Environmental benign RP-HPLC method for the simultaneous estimation of anti-hypertensive drugs using analytical quality by design

ABSTRACT
 Green analytical procedures replace harmful organic modifiers with green solvents without affecting chromatographic performance, enabling industries and research laboratories to develop green analytical methods. Benidipine hydrochloride (BEN) and Chlorthalidone (CHD) were used to treat hypertension. The literature indicates that no method for determining BEN and CHD combines RP-HPLC with green Analytical Quality by Design (AQbD) for long-term use. This study aimed to develop a green RP-HPLC for determining BEN and CHD by incorporating analytical quality by design with green chemistry principles. A central composite design was used for optimization, with 40% ethanol content and flow rate chosen as critical variables. Separation was achieved using Agilent Eclipse Plus (C18, 250 mm × 4.6 mm i.d, 5 μm) with a mobile phase of ethanol and potassium dihydrogen orthophosphate (orthophosphoric acid to 3.5) in a ratio of 40:60 v/v at 1 ml/min, detection wavelength at 230 nm. Retention times for CHD and BEN were 3.1 and 5.1 min, respectively. The concentration ranges for BEN and CHD were 3.2–4.8 μg/ml and 5.0–7.5 μg/ml, respectively. The proposed method was eco-friendly and assessed using green evaluation tools. Thus, AQBD and green technologies provide regular BEN and CHD analysis in pharmaceutical formulations without environmental impact. GRAPHICAL ABSTRACT


Introduction
Hypertension is becoming more prevalent globally and is associated with an increased risk of stroke, cardiovascular and renal illness, cognitive impairment, and early mortality [1].Therefore, maintaining normal blood pressure is essential.Recent guidelines for treating hypertension promote combination therapy since it improves the number of patients who attain target blood pressure levels.According to American and European standards, combining calcium channel blockers and thiazide-like diuretics is preferable for treating hypertension.This combination can prevent myocardial infarction (MI) and stroke more effectively than other combinations [2][3][4].Benidipine and Chlorthalidone combination is effective in managing hypertension [5].
Reverse phase -High performance liquid chromatography (RP-HPLC) is the most extensively used technique in pharmaceutical quality control; it analyzes both API (active pharmaceutical ingredients) and their impurities [18,19].In quality by design-based context, the quality of HPLC techniques has become more important.Conventional HPLC technique optimization involves the study of one factor at a time while others are kept constant.It leads to numerous experiments with an inadequate understanding of key parameters [20].The Quality by Design (QbD) strategy truly overcomes the drawbacks of conventional one factor at a time by offering thorough interaction of several variables simultaneously on method response [21][22][23][24][25].According to International Council for Harmonization (ICH) guidelines Q14, AQbD is now mandatory [26,27].The principles of quality by design in analytical method development have been increasingly popular to attain high robustness and improved method performance [28][29][30][31].Central composite design (CCD) is most commonly used in HPLC method optimization [32,33].
Meanwhile, the RP-HPLC technique frequently requires enormous quantities of toxic organic solvents, such as acetonitrile and methanol.It generates large amounts of waste that must be disposed of, which cause significant problems in the environment and operator safety concerns.Therefore, developing eco-friendly HPLC procedures becomes particularly desirable [18,[34][35][36][37].The goal of green analytical chemistry (GAC) is to eliminate hazardous chemicals and replace them with safe alternatives and environmentally friendly ones to develop a multi-green analytical methodology for sustainable development [38][39][40][41].The guidelines provide a good overview of green chemicals that must be considered when developing green analytical methods [34,39,42,43].Most recommendations classify solvents depending on their net cumulative energy demand (CED) and environmental hazard safety (EHS).Despite the availability of green solvents with lower CED values, such solvents could not be directly utilized as organic modifiers in drug testing due to low drug compatibility and high instrument noise; this led to the conclusion that ethanol is a safe solvent that is an excellent alternative to toxic methanol and acetonitrile [44,45].By incorporating the GAC principles into the AQbD framework, one might ensure that an analytical method verifies the intended performances through risk assessment and complies with environmentally friendly specifications [46][47][48][49][50].The suggested study represents a novel chromatographic technique that uses ethanol, a less dangerous solvent than acetonitrile and methanol, which is considered more environmentally friendly.Hence, the prime focus of this research was to provide a novel methodology integrating the concept of AQbD with GAC ideas into practice.This combination framework was employed for the first time to establish an environmentally friendly and robust HPLC method for evaluating two drugs in bulk and their commercial formulations.The proposed method was validated according to ICH Q14 recommendations.

