Green and sustainable RP-UPLC and UV strategies for determination of metformin and dapagliflozin: Evaluation of environmental impact and whiteness

2.1 Chemicals and reagents

MSN Laboratories Private Limited (Kondapur, India) supplied a DAP standard, while Wanbury Limited (Maharashtra, India) provided an MET standard. Hikma Pharmaceuticals (Giza, Egypt) provided Forminodab XR 10/1,000 mg FCT in a generic formula. Merck (Taufkirchen, Germany) provided HPLC-grade ethanol and bi-distilled water.

2.2 Apparatuses

Waters’ ACQUITY UPLC System (Milford, USA) is intended to distinguish intricate materials with a high degree of sensitivity and resolution. It has a three-pump configuration for sophisticated 2D LC workflows, a column manager for two independent temperature-controlled columns, high working pressures of up to 18,000 psi, and integrated vacuum degassing. It can handle up to four solvents. Leak sensors and extensive Empower 3 software are included for diagnosis.

UV Shimadzu 1900 (Tokyo, Japan) is well known for its cutting-edge and exquisite design, guaranteeing accurate and dependable results for various applications. The investigations were conducted using two quartz cuvettes with a route length of 1 cm. Spectra were recorded and processed using UVProbe (ver 2.71, Shimadzu) on an UV spectrophotometer. Its distinctive qualities and cutting-edge technology distinguish it from its competitors.

2.3 Buffer preparation

A buffer solution was prepared by dissolving 2.7 g of H₂NaO₄P and 0.6 g of NaSO₄C₁₂H₂₅ in 1 L of ultra-pure water. The mixture was sonicated until complete dissolution. The pH of the solution was adjusted to 7.0 using a 1 N NaOH solution.

2.4 Solvent preparation for UPLC technique

A solvent mixture was prepared by combining purified water and ethanol in a 70:30 (v/v) ratio.

2.5 Solvent preparation for the UV technique

Ultra-pure water was used as a diluent for the spectrophotometric method.

2.6 Chromatographic system

In this experiment, reversed-phase ultra-performance liquid chromatography (RP-UPLC) was configured to work in isocratic mode. A Waters XSelect HSS T3 C18 column (10 cm × 0.21 cm, 5 μm) was used for analysis. A sample oven temperature of 5°C and a column oven temperature of 35°C were set. There was a 55:45 v/v proportion of phosphate buffer (pH 7.0) and ethanol in the mobile phase. It was run for 2 min, with a 2 μL injection volume, a 0.5 mL·min⁻¹ flow rate, and UV detection at 225 nm.

2.7 Standard stock solutions

Standard stock solutions of DAP (5 mg) and MET (500 mg) were prepared by accurately weighing the compounds and transferring them to 100 mL volumetric flasks. A diluent was added to approximately 70% of the final volume. The solutions were sonicated for 10 min and then brought to volume with additional diluent.

2.8 Working standard solutions

Working standard solutions were prepared by transferring 1 mL of the stock solution to a 10 mL volumetric flask and diluting it to the required volume.

2.9 Analysis of commercially traded formulas

Twenty Forminodab XR 10/1,000 mg FCT tablets were weighed, and their average weight was calculated. One tablet was ground, weighed, and transferred to a 100 mL volumetric flask containing 70% diluent. The volume was completed with the same diluent, and the solution was mixed, shaken, and sonicated for 1 h. A 5 mL aliquot of the stock sample solution was transferred to a 100 mL volumetric flask and filtered using a syringe membrane filter.

2.10 Construction of calibration curves

Serial dilutions were made and transferred into a 10 mL volumetric flask, utilizing a stock solution of standard concentration. We obtained concentration ranges of 0.0025–0.05 and 0.06–0.6 mg·mL⁻¹ for DAP and MET in the UPLC method, respectively, and 0.005–0.025 mg·mL⁻¹ for each pharmaceutical drug using UV analytical methods. The process involved is as follows:

• Calibrating at two points,

• Conducting a test setup,

• Performing a recovery injection to verify the standard,

• Conducting five injections to assess the system’s suitability, and

• Employing a diluent to evaluate potential carryover.

The association between the concentration and the integrated peak areas was determined through regression equations.

