Development Of Nirmatrelvir and Ritonavir Simultaneous Estimation Method Using Quality by Design and Analytical Greenness Assessment

QbD, as per ICH Q8(R2), ensures pharmaceutical development through predefined objectives, process understanding, and quality risk management. It emphasizes flexibility, scientific advancement, superior design, QRM, DoE, PAT tools, lifecycle management, and continuous improvement.(Bhutani et al., 2014) By prioritizing risk assessment over conventional techniques, Quality by Design (QBD) guarantees consistent quality and produces more reliable analytical techniques. It entails using methodical trials to comprehend variable interactions. Using a risk-based methodology, this study aims to develop and validate an HPLC technique for nirmatrelvir and ritonavir. ¹

Optimization involves stages like screening to identify key factors and improvement to refine them. Response Surface Methodology (RSM) quantifies relationships between responses and inputs, while Central Composite Design (CCD) helps determine optimal conditions. 2 The entire product lifecycle incorporated analytical science since it is regarded as an essential component of pharmaceutical product development. The term “analytical QbD” refers to a science and risk based approach for the development of analytical procedures that aims to attain improved method performance, high robustness, ruggedness and flexibility for continuous improvement by comprehending and predetermined objectives to control the critical method variables affecting the critical method attributed. ³

Chemically, nirmatrelvir is known as “(1R,2S,5S)-N-[(1S)-1-cyano-2-oxopyrrolidin-3-yl]-3-[(2S)-3,3-dimethyl-2-[(2,2,2-trifluroacetyl)amino]butanoyl]-6,6-dimethyl-3-azabicyclo hexane-2-carboxamide”. 499.54 is its atomic mass. The formula for it is C23H32F3N5O4. Nirmatrelvir, an oral antiviral coronavirus illness therapy, is a strong and specific inhibitor of the primary protease of the severe acute respiratory syndrome coronavirus 2 (SARS-Co-2). ⁴

The compound 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}})carbamoyl]amino}butanamido]-1,6-diphenylhexan-2-yl]carbamate. C37H48N6O5S2 is its chemical formula, and its atomic weight is 720.94. One of the few antivirals used to treat COVID-19 and HIV. For a synergistic impact, ritonavir, a strong inhibitor of the HIV protease, is used in combination with other antivirals. To maintain effective systemic medication levels, 100 mg of ritonavir is added to nirmatrelvir as a pharmacokinetic enhancer. ⁵

In order to treat and prevent post exposure prophylaxis for the global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Pfizer has created nirmatrelvir plus ritonavir (Paxlovid^TM), an antiviral medication.(Lamb,2022) Nirmatrelvir is a strong and specific inhibitor of Mpro that activates the 3CL protease in a wide range of the Coronaviridae family.⁶ When taken orally, nirmatrelvir inhibits SARS-CoV-2 Mpro, inhibiting the cleavage of viral polyproteins and the synthesis of vital proteins required for viral transcription and replication.⁷ Ritonavir, a CYP3A inhibitor, increases the systemic exposure and half-life of nirmatrelvir. With a combined half-life of seven hours, the suggested dosage is 300 mg of nirmatrelvir (two 150 mg tablets) and 100 mg of ritonavir (one tablet).⁸

///There were few effective techniques for UV-spectrophotometric⁹ HPLC ^10,11, UPLC¹², and UHPLC-LC/MS¹³ examination of various medication combinations, according to the literature evaluation. Regarding QbD-based RP-HPLC estimation of Nirmatrelvir and Ritonavir , there are no publicly available data. By optimizing critical parameters and validating in accordance with ICH requirements, this study creates a precise, economical approach for their tablet dosage form, improving quality control.

