Analytical Quality by Design Approach to RP-HPLC Method Development and Validation of Ritonavir and Nirmatrelvir

Sandip Rathod, Amol Darade, Amol Darwade, Amol Gayake, Dr. Kailash Rathod,

doi:10.5281/zenodo.17432857

Research Paper | Open Access
Volume 03 | Issue 10 | Article Id IJPS/250310347

Analytical Quality by Design Approach to RP-HPLC Method Development and Validation of Ritonavir and Nirmatrelvir
Sandip Rathod* Amol Darade Amol Darwade Amol Gayake Dr. Kailash Rathod
SND College Of Pharmacy Babhulgaon, Yeola

Abstract

RP-HPLC method was developed by implementing QbD methodology on analytical column- Reversed Phase Agilent C18 (250mm×4.6mm×5 µm), with mobile phase Methanol: (0.1% OPA) Water (41:59 v/v). The flow rate used was 0.7 mL /min and UV detection was carried out at 234 nm. The retention time for Ritonavir & Nirmatrelvir was found to be 4.039 min & 5.653 min respectively. The study was done by using 32 full fraction response surface designs. In this study interaction of 2 factors; flow rate, mobile phase composition at 3 levels.Method Operable Design Region (MODR) was developed to achieve the region of operation for drug and Nirmatrelvir. The Limit of Detection (LOD) and Limit of Quantitation (LOQ) were established at a signal-to-noise ratio. LOD and LOQ were calculated as 3.3×?/S and 10×?/S respectively as per ICH guidelines.System suitability test ensures that the analytical system is working properly and can give accurate and precise results. System suitability tests includes tailing factor, number of theoretical plates, area etc. The results of all system suitability parameters were acceptable in their limits defined by official guidelines.The proposed HPLC method has also been evaluated for accuracy, precision and robustness and proved to be convenient and effective for the quality control of Ritonavir & Nirmatrelvir.Moreover, the lower solvent consumption along with the short analytical run time of10 min leads to a cost effective and environmentally friendly chromatographic procedure.

Keywords

RP-HPLC, Ritonavir, Nirmatrelvir Method Development, Validation, Qbd

Introduction

Quality by Design (QbD) Approach Quality by Design (QbD) is a systematic approach to pharmaceutical development and manufacturing that emphasizes understanding the product and process to ensure quality throughout the product lifecycle. It originated as a regulatory initiative to shift from traditional trial-and-error methods to a science-based approach that integrates quality into every stage of product development and manufacturing (31).

Principles of QbD: Principles encompass a series of systematic steps and methodologies aimed at enhancing product quality and ensuring consistent performance. These principles are broadly applicable across various industries,specific guidelines and frameworks tailored to pharmaceutical development under regulatory bodies such as theInternational Council for Harmonisation (ICH.

Designing Quality into the Product: QbD begins with defining the target product quality profile (TPQP), which outlines the desired attributes and performance criteria of the drug product based on patient needs and regulatory requirements. The TPQP encompasses critical quality attributes (CQAs), which are the measurable physical, chemical, biological, or microbiological characteristics that define the product's quality and performance. Establishing CQAs early in development allows for the identification of critical process parameters (CPPs) that impact these attributes (32). 1.7.1.2. Understanding the Product and Process: A thorough understanding of the product and process is fundamental to QbD. This involves using scientific knowledge, risk assessment, and experimental design to identify and control sources of variability that may affect product quality. Quality risk management (QRM) tools such as Failure Mode and Effects Analysis (FMEA) and Process Analytical Technology (PAT) are employed to systematically evaluate and mitigate risks throughout the product lifecycle (33).

Establishing a Design Space: The design space defines the range of process parameters within which the product meets the desired quality attributes. It is established through systematic experimentation and statistical analysis to ensure robust product performance. By defining a design space, manufacturers gain flexibility in process optimization and scale-up while maintaining product quality and consistency Using Quality Risk Management (QRM)

Approaches: QRM is integral to QbD, focusing on identifying, evaluating, and controlling risks to product quality. It involves a proactive approach to risk assessment and mitigation throughout development, manufacturing, and distribution. QRM tools aid in prioritizing risks based on severity, probability, and detectability, allowing resources to be allocated effectively to manage high-risk areas.

