Skip to main content
Download PDF
Download ePub

Integrating green analytical chemistry and analytical quality by design: an innovative approach for RP-UPLC method development of ensifentrine in bulk and inhalation formulations

BMC Chemistry volume 19, Article number: 70 (2025) Cite this article

Abstract

Background

Chronic obstructive pulmonary disease (COPD) is a significant global health issue, worsened by pollution and modernisation. Ensifentrine (EFT), a new dual inhibitor of phosphodiesterase PDE3 and PDE4, is being developed for inhalation to target airway inflammation, bronchodilation, and ciliary function in COPD treatment.

Objective

This study aims to develop and validate a new quantification method for Ensifentrine, as no previous techniques are available, by integrating analytical quality-by-design (AQbD) and green analytical chemistry (GAC) principles.

Methods

An AQbD framework, utilizing Design-expert® software and a central composite design, optimized the RP-UPLC method. The optimized conditions involved isocratic separation on an ACQUITY UPLC HSS C18 SB column at ambient temperature, with a mobile phase of 0.01 N KH2PO4 (pH 5.4) and acetonitrile (66.4:33.6 v/v), a flow rate of 0.27 mL/min, and PDA detection at 272.0 nm.

Results

The statistical analysis confirmed the model’s significance and normal distribution. The method, validated according to ICH guidelines, showed good linearity (r2 = 0.9997) over a range of 3.75–22.5 μg/mL, with an LOD of 3.3 μg/mL and LOQ of 10 μg/mL. It was successfully applied to bulk materials and pharmaceutical formulations with statistical comparisons.

Green chemistry assessment

The greenness of the developed method was evaluated using tools such as ComplexMoGAPI, AGREE, BAGI, Green certificate-modified Eco-scale, and ChlorTox Scale. Additionally, the EVG method evaluation tool was also used to assess environmental impact, with the results shown in a radar chart.

Conclusion

This study presents a sensitive and robust RP-UPLC method for quantifying Ensifentrine, combining AQbD and GAC principles. The method, validated according to ICH guidelines, also ensures environmental sustainability. This approach sets a precedent for future analytical method development in pharmaceutical sciences with a focus on sustainability.

Graphical abstract

Peer Review reports

Introduction

Chronic obstructive pulmonary disease (COPD) is characterized by chronic respiratory inflammation, airway remodelling, acute exacerbations, and excessive mucus production, leading to irreversible airflow blockage. Despite available treatments, new therapies are needed to alleviate symptoms, provide bronchodilation, and reduce exacerbations without the adverse effects of corticosteroids or oral PDE4 inhibitors [1, 2]. Many patients continue to experience symptoms with existing treatments. Phosphodiesterase (PDE) 3 and PDE4 inhibitors play crucial roles in respiratory functions, as PDE3 regulates cAMP and cGMP in airway smooth muscle, influencing bronchial tone, while PDE4 modulates cAMP in inflammatory cells. Dual inhibition of PDE3 and PDE4 may have additional bronchodilator and anti-inflammatory effects [3,4,5]. Ensifentrine (RPL554), a novel dual PDE3 and PDE4 inhibitor designed for inhalation, targets airway inflammation and ciliary function [6]. Two Phase 3 maintenance therapy trials for COPD have been completed, and it is now in late-stage clinical development [7, 8]. Ensifentrine (EFT), developed by Verona Pharma plc, inhibits PDE3 and PDE4 and focuses on treating respiratory diseases such as COPD. Branded as OHTUVAYRE™, it was approved by U.S. regulatory authorities in June 2024 for COPD maintenance treatment in adults, marking a significant milestone in COPD management [9]. Figure 1 illustrates the structure of the drug EFT. The IUPAC name of EFT is 2-(9,10-dimethoxy-4-oxo-2-(2,4,6-trimethylphenyl) imino-6,7-dihydropyrimido(6,1-a) isoquinolin-3-yl) ethylurea, with a molecular formula of C26H31N5O4 and a molecular weight of 477.6 g/mol [10]. EFT, derived from the trequinsin nucleus, is a potent small molecule with dual selectivity for PDE3 and PDE4. It shows a high affinity for PDE3, approximately 3700 times greater than for PDE4, making it a promising candidate for treating diseases associated with dysregulated PDE activity [11].

Fig. 1
figure 1

The structure of the drug Ensifentrine (EFT)

Full size image

The conventional method development approach relies on molecular properties like polarity, pKa, and solubility, ofteemploying trial and error or one-variable-at-a-time techniques. However, these methods fail to reveal relationships between multiple variables. In contrast, a statistical QbD methodology offers significant advantages by understanding variable interactions and identifying potential risks and failures [12,13,14]. This research employed a statistical optimization process to determine the most optimal parameters for a reliable analytical method, utilizing AQbD-driven development to ensure precise and accurate quantification of EFT [15, 16]. Moreover, to implement GAC principles while maintaining efficiency, it is crucial to monitor and understand the interrelationships of various analytical factors [17]. The AQbD technique, part of ICH Q8 (R2) standards, facilitates this by establishing a thorough foundation for method variables from the outset [18]. This approach reduces time and resources, enables seamless method transfers, and integrates GAC and AQbD to develop efficient, environmentally friendly, and flexible technologies [19,20,21].

As there are no stability study reports, analytical method validation, or development for EFT using UPLC with AQbD and GAC, this study aims to develop a robust analytical method by creating a UPLC method for EFT that integrates GAC and AQbD using eco-friendly solvents. It will conduct a forced degradation study of EFT following ICH Q1A and Q1B standards, validate the method for specificity, precision, accuracy, and detection limits per ICH Q2 guidelines, and apply the method to various matrices to confirm its practical applicability. The study will also assess the method's robustness by introducing intentional changes in parameters such as mobile phase composition, flow rate, and column temperature, measure reductions in solvent usage and waste production to highlight sustainable practices and explore potential applications for other pharmaceutical compounds. This research aims to establish a robust, validated analytical approach and contribute to understanding sustainable analytical practices in drug development [22, 23].

Materials and methods

Chemicals and reagents

Pure Ensifentrine (> 99%) and Ohtuvayre 3 mg/2.5 mL were provided by Spectrum Pharma Labs Pvt. Ltd., Hyderabad, India. The Nebulizer Inhalation Solution was purchased from a local market. All AR-grade chemicals, including acetonitrile, potassium dihydrogen orthophosphate, H2O2, orthophosphoric acid, HCl, and NaOH, were sourced from Rankem in Gurugram, Haryana, India. The Milli-Q water from Millipore Technologies in Mumbai, India was used for solution preparation.