Green chemistry is being used more and more in the pharmaceutical sector to reduce waste, energy use, and hazardous solvents. In order to promote safer and more sustainable methods for the environment and analysts alike, green analytical chemistry aims to minimize the use of solvents and their toxicity during sample preparation and analysis.¹⁴ The Green Analytical Procedures Index (GAPI) evaluates analytical techniques according to waste, solvents, equipment, and sample preparation using pictograms. The greenness of the suggested RP-HPLC process was assessed using GAPI in addition to AGREE and HPLC EAT. A score, color scale, and images are used in the more recent BAGI measure to evaluate environmental impact and feasibility. BAGI still needs improvement even though it improves on current green analytical chemistry (GAC) techniques like AGREE and ComplexGAPI.^15,16 A specialized instrument for evaluating the environmental friendliness of chromatographic procedures, HPLC-EAT concentrates on the kind and amount of solvent used in the mobile phase. It's limited to chromatography but provides a free and easy method. ¹⁷

MATERIALS AND METHODS

MATERIALS:

Cipla Private Limited, Patalganga unit II, Rasayani, Raigad, provided nirmatrelvir and ritonavir as gift samples. Acetonitrile and methanol utilized were HPLC grade. The supplier of all the chemicals was Aceto Pharma Private Ltd. In Ahmedabad, Gujrat. For assay and accuracy, the commercially available formulation PRIMOVIR which contains 150 mg of Nirmatrelvir and 100 mg of Ritonavir was utilized.

Instrument:

A JASCO Extrema LC-4000 RP-HPLC instrument equipped with chromNAV, software with photodiode array detector and auto vial injector, column of BDS Hypersil C18 column (250 × 4.6 mm, 5 μm particle size) in an isocratic mode is employed. The UV-Visible spectrophotometer used was of Shimadzu UV 1800 model. All the glassware employed is of Borosil make and ultra sonicator of MH-010S, Digital Pro. Weighing balance of Aczet is used in developing the analytical method.

Chromatographic Conditions:

At 25°C and 1 mL/min flow rate, an acetonitrile-water (60:40) mobile phase was employed with the BDS Hypersil C18 column (250 mm × 4.6 mm, 5.0 μm). A PDA detector was used to detect at 225 nm with an injection volume of 10 μL and a run time of 8 minutes. The method produced NTP, peak symmetry, and separation that were satisfactory. A central composite design was used to optimize run time, column temperature, and flow rate.

Selection of detection wavelength:

Wavelength maxima of 215 nm for Nirmatrelvir and 240 nm for Ritonavir were chosen by scanning 10 μg mL^-1 of both drugs in the 200-400 nm range. Moreover, 15 μg mL^-1 of Nirmatrelvir and 10 μg mL^-1 of Ritonavir were scanned to determine the isosbestic point, and 225 nm was chosen as the detecting wavelength based on the overlay spectra of both medications.

HPLC method development by QbD approach:

HPLC method development by Analytical QbD wa as follows:

Selection of quality target product profile:

In QbD, the analytical target product profile (ATPP) identifies the variables influencing analytical results. In the suggested HPLC approach, ATPP focused on peak area and theoretical plates for both drugs.¹⁸

Determine critical quality attributes:

The QbD technique affects product quality, safety, and efficacy by managing important quality parameters, which guarantees the method's dependability. Optimization of run time, flow rate, and column temperature was done to keep performance consistent within the appropriate limits.¹⁹

Factorial design:

Three important HPLC parameters run time, column temperature, and flow rate were optimized using a central composite design based on QTPP and CQA. Interactions between these variables and their impacts on theoretical plates, peak area, and retention time were examined using Design Expert® (v8.0.7.1). Retention time, peak area, and theoretical plates were dependent variables, whereas column temperature, run time, and mobile phase were independent variables. (Patil & Chalikwar,2024) Column temperature, run time and mobile phase were selected as independent variables and are displayed in Table I.

Table I. Coded values for independent variables

Evaluation of experimental results and selection of final method conditions:

The CCD technique evaluated peak area, theoretical plates, and retention time to optimize chromatographic settings for Ritonavir and Nirmatrelvir. Method robustness is ensured by established acceptable ranges, which stop variability from compromising quality during validation.²⁰ The design Expert tools will be used to optimize the most appropriate chromatographic settings.

