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Recent application of green analytical chemistry: eco-friendly approaches for pharmaceutical analysis



The substantially operated analytical instruments dealing in the area of analytical chemistry are traditional methods like high-performance liquid chromatography (HPLC) and gas chromatography (GC). Since they use solvents, produce trash, and require energy, these methods seriously compromise the natural milieu. The excessive consumption of an enormous number of organic solvents, along with the trash created from it, can contaminate the environment. As a result, researchers are now creating novel Green Analytical Chemistry approaches to address these environmental problems and create an ecologically preferable replacement.

Main body of the abstract

Ecologically preferable replacement can be accomplished by using a green solvent, such as ethanol or water, in place of harmful solvents. Additionally, the need for solvent can be decreased by omitting the sample preparation stage wherever possible or by utilising alternate green extraction methods. Adoption of compact methods like ultra-high-performance liquid chromatography (UHPLC) may also result in a decrease in the amount of energy used and trash produced during analysis.

Short conclusion

This review features information on using sustainable practises in analytical chemistry as well as details on using green solvents and sample preparation methods such as Solid Phase Extraction (SPE), Qucheers. It also provides information related of application of green analytical techniques such UHPLC, High-Performance Thin Layer Chromatography (HPTLC), and Thin Layer Chromatography (TLC).


Implementing green chemistry and placing a focus on it in the laboratory are two themes that are becoming more and more popular in the chemical world. Chemists all across the world are striving to adapt more sustainable, energy-efficient processes and reactions for conventional ones. Green chemistry can take many different shapes, such as redesigning experiments with more environmentally friendly ingredients or actively managing waste [1]. Anastas initially defined "green chemistry" as a field that aims to eliminate or significantly reduce the production of dangerous compounds during any type of chemical process [2]. Although Anastas coined the phrase “green chemistry,” Badami, Nameroff, and Keltz are responsible for starting and maintaining the notion [3, 4]. The first steps in the exertion of green analytical chemistry (GAC) in the analytical chemistry field were taken in 1995 when Anastas highlighted the need to create GAC ideologies in an editorial that was published in a special issue of the Analyst journal that was devoted to the miniaturisation, containment, and reagent replacement in analytical methods [5].

To compare the efficiency of green chemistry approaches to conventional methods, many systems have been devised. This tactic is based on the twelve guiding Principles of Green Analytical Chemistry, which are listed in Table 1 [6,7,8,9,10] and may be remembered by the term significance.

Table 1 12 Principles of green analytical chemistry

One instance area that is making an effort to incorporate green chemistry ideas into equipment and analytical procedures is analytical chemistry. Creating more effective and resilient sample preparation protocols is one of the biggest concerns in analytical chemistry [11,12,13]. Therefore, as shown in Table 1, the first principle specifies that introducing the sample with minimal or no preparation is the most viable approach to decrease waste. However, most analytes require matrix extraction prior to analysis. The second, fifth, seventh, ninth, tenth, eleventh, and twelve principles may and should be used in situations when sample preparation is required. Techniques for preparing samples should be improved to the greatest extent feasible to decrease the amount of energy needed, solvent usage, waste output, and employee contact. This may be done in an assortment of manners, such as by limiting the amount and quantity of representative, using utile sample extraction equipment, and staying away from organic solvents that are hazardous [14]. Chromatographic strategies, which are used for distinguishing between the components of complicated mixtures in diverse matrices, are one of the topics of chemical analysis that GAC is passionate about. In the procedure of chromatographic analysis, the detrimental effects of harmful solvents to the health of humans and the environment must be ignored. The trash produced by a traditional chromatographic process, that demands a lot of organic solvent and can produce 1–1.5 L of scrap per day, must be dismissed of [15]. Since the majority of utilised organic solvents are volatile, they can quickly disperse and damage the surroundings. Thus, it is the duty of analytical scientists to reduce the detrimental impact produced by their work and to prevent, or at the at least, minimise it.

This systemic analysis review will give information on implementation of sustainable practises in analytical chemistry as well as details on using green solvents and sample preparation methods. Additionally, we have described the execution of GAC principles for the validation and development of analytical methods utilising HPLC, UHPLC, Ultraviolet–Visible spectroscopy (Uv–Visible Spectroscopy), HPTLC, and TLC.

Main text

Outline of sample preparation approaches and the effects they have on analytical green techniques

Since the processes shown in Fig. 1 may entail the employ of hazardous substances and/or volatile solvents, the sample preparation stage tends to be the analytical method that causes the greatest pollution [Grob, 1984].

Fig. 1
figure 1

Analytical methods for measuring an analyte in matrix sample

The use of direct chromatographic methods in the absence of sample preparation

Based on the perspective of the GAC, direct analytical techniques—i.e. those that do not need sample preparation—are especially preferred. It is vital to adopt direct chromatographic procedures as the vast majority of chromatographic studies performed and the likelihood to produce residues during sample preparation. Direct injection of both liquid & solid samples may be effectively carried out by GC or liquid chromatography (LC) analysis, according to Tobiszewski and Namiesnik's 2009 statement [16]. It used to be discouraged to add the sample straight into the chromatographic capillary column, which may be water or a water–ethanol combination. A thick, nonpolar column stationary phase is utilised because water is thought to promote column bleeding, specifically when polar stationary phases are used. Significant improvements in column stationary phase quality and state-of-the-art cross-linking tactics have boosted resistance to deterioration triggered by water. When environmental water is injected into a column, notably when the water carries an immense amount of salt, performance issues with the column and decreased detector sensitivity can arise. In order to keep organic non-volatile & inorganic salts substances out of the analytical column, deactivated pre-columns must be positioned in front of it. The initial on-column injection techniques were the primary aim of the direct gas chromatographic approaches Grob et al. developed in 1978 [17]. In order to identify halogenated chemicals in sample of water by direct aqueous injection, Grob et al. developed an amalgamation of on-column injection and electron capture detection in 1984 [18]. A different approach was created using direct aqueous injection-gas chromatography and mass spectrometric detection to identify polar and nonpolar volatile chemical molecules in sample of water [19]. High-boiling volatile organic chemicals in surface water have recently been identified using greater volume direct aqueous injection-gas chromatography with flame ionisation detection, electron capture detection, and flame photometric detection [20]. The main drawback of these approaches is that they are restricted to the sample with generally pure matrices free of suspended debris [21]. The degradation of the chromatographic column might happen quickly if the analysed sample is not sufficiently clean due to an accumulation of particulates or non-volatiles that cannot release out of the column. Spirits & petroleum portions are illustrative of clean matrix that may be infused onto chromatography columns without any preparation. Since they merely call for straightforward sample preparation methods like filtering, dilution, or centrifugation, several approaches may be categorised as almost direct [22,23,24]. Thus, there are two ways that direct chromatographic procedures connect to green chemistry. They adhere to the principles of waste reduction and decreased usage of solvents, auxiliaries, and energy by skipping the sample preparation stage.

Green sample preparation

  1. 1.

    Solid Phase Extraction (SPE)

The substantially operated methods for preparing samples is SPE. In SPE, solutes are adsorbed onto a short column of a compatible solid sorbent after an aqueous sample is run over it. The analysed substances are retrieved from the absorbent in minuscule amounts of highly eluting organic solvents, which results in their enrichment [25]. Small quantities of solvent are used during solid phase extraction, which also produces minimal waste. It is therefore regarded as an environmentally beneficial method. SPE may be mechanised with the use of basic, low-cost apparatus, increasing laboratory throughput while increasing accuracy and precision. Despite this method's advantages, there are certain possible drawbacks that should be taken into account to prevent ineffective analyte extraction. One of the problems is the unevenness of the packing material’s bed, that might result in a reduction in efficiency. A secure solution to this issue is through the utilisation of commercial cartridges. Insufficient retention of extremely polar molecules may also result from certain traditional sorbents' low selectivity. Another problem is the conflict over retention between analytes and sample matrix, which has a significant negative influence on analyte recovery. As a result, rigorous process optimisation is necessary to guarantee successful analyte extraction [26].

  1. 2.

    QuEChERS Extraction Methodology

Popular extraction technique QuEChERS is known for being rapid, simple, inexpensive, efficient, robust, and safe. Anastassiades et al. [27] established the comprehensive QuEChERS technique in 2002. This method is regarded as a green extraction method since it only adopts a tiny number of organic solvents in comparison with other extraction means. Two essential phases are included in QuEChERS procedures: the extraction of solvents and sample clean-up. The sample is vigorously shaken throughout the solvent extraction process together with buffer (to safeguard base-sensitive analytical agents inside the sample), anhydrous magnesium sulphate, sodium chloride (to salt out). In order to get rid of interfering matrix components such carbohydrates & fatty acids, the sample cleansing phase is essential. Implementing magnesium sulphate (to soak any remaining water) and a weak anion exchanger known as “primary secondary amine” (PSA) absorbent as the dispersive solid-phase extraction agent, rapid dispersive SPE is enhancing purity of the extract [28]. Several applications used QuEChERS for extracting analytes of interest from blood specimens, including the eradicating a variety of pollutants from human specimens of blood [29], and removing amphetamine, opiate, and cocaine from blood through tandem mass spectroscopy and liquid chromatography [30], and the recently completed extraction of tetrahydrocannabinol (THC) [31].

  1. 3.

    Solid Phase Microextraction (SPME)

The process of extraction & enrichment might be combined using SPME, resulting in solvent-free sample preparation. The initial version of this approach was created in 1990 by Arthur and Pawliszyn. This method makes use of silica fibre that has been coated with the appropriate adsorbent phase. Direct extraction of the analyte from the solution and concentration on the fibre layer are both used [32]. In the SPME approach, the effectiveness of the analyte preliminary concentration might vary depending on a no. of facets, along with the fibre type, sample stirring, extraction duration, etc. [33]. By using SPME in conjunction with, high-performance liquid chromatography (HPLC), GC/mass spectrometry (MS), GC, and LC–MS, substances in food samples could be isolated. Minimal expenditure, ease of use, the removal of solvent disposal expenses, quick sample duration of preparing samples, dependability, susceptivity, and perceptiveness are certain main benefits of SPME. However, there are some shortcomings that need to be identified to prevent adverse outcomes. They include the fibre's brittleness, which makes it potentially breakable when handled, the risk of coating damage from recurrent Competitive absorption when sorbent-type coatings are put to use, the profound impact of mass & temperature transmit factors on equalisation, matrix effects, and so on. [34].