Chemicals and reagents
The standard pure drug BEN and CHD was purchased from (Yarrow chem products, Mumbai-India).HPLC grade organic phase ethanol was procured from Hayman Group Ltd, East Ways Park Witham, UK.Potassium dihydrogen orthophosphate (KH 2 PO 4 ) and sodium hydroxide (NaOH) were obtained from Sisco Research Laboratories Pvt.Ltd, Maharastra, India.Analytical-grade hydrochloric acid (HCl), orthophosphoric acid (OPA), and hydrogen peroxide (H 2 O 2 ) 30% (Rankem, New-Delhi, India) were used.Milli Q filter system (ELGA Lab Water, Lane End, High Wycombe, UK) was utilized to produce HPLC-grade water.Benidin CH (BEN 4 mg and CHD 6.25 mg) was acquired from the local pharmacy.

Instrumentation and optimized HPLC conditions
Method development and analyzes by isocratic separation were performed using HPLC Agilent 1220 infinity II (Santa Clara, California, USA) with a binary solvent delivery pump, autosampler injector, and a diode array detector.Separation was achieved using Agilent Eclipse Plus (C18, 250 mm 4.6 mm i.d, 5 μm).The buffer solution's pH adjustment was carried out using the ELICO INDIA model LI 120 pH meter (Hyderabad, Telangana, India).Filtration was carried out by using 0.45 μm nylon membranes.The solvents were degassed using an ultrasonicator (Maharastra, India).The optimum mobile phase comprised of ethanol and potassium dihydrogen orthophosphate (KH 2 PO 4 ) buffer (the pH was adjusted to 3.5 using 1% OPA) in the proportion of 40: 60 v/v; rate of flow at 1 ml/min; the temperature of a column at 35 ± 1; injection volume 10 μl, and detection wavelength at 230 nm.

Software
The data was collected and processed using the Agilent Open Lab CDS chemstation (version 2.6).Design-Expert® trailed version 12 (Stat-Ease Inc., Minneapolis-USA) software was used in experimental design for optimization of various chromatographic parameters as well as to create design space.
2.4.Preparation 2.4.1.Preparation of buffer solution 3.4 grams of potassium dihydrogen orthophosphate (KH 2 PO 4 ) were weighed and placed into a 1000 ml standard flask, then dissolved by HPLC grade water, and then the solution was made up to the required level.Further, 1% OPA was utilized to adjust the pH to 3.5.The buffer solution was filtered and degassed using sonication.

Preparation of standard stock solution
The BEN and CHD (1000 μg/ml) stock solutions were prepared in a separate volumetric flask, dissolved using ethanol, and made up to the mark.Suitable dilutions were prepared using ethanol to get the concentration of BEN 4.0 μg/ml and CHD 6.25 μg/ml.

Preparation of sample analysis
Twenty tablets of Benidin CH were weighed accurately and grind finely.An equivalent amount 40 mg of BEN and 62.5 mg of CHD was placed in a 100 ml standard flask, followed by 50 ml of ethanol was added; the solution was sonicated for around 20 min and made up to the required level.Filtered the solution with whatmann filter paper, then the filtrate was diluted accordingly to obtain the concentration of BEN 4.0 μg/ml and CHD 6.25 μg/ml to be injected in HPLC for estimation of its drug content.

System suitability
The standard solutions comprising 4.0 μg/ml for BEN and 6.25 μg/ml for CHD were injected in six replicate injections to determine the system appropriateness parameters.All percent relative standard deviations (RSD) were calculated for resolution, retention time (Rt), and theoretical plates.Figure 2 shows the standard chromatogram for BEN and CHD.

Solution stability
The standard solutions BEN and CHD were kept at room temperature and checked for stability over 72 h.The percent assay value of the standard samples was compared with the newly prepared samples.