Risk assessment:

Because of its effectiveness and long-term dependability, the ultimate approach was chosen. In accordance with ICH Q8 and Q9, a QbD-based risk approach assessed robustness and ruggedness in a range of settings, including labs, analysts, tools, and reagents.

Implement a control strategy:

Method performance and output quality are guaranteed by a control approach that is based on the analytical target profile. It covers measurement, replication control, and sample preparation and integrates risk management, analytical technique, and fitness for purpose.²¹

Continual improvement for managing analytical lifecycle:

The best method for managing the analysis lifecycle is to implement continuous improvement, which can be done in the laboratory by keeping an eye on the consistency of quality and performing routine maintenance on computers, HPLC instrument, software and other related equipment.²²

Analytical method validation:

Method validation is written proof that gives a particular method a high level of assurance the procedure used to verify the analytical process is appropriate for its intended usage. In accordance with ICH Q2(R1) recommendations, the developed HPLC technique for the simultaneous analysis of nirmatrelvir and ritonavir was verified.

System suitability studies:

The system suitability characteristics, including peak tailing, resolution and plate count, were determined by creating standard solutions of nirmatrelvir (45 μg/mL) and ritonavir (30 μg/mL) and injecting the solutions six times. The percsntage RSD for the outcomes of six standard injections should not be higher than 2%.

Linearity:

In order to determine the regression equation and correlation coefficient, the linearity of Nirmatrelvir (15-75 μg/mL) and Ritonavir (10-50 μg/mL) was assessed by creating a calibration curve by graphing peak area versus concentration.

Precision:

Six samples containing 45 μg/mL nirmatrelvir and 30 μg/mL ritonavir were measured in order to determine repeatability. By comparing this convcentration on the same day and following day, the intraday and interday precisions were ascertained. Less than two was the acceptable limit for %RSD.

Accuracy:

Using recovery studies at 80%, 100%, and 120% of the standard solution, accuracy was evaluated. Recoveries for Ritonavir and Nirmatrelvir fell between 98 to 102%, which is the range that the ICH has approved.

LOD and LOQ :

The term “quantification limit,” or “LOD,” refers to the lowest drug concentration that can be quantified, and the term “detection limit,” or “LOD”,” refers to the lowest drug concentration that can be reliably identified and distinguished from the background. The following formula was used to calculate LOD and LOQ in accordance with ICH recommendations.

LOD = 3.3×σ/SD
LOQ = 10 × σ/SD

Where SD is the calibration curve’s slope and is the regression line’s y-intercept standard deviation.

Robustness:

By altering the detection wavelength (223–227 nm), column temperature (20–30°C), and flow rate (0.8–1.2 mL/min), robustness was assessed. With system suitability parameters unaltered %RSD within acceptable limits, the results stayed within ICH limitations.

Assay:

A precisely weighed amount of twenty powdered tablets (150 mg of Nirmatrelvir and 100 mg of Ritonavir) was dissolved in 25 mL of methanol, sonicated for fifteen minutes, and then diluted to 100 mL. Following filtration, a solution containing 150 μg/mL and 100 μg/mL was obtained by diluting 1 mL of the filtrate to 10 mL. Using the average of six assays for calculation, HPLC analysis was carried out in the same chromatographic conditions as linearity.

Greenness Assessment:

The GAPI pictogram assesses an analytical assay's greenness at every stage, from sample collection to final analysis. The three-color scale (green, yellow, and red) is used to evaluate the effects on the environment and human health. Energy consumption, chemical risks, and general sustainability are all taken into account by GAPI, a trustworthy GAC tool that makes it simple to compare approaches and select the most  green one.

AGREE is a comprehensive methodology that uses 12 GAC principles to evaluate analytical results from 0 to 1 for greenness. These concepts are graphically represented by the AGREE symbol, whose parts are color-coded to reflect environmental effect. By assessing assay viability and environmental impact using ten characteristics.

