Development and validation of a sustainable UHPLC method for quantification and impurity profiling of carfilzomib

Abstract

Carfilzomib, a second-generation proteasome inhibitor, is widely used in the treatment of multiple myeloma. This synthetic tetra-peptide epoxyketone derivative binds irreversibly to the 20 S proteasome, leading to the accumulation of misfolded proteins and apoptosis of malignant cells. Despite its FDA approval, maintaining the purity of carfilzomib remains a challenge due to potential impurities affecting its stability, efficacy, and safety. Various studies have identified key carfilzomib related substances, including Epoxy amine, Epoxy RR isomer, Epoxy SS isomer, Epoxy RS isomer, N-BOC Epoxy impurity, Alkene impurity, D-Phenyl alanine, Diol impurity and N-Oxide impurity, which may arise during synthesis, storage, or degradation. Although mass spectrometry and NMR techniques have been used to identify impurities, a robust analytical method for comprehensive impurity profiling is still lacking. In this study, a gradient UHPLC-UV analytical method was developed to separate and quantify carfilzomib and its impurities efficiently. The method achieved precise separation by resolving impurities and carfilzomib peak within 22 min. Linearity for related substances was observed from LOQ to 150% of the specification level (0.5 µg/mL to 2.25 µg/mL), while the assay method exhibited a correlation coefficient of 0.9983 within a range of 25.54 µg/mL to 76.64 µg/mL. A forced degradation study and mass balance confirmed the accuracy of impurity quantification. Additionally, the method demonstrated superior specificity, minimizing interference from structurally similar contaminants. Environmental sustainability was assessed using GAPI, AGREE, and BAGI tools, validating the method’s eco-friendliness and improved analytical performance.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-33731-y.

Introduction

Multiple myeloma, a malignant plasma cell disorder, accounts for approximately 10% of all hematologic malignancies. Carfilzomib exerts its therapeutic effect through selective proteasome inhibition. Proteasome is a self-regulated protease complex responsible for the ubiquitin-dependent degradation of cellular proteins. Its inhibition leads to the accumulation of substrate proteins, ultimately triggering cell death¹. Carfilzomib is a tetra-peptide epoxy ketone derivative used in the treatment of multiple myeloma and is structurally and mechanistically distinct from bortezomib. It selectively and irreversibly inhibits the chymotrypsin-like activity of the 20 S proteasome, thereby disrupting its function. This inhibition halts cell proliferation and induces apoptosis, the programmed cell death process. Chemically, carfilzomib is known as (2S)-N-((S)-1-((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-ylcarbamoyl)-2-phenylethyl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-4-methylpentanamide, and its molecular structure is depicted in Fig. 1².

Fig. 1.

Structure of carfilzomib.

The chemical stability of a drug substance directly impacts the efficacy of its drug product. As a peptide-based molecule, carfilzomib is prone to the formation of various diastereomeric and process-related impurities. Therefore, it is critical to monitor related substances in the drug substance. However, limited information is currently available on the quantification of these impurities, and carfilzomib has not yet been included in the United States Pharmacopoeia (USP) or British Pharmacopoeia (BP).

A stability-indicating HPLC method utilizing the DoE approach has been reported for the quantification of diastereomeric and process-related impurities of carfilzomib. However, this method has an extended run time of 100 min and a flow rate of 0.9 mL/min, leading to high solvent consumption and reduced cost-effectiveness³. A UHPLC-UV-QTOF method was published to analyze carfilzomib within the concentration range of 10–250 µg/mL and evaluate the stability of carfilzomib through forced degradation studies. Although this method features a 6 min run time and enables monitoring of five impurities which are eluted in close proximity to one another, Q-TOF is required for identification of impurities⁴. High-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) were used to characterize two impurities of carfilzomib⁵; however, these advanced techniques necessitate high-end instrumentation, making them impractical for routine analytical applications. LC-MS/MS method was reported for quantification of carfilzomib in mouse plasma with pharmacokinetic application⁶. A bioanalytical method employing HPLC has been published for monitoring carfilzomib levels in human plasma during preclinical studies⁷. Considering these factors, there is a need for an Ultra-high performance liquid chromatography (UHPLC) method that is accurate, precise, robust, and cost-effective for determining related substances in the carfilzomib drug substance. UHPLC focuses on three key aspects: speed, resolution, and sensitivity. Utilizing a UHPLC method with a column packed with sub-2 μm particles offers multiple advantages, including improved resolution, high sensitivity, reduced analysis time, and minimized solvent consumption. As a result, UHPLC has become a standard approach for both laboratory and industrial analysis of drug substances and products, providing benefits such as lower costs, faster results, and higher throughput⁸.