Stress degradation study
The stock solutions of BEN (4.0 μg/ml) and CHD (6.25 μg/ ml) were exposed to various stress conditions, which include acid, alkali, oxidation, and thermal, and the level of degradation was studied concerning the period of time.The decrease in the area of the peak or the appearance of additional peaks was considered as degradation.The extent of degradation was measured by percentage recovery.Acid degradation: Hydrochloric acid (HCl) at a concentration of 1M was utilized to execute acid degradation.1 ml of stock solution was added to a 10 ml standard flask, and then 1 ml of 1M HCl was added and made up to the required volume using a diluent.Base degradation: The drugs were treated with 1M sodium hydroxide (NaOH) for alkali degradation. 1 ml of stock solutions was placed in a 10 ml standard flak, and add 1M NaOH then made up to the mark and maintained at 60°C for 1 h.Oxidation with hydrogen peroxide (H 2 O 2 ): 3% v/v hydrogen peroxide was used for peroxide degradation.Add 1 ml of standard solutions followed by 1 ml of 3% v/v peroxide solution in a 10 ml volumetric flask; the solution was then brought to the required level and maintained at 60 °C for 1 h.Thermal degradation study: The standard drug solutions were exposed to 60 °C in an oven for 8 h.The prepared solutions were injected into the HPLC system under ideal chromatographic circumstances.

Method validation
The suggested technique was validated as per ICH Q14 recommendations.

Linearity, LOD, and LOQ
The aliquots were taken from the stock solutions of (BEN and CHD) was placed in a 10 ml standard flask, and serial dilutions were prepared with the diluent to yield the concentration over the range of 3.2-4.8μg/ml for BEN and 5.0-7.5 μg/ml for CHD.The calibration curve was constructed by graphing concentration versus peak area.The regression correlation coefficient and intercept value were used to evaluate linearity.The method's sensitivity was assessed by the limit of detection (LOD) and the limit of quantification (LOD).

Results and discussion
Eco-friendly, hazardous-free, and less environmentally impacting analytical techniques can be developed with minimal trials and optimum results in the field of pharmaceutical analysis.However, developing a greener analytical technique without applying AQbD may suffer from method performance and necessitates constant revalidation.Consequently, incorporating both the GAC and AQbD principles in a single technique helps to enhance the robustness and sustainability of the method.An HPLC method optimization simultaneously adjusts various factors to achieve the desired separation.The optimization process using central composite design (CCD) has numerous benefits as an experimental design demonstrating the influence of independent variables on dependent variables at varying levels.The current study explains AQbD step by step using GAC principles for developing an analytical method.

Selection of mobile phase
The most frequently employed organic solvents in the HPLC method include methanol and acetonitrile; they should be ignored due to their toxic effects on the environment and health risks.Choosing an environmentally friendly liquid chromatography mobile phase that keeps the principles of GAC is challenging due to its existence and compatibility.Several agencies have provided solvent selection guides that indicate certain solvents can be used as a substitute for the organic phase.Ethanol is considered a perfect substitute for methanol.For better separation, various mobile phase ratios were tested by solvents such as acetonitrile, methanol, and ethanol in the organic phase, and different buffers like potassium dihydrogen orthophosphate buffer, ammonium formate of different pH, disodium hydrogen phosphate, acetate buffers and 1% acetic acid in the aqueous part; among them, potassium dihydrogen orthophosphate (KH 2 PO 4 ) along with ethanol was found to have better system suitability parameters for both drugs.The buffer solution (pH adjusted to 3.5 using 1% OPA), which elutes analytes faster from the stationary phase, requires minimal time and results in optimal retention time with excellent peak symmetry, less tailing, capacity factor, and theoretical plates.

AQbD aided method development.
The RP-HPLC method development for estimating BEN and CHD was performed by implementing AQbD principles to decrease the variability in the method's performance.The prime goal is to define the analytical target profile (ATP) in method development.The ATP was to develop a robust, accurate, specific, and environmentally friendly HPLC technique using greener chemicals.Several preliminary experiments were carried out by trial and error to improve method performance and identify critical, independent parameters and their effects on dependent variables.Selecting critical variables and responses is crucial in developing an HPLC technique using the AQbD strategy with preliminary experiments and risk assessment.In addition to the preliminary tests, the Ishikawa fishbone diagram (Figure 3) was used to identify and assess critical factors that pose an overall risk to the method's performance.Further, the experimental design approach was utilized to optimize the method factors in their specific ranges.

