Environmental benign analytical quality by design and UPLC method development for Betamethasone and Calcipotriene in ointment

The present study aimed to estimate Betamethasone and Calcipotriene in bulk and ointment formulation using a validated UPLC method by applying analytical quality design with green analytical chemistry principles. The optimal method resulted with Dikma Endeversil C18 ODS (2.1 × 50 mm, 1.8 µm) column at a UV detection of 254 nm encompassed ethanol and potassium dihydrogen phosphate (3.0 pH) buffer 51:49 (v/v), with a flow rate of 0.31 mL/min. The detector response was linear at 125–750 and 12.5–75 µg/mL for Betamethasone and Calcipotriene with a detection limit and quantification of 12.484, 37.831 and 3.229, 9.785 µg/mL, respectively. The % recovery was found to be within limits of less than 1.5%. Overall, the AQbD based developed method was greener and confirmed by the greenness evaluation tools. Hence, the optimized technique is environment friendly, simple, robust for the concurrent assessment of Betamethasone and Calcipotriene in bulk and ointment formulation. GRAPHICAL ABSTRACT

The available marketed ointment formulations containing BMS and CPT are well known for their synergistic effect for treating plaque psoriasis in elderly patients [3]. There is a rising concern across the toxic effect of several chemicals in commercially used analytical methods, especially by the industry, on the environment. Analytical approaches for drug study also include toxic and hazardous solvents with massive volumes of waste production and treatment [4]. Nowadays, the pharmaceutical and chemical industries aim to improve an eco-friendly analytical method for drug and chemical analysis with their regulatory commitments by applying the Green Analytical Chemistry (GAC) concepts [5][6][7][8]. Eleven out of twelve green analytical principles could be covered [9,10] (sample size, eco-safety chemicals, renewable solvents, waste reduction, insitu measurement, miniaturization, hyphenated techniques, multiple analysis, energy consumption, derivatisation, and analyst safety) by utilizing a reliable UPLC instrument.
Analysis of Ointments requires hazardous solvents and may the extract needs to be partitioned between hexane and methanol or methanol/water combinations; the highly lipophilic substance is removed into the hexane layer [11].
To analyses, an ointment, in general, requires a minimum of 50-100 mL of organic solvent. This may raise a serious concern over to the environment and analyst occupational hazards by these chemicals. The exploitation of toxic solvents like methanol, hexane may benefit the analysis but not the environment. So, this raised concern over the development of hazardous methods for the study of pharmaceutical and chemical substances and made to initiate a new approach that is entirely eco-friendly and nullifies the occupational hazard towards the analyst.
There are few analytical methods reported for individual drugs based on varied techniques for detecting BMS and CPT, such as spectrophotometric [12], HPLC [13][14][15][16][17], HPTLC [18], and UPLC [19]. However, few HPLC methods are available for analysing BMS and CPT. Still, they possess disadvantages like using more hazardous chemicals and need to develop AQbD based strategies. That help detects and reduces sources of variability that may result in inadequate method robustness and ensure that the method fulfils its intended performance criteria throughout the product and method lifetime.
As per the literature, no combined UPLC, AQbD, and green analytical-based methodology were available to analyse any drug combinations. Combining the GAC with UPLC and Analytical Quality by Design (AQbD) [20,21] significantly benefits when constructing an innovative method. It creates a synergistic setting for developing environmentally sustainable, effective, and adaptive eco-friendly analytical procedures.
Hence, this work aimed to conduct a novel combined framework for applying GAC principles along with AQbD principles. This novel combination of analytical approach was used for the first instance to develop a green and robust UPLC-PDA study to analyse BMS and CPT in their bulk and marketed ointment formulations. The rotatable central composite design was utilized to determine the critical method parameters and their effect on critical quality attributes of the analytical method.

Chemicals reagents
Ortho Phosphoric acid, HPLC grade Ethanol, HPLC grade water (Milli Q or equivalent), Pure BMS, and Pure CPT. (gifted by Glenmark Pharmaceuticals Ltd., Mumbai-India). Marketed formulations Heximar B ointment, with a label claim of 0.05% w/w BMS and 0.005% w/w CPT manufactured by Marini India Pvt Ltd pharma and acquired from the local pharmacy.