2.11 Greenness and whiteness tools for measuring

2.12 MCR spectrophotometric method

MCR is the foundation for the suggested MCR approach, whose mathematical justification was provided [32]. This technique solved ternary and binary mixtures in intricate samples whose matrices were unknown.

Abs(Mix) indicates the absorption vector used for mixtures, while C _MET and C _DAP represent the concentrations of individual drugs. Th molar absorption coefficients of these medicines are denoted by α _MET and α _DAP. When the absorbance vectors of the mixtures are divided by α _DAP, it is possible to derive the initial ratio spectrum, denoted as A, as follows:

To divide the data, it is necessary to disregard the zero values of α _DAP. It is possible to obtain a zero value by applying the mean centering to a constant. In light of this,

Through utilization of the mean that was discussed earlier, along with Eq. 2, we obtain

Eq. 4 can be used to analyze binary mixtures statistically. This is accomplished by measuring the concentration of each medication in the mix without allowing the other drug to interfere with the measurement. First, one must plot the MC (MET) concentration against the MET concentrations separately or in binary mixtures to generate calibration curves. For the DAP calibration curves, a process comparable to the one employed for the MET was utilized.

3.1 Method development and optimization

DAP and MET were obtained and measured in medicine tablets and plasma using different mobile phase compositions to develop a new RP-UPLC method. Initial tests were conducted to determine the best conditions for developing an effective process for pharmaceutical analysis. Changes to the detector wavelength, column temperature, and mobile phase composition were made to maximize separation and precision. Several experiments were carried out to manipulate the mobile phase composition experimentally. The experiment was started with a 50:50 volumetric ratio of water to methanol. After that, a different strategy was created using acetonitrile and pH 4 phosphate buffer in a 50:50 volumetric ratio. Flow rates, pH levels, and compositions of the mobile phase influenced the retention durations of the analytes. Analyte retention times first decline as the pH falls below 7.0, but they eventually rise as the pH falls. At pH 7.0, it was possible to obtain crisp peaks with acceptable retention durations and satisfactory symmetry. Similar peak behavior was seen when the composition and flow rate of the mobile phase were altered after utilizing the green solvent of ethanol. The optimal composition was 55:45 v/v phosphate buffer and ethanol, with a 0.5 mL·min⁻¹ flow rate. Additionally, columns of various lengths and packing materials were used in the investigation. The columns included C8, phenyl, and cyano; their lengths varied from 10 mm to 100 mm. The most suitable option was the Waters XSelect HSS T3 C18 column (10 cm × 0.21 cm, 5 μm). The heating of the column took place between 20 and 40°C. Elevated temperatures play a significant role in drug separation. Drug degradation and separation, a critical process, are also affected. As a result, the recommended methods established that 5°C and 35°C – sample and column oven temperatures, respectively – were the optimal separation temperatures. As can be seen in Figure S1, we looked into wavelengths between 200 and 400 nm using the spectrophotometric method. DAP and MET were chosen for their maximum sensitivity and remarkable intensity at 225 nm while having the largest absorbances at 223.2 and 233 nm (Figure S2). To attain clear, discrete peaks with elevated resolution, the subsequent chromatographic parameters were ascertained: For RP-UPLC processes, a Waters XSelect HSS T3 C18 column (10 cm × 0.21 cm, 5 μm) was utilized. The sample and column oven temperatures were adjusted to 5°C and 35°C, respectively. The mobile phase in an RP-UPLC procedure was phosphate buffer and ethanol (55:45, v/v) adjusted to pH 7.0 with a 2 min duration, 2.0 μL injection volume, 0.5 mL·min⁻¹ flow rate, and 225 nm UV detection. Figure 1a and b shows how these issues can be resolved more effectively and quickly following the chromatographic condition with the shortest run time of less than 2 min.

Figure 1

UPLC charts of MET and DAP in (a) binary standard solution and (b) test solution.

3.2 Employing the MCR methodology

The MCR method has been claimed to effectively separate the binary mixture of pharmaceuticals without pre-separation. To enhance the technique, tests were conducted with various divisor concentrations, and the most appropriate concentration was determined to be µg·mL⁻¹. Figure 2a and b shows a spectrum of a substance produced within the 200–290 nm range. A laboratory-prepared combination of drugs, with a concentration range of 525 µg·mL⁻¹, was imported into MATLAB software. The MCR for each drug was calculated using Eqs. 1–4. As illustrated in Figure 2c–f, the 257.7 and 240.9 nm wavelengths were appropriate for estimating MET and DAP. The calibration profiles were established by plotting the amplitudes obtained against their respective concentrations.