The present study aimed to develop and validate an analytical UHPLC method for carfilzomib and its nine impurities. The method development prioritized minimizing time and mobile phase consumption through optimized chromatographic conditions. In the gradient system, carfilzomib and its impurities including diol impurity, N-oxide impurity, epoxy amine impurity, epoxy SS isomer impurity, epoxy RR isomer impurity, D-phenyl alanine impurity, epoxy RS isomer impurity, alkene impurity, and N-BOC epoxy impurity were efficiently separated.

Additionally, this study emphasized the environmental sustainability of newly developed methods by incorporating eco-friendly solvents. The ecological impact of these solvents was assessed within the framework of Green Analytical Chemistry metrics, including GAPI, AGREE, and BAGI, reinforcing the sustainability and feasibility of the proposed approach^9–11.

Experimental

Materials and methods

Carfilzomib with purity of 99.60% and its impurities (Purity > 80%) were obtained from Zydus Lifescience Ltd (Ahmedabad, India). Acetonitrile and methanol, utilized as solvents, were of high-performance liquid chromatography (HPLC) grade (Make – J. T. Baker), whereas potassium dihydrogen phosphate and potassium hydroxide, employed as reagents, were of analytical reagent (AR) grade (Make – Merck). All chemicals were obtained from Zydus Lifesciences Ltd (Ahmedabad, India). Ultrapure water (Resistivity of 18.2 MΩ⋅cm at 25 °C and a Total Organic Carbon (TOC) level of ≤ 5 ppb) from the Millipore purification system was used throughout the experiments to ensure the accuracy and reliability of the results. All reagents were stored according to the manufacturer’s specifications to maintain their integrity before use.

Instrumentation

The liquid chromatographic analysis was conducted on Agilent 1290 Infinity II (UHPLC) system from Agilent Technologies. It was equipped with an ultraviolet (UV), photo diode array (PDA) detector, auto sampler, and gradient pump with degasser. Acquisition of chromatographic data was done using Chromeleon software version 7.2. Weighing of standards and sample was performed using Sartorius Secura225D-10IN analytical balance and Cubis MCA6.6-20IN-M micro balance. The adjustment of pH was performed using Thermo Scientific orion star A211 Benchtop pH meter.

Method for related substances of carfilzomib

Preparation of mobile phase

Buffer solution of 0.02 M Potassium dihydrogen phosphate was prepared and adjusted pH 5.5 using 10% KOH solution. Mobile phase A consisted of buffer solution, acetonitrile and methanol in a ratio 750:200:50 (v/v/v). Mobile phase B consisted of water, acetonitrile and methanol in a ratio 200:750:50 (v/v/v).

System suitability and sample preparation

The diluent utilized for both the sample and standard preparations was prepared by mixing water and acetonitrile in a 40:60 (v/v) ratio.

Each impurity Carfilzomib Epoxy amine, Carfilzomib Epoxy RR isomer, Carfilzomib Epoxy SS isomer, Carfilzomib Epoxy RS isomer, N-BOC Epoxy impurity, Alkene impurity, D-Phenyl alanine, Diol impurity and Carfilzomib N-Oxide impurity were weighed and dissolved using diluent to prepare each impurity stock solution of 75 µg/mL. The structures of carfilzomib impurities are presented in Fig. 2.