Preparation of stock and calibration solutions
The HPLC grade ethanol made the BMS and CPT standard stock solutions (1 mg/mL). Further, the mobile phase is utilized as a diluent; separate serial dilutions (125-750 and 12.5-75 μg/mL) were achieved using the corresponding standard stock solutions for developing calibration curves. The BMS and CPT mixture was made by adding various aliquot portions from the standard stock solution and filling the volumes with diluent to obtain 500 and 50 μg/mL concentrations.

Precision and trueness
The six sample solutions for precision and three separate concentration ranges (80, 100, and 120 μg/mL) for trueness prepared and analysed for intra-day precision and trueness.

Forced degradation studies
To a series of 10 mL volumetric flasks, containing 1 mL of standard BMS and CPT, then 1 mL of 0.1 N HCl, 0.01 N NaOH, and 3% H 2 O 2 were added separately, and made up to volume with diluent. The final solutions were injected into the UPLC system for the chromatographic analysis, with a time range of 0, 30 min, and 1 h and the amount of degradation was compared with the control.

Photodegradation
Standard solutions of BMS and CPT were prepared and placed in Ultraviolet rays for 3 h and injected into the UPLC system for studying the photodegradation studies.

Assay of pharmaceutical formulation
An accurately weighed sample from the tube's top, middle, and bottom positions, equivalent to 5 and 0.50 mg of BMS and CPT, respectively, has been transferred to a 10 mL volumetric flask. The HPLC grade ethanol (5 mL) was added, sonicated for 15 min, makeup with diluent, and filtered through 0.45μ Polyvinylidene fluoride (PVDF) filter. Final Aliquots of 4 μL infused in as triplicate, the described earlier procedures have been used to determine the concentration of the investigated drugs in their formulations.

Results and discussion
Although pharmaceutical researchers have established several analytical techniques for evaluating drugs and chemical substances existing in various samples, most of these methodologies possess environmental and health consequences when transferring a method to commercial exploitation on an industrial scale. Developing a strategy should focus on enriching eco-friendly concepts so that it helps the method to sustain for longterm usage. The preliminary step in the liquid chromatographic methods was the selection of the most appropriate solvents. There are several agencies [22,23] that have listed the solvent-based on the cumulative energy demand (CED) calculations. Still, all these solvents cannot be applied in liquid chromatography due to several constraints like solvent compatibility with either chemicals (solubility) or instruments (high noise). From these constraints, very few solvents were picked for the column chromatography, like ethanol, a good alternative for methanol, propylene carbonate for acetonitrile, and ethyl lactate for ethyl acetate. From the initial trials of the solubility of the drugs, it is found that the drugs were completely soluble in ethanol. So, ethanol, a biodegradable solvent with significantly less CED, has been selected for further analysis.
Selecting a pH for separating two analytes is the primary task for an analyst, and it is based on two main criteria. Firstly, column-like silica-based type should be operated at a pH range of 2-8. No decrease or increase in the pH outside this range will damage or solubilize the bonded silica phase. The second criteria are based on the analyte's need to be separated, where BMS and CPT have a pKa value of 13.4 and 14.39 and ionize in lower pH (acidic range). The basic principle of chromatography states that the ionized form of analytes is more polar and less retained in the reverse phased columns, so the mobile phase pH selected at 3.0 to elute analytes with short runtime to enhance the green analytical principles.
The next factor for consideration was the composition of the two drugs as pharmaceutical dosage form of BMS and CPT was in a ratio of 1: 10. There should be a definite check in the interaction of conditions and parameters with the responses. So instead of checking the parameters and their interaction with each other by using one factor-at a time and wasting the solvent, energy, and time AQD technique will simplify this problem and helps to find the interaction between the constraints and responses.
Considering this, new approaches have been developed to integrate UPLC, AQbD, and GAC concepts into a coherent model to improve the long-term sustainability and robustness of BMS and CPT estimation. Following the five components of the AQbD paradigm makes it possible to understand method parameters and their relationships better, identify elements that significantly influence performance characteristics, and allocate permissible limits of variation. In this article, a thorough overview of AQbD, with GAC principles, has been applied to develop a sustainable analytical procedure to estimate a drug combination BMS and CPT, demonstrating the technological advances in a chemical compound or drug analysis.