Method optimization by CCD
Optimization was performed by response surface methodology by utilizing a rotatable central composite design.CCD was utilized to fit a second-order model.The application of CCD is advantageous in sequencing studies since the prior center, and axial positions in  factorial trials were frequently determined.The factors were chosen for optimization to achieve the chromatographic separation of BEN and CHD, including the mobile phase composition (ethanol content: 35-45% v/ v) and rate of flow (0.8-1.2 ml/min), at five distinct levels.The design's actual values, along with their outcomes, are depicted in Table 1.CCD was performed with thirteen experimental runs with five center points.The responses of the midpoint of each variable at zero level were performed five times to determine the errors in the experiment.The individual response was analyzed using statistical methods, including analysis of variance, lack of fit (LOF), perturbation plots, 2D contour, 3D surface plots, and design space were produced.The experimental model's significance was determined using an analysis of variance, and their results are reported in Table 2.The model coefficients employed in this section are statistically significant (P values less than 0.0001).Furthermore, the Fischer ratios (F value) illustrate the significance of each coefficient in the model.Larger R 2 and lower lackof-fit values suggest the model is well-fitting, while a high F ratio shows that the analytical model equation is statistically significant.
The effect of variables on each response was investigated by graphical data interpretation, including perturbation, contour, and 3D surface graphs.The perturbation plot Figure 4a shows the effect of ethanol and flow rate on a resolution indicating that an increase in the amount of ethanol leads to a decrease in resolution between two drugs; the rate of flow also exhibits the same interaction; it does not offer steep slope like ethanol, but it has an interactive influence on resolution.The perturbation plot (Figure 4b) indicates the higher percentage of ethanol showed a decrease in the retention time of BEN peak,  whereas the flow rate has minimal effect.Perturbation plot Figure 4c represents that the increase in ethanol percent positively affects the theoretical plates of peak 2, and the higher flow rate leads to a reduction in the number of theoretical plates.The contour plots in Figure 4(d-f) generated show a similar interpretation consistent with the conclusions obtained from response surface findings.The 3D surface plots interaction is depicted in Figure 4(g-i The responses were optimized by the derringer's desirability function with various targets.The desired goal is to obtain suitable system suitability parameters like resolution to attain good separation of peaks; the second peak's retention time was kept to a minimum to get less time for analysis and maximum theoretical plates for column efficiency.Figure 5 depicts the overlay plot with optimum design space region.The predicted solution chosen was ethanol 40% v/v, a flow rate of 0.91, resolution of 6.781, the outcome value for Rt second peak is 5.239, and theoretical plates for the second peak is 3839.093with a desirability value of 1.000.Predicted experimental conditions determined the control strategy.The system suitability parameters were computed, and the values were obtained within acceptable limits.

System suitability
The outcomes of the system suitability test are represented in Table 3.The % RSD obtained for all parameters was less than 2.

Method validation
3.5.1.Linearity, LOD, and LOQ A significant linear relationship was attained among the concentration and peak areas of BEN and CHD over the ranges 3.2-4.8μg/ml and 5.0-7.5 μg/ml, respectively, under optimized chromatographic conditions with the correlation coefficient R 2 = 0.9982 for BEN and R 2 = 0.9986 for CHD.The analytical outcomes for linearity with slope and intercept are depicted in Table 4.The values of LOD and LOQ were computed using the standard deviation (SD) of response and regression line slope, which indicates the high sensitivity of the proposed method.The values for LOD and LOQ were obtained to be 1.812 and 5.493 μg/ml, respectively,   for BEN and 0.672 and 2.037 μg/ml, respectively, for CHD.

Precision and accuracy
All the precision studies were conducted, and their outcomes are portrayed in Table 5.The percentage RSD was not more than 2%, indicating reliable precision.The accuracy results of the developed method show a good range of percent recovery, representing great accuracy for the recommended method.The outcomes of accuracy are depicted in Table 4.

Solution stability
The related chromatograms for solution stability exhibited no deterioration peak and no significant change in the area of peak over 72 h.Compared to the fresh solution, the test findings were within 2%.

Outcomes of the degradation studies
The forced degradation studies were carried out on BEN and CHD drug combinations.The 1 M NaOH, 1 M HCl, 3% H 2 O 2 , and thermal at 60°C were chosen as primary degradation conditions; this generates relevant information on the stability of BEN and CHD.The drug CHD was more prone to degrade under acidic and alkali conditions; one additional peak was observed in acidic and alkali stress conditions at Rt 4.0 (Figure 6a and b), while BEN was stable to acid and alkali under experimental conditions.CHD was degraded instantly after adding the acid 1M HCl, and in alkali conditions, the degradation was observed after one hour.On exposure to 3% hydrogen peroxide, the degradation rate for CHD was considered very slow, while BEN was susceptible to hydrogen peroxide; the degradation was observed after one hour the percent drug recovery was found to be 85.41 for both drug degradation was observed under thermal conditions.The outcomes of the degradation study and their chromatogram are presented in Figure 6 and Table 6.