  1. 4.

    Stir-Bar Sorptive Extraction (SBSE)

As a substitution to SPME, SBSE is an environment friendly, free of solvent extraction method that was launched in 1999. The isolation and purification of volatile analytes from the water-based matrix was the original purpose of SBSE. Later, its uses were expanded to include non-volatile analytes in conjunction with HPLC and the examination of the headspace over liquid/ solid samples, or gaseous samples, and other samples. SBSE uses sorptive extraction, just as SPME. Analytical substances get absorbed into a polymer (such as polydimethylsiloxane) matrix wrapped around a magnetic stir rod in this instance as opposed to a fibre, however. The substantially higher amount of the sorptive phase employed in SBSE compared to SPME is the primary distinction between the two. Better sensitivity is the outcome, particularly when utilising high sample volumes or substances with low partition coefficients. This approach is entirely without solvent in this instance since the coating's ability to capture volatile analytes allows them to be thermally desorbed right into a GC. Non-volatile analytical substance is desorbed using tiny amounts of a diluent, upholding the method's environmentally friendly nature [35].

  1. 5.

    Dispersive Liquid–Liquid Microextraction (DLLME)

The method uses three-phase solution that includes an aqueous sample, an extracting solvent that is immiscible with water, & some dispersive fluid which is solvable with in all the phases. The two distinct solvents are combined, & resulting solutions quickly introduced in the samples to create an extremely fine emulsion. As a result, the analytes may be transferred in the dispersed extraction solution extremely quickly. After the emulsion is created, it is centrifuged. The phase of increasing extraction density is retrieved by using a micro syringe and added to a preferred analytical device. DLLME has a number of benefits, including a small sample volume, little solvent consumption, high enrichment factor, excellent reproducibility, and high recovery. Enormous areas of proximity among the watery part & the solvent mixture are produced by the emulsion created following adding it to the fluid amalgam. As a result, the extraction is effective and the equilibration happens quickly [36].

  1. 6.

    Pressurised fluid extraction (PFE)

This methodology, referred as accelerated solvent extraction (ASE), involves doing the extracting using high pressures & temperatures that are beyond the solvent's boiling point at atmospheric pressure. The analytes’ solubility and diffusion rate rise at the higher temperature, while the solvent’s viscosity and surface tension decrease. Increased pressure makes it easier for the extractant to enter the matrix pores. PFE is regarded as a green technology since it uses less solvent, enables utilizing eco-friendly solvents like ethanol, methanol, and achieves quick and effective extraction in a lesser amount of period with less energy [36].

  1. 7.

    Microwave-Assisted Extraction (MAE)

Microwave radiation is employed in MAE to speed up the extraction procedure. The MAE principle relies on heating the system as a result of polar molecules absorbing microwaves, which heats the solvent that interact with the material to enable the extraction of analytes into a solvent or a water-based solution. It is possible to use the entire range of the microwave frequency, which ranges from 300 MHz to 100 GHz; traditional ovens should only function at 2.45 GHz. The major benefits of the MAE are its quick heating, elevated temperatures, and simplicity of use. Its one drawback is the restricted heating up investigated solvent caused by the dielectric constant [37].

  1. 8.

    Ultrasound-Assisted Extraction (UAE)

Since there are several drawbacks associated with traditional or other newer procedures, such as costly capital investment, the use of energy, and the use of harmful organic compounds for extraction, the use of ultrasound for extraction has grown over the past ten years. Ultrasound-assisted extraction (UAE) is a technique that uses ultrasound during the preapplication processes, and it might be favoured since it is a clean and ecologically friendly extraction approach [38]. As a result, the usage of UAE is now acknowledged as a safe and practical alternative to traditional food production methods. Therefore, when compared to other extraction procedures, ultrasound is a simple to use, multidirectional, versatile, and little investment required technology. Application of ultrasound broadens the range of commonly regarded as safe solvents.

  1. 9.

    Supercritical Fluid Extraction (SFE)

Supercritical fluid extraction (SFE) is an environmentally friendly, simple, and fully automated analytical technique. The term “environmentally benign sample preparation method” (e.g. “typical SFE”) refers to a technique that employs water or other non-toxic solvents. Short extraction times, lower pressures, and temperatures, together with the ability to preserve the integrity of food's functional components, allow SFE to almost minimise the possibility of activity loss [39]. The benefits of SFE include clean extract since less solvent is used and the extraction process takes less time. The extraction of the analytes is completed with no additional clean-up procedures. The sample pretreatment step of this approach lends itself to the most widespread usage of nontoxic and non-polluting extraction fluids [40]. Carbon dioxide, nitrous oxide, ethane, propane, n-pentane, ammonia, and sulphur hexafluoride are some of the most popular supercritical fluids. Due to its lack of abrasive and explosive properties, CO2 is the most often used gas. Quantitative, straightforward, quick, selective, and ecologically friendly are the SFE's key benefits [41] (Table 2).

Table 2 Green sample preparation technique

Greener organic solvents in analytical chemistry

The most ecologically conscious and long-lasting solvent that might be used in analytical chemistry is unquestionably with zero solvent. But in order to make samples liquid and make it easy to separate useful components, both sample preparation and analytical procedures need solvent. In order to alter traditional organic solvents, which are recognized due to their great volatility, combustibility, and toxicity, current developments in embracing the concepts of green chemistry into analytical procedures leads to the invention and use of novel solvents [42]. Solvent selection guides (SSGs) are instruments that help in the choosing of green organic solvents [43]. A replacement to traditional organic solvents, emphasising the three most significant green solvent types: amphiphilic solvents (encompassing alcohols, carboxylic acids and surfactants), ionic liquids (ILs) and analogues [44], and deep eutectic solvents (DESs) [45]. All of these solvents have been applied to the preparation of analytical samples (as extraction solvents) as well as to liquid chromatography (LC) (mostly as mobile phase enhancers or in the form of pseudo stationary phases).

  1. 1.

    Alternative solvents for sample preparation

Water is the most ecologically friendly, economically viable, ecologically sound, and harmless solvent. Maceration is the most basic and widely employed method for doing extraction with water as the solvent. Specific additives can be used to subtly alter the characteristics of water. Water plus an additional substance—a surfactant or a hydrotrope, respectively—are necessary for both micellar and hydrotropic extractions. By producing micelles and aggregates, respectively, these amphiphilic molecules can increase the dissolution of water hating substances in aqueous conditions [46, 47]. Micellar extraction mimics a method of two-liquid-phase but hydrotropic extraction simply includes a continuous liquid phase. Additionally, nonpolar molecules can be extracted with water using subcritical water extraction (SWE).

Natural Deep Eutectic Solvents (NADESs) are combinations of naturally occurring solids with melting points that are far less than any of their separate constituents. The primary cause of this specificity is the formation of between molecules hydrogen bonds connecting hydrogen bond donors & acceptors. Since NADESs are solvent that are ecologically beneficial and easily recyclable [48, 49].

According to the agro-sector from where they were created, the "bio-based solvents" collect an enormous number of biomolecules that may be splited into three groups: wood, oleo-proteaginous, and cereal/sugar. Representatives of bio-based solvents include ethanol, ethyl acetate, lactic acid esters, succinic acid derivatives, and furfural and its derivatives, such as 2-methyl-tetrahydrofuran, cyclopentyl methyl ether (CPME), and others [50, 51].

Gases that may liquefy in its vapour pressure inside of a pressurised vessel at a pressure (1–100 bar) are referred to be liquefied gases. Due to their volatility, they make it simple to separate them from the extracts and have the benefits of dissolving natural ingredients at temperatures that are comparatively low, protecting delicate components from degradation. Increasing numbers of research document, the usage of liquefied gases as eco-friendly solvents for extraction such as dimethyl ether, n-propane & n-butane [52]

Substances at temperatures and pressures over their vital points are referred to as supercritical fluids (SFs). The major benefit of an SFs is that it can be tailored for density by adjusting temperature and/or pressure, allowing for selective extraction of specific analytes. Additionally, SFs have virtually zero surface tension, which enables them to enter microporous surfaces and leave essentially little solvent residue in the final extract. The most often used SF is carbon dioxide (CO2) because of its modest significant pressure and temperature, which are 31 °C and 74 bar, respectively. Additionally, this fluid is tasteless, odourless, affordable, not combust ecologically friendly, and inert [53].

  1. 2.

    Using more sustainable solvents as mobile phase

High-purity organic solvents are needed in quite enormous quantities as the mobile phase in chromatography. The transition to reversed phase chromatography from normal phase, which used nonpolar, poisonous solvents, was the initial step towards environmentally friendly chromatography [54]. Methanol (MeOH), Ethanol (EtOH), Acetonitrile (ACN), acetone, ethyl acetate, or their combinations, or their mixes with water, are often used in reversed phase liquid chromatography. Among these solvents, EtOH, acetone, and ethyl acetate are preferred in terms of environmental friendliness, and some attempts are made to replace the more hazardous acetonitrile and methanol with them [55]. The disadvantage of ethanol compared to acetonitrile is that it has a somewhat high viscosity [56]. Ethanol is slightly harmful organic mobile phase ingredients. While the separation efficiency is statistically equivalent, acetone is another more environmentally friendly alternative to acetonitrile [57]. With water containing 0.04% triethylamine as the mobile phase, the use of polyethylene glycol as the stationary phase enabled successful partition of all 4 basic and acidic chemicals [58]. Only 0.85 mL EtOH was used per chromatographic run when a water-based mobile phase was used in extreme temperature liquid chromatography [59, 60].

Normal phase systems are used to determine some samples which are nonpolar and non-volatile (such as lipids), hence it's important to focus on applying more environmentally conscious solvents in this field. As mobile phase ingredients, cyclopentyl methyl ether, hexamethyldisiloxane, isopentyl acetate & 2-methyltetrahydrofuran, have all been employed satisfactorily [61, 62] (Figs. 2, 3 and Table 3).