BAGI, which focusses on White Analytical Chemistry, enhances green metrics. Scores range from 25 to 100, where higher scores signify better sustainability.²³

HPLC EAT assesses the effects of solvents in liquid chromatography on the environment, human health, and safety. It ignores things like energy, equipment, and sample preparation in favor of concentrating only on solvents. Solvent use determines the overall greenness score; a more environmentally friendly approach is indicated by lower EAT values.²⁴

RESULTS AND DISCUSSION:

Initial trials with methanol-water and acetonitrile-water (50:50) failed to produce peaks. A slight improvement was seen at 55:45, with correct peaks at 60:40. The central composite design optimized key parameters within the design space.

HPLC method development by QbD approach

Quality target product profile

In order to optimize HPLC chromatographic conditions the quality target product profile included the peak area of both drugs, retention time and number of theoretical plates.

Flow rate, column temperature and run time were identified as critical quality attributes.

The central composite design was selected for proposed HPLC method development. The optimization of various parameters shows in table IV.

Twenty runs of the central composite design, a sort of response surface study were employed. Applying the suggested CCD experimental design the flow rate, column temperature and run time were evaluated in relation to six responses , retention time, NTP and peak area of nirmatrelvir and ritonavir. And a summary of the outcome was provided.

1.                                                                              (B)

From figure 3.  (B)and equation for retention time of ritonavir (for actual values) = +19.48716 -20.32834 * A+0.023957 * B -0.10830 * C +0.026250 * A * B +0.011875 * A * C +0.00343750* B * C +6.54497 * A² -0.00185798* B² +0.000661083* C², it was concluded that as β1 negative coefficient (-20.32834) suggests that as flow rate(A) decreases, β2 positive coefficient (+0.023957) suggests that as the column temperature(B) increases and β3 negative coefficient(-0.10830) suggests that as the run time(C) decreases, the value of retention time of ritonavir was increases.

1.                                                                                    (B)

From figure 4. (B) and equation for theoretical plates of ritonavir(for actual values) =+22948.75526 -373.37764 * A -316.48718 * B -1030.89274 * C +35.62500 * A * B -70.62500 * A * C +23.87500 * B * Run time -3012.80149 * A² -0.34114 * B² +26.57311 * C² ,it was concluded that as β1 negative coefficient (-373.37764) suggests that as the flow rate (A) decreases, β2 negative coefficient (-316.48718) suggests that as the column temperature(B) decreases and  β3 negative coefficient (-1030.89274)  suggests that as the run time(C) decreases, the value of theoretical plates of ritonavir was increased.

1.                                                                        (B)

Figure 5. 3D surface plot for effect of combination of factors of (A)peak area of nirmatrelvir (B)peak area of ritonavir by using central composite design

From figure 5. (A)and equation for peak area of nirmatrelvir(for actual values)= +1848720-4004180* A +140243* B +26295.96801 * C -5321.87500 * A * B +28521.25000 * A * C -1790.68750 * B * A +1.30863E+006 * A² -2444.76905 * B² -205.58285 * C² , it was concluded that as β1 negative coefficient (-4004180) suggests that as the flow rate (A) decreases, β2 positive coefficient (+140243) suggests that as the column temperature(B) decreases and  β3 positive coefficient (+26295.96801)  suggests that as the run time(C) decreases, the value of retention time of ritonavir was increased.

From figure 5. (B)and equation for peak area of ritonavir(for actual values) = +2351760-4655090* A +1.45123E+005 * B +34064.06029 * C -2570.00000 * A * B +22477.50000 * A * C -969.87500 * B * C +1518280* A² -2702.92232 * B² -1260.77804 * C² , it was concluded that as β1 negative coefficient (-4655090) suggests that as the flow rate (A) decreases, β2 positive coefficient (+145123) suggests that as the column temperature(B) increases and  β3 positive coefficient (+34064.06029)  suggests that as the run time(C) increases, the value of peak area of ritonavir was increased.

Optimized condition obtained

It was obtained by studying all responses in different experimental conditions using the design expert 8.0.7.1 software and optimized HPLC conditions and predicted responses are shown in Table no.3.