Continuous Improvement and Lifecycle Management: QbD promotes a lifecycle approach to product development and manufacturing, emphasizing continuous improvement and optimization. Lifecycle management involves monitoring product performance through real-time data analysis and feedback loops, enabling proactive adjustments to maintain quality standards.

Application of Science and Risk-Based Approaches: Central to QbD is the application of scientific principles and risk-based approaches to decision-making. This includes using advanced analytical techniques, mathematical modeling, and simulation studies to understand process behavior and predict outcomes. By integrating scientific knowledge with risk assessment, manufacturers can make informed decisions that enhance product quality and compliance with regulatory

3. DRUG PROFILE :

3.1 Ritonavir

Chemical Name	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] butanoyl]amin o]-1,6-diphenylhexan-2-yl]carbamate
Molecular Formula	C37H48N6O5S
Molecular weight	720.944g/mol
Appearance	White crystalline powder
Solubility	Solubility Slightly soluble in water, Methanol, DMSO
Category	HIV agent

Mechanism of Action: Ritonavic inhibits the HIV viral proteinase enzyme that normally cleaves the structural and replicative proteins that arise from major HIV genes, such as gag and pol. Gag encodes proteins involved in the core and the nucleocapsid, while pol encodes the the HIV reverse transcriptase, ribonuclease H, integrase, and protease . The pol-encoded proteins are initially translated in the form of a larger precursor polypeptide, gag-pol, and needs to be cleaved by HIV protease to form other complement proteins 1. Ritonavir prevents the cleavage of the gag-pol polyprotein, which results in non-infectious, immature viral particles. Ritonavir is a potent inhibitor of cytochrome P450 CYP3A4 isoenzyme present both in the intestinal tract and liver . It is a type II ligand that perfectly fits into the CYP3A4 active site cavity and irreversibly binds to the heme-iron via the thiazole nitrogen, which decreases the redox potential of the protein and precludes its reduction with the redox partner, cytochrome P450 reductase

Nirmatrelvir Structure:

Drug Profile Of Nirmatrelvi

Chemical Name	1R,2S,5S)-N-[(1S)-1-cyano-2-[(3 S)-2-oxopyrrolidin3-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
Molecular Formula	C23H32F3N5O
Molecular weight	720.944g/mol
Appearance	White crystalline powder
Solubility	Solubility Slightly soluble in water, Methanol, DMSO
Category	oral protease inhibitor

Mechanism of action Nirmatrelvir is an inhibitor of a cysteine residue in the 3C-like protease (3CLPRO) of SARS-CoV2.This cysteine is responsible to the activity of the 3CLPRO of SARS-CoV-2 and potentially other members of the coronavirus family. The 3CLPRO, also known as the main protease or non-structural protein 5, is responsible for cleaving polyproteins 1a and 1ab.1 These polyproteins contain the 3CLPRO itself, a papain-like (PL) cysteine protease, and 14 other non-structural proteins.3 Without the activity of the 3CLPRO, non-structural proteins (including proteases) cannot be released to perform their functions, inhibiting viral replication.

MATERIAL AND METHODS

Selection and Procurement of Drug

Name of Drug	Drug Supplier
Ritonavir	Swapnroop pharmaceutical drug
Nirmatrelvir	Swapnroop pharmaceutical drug

List of reagents & chemicals used

Acetonitrile (HPLC grade)	Merck Ltd., India 2
Methanol (HPLC grade)	Merck Ltd., India 3.