Apparatus and equipment

A Water Acquity UPLC system with a PDA detector was used to develop, optimize, and validate the ensifentrine method. A HSS C18 SB column (2.1 × 100 mm, 1.8 μm) and Empower-2 software were utilized for chromatographic separation. The Design-Expert® software version 13.0.0 facilitated parameter optimization. For spectrophotometric measurements, a PG Instruments T60 UV–VIS spectrophotometer was used. An HTLP Engineering forced-air circulating oven was used for thermal degradation studies. An Ultrasonic bath sonicator from Analab Scientific Instruments was used to dissolve samples, and a BVK enterprises pH meter measured the mobile phase pH. A hot plate from Optima Laboratory Hot Plates was used for forced degradation studies. The pure EFT drug and other chemicals were weighed using a Denvar DWS-124C balance from New Delhi, India.

The RP-UPLC method optimization and development via AQbD

The method development strategy was based on AQbD principles, involving the creation of an analytical target profile (ATP) to define performance requirements. Critical analytical attributes (CAT) were identified, and a risk assessment was conducted. Preliminary method development and design of experiments established the design space, with a robustness study identifying key high-risk factors such as column flow rate, column temperature, and buffer pH, while other factors were deemed low-risk.

Sample and standard preparation

Diluent

The diluent, a 50:50 acetonitrile–water mixture, was chosen to ensure the drug’s solubility.

KH2PO4 (0.01N) Buffer

1.36 g of potassium dihydrogen orthophosphate was weighed and added to a 1000 mL volumetric flask containing approximately 900 mL of milli-Q water. The solution was degassed using a sonicator, and then was filled to the final volume. The pH was adjusted to 5.4 using dilute H3PO4.

Standard stock solutions

Weighed 7.5 mg of EFT, transferred it to a volumetric flask of 50 mL, and added a portion of the (25 mL) diluent. Sonicated for 10 min, then filled the flask with diluent to create a 150 µg/mL EFT standard stock solution.

Standard working solutions

In addition, to achieve a 15 µg/mL EFT concentration, 1 mL from the standard stock solution was taken and diluted to 10 mL with diluent in a volumetric flask.

Sample stock solutions

The suspension liquid weight (Ohtuvayre 3 mg/2.5 mL EFT) was transferred to a 10 mL volumetric flask. The volume was diluted with 1 mL of diluent and then sonicated for 25 min. The flask was filled to the mark with diluent to a total volume of 10 mL, and the solution was passed through UPLC filters to achieve a 300 µg/mL EFT concentration.

Sample working solutions (100% solution)

The filtered sample stock solution (0.5 mL) was added to a 10 mL volumetric flask and diluted with a solvent to achieve a 15 µg/mL EFT concentration.

Stress degradation studies

EFT was assessed under various stress conditions, including thermal, oxidation (peroxide), hydrolytic (alkaline and acidic), neutral (diluent), and photolytic, following ICH guidelines Q1A (R2) and Q1B. All the stress experiments were conducted at a final concentration of 15 ppm [24, 25].

Investigation of hydrolytic degradation

For the acid and base degradation studies, the following procedures were followed:

Acid degradation: 1 mL of the stock EFT solution was mixed with 1 mL of 2 N HCl and refluxed at 60 °C for 30 min. The resulting solution was then neutralized with 2 N NaOH, made to volume with the diluent, and further diluted to a concentration of 15 µg/mL. A 10 µL aliquot of this final solution was injected into the UPLC system for stability assessment.

Base degradation: In a separate procedure, 1 mL of the stock EFT solution was combined with 1 mL of 2 N NaOH and refluxed at 60 °C for 30 min. The solution was subsequently neutralized with 2 N HCl, made to volume with the diluent, and diluted to a concentration of 15 µg/mL. A 10 µL aliquot of this final solution was also injected into the UPLC system for stability evaluation.

Studies on oxidative, photolytic, neutral, and thermal degradation

1 mL EFT stock solution was combined with 1 mL of 20% H2O2 and left at 60 °C for 30 min. After diluting the resultant solution to 15 µg/mL, 10 µL was added to the UPLC system to test for stability. For photochemical stability, in a UV chamber, a 400 µg/mL solution was exposed to Ultra Violet radiation for 200-W hrs/m2 or 7 days in a photostability chamber [26]. Following a 15 µg/mL dilution of the solution, 10 µL was added to the UPLC system to record chromatograms and assess stability. Under neutral conditions, for 6 h, the drug was refluxed in water at 60 °C. The standard drug medication solution was heated for 6 h at 105 °C to undergo thermal degradation.

Method validation

The finalized analytical method validation (AMV) evaluated specificity, system suitability, accuracy, robustness, precision, solution stability, Range, LOD, LOQ, and linearity according to the protocols outlined in the ICH guidelines Q2 (R1) [25, 27].

System suitability test

Parameters for standard injections included the resolution (R) and theoretical plate count (N) between the degradation product and EFT peak, and peak asymmetry (T) of the EFT peak.

Specificity and stress testing analysis

Moreover, to find any interference from blank injections, the specificity was assessed. The drug substance was subjected to stress in solution under neutral, alkaline, acidic, and oxidative conditions, as well as humidity, thermal, and photolytic conditions. The method's specificity was assessed using purity threshold and purity angle values. These stress studies optimized the analytical method for EFT under various conditions, establishing it as a stability-indicating assay method.

Analysis of precision and accuracy

The precision of X-ray analysis was evaluated by injecting the sample solution six consecutive times at a concentration of 15 ppm. The intermediate precision (interday precision) was assessed by injecting a freshly prepared sample solution six times on a different day using a different apparatus. For intra- and interday analysis, the relative standard deviation (%RSD) was calculated. The system precision was determined through 6 injections from the same standard solution. The accuracy was evaluated using the standard addition method, where the reference standard was spiked at 50%, 100%, and 150% of the 15 ppm test concentration. The findings were presented as the percentage of the expected finding (EFT) from the sample matrix which is recovered.

Linearity and calibration range

The linearity of this method was evaluated over a concentration range that spanned approximately 25% to 150% of the expected test concentrations for EFT. A linear regression model was used to establish the relationship between the area under the curve and the concentration of EFT. This evaluation was conducted across a concentration range of 3.75–22.5 μg/mL, and the results were graphically represented to demonstrate the linearity of the method. The correlation coefficient, slope, and y-intercept of the regression line were calculated to confirm the direct proportionality between the analyte concentration and the signal produced.

Limit of detection (LOD) and limit of quantification (LOQ) determination

The LOD and LOQ were determined using the standard deviation (σ) and slope (S) of the calibration curve, according to the following formulas: LOD = 3.3 × σ/S, LOQ = 10 × σ/S. These calculations are based on the guidelines provided by the International Conference on Harmonisation (ICH) and utilize the standard deviation of the response and the slope of the calibration curve to establish these critical limits.

