Sustainable HPLC technique for measurement of antidiabetic drugs: Appraisal of green and white metrics, content uniformity, and in vitro dissolution
-
Asma S. Al-Wasidi
and
Hossam F. Nassar
Abstract
Green chemistry and white chemistry are two approaches to chemistry that prioritize sustainability and environmental protection. The pursuit of green chemistry is to develop chemical processes and products that decrease or stop the use and generation of dangerous materials. In contrast, white chemistry focuses on developing energy-efficient, sustainable chemical processes that produce minimal waste. Our study evaluated the environmental friendliness of the suggested approach, using eight greenness appraisal techniques, including analytical eco-scale, analytical method volume intensity, HPLC-environmental assessment tool, analytical method greenness score, analytical greenness, analytical greenness metric for sample preparation, green analytical procedure index, and complexgreen analytical procedure index, in addition to the unique metric blue applicability grade index of white chemistry. We have developed and validated a highly effective and reliable method for concurrently analyzing designated pharmaceutical medications characterized in metformin (MET) and empagliflozin (EMP) formulations, including their degraded products. This method is cost-effective, specific, and environmentally friendly, utilizing reversed-phase high-performance liquid chromatography with an XBridge BEH C8 column (150 mm × 4.6 mm, 5 μm) at a flow rate of 1.0 mL·min−1, an injection volume of 5.0 μL, a column oven temperature of 50°C, a wavelength of 224 nm, and a mobile phase comprised of phosphate buffer adjusted at pH 6.8 and acetonitrile in gradient mode. In the HPLC method, linearity has been achieved over the concentration range of 10–106 and 30–1,050 µg·mL−1 for EMP and MET, respectively, with correlation coefficients more than 0.999 and good recoveries within 98–102%. An assessment of the content uniformity of finished products confirmed that they met the declaration’s acceptance standards (85–115%). A comparative study has been successfully conducted on generic and reference products, demonstrating their similarity. The suggested approach was validated by adhering to international council for harmonisation criteria.
Graphical abstract
1 Introduction
Green chemistry and white chemistry are two fundamental concepts essential for the chemical industry to adopt to achieve sustainable and eco-friendly practices. Green chemistry strives to develop and apply chemical processes and products that reduce the usage and production of hazardous materials [1,2,3]. On the other hand, white chemistry emphasizes using renewable resources and the development of biodegradable products. Both concepts are vital in reducing chemical production’s environmental impact while promoting economic growth [4,5,6]. HPLC is a widely adopted technique in analytical chemistry. However, the resource-intensive process raises ecological concerns. To address this, researchers have been developing sustainable HPLC techniques that minimize waste and reduce the use of hazardous solvents. One of the most promising approaches involves using supercritical green solvents as a mobile phase instead of traditional ones, which provides better selectivity while reducing waste. Another strategy is to use microscale HPLC, which requires fewer samples and solvents and effectively minimizes environmental impact. These sustainable HPLC techniques are becoming increasingly popular among researchers committed to reducing their ecological footprint while ensuring analytical performance [7,8]. Environmental sustainability is a crucial component in the advancement of chromatographic technology. A comprehensive evaluation of HPLC techniques’ potential environmental impacts is necessary to determine their ecological benefits and eco-friendliness [9]. We can use many tools, such as the blue applicability grade index (BAGI) tool, to facilitate this evaluation. Several other metrics have also been developed for ecologically sustainable analytical methodologies, including analytical GREEnness (AGREE), green analytical procedure index (GAPI), analytical greenness metric for sample preparation (AGREEprep), complexgreen analytical procedure index (ComplexGAPI), analytical method greenness score (AMGS), HPLC-environmental assessment tool (HPLC-EAT), analytical method volume intensity (AMVI), and the analytical eco-scale analytical eco-scale (ESA) [10,11,12].
Metformin (MET) (Figure 1a) is a commonly prescribed medication for people with type 2 diabetes. By decreasing the quantity of glucose, the liver generates and enhancing insulin sensitivity in the body, type 2 diabetes lowers blood sugar levels. It is usually taken orally and is often combined with other diabetes medications or lifestyle changes to manage blood sugar levels effectively [13]. One drug used to treat type 2 diabetes is empagliflozin (EMP) (Figure 1b). It lowers blood sugar levels, increases urine excretion of glucose, and prevents the kidneys from reabsorbing glucose. It is often prescribed along with diet and exercise [14]. EMP and MET are highly effective combination drugs used in tandem with diet and exercise to help effectively manage blood sugar levels in individuals diagnosed with type 2 diabetes. This potent medication combination significantly modifies the body’s insulin response, increases sugar secretion through the kidneys, reduces sugar production in the liver, and effectively decreases sugar absorption from the stomach and intestines [15].
Chemical structures of (a) EMP and (b) MET.
In vitro dissolution is a process used to study the rate at which a drug dissolves in a medium under controlled conditions. It is an essential step in drug development and helps determine its bioavailability. The dissolution rate of a drug can be affected by various factors, such as the pH of the medium, temperature, agitation, and the presence of enzymes. In vitro dissolution testing is conducted using a dissolution apparatus that mimics the conditions of the human gastrointestinal tract. The results of in vitro dissolution testing are used to optimize drug formulations and establish appropriate dosing regimens for clinical use [16]. Comparative dissolution profiles compare and evaluate the performance of different drug formulations, such as generic drugs and their branded counterparts. These profiles provide information on the rate and extent of drug dissolution under standardized conditions. Comparative dissolution testing is an essential step in drug development, as it helps to ensure that the generic drug is bioequivalent to the branded drug. This testing is conducted using a dissolution apparatus, and the results are compared to determine if there are any significant differences in the dissolution profiles of the two formulations. By comparing dissolution profiles, pharmaceutical companies can ensure that their generic drugs are safe, effective, and equivalent to their branded counterparts [17].