Fig. 2
figure 2

Methodological introduction of green analytical chemistry principles

Fig. 3
figure 3

Green Analytical Techniques

Table 3 A few instances of alternate analysis solvent use [63,64,65,66,67,68,69,70,71]

Recent applications of green technology

By using green solvents in HPLC

Elsheikh et al. have developed and optimised straightforward, accurate, and reliable stability-indicating chromatographic methods, such as the HPLC method supported with the Central Composite Design, to achieve an environmentally conscious time- as well as money-saving approach. He attempted to employ benign solvents here to detect zonisamide (ZNS) even in the presence of its breakdown product. By using a Kromasil MS C18 (150 mm, 4.6 mm, i.d. 5 μm) column at 35 °C with a green and safe mobile phase (MP) containing EtOH: H2O (30:70%v/v), at (1 mL/min), and for detection, a photodiode array detector was used at 280 nm, ZNS was chromatographically separated from its degradation product. ZNS 50 g/mL HPLC chromatogram (retention time, Rt-2.806) and its degraded form 10 g/mL (Rt-1.553). Reproducible results were obtained in the range of (2–10 μg/band). The newly developed HPLC procedures have ECO Scale scores of 91 with modest green colours. The comparison of the results with the official USP procedure (81 eco scale score) demonstrates that the recommended methods were more environmentally friendly [72].

Kokilambigai et al. had optimised the HPLC technique in evaluation of atorvastatin calcium using analytical Quality by design (AQbD) and more environmentally conscious solvent approaches. The Central Composite Design was employed to filter out factors that could have an impact on method development. The analysis was done using a Zorbax Eclipse plus C18 column (150 × 4.6 mm, 5 μm). Since neat EtOH along water as mobile phase produces broad peaks with unnecessary fronting and asymmetry, the best chromatographic analysis was accomplished utilising 0.5% v/v aqueous acetic acid: EtOH (42.5: 57.5%v/v) with a flow of 0.91 mL/min. Detection was performed at 246 nm using photodiode array. For atorvastatin, the Rt was 6.27 min, while the run-time was 12 min. There was good linearity between 10 and 150 μg/mL, with a correlation coefficient (R2 > 0.9999). According to a forced degradation research, atorvastatin is more vulnerable to degradation when exposed to acidic stress. The created approach received a total score of 90, indicating that it adhered to all green standards and was therefore ecologically benign [73].

By using a Quality by Design-based HPLC approach, Perumal et al. discussed a rapid, simple, specific, accurate, reproducible, and environmentally friendly approach for determining the existence of escitalopram (ESC) & etizolam (ETZ) in the formulation. Utilising Phenomenex column C18 with EtOH and phosphate buffer (60:40%v/v), with a flow rate maintained at 1 mL/min, and detected at 254 nm using a dual absorption detector, is the best method for separating ESC and ETZ and the products of their deterioration. It was discovered that the ESC and ETZ had retention durations of 3.5 and 6.5 min, respectively. 99.55 and 99.94 percent, respectively, were found to be the percentage recovered for ESC & ETZ. This technique was tested and shown linearity ranges from 5 to 30 μg/mL for ESC, 2–12 μg/mL for ETZ, precision, repeatability, and selectivity with R2 values of 0.9975 and 0.9987 for ESC and ETZ, respectively. In the forced degradation trials, ESC deteriorated significantly in an acidic environment, then most under photodegradation stress. In alkaline condition, ETZ's % recovery decreased, indicating the drug’s susceptible to alkali. According to the analytical greenness metric approach (AGREE), the method's greenness was assessed, and the result was a score of 0.78 [74].

In order to quantify zidovudine (ZDV), lamivudine (3TC), and nevirapine (NVP), Vieira-Sellai et al. devised an environmentally sustainable HPLC approach. This method employs ethanol as the sample preparation solvent and as the MP for the analysis of the drugs. The analysis was carried by lowering the prepared solution's solvent volume and analysis time, as well as the column's diameter (3 mm as rather than 4.6 mm). Utilizing C18 column (ARV4 5 µm 250 × 3.0 mm, Interchim), the analysis was done in a gradient mode by injecting volume of 10 µL, with a flow rate 0.4 mL/min, while detection is done at 270 nm on a photodiode array (PDA) detector. Linearity was evaluated by preparing five standard solutions in the range from 80 to 120% of nominal concentration (37.5 μg/mL for 3TC, 75.0 μg/mL for ZDV and 50.0 μg/mL for NVP). As the mean recoveries fall between the range of 99.95% and 100.27%, the approach assures good accuracy. This technique obtains a count of 75 on the ECO Scale, indicating that it is environmentally friendly [75]

Dharuman et al. show the novel inclusion of analytical quality by design with GAC in the establishment of analytical approach for measurement of Benidipine hydrochloride (BEN) & Chlorthalidone (CTD) in bulk and formulations. For optimisation, a central composite design was employed. Using a MP of ratio 40:60%v/v of EtOH & potassium dihydrogen orthophosphate analysis was accomplished utilising an Agilent Eclipse Plus (C18, 250 mm 4.6 mm i.d, 5 μm) at 1 ml/min, and was detected via a diode array detector at 230 nm. The buffer solution (pH adjusted to 3.5) was chosen because it elutes analytes from the stationary phase more quickly, needs fewer time, and yields the best retention period with excellent peak symmetry, little tailing, and theoretical plates CTD and BEN had retention times of 3.1 and 5.1 min, respectively. Linearity range of 3.2–4.8 μg/mL for BEN and 5.0–7.5 μg/mL for CTD was obtained. The values for LOD and LOQ were obtained to be 1.812 and 5.493 μg/ml, respectively. The medication CTD was more likely to decompose under acidic and alkaline circumstances, whereas BEN was more vulnerable to hydrogen peroxide, according to the forced degradation trials. The developed techniques' ECO Scale score is 93, with mild green colours [76].

Sukumar et al. proposed an easy, effective, and reproducible approach to analyse Atorvastatin (ATO), Ezetimibe (EZB), and Fenofibrate (FF), utilising stress studies and RP-HPLC green analytical chemistry principles. The buffer (0.1% triethanolamine in water) and EtOH was used to separate these medicines in the ratio of 90:10 on MP, whereas stationary phase (SP) used is Inertsil ODS 3 (250 mm × 4.6 mm), 5 μm column, employing a photodiode array detector (PDA) detector at a wavelength of 256 nm. The forced degradation investigations revealed that EZB decay greater than 15% at alkali hydrolysis, while the ATR has > 5% deterioration in acid, peroxide, and heat deterioration. The Rt of ATO, EZB, and FF are 2.86, 6.723, and 11.13, respectively. ECO Scale of the developed methods show a score of 91 with faint green colours [77].

By combining UV spectrophotometric & HPLC techniques, Perumal et al. devised an analytical quality by design approach that is quick, affordable, accurate, precise, and environmentally friendly. Analysis of Molnupiravir and its breakdown products was done on C18 column having a MP of EtOH and phosphate buffer in ratio of 42.5:57.5% v/v, at flow rate of 0.9 mL/min, and detected at 235 nm. Utilizing the UV and HPLC techniques, the molnupiravir linearity ranges were 4–9 and 10–150 g/mL; also, the R2 values showed 0.9999 and 0.9999. For molnupiravir, the recovery rate ranged from 98 to 102%. The proposed strategy received a 94 on the ECO Scale, signifying that it is beneficial to the environment [78].

By combining analytical quality design with green analytical chemistry concepts, Kannaiah et al. devised a green, straightforward, and trustworthy approach for estimation of betamethasone dipropionate (BD) and calcipotriene (Cal) through a UHPLC method. The Central Composite Design (CCD) was utilised to filter out factors that could have an impact on method development. Dikma Endeversil C18 ODS (2.1 × 50 mm, 1.8 µm) column was used for separation. For best chromatographic separation, EtOH and potassium dihydrogen phosphate (3.0 pH) buffer were mixed 51:49 (%v/v), at a flow of 0.31 mL/min, and detected at 254 nm. Betamethasone and Calcipotriene had linear detector responses at 125–750 and 12.5–75 μg/mL, with detection limits and quantifications of 12.484, 37.831 and 3.229, 9.785 μg/mL, respectively. The recovery percentage was shown to be within tolerances of less than 1.5%. BD showed a degrades of more than 15% in the alkali, while degradation in Cal was nearly 15% in oxidation, according to a forced degradation research. AGREE metrics software program shows the total score of 0.89, greenest in all respects [79].

A single reversed-phase HPLC technique devised by Vijaykumar et al. for the estimation of apremilast, its enantiomer and its seven impurities in the medicinal material. Apremilast chromatographic separation from its enantiomer product was achieved by utilising immobilised chiral SP having a chiral selector "tris (3,5-dimethyl phenyl carbamate) derived from amylose-Chiralpak IA-3 (250 mm × 4.6 mm, 3 μm) column at 25 °C with green MP consists of buffer (0.01 M NH4HCO3, PH 8.0) and ACN in equal ratio at (0.4 mL/min). Two contaminants, impurity-2 (open ring acid impurity) and impurity-5 (deacetylated impurity), were discovered through forced degradation research. The impurity recoveries range from 96.1 to 102.1%. It was discovered that the suggested method's AGREE matric score was 0.66 as its consumption of ACN is comparatively less than previous established methods [80].

Kowtharapu et al. devised a linear, exact, accurate, particular, robust, environmentally friendly and sustainable LC method for determining olopatadine hydrochloride. A Boston eco-friendly C8 column (150 × 4.6 mm, 5 μm i.d.) was used to optimise and validate the isocratic chromatography procedure. With flow rate of 1.0 mL/min and a column temperature of 30 °C, a MP of sodium dihydrogen phosphate buffer of pH 3.5 and ACN in the ratio of 75:25 (%v/v) was utilised. The detection was carried out at 299 nm. The accuracy outcomes varied from 99.9 to 100.7%, the relative standard deviation (RSD) from the precision was 0.5, and the correlation coefficient from the linearity experiment was more than 0.999. Analytical eco scale tool was used which gave an outstanding analytical eco-score of 77 was discovered [81].

Kowtharapu et al.; optimised the High-Performance Liquid Chromatographic technique for evaluating ondansetron hydrochloride utilising AQbD design approaches. The devised approach is straightforward, considerate and beneficial to the environment. A Discovery (250 × 4.6) mm, 5 μm column using phosphate buffer pH 5.7 and acetonitrile as the MP, at flow rate fixed at 0.9 mL/min, and detected at 216 nm were used to achieve separation. The relative standard deviation for the accuracy findings ranged between 0.55 and 2.72% for all contaminants. The linearity experiment yielded correlation coefficients greater than 0.995 for both contaminants and analyte. The accuracy ranged from 88.4 to 113.0% for all impurities. Results for accuracy varied from 99.9 to 100.7% [82].