Robustness assessment

The method’s robustness was assessed by systematically altering parameters such as flow rates (between 0.1 and 0.3 mL/min), mobile phase compositions (between 62B:38A to 69B:34A), and column temperatures (between 24 °C and 35 °C).

Solution stability

Standard and sample solutions were assessed by injecting freshly prepared solutions at different time intervals.

Statistical research

The data were presented in the form of mean ± SD. In Excel, the regression coefficient, mean, SD, and % RSD were calculated. A p-value of < 0.05 was considered statistically significant, and ANOVA was utilized to evaluate the model’s and its terms’ significance.

Results and discussion

Preliminary method development investigation

This study optimized the UPLC technique for analysing (EFT) in its inhalation form. The optimal wavelength was identified as 272.0 nm, enhancing sensitivity and reducing noise. After testing various columns, the ACQUITY UPLC HSS C18 SB column (2.1 × 100 mm, 1.8 μm) was found to be the most suitable. The optimal mobile phase consisted of 0.01 N aqueous KH2PO4 (pH 5.4) and acetonitrile in a 66.4:33.6 ratio. Adjustments to the acetonitrile-to-phosphate buffer ratio, pH, and flow rate significantly impacted the tailing factor, retention time, and theoretical plate count, highlighting the importance of carefully controlling these parameters.

Method development by AQbD

Using AQbD, three key parameters were optimized for the analytical method: column temperature (24.95–35.05 °C), flow rate (0.2495–0.3505 mL/min), and organic solvent percentage in the mobile phase (21.59–38.41%). The organic solvent ratio was a high-risk factor requiring meticulous optimization. The optimized UPLC method involved a flow rate of 0.27 mL/min, a mobile phase of 66.4:33.6 acetonitrile to 0.01 N aqueous KH2PO4 buffer at pH 5.4, and a column temperature of 29.6 °C. The optimal UV absorption wavelength was 272.0 nm. Sample preparation used a 50:50 ACN and H2O mixture as a diluent, with a sample temperature of 15 °C and an injection volume of 2 μL. This method was applied to both stable and stressed samples, ensuring robust development for accurately assessing EFT quality in the presence of its degradation products.

Optimizing methods using experimental design

In this study, the effects of three independent variables on three dependent variables were investigated using 20 trials of central composite design. The independent variables included flow rate (Factor 1, A), the percentage of organic phase in the mobile phase (Factor 2, B), and temperature (Factor 3, C). The dependent variables analysed included retention time (RT) (Response 1, Fig. 2a), the number of theoretical plates (NTP) (Response 2, Fig. 2b), and tailing factor (Response 3, Fig. 2c), with the results summarized in Table 1. Second-order polynomial equations were derived by fitting the gathered results to different mathematical models to examine the effects of the independent variables on each dependent variable. ANOVA was utilized to evaluate the statistical significance of the model terms. Additionally, to illustrate relationships between the dependent and independent variables, 2D contour plots and 3D response surface plots were generated. Lastly, based on predefined acceptance criteria, methods for numerical optimization were used to forecast the optimum chromatographic. The results of the ANOVA tests for Retention Time, Number of Theoretical Plates (NTP), and Tailing Factor (Symmetry) (TF) using Central Composite Design (CCD) for different responses are shown in Table 2a, b, and c.

Fig. 2
figure 2figure 2

a 3D-response surface (1–2–3) and 2D-contours (4–5–6) plots showing the influence of CMPs, i.e., flow rate (A), mobile phase concentration (B), temperature (C) on retention time (Response 1) as the CAA. b 3D-response surface (1–2–3) and 2D-contours (4–5–6) plots showing the influence of CMPs, i.e., flow rate (A), mobile phase concentration (B), temperature (C) on theoretical plates (Response 2) as the CAA. c 3D-response surface (1–2–3) and 2D-contours (4–5–6) plots showing the influence of CMPs, i.e., flow rate (A), mobile phase concentration (B), temperature (C) on the tailing factor (Response 3) as the CAA. d Overall Desirability (1, 2, and 3) 2D plot for optimized conditions to predict retention time, theoretical plates, and peak area

Full size image
Table 1 The central composite design and the observed values are used to optimize the RP-UPLC method for Ensifentrine optimization
Full size table
Table 2 ANOVA test results for Retention Time (a), Number of Theoretical Plates (NTP, b), and Tailing Factor (Symmetry) (TF, c) using CCD for various responses
Full size table

Calibrating the model to response data

The Design-Expert tool was used to fit the observed responses from 20 runs to various mathematical models, with the quadratic model selected as the best fit based on high R2 values, low SD, and close matches between adjusted and predicted R2 values. The relationships between responses (retention time, NTP, and tailing factor) and factors were visualized in 3D response surface plots and 2D contour plots (Fig. 2A, B, and C). A composite desirability function identified optimal conditions, with desirable values ranging from 0 to 1. For asymmetry, retention time, and peak area, a composite desirability of 1 indicated an optimal flow rate of 0.27 mL/min. The validation involved six replicate injections of a 15 µg/mL EFT solution, confirming that the observed retention time, asymmetry, and theoretical plates were within the predicted ranges, with differences of less than 5%. This validated the accuracy of the optimum conditions.

Analytical method validation (AMV)

System suitability

Establishing the system's suitability data is crucial before moving further with the analytical method's validation. The outcomes for these parameters met ICH guidelines and were satisfactory: the tailing factor was < 2, the resolution was > 2, and the theoretical plate count exceeded 2000.

Specificity

The PDA detector was utilized to estimate peak purity and verify the UPLC method's specificity. The developed UPLC method exhibited selectivity and specificity, as there was no interference detected from the blank and placebo solutions at the retention time of EFT (1.207 min). Figure 3 illustrates the chromatograms for the blank, placebo, and EFT drug samples.

Fig. 3
figure 3

The Chromatogram of A Blank, B Placebo, C Drug Ensifentrine

Full size image

Linearity and range

A calibration curve was generated to relate area to concentration over the range of (3.75–22.5) μg/mL. The linear regression equation for this curve is (y = 59680x + 4269.8), with a correlation coefficient of 0.9997 for EFT, showing a highly linear response over the concentration range of the test. The linearity results are depicted in Fig. 4.

Fig. 4
figure 4

Calibration curve of Ensifentrine (3.75–22.5 µg/mL)

Full size image

Method precision

Repeatability (Intra-day)

The intra-day repeatability was evaluated by determining %RSD from six measurements of a 15 μg/mL solution, all performed on the same day and within the same laboratory setting. The calculated RSD was 0.40%, which signifies the method's high reliability.

Intermediate precision (Inter-day)

The evaluation of inter-day precision was performed by measuring the % RSD from six distinct determinations of a 15 μg/mL solution. This assessment involved inter-day precision tests carried out on various days, utilizing different instruments, columns, and sample preparations, all within the same lab setting. The % RSD for the inter-day precision data was found to be 0.50. The precision outcomes of the method developed are shown in Table 3.