Fig. 2.

Structure of carfilzomib impurities.

The resolution solution was prepared by dissolving Carfilzomib at a concentration of 1000 µg/mL, with each individual impurity spiked into the solution at a concentration of 1.5 µg/mL.

A sensitivity solution containing carfilzomib at a concentration of 0.5 µg/mL was prepared.

The sample solution of 1000 µg/mL was prepared by accurately weighed and transferred 10 mg carfilzomib API into 10 mL volumetric flask. It was dissolved in 5 mL of diluent using sonication. The solution was made up to the mark using diluent and mixed well.

Chromatographic condition

Chromatographic separation was performed using a Hypersil GOLD™ C18 Selectivity reversed-phase column (150 mm × 2.1 mm, 1.9 μm; Part No. 25002–152130), supplied by Thermo Scientific, which served as the stationary phase. A linear gradient elution was employed using mobile phase A and mobile phase B. The gradient program was set as follows: 0 min, 50% B; 5.0 min, 50% B; 15.0 min, 55% B; 20.0 min, 100% B; 23.0 min, 100% B; 24.0 min, 50% B; and 30.0 min, 50% B. The flow rate was 0.25 mL/min, and the injection volume for sample was 5 µL. Sample temperature and column temperature were maintained at 5 °C and 30 °C, respectively. The acquisition was performed at 210 nm using UV detector.

Procedure for assay of carfilzomib

Preparation of mobile phase

Buffer solution of 0.01 M Potassium dihydrogen phosphate was prepared and adjusted pH 5.5 using 10% KOH solution. Mobile phase A consisted of buffer solution, acetonitrile and methanol in a ratio 700:200:100 (v/v/v). Mobile phase B consisted of water, acetonitrile and methanol in a ratio 200:700:100 (v/v/v).

Standard and sample preparation

The diluent utilized for both the sample and standard preparations was prepared by mixing water and acetonitrile in a 40:60 (v/v) ratio. Standard and sample solutions of carfilzomib were prepared at a concentration of 50 µg/mL.

Chromatographic condition

Chromatographic separation was performed using a Hypersil GOLD™ C18 Selectivity reversed-phase column (100 mm x 2.1 mm, 1.9 μm, part no. − 25002–102130) supplied by Thermo Scientific which served as the stationary phase. A linear gradient elution was employed using mobile phase A and mobile phase B. The gradient program was set as follows: 0 min, 55% B; 4.0 min, 60% B; 4.1 min, 100% B; 5.5 min, 100% B; 6.0 min, 55% B; and 10.0 min, 55% B. The flow rate was 0.4 mL/min, and the injection volume for sample was 5 µL. Sample temperature and column temperature were maintained at 5 °C and 30 °C, respectively. The acquisition was performed at 210 nm using UV detector.

Method validation

Method validation was performed in accordance with the validation guidelines^12–14 for parameters such as specificity, linearity, LOD, LOQ, precision, accuracy, robustness, and stability of the analytical solution.

Forced degradation

Forced degradation studies of the carfilzomib drug substance were conducted to evaluate its stability under various stress conditions. The drug was subjected to acid hydrolysis, alkali hydrolysis, oxidative degradation, thermal degradation, and photolytic degradation, in accordance with ICH guidelines.

Results and discussion

Optimization of chromatographic condition

The major objective of mobile phase and column optimization was to obtain good separation with acceptable resolution, peak shape, and sensitivity within shorter run time. Based on literature, the mobile phase buffer was prepared using potassium dihydrogen phosphate, with the pH-5.5 precisely adjusted³. This buffer system was selected due to its effective buffering capacity in the mildly acidic range, making it suitable for maintaining consistent chromatographic conditions and ensuring analyte stability during separation. Multiple mobile phase compositions were systematically evaluated using change in the mobile phase composition by adjusting buffer, acetonitrile, methanol and water through gradient elution mode. To enhance chromatographic resolution and improve peak selectivity, multiple columns from different manufacturers, each with varying stationary phase chemistries, particle sizes, and dimensions were systematically evaluated. This comparative approach select Hypersil GOLD™ C18 reversed phase column that provided optimal separation and selectivity between closely eluting peaks ensuring reliable quantification and reproducibility of the method.