For the simultaneous analysis of the innovative combination of metformin hydrochloride (MTF), pioglitazone hydrochloride (Pio), and glibenclamide (GBC) and also with MTF toxic impurities, Fawzy et al. proposed a reliable, specific, eclectic, and environmentally conscious HPLC–UV technique. With a short run time and less environmental risks, the suggested approach is green. On a VDSpher Pur 100 C18-E (250 mm × 4.6 mm, 5 m) column, contaminants and antihyperglycemic medications were separated and quantified using gradient elution with a mobile phase made up of 0.1 M heptane sulfonic acid having a pH 2.2 and ACN under a flow of 1.5 mL/min and detection done at 225 nm using photodiode array detector (PDA). Retention intervals for MTF = 3.640, Pio = 5.062, and GBC = 7.788 min, respectively. Analytical AGREE tool reveal the score of 0.76 suggesting it as green [83].

The four medications, tamsulosin HCl (TAM), tadalafil, alfuzosin HCl, and solifenacin succinate (SOL), were examined by Abdel-moety et al. utilising a mix of GAC and AQbD techniques using HPLC–DAD methodologies. Derringer's desirability function and CCD was utilised to identify and optimise key technique limits. Optimal chromatographic separation was achieved using EtOH and phosphate buffer (pH 4.0) in a ratio of 40:60 (%v/v) for mixture I and in equal ratio for mixture II flowing at 2.3 mL/min, and detected through UV detector at 210 nm. Separation was performed on Agilent HPLC–DAD system C18 column (100 mm × 4.6 mm). The suggested approaches also make effective utilization of monolithic-based columns by enabling high throughput analysis with just a little under 4 millilitres of ethanol and a roughly 3-min run time. The percentage recoveries ranged between 98 and 102%. Linear concentration was in a range of 3–70 µg/mL for TAM, 5–110 µg/mL for tadalafil and 5–90 µg/mL for both alfuzosin & SOL. The Analytical Eco Scale was employed to measure the method's greenness, and the result was a score of 94[84].

For the determination of ketoconazole along with beclomethasone utilising RP-HPLC & multi-analytical UV spectrophotometric approach, Kannaiah et al. developed a straightforward, dependable, exact, effective, and ecologically friendly technique. The absorption ratio, first-order the absorption ratio, and area under curve approaches are the three environmentally friendly approaches covered by the spectroscopy technique for estimate, which is the initial approach. The second method employed an eco-friendly rotatable CCD analytical quality via a design-based RP-HPLC technique and an ODS reversed-phase column (250 × 4.6 mm, 5 µm). The best chromatographic separation was obtained through a MP of EtOH: 0.1 M potassium dihydrogen phosphate buffer (pH 2.5) 33: 67%v/v with a flow of 1.0 mL/min. Analytical eco scale, green analytical procedure index (GAPI), and AGREE were used to examine the technique's greenness, and it was determined that the approach is green [85].

Badr-Eldin et al. suggested a simple, recursive, quick, accurate, exact, repeatable, resilient, specific, and stability-indicating RP-HPLC technique for identifying clonazepam and its associated compounds. With an ODS column and a mobile phase made up of 2% sodium dodecyl sulphate, 0.05 M sodium acetate buffer pH 3.5, and isopropanol in the ratio (25:55:20%v/v/v), with a flow of 1.5 mL/min the chromatography process was optimised and verified. Detected at 254 nm using a DAD detector. The recovered percentage ranged from 101.33 to 99.40%. Linearity for clonazepam and its associated compounds was between the range of 4–140 μg/mL and 4–64 μg/mL, respectively. Clonazepam and its related compound have LOD of 0.024 μg/mL and 0.380 μg/mL while LOQ of 0.0799 μg/mL and 1.2870 μg/mL for, respectively. It was discovered that clonazepam breaks down more quickly in oxidative environments. The approach is also straightforward in terms of susceptibility, utilisation of an ecologically friendly MP, straightforward extraction techniques, considerably shorter Rt [86].

For the simultaneous testing of a combination of sofosbuvir and ledipasvir, El-Shorbagy et al. proposed a straightforward, quick, prudent, linear, exact, accurate, and environmentally conscious RP-HPLC–UV-Fluorescence approach. On C18 (250 mm × 4.6 mm, 5 μm in particle size), isocratic elution at a flow rate of 1.2 mL/min was used at room temperature. The UV and fluorescence detectors are both installed in the column. The MP contained 35: 35: 30 (%v/v/v) acetonitrile, methanol, and 0.01% triethylamine that had been pH-adjusted to 3. The UV detector was operated at 261 nm until the SOF was eluted, at which point it switched to 333 nm (the LED maximum) for five minutes. By using a UV detector, it was discovered that the mean % recovery of sofosbuvir and ledipasvir was 100.33 and 100.06, respectively. Linearity for sofosbuvir was between the range of 1 and 40 μg/mL while for ledipasvir 0.4–20 μg/mL. LOD for sofosbuvir was 0.012 μg/mL while for ledipasvir was 0.011 μg/mL. LOQ were 0.040 μg/mL and 0.036 μg/mL for sofosbuvir and ledipasvir, respectively [87].

Subhadip et al. devised an ecologically sound, resilient, quick, accurate, specific, linear, and exact RP-HPLC technique for predicting favipiravir in its medicinal dosage form, and compared it using ANOVA and in-vitro dissolution studies. With a C18 column (4.6 mm × 150 mm, 3 µm spherical particles), MeOH, EtOH, and H2O (25:35:40%v/v/v) as the MP, with a flow of 0.80 mL/min, a Rt of 7.216 min, at temp of 25 °C, and detected at 236 nm, in isocratic mode, with a run-time of 10 min, separation was accomplished. Oxidative stress was shown to cause the greatest deterioration percentage. The average recovery rate was more than 98%. The created procedures have an eco scale score of 92, which means they are more considerably secure as well as environmentally friendly [88].

A green and environmentally friendly HPLC technique was created by Khaled et al. to measure vitamin D3 and calcium levels in atorvastatin. In less than 10 min, the two medicines were separated using a gradient of 0.1% orthophosphoric acid (OPA) having pH = 2.16 and EtOH as the MP & a photodiode array detector. The column was Symmetry column C18 (100 × 4.6 mm, 3.5 µm). The column temperature had been set 40 °C while the MP was introduced at a rate of 1 ml/min. Atorvastatin calcium & vitamin D3 were detected at wavelengths of 246 and 264 nm, respectively. The average recovery for atorvastatin calcium & vitamin D3 was determined to be 102.87% & 101.35%, respectively. Linearity of the method was verified in the range from 5 to 40 μg/mL for atorvastatin calcium and 1–8 μg/mL for vitamin D3 while LOD are 0.475 µg/mL and 0.041 µg/mL for atorvastatin calcium and vitamin D3. They evaluated the greenness of our suggested procedure such as AGREE & GAPI tools. The developed approach has a score of 0.75 and is a semi-automatic, miniature method [89].

Using a Teknokroma C18 (150 mm × 4.6 mm, 5 µm particle size) reversed-phase guard column, Sepideh et al. devised a straightforward, quick, economical, and environmentally friendly HPLC test for estimating capecitabine in plasma. An oral 5-fluorouracil (5-FU) prodrug called capecitabine is frequently administered to people with colorectal and breast cancer. Formic acid solution (pH = 3): EtOH (55:45%v/v) flowing in a flow rate of 1.0 mL/min along with UV detection at 310 nm was used as the MP for the extraction. The temperature inside the column was fixed at 50 °C. Protein was precipitated from the sample using a zinc sulphate-ethanol solution. This approach yields a high capecitabine recovery in human plasma, which can vary from 95.98 to 102.50% [90].

For determining domperidone aspirin and in bulk or formulation using HPLC, Sneha et al. devised an analytical approach that is affordable, quick (short retention time), straightforward, precise, reliable, and sustainable. On a Prontosil C-18 column (4.6 × 250 mm, 5 μ particle size), separation was carried out isocratically using a MP of 10 mM KH2PO4: ACN (20:80%v/v) pH 3.5. Chromatograms were taken at 231 nm by a UV–visible detector [91].

Nazrul et al. devised a simple, rapid, reliable, precise, robust, specific, environmentally friendly & stability determining technique for quick measurement of rosuvastatin calcium in conventional medicines utilising HPLC–UV. As a sustainability-conscious green eluent, a mixture of EtOH, MeOH, and ethyl acetate (6:3:1%v/v/v) at a rate of 1.0 mL/min was chosen to produce a quick and straightforward assay with a reasonable run time (5 min), which was the most effective to detect rosuvastatin calcium & its separation. A NUCLEODUR 150 mm 4.6 mm RP C8 column filled with 5 m filler as the SP was used to identify rosuvastatin calcium, with detection taking place at 254 nm. The estimated Rt for rosuvastatin calcium is 1.52 min. Linear concentration was found in range of 0.1–100 µg/mL with correlation coefficient of 0.996. The % recovery was found to be 99.99%. According to investigations on forced degradation, rosuvastatin calcium deteriorated under oxidative stress in the presence of H2O2 and in 0.1 M NaOH [92].

For the measurement of clorsulon, albendazole, triclabendazole, and ivermectin utilising monolithic columns, Rashed et al. devised a straightforward, quick, environmentally friendly, and highly sensitive micellar liquid chromatographic approach. With a MP made up of 120 mM sodium dodecyl sulphate, 15% propanol, & 15 mM phosphate buffer (pH 5.5), separation was carried out on an Anonyx monolithic C18 column (100 × 4.6 mm) at 40 °C using UV detection at 225 nm. The average recoveries varied from 99.2 to 101.2%. Linearity of the method was verified in the range from 0.625 to 25 μg/mL for clorsulon, albendazole, triclabendazole and 30–300 μg/mL for ivermectin. LOD are 0.16, 0.18, 0.18 and 6.15 µg/mL and LOQ are 0.54, 0.60, 0.60 and 20.50 µg/mL for clorsulon, albendazole, triclabendazole and ivermectin, respectively. The recommended method's analytical Eco Scale value was determined, and it received a score of 75, which is very near to being an exceptional green analysis [93].