Identifying attribute and risk assessment
The Analytical Target Profile (ATP) contains most of the attributes necessary to validate the analytical method's quality and purpose. Table S1 illustrates the numerous chromatographic components that comprise the ATPs required for effective method development. The Critical Quality Attributes (CQAs) are often detected during the formulation development process as a part of Quality by Design-based manufacturing process characteristics. In AQbD assessment, resolution, precision, retention, peak shape, and drug sensitivity are probable CQAs. The Ishikawa Fishbone Diagram is utilized to identify risks ( Figure 2); risk evaluation is the fundamental ideology with varying criteria on various responses. This approach helps investigate the cause-and-effect relationship of the experimental conditions in our study design; thus, much less work and time were consumed, and quicker optimization processes were attainable. The separation criticality among a set of closely eluted peaks (BMS and CPT) was investigated using isocratic mobile phase elution by trails of the one-time factor were utilized and found that second compound peak tailing (PT 2 ), emphasizing the tailing of these combinations. Trails also show that variation of other factors like temperature, injection volume leads to a negligible effect on CMP's. Few materials attribute like flow rate, pH, and mobile phase composition are considered significantly impacting the parameters.
Consequently, minor changes in the above factors significantly affect the output in the system suitability parameters. The essential CAAs were chosen, taking into account factors such as resolution (Rs), second peak tailing (PT 2 ), and the retention time of the second peak (RT 2 ) were considered as critical attributes for the present method based on the one-time factor analysis. A rotatable central composite design was applied for the method development ensures overall consistency of prediction error and attained through the proper choice of α values gives more detailed interaction ranges and the effect of CMP on CAA.

Rotatable central composite design (rCCD) applied methods optimization
The entire optimal state was evaluated with rCCD, the combination when defining its design space for rigorous analysis; the three CMPs were constrained to pilot study examination using rotatable central composite design (rCCD). The preliminary analysis determined the levels of each parameter, and the chosen CAAs were employed to get the best results. (Table S2). At the same time, high ethanol percentages (55% for the mixture) and flow rates (0.35 mL/min) improved peak shape but negatively impacted peak resolution. Furthermore, at low ethanol (45%) and low pH 2.5, a resolution has been achieved with a longer run time which is not recommended according to green analytical principles. The mixture was measured in three separate ranges for ethanol % (-α, 0, +α), pH (-α, 0, +α), and flow rates (-α, 0, +α). Six centre points for accurate experimental error estimation and the resulting RT 2 , Rs, and PT 2 values were recorded after a sequence of twenty experiments were performed in chronological order. After the generated frameworks were analysed, the method was discovered to obey polynomial equations with wellconsidered linear effects, quadratic effects, and factor interactions (Table S3).

Mathematical validation, interpretation, and desirability function
The predictive viability of the regression models for the three responses was then ensured with the obtained ANOVA data, lack of fit non-significance, reasonable standards of R 2 , adjusted and predicted R 2 (Table S3). Graphical data interpretation techniques such as Perturbation, contour, and 3D surface plots were used to investigate the impacts of CMPs like ethanol (X1), Flow rate (X2) and pH (X3) on retention of the second peak (Y1), Resolution (Y2) and Tailing factor of the second peak (Y3) (CAA's). The model's p-value of < 0.0001 for (Y1,2,3). The Model F-values of 1444.94 (Y1), 72 (Y2), 29.76 (Y3) implies the model is significant. There is   Table S4 demonstrate that changing the CMA in the design space shows a prominent effect on three attributes and the need to combine these variations in the determination of BMS and CPT. CMA's role on CMP's as increasing in ethanol concentration increases the RT 2 , indicating that CPT retains in the column on the increase in ethanol concentration and showed a steep rise in Rs but decreased in PT 2 . The second-factor flow rate shows increased RT 2 and Rs but showed a parabolical decrease in PT 2 . Finally, pH as the third factor impacts RT 2 as an increase in pH increases RT 2 and resolution but had a minimum effect on the PT 2 .
The present study's objective was to establish a procedure that did not affect green analytical principles and was primarily focused on waste reduction via UPLC to avoid interfering with validation standards, as previously stated. The obtained results with a PT 2 limit of less than 2 for an acceptable peak elution, the ethanol ratio, flow rate, and pH were maintained, which might not affect PT 2 . Regarding RT 2 , even if ICH guidelines impose no limits, but according to GAC principles, the delayed RT 2 was considered. Finally, there is a criterion for the resolution, which must be more than 1.5. The three responses were interconnected, demonstrating the need to select a technique that did not interfere with the other responses. Derringer's desirability (Table  S5) methodology was used to determine the ideal combination of circumstances based on the importance and limitations of each response. For instance, as seen in Figure 4, the desirability methodology proved specific goals within predefined criteria. Some of the parameters like pH were set as a target. The resolution responses were kept maximum for better separation, and the tailing factor was developed to minimize to attain better elution. A specific experimental range was evaluated for compositions containing an ethanol ratio of 52.04 with a flow rate of 0.318 and buffer pH of 3 showed desirability of 0.832, which is near to 1. The same parameters were selected as a target result which was compared practically and showed a deviation of less than 10% that met the set limitations to the entire degree possible, which would have been the limit of consistency. The overlay plot for the design space was depicted in Figure 5, which shows any changes in the yellow region do not require further revalidation. The following parameters were chosen for the final optimum RP-UPLC chromatographic setup: mobile phase is ethanol and buffers 51: 49, flow rate (0.31 mL/min), and pH 3.0.