Table 3 Method precision results presentation
Full size table

Accuracy

The developed method’s accuracy was assessed using the standard addition method. EFT was spiked at concentrations equivalent to 50%, 100%, and 150% of the test concentration (7.5, 15, and 22.5 ppm) in the test solution. The mean recovery rate was reported to be 100.17%, with a % RSD of less than 2.0, which indicates satisfactory accuracy. The accuracy outcomes are presented in Table 4.

Table 4 The accuracy results
Full size table

Detection limit (DL) and quantification limit (QL)

DL and QL were determined by the slope method based on linearity, yielding DL values of 3.3 μg/mL or less and QL values of 10.0 μg/mL or less.

Robustness

The robustness of the developed method was verified under various conditions, including different flow rates, mobile phase compositions, and column temperatures, with the method remaining unaffected by changes in flow rate from 0.1 to 0.3 mL/min, column temperature from 24 to 35 °C, and mobile phase composition from 62B:38A to 69B:34A, demonstrating its robustness. The system suitability parameters were largely unchanged, with all parameters meeting the required standards and the percentage relative standard deviation (% RSD) remaining within acceptable limits. The method’s robustness details are shown in Table 5.

Table 5 Results of the developed method’s Robustness (n = 6, 15 µg/mL)
Full size table

Solution stability

The samples and the standard solutions' stability in a diluent made of a 50:50 (v/v) ACN: H2O mixture was assessed and determined to remain stable for 48 h when stored at 5 °C.

Assay

The inhalation product designated as Ohtuvayre, labelled with the claim of EFT at a dosage of 100 mg, underwent an assay utilizing the aforementioned formulation. The analysis produced an average assay result for EFT, which was determined to be 99.93%. These assay results have been systematically compiled and are displayed in Table 6.

Table 6 Results of the assay for ensifentrine inhalation formulation (Ohtuvayre)
Full size table

Studies of forced degradation

The forced degradation studies of EFT revealed that the drug exhibits minimal degradation (less than 2%) under photolytic and neutral conditions. In conditions of acid hydrolysis, it experienced a degradation of 5.15%, with a degradation peak observed at 0.747 min. Conversely, under basic hydrolysis, the drug showed a degradation of 4.38%, corresponding to a degradation peak at 0.790 min. For oxidative and thermal degradation, EFT demonstrated degradations of 4.81% and 3.23%, respectively. Importantly, the analysis revealed no interference from the degradation peaks. The forced degradation studies observations are presented in Table 7, with corresponding chromatograms displayed in Fig. 5.

Table 7 Stability studies of ensifentrine
Full size table
Fig. 5
figure 5

Chromatograms displaying the degradation profile of ensifentrine during conditions. A acid, B alkali, C oxidative, D thermal, E neutral, and F photolytic degradation

Full size image

Green analytical metric tools assessment

Ideal green analysis seeks to make lab processes eco-friendly by minimizing or eliminating organic solvents, reducing energy use, simplifying sample prep, and avoiding waste. In liquid chromatography, while it is impossible to fully eliminate solvents, strategies such as using safer, biodegradable alternatives, minimizing waste hazards, streamlining processes, and employing miniaturized sample prep can enhance sustainability [28]. Evaluating the ecological friendliness of an analytical procedure is complex, requiring careful consideration of multiple factors. The developed method’s sustainability was evaluated using five tools to assess its environmental impact: ComplexMoGAPI, ChlorTox Scale, AGREE, Analytical Eco-Scale, and BAGI.

Complex modified GAPI (ComplexMoGAPI)

The ComplexMoGAPI (Complex Modified GAPI) is an advanced hazard assessment tool that enhances traditional GAPI methodologies. It offers a detailed evaluation of chemical hazards by incorporating factors such as environmental persistence, bioaccumulation, and toxicity, resulting in a comprehensive risk profile. This model is vital for regulatory assessments where precise hazard evaluations are necessary. The ComplexMoGAPI merges the visual elements of ComplexGAPI with accurate scoring. Researchers such as Tobiszewski and Namieśnik emphasized the value of thorough hazard assessments in chemical risk management. Moreover, by utilizing these advancements, the ComplexMoGAPI improves hazard assessment accuracy, supporting informed decisions in chemical safety and environmental protection [29,30,31,32].

ComplexMoGAPI offers a more detailed assessment compared to the traditional red/green/yellow icons of ComplexGAPI. It evaluates the overall environmental sustainability of an analytical method by generating a cumulative score, considering a wide range of choices within each category. For the developed method, ComplexMoGAPI calculated a score of 71, indicating room for significant environmental sustainability due to the presence of yellow and green icons. Figure 6a visualizes these results with greenness metrics, and Supplementary Table 1 provides detailed method scores. This table allows for a thorough comparison and analysis of the environmental impact of different aspects of the method, helping researchers identify areas for improvement to enhance ecological sustainability.

Fig. 6
figure 6

Evaluation of the proposed UPLC method’s greenness, a ComplexMoGAPI pictogram; b AGREE pictogram score; c Green certificate modified Eco-scale score; d BAGI asteroid pictogram; e ChlorTox Scale; f EVG Radar chart

Full size image

Chloroform-oriented Toxicity estimation scale (ChlorTox Scale)

The ChlorTox Scale is a tool for assessing chemical risks related to laboratory analytical methods. It evaluates the toxicity and reactivity of chemicals, aiding in the identification of safer alternatives and reducing exposure to hazards. Moreover, by using chloroform, a well-studied reference with known toxicity, the scale offers a reliable risk assessment framework. This comprehensive evaluation considers chloroform's significant hazards, including acute toxicity and carcinogenic potential, which is supported by abundant safety data. The scale emphasizes the importance of personal protective equipment (PPE) and safety measures, facilitating comparisons of risks between different chemicals. Overall, the ChlorTox Scale standardizes chemical risk estimation, offering a broader view of potential hazards. The methodology for the WHN and CHEMS-1Hazard Assessment models uses safety data sheets (SDS) for the chemicals studied. The WHN model condenses hazard assessment by assigning weights to different hazard categories based on severity, yielding a single score for overall hazard. In contrast, the CHEMS-1 model, built on earlier work by Tobiszewski and Namieśnik, provides a more thorough evaluation, considering various factors like toxicity, flammability, reactivity, persistence, and bioaccumulation. CHEMS-1 was preferred over WHN for its precision in detailing chemical hazards, improving risk differentiation and management. This method captures the complexities of chemical risk more effectively than simpler models like WHN [33,34,35,36,37,38]. Table 8 and Fig. 6e displays results from the ChlorTox WHN & CHEMS-1 models. The ChlorTox Scale effectively evaluates chemical risks by utilizing extensive knowledge of chloroform, enhancing assessment accuracy compared to traditional methods. The ChlorTox Scale GAC metric calculation formulas are shown in Equations (1 and 2) [39]. The ChlorTox Scale, Equation (1) uses the value of chloroform, which is a global standard for determining the relative hazard of other compounds, such as CHCHCl3.

$$ChlorTox={\frac{{CH}_{sub}}{{CH}_{{CHCI}_{3}}} . m}_{sub} $$
(1)
$${m}_{sub}=\frac{mN+{m}^{{\prime}}}{N}$$
(2)

Where: CHsub: Overall hazard level of the chemical in question; msub: Amount of substance used in a single analysis; mN: The mass of all the substances used in N analyses; m': The amount of substance used in additional mandatory procedures; N: The most extensive series in the analysis.