Result of different trials:

Fig. No.	Column used	Mobile phase, Flow Rate and Wavelength	Inj. Vol.	Observation	Conclusion
1	C18 (AGILE NT) (250×4.6mm, 5μ)	90% methanol: 0.1% OPA 234 nm, Flow rate 0.7ml.	20 μl	Sharp peaks were not obtained	Hence rejected
2.	C18 (AGILE NT) (250×4.6mm, 5μ)	80% Methanol: 30% Water (0.1%OPA) 234 nm, Flow rate 0.7ml.	20 μl	Sharp peaks were not obtained	Hence rejected
3	C18 (AGILE NT) (250×4.6mm, 5μ)	70% Methanol: 30% Water (0.1%OPA) 234 nm, Flow rate 0.7ml.	20 μl	Sharp peaks were not obtained	Hence rejected
4	C18 (AGILEN T) (250×4.6mm, 5μ)	60% Methanol: 40% Water (0.1%OPA) 234 nm, Flow rate 0.7ml.	20 μl	Sharp peaks were not obtained.	Hence rejected
5	C18 (AGILEN T) (250×4.6mm, 5μ)	40% Methanol: 60% Water (0.1%OPA)- 234 nm, Flow rate 0.7ml	20 μl	Sharp peaks were not obtained.	Hence rejected
6	C18 (AGILEN T) (250×4.6mm, 5μ)	40% Methanol: 60% Water (0.1%OPA) – 234 nm, Flow rate 0.7ml	20 μl	Sharp peaks were obtained.	Hence Method selected

Chromatogram Trail :1

Chromatogram of Trial 1:

No.	RT [min]	*Area [mVs]**	TP	TF	Resolution
1	2.137	1243.6527	911	0.24	-
2	3.958	426.5007	7135	1.53	7.73
3	4.321	5917.76123	991	2.59	0.98
4	4.445	2557.45068	6676	0.19	0.32
5	6.473	74.41815	1081	0.52	4.03

Full factorial design of DOE

	Factor 1	Factor 2	Factor 3
Run	A: Methanol	B: Flow rate	C: Wavelength
	%	ml/min	nm
1	41	1.1	234
2	41	1.2	235
3	41	1	233
4	41	1.2	233
5	41	1.1	234
6	42	1.1	235
7	41	1.1	234
8	42	1.1	233
9	42	1.2	234
10	42	1	234
11	41	1.1	234
12	40	1.1	233
13	40	1.2	234
14	41	1.1	234
15	40	1	234
16	40	1.1	235
17	41	1	235

RESULT & DISCUSSION:

UV Spectroscopy: UV absorption of 10 µg/mL solution of Ritonavir and Nirmatrelvirin λm of Ritonavir and Nirmatrelvir in Methanol was found to be 259nm and 210nm .

UV Spectrum of Ritonavir

Studies on the Chromatographic Behaviour of Ritonavir and Nirmatrelvir.

Fig. No.	Column used	Mobile phase, Flow Rate and Wavelength	Inj. Vol.	Observation	Conclusion
1	C18 (AGILEN T) (100×4.6mm, 2μ)	90% Methanol: 10% water (0.1% OPA), 234 nm, Flow rate 0.7ml.	20 μl	Sharpe peaks were not obtained	Hence rejected
2.	C18 (AGILE NT) (100×4.6mm, 2μ)	80% Methanol: 20% Water (0.1% OPA) 234 nm, Flow rate 0.7ml.	20 μl	Sharpe peaks were not obtained	Hence rejected
3	C18 (AGILE NT) (100×4.6mm, 2μ)	70% Methanol: 30% Water (0.1% OPA) 234 nm, Flow rate 0.7ml.	20 μl	Sharpe peaks were not obtained	Hence rejected
4	C18 (AGILEN T) (100×4.6mm, 2μ)	60% Methanol: 40% Water (0.1% OPA) 234 nm, Flow rate 0.7ml.	20 μl	Sharpe peaks were not obtained	Hence rejected
5	C18 (AGILEN T) (100×4.6mm, 2μ)	40% Methanol: 0% Water (0.1%OPA)- 234 nm , Flow rate 0.7ml	20 μl	Sharpe peaks were not obtained	Hence rejected
6	C18 (AGILEN T) (100×4.6m)	40% Methanol: 60% Water (0.1%OPA)-	20 μl	Resolved Sharpe peaks	Hence Selected

Chromatogram of Final Trial:

Chromatogram of Ritonavir and Nirmatrelvir

No.	RT [min]	*Area [mVs]**	TP	TF	Resolution
1	4.039	684.33228	7204	0.77	-
2	5.653	2251.54126	8821	0.77	7.48