Specificity
The technique's specificity was tested by injecting a laboratory-prepared extracted placebo to verify the lack of interference with the analyte's elution. The absence of additional peaks in the chromatogram demonstrates that the approach was specific for drug analysis. Figure 6 shows the blank, placebo, and Peak purity plot chromatograms of the BMS and CPT method.

System suitability parameters
After six duplicate injections, the system suitability tests indicated no statistically significant difference between BMS and CPT in peak area, retention time, theoretical plates, and peak tailing. The % RSD values were determined to be < 1.5%, validating the high degree of trueness of the chromatographic instrument. The validation results for system specificity are summarized in Table 1.

Linearity LOD and LOQ
The linearity for BMS and CPT was determined by constructing calibration curves in the concentration ranges of 125-750 and 12.5-75 μg/mL, respectively (Figure 7). Each solution of different concentrations was injected in triplicates, and peak areas were observed for each solution. The calibration curves were constructed by plotting the mean area of a peak versus the concentration (μg/mL). The correlation coefficient results suggested that the linearity was excellent in this case (0.999 for BMS and 0.9992 for CPT). The regression models were derived in the way shown in Table 1. The LOD and LOQ of the approach were determined theoretically to be 12.484, 37.831 μg/mL for BMS, and 3.229, 9.785 μg/mL for CPT, respectively, and then applied in practice.

Precision and trueness
The precision has been calculated at three different concentrations, and the results are portrayed in Table 1. The % RSD for inter-day and Intra-day precision ranges from 0.59, 0.70 and 0.48, 0.49 for BMS and CPT. The trueness of the sample was validated at three different concentrations of 80, 100, and 120%; results were   Tailing factor Mean ± SD * * 1.18 ± 0.01 1.57 ± 0.02 % RSD 1.10 0.81 * Mean of six determinations, * * Set of five determinations, * * * Set of three determinations reported in Table 1, and results were within reasonable limits compared to the reported methods in HPLC.

Forced degradation results
The forced degradation studies of BMS and CPT were studied in various stressed conditions and found to be stable in acid condition with around 5%. BMS degrades in the alkali with more than 15% and CPT degrades up to 15% in oxidation however, the other drugs (CPT in alkali and BMS in Oxidation) shows to be stable in those conditions. Photo degradation results illustrates that the drugs were stable and shows less than 2% degradation after 3 h of exposer to the UV light. Figure 8a, b, c, d and Table 2 illustrates the total degradation of the two drugs in their respective stressed conditions.

Assay of marketed formulation
The efficacy of the newly developed UPLC method was proven by examining the mixture of commercially available pharmaceutical products (Figure 7). The drugs, as mentioned above, were estimated in such a selective manner, with high recovery values in their combination formulation. Furthermore, the efficacy of the extraction and the relationship between the excipients were investigated utilizing the conventional addition methodology. The findings reveal that satisfactory recoveries were achieved with no influence from the common ointment excipients used in the preparation. The student's t and F-tests were used to compare the findings obtained by the designed approach to the results obtained by other methods for assessing each of the studied substances. None of the obtained results was more significant than any calculated results, indicating no statistically significant variations in the two processes' trueness and precision (Table 3).