The forced degradation tests produce degradation products more quickly than accelerated and extended stability assessments because they produce degradation products more rapidly. Drug ingredients and products are degraded more efficiently under stress. Stress parameters must be evaluated to evaluate their impact on drug stability [18]. It is becoming increasingly common in modern analytical chemistry to perform simultaneous estimation of mixed binary analytes using identical reagents and columns. Decreased work for evaluating and approving items before the planned release date can save money and time for the quality control lab [19]. In each pharmacopeia, the British Pharmacopoeia (BP) and the United States Pharmacopeia (USP), an HPLC method is individually described for MET and EMP quantification [20,21]. However, after conducting a comprehensive literature review, it has been found that only a limited number of UPLC and HPLC methods are available for determining EMP and MET [22,23,24,25,26,27]. It is evident that no HPLC method currently exists for the simultaneous evaluation of MET and EMP and their degraded products in a single run while also utilizing eight greenness appraisal techniques, such as ESA, HPLC-EAT, AGREEprep, GAPI, AMGS, AGREE, AMVI, and ComplexGAPI, alongside the unique metric BAGI of white chemistry and implementing content uniformity and in vitro dissolution studies.
This study stands out for developing a highly robust HPLC method that simultaneously estimates both MET and EMP and their degraded products in a single run. In addition, the study employs not one but eight different greenness appraisal techniques, including ESA, HPLC-EAT, AGREEprep, GAPI, AMGS, AGREE, AMVI, and ComplexGAPI, which all contribute to the study’s overall confidence. Furthermore, the researchers used the highly reliable BAGI metric of white chemistry. They conducted thorough investigations of content uniformity and dissolution in vitro, all adding to the study’s impressive thoroughness.
2 Experimental
2.1 Materials and reagent
The MET working standard is purchased from Wanbury Limited (Mumbai, India) with a potency of 99.8% as an anhydrous base. In contrast, the EMP working standard is purchased from Optrix Laboratories Private Limited (Telangana, India) with a potency of 98.8%. Hikma Pharmaceutical Company (6th of October City, Egypt) supplied Empagliform XR 25/1,000 mg film-coated tablets (FCT). Synjardy XR (25/1,000 mg tablets), extended-release which was Licensed from Boehringer Ingelheim International GmbH, (Ingelheim, Germany) and marketed by Boehringer Ingelheim Pharmaceuticals, Inc. (Ridgefield, USA) and Eli Lilly Company (Indianapolis, USA). A variety of chemicals were purchased from Scharlau (Barcelona, Spain) including monopotassium phosphate, octane-1-sulfonic acid sodium salt, orthophosphoric, sodium hydroxide, hydrogen peroxide, hydrochloric acid analytical grade, acetonitrile, and methanol HPLC grade.
2.2 Instrumentation and software
The integrated Alliance HPLC system used in this experiment from Waters Company (Milford, USA) includes an autosampler and an E2695 quaternary gradient pump. In addition to the column oven that functioned as a heater and a cooler, the system was equipped with a photodiode array detector. Computer software Empower 3 was used to obtain and process the chromatograms.
Pharma Test’s PTWS 1420 tablet dissolution tester is an advanced device for testing tablet dissolution (Hainburg, Germany).
HPLC-EAT is a software application specifically developed to evaluate liquid chromatography procedures. The software can be accessed at no cost using the URL: http://www.biotek.lu.se/hplc-eat/. The AMGS program offers a website accessible via ACS-GCI-PR, found at https://www.acsgcipr.org/amgs. The ComplexGAPI-based software is freely accessible at mostwiedzy.pl/complexgapi. The AGREE software can be accessed at https://mostwiedzy.pl/AGREE, with the latest version available at https://git.pg.edu.pl/p174235/AGREE. AGREEprep, available at https://agreeprep.anvil.app, allows us to evaluate the environmental sustainability of sample preparation during analytical operations. A straightforward, open-source program (mostwiedzy.pl/bagi) was developed to streamline the utilization of the BAGI measure. The web application can be accessed at bagi-index.anvil.app.
2.3 Procedures
The diluent is formed of buffer pH 6.8: organic solution with a ratio of 40:60, where the organic solution is formed of acetonitrile and methanol (50:50).
2.4 Dissolution media used
Acidic medium (pH 1.2), acetate buffer (pH 4.5), and phosphate buffer pH 6.8 (the optimal medium) were created by combining a 0.05 M solution of monobasic potassium phosphate with a 0.02 M solution of sodium hydroxide in water. The pH was then adjusted to 6.8 using either 0.2 N sodium hydroxide or 1 M phosphoric acid after diluting the mixture with water to a total volume of 1,000 mL.
2.5 Standard solution preparation
2.5.1 Standard stock solution (A)
Accurately weigh approximately 20 mg of the MET working standard and then transfer it completely into a 10 mL volumetric flask using water as a diluent. Sonicate for approximately 10 min, then fill up to the mark with water, and mix thoroughly [28].
2.5.2 Standard stock solution (B)
The 10 mg of the EMP working standard was accurately weighed using a sensitive balance. After weighing, the entire amount was transferred into a 10 mL volumetric flask and dissolved in water by sonication and mixing.
2.5.3 Standard solution (for assay and content uniformity methods)
The 1.0 mL each from stock solutions A and B were accurately transferred into a 20 mL volumetric flask and dissolved in diluent by sonication and mixing to achieve concentrations of 0.05 mg·mL−1 for EMP and 0.100 mg·mL−1 for MET.
2.5.4 Standard solution (for dissolution method)
The 5 mL from stock solution A and 0.25 mL from stock solution B were accurately transferred into a 10 mL volumetric flask and dissolved in dissolution media by sonication and mixing to obtain a concentration of 0.025 mg·mL−1 for EMP and 1.0 mg·mL−1 for MET.