Statistical data analysis (DOE)

Layout of Actual Design of DOE

	Factor 1	Factor 2	Factor 3	Response 1	Response 2	Response 3
Run	A:Methanol	B:Flow rate	C:Wavelength	RT	PA	TP
	%	ml	nm
1	41	1.1	234	3.9	637.57	7030
2	41	1.2	235	3.793	573.73	6833
3	41	1	233	3.739	746.51	7490
4	41	1.2	233	3.649	614.86	6648
5	41	1.1	234	3.828	639.2	7135
6	42	1.1	235	3.695	589.48	6991
7	41	1.1	234	3.813	635.95	7080
8	42	1.1	233	3.685	684	6955
9	42	1.2	234	3.459	618.18	7093
10	42	1	234	3.927	704.41	7323
11	41	1.1	234	3.794	635.11	7008
12	40	1.1	233	3.72	673.52	7090
13	40	1.2	234	3.468	604.08	6814
14	41	1.1	234	3.761	635.16	7010
15	40	1	234	4.047	706.53	7591

Layout of Actual Design of DOE of Nirmatrelvir

	Factor 1	Factor 2	Factor 3	Response 4	Response 5	Response 6
Run	A:Methanol	B:Flow rate	C:Wavelength	RT 2	PA 2	TP 2
	%	Ml	nm
1	41	1.1	234	5.209	2318.57	8903
2	41	1.2	235	5.23	2297.03	8523
3	41	1	233	5.795	2449.58	9169
4	41	1.2	233	4.998	2133.03	8404
5	41	1.1	234	5.212	2315.27	8680
6	42	1.1	235	4.931	2350.17	8321
7	41	1.1	234	5.187	2305.54	8819
8	42	1.1	233	4.922	2233.38	8588
9	42	1.2	234	4.474	2237.3	8635
10	42	1	234	5.102	2509	8988
11	41	1.1	234	5.166	2301.73	8748
12	40	1.1	233	5.04	2209.94	8770
13	40	1.2	234	4.613	2189.41	8418
14	41	1.1	234	5.102	2294.04	8534
15	40	1	234	5.3502	2529.63	9382
16	40	1.1	235	5.253	2369	8599
17	41	1	235	5.645	2577.01	9225

ANOVA for response surface Quadratic model

The analysis of variance (ANOVA) was performed to identify the significant and insignificant factors. The results of ANOVA for the retention time of DOE are as following Table no.31.

Response 1: RT

Fit Summary of Peak Area

Source	Sequential p- value	Lack of Fit p- value	Adjusted R²	Predicted R²
Linear	0.0009	0.0553	0.6377	0.4194	Suggested
2FI	0.5901	0.0407	0.6077	-0.1117
Quadratic	0.3708	0.0318	0.6322	-1.2653
Cubic	0.0318		0.9142		Aliased

Sequential Model Sum of Squares [Type I]

Source	Sum of Squares	df	Mean Square	F-value	p-value
Mean vs Total	242.90	1	242.90
Linear vs Mean	0.3509	3	0.1170	10.39	0.0009	Suggested
2FI vs Linear	0.0245	3	0.0082	0.6690	0.5901
Quadratic vs 2FI	0.0419	3	0.0140	1.22	0.3708
Cubic vs Quadratic	0.0694	3	0.0231	8.67	0.0318	Aliased
Residual	0.0107	4	0.0027
Total	243.40	17	14.32

Select the highest order polynomial where the additional terms are significant and the model is not aliased.

Graphical Presentation:For RT of Ritonavir:

Color Point by Value of Peak Area Residuals Vs Run

Final Equation in Terms of Coded Factors

RT2=+5.18-0.1034A-0.3221B+0.0380C+0.0273AB-0.0510AC+0.0955BC- 0.3355A²+0.0450B²+0.1968C²

The equation in terms of coded factors can be used to make predictions about the response for given levels of each factor. By default, the high levels of the factors are coded as +1 and the low levels are coded as -1. The coded equation is useful for identifying the relative impact of the factors by comparing the factor coefficients.

