Promoting Green Chemistry in Analysis: An Assessment of Analytical Methodologies and Their Environmental Implication

In the field of pharmaceutical sciences, it is very important to accurately estimate drug concentrations in order to make sure that pharmaceutical products are safe, effective, and of high quality. There are many different analytical methods for estimating drugs, and each one is best for the specific needs and legal requirements of the drug. These methods are very important for finding pollutants, degradation products, and excipients, and they also help with the quantitative analysis of active pharmaceutical ingredients (APIs). The development of reliable and verified analytical methods is very important at many points in the drug development process, including formulation, manufacturing, and quality control. These methods are the basis for stability testing, bioavailability assessments, and pharmacokinetic and pharmacodynamic studies⁽¹⁾.

Efavirenz (efv) and hiv : a therapeutic and analytical perspective

HIV leads to “Human Immunodeficiency Virus”. It is  usually  a  sexually  transmitted  infection  that usually occurs during intimation, blood transfusion, transfer of semen and vaginal fluids. HIV  is  the  virus  that  is  responsible  for  AIDS  in human.  The  immune  system of  the  human  body is designed in such a way that it can protect from various bacterial as well as viral infections. White blood cells of the immune system contains CD4+ cells  which  are  also  known  as  helper  T  cells. During  HIV  infection  the  count  of  CD4+  cells decreases.  The  diagnosis as well as testing  of  HIV  is  very  crucial  as  the diagnostic  strategies  need  to  be  continuously revised  according  to  any  new  discoveries  on replication  as  well  as  pathogenic  mechanism  of the  infection. There  are  mainly  two  types  of  HIV virus grouped so far, HIV-1 and HIV-2 are the two main types of HIV, and HIV is the main cause of AIDS.HIV is a type of retrovirus. HIV-1 and HIV-2 have the same basic structure, but their genomes are set up in different ways. This virus usually doesn't affect the central nervous system. HIV-2 is more likely to cause diseases of the central nervous system, but both viruses can cause AIDS. HIV-2 is not as harmful as HIV-1 and takes longer to turn into AIDS. The first-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) for treating HIV type 1 infection or stopping the virus's transmission is efavirenz^(2,3).

1.1.MECHANISM OF ACTION:

As a non-nucleoside reverse transcriptase inhibitor (NNRTI), efavirenz binds to the HIV reverse transcription enzyme's non-catalytic site, preventing its action and causing DNA chain termination. HIV replication and other DNA polymerase activities are inhibited by this effect. Efavirenz does not require therapeutic drug concentration monitoring, however achieving appropriate serum concentrations is crucial. While larger concentrations are linked to more negative consequences such sleep disturbances, lower concentrations are linked to virologic failure. Efavirenz can be taken in conjunction with other antiviral drugs. It has been demonstrated that efavirenz treatment with food raises serum concentrations and the frequency of side effects. Patients shouldn't take efavirenz with food because of this.After a single oral dosage, peak concentrations are reached in five hours, and steady-state plasma concentrations are reached in six to seven days. Efavirenz has a half-life of around 45 hours, thus once-daily dose is appropriate. Efavirenz is well linked to human plasma proteins, primarily albumin. The CYP3A4 enzyme transforms efavirenz into inactive hydroxylated metabolites⁽⁴⁾.

1.2.PHARMACOKINETICS:

Absorption: Peak concentrations of efavirenz are reached five hours after oral treatment, and the drug is well absorbed. It takes six to ten days to reach steady-state plasma concentrations.

Distribution: Efavirenz has a distribution volume of about 252 L.

Metabolism: Efavirenz is mostly metabolized into hydroxylated metabolites with subsequent glucuronidation by the hepatic CYP450 enzyme system, particularly CYP2AB. The medication is a major substrate of CYP3A4.

Elimination: Efavirenz is excreted in urine (14% to 34%) and faeces (16% to 61%), with less than 1% remaining unaltered. The half-life of the medication ranges from 40 to 55 hours.

Chemical and pharmacokinetic profile:

Efavirenz, (S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one,1 is an inhibitor of reverse transcriptase that is not nucleoside-based One.

2.OVERVIEW OF REPORTED ANALYTICAL METHODS FOR EFAVIRENZ

2.1UV - spectroscopy methods

To estimate Efavirenz in bulk and tablet dose form, a straightforward, accurate, and exact UV-spectrophotometric approach has been devised and validated. Methanol and water (30:70) exhibit the highest absorption at 320 nm. A(1%1cm) and comparison with the standard were used for estimation. Over the concentration range of 1-4 μg/ml, the calibration graph was determined to be linear (r2 = 0.09657). When tested for factors including accuracy, precision, and limit of detection for routine determination of Efavirenz in bulk as well as in tablet form, the suggested procedures seem straightforward, sensitive, and repeatable. The techniques can be used for routine analysis.