Table 8 The developed UPLC Method for the forced degradation studies of ensifentrine is evaluated for greenness using the ChlorTox Scale
Full size table

The ChlorTox values, which quantify the potential risk associated with different substances, can be aggregated to represent the total chemical risk anticipated for an entire analytical method. This cumulative measure is referred to as the Total ChlorTox value. It is important to note that the ChlorTox value has a purely theoretical significance and does not directly reflect real-world conditions. Instead, it serves as an indicator of the general scale of potential risk involved. In the context of the developed method, the Total ChlorTox values were calculated using two different models: the WHN model and the CHEMS-1 model. According to these calculations, the Total ChlorTox value for the developed method is 1.24 g when using the WHN model and 1.32 g when using the CHEMS-1 model. To put this into perspective, these values suggest that the risk associated with this method is equivalent to using approximately 1.49 g of pure chloroform as the sole hazardous chemical reagent in an alternative method. This comparison helps in understanding the relative risk profile of the developed method in terms of its chemical hazards.

Analytical greenness (AGREE) tool

AGREE is a comprehensive system for measuring greenness, integrating all 12 GAC principles [25, 50] and offering results in both colour and numerical formats. Its assessment process is straightforward, though it shares drawbacks with GAPI. AGREE overlooks compounds, solvents, energy use, waste from pre-extraction, and sample preparation greenness [40, 41].

The RP-UPLC method developed in this research exhibits environmentally friendly characteristics, as evidenced by an overall score of 0.55. Figure 6b presents the AGREE tool pictogram for the method, and Supplementary Table 2 provides a detailed breakdown of the method scores and values obtained using the AGREE tool. These assessments offer valuable insights into the environmental friendliness of the RP-UPLC method, demonstrating its compliance with Green Analytical Chemistry (GAC) criteria and highlighting the accepted greenness of the proposed approaches.

Analytical Eco-scale (AES)

The AES, which was introduced in 2012 [42], is a key tool in GAC for assessing the environmental and health impacts of analytical methods. For ideal green analysis, a maximum of 100 points are awarded, deducting points for associated hazards. The AES allows for a semi-quantitative assessment of methods, making it easy for researchers to calculate and compare scores [43]. However, it lacks detailed insights into specific hazards and sources of environmental impacts, limiting its effectiveness in improving methods during design. To overcome these issues, enhancements like the Green Certificate Modified Eco-Scale have been suggested, which categorize eco-scale values and use colours for visualization [44,45,46,47,48,49,50].

The proposed RP-UPLC method integrates several principles of Green Analytical Chemistry (GAC), enhancing its environmental friendliness and sustainability. The method achieves an eco-score of 58 on the Eco-Scale, indicating a favourable environmental profile. Despite using acetonitrile, a solvent with environmental and health concerns, its use is optimized to minimize waste and reduce overall environmental impact, aligning with GAC principles of using safer solvents. For forced degradation studies, the method employs Sodium hydroxide, hydrochloric acid, and hydrogen peroxide in minimal quantities, reducing their toxicity and occupational hazard score. This approach adheres to GAC principles by preventing waste generation, using safer chemistry, and designing for energy efficiency. The method's sustainability is further enhanced by its instrumental energy efficiency and reduced sample and reagent volumes, aligning with GAC's goals of minimizing sample size and waste generation. Figure 6C illustrates the modified Eco-scale scores, and Supplementary Table 3 details the penalty points, providing a comprehensive view of the method's sustainability metrics. Overall, the method demonstrates acceptable sustainability and compliance with GAC principles, making it a more environmentally friendly approach in analytical chemistry.

BAGI (Blue applicability grade index)

Manousi et al. developed the BAGI [51], a metric in GAC for assessing the practicality and greenness of analytical methods. Based on White Analytical Chemistry, BAGI has a grading system, a colour scale, and an asteroid graphic to denote greenness and practicality, with scores from 25 to 100. It evaluates methods based on 10 criteria, including analysis type, analyte count, techniques, reagent types, and more. BAGI is accessible at its official website (https://bagi-index.anvil.app) for quick assessments. While a useful GAC tool alongside AGREEprep and ComplexGAPI, it lacks a Safety, Health, and Environmental (SHE) evaluation for reagents and waste [52,53,54,55,56,57].

The BAGI tool was utilized to employ UPLC-PDA detection for the quantification of EFT in both bulk and inhalation formulations. The analysis was conducted using a PDA detector set at 272.0 nm, with the equipment being straightforward and commonly available in laboratories. The process involved the simultaneous preparation of samples with varying concentrations using volumetric flasks. After preparing 20 samples, which took approximately 2 h, the total analysis time per sample using UPLC-PDA was 10 min. This resulted in a sample throughput of 12 samples per hour. The results indicated that no preconcentration was necessary, as the required sensitivity was achieved directly. However, the manual treatment and analysis steps could be considered a limitation, which might be mitigated by automating specific analytical steps in the future. Miniaturized extraction was employed for sample preparation, utilizing a sample volume of 100 μL for the analytical matrix. Consequently, the method achieved a BAGI score of 70, suggesting promising applicability potential for the entire protocol. Figure 6d illustrates the method's environmental friendliness in the BAGI pictogram, with detailed information provided in Supplementary Table 4, including the UPLC method and its corresponding BAGI score.

The results from these greenness assessment tools collectively indicate that our analytical method exhibits strong environmental sustainability and compliance with Green Analytical Chemistry principles. The detailed scores and color-coded representations from each tool provide valuable insights into the method's strengths and areas for potential improvement, ensuring that the method is both effective and environmentally benign.