Graphical Presentation: For RT 2 of Nirmatrelvir:

Color point by value of RT2 Residuals vs Run

Final Equation in Terms of Coded Factors

TP2=+8747.41-47.63A-348.00B-32.87C

Linearity data for Ritonavir

Method	Conc. µg/ml	Peak area(µV.sec)		Average peak area (µV.sec)	S.D. of Peak Area	% RSD of Peak Area
Method	Conc. µg/ml	1	2	Average peak area (µV.sec)	S.D. of Peak Area	% RSD of Peak Area
RP- HPLC Method	10	130.6188	130.4198	130.5193	0.14	0.11
	20	262.0044	262.6045	262.3045	0.42	0.16
	30	400.6041	399.8734	400.2388	0.52	0.13
	40	532.3975	531.4891	531.9433	0.64	0.12
	50	654.0488	652.0862	653.0675	1.39	0.21
	Equation		y = 13.147x-1.194
	R2		0.999

Fig: Calibration curve of Ritonavir

The RP-HPLC Method for respective linear equation for Ritonavir was y = 13.147X+1.194where x is the concentration and y is area of peak. The correlation coefficient was 0.999. The calibration curve of Ritonavir.

Linearity data for Nirmatrelvir

Method	Conc. µg/ml	Peak area(µV.sec)		Average peak area (µV.sec)	S.D. of Peak Area	% RSD of Peak Area
Method	Conc. µg/ml	1	2	Average peak area (µV.sec)	S.D. of Peak Area	% RSD of Peak Area
RP- HPLC Method	15	480.4581	475.4668	477.9625	3.5294	0.7384
	30	965.4802	965.4014	965.4408	0.0557	0.0058
	45	1457.3192	1453.3624	1455.3408	2.7979	0.1922
	60	1954.6055	1957.5051	1956.0553	2.0503	0.1048
	75	2415.5266	2409.8601	2412.6934	4.0068	0.1661
	Equation		y = 32.401x + 4.524
	R2		0.999

Fig: Calibration curve of Nirmatrelvir

The RP-HPLC method for respective linear equation forNirmatrelvirwas y = 32.401 x- 4.524where x is the concentration and y is area of peak. The correlation coefficient was 0.999. The calibration curve of Nirmatrelviris.

Calibration Curve for HPLC Method Regression Equation Data for Ritonavir

Regression Equation Data Y=mx+c
Slope(m)	13.147x
Intercept(c)	1.194
Correlation Coefficient	0.999

Linearity of Nirmatrelvir

Concentration μg/ml	Area Nirmatrelvir
15	477.9625
30	965.4408
45	1455.3408
60	1956.0553
75	2412.6934

Result of Recovery data for Ritonavir and Nirmatrelvir

Drug	Level (%)	Amt. taken (μg/ml)	Amt. Added (μg/ml)	*Absorbance Mean ± S.D.**	*Amt. recovered Mean±S.D.**	*%Recovery Mean ± S.D.**
RITO	50%	10	5	15.00±0.048	5.00±0.048	99.97±0.97
	100%	10	10	19.93±0.043	9.93±0.043	99.34±0.43
	150%	10	15	24.91±0.041	14.91±0.041	99.40±0.27
NIRM A	50%	15	7.5	22.55±0.031	7.55±0.031	99.65±0.06
	100%	15	15	29.89±0.037	14.89±0.03	100.27±0.75
	150%	15	22.5	37.68±0.040	22.68±0.040	100.81±0.18

*mean of each 3 reading for RP-HPLC method

Statistical Validation of Recovery Studies Ritonavir and Nirmatrelvir

METHOD	Level of Recovery (%)	Drug	Mean % Recovery	Standard Deviation*	% RSD
Rp-HPLC Method	50%	RITO	99.97	0.97	0.97
	50%	NIRMA	100.64	0.41	0.40
	100%	RITO	99.34	0.43	0.43
	100%	NIRMA	99.24	0.24	0.25
	150%	RITO	99.40	0.27	0.27
	150%	NIRMA	100.81	0.18	0.17

System suitability parameters: (Repeatability)

To ascertain the resolution and reproducibility of the proposed chromatographic system for estimation of Ritonavir and Nirmatrelvir system suitability parameters were studied. The result shown in below.