Table 6. Chromatographic conditions

Figure 6. 3D surface plot of desirability for optimized method

Figure 7. Optimized Method Chromatogram

Five aliquots from the stock solution were analyzed, showing linearity for Nirmatrelvir (15–75 μg/mL) and Ritonavir (10–50 μg/mL). The regression equations were y = 7617.3x - 20.8 (R² = 0.9998) for Nirmatrelvir and y = 13787 + 2887.7x (R² = 0.9995) for Ritonavir.

Precision

Six replicates were used to measure intraday and interday precision (45 μg/mL Nirmatrelvir, 30 μg/mL Ritonavir). The correctness of the approach was confirmed by the %RSD for peak area, NTP, tailing factor, and retention time being less than 2%.

Accuracy

The accuracy was done by measuring the recovery of drug from marketed formulation. Recovery was checked at 3 levels which are 80%, 100% and 120%. The % recovery found to be within 98 to 102% and so as per ICH guidelines it justify that the optimized method is accurate.

Robustness

Robustness study was studied by slight but deliberate change in method parameters such as flow rate, column temperature and detection wavelength %RSD for retention time, NTP and peak area of both drugs were less than 2%.

Limit of detection

Limit of detection for nirmatrelvir were found to be 1.0034 μg mL^-1 and for ritonavir 1.3642 μg/mL .

Limit of quantification

Limit of quantification for nirmatrelvir were found to be 3.3449 μg/mLand for ritonavir 1,3642 μg/mL.

Assay

Nirmatrelvir and Ritonavir had retention times of 3.82 and 5.87 min, respectively, according to the optimized assay. Purity values of 98.95% and 99.31%, respectively, demonstrated that the technique could identify the medications among tablet excipients.

(A)                                                                          (B)

Table 9. Precision data of nirmatrelvir and ritonavir

Table 10. Accuracy data of Nirmatrelvir and Ritonavir

Figure 6. Chromatograms for accuracy of 80%

Figure 7. Chromatogram for accuracy of 100%

Figure 8.Chromatogram for accuracy of 120%

GAPI:

The GAPI pictogram uses a color scale to evaluate analytical greenness. With the exception of sections 7, 10, 11, and 14, which were yellow, the majority of the parameters in the created approach were green. Acetonitrile, an organic modifier and green solvent, was the cause of Section 7's yellow color. Its mild toxicity (NFPA health hazard rating 2) was indicated in Section 10, and its flammability (rating 3) was emphasized in Section 11. Since waste generation ranged from 1 to 10, Section 14 was yellow. Furthermore, even though no waste treatment was used, the yellow hue suggested that the sample needed to be filtered.²⁵

AGREE:

Based on GAC principles, the Analytical GREEnness calculator (AGREE) is a versatile instrument for evaluating analytical sustainability. It assigns a score between 0 and 1, where higher scores denote greater greenness. The final score is shown at the center of a pictogram that visualizes the evaluation and has 12 color-coded segments. With the degree of environmental impact shown by different shades of green, the suggested approach received a strong sustainability score of 0.86. When evaluating the sustainability of analytical methods, the AGREE pictogram is a useful instrument.²⁶

BAGI:

Four weighted scores white (2.5), light blue (5.0), blue (7.5), and dark blue (10), respectively are used in the BAGI examination. For a method to be deemed practical, it must receive a minimum score of 60. Our approach demonstrated its viability and suitability in a bioanalytical context with an 85.0 BAGI score.²⁷

HPLC EAT:

This method provides a dependable and effective means of assessing liquid chromatographic procedures by analyzing HPLC EAT in relation to the criteria explained in figure 10. This method’s capacity to evaluate the impacts of all solvents employed in the process makes it a useful tool for guaranteeing the safety and quality of chromatographic operations.²⁸ The method’s ability to evaluate each solvent’s effects on safety, the environment and human health is demonstrated in figure 12.

Figure 11. BAGI (Blue Applicability Grade Index)

Figure 12. HPLC EAT score