Balance point display using the EVG tool

The proposed chromatographic method employs the EVG tool to assess three critical dimensions: validation, efficiency, and greenness—each rated on a scale from 0 to 3 using five assessment criteria. A radar chart in Fig. 6f illustrates the performance of the EVG-UPLC method in these aspects. Efficiency focuses on the development of the method in isocratic mode, utilizing advanced columns for UPLC to achieve effective separation of EFT from its impurities. Validation looks at essential parameters that ensure the reliability of the method, including precision, LOQ, robustness, and system suitability. Lastly, the greenness component in analytical chemistry highlights the application of five advanced greenness tools, taking into account factors such as sample treatment, reagents used, solvent choice, instrumentation efficiency, energy consumption, and waste production. This comprehensive evaluation aims to strike a balance among these aspects, ensuring that the method developed is not only effective but also environmentally friendly and scientifically valid [58,59,60]. The scoring for the EVG aspects is detailed in the Supplementary material file.

Conclusion

The principles of the GAC and AQbD approach were utilized to develop a robust analytical method and validation of EFT. The greenness was evaluated utilizing tools such as ComplexMoGAPI, AGREE, ChlorTox Scale, and Analytical Eco-scale, resulting in a greenness certificate. The AQbD method, created with Design Expert software, involved a central composite design to assess three responses with three factors, revealing that flow rate and mobile phase composition significantly impacted EFT peak shape and resolution. Validation of the method was performed for specificity, accuracy, precision, robustness, LOD, and LOQ according to guideline ICH Q2 and met all acceptance criteria. Stability studies under different stress conditions, following ICH Q1A and Q1B guidelines, identified one degradation product under acidic and alkaline conditions. In addition, to quantify ETS, a robust, simple, and validated method was developed using greener solvents, minimizing hazardous solvents, and adhering to green analytical principles and AQbD for routine and quality control analysis of EFT samples. An Efficiency, Validation, Greenness (EVG) radar chart framework was introduced for chromatographic method development to evaluate EFT in pure and pharmaceutical inhalation formulations.

Availability of data and materials

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

Abbreviations

COPD:

Chronic obstructive pulmonary disease

EFT:

Ensifentrine

AQbD:

Analytical quality-by-design

GAC:

Green analytical chemistry

PDA:

Photodiode Array

LOD:

Limit of detection

LOQ:

Limit of quantitation

PDE:

Phosphodiesterase

AR:

Analytical Reagent

ATP:

Analytical target profile

CAT:

Critical analytical attributes

AMV:

Analytical method validation

RSD:

Relative standard deviation

EFT:

Expected finding test

PRESS:

Predicted residual sums of squares

NTP:

Number of Theoretical Plates

AGREE:

Analytical Greenness

BAGI:

Blue Applicability Grade Index

SHE:

Safety, Health, and Environmental

References

  1. Donohue JF, Rheault T, MacDonald-Berko M, Bengtsson T, Rickard K. Ensifentrine as a novel, inhaled treatment for patients with COPD. Int J Chron Obstruct Pulmon Dis. 2023;18:1611–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease 2023 report. Global initiative for chronic obstructive lung disease (GOLD). 2023. https://goldcopd.org/. Accessed 5 July 2024.

  3. Cazzola M, Rogliani P, Matera MG. The future of bronchodilation: looking for new classes of bronchodilators. Eur Respir Rev. 2019;28: 190095.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Lipworth et al. Phosphodiesterase 3 and 4 inhibitors in chronic obstructive pulmonary disease. 2017.

  5. Abbott-Banner KH, Page CP. Dual pde 3/4 and pde 4 inhibitors: novel treatments for copd and other inflammatory airway diseases. Basic Clin Pharmacol Toxicol. 2014;114:365–76.

    Article  CAS  PubMed  Google Scholar 

  6. Singh et al. Ensifentrine (RPL554) in patients with chronic obstructive pulmonary disease: a randomized, double-blind, placebo-controlled phase 3 trial. 2022.

  7. Giembycz et al. Dual Phosphodiesterase 3 and 4 Inhibitors: A New Therapeutic Approach For Chronic Obstructive Pulmonary Disease. 2018.

  8. Anzueto A, Barjaktarevic IZ, Siler TM, Rheault T, Bengtsson T, Rickard K, et al. Ensifentrine, a novel phosphodiesterase 3 and 4 inhibitor for the treatment of chronic obstructive pulmonary disease: randomized, double-blind, placebo-controlled, multicenter phase III trials (the ENHANCE Trials). Am J Respir Crit Care Med. 2023;208:406–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Keam SJ. Ensifentrine: first approval. Drugs. 2024;84:1157–63.

    Article  PubMed  Google Scholar 

  10. Boswell-Smith V, Spina D, Oxford AW, Comer MB, Seeds EA, Page CP. The pharmacology of two novel long-acting phosphodiesterase 3/4 inhibitors, RPL554 [9,10-dimethoxy-2(2,4,6-trimethylphenylimino)-3-( N -carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2 H -pyrimido[6,1- a ]isoquinolin-4-one] and RPL565 [6,7-Dihydro-2-(2,6-diisopropylphenoxy)-9,10-dimethoxy-4 H -pyrimido[6,1- a ]isoquinolin-4-one]. J Pharmacol Exp Ther. 2006;318:840–8.

    Article  CAS  PubMed  Google Scholar 

  11. Hubert S, Kök-Carrière A, De Ceuninck F. Ensifentrine (Ohtuvayre™) for chronic obstructive pulmonary disease. Trends Pharmacol Sci. 2024;45:941–2.

    Article  CAS  PubMed  Google Scholar 

  12. Ahmed DA, Hussein OG, Rezk MR, Abdelkawy M, Rostom Y. Impressive merger between green analytical approaches and quality-by- design for alcaftadine determination in eye drops and rabbit aqueous humor; application to stability study by two validated chromatographic methods. Microchem J. 2024;196: 109717.

    Article  CAS  Google Scholar 

  13. Hussein OG, Ahmed DA, Rezk MR, Abdelkawy M, Rostom Y. Exquisite integration of quality-by-design and green analytical approaches for simultaneous determination of xylometazoline and antazoline in eye drops and rabbit aqueous humor, application to stability study. J Pharm Biomed Anal. 2023;235: 115598.

    Article  CAS  PubMed  Google Scholar 

  14. Abdel-Moety EM, Rezk MR, Wadie M, Tantawy MA. A combined approach of green chemistry and quality-by-design for sustainable and robust analysis of two newly introduced pharmaceutical formulations treating benign prostate hyperplasia. Microchem J. 2021;160: 105711.

    Article  CAS  Google Scholar 

  15. Patra SR, Bali A, Saha M, Singh J, Shekhar S. Method validation and characterization of stress degradation products of gefitinib through UPLC-UV/PDA and LC–MS/TOF studies. Int J Mass Spectrom. 2023;490: 117070.

    Article  CAS  Google Scholar 

  16. Lakka NS, Kuppan C, Vadagam N, Reddamoni SY, Muthusamy C. Degradation pathways and impurity profiling of the anticancer drug apalutamide by HPLC and LC–MS/MS and separation of impurities using Design of Experiments. Biomed Chromatogr. 2023;37: e5549.