Fig: Chromatogram of System suitability -1(30+45 mcg)

Table: Chromatogram of System suitability -1(30+45 mcg)

No.	RT[min]	*Area[mVs]**	TP	TF	Resolution
1	3.541	401.22311	6766	0.76	0.000
2	4.692	1414.52502	8240	0.76	6.06

Fig: Chromatogram of System suitability No- 2(30+45 mcg)

Table: Chromatogram of System suitability No- 2 (30+45 mcg)

No.	RT [min]	*Area [mVs]**	TP	TF	Resolution
1	3.541	411.1132	6916	0.79	-
2	4.692	1516.4256	8150	0.78	6.06

Table: Repeatability studies on RP-HPLC for Ritonavir and Nirmatrelvir

Method	Concentration of Ritonavir and Nirmatrelvir(mg/ml)	Peak area	Amount found (mg)	% Amount found
RP- HPLC Method for RITO	30	401.2231	30.42	101.41
	30	401.1132	30.40	101.39
		Mean	30.41	101.40
		SD	0.08	0.08
		%RSD	0.02	0.02
RP- HPLC Method for NIRMA	45	1414.5200	45.37	100.82
	45	1516.4200	45.40	100.80
		Mean	45.39	100.81
	45	SD	72.05	72.05
	45	%RSD	4.92	4.92

Repeatability studies on RP-HPLC method for Ritonavir and Nirmatrelvir was found to be 101.40 and 100.81, The %RSD was less than 2%, which shows high percentage amount found in between 98% to 102% indicates the analytical method that concluded (Table No.88).

Precision:-

The method was established by analyzing various replicates standards of Ritonavir and Nirmatrelvir. All the solution was analyzed thrice in order to record any intra-day & inter-day variation in the result that concluded. The result obtained for intraday is shown in( Table No. 92) respectively.

Chromatogram of Precision:

Fig No.: Chromatogram of Precision

Table no: Chromatogram of Precision

No.	RT [min]	*Area [mVs]**	TP	TF	Resolution
1	3.546	129.94199	6440	0.77	-
2	4.859	477.02600	7941	0.77	6.64

Chromatogram of Intraday Precision:-

Fig No.: Chromatogram of intraday Precision (10+15 mcg)

Table No : Chromatogram of intraday Precision (10+15 mcg)

No.	RT [min]	*Area [mVs]**	TP	TF	Resolution
1	3.524	131.90094	6527	0.77	-
2	4.808	476.78275	7983	0.77	6.58

*Each 3 concentration they have 2 reading forintraday Precision (10+15 mcg),(40+45 mcg,50+75 mcg)

Result of Intraday and Inter day Precision studies on RP-HPLC for Ritonavir and Nirmatrelvir

Drug	Concⁿ(µg/ml)	Intraday Precision		%	Interday Precision		%
Drug	Concⁿ(µg/ml)	Mean ± SD	% Amt Found	RSD	Mean ± SD	% Amt Found	RSD
RITO	10	130.9± 1.40	98.66	1.07	130.41± 1.20	98.28	0.92
	30	399.36±0.6	100.95	0.15	399.03±0.38	100.87	0.09
	50	666.2±3.77	101.17	0.57	5182.53±3.29	100.48	0.09
NIRMA	15	476.6± 0.53	99.00	0.11	478.66±0.64	99.42	0.13
	45	1459.6±0.5	100.42	0.04	1466.07±0.14	100.86	0.01
	75	2420.6±16.	99.80	0.68	2434.19±8.22	100.36	0.34

*Mean of each 3 concentration they have 2 reading.