Method validation for related substances of carfilzomib

Analytical method for Carfilzomib and its related substances was developed and validated as per ICH Q2 (R2) guidelines. The parameters covered for method validation included system suitability, specificity, linearity, accuracy, precision, robustness and stability of analytical solution.

System suitability was assessed by injecting sensitivity solution and resolution solution. Signal to noise(S/N) ratio was measured in sensitivity solution for Carfilzomib peak and it should not be less than 10. S/N ratio was found 520 for Carfilzomib peak. Resolution between carfilzomib peak and D-Phenyl alanine Impurity peak should not be less than 1.5. Resolution between carfilzomib peak and D-phenyl alanine Impurity peak was found 3.2 indicate carfilzomib peak and D-phenyl alanine impurity peaks are well resolved and system is suitable for analysis. Figure 3 illustrates the chromatogram of resolution solution.

Fig. 3.

Chromatogram of resolution solution for carfilzomib and its related substances.

Specificity was assessed by injecting the mobile phase, diluent and each impurity at a concentration of 1.5 µg/mL. No interference was observed at the retention times corresponding to the impurities and the Carfilzomib peak.

Linearity for Carfilzomib and its impurities was evaluated over a concentration range from the limit of quantitation (LOQ) to 150% of the specification limit. The correlation coefficient for each analyte was required to be not less than 0.990.

Table 1 summarizes the results of linearity and Fig. 4 illustrates graph between concentration (µg/mL) vs. Area (mAU*sec).

Table 1.

Linearity equation, LOD and LOQ for Carfilzomib and related substances.

Analyte	RRT	Linearity range (µg/mL)	Linearity equation	Correlation coefficient	LOD (µg/mL)	LOQ (µg/mL)
Carfilzomib	1.00	0.488–2.438	y = 42.500x − 0.222	0.9999	0.163	0.488
Diol impurity	0.44	0.458–2.290	y = 37.709x − 0.130	0.9999	0.153	0.458
N-oxide impurity	0.49	0.466–2.332	y = 42.679x − 0.244	0.9999	0.155	0.466
Epoxy amine Impurity	0.60	0.476–2.379	y = 41.957x − 1.578	0.9998	0.159	0.476
Epoxy SS isomer impurity	0.81	0.461–2.307	y = 33.123x − 3.231	0.9988	0.154	0.461
Epoxy RR isomer impurity	0.87	0.430–2.150	y = 37.064x − 0.525	0.9986	0.143	0.430
D-phenyl alanine Impurity	1.09	0.428–2.139	y = 42.677x − 0.154	0.9998	0.143	0.428
Epoxy RS isomer impurity	1.11	0.466–2.328	y = 43.363x − 0.304	0.9996	0.155	0.466
Alkene impurity	1.18	0.487–2.434	y = 48.284x − 0.291	0.9999	0.162	0.487
N-BOC epoxy impurity	1.63	0.447–2.223	y = 34.554x − 1.159	0.9998	0.149	0.447

Fig. 4.

Linearity plot for carfilzomib and its related substances.

Accuracy was assessed by determining recovery for spiked samples at three concentration levels (50%, 100% and 150%) from target concentration (100%). The acceptance criteria for the recovered spiked level shall remain within 80% – 120% and the % related standard deviation (RSD) shall not exceed 10%. Table 2 summarizes the results for mean recovery of the related substances.

Table 2.

Results of accuracy (recovery) study for carfilzomib related substances.