2.6 Test solution for assay and content uniformity methods
Weigh 10 tablets of Empagliform XR 25/1,000 mg FCT and Synjardy XR 25/1,000 mg tablet to calculate the average weight. Grind 10 tablets, weigh an equivalent of one tablet, and transfer the powder into a 100 mL volumetric flask with buffer pH 6.8 as the diluent. Sonicate for 10 min, fill up to volume with water, and mix thoroughly. Transfer 25 and 1.0 mL from the stock test solution into two separate 100 mL volumetric flasks. Fill to volume with diluent and mix thoroughly to achieve concentrations of 0.05 mg·mL−1 for EMP and 0.100 mg·mL−1 for MET. The sample was put into an HPLC vial and analyzed after the first 5 mL of filtrate from the 0.2 µm PTFE filter was discarded [29].
2.7 Test solution for dissolution method
Place one tablet in each dissolution vessel and operate with the specified parameters. This involves using 900 mL of USP dissolution medium, setting the apparatus to USP I with a basket mesh size of 40, running at 100 rpm, maintaining a temperature of 37°C, and running for 10, 15, 20, 30, 45, and 60 min for EMP and 1, 2, 4, 6, 8, 10, and 12 h for MET.
2.8 Chromatographic conditions
In alliance with HPLC waters, we developed a gradient system for simultaneous estimating EMP and MET using an XBridge BEH C8 column (150 mm × 4.6 mm, 5 μm). The mobile phase comprised phosphate buffer adjusted at pH 6.8 and acetonitrile in gradient mode. It was applied at a flow rate of 1.0 mL·min−1, a column temperature of 50°C, and an injection volume of 5.0 µL. Moreover, all solvent lines and seal wash were primed to minimize the formation of air bubbles and buffer precipitations. The mobile phase was filtered and degassed for 10 min in an ultrasonic bath before loading onto the system with the following sequence: (i) 0–1.5 min with mobile phase (80% Sol(A): 20% Sol(B)); (ii) 10 min with (50% Sol(A): 50% Sol(B)); and (iii) 10.1–15 min with (80% Sol(A): 20% Sol(B)) [28,29].
2.9 Calibration curves construction
The final concentration range of EMP and MET in the HPLC method was determined by dilutions of 10 mL volumetric flasks from the standard stock solution. During the procedure for the HPLC technique, calibration curves were established, and regression equations were calculated using Empower 3 software to process data and generate sign-off reports.
2.10 Sustainability assessment methods
2.10.1 AMVI
Regarding evaluating chromatographic procedures with a high degree of professionalism, AMVI is widely considered the most reliable and resilient approach. Quantifying solvents and waste products by analyzing the generated waste during the analytical process is possible. With AMVI, highly accurate results are obtained through specialized HPLC analysis. To determine the amount of solvent used in chromatographic processes and sample preparation, all solvents and chemicals must be recorded. A simple equation can determine the solvent used throughout the analytical procedure. Precision and accuracy are guaranteed. HPLC and sample preparation solvents are combined to determine an aggregate solvent consumption for a method. HPLC and sample preparation are combined to determine the total solvent consumption of a procedure. One can obtain the solvent consumption (in mL) for an HPLC analysis by multiplying the injections by the number and flow rate of analytes [30].
Sample preparation requires a total volume of solvent equal to a standard preparation plus each preparation multiplied by the number of preparations plus system suite preparations multiplied by the volume of solvent used. By multiplying the combined solvent usage of peak areas of interest by the solvent usage of peak areas of interest, the AMVI is calculated. A lower AMVI number increases the probability of achieving sustainability. An analytical approach can be adequately evaluated with the help of this equation. To calculate volume intensity, multiply the total amount of solvent used by the total number of analytes. Solvent consumption for HPLC or sample preparation is expressed as a percent of the total solvent consumption of a method [31].
2.10.2 ESA
Eco-scale analytical tools are required to conduct sustainability assessments. Sustainable systems consider waste generation, energy consumption, reagent consumption, and other risks when determining sustainability. The actual score is lower than 100, despite the assumption of obtaining a perfect score [32]. A system with fewer than 50 points is classified as “insufficient green,” one with 50–75 points as “ideal green,” and one with 75 points or more is classified as “superior green.” A greenness profile can be used to measure sustainability in the environment.
2.10.3 AGREE tool
A comprehensive and ecological assessment of sustainability criteria is conducted as part of the AGREE evaluation process. A scale of 0–1 rates green analytical chemistries (GAC’s) 12 essential principles. Ecological sustainability is assessed for various sustainability concepts. The graphic displays several red, yellow, and green gradients, each representing the benchmark’s performance level. Therefore, all parameters directly relate to section diameters [33]. AGREE evaluates companies’ ecological footprints based on their sustainability. GAC’s 12 principles guide the development of this technique.
2.10.4 AGREEprep tool
It is crucial to prepare a sample properly for analysis since its strength is determined by how it is prepared. AGREEPrep minimizes its ecological footprint in the sample preparation process. Sample handling and environmental analysis are conducted sustainably. In contrast to the 12-point guidance provided by the GAC, AGREEprep adopted a different approach. green sample preparation stipulates the 10 principles to which AGREEPrep adheres. A sustainable approach is demonstrated by Wojnowski et al. through environmentally conscious sample preparation. Several sample preparation techniques are assessed for their ecological impact as part of AGREEprep. The AGREEprep system combines sample preparation and environmental responsibility for optimal sample preparation. It consists of 10 discrete stages that assess a person’s capability. The ideal score is 1, with 0 representing the minimum and 1 representing the maximum. Graphic icons are included in each region. AGREEprep’s success depends on several essential factors. Before sending samples from the lab, hazardous compounds, trash production, and sample consumption per hour must be considered. Preparation for the examination should consider all these factors [34].