Magdy et al. devised a unique and environmentally friendly reversed-phase HPLC technique with fluorescence detection, as well as an analytical QBD approach, for the simultaneous quantification of lesinurad & febuxostat (FBX) with diflunisal (DIF). On the Hypersil BDS C18 column, the separation via chromatography is carried out using isocratic elution at 40 °C. ACN: potassium phosphate buffer (30.0 mM; pH 5.5, 32.2:67.8% v/v), pumped at 1.0 mL/min with an injection volume of 20.0 µL, was the MP. Fluorescence detector (FLD) detection was carried out at 280/370 nm. The ternary mixture may be separated using the suggested procedure in about 10 min. The Rt was determined to be 6.599, 7.641, and 9.155 min for DIF, lesinurad, and FBX, respectively. Linear concentration was found in range of 50–500 ng/mL, 50–700 ng/mL and 20–700 ng/mL, respectively, for DIF, lesinurad and FBX. The recoveries for DIF, lesinurad, and FBX were 99.32%, 98.8%, and 99.65%, respectively. The relative standard deviation was found to be less than 2. The recommended method's analytical Eco Scale value was computed, and a score of 87 was discovered [94].

By combining the concepts of GAC, Saroj et al. established a reliable & environmentally friendly RP-HPLC technique for determination of fenoverine. With a mobile phase of methanol and ammonium acetate buffer 20 mM (81:19% v/v), on a flow rate of 1.0 mL/min, and a column oven temperature of 33ºC, while employing UV detection at 262 nm, separation was accomplished on a Spherisorb C18 column (150 × 4.6 mm, 3 µm). The peak of fenoverine in the unstressed condition was seen in the chromatogram at 7.8 min. According to a forced degradation research, considerable degradation occurs under 10% H2O2 and hydrolytic conditions. Linear concentration was found in range of 0.5–160 µg/mL with a limit of detection and limit of quantitation of 0.1 and 0.3 µg/mL, respectively. The % recovery was found to be 99.7%. The developed HPLC method had an efficiency analysis trees (EAT) value of 41.82, while the literature method had a score of 44.01, which concluded that the approach that was created had a smaller effect on the nature because it was developed entirely without using ACN [95].

A brand-new, straightforward liquid chromatographic method for brivaracetam detection together with piracetam and carbamazepine was devised and validated by Mansour et al. On a Promosil C18 column (100 mm × 4.6 mm, 5 µm particle size), separation was accomplished using a MP of ACN: H2O containing 0.1% triethylamine at a ratio of 30:70%v/v at pH 6.5 corrected with orthophosphoric acid with a column temperature of 25 °C. The flow rate is 0.6 mL/min, the run period was around 9 min, and the UV detector used a 215 nm wavelength for detection. Piracetam, brivaracetam, and carbamazepine were shown to have retention times of 1.4, 4 and 8 min, respectively. The recovery rates were found to be between 94.8 and 101.05%. Values of the limit of quantification were 3.7, 2.3 and 1.8 µg/mL for the piracetam, the brivaracetam and the carbamazepine, respectively. The recommended method's analytical Eco-Score was discovered to be 85[96].

For the simultaneous detection of amlodipine (AML) & metoprolol (MET), Mabrouk et al. devised and validated a rapid, sensitive, and accurate green micellar liquid chromatographic technique. The XBridge ODS column (1150 × 4.6 mm, 5 μm particle size) was used for separation. The mobile phase was composed of SDS and sodium dihydrogen phosphate in water, while the pH was adjusted to pH 3.0 using phosphoric acid. The injection volume was 50 µL, and the flow rate was 1.5 mL/min at 40 °C. For MET and AML, the fluorescence detector was adjusted to emit light at 275/303 nm and excite light at 364/455 nm, respectively. The MET Rt was 3.46 min, and AML was at 6.35 min. Linearity of the method was verified in the range from 0.1 to 10 μg/mL for MET and 0.2–2 μg/mL for AML. The recovery was close to 100.156%. RSD values were below 2%, which demonstrates adequate precision. LOD was 20 ng/mL and 50 ng/mL for MET and AML while LOQ was 61 ng/mL and 81 ng/mL for MET and AML. The developed method was found to have an analytical Eco Scale value of 80 indicating an excellent green HPLC method [97].

A quick, secure, and ecologically conscious analytical technique for determining daptomycin in lyophilized powder was developed by Totoli et al. Using a MP of EtOH-H2O (55:45%v/v) around pH 4.5, pumped at a flow rate of 0.6 mL/min for an 8-min run period, and UV detection at 221 nm with a DAD detector, separation was accomplished on an Agilent Zorbax C18 analytical column (4.6 × 150 mm, 5 μm). Daptomycin had a retention period of 5.8 min. Linearity of the method was verified in the range from 20.0 to 70.0 μg/mL. The recovery was close to 100%. RSD values were below 2%, which demonstrates adequate precision. LOD was 1.87 μg/mL and the LOQ was 5.68 μg/mL. According to a study on force degradation, the sample was less stable under basic circumstances [98].

Bhukya et al., had developed and validated stability indicating HPLC method for separation and identification of Panobinostat and its degradant product. The analysis was done using a column Waters X bridge C18 3.0 µm (50 × 4.6 mm) was used with mobile phase A as 10 mM NH4COOH buffer adjusted to pH 3.0 with HCOOH and Ethanol as mobile phase B with gradient elution mode. The mobile phase flow rate is 0.5 mL/min, injection volume are 3 µL and at 277 nm as UV wavelength. The method found linear from 12 to 300 µg/mL with R2 ≥ 0.998. The % recovery for analyte was in between 99.28 and 100.36 with 0.60% RSD. The %RSD for the analyte in Method precision and intermediate precision is below 1.0. According to the AGREE, the method's greenness was assessed, and the result was a score of 0.74 [99].

Hammad et al., had developed a solvent free, robust, sensitive, selective and fast chromatographic method for determination of metronidazole (MTR) and ciprofloxacin (CIP). The analysis was done using a X-Bridge (150 × 4.6 mm, 5 μm) column. The separation was achieve using 0.13 M sodium dodecyl sulphate and 0.02 M polyoxyethylene lauryl ether (Brij-35) solution to pH 2.5 using phosphoric acid at a flow rate of 1.3 mL/min and 280 nm wavelength in UV detector. The method was said to be greener as it eliminates the use of harmful organic solvent as well as it minimises the run time (8 min) to reduce cost and energy consumption. The method found linear from 0.5 to 50 µg/mL with R2 ≥ 0.9998 for MTR and 0.9997 for CIP. The % recovery for MTR and CIP was to be 99.45 and 100.13%, respectively. The %RSD for the analyte in Method precision and intermediate precision is below 2.0. The AGREE software was employed to evaluate the technique's greenness, and the resultant score of 0.81 [100].

Michael et al., had developed green, accurate, selective and stability-indicating method for determination of levetiracetam using HPLC. The analysis was done using Lichrosorb RP-18 (250 × 4.6 mm i.d., 5 μm particle size) column. The separation was achieve using phosphate buffer (pH 3.1) and acetonitrile (87:13%v/v) mobile phase with the flow rate of 1.0 mL/min, and UV detection was carried out at 210 nm. The Rt of drug was found to be at 5.6 min. The AGREE software was employed to evaluate the technique's greenness, and the resultant score of 0.82 [101].

Yabre, M., et al., developed an easy, green, and stable RP-HPLC methodology for measuring contaminants in artesunate and amodiaquine using a Quality by Design (QbD) methodology. It effectively handled the difficulty of evaluating nine compounds under a single chromatographic condition by using a gradient elution with EtOH, resulting in a 95% confidence level for identifying the method's operational domain region. To achieve greenness, EtOH and 10 mM acetic acid were used as the mobile phase, which was tested using accuracy profiles and successfully used to raw materials and fixed-dose combination tablets. The technique used an Xbridge BEH C18 column (150 mm × 3 mm, 5 µm) with a flow rate of 0.4 mL/min. [102]

Megahed et al.; described an experimental approach to establish a green, gradient chromatographic technique for simultaneously analysing metronidazole (MTR) and spiramycin (SPR). The optimal chromatographic conditions involved a mobile phase of ethanol and 20 mM sodium dihydrogen phosphate solution (pH adjusted to 2.5) in a 2:98 (%v/v) ratio for 2 min, followed by a change to 30:70 (%v/v). The flow rate was 1.3 mL/min, and the separation and analysis were done on an X-bridge C18 column (150 mm × 4.6 mm × 3.5 μm). The detection wavelength was 230 nm, and the column oven temperature was set at 40 °C. The approach showed a linear response for both drugs in the range of 5–125 μg/mL. [103]

Duan et al.; developed and validated a green HPLC technique for the simultaneous measurement of nine sulphonamides utilizing EtOH as the green alternative solvent, and it was effectively applied to beef and milk samples. The Venusil XBP C18 column (Lanzhou Acetch Technologies Co., Ltd., Lanzhou, China) consisting of the following features: 250 mm, 4.6 mm in diameter, 5 µm particle size was used. The pH of the mobile phase was around 3. Furthermore, no more than 30 mL of trash was created each sample. The four quadrants of the greenness profile are represented by the following criteria: persistent, bio accumulative and toxic (PBT), hazardous, corrosive, and waste. The score of the analytical Eco Scale was 86, indicating an excellent value for the proposed method [104].

Habib et al.; used quality by design principles and green analytical chemistry which aided in the creation of a green micellar HPLC technique for assessing atorvastatin calcium and amlodipine besylate in binary combinations and tablet dosage forms in 8 min. The optimal conditions were a mobile phase of 0.17 M sodium dodecyl sulphate solution (pH 2.9) with 10%v/v n-butanol, a flow rate of 1.5 mL/min, and a column temperature of 45 °C utilizing an X-Bridge™ (150 mm × 4.6 mm, 5 μm). Fluorescence detection was set at 276/378 nm for atorvastatin and 366/442 nm for amlodipine. The approach showed linear responses for both medicines in the range of 0.2–25 μg/mL and was verified for tablets. [105]

Elmansi et al.; developed the method for separation of three BDZs (diazepam, clonazepam, and bromazepam) utilizing a green micellar mobile phase. Brij-35, a non-ionic surfactant, substitutes organic solvents, resulted in much shorter retention times and more ecologically friendly separation. The procedure, was carried out on a monolithic C18 column, uses a 50:50% v/v mixture of sodium dodecyl sulphate (SDS) and Brij-35 as the mobile phase. Using UV detection at 240 nm and a flow rate of 1.0 mL/min, separation was accomplished in less than ten minutes, even without an organic modifier. Furthermore, the method's greenness was evaluated using the analytical eco scale, demonstrating its suitability for testing the three medications. It was found that the developed micellar HPLC method, had an Eco scale value of 95 [106].