Robustness:

Flow rate change 1.1ml

No.	RT[min]	*Area[mVs]**	TP	TF	Resolution
1	3.191	640.47089	6035	0.75	-
2	4.195	2334.12964	7371	0.76	5.57

Flow Rate Change 0.9ml

Chromatogram of flow change 0.9 ml T Chromatogram of flow change 0.9ml

No.	RT [min]	*Area [mVs]**	TP	TF	Resolution
1	3.620	730.8396	6888	0.75	-
2	4.670	2601.26318	8391	0.74	5.54

Parameters	Conc. (µg/ ml)	Amount of detected (mean±SD)	% RSD
Chromatogram of flow change 0.6 ml	50	666.69±2.51	0.38
Chromatogram of flow change 0.8 ml	50	640.91±0.62	0.10
Chromatogram of comp change 40 MEOH + 60 WATER	50	731.4±0.73	0.10
Chromatogram of comp change 42 MEOH + 58 WATER	50	704.47±2.64	0.38
Chromatogram of comp change wavelength change 233nm	50	665.5±4.18	0.63
Chromatogram of comp change wavelength change 235 nm	50	645.33±3.85	0.60

Robustness Study of Ritonavir:

Parameters	Conc. (µg/ml)	Amount of detected (mean ± SD)	% RSD
Chromatogram of flow change 0.6 ml	75	2523.73±14.5	0.58
Chromatogram of flow change 0.8 ml	75	2323.50±15.03	0.65
Chromatogram of comp change 40Meoh +60 WATER	75	2597.7±4.99	0.19
Chromatogram of comp change 42MEOH + 58 WATER	75	2527.13±15.57	0.62
Chromatogram of comp change wavelength change 233nm	75	2440.1±0.01	0.00
Chromatogram of comp change wavelength change 235 nm	75	2571.63±0.90	0.002

Limit Detection

The LOD is the lowest limit that can be detected. Based on the S.D. deviation of the response and the slopethe limit of detection (LOD) may be expressed as:

LOD = 3.3 (SD)/S

Where, SD = Standard deviation of Y intercept

S = Slope

Limit of detection = 3.3X0.62/13.147= 0.1562 (μg/mL)

Limit of Quantitation= 10X 0.62/13.147= 0.4733 (μg/mL)

The LOD and LOQ of Ritonavir was found to be 0.1562 (μg/mL) and 0.4733 (μg/mL), analytical method that concluded.

Limit Quantification

The LOQ is the lowest concentration that can be quantitatively measured. Based on the S.D. deviation of the response and the slope,

The quantitation limit (LOQ) may be expressed as:

LOQ = 10 (SD)/ S

Where, SD = Standard deviation Y intercept

S = Slope

Limit of detection = 3.3 X 2.49/32.401 = 0.2534 (μg/mL)

Limit of Quantitation= 10 X 2.49/32.401 = 0.76788 (μg/mL)

The LOD and LOQ of Nirmatrelvir was found to be 0.2534 (μg/mL) and 0.76788 (μg/mL), analytical method that concluded.

Analysis of tablet formulation:-

Procedure:

Weigh 20 Ritonavir and Nirmatrelvir combination Tablets and calculated the average weight, accurately weigh and transfer the sample equivalent to 10 mg Ritonavir and 15 mg Nirmatrelvir into 10 ml volumetric flask. Add about 10ml MEOH of diluents and sonicate to dissolve it completely and make volume up to the mark with diluent. Mix well and filter through 0.45 µm filter. Further pipette 0.2 ml of the above stock solution into a 10 ml volumetric flask and dilute up to the mark with diluents.(20+30µg/ml). The simple chromatogram of test Ritonavir and Nirmatrelvir Shown in (Fig No:87) The amounts of Ritonavir and Nirmatrelvir per tablet were calculated by extrapolating the value of area from the calibration curve. Analysis procedure was repeated five times with tablet formulation. Tablet Assay for % Lable claim for %RSD Calculated, Result was shown in (Table No. 101,102).

Brand Name: Shytomel Duo47 (Derma medicine Point)

Total weight of 20 Tab wt. = 11.98 Gms - Avgr Weight = 0.599Gms./Tab

Eq. wt for 15 mg = 15 X 599/150 = 59.99 mg

Take 59.99 mgs in 10 ml water Sonicate 10 min i.e. 1000 µgm/ml Ritonavir and 1500 µgm/ml Nirmatrelvir ------ STOCK -I

Take 0.2 ml in 10 ml meoh = 20 µgm/ml RITO and 30 µgm/ml NIRMA.