2.10.5 GAPI tool
As a result of a study conducted in 2018, Potka-Wasylka discovered that the GAPI technique can produce notable results when different analytical techniques are utilized in conjunction with it. A key aspect of evaluating analytical methods is their ecological viability, and GAPI is one of the most reliable methods available. Five essential components make up the technique, including sample preparation and result interpretation. GAPI is considered an excellent method for evaluating various analytical techniques once all relevant factors and considerations have been considered. Tools for assessing environmental sustainability must be evaluated to evaluate proposed techniques. To assess their ecological impact, it is essential to classify and analyze the prepared samples. It is crucial to categorize impact levels according to their significance level. Our analytical methods must be evaluated concerning their ecological impact to create informed evaluations. The use of this approach is necessary to evaluate ecological sustainability. All stages of the process are involved in the preparation of samples and the final determination. The procedure has five distinct characteristics depicted in the pictogram [35].
2.10.6 ComplexGAPI tool
A hexagonal layer is incorporated into the conventional GAPI metric as part of the ComplexGAPI metric, improving the pre-analysis process. It comprises several components, including the outcome of the procedure, the conditions under which it was carried out, the substances and solvents used, the environment suitability, the equipment used, and any additional steps that may have been required for the materials to be processed and purified. As part of the GAPI evaluation, various components are color-coded based on their durability. There is a difference between levels of environmental concern represented by the various shades of green, yellow, and red. Red flags signify severe challenges, while yellow flags indicate moderate troubles. On the other hand, a green signal indicates more serious issues [36]. To accommodate pre-analysis methods, ComplexGAPI incorporates an additional hexagon in the GAPI graph to evaluate GAC qualities. Several crucial attributes are incorporated into this enhancement to ensure that analytical techniques are accurate. Analyzing data with ComplexGAPI is easy and reliable.
2.10.7 BAGI tool
The BAGI is a critical measure for evaluating analytical methods. By leveraging white analytical chemistry, this tool will enhance well-known green metrics. Analytical techniques are evaluated according to ten critical characteristics: their effectiveness, automation, sample preparation, sample quantity, reagents, materials, sample analysis capacity per hour, concurrent analyte determination capacity, instrumentation requirements, and simultaneous sample treatment capacity, among others. BAGI uses this information to generate pictograms and scores of asteroid candidates. The BAGI complements green evaluation technologies such as AGREE, ComplexGAPI, GAPI, and AGREEprep. Due to its convenience and incorporation of all analytical chemistry fundamentals, it is easy to use [37].
2.10.8 HPLC-EAT tool
The HPLC-EAT tool, developed in 2011, assesses an HPLC-EAT tool designed for liquid chromatography methods; this application calculates environmental health and safety scores for solvents used in liquid chromatography. A score comparison technique can be used to compare HPLC methods for methods of greenness [38].
2.10.9 AMGS tool
The AMGS tool scores each instrument and solvent and their environmental impact, as well as the environmental impact of the solvents. Consequently, the method with a lower green score is greener than the one with a higher green score [39]. We used the AMVI and HPLC-EAT tools to design the AMGS spreadsheet. The instrument solvent energy and EHS scores will be combined to generate a greens score. AMGS spreadsheets only apply to chromatography techniques, regardless of their simplicity and ease of use.
3 Results
The HPLC method was utilized in this study to simultaneously identify EMP and MET in a quantitative assay, even in the presence of their degraded products. This allowed evaluation of the drug’s effectiveness in pure and tablet dosage forms. The study also assessed the product’s chemical, oxidative, acidic, and thermal stability and its essential hydrolytic stability.
3.1 An initial assessment
The mobile phase was optimized multiple times to achieve ideal HPLC conditions. Initial trials used methanol and acetonitrile at a pH of 2.5–7.5, with aqueous percentages ranging from 10% to 50%. The chromatogram showed a clear separation of the MET and EMP medications. Different buffers like phosphate, acetate, and perchlorate were tested at concentrations of 0.05–0.5 M and pH levels of 2.5–7.5 on various stationary phases (C8 and C18). All medication peaks were well separated, but degradation products could not be distinguished. Acid drops and bases were added at various concentrations, affecting peak shape and capacity. Adding octane-1-sulfonic acid sodium salt improved peak form and resolution. The best separation solution was 0.01 M octane-1-sulfonic acid sodium salt in 1 L of purified water. MET retention time was influenced by the percentage of octane-1-sulfonic acid in the mobile phase. A pH of 6.8 was used in the reversed-phase high-performance liquidchromatography (RP-HPLC) buffer to increase theoretical plates based on medication properties.
The column was heated between 25 and 55°C. Higher temperatures significantly affected EMP and MET, separating degradants. EMP was not as affected by heat, but low temperatures delayed and separated the signal. The ideal temperature for separation was found to be 50°C. An XBridge BEH C8 column (150 mm × 4.6 mm, 5 μm) with PDA detection was used with a flow rate of 1.0 mL·min−1 and a column oven temperature of 50°C. The mobile phase consisted of phosphate buffer adjusted to pH 6.8 and acetonitrile in gradient mode. Waters HPLC methods produced optimal peak shapes for standard solutions in Figure 2a and b.
An HPLC chromatogram of (a) standard solution and (b) sample solution of Empagliform XR 25/1,000 mg FCT.
The USP Uniformity of Dosage Units general chapter ensures consistency in tablets with a drug-substance ratio of 25%. Dissolution profiles are crucial for drug formulation development, as recognized in pharmacopeias for identifying production irregularities and measuring medication release. The paddle procedure was conducted at 100 rpm with a USP dissolution medium (acidic water pH 1.2, acetate buffer pH 4.5, and phosphate buffer pH 6.8) at 37°C, simulating gastric fluid conditions.