Kokilambigai et al.; used analytical quality by design (AQbD) and greener solvent technologies to improve a High-Performance Liquid Chromatographic (HPLC) method for detecting atorvastatin calcium in bulk and pharmaceutical dose forms. A rotating central composite design was used, with EtOH volume and flow rate as essential method variables that influenced analytical characteristics. Using a Zorbax Eclipse plus C18 column (150 × 4.6 mm, 5 µm), optimum separation was achieved using a mobile phase of 0.5% v/v aqueous acetic acid: EtOH (42.5:57.5%v/v) at 0.91 mL/min. Atorvastatin was detected at 246 nm, resulting in a retention time of 6.27 min and a total run duration of 12 min. The technique demonstrated linearity from 10 to 150 µg/mL, and validation trials produced good findings. The method's greenness and ecological effect were assessed using a variety of indicators, which validated its great environmental profile. When evaluated using the analytical eco scale, the recommended HPLC method received a score of 90 [73].

Stability-indicating techniques for modafinil (MDF) and the by-product of its acid-induced deterioration have been devised and confirmed by Elsheikh et al. The procedure was an environmentally conscious (HPLC) procedure that used an XTERRA MS C-18 column (100 mm × 4.6 mm, 5 μm id), EtOH-H2O (30:70, %v/v) as the MP at 40 °C at a flow of 1 mL/min, and UV scanning at 220 nm. With a lowest time to retention of 3.6 min, the HPLC chromatogram of MDF at 50 g/mL has a Rt of 3.628 while that of its degradation product at 10 g/mL has a Rt of 1.129. Linearity was in ranges of 2–10 μg/mL. Accuracy was found to be 100.01%. LOD and LOQ was 0.127 and 0.384 μg/mL. The created HPLC Eco Scale procedures yield scores of 90 [119] (Table 4).

Table 4 Summary of applications of green solvent in HPLC

By minimising solvent consumption

  1. (A)

    UHPLC (Ultra high-performance liquid chromatography)

A QbD-compliant simple, quick, robust, sensitive, efficient, economical, and ecologically conscious UHPLC approach for chrysin quantification has been developed by Sharma et al. Utilising Box Behnken Design, essential parameter optimisation was completed. The best technique conditions are a C8 column with an ACN and H2O mobile phase that flow at a rate 0.5 mL/min by injecting volume of 5 µL at a maximum wavelength of 267 nm via a PDA detector. Here, ethanol is not used as the mobile phase because it produces high pressure, which can be reduced by raising the column temperature, but this was avoided because doing so would increase energy requirement, which is in contradiction of the GAC principles, so ACN along with water were chosen as the mobile phases instead. Additionally, the UHPLC method is referred to as a “greener” method because it minimised the amount of waste generated and operator and ecological safety risks associated with exposure to organic solvents. Other benefits of the method include increased method effective; decreased back pressure, and low solvent consumption. Chrysin had a retention time of 4.802 min. The accuracy testing revealed that the percent recovery ranged from 98.34 to 104.80%, proving that the procedure is very accurate. The method linearity for chrysin concentration ranging from 0.1 to 50 µg/mL. The method showed 0.028 µg/mL as LOD and 0.075 µg/mL as LOQ. The Analytical Eco Scale employed to evaluate the technique's greenness, and the resultant score of 81 [107].

An environmentally friendly AQbD based UHPLC technique has been proposed by Chanduluru et al. for the environmentally friendly measurement of isosorbide dinitrate (ISDN) and hydralazine hydrochloride (HHC). On a Phenomenex C18 (50 × 2.1 mm, 2 μm) column with EtOH and 0.1% trifluoroacetic acid (60:40% v/v) at a flow rate of 0.5 mL/min, separation was carried out. The devised approach discovered a runtime of just 3.5 min and an excellent resolution of 5.4 between the two medications. The recovery rate falls between 98.4 and 101.9%. Linearity ranges from 10–60 μg/mL and 18.75–112.5 μg/mL, with R2 of 0.9998 and 0.9992 for ISDN and HHC, respectively. LOD was found to be 0.923 μg/mL and 0.549 μg/mL while LOQ was found to be 2.797 μg/mL and 1.666 μg/mL for ISDN and HHC, respectively. When the technique's greenness was evaluated through the ECO Scale tool, the score was discovered to be 96, which is very excellent [108].

Kannaiah et al. suggested an environmentally conscious, simple, and reliable technique for estimating Crotamiton (CTM) and Hydrocortisone (HCT) by combining GAC principles with an AQbD-rotatable CCD methodology employing RP-UHPLC. The Waters X-Bridge (C18 50 × 2.5 mm i.d, 1.7 µm) column were utilisd for estimation, and the MP composition was ethanol: 0.01 M ammonium acetate buffer (pH 3.0) 30:70%v/v with the flow of 0.3 ml/min. Linearity concentration was made in range of 70–130 µg/mL for CTM and 1.75–3.75 µg/mL for HCT. Here, UHPLC was chosen over HPLC because it requires less energy as well as solvent, and analytical time. Using the AGREE tool, the method's greenness was evaluated, and the analytical score came out to be 0.83[109].

The UHPLC technique was proposed by Chanduluru et al. to measure pitavastatin and ezetimibe (EZB). Rotatable central composite design, which is modelled after the Ishikawa fishbone diagram, was utilised for optimisation. The satisfactory chromatographic estimation was done on Agilent UHPLC-PDA system (1290 infinity II LC system), alongside Kinetex phenyl hexyl (50 × 4.6 mm, 2.6 µm) column and MP of 72: 28% v/v EtOH and 0.1% orthophosphoric acid (pH 3.5), at a 0.31 mL/min flow rate with detection on 240 nm wavelength through photodiode array (PDA) detector. Rt for pitavastatin was 0.85 and for EZB was 1.91 min. Recovery for pitavastatin and EZB was determined to be 99.1%-100.94% and 99.9%-101.52%, respectively. Linearity ranging from 2–30 and 10–150 μg/mL with an R2 of 0.9999 and 0.9997, respectively, for pitavastatin and EZB. LOQ for pitavastatin and EZB was 0.0584 µg/mL and 0.0233 µg/mL, respectively, while LOD was 0.0192 µg/mL and 0.0076 µg/mL, respectively. Studies on forced deterioration have revealed that whereas EZB was hydrolysed under basic conditions, pitavastatin was vulnerable to acid hydrolysis. The result of the ECO Scale was found to be 97 [110].

Muzaffar et al. presented a UHPLC-MS/MS approach for fast measurement of umifenovir in plasma samples and measured greenness. Using the ACQUITY UPLC BEH C18 column (2.1 × 100; 1.7 m) and a MP made up of 15 mM ammonium acetate and ACN in an 80:20 (%v/v) ratio while being pumped at 0.3 mL/min, umifenovir and the IS were separated in under 2.5 min. The auto-sampler and column oven were each set to a temperature of 10 and 40 °C, respectively. The material was ionised using positive electrospray ionisation. The developed method showed excellent linearity in the concentration range of 1.32–625 ng/mL. Accuracy was in the ranges of 90.5–105.8%. The score, which ranges from 0.75 to 1.00 and indicates an environmentally favourable procedure, was determined to be 0.77 [111].

A unique UHPLC-MS/MS assay was created by Muzaffar et al. for the quick measurement of delafloxacin. Delafloxacin (DFX) and the internal standard (losartan) were separated on a UHPLC BEH C18 column (50 × 2.1 mm; 1.7 m) using gradient programming of an MP containing 0.1% formic acid in acetonitrile and 0.1% formic acid in water, with a flow rate of 0.3 mL/min, injection volume of 5 µL (partial loop mode), column oven temperature at 35 °C. A positive mode triple quadrupole mass detector was used for the quantification at an electrospray ionisation interface. The Rt of DFX and IS were, respectively, 1.72 and 1.79 min. Linearity was between concentration ranges of 2.92–6666 ng/mL with an acceptable correlation coefficient of R2 ≥ 0.995. The accuracy values ranged between 94.4 and 106.1%. The approach’s greenness evaluation was evaluated by using AGREE software having a score of 0.78, confirming excellent greenness of the approach [112].

Kamal et al.; developed method using The Waters Acquity UHPLC BEH C18 column (100 × 2.1 mm, 1.7 µm) which efficiently removed Meropenem MPM, contaminants, and peaks from the sample matrix. Mobile phase A was a 25 mM monobasic potassium phosphate buffer adjusted to pH 4.05 using orthophosphoric acid (OPA) solution, while mobile phase B was a 30:70 (%v/v) combination of water and ACN. A simple binary gradient program (0.0–12.0 min, 87% → 0% A, 13% → 100% B) with a flow rate of 0.4 mL/min allowed for component separation in 15 min. The method's greenness was assessed with GAPI, AGREE, and analytical eco scale methods. Each sample preparation used 40.0 mL of ACN, resulting in a total consumption of < 50 mL per sample analysis and a penalty point score of 15. The approach achieves an eco scale score of 85, indicating exceptional greenness. [113]

Muchakayala et al. devised a green, robust, and speedy stability indicating chromatographic approach for the simultaneous measurement of fluorescein sodium, benoxinate hydrochloride, and their degradation products within 4 min. Fractional factorial and Box-Behnken designs were used for screening and optimization, respectively. The ideal chromatographic conditions included a mobile phase of isopropanol and 20 mM potassium dihydrogen phosphate solution (pH 3.0) in a 27:73%v/v, a flow rate of 1.5 mL/min, and a column oven temperature of 40 °C. The analysis was done on an Eclipse plus C18 column (100 mm × 4.6 mm × 3.5 μm) with a DAD detector tuned to 220 nm. Linear responses were obtained between 2.5–60 μg/mL for benoxinate and 1–50 μg/mL for fluorescein. The approach proved successful in quantifying pharmaceuticals in ophthalmic solution, with mean %recovery ± SD of 99.21 ± 0.74 for benoxinate and 99.88 ± 0.58 for fluorescein. Compared to previously described approaches, the suggested method is quicker and more environmentally friendly. Its greenness was proven by a high analytical eco scale score of 90 and a low EAT score of 4.21 [114] (Table 5).

