODS:

CHEMICALS AND REAGENTS:

Pharmaceutical grade Nirmatrelvir working sample was a kind gift from Hetero Labs, Mumbai, India, and methanol was purchased from SRL Pvt. Ltd., Mumbai, India.

TABLET FORMULATION¹⁰:

Tablets were prepared in the laboratory using the ingredients described in Table 1.

INSTRUMENTATION:

Spectral analysis was performed using Lab India Analytical UV 3092 dual chamber with 10mm Optics. way UV-Vis spectrophotometer with long quartz cell beam is used for analysis purpose using methanol as diluent and lambda max as shown in Figure 2.

PREPARATION OF STANDARD STOCK SOLUTIONS:

Dissolve 100mg in 100mL of methanol (1000µg/mL) and prepare a standard methanol stock of 100µg/mL NRT from this solution. Pipette 1, 2, 3, 4, 5 and 6ml of solution from this solution set to obtain NRT concentrations of 10, 20, 30, 40, 50 and 60µg/ml.

ANALYSIS OF IN-HOUSE TABLET FORMULATION:

In-house Twenty tablets were prepared; As shown in Table 1, each tablet contains 150mg of NRT with an average weight of 500.34mg. Measure the contents of 20 tablets and grind them to a fine powder. Transfer the powder equivalent to 30mg (100.06mg) into a 100mL container volume, dilute to volume with methanol, ultrasonicate for 20min, and filter with Whatmann filter paper. Finally, dilute 1 ml of the above solution in a 10 ml volumetric flask to obtain a 30 μg/mL solution and adjust the volume with methanol Table 3.

METHOD VALIDATION^11-23:

STUDY OF THE LINEARITY CURVE:

The linearity of NRT was examined in the concentration range of 10-60 µg/ml. The drug showed good linearity over the experimental range. The regression equation for NRT is y = 0.014x+0.065, and the regression coefficient value (r²) for NRT is 0.999. Table 2, Figure 3.

PRECISION:

INTRA-DAY AND INTER-DAY PRECISION

Intra-day precision: On the same day analysis of 3 different concentrations of NRT for 3 times was conceded.

Inter-day precision: The analysis of standard drugs at three different concentrations in the linearity range of the NRT for the three days (30, 40, and 50µg/mL) and %RSD was calculated for both methods.

RUGGEDNESS:

The evaluation of ruggednesscarried out using two different UV spectrophotometer using a concentration of 30µg/mL and calculating the % RSD.

DETECTION LIMIT AND QUANTIFICATION LIMIT:

For these studies, solutions of different concentrations in the range of 5-30µg/ml were run in UV. The limit of detection (DL) and limit of quantification (QL) of NRT were tested and calculated using the formula DL = (3.3 x A.S.D.)/m. The calculation limit is calculated as (10 x A.S.D)/m; where “A.S.D” is the mean standard deviation of the peak area (n = 3) and “m” corresponds to the line at the bottom of the diagram shown in Figure 4.

SPECIFICITY:

Specificity is the ability to is the ability to unambiguously analyze an analyte in the presence of desired substances that may be present in the sample matrix.

ACCURACY:

The accurate (% recovery) study of NRT was performed according to ICH guidelines, in which the drug was added to the drug sample at levels of 80%, 100% and 120%. Determine the total concentration of the solution and calculate the %RSD.

RESULT AND DISCUSSIONS:

LINEARITY AND RANGE:

Linearity and Range There is a relationship between the absorbance of λmax and the concentration of NRT. This graph is described by the inverse equation: Y = mx + c (where Y = absorbance of drug solution; c = intercept; m = slope; X = drug concentration (μg/ml)). Calculate the slope (3), intercept (c) and correlation coefficient (r) using the least squares method. A good linear correlation was obtained between absorbance and standard drug concentration in the range of 10-60 μg/ml. Parameters such as slope 0.014, intercept 0.065, and correlation coefficient 0.999 are shown in Table 2.

LIMIT OF DETECTION (LOD) AND LIMIT OF QUANTIFICATION (LOQ):

Relative standard deviation and slope method specified in the International Conference on Harmonization (ICH) guidelines. The limit of detection (LOD) and limit of quantification (LOQ) of NRT are 0.46 μg/ml and 1.38 μg/ml, respectively; This indicates the high sensitivity of the UV method. The regression and analysis results are shown in Table 4.

ACCURACY:

The accuracy of the analysis method refers to the closeness of the measured value to reality. The accuracy of the method was evaluated at three levels using additional methods. Values equal to 80%, 100% and 120% should be added to the formula. The results reported in Table 4 range from 99.07–100.36% to ensure the accuracy of the method and demonstrate that there is no interference with additional equipment.

ROBUSTNESS:

The robustness of the method was tested as a function of change in wavelength. Experimental results showed that the change in %RSD value was 0.8% (less than the acceptance limit of <2% RSD). The UV strategy appeared to be durable. The results obtained are presented in Table 4.

PRECISION:

For RSD% repeatability, intraday and intraday accuracy is 0.55%, between 0.58% and 0.95%, and between 0.26% and 0.73%. The results showed that the method had good repeatability, intraday accuracy, and interday accuracy. The results obtained are shown in Table 4.

CONCLUSION:

The results and statistical data show that the proposed UV spectrophotometric method is simple, fast, reliable, accurate and clear.

Figure 1. Structure of Nirmatrelvir

Figure 2. UV Spectrum of Nirmatrelvir

Figure 3. Linearity Graph of Nirmatrelvir

Figure 4. Sensitivity Study of Nirmatrelvir

Table1. Formulation of In-Housetablets

Each NRT tablets contains:
Components	mg/tablet
Nirmatrelvir	150
Lactose Monohydrate	176
Microcrystalline cellulose	174
Total	500

Table 2. Linearity of Nirmatrelvir

Parameter	Result
Wavelength (nm)	210
Linearity range (ng/band)	10-60μg/ml
Correlation coefficient	0.999

Table 3. Analysis of In-houseTablet

Conc μg/ml (n=3)	SD	%RSD	% Amount Found
30	0.246	0.247	99.37

n- number of determinations

Table 4. Validation Parameters

Precision
Parameters		Cone (μg/ml)		Amount Found		%RSD
Intra-day (n=3)		30		100.24		0.48
		40		101.49		0.37
		50		101.14		0.29
Inter-day (n=3)		30		100.40		0.60
		40		101.43		0.36
		50		101.14		0.25
Robustness and Ruggedness
Parameter		Amount Found		% RSD
Instrument Change		101.23		0.78
Limit of Detection and Limit of Quantification
Limit of Detection			0.46
Limit of Quantification			1.38
Accuracy
Label Claim (mg/tablet)	Level of Recovery		Amount Found		%RSD
150	80		99.25		0.20
	100		100.20		0.18
	120		99.78		0.22

n- number of determinations

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ABBREVIATIONS:

· ml - Millilitre

· μg – Microgram

· UV- Ultra Violet

· NRT- Nirmatrelvir

· AR- Analytical reagent

· mm- Millimetre

· mg- Milligram

· nm- Nanometre

· RSD- Relative Standard Deviation

· LOD- Limit ofDetection

· LOQ- Limit of Quantification

· SD- Standard Deviation

· ASD-AverageStandard Deviation

· ICH-International Conference on Harmonization

Received on 28.07.2023 Modified on 18.11.2023

Asian J. Pharm. Ana. 2024; 14(3):127-130.

DOI: 10.52711/2231-5675.2024.00022