Figure 2

Drug studies with absorbances ranging from 5 to 25 µg·mL⁻¹ for (a) DAP and (b) MET, as well as (c) and (d) drug first ratio spectra and (e) and (f) drug first ratio spectra with spectrum’s MC.

3.3 Evaluation of the whiteness and greenness of the recommended methods

An assessment of the impact of analytical methods on the environment can be made more robust and comprehensive by utilizing multiple approaches. There are a variety of tools focused on sustainability, such as AGREE, GAPI, AMGS, BAGI, and HPLC-EAT. Combining these tools allows analysts to examine all critical aspects of environmental performance thoroughly. As a result of this multi-tool approach, results can be cross-validated, findings become more credible, and specific areas for improvement can be identified. Additionally, a green and white standard for analytical chemistry aligns with evolving industry requirements. Through this approach, not only are scientific findings strengthened but also more sustainable practices are promoted in pharmaceutical and analytical research.

3.3.1 AGREE tool

AGREE is a contemporary approach to assessing green profiles based on the 12 elements of the Green Appraisal Guidelines (Figure S3a and b). A pictogram representing an environmentally friendly product was created using this method, as indicated in Table 1. It shows an AGREE score ranging from 0.7 to 0.74 in the middle for UPLC and UV strategies, along with various intensifying green color combinations.

Table 1

Comparison of the recommended UPLC and UV strategies for ecological evaluation

Method	Suggested UPLC	Suggested UV
AGREE
AGREEprep
GAPI
ComplexGAPI
RGB12
BAGI

3.3.8 AMGS

AMGS compares and evaluates different strategies according to how they might impact the environment feasible. Figure S5a shows the analytical analysis process for components, instrumental energy use, solvent waste production, and waste disposal. A perspective-based methodology is used in AMGS inspections, as shown in Figure 3a.

Figure 3

A sustainable UPLC procedure was evaluated using graphs (a) AMGS, (b) HPLC-EAT, and (c) RGB12.

3.4 Method validation

The processes have undergone a thorough evaluation following international council for harmonisation guidelines, and the conclusive results validate their exceptional efficacy and dependability [33].

3.4.8 Specificity

3.4.8.1 Selectivity

The method’s selectivity was evaluated by analyzing the possible impacts of coating agents, degradation products, and preservatives on the analyte. As shown in Figure S6a–e, no significant difference exists between the two active components when diluted with diluents, placebos, or other medications. These methods could be used to attain a significant degree of selectivity.

3.4.8.2 Forced degradation

Various methods of specificity testing were used, including heat and light as well as acids, bases, and oxidation, to guarantee consistent findings. Figures 4(a–e) and 5(a–d) show that the pharmaceutical compounds and products were subjected to various stressors to assess their forced degradation. Both high and low base concentrations caused MET to degrade completely. Nevertheless, MET and DAP deteriorated by 0.6% and 0.7% when exposed to 1 N HCl for 2 h at 80°C. The degradation percentage in MET increased to 73.2% when exposed to 1 N NaOH for 2 h at 80°C, while DAP degraded slightly by 0.4%. Heating for 2 h at 85°C caused MET and DAP to degrade slightly by 0.2% and 0.8%, respectively. Table 6 illustrates how oxidation treatment with 30% H₂O₂ for 2 h at 80°C reduced the efficacy of MET and DAP and with the test drug by 10.3% and 4.5%, respectively. There were no interactions between the peaks of MET and DAP, which meant each degradation product could be identified. As MET and DAP had similar peak purity angles, the proposed method had reasonable specificity, as demonstrated by their non-significant differences from the peak purity threshold.

Figure 4

UPLC charts of DAP under (a) normal conditions and forced degradation, including (b) heat, (c) acid, (d) base, and (e) oxidation conditions.

Figure 5

UPLC charts of MET for forced degradation, including (a) heat, (b) acid, (c) base, and (d) oxidation conditions.