Analyte	Level	% Recovery (n = 3)	Standard deviation	% RSD
Diol impurity	50%	89.367	0.032	0.036
	100%	89.653	0.105	0.117
	150%	91.830	0.166	0.181
N-oxide impurity	50%	113.000	2.695	2.385
	100%	112.203	0.794	0.708
	150%	111.940	0.334	0.299
Epoxy amine impurity	50%	97.203	0.413	0.425
	100%	97.180	0.201	0.207
	150%	99.197	0.289	0.292
Epoxy SS isomer impurity	50%	83.933	3.958	4.715
	100%	92.567	0.656	0.709
	150%	97.930	0.584	0.596
Epoxy RR isomer impurity	50%	104.333	2.619	2.510
	100%	94.487	0.816	0.863
	150%	96.523	0.373	0.386
D-Phenyl alanine Impurity	50%	111.433	3.845	3.450
	100%	107.407	0.662	0.617
	150%	105.817	0.936	0.885
Epoxy RS isomer impurity	50%	94.970	2.700	2.843
	100%	91.690	0.765	0.834
	150%	94.457	0.768	0.813
Alkene impurity	50%	95.717	0.380	0.397
	100%	94.987	0.324	0.341
	150%	96.537	0.243	0.252
N-BOC epoxy impurity	50%	104.877	0.487	0.464
	100%	103.017	0.267	0.259
	150%	103.883	0.060	0.058

Method precision was assessed by preparing six replicate of spiked sample preparation. % RSD for each impurity of six replicate injections should not be more than 10%. Table 3 represents method precision for carfilzomib related substances.

Table 3.

Method precision for carfilzomib related substances.

Name of impurity	Diol impurity	Carfilzomib N-oxide impurity	Epoxy amine impurity	Epoxy SS isomer impurity	Epoxy RR isomer impurity	D-Phenyl alanine impurity	Epoxy RS isomer impurity	Alkene impurity	N-BOC epoxy impurity
% RSD	3.5	2.7	3.1	4.1	3.0	2.9	2.8	2.2	3.8

Stability of analytical solution was evaluated at 5 °C in vial thermostat for sample preparation and it was found stable for about 30 h.

Robustness was performed by deliberate variation in analytical method parameter like column temperature (± 5 °C), Flow rate (± 10%) and pH of buffer in mobile phase (± 0.2). The system suitability parameters were evaluated and found to be compliant with all specified conditions. The results of system suitability for each condition are detailed in the below Table 4.

Table 4.

Robustness for carfilzomib related substances.

Changed method parameter	S/N ratio	Resolution between carfilzomib and D-Phenyl alanine peak
Increase in flow rate (0.275 ml/min)	311	2.8
Decrease in flow rate 0.225 ml/min)	345	2.9
Increase in column temperature (35 °C )	467	3.2
Decrease in column temperature (25 °C )	670	3.1
Increase in PH (pH − 5.7)	303	2.9
Decrease in PH (pH − 5.3)	311	3.0

Method validation for assay of carfilzomib

Specificity was checked by injecting diluent and impurities along with carfilzomib to check interference at carfilzomib retention time. There was no interference from diluent and known impurities at retention time of carfilzomib. Figure 5 shows the chromatogram of the carfilzomib standard for the assay method.

Fig. 5.

Chromatogram of standard of carfilzomib.

The analytical method was found linear between concentration 25.54 µg/mL to 76.64 µg/mL. The increment in area was in accordance with the increase in concentration. The correlation coefficient (r) was found to be 0.9991. Figure 6 display the linearity overlay chromatogram of the carfilzomib standard. The intraday and intermediate precision was performed at 50 µg/mL concentration for carfilzomib. The %RSD for method precision was assessed and was found to be 0.87% and 0.99% for Intraday and intermediate precision, respectively and results are summarized in Table 4. System suitability study was performed with replicate injections (n = 5). The %RSD was found to be well within the limit, which confirms the system suitability. The robustness for the analytical method was evaluated by making deliberate changes in column temperature, flow rate and pH. The %RSD for multiple injections (n = 6) at concentration 50 µg/mL was evaluated. The solution stability study was performed till 86 h and was found stable. The summary of validation parameters is presented in Table 5.