Application of pharmaceutical formulation
The application of the developed green HPLC method was tested by determining BEN and CHD content in the marketed formulation Benidin CH.The suggested method was not affected by the interference of excipients in the manufactured tablets.The amount of drug percentage was determined by triplicate analysis by standard addition method or within the acceptable range.The assay outcomes are depicted in Table 5.

Assessment of greenness profile for the developed method
Today's analysts have a great desire to develop innovative, green, more eco-friendly methods of analysis, which necessitates the use of safer solvents in place of dangerous and toxic ones.For developing sustainable analytical techniques, GAC has suggested many strategies as a part of the 3R's principles (replace, reduce, and reuse) in which toxic solvents are replaced with environmentally friendly and less expensive ones or reduced if their role is essential.A proposed approach can only claim to be environmentally benign if appropriate evaluation tools assess it.Here, the approach was evaluated by three green metrics, namely, the Analytical eco scale (AES), the Green analytical procedure index (GAPI), and the software-based Analytical greenness metric (AGREE).GAPI is widely recognized as one of the most popular greenness matrices.It is a quick, easy, and reliable tool for assessing the analytical method's greenness.GAPI is an excellent semi-quantitative tool for laboratory practice and education.It examines and quantifies the environmental effect associated with each step of an analytical method.Depending on the technique employed, the pictogram is coded with three different colors, red, yellow, and green assessing the green nature of the overall analytical procedure from sample collection to result from interpretation, providing qualitative information and making it simple to compare various analytical procedures.The output of the GAPI diagram is depicted in Figure 7a.Analytical eco scale (AES) is another tool for greenness assessment.It is a semi-quantitative tool to calculate the method's penalty points (PP).The environmental impact of the technique is evaluated based on the four criteria, which include chemicals reagents PP scored, instrumental energy, occupational risk, and waste produced by the total method.Calculate the total penalty points for the above four steps; then, it must be subtracted from the ideal green analysis score of 100.The results of the overall analytical eco-scale calculation are portrayed in Table 7.

AGREE metrics
The most recent tool for evaluating the greenness profile is software-based AGREE metrics that address all 12 green analytical principles.Each principle or factor is assigned a score ranging from 0 to 1 according to the risk of greenness principles.A value that is nearer to 1 represents the greenness of the method.Greenness is represented by the AGREE tool as a classic clock design having numbers 1-12 on the circle's side, representing the philosophy of 12 GAC principles.The developed technique has a total score of 0.85, as portrayed in Figure 7b, which shows that it was environmentally  friendly concerning all green principles.The comparison of the greenness between the reported and suggested techniques using GAPI, AES, and AGREE tools were depicted in Table 7.

Conclusion
This study demonstrates the innovative incorporation of AQbD and green analytical chemistry (GAC) through the analytical method development for the determination of BEN and CHD in bulk and its formulations.By implementing AQbD, method variables can be visualized, resulting in stable and robust methods that can be efficiently employed in quality control laboratories without further revalidation.An experimental design, such as a central composite design, has been employed for the statistical optimization investigations.The acquired results established the best-operating conditions for the drug combination within the design space region, which were validated practically with additional runs.Concerning the GAC principles, ethanol was used as an organic solvent rather than a potentially hazardous solvent.Finally, the findings of the green evaluation tools demonstrated that the procedure was most environmentally friendly and readily applicable for industry and routine quality control.Table 7.Comparison of green assessment between proposed vs reported HPLC method.

Figure 7 .
Figure 7. Pictogram represents the green assessment results for the proposed method a) GAPI, b) AGREE metrics.

Table 1 .
Experimental design actual and measured responses.

Table 2 .
ANOVA and regression summary of models.

Table 3 .
System suitability results for the developed HPLC method.

Table 4 .
The results of Linearity parameters for BEN and CHD.

Table 5 .
Accuracy and Precision results for BEN and CHD.

Table 6 .
Forced degradation studies of BEN and CHD.