3.2 Quantifying environmental sustainability through assessment of techniques
3.2.1 Evaluating AMVI
Liquid chromatography applications can calculate solvent utilization accurately using AMVI. Liquid chromatography protocols can be incorporated into the procedures above. Sophisticated laboratory equipment can be used to conduct various analytical procedures. Based on the HPLC result, Table 1 illustrates AMVI’s environmental friendliness with a 160 score.
The AMVI assessment of the recommended technique
| Specification | Recommended HPLC technique |
|---|---|
| Solvent consumption HPLC (mL) | 180 |
| Flow rate | 1.0 |
| Run time | 15 |
| Number of injections for 1 complete analysis | 12 |
| Number of analytes | 2 |
| Solvent consumption sample prep (mL) | 140 |
| Volume (mL) for standard preparation | 20 |
| Standard preparations numbers | 1 |
| Volume (mL) for test preparation | 100 |
| Test preparations numbers | 2 |
| Volume (mL) for system suitability | 20 |
| System suitability numbers | 1 |
| Total method solvent consumption | 320 |
| Analytical method volume intensity | 160 |
| % Consumption HPLC | 56.25 |
| % Consumption preparations | 43.75 |
3.2.2 Evaluating ESA
Greenness profiles are widely recognized and follow strict protocols for measuring environmental sustainability. The current methods were evaluated using a precision penalty point as part of the ESA tool. A score of 71 demonstrates the method’s strong ecological sustainability and exceptional environmental friendliness. Table 2 summarizes the evaluation outcomes, providing a clear assessment of penalties.
Penalty for computing the ESA score employing prescribed methodologies
| Analytical eco-scale | Penalty points | |
|---|---|---|
| HPLC method | ||
| Reagents | Purified water | 0 |
| Octane-1-sulfonic acid sodium salt | 0 | |
| Potassium phosphate monobasic | 0 | |
| Orthophosphoric | 2 | |
| Sodium hydroxide | 2 | |
| Acetonitrile | 4 | |
| Methanol | 6 | |
| Hydrogen peroxide | 6 | |
| Hydrochloric acid | 4 | |
| Instruments | Energy for HPLC ≤1.5 kWh/sample | 1 |
| Occupational hazard | 0 | |
| Ultrasonic | 1 | |
| Waste | 3 | |
| Total penalty points | 29 | |
| Eco-scale total score | 71 |
3.2.3 Evaluating AGREE
GAC is a method for assessing the environmental impact of chemical processes using the AGREE instruments outlined in Figure S1a. The AGREE pictograms in Figure 3a indicate several levels of environmental sustainability. The pictograms have a central score of 0.66.
Seven pictograms (a) AGREE, (b) AGREEprep, (c) GAPI, (d) ComplexGAPI, (e) BAGI, (f) AMGS, and (g) HPLC EAT are used to evaluate the environmental sustainability of the suggested HPLC method.
3.2.4 Evaluating AGREEprep
A metric such as AGREEprep can be used to assess the ecological impact of sample preparation processes. Green sample preparation complies with ten fundamental criteria to streamline the assessment process. AGREEprep’s ten discrete steps result in performance assessment, with one representing the highest level of achievement. Pictograms depict the ten sectors in Figure S1b. The existing approach (Figure 3b) scores highly on environmental sustainability, with 0.56.
3.2.5 Evaluating GAPI
GAPI can be used to evaluate analytical methods’ environmental friendliness. Figure S2a illustrates that the approach comprises five critical components, from sample preparation to result interpretation. When considering all pertinent elements and considerations, GAPI can be a beneficial approach to evaluating various analytical techniques. As shown in Figure 3c, this approach yields environmentally beneficial outcomes.
3.2.6 Evaluating ComplexGAPI
GAC characteristics are used in ComplexGAPI to assess analytical methods. The GAPI graph is extended to include pre-analysis procedures by adding an extra hexagon. Figure S2b illustrates this situation. Figure 3d and h shows that this process encompasses multiple aspects.
3.2.7 Evaluating BAGI
The ten BAGI standards are illustrated by a precise pictogram and score illustrating the utility and effectiveness of an analytical procedure. Figure S2c illustrates the final HPLC score ranging from dark blue to light blue to dark blue. The color of an approach indicates its closeness to meeting the requirements. An analytical technique is considered applicable if it scores 60 or higher. Figure 3e shows suggested pictograms of BAGI indexes.
3.2.8 Evaluating HPLC-EAT
By analyzing HPLC-EAT concerning the points clarified in Figure S3a, this technique offers a reliable and efficient way of evaluating liquid chromatographic techniques. A valuable tool for ensuring the quality and safety of chromatographic operations is this method’s capability to assess the effects of all solvents used in the process. Figure 3f illustrates how the method can assess the effects of all solvents used in the process regarding health, environmental, and safety aspects.
3.2.9 Evaluating AMGS
Different methods can be evaluated and compared using AMGS regarding their environmental implications. As illustrated in Figure S3b, a quantitative approach is used to analyze additives, instrumental energy consumption, solvent waste generation, and waste management. Figure 3g shows that AMGS assessments evaluate sustainability via a perspective-based approach.
3.3 Method validation
ICH criteria were used as the basis for optimizing and validating the developed HPLC technique [40].
3.3.1 Linearity and range
An analytical method that directly correlates the analyte concentration and the data obtained is termed linear. This calibration graph has significant correlations (r) between peak area and concentration of EMP and MET bulk forms. A detailed description of calibration curve characterization is presented in Table 3.