Fig. 6.

Linearity overlay chromatogram of carfilzomib standard.

Table 5.

Results of validation parameters for assay of carfilzomib.

Parameter	Observation
Linearity	Range (µg/mL) 25.54 µg/mL – 76.64 µg/mL								Linearity equation Y = 22.79x + 16.06
Recovery	Level			% Assay ± SD					% RSD
	50%			100.22 ± 1.28					1.27
	100%			98.57 ± 0.29					0.30
	150%			99.77 ± 1.06					1.06
Precision	Type			% Assay ± SD					% RSD
	Intraday (n = 6)			99.67 ± 0.87					0.87
	Intermediate (n = 6)			100.66 ± 1.00					0.99
System suitability	Area ± SD						% RSD (< 2.0%)
	1199.512 ± 4.90						0.4
Robustness	Optimum parameter			Column temperature (°C)			Flow rate (mL/min)				pH
				35	25		0.44	0.36			5.7	5.3
	Average Area ± SD	1184.31 ± 4.7		1182.75 ± 1.9	1185.26 ± 5.1		1079.47 ± 2.4	1319.88 ± 3.4			1195.71 ± 0.31	1192.10 ± 2.5
	% RSD	0.39		0.16	0.43		0.22	0.26			0.31	0.21

Forced degradation of carfilzomib

Degradation condition samples were injected in related substance method for quantification of degradation impurities. Same samples were diluted to assay method sample concentration and injected to assay method for quantification of carfilzomib. Mass balance was evaluated during forced degradation studies and found to be within the acceptable range of 95% to 105%.

Forced degradation study of Carfilzomib was performed using degradation condition given in below Table 6.

Table 6.

Results of forced degradation study for carfilzomib.

Degradation condition	Assay (%w/w)	Total degradation impurities (%)	Mass balance (%)
Sample as such	99.9	0.183	--
Acid degradation (Added 0.1 mL 0.1 N HCl, kept at RT for 90 min)	98.3	0.251	98.5
Alkali degradation (Added 0.1 mL 0.1 N NaOH, kept at RT for 90 min)	91.4	9.795	101.0
Oxidative degradation (Added 0.5 mL 0.3% H2O2, heat at 80 °C for 30 min)	75.3	23.197	98.4
Photolytic degradation (samples exposed to light providing an overall illumination of not less than 1.2 million lux hours and an integrated near ultraviolet energy of not less than 200 W hours/meter2)	96.1	0.205	96.2
Thermal degradation (Sample heat at 80 °C for 120 min in oven)	99.5	0.234	99.6

The forced degradation studies of carfilzomib under various stress conditions reveal its susceptibility to different degradation pathways. Oxidative degradation results in the formation of N-Oxide impurity and other oxidation-related products. Carfilzomib undergoes alkali degradation primarily due to the hydrolysis of its beta-lactone ring structure. The alkaline environment breaks the bond in the beta-lactone ring, leading to the degradation of the molecule. This degradation can also result in the formation of various impurities, including the Epoxy isomer and other degradation products. Thermal degradation at 80 °C leads to the development of impurities such as Epoxy amine, indicating heat-induced instability. Photolytic degradation under UV light exposure produces significant amounts of N-Oxide and D-Phenyl alanine impurities, confirming its sensitivity to light. Acid degradation in 0.1 N HCl generates multiple degradation products, including Epoxy isomers, affecting its stability. These findings emphasize the need for a robust stability-indicating method to ensure the efficacy and safety of carfilzomib throughout its shelf life. These findings highlight the importance of proper storage and handling conditions to maintain the drug’s stability. The developed UHPLC method effectively separates and quantifies carfilzomib and its impurities, making it a reliable stability-indicating tool. This study ensures the drug’s efficacy and safety by providing a comprehensive impurity profiling approach, essential for regulatory compliance and quality assurance. Figure 7 represents the chromatograms in various stress conditions including acidic, alkali, oxidative, photolytic and thermal degradation.