Table 6

Degradation profile and purity assessment of DAP and MET under stress conditions

	Condition	% Degradation	Purity angle	Purity threshold
DAP	Heat	0.8	1.414	9.622
	Acidic	0.7	1.235	11.922
	Basic	0.4	1.849	29.543
	Oxidation	4.5	53.324	90.000
MET	Heat	0.2	12.264	18.572
	Acidic	0.6	10.271	28.318
	Basic	73.2	27.744	63.438
	Oxidation	10.3	19.501	47.619

3.5 Evaluation of our methodology

Literature analysis suggests that neither UPLC nor MCR approaches are suitable for determining MET, DAP, and their byproducts in drug formulations that incorporate eight environmental metrics, BAGI, and RGB12, which is a characteristic of sustainable chemistry. Several green criteria were used in our study to evaluate the proposed approach’s ecological sustainability. The tools and software used were AMVI, GAPI, AGREEprep, HPLC-EAT, AGREE, ESA, AMGS, and ComplexGAPI. By using a short column, waste generated during the separation of mobile phases was reduced as well as the retention time. In the current study, purified water and ethanol were used instead of hazardous chemicals for the solvent. Compared to the prior strategy, the new one resulted in higher accuracy and sustainability.

Our cutting-edge UPLC and MCR procedures adhere to sustainability and ecologically friendly chemistry principles most effectively and efficiently. The accuracy and reliability of this method are improved over previous methods [14,15,16,20,21]. The current approaches are the most environmentally friendly and efficient solution for chemical processes. The recommended strategies surpass the previous ones in the literature following a comprehensive evaluation of sustainability strategies [14,15,16,20,21]. Table S4 provides a summary of the results.

As part of our extensive assessment of environmental approaches, we carried out a comparative analysis of the presented techniques via those illustrated in the literature. Table S5 summarizes the findings. We used five green tools AGREE, GAPI, AGREEprep, BAGI, and ComplexGAPI to reach a comprehensive evaluation. Its performance in the AGREE and AGREEprep tools was superior to those of the published techniques [14,15,16,20,21] for several reasons, including the fact that the waste generated was less than 1.0 mL, the hazardous chemical was replaced by ethanol, and the number of samples examined per hour was maximized. Table S5 demonstrates this concept clearly with the AGREE and AGREEprep pictograms, which have central scores of 0.78 and 0.77 for AGREE and 0.77 and 0.76 for AGREEprep, respectively. Likewise, different green hues indicate different levels of environmental sustainability achieved with UPLC and UV. GAPI shows (10) green, (3) yellow, (1) white, and (1) red for the proposed UPLC method, surpassing the previously reported method [15], which scored significantly higher than the other methods [14,15,16,20,21]. We used ethanol as the green solvent and generated waste less than 1.0 mL, as opposed to the waste in the published methods [20,21], which used hazardous reagents and chemicals. The proposed method, however, does not require samples to be preserved, transported, stored under special conditions, or otherwise treated. Table S5 illustrates our investigation’s results, which prove the efficacy of this approach.

The presented technique exceeded those reported [14,15,16,20,21] for the same factors as in the GAPI tool. An eco-friendly reagent and a straightforward technique produce a 99.8% purity. Additionally, an isocratic system and environmental friendly solvent (ethanol and water) are combined with a lower flow rate and a short run time. Several aspects are covered in the strategies, as illustrated in Table S5. With the ComplexGAPI metric, one can evaluate methodologies comprehensively incorporating GAC attributes.

Our investigation has discovered that our technique has a high BAGI score, which indicates that it is more practicable and more applicable than published methods [14,15,16,20,21] with scores of 77.5, 80, 75, 77.5, and 80. Along with its high score, our method performed better on key BAGI attributes such as sample throughput, reagent usage, sample preparation, and automation. In addition to analyzing more samples per hour, reducing reagent and material usage, simplifying sample preparation, and increasing automation, our method offers several other advantages. Several benefits result from these improvements, including increased efficiency, sustainability, and user-friendliness. Compared with existing methods, these methods may have limitations in one or more of these areas, limiting their practicality. For example, Suman and Chaubey [16] used hazardous solvents like methanol, decreasing the environmental impact. Consequently, its score of 75.5 will likely be lower due to this dangerous solvent requirement.

In this study, a simplified method of analytical chemistry was developed, demonstrating a significant breakthrough in the field. In addition to being efficient, sustainable, and easy to use, we address key aspects of the field through this method.