    Article  CAS  PubMed  Google Scholar 

  17. Bhukya VN, Beda DP. Implementation of green analytical principles to develop and validate the HPLC method for the separation and identification of degradation products of Panobinostat, and its characterization by using LC-QTOF-MS/MS and its in-silico toxicity prediction using ADMET software. Green Anal Chem. 2024;8: 100090.

    Article  Google Scholar 

  18. Fares MY, Hegazy MA, El-Sayed GM, Abdelrahman MM, Abdelwahab NS. Quality by design approach for green HPLC method development for simultaneous analysis of two thalassemia drugs in biological fluid with pharmacokinetic study. RSC Adv. 2022;12:13896–916.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Muchakayala SK, Katari NK, Saripella KK, Schaaf H, Marisetti VM, Ettaboina SK, et al. Implementation of analytical quality by design and green chemistry principles to develop an ultra-high performance liquid chromatography method for the determination of fluocinolone acetonide impurities from its drug substance and topical oil formulations. J Chromatogr A. 2022;1679: 463380.

    Article  CAS  PubMed  Google Scholar 

  20. Yabré M, Ferey L, Somé IT, Gaudin K. Greening reversed-phase liquid chromatography methods using alternative solvents for pharmaceutical analysis. Molecules. 2018;23:1065.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Elsheikh SG, Hassan AME, Fayez YM, El-Mosallamy SS. Green analytical chemistry and experimental design: a combined approach for the analysis of zonisamide. BMC Chem. 2023;17:38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Shaik MA, Manchuri KM, Nayakanti D. A novel UHPLC-MS/MS method for trace level identification and quantification of genotoxic impurity 2-(2-chloroethoxy) ethanol in quetiapine fumarate. J Liq Chromatogr Relat Technol. 2023;46:325–34.

    Article  CAS  Google Scholar 

  23. Ali SM, Moorthy MK, Devanna N. A novel liquid chromatography with quadrupole time-of-flight-tandem mass spectroscopy method for ultra-trace level identification and quantification of the genotoxic impurity 2,6-diamino-5-nitropyrimidin-4(3H)-one in valganciclovir hydrochloride. Biomed Chromatogr. 2024;38: e5805.

    Article  CAS  PubMed  Google Scholar 

  24. ICH Topic Q 1 A (R2). Stability testing of new drug substances and products. European medicines agency. 2003.

  25. Bakshi M, Singh S. Development of validated stability-indicating assay methods—critical review. J Pharm Biomed Anal. 2002;28:1011–40.

    Article  CAS  PubMed  Google Scholar 

  26. Center for drug evaluation and research, center for biologics evaluation and research. Q1B photostability testing of new drug substances and products. U.S. Food and Drug Administration. 1996.

  27. Nowak PM. What does it mean that “something is green”? the fundamentals of a Unified greenness theory. Green Chem. 2023;25:4625–40.

    Article  CAS  Google Scholar 

  28. Tobiszewski M, Namieśnik J. PAH diagnostic ratios for the identification of pollution emission sources. Environ Pollut. 2012;162:110–9.

    Article  CAS  PubMed  Google Scholar 

  29. Mansour FR, Omer KM, Płotka-Wasylka J. A total scoring system and software for complex modified GAPI (ComplexMoGAPI) application in the assessment of method greenness. Green Anal Chem. 2024;10: 100126.

    Article  Google Scholar 

  30. Wojnowski W, Tobiszewski M, Pena-Pereira F, Psillakis E. AGREEprep—analytical greenness metric for sample preparation. TrAC Trends Anal Chem. 2022;149: 116553.

    Article  CAS  Google Scholar 

  31. Sheldon RA. The E factor 25 years on: the rise of green chemistry and sustainability. Green Chem. 2017;19:18–43.

    Article  CAS  Google Scholar 

  32. Nowak PM, Wietecha-Posłuszny R, Płotka-Wasylka J, Tobiszewski M. How to evaluate methods used in chemical laboratories in terms of the total chemical risk?—A ChlorTox Scale. Green Anal Chem. 2023;5: 100056.

    Article  Google Scholar 

  33. Armenta S, Garrigues S, De La Guardia M. Green analytical chemistry. TrAC Trends Anal Chem. 2008;27:497–511.

    Article  CAS  Google Scholar 

  34. El Deeb S. Enhancing sustainable analytical chemistry in liquid chromatography: guideline for transferring classical high-performance liquid chromatography and ultra-high-pressure liquid chromatography methods into greener, bluer, and whiter methods. Molecules. 2024;29:3205.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Tobiszewski M, Namieśnik J, Pena-Pereira F. Environmental risk-based ranking of solvents using the combination of a multimedia model and multi-criteria decision analysis. Green Chem. 2017;19:1034–42.

    Article  CAS  Google Scholar 

  36. Swanson MB, Davis GA, Kincaid LE, Schultz TW, Bartmess JE, Jones SL, et al. A screening method for ranking and scoring chemicals by potential human health and environmental impacts. Environ Toxicol Chem. 1997;16:372–83.

    Article  CAS  Google Scholar 

  37. Tobiszewski M, Namieśnik J. Scoring of solvents used in analytical laboratories by their toxicological and exposure hazards. Ecotoxicol Environ Saf. 2015;120:169–73.

    Article  CAS  PubMed  Google Scholar 

  38. Nowak PM, Bis A, Zima A. ChlorTox Base—a useful source of information on popular reagents in terms of chemical hazards and greenness assessment. Green Anal Chem. 2023;6: 100065.

    Article  Google Scholar 

  39. Hussein AR, Gburi MS, Muslim NM, Azooz EA. A greenness evaluation and environmental aspects of solidified floating organic drop microextraction for metals: a review. Trends Environ Anal Chem. 2023;37: e00194.

    Article  CAS  Google Scholar 

  40. Yin L, Yu L, Guo Y, Wang C, Ge Y, Zheng X, et al. Green analytical chemistry metrics for evaluating the greenness of analytical procedures. J Pharm Anal. 2024;14:101013.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Gałuszka A, Migaszewski ZM, Konieczka P, Namieśnik J. Analytical Eco-Scale for assessing the greenness of analytical procedures. TrAC Trends Anal Chem. 2012;37:61–72.

    Article  Google Scholar 

  42. Turner C. Sustainable analytical chemistry&mdash;more than just being green. Pure Appl Chem. 2013;85:2217–29.

    Article  CAS  Google Scholar 

  43. Gallart-Mateu D, Cervera ML, Armenta S, De La Guardia M. The importance of incorporating a waste detoxification step in analytical methodologies. Anal Methods. 2015;7:5702–6.

    Article  CAS  Google Scholar 

  44. Kokosa JM, Przyjazny A. Green microextraction methodologies for sample preparations. Green Anal Chem. 2022;3: 100023.