Parameters for regression and validation of the suggested techniques for estimating EMP and MET
| Parameters | HPLC | |
|---|---|---|
| EMP | MET | |
| Linear | ||
| Range (µg·mL−1) | 10–106 | 30–1,050 |
| Wavelength (nm) | 224 | 224 |
| Slope | 12,992,802.07 | 17,187,076.75 |
| Intercept | 8,327.10 | 19,431.36 |
| Correlation coefficient | 0.99996 | 0.99968 |
| System precision | 0.3 | 0.4 |
| LODa (µg·mL−1) | 0.25 | 0.42 |
| LOQa (µg·mL−1) | 0.76 | 1.25 |
aLimit of detection (3.3 × σ/Slope) and a limit of quantitation (10 × σ/Slope).
3.3.2 Detection and quantitation limits
The validated Excel sheet utilized formulas (3.3σ/S) and (10σ/S) to perform limit of detection (LOD) and limit of quantitation (LOQ), respectively. These formulas are highly sensitive, as they consider the standard deviation of the intercept (σ) and the slope of the calibration curve (S) to compute LOD and LOQ. The results in Table 3 demonstrate that the suggested approaches are most effective when the values for LOQ and LOD are lower.
3.3.3 Precision
3.3.3.1 System precision (repeatability)
Each drug was tested with six replicates of a known concentration to assess repeatability. Table 3 shows that the system’s relative standard deviation (RSD) was less than 2.0% for six replicates.
3.3.3.2 Intermediate precision (reproducibility)
Ruggedness is evident when laboratory conditions vary, days differ, analysts differ, and equipment differs. As shown in Table S1, there were good results.
3.3.4 Robustness
The robustness of the suggested analytical methods was verified based on the data shown in Table S1 to ensure that the methods remain effective and are not affected by minor intended adjustments to the parameters of the methods, including pH, column, temperature, wavelength, and flow rate.
3.3.5 Standard solution stability
The experimental results demonstrate the high quality of the standard solution. Despite being stored for 72 h at room temperature and in a refrigerator, the newly prepared solution showed excellent agreement with a previously made solution. The recovered stored standard also matched well with the freshly prepared one, with a range of 100% ± 2.0% and an RSD of less than 2.0. These findings suggest that the standard solution is highly stable and reliable, making it an excellent choice for any analytical application, as clarified in Table S1.
3.3.6 Accuracy and recovery
The method’s accuracy was calculated using the following regression equations based on percentage recoveries at three concentrations crossing the linearity curve. Results in Table 4 show that 98–102% is an acceptable range for testing drug samples using the suggested procedures.
Accuracy and recovery of the suggested techniques for estimating EMP and MET
| Parameters | HPLC | |
|---|---|---|
| Relative concentrations (%) | EMP | MET |
| Recovery (%) | Recovery (%) | |
| 50 | 99.8 | 99.0 |
| 100.8 | 98.4 | |
| 100.5 | 100.6 | |
| Mean ± RSD | 100.4 ± 0.51 | 99.3 ± 1.14 |
| 100 | 98.2 | 98.1 |
| 98.1 | 98.5 | |
| 98.8 | 98.0 | |
| Mean ± RSD | 98.4 ± 0.38 | 98.2 ± 0.27 |
| 150 | 99.5 | 98.2 |
| 100.2 | 99.4 | |
| 99.1 | 98.7 | |
| Mean ± RSD | 99.6 ± 0.56 | 98.8 ± 0.61 |
3.3.7 Specificity
3.3.7.1 Selectivity
An evaluation of the approach’s specificity involved examining the ability of excipients, coating agents, and degradation products to interfere with the analyte. Figure S4a–e shows no appreciable variation between the two active components when diluted with diluents, placebos, and other compounds. These techniques can achieve remarkable selectivity.
3.3.7.2 Forced degradation
Heat, acid, base, and oxidation were used to verify that the assay test method could provide stable results. To evaluate the forced degradation of pharmaceutical compounds and products, different stresses were applied to the compounds and products in Figure S5a–e. Temperatures of 60°C and lower concentrations of acid and base did not facilitate the degradation of EMP and MET. EMP and MET, however, degraded by 37.4% and 83.1% after 1 h of exposure to 80°C at 12 N in an alkaline condition, respectively. After exposure to a temperature of 80°C or more within 1–2 h in thermal and acid conditions, EMP and MET showed a slight degradation of 0.1%, 1.3%, 0.8%, and 1.4%, respectively. As a result of oxidation, 30% H2O2 treatment effectiveness and drug test effectiveness decreased by 10.2% and 10.1%, respectively as displayed in Table 5. There were no interferences between the peaks of EMP and MET and all degradation products of EMP and MET were well resolved. The peak purity angles for EMP and MET in all states were no greater than the peak purity threshold, confirming the proposed technique’s high specificity.
Stability indicates the capability of EMP and MET by the suggested techniques
| Condition | % Degradation | Purity angle | Purity threshold | |
|---|---|---|---|---|
| EMP | Normal | – | 0.258 | 1.176 |
| Thermal | 0.1 | 0.301 | 1.093 | |
| Acidic | 0.8 | 0.302 | 1.178 | |
| Basic | 37.4 | 0.089 | 2.630 | |
| Oxidative | 10.1 | 0.093 | 2.045 | |
| MET | Normal | — | 0.174 | 0.375 |
| Thermal | 1.3 | 0.220 | 0.359 | |
| Acidic | 1.4 | 0.122 | 0.362 | |
| Basic | 83.1 | 0.088 | 0.727 | |
| Oxidative | 10.2 | 0.158 | 0.375 |
3.3.8 System suitability
By applying these techniques, parameters defining the system’s suitability were determined. The tailing factor, peak resolution, retention period, and theoretical plate count are included. These innovative analytical techniques meet ICH and USP standards for system appropriateness. Significantly, the number of theoretical plates that measure column efficiency must exceed 2,000. Furthermore, the minimal resolution required between MET and EMP is 1.5. According to Table S2, the tailing factor is not recommended to surpass 2. Complying with these factors is crucial in verifying the system’s appropriateness for its intended objective.
