Ritonavir is a protease inhibitor designed to treat HIV/AIDS. It works by inhibiting the HIV 1 protease, which is important for the growth of the virus. By blocking this enzyme, ritonavir prevents the virus from multiplying in the host. Although ritonavir was first used to treat HIV, it has been discovered that it can be used in combination with other protease inhibitors based on pharmacokinetics because it can inhibit the cytochrome P450 enzyme (CYP3A4), which metabolizes many drugs. In the treatment of Covid 19, ritonavir is often used as a booster for nimatrelvir, helping to break down nirmatrelvir and maintain it at higher levels in the body. 2. This virus is responsible for SARS CoV 2. It targets the primary protease (Mpro) of SARS CoV 2, an enzyme needed by the virus to convert its polyprotein into a functional virus. By inhibiting this protease, nivolumab can prevent the virus from growing and multiplying, thereby reducing a decreased rate of infection and contribute in decreasing the flow of the infections. Nimaprevir acts directly on the virus by inhibiting the activity of the viral protease, while ritonavir slows down the metabolism of nimaprevir, increasing its effectiveness and thus maintaining healthy nimaprevir levels. Clinical studies have shown that this combination can reduce the risk of hospitalization and death in patients at high risk of Covid 19. Structure of ritonavir and nirmatrelvir shown in Figure 1. Studies have shown that the RP HPLC method is better for ritonavir (Gandlaet al., 2024; Gandhiet al., 2016; Badaret al., 2019; Bhirudet al., 2015) and the combination of ritonavir and nimaprevir (Kaleet al., 2024) and the Rp HPLC method is better for the other drugs (Godgeet al., 2020).

Figure 1:
Structure of Ritonavir and Nirmatrelvir.

The supplier of Ritonavir and Nirmatrelvir (API) were to be Swapnaroop Drugs and Pharmaceuticals, sambhajinagar, Among solvents used were HPLC quality. The chemicals and reagents employed were of analytical grade.

The HPLC system applied was purchased from Analytical Technologies Ltd., HPLC Workstation Software and UV-DAD detector.

Column used was C18 (250×4.6 mm id, particle size: Sample temperature: The sample temperature is 22°C Concentration: 10 µM) absorptivity (Α): 0.368 (±0.005) Wavelength: 5 µ, 234 nm Acetonitrile: 0.1% OPA (49:51) in water/acetonitrile was used as a mobile phase, with a flow rate of 0.7 mL/min. The Injection volume is 20 µL.

0.1% OPA Buffer-Take 1 mL orthophosporic acid in 1000ml volumetric flask and add 1000 mL of HPLC grade water to give 0.1% OPA.

Assay: 15 mg nimatravir added in Methanol was added to a 100 mL volumetric flask until the final content was 100 mL. gives a concentration of 1500 μg/mL. Dilute the drug in serial manner to achieve nimaprevir concentration of 15-75 μg/mL (Madhuriet al., 2024).

Add 10 mg of ritonavir into 100 mL volumetric flask, dissolve the content with methanol to 100 mL to give a stock solution containing 1000 μg/mL of the substance. Dilute the drug in serial manner to achieve ritonavir concentration of 10-50 μg/mL (Bhaskaranet al., 2022; Jyothiet al., 2019).

The mobile phase was made by combining 410 volumes of acetonitrile with 590 L of 0.1% OPA (41:59), which was then diluted in distilled water to create a 1000 mL volumetric solution in a 1000 mL volumetric flask. The mobile phase was subjected to ultrasonication for 10 min to release dissolved gases and later filtered through 0.45 µ membrane filter using vacuum filtration.

To determine the appropriate analytical wavelength for this complex matrix, it is crucial to consider the presence of metal ions. Preparing the suitable diluted standard stock solution of drug with its UV spectrum of 10 µg/mL of ritonavir and nirmatrelvir. For RP-analysis, these diluted solutions were scanned in the range from 200 to 400 nm. Both drugs had significant absorbance at 234 nm. It was opted for as determination wavelength.

The development method for ritonavir and nirmatrelvir were tested at various mobile phases including different concentrations of acetonitrile, methanol and buffers; the final ratio was 49:51% v/v acetonitrile and 0.1% OPA, preferring to deliver ritonavir and Nirmatrelvir. Appropriate studies were performed to determine the peak asymmetry, theoretical plates and resolution of the sample as shown in Table 1. Chromatographic separation was performed using the AGILENT (1100) equipped with a 20 µL sample injection loop and UV visible detector. A mixture of acetonitrile and 0.1% orthophosphoric acid was used. 234 nm was the detecting wavelength, and 0.7 mL/min was the flow rate. Pass the cells through a 0.2 μM filter and degas before use. Analysis was conducted at room temperature in a 20 μL volume.