Chromatogram for Marketed Formulation (20+30 mcg)

Table. No: Result Chromatogram of Marketed Formulation (20+30 mcg)

No.	RT[min]	*Area[mVs]**	TP	TF	Resolution
1	3.554	265.14239	6811	0.77	-
2	4.873	965.3200	8198	0.77	6.80

Analysis of marketed formulation.

Assay	Drug	conc	Amt. Found	% Lable Claim	SD	%RSD
Rp-HPLC Method	RITO	20	20.07	100.38	0.264	0.26
	NIRMA	30	29.93	99.78	0.081	0.082
	RITO	20	20.1510	100.76	0.264	0.0262
	NIRMA	30	29.89	99.66	0.082	0.082

Analysis of marketed formulation were also % Lable Claim was found to be 98-102 % Satisfactory are concluded.

FORCED DEGRADATION STUDIES

Forced degradation study was performed to evaluate the stability of the developed method using the stress conditions like exposure of sample solution to acid (0.1 N HCl), base (0.1 N NaOH), Hydrogen peroxide (H2O2) and Neutral. Investigation was done for the degradation products.

Results of Forced degradation studies

Stress conditions	RITO	NIRMA
Stress conditions	(%) Degradation 2 Hr	Degradation (%) 2 Hr
Acetic hydrolysis	6.17	3.80
Alkaline hydrolysis	100.00	18.67
Peroxide Degradation	17.89	4.45
Neutral Degradation	10.63	4.16

Degradation Nirmatrelvir and Ritonavir Acid hydrolysis Degradation

Chromatogram of Acid hydrolysis RITO+NIRMA AFTER 1hr

Chromatogram of Acid hydrolysis RITO+NIRMA AFTER 1 hr

No.	RT[min]	*Area[mVs]**	Area %
1	1.928	8.1365	0.4222
2	2.165	1.44051	0.0748
3	2.526	0.000	0.0
4	2.593	1.82364	0.0946
5	3.544	375.53690	21.5633
6	4.624	1400.11890	77.8451

In this chromatogram of acid degradation has lead to formation of degrading and calculate % Degradation of drug6.17 % and 3.80%.

Alkali hydrolysis degradation

Chromatogram Alkali hydrolysis RITO + NIRMA 1Hr

Table no. Chromatogram Alkali hydrolysis RITO + NIRMA 1Hr

No.	RT[min]	*Area[mVs]**	Area%
1	2.250	16280.2	42.22
2	2.479	13468.7	34.93
3	2.659	8161.9355	21.1600
4	4.634	1183.63513	1.6694

In this chromatogram of alkali degradation has lead to formation of degradant and calculate % Degradation of drug 100.0-18.67%.

Hydrogen Peroxide Degradation

Fig. No. Chromatogram of Hydrogen Peroxide RITO+NIRMA1Hr

Table no Chromatogram of Hydrogen Peroxide RITO+NIRMA 1Hr

No.	RT[min]	*Area[mVs]**	Area %
1	1.907	5.80332	0.0974
2	2.483	3802.07495	63.8352
3	3.482	328.63354	6.6929
4	4.629	1390.57678	25.0261
5	5.380	246.26793	4.1347
6	6.388	12.72714	0.2137

In this chromatogram of H2O2 degradation has led to formation degrading and calculate % Degradation of drug 17.89 and 4.45 %.

CONCLUSION

The study was done by using 3² full fraction response surface designs. In this study interaction of 2 factors; flow rate, mobile phase composition at 3 level he study was done by using 3² full fraction response surface designs. In this study interaction of 2 factors; flow rate, mobile phase composition at 3 levels. The Limit of Detection (LOD) and Limit of Quantitation (LOQ) were established at a signal-to-noise ratio. LOD and LOQ were calculated as 3.3×δ/S and 10×δ/S respectively as per ICH guidelines.

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https://www.rxlist.com/Ritonavir
https://www.rxlist.com/Nirmatrelvir
http://en.wikipedia.org/wiki/Nirmatrelvir
http://en.wikipedia.org/wiki/ Ritonavir
The Merck Index: An encyclopedia of chemical drugs and biological mono
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