3.3.9 Application of assay test
Analyses of Empagliform XR 25/1,000 mg FCT and Synjardy XR 25/1,000 mg tablet samples were compared with working standards to estimate EMP and MET assay results in the different formulations. After the automatic computation of the processing data in the same order, all results were consistent and within limits. Assay results of EMP and MET in Empagliform XR 25/1,000 mg FCT and Synjardy XR 25/1,000 mg tablet are shown in Table 6.
Assay findings for estimating EMP and MET in their various marketed formulation dosage form by the suggested techniques
| Pharmaceutical formulation | HPLC | Limit | |
|---|---|---|---|
| Assay% | |||
| Empagliform XR 25/1,000 mg FCT | EMP | MET | 90–110% |
| 97.80 | 98.01 | ||
| 97.68 | 98.45 | ||
| 97.49 | 97.51 | ||
| Mean ± RSD | 97.66 ± 0.16 | 97.99 ± 0.48 | |
| Synjardy XR 25/1,000 mg tablet | 98.77 | 99.38 | |
| 98.58 | 98.81 | ||
| 98.43 | 98.97 | ||
| Mean ± RSD | 98.59 ± 0.17 | 99.05 ± 0.29 | |
3.3.10 Application of content uniformity test
Based on a thorough investigation, the content uniformity is within an acceptable range of 75–125%. These results are consistent with the acceptance value (AV) formula and the BP pharmacopeia, which specify a range of 85–115% and an AV NMT of 15%, as shown in Table S3. Therefore, it can be concluded that the content uniformity meets the required standards.
3.3.11 Application of comparative in vitro dissolution test
The results of this study confidently demonstrate that both generic and novel dosage forms dissolve over 85% of the prescribed amount in under 15 min, regardless of the medium used. This remarkable finding indicates that the dissolution profiles are highly similar. The data presented in Tables S4 and S5 indicate the effectiveness and efficiency of these dosage forms (Figures 4 and 5).
The comparative dissolution profile of EMP from Empagliform XR 25/1,000 mg FCT (Pharmaceuticals, Egypt), the generic product and Synjardy XR 25/1,000 mg tablet, extended-release (Boehringer Ingelheim Pharmaceuticals and Eli Lilly, USA), the reference product at various USP dissolution media (a) pH 1.2, (b) pH 4.5, and (c) pH 6.8.
The comparative dissolution profile of MET from Empagliform XR 25/1,000 mg FCT (Pharmaceuticals, Egypt), the generic product, and Synjardy XR 25/1,000 mg tablet, extended-release (Boehringer Ingelheim Pharmaceuticals and Eli Lilly, USA), the reference product at various USP dissolution media (a) pH 1.2, (b) pH 4.5, and (c) pH 6.8.
4 Discussion
According to the literature analysis, no HPLC approach is available for assessing EMP, MET, and their degradation products in pharmaceutical formulations incorporating eight environmentally friendly metrics and the distinctive BAGI related to sustainable chemistry. Our study employed eight distinct green criteria to assess the ecological sustainability of the proposed approach. The following software and tools were included: GAPI, ESA, HPLC-EAT, AGREE, AMVI, AMGS, AGREEprep, and ComplexGAPI. Furthermore, content uniformity and comparative dissolution studies were applied to evaluate the product formulation and their similarity with the reference one. In addition, we utilized a short column, resulting in a decrease in retention time and a reduction in the generation of mobile phase waste. The new strategy surpassed the prior one in terms of performance. Our state-of-the-art HPLC process is the most efficient and effective method for complying with environmentally friendly and sustainable chemistry principles. It outperforms earlier techniques regarding dependability and accuracy. This system suits chemical process in the most efficient and eco-friendly way. Subsequently, the suggested approach was opposed to existing ones in the literature after conducting a thorough assessment of sustainability approaches. The findings are summarized in Table 7.
Comparison of the proposed approach with the previously reported methods using nine ecologically friendly metrics
| Method | Proposed method | Reported method [22] | Reported method [23] | Reported method [24] | reported method [25] | Reported method [26] | Reported method [27] |
|---|---|---|---|---|---|---|---|
| AGREE |
 |
 |
 |
 |
 |
 |
 |
| AGREEprep |
 |
 |
 |
 |
 |
 |
 |
| GAPI |
 |
 |
 |
 |
 |
 |
 |
| ComplexGAPI |
 |
 |
 |
 |
 |
 |
 |
| BAGI |
 |
 |
 |
 |
 |
 |
 |
| HPLC-EAT score |
 |
 |
 |
 |
 |
 |
 |
| 15.342 | 13.948 | 15.047 | 15.02 | 7.968 | 6.437 | 4.202 | |
| AMVI | 160 | 222 | 316 | 217.3 | 205.3 | 169.3 | 206.4 |
| Eco-scale score | 71 | 71 | 81 | 67 | 73 | 65 | 75 |
| AMGS score | 657.82 | 756.76 | 455.54 | 924.48 | 350.47 | 349.07 | 346.01 |
The suggested methodology’s superior performance in the AGREE and AGREEprep tools can be attributed to several factors, such as its capacity to produce less than 3.0 mL of waste, minimize the presence of hazardous compounds, and maximize the number of samples analyzed each hour. Table 7 displays the AGREE and AGREEprep pictograms, effectively depicting the idea. The center scores for AGREE and AGREEprep are 0.66 and 0.56, respectively. The varying shades of green in the pictograms represent different levels of environmental sustainability. The suggested HPLC approach demonstrates superior performance in detecting (7) green, (6) yellow, and (2) red compared to the reported techniques [22,23,24,25,26,27]. Additionally, it generates less waste (less than 3.0 mL) than the reported approaches [22,23,24,25] without requiring pretreatment or extraction. An essential distinction between the suggested and previous approaches is their lack of necessity for the preservation, transportation, or specific storage conditions of materials. According to our research, as indicated in Table 7, this method is environmentally feasible. For reasons like those in the GAPI tool, the suggested strategy surpassed the methods used in ComplexGAPI. This was achieved by utilizing a little risky reagent and a simple process with a purity level of 98% or more. Table 7 illustrates many facets of the techniques. ComplexGAPI enables the evaluation of techniques that include GAC features thoroughly and inclusively. The method was utilized to quantify the levels of two compounds and their metabolites using common reagents and simple apparatus. This method facilitated processing over ten samples per hour, with a preparation capacity of about 12. Preconcentration was not required to achieve the desired sensitivity level. The introduction of an autosampler enabled partial automation. Aiming to maintain a simple and cost-effective sample preparation procedure, a sample volume of under 10 mL was chosen. As a result, the method received a BAGI rating of 82.5, reflecting its excellence in practicality and application compared to current techniques [22,23,24,25,26,27]. While the suggested method has a high score in terms of HPLC-EAT, the other described methods [22,23,24,25,26,27] have lower HPLC-EAT scores. The principles of HPLC-EAT do not encompass using fewer toxic solvents. The findings from the software presented in Table 7 indicate that a lower value indicates a higher eco-friendly.