Linearity studies of the ritonavir and nirmatrelvir were done by dilution of the stock solution to get the nimaprevir concentration of 15-75 μg/mL and 10-50 μg/mL for ritonavir. The calibration curves are constructed to illustrate the variation of the peak area against the concentration of ritonavir and nimaprevir to demonstrate in Figures 2 and 3, respectively. The regression equations were y=34.858x 59.097 (R²=0.9992) for ritonavir and y=42.271x 33.657 (R²=0.9986) for nimaprevir. Diluting the stock solution to obtain a nimaprevir concentration of 15 75 μg/mL and 10 50 μg/mL for ritonavir.

Figure 2:
Calibration Curve of Ritonavir.

Figure 3:
Calibration Curve of Nirmatrelvir.

Weigh 20 tablets, then powder the tablets, accurately transfer 599.99 mg of the powder into a 100 mL volumetric flask, treat the flask with methanol and ultrasonically shake for 15 min. The solution were finally filtered through Millipore syringe filter (0.42 μ). Duplicated portion of the pure solution was then run into the HPLC. It was demonstrated that the preparation is selective and its preparation is relatively unaffected by capsules such as lactose. A linear regression equation was used to quantify the assay. The result analysis revealed that there is a correlation between the labelled claim concentration and the drug content quantified in the study. This relationship confirms that the content of the formulations being studied must be analyzed using the analytical method to show that it is accurate and reproducible. Like quantity, detection, linearity, accuracy, precision and robustness as per the guideline from the ICH. Altogether, these tests proved that the model is appropriate for use and can provide good, accurate, and reliable outcomes across multiple test facilities. Chromatogram of ritonavir and Nirmatrelvir shown in Figure 4.

Figure 4:
Chromatogram of Ritonavir and Nirmatrelvir.

The validation of this method includes the evaluation of parameters such as quantity, detection, linearity, accuracy, precision and flexibility as recommended by ICH. Together, these tests confirm that the model is suitable for use and can produce good, accurate and reliable results at multiple test sites.

The linearity of the ritonavir was establish at concentration ranges of 10-50 µg/mL and calibration curve is y=34.858x-59.097) with (R²=0.9992) and for nirmatrelvir establish at concentration ranges of 15-75 µg/mL and calibration curve is y=42.271x-33.657) with (R²=0.9986).

Studies on the difference between day and midday were accurate. In a study involving three consecutive injections of a sample drug on the same day. The %RSD value was obtained by testing the difference between days when the sample and the sample solution were injected three times in succession over several days shown in Table 2. The data collected showed that the RPPHLC method produced high accuracy with an RSD percentage of less than 1.5%. The recovery results are within 100±2%, supporting the precision of this approach. Accuracy of the method is evaluated by testing two drugs at three different recovery levels at 80%, 100% and 120% of the recovery percentage are given in Tables 3 and 4 for ritonavir and nirmatrelvir respectively.

Robustness assesses the reliability of an analytical method in response to minor alterations in method parameters. It constitutes a component of the method validation procedure. Strength of drug evaluated by making small targeted changes to the test protocol like pH, wavelength, and flow rate are deliberately varied and results are recorded. The results are presented in Table 5.

In addition, the Limit of Quantification (LOQ) and the Limit of Detection (LOD) were determined. LOD=(3.3×standard deviation/slope of the calibration curve) is the formula used to calculate the LOD. This is the lowest test level that produces visible results. The LOD values for ritonavir and nirmavir were found to be 0.4382 µg/mL and 0.5618 µg/mL, respectively. The lower limit of analytical precision (LOQ) is expressed as: LOQ=(10×standard deviation/slope of the calibration curve). The LOQ values for ritonavir and nirmavir were 1.3278 µg/mL and 1.7025 µg/mL, respectively.

The proposed approach was determined to be simple, exact, accurate, and rapid for the analysis of Ritonavir and Nirmatrelvir, individually from pure pharmaceutical formulations. The mobile phase was simpler to prepare, and the run time was under 6 min, and the consumption of less than 6 mL of the mobile phase only proved that the method was inexpensive. The observed sample recoveries in all formulations enhanced well in label claims; this implied non-interference in the estimation. Consequently, the approach can be consistently employed for analysis of Ritonavir and Nirmatrelvir in their combined dosage forms. This degree of simplicity renders the RP-HPLC approach accessible for the quantification of Ritonavir and Nirmatrelvir in tablet formulations and pure pharmaceutical preparations. The mobile phase is easy to produce, and the runtime was under 6 min, utilizing less than 6 mL of mobile phase. The phase indicates that the strategy was cost-effective. The sample recoveries in all formulations were consistent with their individual label claims, indicating no interference in the estimation. Consequently, the method can be readily and conveniently employed for the regular analysis of Ritonavir and Nirmatrelvir in mixed dosage forms. The method’s simplicity guarantees its applicability for the measurement of Ritonavir and Nirmatrelvir in tablet dosage forms using RP-HPLC. The aforementioned chromatographic peak resolutions indicate that the procedure was accurate, precise, linear, robust, and rugged.

The authors would also like to thank the Principal and Management for supporting our efforts in developing analytical methods.