Table 5 Summary of green technique by reducing solvent consumptions using UHPLC
  1. (B)

    HPTLC (High-performance thin layer chromatography)

Using the AQbD methodology, Prajapati et al. developed a reliable and ecological conscious HPTLC approach for the measurement of thiocolchicoside (THC) in its medicinal dose forms. Utilising Box Behnken Design, essential parameter optimisation has been accomplished. In contrast to HPLC, HPTLC uses a little quantity of MP to analyse several samples at once, decreasing the quantity of time and money required for each analysis. Chromatographic separation was carried out using toluene-acetone–water (1.5:7.5:1.0, %v/v/v) as the MP and 10 cm × 10 cm aluminium plates pre-coated using 250 µm of silica gel 60 F254 at 25 °C, 35% relative humidity, 15 min chamber saturation time, and 90 mm displacement distance. According to the degradation research, thiocolchicoside is more susceptible to oxidative degradation and acidic-alkaline hydrolysis, but less susceptible to photolysis, dry-heat, and water hydrolysis. thiocolchicoside, acidic deteriorated product DP1, and acidic deteriorated product DP2 have spots at retardation factor, Rf = 0.53, 0.65, and 0.80, respectively. The recovery rate falls between 99.39 and 101.65%. Linearity ranges from 100 to 500 ng/spot with R2 of 0.9979. LOD was found to be 4.03 µg/band while LOQ was found to be 12.21 µg/band [115].

In order to determine a novel blend of mebendazole (MBZ) & quinfamide formulations, Ibrahim et al. devised a simple, quick, inexpensive, sensitive, selectivity, broader linearity ranges, ecological, and short analytical time technique. The first approach is HPTLC, in which at 254 nm, distinct bands were scanned. After being separated employing silica gel HPTLC F254 plates & a simple MP made of MeOH: toluene (2:6, %v/v). It was discovered that the values of the Rf for MEB and quinfamide were 0.45 and 0.75, respectively. Linearity was in ranges of 0.2–2.5 and 0.1–2 μg/band for quinfamide and MEB, respectively. Accuracy was found to be 99.62% and 100.10% for QF and MBZ, respectively. LOD and LOQ for quinfamide was 0.055 and 0.168 μg/band, for MBZ was 0.031 and 0.094 μg/band. The second technique used RP-HPLC and involved isocratic separation of both pharmaceuticals on a Phenomenex C18 column with a green MP made of double distilled H2O: MeOH (30:70%v/v) with a flow rate of 0.8 mL/min and a run duration of 4 min and UV detection at 254 nm. The volume of trash was reduced to 3.2 mL/run using a short column (100 mm) having tiny particle size (3.5 µm), which is thought to be a significant indicator of greenness of process. MBZ and quinfamide had retention times of 2.69 and 3.43, respectively, Linearity was in ranges of 1–60 and 2–80 μg/band for QF and MBZ, respectively. Accuracy was found to be 100.04% and 99.87% for QF and MBZ, respectively. LOD and LOQ for quinfamide was 0.323 and 0.979 μg/band, for MBZ was 0.643 and 1.948 μg/band [116].

A novel technique for the estimation of furosemide (FRS), spironolactone (SPL), & canrenone (CAN) is being researched by Ibrahim et al. Alumina plates (20 × 20 cm) coated by 0.25 mm silica gel 60 F254 plates were utilised for Method 1's HPTLC separation, with ethyl acetate, triethylamine, and acetic acid (9: 0.7: 0.5, by volume) serving as the forming system & UV detection at 254 nm serving as the detection wavelength. For the three distinct components, FRS, SPL, and CAN have the Rf values of 0.320.02, 0.640.02, and 0.790.02, respectively. Linearity was in ranges of 0.2–2, 0.05–2.6 and 0.05–2 μg/band for FRS, SPL and CAN, respectively. Accuracy was found to be 100.11%, 99.65% and 99.98% for FR, SP and CN, respectively. LOD and LOQ for FRS was 0.059 μg/band and 0.195 μg/band, for SPL was 0.015 μg/band and 0.048 μg/band and for CAN was 0.014 and 0.045. Using a ZOBRAX Eclipse Plus C18 (4.6 × 100 mm) column and a MP made up of EtOH and deionized water (45:55%v/v), Method 2 was developed that uses green isocratic RP-HPLC with UV estimation at 254 nm. The flow rate was adjusted to 1 ml/min and the pH to 3.5 using glacial acetic acid. Within 5 min, the three chemicals had been successfully eluted with satisfactory separation. The duration of retention values for SPL, FRS, and CAN were determined to be 3.36, 1.64, and 4.42 min, respectively. Linearity was in ranges of 5–60, 2–60 and 2–60 μg/mL for FRS, SPL and CAN, respectively. Accuracy was found to be 100.10%, 100.03% and 100.01% for FRS, SPL and CAN, respectively. LOD and LOQ for FRS was 1.333 μg/mL and 4.00 μg/mL, for SPL was 0.485 μg/mL and 1.60 μg/mL and for CAN was 0.60 μg/mL and 1.80 μg/mL [117].

Bang et al., had developed and validated green HPTLC technique for estimation of Lenvatinib. He had developed two green methods, i.e. RP-HPTLC and NP-HPTLC, mobile phase used here are ethanol and water (60:40, %v/v), and 50:50%v/v of ethanol and ethyl acetate, respectively. The detection was carried out at 243 nm. Analytical greenness (AGREE) scores were estimated as 0.88 and 0.82, respectively, for the RP and NP [118] (Table 6).

Table 6 Summary of green technique by reducing solvent consumptions using HPTLC
  1. (C)

    TLC (Thin layer chromatography)

Stability-indicating techniques for modafinil and the by-product of its acid-induced deterioration have been devised and confirmed by Elsheikh et al. using TLC-Densitometric, which uses plates that are precoated with silica gel G60 F254 and are 10 × 20 cm in size and 0.25 mm thick. Dichloromethane-methanol (90:10, %v/v) was employed as the growing solution, and the chromatograms are scanned at 254 nm afterwards. Modafinil had a Rf of 0.48 and its degradation product had an Rf of 0.77. Linearity was in ranges of 1–10 μg/band. Accuracy was found to be 100.11%. LOD and LOQ was 0.110 and 0.335 μg/band. The created TLC-densitometry analytical Eco Scale procedures yield scores of 80 [119].

Elsheikh et al. have developed and optimised straightforward, accurate, and reliable stability-indicating TLC-densitometric technique for zonisamide (ZNS) in the presence of its breakdown product, which was carried out using aluminium plates covered with silica gel 60F254 as a SP, while chloroform, MeOH, and acetic acid (80:15:5%v/v/v) as a growing solution, and detection at 240 nm under a UV lamp. ZNS had an Rf value of 0.76 and its degradation product had an Rf value of 0.11. The newly developed TLC-densitometry procedures have ECO Scale scores of 84, with modest green colours which shows that the method is eco-friendly [72].

Green spectroscopy methods

An eco-sustainable spectrophotometric simultaneous equation approach was suggested by Kokilambigai et al. for the measurement of paracetamol (PCM), aceclofenac (ACF), and thiocolchicoside (THC) pharmaceuticals in their combined dose form. Instead of methanol, the diluent phosphate buffer with pH 7.8 was employed as an ecological solvent since it has no effect on the medicines' UV spectral properties. PCM, ACF, and THC all had maximum absorptions (λmax) at wavelengths of 243, 274, and 259 nm, respectively. The devised procedure was accurate since the recovery amount varied from 99.56 to 100.99% w/w. The linear range was established between 5 and 15 µg/mL for paracetamol, 1–5 µg/mL for aceclofenac, and 1–5 µg/mL thiocolchicoside. The LOD values for PCM, ACF and THC were in the ranges of 0.24 µg/mL, 0.33 µg/mL and 0.32 µg/mL, respectively. The LOQ value were in the ranges of 0.78 µg/mL, 1.01 µg/mL and 0.95 µg/mL, respectively. The ECO Scale tool was used to assess the greenness profile score, and it revealed a greenness score of 100 [120].

For the simultaneous quantification of Paracetamol (PCM), Aceclofenac (ACF), and Eperisone Hydrochloride (ES) Rathinam et al. have devised an environmentally safe, user-friendly, quick, sensitive, and affordable UV spectrophotometric approach. The diluent phosphate buffer pH 7.8 is employed as a green solvent since it has no effect on the medicines' UV spectral properties. When the wavelengths of the PCM, ACF, and ES were scanned across 200–400 nm with phosphate buffer pH 7.80 serving as the reference, their λmax were 243, 272, and 262 nm, respectively. Linearity concentration ranges between 12 and 18 µg/mL for paracetamol, 3.69–5.53 µg/mL for Aceclofenac, and 2.76–4.15 µg/mL Eperisone hydrochloride, and the LOD values were in the ranges of 0.48 µg/mL, 0.20 µg/mL and 0.13 µg/mL, respectively. The LOQ value were in the ranges of 1.44 µg/mL, 0.61 µg/mL and 0.38 µg/mL, respectively. The %recovery for PCM, ACF and ES was 100.48%, 100.33 and 100.07%. Software called ECO Scale determined that the simultaneous equation score was 96 [121].

Using Ultra-Violet (UV) spectrophotometric techniques, Chanduluru et al. have presented a straightforward, distinctive, economical, environmentally conscious, stability-indicating test approach to evaluate Chlorthalidone (CTD) and Cilnidipine (CIL). This approach uses propylene carbonate rather than methanol as a green solvent for CIL and CTD analyses because propylene carbonate is harmless in terms of influence on water, air, health risks, combustibility, stability. According to area under curve approaches, the absorbance ranges for CTD and CIL were 218–227 nm and 224–232 nm, respectively. Linearity concentration range of 7–13 μg/mL for CTD and 8.75–16.25 μg/mL for CIL. According to the agree programme, the simultaneous equation score was 0.91[122].

By utilising spectrophotometric techniques, Fawzy et al. produced accurate, eclectic, affordable, simple, and sensitive spectrophotometric tactics for the simultaneous estimation of saxagliptin hydrochloride and dapagliflozin propanediol monohydrate. The initial strategy, known as the induced dual-wavelength approach (IDW), mainly depended upon employment of substitute equality factors (F) to exclude the influence of dapagliflozin propanediol monohydrate while determining saxagliptin hydrochloride and vice versa. The second approach, known as the ratio difference method (RDM), determined the amplitude difference on the ratio spectrum of saxagliptin hydrochloride and DAG, respectively, using divisors of 25 μg/mL of dapagliflozin propanediol monohydrate and 20 μg/mL of saxagliptin hydrochloride. The third approach, plateau subtraction followed by multiplication by the division of dapagliflozin propanediol monohydrate 25 μg/mL, was used to compute SAG at λmax 221 nm. The ECO Scale score for given technique was 88[123].