The investigation has identified three crucial factors that substantially impact AMGS’s final score: instrument quality, solvent energy, and environmental health and safety. The AMGS values exhibit an inverse relationship with the level of process sustainability. A notable differentiation between the proposed and recorded AMGS values can be observed. Table 7 illustrates that the proposed approach is more ecologically sustainable than the approaches described in references [22,24], although the alternative ways [23,25,26,27] surpass the recommended one. According to research investigations [24,26], the ESA scores were 67 and 65, respectively. These procedures’ scores are lower than those of the published and proposed methods, which achieved a minimum score of 71. The proposed method incorporated a forced deterioration inquiry, in contrast to the existing methods [23,25,27]. Notably, penalty points for reagents were imposed during forced degradation, resulting in a decrease in the overall ESA score. However, the instrument assessed the system and granted it a score of 71, suggesting it had a significant degree of ecological sustainability. The penalty point results are presented in Table 7.
Based on the data analysis, the proposed methodology in the AMVI tool offers numerous benefits compared to existing methodologies. When using the AMVI instrument, sample preparation necessitates a volume of less than 10 mL because of solvent use. Although this approach has a solvent consumption value of 169.3 [26], it does not perform well regarding AMVI since it uses more solvent during sample preparation, leading to a lower AMVI score. According to Table 7, the proposed approach is deemed more environmentally beneficial due to its AMVI value of 160.
5 Conclusion
Following ICH recommendations, a unique, prompt, highly reliable, and environmentally friendly RP-HPLC technique was developed and validated for the simultaneous quantification of a binary mixture of EMP and MET and their degradation products in tablet dose form. The specific stability-indicating procedures assessed a wide range of conditions. An analytical method was successfully developed to assess the suitability of BAGI, one of the critical metrics utilized in the study. Furthermore, in addition to the GAPI and ComplexGAPI, it was found that the BAGI provides complementary insights to other metrics such as AMVI, AGREEprep, HPLC-EAT, AGREE, AMGS, and ESA. It is worth noting that “blue” is an indispensable element in white analytical chemistry, and its practical application is highly emphasized. The proposed methodology has been used successfully to determine the assay of dosage forms, analyze content uniformity, and conduct in vitro dissolution experiments to develop promising formulations. Bioequivalence centers, research labs, and routine quality control can all benefit from their use. The dissolution analysis for all studied drugs showed a greater than 85% medication release percentage in all dissolution media, confirming that all studied drugs passed the uniformity test and dissolution studies. It has been demonstrated that the proposed HPLC procedure can selectively detect EMP and MET in the presence of the degradants, indicating that the HPLC analytical process is reliable.
Acknowledgements
The authors thank Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2024R35), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.
-
Funding information: This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2024R35), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.
-
Author contributions: Asma S. Al-Wasidi: data curation, software, visualization, investigation (equal), writing – review and editing (equal); Hoda Ahmed: supervision, writing – review and editing, investigation, conceptualization; Samar M. Mahgoub: methodology, investigation (equal), writing – review and editing (equal); Mahmoud A. Mohamed: project administration, writing – original draft, software(equal), investigation (equal), conceptualization (equal). Hossam F. Nassar: formal analysis, software (equal), data curation (equal), conceptualization (equal).
-
Conflict of interest: Authors state no conflict of interest.
-
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Articles in the same Issue
- Research Articles
- Microfabricated potentiometric sensor based on a carbon nanotube transducer layer for selective Bosentan determination
- A novel Six Sigma approach and eco-friendly RP-HPLC technique for determination of pimavanserin and its degraded products: Application of Box–Behnken design
- Enantiomeric separation of four pairs of alkaloids by using a C18 column tandem polysaccharide-based chiral column
- Sustainable HPLC technique for measurement of antidiabetic drugs: Appraisal of green and white metrics, content uniformity, and in vitro dissolution
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- Review Articles
- Recent advance in electrochemical immunosensors for lung cancer biomarkers sensing
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- Quantitative methods in the analysis of clozapine in human matrices – A scoping review
- Review of potentiometric determination of cationic surfactants
- Surface-enhanced Raman spectroscopy in forensic analysis
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- KPI-based standards benchmarking for the preference of different analytical approaches developed for simultaneous determination of ciprofloxacin and hydrocortisone: A SWOT case study