    Article  Google Scholar 

  45. Van Aken K, Strekowski L, Patiny L. EcoScale, a semi-quantitative tool to select an organic preparation based on economical and ecological parameters. Beilstein J Org Chem. 2006;2:3.

    PubMed  PubMed Central  Google Scholar 

  46. Garrigues S, Armenta S, Guardia MDL. Green strategies for decontamination of analytical wastes. TrAC Trends Anal Chem. 2010;29:592–601.

    Article  CAS  Google Scholar 

  47. Ramos L, Ramos JJ, Brinkman UAT. Miniaturization in sample treatment for environmental analysis. Anal Bioanal Chem. 2005;381:119–40.

    Article  CAS  PubMed  Google Scholar 

  48. UNECE. Globally harmonized system of classification and labelling of chemicals (GHS).

  49. Dunn PJ, Wells AS, Williams MT. Future trends for green chemistry in the pharmaceutical industry. In: Dunn PJ, Wells AS, Williams MT, editors. Green chemistry in the pharmaceutical industry. 1st ed. Hoboken: Wiley; 2010. p. 333–55.

    Chapter  Google Scholar 

  50. Wardencki W, Curyło J, Namieśnik J. Green chemistry–current and future issues. PolJ Env Stud. 2005;14:389–95.

    CAS  Google Scholar 

  51. Manousi N, Wojnowski W, Płotka-Wasylka J, Samanidou V. Blue applicability grade index (BAGI) and software: a new tool for the evaluation of method practicality. Green Chem. 2023;25:7598–604.

    Article  CAS  Google Scholar 

  52. Henrique Petrarca M, Vicente E, Amelia Verdiani Tfouni S. Single-run gas chromatography-mass spectrometry method for the analysis of phthalates, polycyclic aromatic hydrocarbons, and pesticide residues in infant formula based on dispersive microextraction techniques. Microchem J. 2024;197:109824.

    Article  CAS  Google Scholar 

  53. Olayanju B, Kabir A, Manousi N, Furton KG. Application of sol-gel universal sorbent coated fabric phase sorptive extraction membranes in combination with high-performance liquid chromatography-ultraviolet detection to monitor endocrine-disrupting chemicals in milk and environmental water samples. Sep Sci PLUS. 2024;7:2300101.

    Article  CAS  Google Scholar 

  54. Essawy AA, Alsohaimi IH, Hassan HMA, El Agammy EF, Hussein MF, Hasanin THA, et al. Basic Fuchsin dye as the first fluorophore for optical sensing of morpholine in fruits crust and urine samples. Anal Chem. 2024;96:373–80.

    Article  PubMed  Google Scholar 

  55. Godela R, Venugopal M, Yagnambhatla R, Gugulothu S, Mayasa V, Maddukuri S, et al. Estimation of lexisinatide in bulk and tablets by RP-UPLC method developed by quality by design concept. Macromol Symp. 2024;413:2300142.

    Article  CAS  Google Scholar 

  56. Boussès C, Ferey L, Vedrines E, Gaudin K. Using an innovative combination of quality-by-design and green analytical chemistry approaches for the development of a stability indicating UHPLC method in pharmaceutical products. J Pharm Biomed Anal. 2015;115:114–22.

    Article  PubMed  Google Scholar 

  57. Nuli MV, Seemaladinne R, Tallam AK. Analytical quality by design (AQbD) based optimization of RP-UPLC method for determination of nivolumab and relatlimab in bulk and pharmaceutical dosage forms. Fut J Pharm Sci. 2024;10:86.

    Article  Google Scholar 

  58. Saleh SS, Lotfy HM, Elbalkiny HT. An integrated framework to develop an efficient valid green (EVG) HPLC method for the assessment of antimicrobial pollutants with potential threats to human health in aquatic systems. Environ Sci Process Impacts. 2023;25:2125–38.

    Article  CAS  PubMed  Google Scholar 

  59. Abd El-Fatah NA, Elbalkiny HT, Hegazy MA, Fouad MM, El-Sayed GM. Green analytical chemistry and quality by design: a combined approach towards simultaneous determination of Letrozole with its co-administered Zoledronic acid for cancer patients. J Pharm Biomed Anal Open. 2024;4: 100036.

    Article  Google Scholar 

  60. Manukonda V, Dandamudi SP, Kusuma PK, Thumma G, Gangarapu K. Development and validation of a UPLC-MS/MS method for the simultaneous estimation of ertugliflozin and sitagliptin in bulk and tablet dosage forms: assessment of greenness and blueness. Green Analyt Chem. 2025;12:100193.

    Article  Google Scholar 

Download references

Acknowledgements

The authors extend their appreciation to the JBR Educational Society, Kampala International University, DPSR University and Deanship of Research and Graduate Studies at King Khalid University for funding this work through Large Research Project under grant number RGP2/530/45.

Funding

The author(s) received for funding this work through Large Research Project under grant number RGP2/530/45.

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Analysis, Joginpally BR Pharmacy College, Hyderabad, Telangana, 500075, India

    Mohan Goud Vanga

  2. Department of Pharmaceutics and Pharmaceutical Technology, Kampala International University, Western Campus, P. O. Box 71, Ishaka-Bushenyi, Uganda

    Sarad Pawar Naik Bukke

  3. Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Delhi Pharmaceutical Sciences and Research University, New Delhi, India

    Praveen Kumar Kusuma

  4. Department of Public Health, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia

    Bayapa Reddy Narapureddy

  5. Department of Pharmacy Faculty of Pharmaceutical Sciences, Assam Down Town University (AdtU), Guwahati, 781026, Assam, India

    Chandrashekar Thalluri

Authors
  1. Mohan Goud Vanga
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Sarad Pawar Naik Bukke
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Praveen Kumar Kusuma
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Bayapa Reddy Narapureddy
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Chandrashekar Thalluri
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

Conceptualisation: V. M. G, S. P. N. B, K. P. K Methodology and investigation: S. P. N. B, V. M. G, K. P. K, C. T Analysis, interpreted the data and writing—original draft preparation: S. P. N. B, V. M. G, K. P. K, B. R. N, C. T Writing—review and editing: S. P. N. B, V. M. G, K. P. K, B. R. N, C. T

Corresponding authors

Correspondence to Sarad Pawar Naik Bukke or Praveen Kumar Kusuma.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Vanga, M.G., Bukke, S.P.N., Kusuma, P.K. et al. Integrating green analytical chemistry and analytical quality by design: an innovative approach for RP-UPLC method development of ensifentrine in bulk and inhalation formulations. BMC Chemistry 19, 70 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13065-025-01448-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13065-025-01448-8

Keywords

BMC Chemistry

ISSN: 2661-801X

Contact us