Fawzy et al. develop ecologically sustainable, easy, and reliable spectrophotometric and chemometric approaches for the simultaneous quantification of Fluticasone propionate and Azelastine. Although methanol is used as a diluent, the procedure is ecologically favourable in terms of a number of other aspects, including the utilization of instruments, chemicals, and disposal of waste. For both medications, the absorbance is at 258 nm (λiso). This approach received an ECO Scale score of 88[124].

For the measurement of ramipril as well as temisartan both coupled with hydrochoryhiazide (HCTZ), Elsonbaty et al. have presented a specific, quick, green, exact, and simple analytical methodology employing a reconstituted FSD aided UV spectrophotometric technique. HCTZ is measured at 276 nm, which is the zero-crossing point for temisartan, whereas temisartan was measured at 257 nm, which was the no contribution point of HCTZ. HCTZ was concurrently measured at 244 nm and ramipril was calculated at 231 nm for ramipril /HCTZ, which is the zero-crossing point of both HCTZ and ramipril. The linear ranges for telmisartan, ramipril, and hydrochlorothiazide were 1–25 µg/mL, 5–35 µg/mL, and 1–10 µg/mL, respectively, and the LOD values were in the ranges of 0.067–0.747 µg/mL. The %recovery for about drugs was 100.82, 99.10, 99.55, respectively. The ease of use, speed, lack of demand for complex software, and increased sensitivity of the new technology over the conventional deconvolution-curve fitting are its advantages. The analytical eco scale's overall score is 88, which is very good [125].

Lofty et al. devised a straightforward, inexpensive, quick, efficient, and environmentally friendly approach for evaluating single-tablet regimens of atenolol (Ate), amiloride (AMD), and chlortalidone (CTD). Zero order spectra, which serve as spectral profiles or fingerprints for pharmaceuticals, were recovered using the Parent Spectrum Restoration Technique (PSRT). This approach used three computing approaches: the well-known smart method spectrum subtraction (SS), the derivative ratio transformation utilising normalised spectra or any other divisor, and the dual amplitude difference (DAD) through factorised spectra. Utilising methanol as both a blank and a solvent, the λmax for Ate, AMD, and CTD were discovered to be 226 nm, 362 nm, and 230 nm, respectively. Linearity for each drug was ranging between 4.0 and 40.0 μg/mL for Ate, 3.0–20.0 μg/mL for both AMD and CTD. The %recovery for AMD, CTD, Ate was 99.61%, 99.92%, and 99.82%, respectively. The UV-methods obtained a better score for greenness 89 due to their use of less energy and solvent than the HPLC described approach, which received a middling score of 84[126].

Kannaiah et al. suggested a simple, sustainable, inexpensive, sturdy, specific, and successful extraction technique, as well as a multivariant green analytical approach, for estimating ketoconazole using UV spectroscopy. The diluent is made by combining water and ethanol (99.9%) in 80:20%v/v. The drug's λmax at 226 nm over a diluent in blank solution was discovered. The linearity was found at concentration of 12.8–19.2 μg/mL with R2 value of 0.9992. LOD and LOQ was found to be 0.338 and 1.026 μg/mL, respectively. The %recovery was 100.31%. Through the ECO Scale tool, the analytical value was discovered to be 96 [127].

Salicylic acid (SA) & Mometasone furoate (MF) were analysed concurrently using a green capillary zone electrophoretic technique (CZE) that was developed & validated by Amira et al. Agilent CE instrument 7100 series along with a Diode Array Detector was utilised for separation. Agilent Technologies provided a capillary made of fused silica with an effective length of 50 cm and an internal diameter (id) of 50 m. Measurements were made in the hydrodynamic mode at a temperature of 25 °C. MF and SA showed good linearity in the series of 0.2–40 µg/mL and 0.1–80 µg/mL, respectively. The detection and quantification limits for the assay of MF were 0.04 and 0.13 μg/mL, and of SA were 0.03 μg/mL and 0.1 μg/mL, respectively. The% recovery was 99.60% and 99.35%, respectively. The discovered approach has various benefits, including little solvent use, low operating expenses, minimal sample time for analysis requirements, and no need for sample preparation. The recommended method's analytical Eco Scale value was computed, and its score of 86 was discovered [128].

For the estimation of remogliflozin (REM) & metformin (MET), Attimarad et al. devised and validated straightforward, precise, sensitive, affordable, and green UV spectrophotometric techniques. The diluent is ethanol. For REM and MET, the peak amplitudes were at 233.0 nm & 252.2 nm, respectively. The assay was found to be in the range of 98.16–101.37% for REM and 98.10–101.76% for MET. REM and MET showed good linearity in the series of 1–20 µg/mL and 2.5–35 µg/mL, respectively, with correlation coefficient of 0.998. The %RSD for the analyte in Method precision and intermediate precision is below 1.0. The recovery rate was shown to be between 98.4 and 101.5%. The new spectroscopic techniques scored 94 on the eco scale, showing their greenness [129].

Megahed et al., had developed a simple, rapid, robust, selective, green and sensitive spectrofluorimetric method for determination of favipiravir. The stock solution of FAV was prepared in distilled water then transfer FAV stock solution followed by 2.5 mL of 0.2 M borate buffer pH 8.0 to make a range of 40–280 ng/mL. The solutions were measured at 432 nm after excitation at 361 nm. The% recovery was found to be 100%. The %RSD for the analyte in Method precision and intermediate precision is below 1.0. The eco scale was found to be 92 [130].

Abdelazim et al., had developed an eco-friendly spectrophotometric method for the determination of Molnupiravir. A standard stock solution of molnupiravir was prepared in ethanol then 0.5 mL of hydrochloric acid [0.5 M] and 0.5 mL sodium nitrite was added for diazotization of molnupiravir. After that 1 mL of 8-hydroxyquinoline [0.35%] was added. Then 0.5 mL sodium hydroxide [2 M] was added and the prepared solutions were allowed to stand for 5 min. Finally, the volume was made with ethanol. The absorbance of the red-coloured azo dye complexes was measured at 515 nm against a blank. The method found linear from 1.0 to 12.0 µg/mL with R2 ≥ 0.9996. The %recovery was 99.82%. The %RSD for the analyte in Method precision and intermediate precision is below 1.0. The AGREE software was employed to evaluate the technique's greenness, and the resultant score of 0.81 [131] (Table 7).

Table 7 Summary of Green Spectroscopy techniques


The insights provided in this review article on green analytical techniques incorporating examples of GAC using HPLC, HPTLC, UHPLC, and TLC lay the groundwork for several future advancements and applications. Firstly, it serves as a comprehensive reference for researchers, analysts, and industry professionals seeking to adopt more sustainable, novel, cost-effective, efficient, and ecologically practices in their analytical workflows. By highlighting the benefits and feasibility of green analytical techniques, this review facilitates the widespread adoption of environmentally friendly methodologies across various sectors, including pharmaceuticals, food analysis, environmental monitoring, and beyond.

Liquid chromatographic equipment is extensively used in pharmaceutical quality control globally, often consuming large quantities of organic solvents and leading to unsustainable waste generation. This has driven considerable interest in greening liquid chromatographic techniques, aiming to protect both analysts and the environment. Solvent use is a significant contributor to waste and poses toxicity concerns, which can be addressed by integrating green analytical chemistry principles into method development. Various tools for assessing the environmental impact of liquid chromatography methods play a crucial role in developing environmentally friendly methods with minimal adverse effects. It's advisable to incorporate suitable green evaluation tools into method design to ensure better environmental consideration. Common aspects considered across assessment tools include evaluating a method's environmental impact in terms of solvents, energy consumption, and waste generation. Incorporating green solvents like Sodium dodecyl sulphate and propylene carbonate into methods can help address environmental concerns. Additionally, adopting miniaturised techniques like UHPLC may reduce energy consumption and waste generation during analysis. This shift towards greener practices in pharmaceutical analysis is essential for minimising adverse impacts while maintaining analytical performance standards.

Additionally, this review fosters ongoing innovation in the field of analytical chemistry by identifying areas for further research and development. It encourages the exploration of new green solvents, alternative separation techniques, and novel instrumentation to enhance the eco-efficiency and analytical performance of green analytical methods. Moreover, the examples provided offer inspiration for the design and implementation of customized green analytical protocols tailored to specific analytical challenges and applications.

Furthermore, this review contributes to the global sustainability agenda by promoting awareness and advocacy for greener practices within the scientific community and beyond. By emphasizing the societal and environmental benefits of adopting green analytical techniques, it encourages policymakers, regulatory agencies, and industry stakeholders to prioritise sustainability in chemical analysis and decision-making processes.

In essence, the future usefulness of this review lies in its ability to catalyse positive change, inspire innovation, and promote collaboration towards a more sustainable and environmentally conscious approach to analytical chemistry.

This review includes developing methodologies, unique technology, and creative approaches that have not garnered much attention. Green extraction technologies, solid-phase microextraction, and supercritical fluid extraction are all revolutionary procedures that utilise fewer organic solvents and generate less waste. The review also examines the use of sophisticated technologies such as microfluidics, nanotechnology, and spectroscopic approaches to create more efficient and environmentally friendly analytical procedures.

Our evaluation provides a unique viewpoint or focus on certain parts of green analytical methodologies. We have prioritised conversations about the application of green chemistry concepts, the use of greener solvents, and the assessment of environmental effect using diverse methodologies. We have included the most recent advances in green analytical approaches, including data from recent research publications.

Availability of data and material

The data that support the findings of this study are available from the corresponding author, upon reasonable request.



Green analytical chemistry


High-performance liquid chromatography


Ultraperformance liquid chromatography




High-performance thin layer chromatography


Thin layer chromatography


Gas chromatography


Liquid chromatography


Mass spectroscopy







CHCl3 :



Mobile phase

Rf :

Retardation factor


Analytical quality by design


Retention time


Photodiode array


Reverse phase


Stationary phase


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We are very grateful to the Faculty of Pharmacy, The Maharaja Sayajirao University of Baroda. This review article is based on used green analytical techniques such as, HPLC with green solvents, UPLC, HTLC, HPTLC, and TLC, to develop a novel, cost-effective, efficient, and ecologically sustainable approach. Also, vast research is going on to creating novel Green Analytical Chemistry approaches to address these environmental problems and create an ecologically preferable replacement.


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Mehta, M., Mehta, D. & Mashru, R. Recent application of green analytical chemistry: eco-friendly approaches for pharmaceutical analysis. Futur J Pharm Sci 10, 83 (2024).

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