Greenness assessment and methods comparison
The GAC offered many advancements for creating stable green analytical methodologies that would be implemented in the future. As part of the 3R principles, hazardous/toxic chemicals are substituted with more environmentally friendly and cost-effective alternatives, or their usage was regulated if excessive (Replace, Reduce, Reuse) [24]. Compared to prior methodologies for analysing drug mixtures, this developed method effectively measured the BMS and CPT in their combination by employing greener ethanol rather than the potentially hazardous methanol or acetonitrile, which was widely used. This suggested UPLC technique uses the least amount of solvent and energy possible. As a result of these characteristics, the proposed methodology seemed more ecologically sustainable than one that had already been reported. Five state-of-the-art metrics were employed to determine if a device was ecologically sustainable: NEMI, GAPI, Analytical Eco-Scale, AMGS, and AGREE. NEMI is a statistic that measures how environmentally friendly a method is. NEMI pictogram is a classic tool for assessing greenness in the technique. NEMI results were represented as a circular pictogram parted into four quadrants with a colour coding with green and colourless and the detailed assessment was explained in supplementary file. Table S6 Shows the NEMI pictogram  for the proposed method. GAPI results represent the colour coding as green, yellow, and red. GAPI covers sample preparation and instrumentation assessment which is a drawback in NEMI [25] detailed explanation was given in supplementary file. Table S6 portrayed the GAPI pictograms for the proposed method and reported using the help of J. Płotka-Wasylka [26] developed software for easy and perfect projections of the GAPI assessment [27].
Analytical eco-scale [28] is signified through a final score of 100. The final score was reduced by the Penalty point (PP) based on the method performance like the chemicals, reagents, instruments, and wastage by the method is considered and deducted. This process involves five different steps; as it was designed for our developed UPLC methodology was well explained in supplementary file.
The approach with a 75 was deemed greener; the recommended approach received a score of 96,   reflecting the approach's influence on future use in terms of environmental acceptability. AMGS amalgams some metrics, including SHE (safety, health, and environmental assessment). It is used to estimate solvent safety using the geometric mean, CED (Cumulative Energy Demand), and AMVI (Analytical Mass Volume Intensity) [29], used for calculating solvent volume waste and instrument and solvent selection. It is a semi-quantitative method for determining environmental sustainability [30]. Once the data required has been administered, it generated a score of 49.38 (Table S6), indicating that the technique's ecological impact is eco-friendly as the decrease in score indicates the greener method.
AGREE metrics is a software-based program based on the 12 principles. Results incorporated in each principle or slot show the impact of the method on the environment, with a score of 1 indicates the process greenest [31][32][33]. The total score of 0.89 in Table S6 shows that the proposed method was the greenest in all respects. In retention time or the waste produced, the greenest results were correlated with the reported HPLC techniques. Table S6 provides comparative findings of the procedures that have been published and those suggested for BMS and CPT.

Conclusion
The incorporation of the AQbD approach with GAC postulates a new eco-friendly and precise analytical methodology. For analysis of two drugs, BMS and CPT, in their bulk and branded formulations, established a novel green AQbD system. A detailed step-by-step overview of this AQbD approach was introduced to provide an accurate image of system variables and develop simple, stable, and robust methods that can be utilized in QC laboratories without further revalidation. Experiment designs like the rotatable central composite design were used for mathematical optimization trials with mobile phase composition, flow rate, and buffer pH as variables and resolution, retention time, and peak tailing as a response. Instead of toxic organic solvents including methanol and acetonitrile, less hazardous ethanol-based mobile phases were used. In UPLC, shorter columns with the finest particle sizes at high pressure allowed for a 2.4-minute analysis indicating that the developed method was much better than previously reported methods in retaining two drugs within less than 1.14. min without compromising the ratio factor. The findings identified the optimum working conditions for the drugs mix and design spaces, experimentally tested with extra runs. The optimized method was further validated and found the trueness results at three different levels, and precision results show that less than 1.5% RSD was within limits. Linearity range and correlation coefficient, which is less than one and under limits. Finally, the green assessment tools results demonstrated that the approach was the most environmentally sustainable and could be readily adapted for industrial and quality control purposes.

Disclosure statement
No potential conflict of interest was reported by the authors.