Fig. 7.

Forced degradation chromatograms.

Evaluation of UHPLC method through green analytical chemistry (GAC) principles

Green analytical chemistry emphasizes the importance of evaluating the environmental friendliness of analytical techniques. Several assessment metrics have been developed, including the analytical eco-scale, Green Analytical Procedure Index (GAPI), Analytical Greenness Metric (AGREE), and Blue Applicability Grade Index (BAGI). The analytical eco-scale assesses the sustainability or greenness of a selected method based on its environmental impact, considering factors such as reagents used, minimal solvent usage, low energy consumption, avoidance of hazards, and reduction of waste generation. An ideal value for green analysis is 100.

GAPI includes parameters representing low, medium, and high levels of environmental impact, represented by hexagonal glyphs. The AGREE index evaluates the analytical method based on the 12 principles of GAC, scored from 0 to 1. The AGREE approach is comprehensive but simple, allowing easy interpretation of results.

The Blue Applicability Grade Index (BAGI) evaluates the practicality of the analysis process, considering attributes such as analysis type, multi- or single-element analysis, analytical technique, simultaneous sample preparation, samples per hour, reagents and materials, pre-concentration, degree of automation, and number of samples. A method is considered green and capable of practical application when the BAGI index reaches at least 60. Figure 8 represents the pictograms of GAPI, AGREE and BAGI.

Fig. 8.

Method greenness tools assessment pictogram (a) GAPI (Green analytical Procedure Index) (b) AGREE (Analytical Greenness Metric Approach) (c) BAGI (Blue Applicability Grade Index).

The current UHPLC method for analyzing carfilzomib drug substance is considered a green analytical method with practical applicability. The method uses water and acetonitrile as the extraction solvent, automated analysis equipment, shorter run time, low flow rate and energy-saving practices. The eco-scale index and GAPI index evaluate the method, indicating its environmental friendliness. The AGREE evaluation tool assigns the method an overall score of 0.73, suggesting it needs to reduce environmental waste. The Blue Applicability Grade Index (BAGI) evaluates the method, scoring 75, indicating its practicality and applicability in real-world settings. Despite some disadvantages, such as using a flammable mobile phase and untreated waste, the method offers significant advantages, such as using an automated analytical equipment, and efficient analysis of multiple analytes in a single sample. Overall, the method is environment friendly and suitable for practical application.

Conclusion

Based on the validation results, it can be concluded that the developed stability-indicating UHPLC method for related substances is reliable for quantifying the specified impurities of carfilzomib. Furthermore, the stability-indicating UHPLC method for the assay has been proven to provide accurate quantification of carfilzomib. The results from the forced degradation study, specifically the mass balance analysis, validate that this method is appropriate for quantifying carfilzomib in drug product in bulk and commercial samples. This methods offers a shorter run time and lower flow rate, which will enable carfilzomib manufacturers and suppliers to quantify purity and related substances more efficiently, reducing both time and costs. Furthermore, its strong alignment with green analytical chemistry principles, as highlighted by AGREE, BAGI, and GAPI assessments, underscores its eco-conscious approach. This innovative approach not only improves pharmaceutical quality control but also establishes a new standard for sustainability, rendering it an invaluable asset in the field of modern analytical science.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Hitesh Patel: Conceptualization, Method development, Validation, Experimental work, Data collection, Data analysis, Writing—original draft.Gayatri Patel: Supervision, Guidance in experimental design, Critical review and editing of the manuscript.Payal Chauhan: Methodology, Validation, Writing—review and editing.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.