UV spectrophotometry represents the simplest and most economical approach for efavirenz quantification. These methods typically require dissolution in organic solvents or surfactant systems due to poor water solubility. While environmentally favorable in terms of instrumentation and energy use, their sensitivity and selectivity are limited, particularly for biological matrices⁽⁵⁾.

2.2.RPHPLC methods

RP?HPLC remains the most widely reported technique for efavirenz analysis in bulk drug, dosage forms, and dissolution samples. Conventional mobile phases commonly include mixtures of acetonitrile or methanol with aqueous buffers. Although these methods provide excellent accuracy, precision, and robustness, they contribute significantly to solvent consumption, toxicity, and laboratory waste generation .

Acetonitrile (ACN) was utilized as the organic phase to create the mobile phase in the RP-HPLC technique development for Efavirenz measurement, while phosphate buffer (10 mM, pH 6.8) was utilized as the aqueous phase. At a 50:50 ratio of ACN to phosphate buffer (10 mM, pH 6.8) and a 1 ml/min flow rate, efavirenz took 20 minutes to elute. After that, efavirenz was separated and evaluated using a mobile phase that included various ratios of ACN:phosphate buffer (10 mM, pH 6.8), including 60:40, 70:30, and 80:20⁽⁶⁾.

2.3.LC-MS methods

Liquid chromatography

Two LC-20AD delivery pumps, a DGU-20A5 Shimadzu vacuum degasser, a SIL-20AC Shimadzu auto sampler, and a CBM-20A system controller made up the Shimadzu HPLC system (Shimadzu Scientific Instruments; Columbia, MD, USA). A Waters Xbridge C18 analytical column (50 mm x 2.1 mm, 3.5 µm 120 Å) was used for HPLC separations. period of mobility Water and 0.1% formic acid made up mobile phase A, while acetonitrile and 0.1% formic acid made up mobile phase B. The gradient looked like this: Mobile phase: 0.0–1.5 minutes 98% to 2%, with the divert valve closed; Mobile phase A 2% for 1.51–3.00 minutes, mobile phase B 98%, then mobile phase A 98% for 3.1–5 minutes. For every analysis, 10 µL of the material was injected at a flow rate of 0.3 mL/min. The autosampler and column were kept at 15°C and 40°C, respectively. For the first minute, while no data capture was occurring, an electronic valve actuator with a Rheodyne selector valve was employed to redirect LC flow to waste^(7,8).

Mass spectrometry analysis

 An AB Sciex 4000 triple quadrupole mass spectrometer with Turbo Ion spray was used to evaluate the samples. This equipment was controlled and data was collected and processed using Analyst version 1.4.2 software (MDS Sciex; Toronto, Canada). For MS/MS analysis, the negative ion mode was chosen since it provided the greatest response. The nebulizer, auxiliary, collision, and curtain gases were all nitrogen. Tandem mass spectrometry was used to detect analytes utilizing multiple reaction monitoring (MRM) with a dwell period of 250 milliseconds. A solution of 1 µg/mL efavirenz or internal standard in mobile phase was directly fed into the ion sources using a Harvard Apparatus syringe pump at a flow rate of 10 µL/min in order to determine the precursor and product of ion spectra. Using negative turbo spray, the highest intensive precursor-to-fragment transitions were 314.20→243.90 for efavirenz and 320.20→249.90 for 13C6-efavirenz (internal standard).

In order to replicate the LC-MS/MS conditions, the conditions for ionization of efavirenz and internal standard were adjusted using individual standard solutions, each at 100 ng/mL, infused by a syringe pump through a Tee device at a flow rate of 10 µL/min into a stream of mobile phase eluting from the LC column through a mixing Tee and subsequently into the turbo spray source. The mass spectrometer's primary operating settings were: source temperature 550°C; collision activated dissociation (CAD) gas, medium; curtain gas, 35; Gas 1 (nebulizer gas), 50; and Gas 2 (heater gas), 30. For Efavirenz, the optimum declustering potential (DP), entry potential (EP), collision energy (CE), and collision cell exit potential (CXP) were set at -55.40, -7.0, -25.3, -4.64 , For 13C6-efavirenz (internal standard), the corresponding values are −53.24, −10, −25.11, and −4.39^(9,10,11).

2.4.HPTLC and Alternative Methods

HPTLC and emerging spectroscopic or electrochemical techniques offer reduced solvent usage and lower operational cost. Although less commonly applied to efavirenz, such alternatives demonstrate potential as greener analytical platforms when appropriately validated(12).

Three Green Analytical Chemistry Fundamentals
The concepts of green chemistry are extended to analytical processes in green analytical chemistry.

Key goals include of:
Minimizing sample size and waste production, reducing or getting rid of dangerous solvents and reagents, and using energy-efficient equipment and processes Utilizing biodegradable or renewable materials improves operator safety and protects the environment. Sustainable pharmaceutical quality control and compliance with international environmental requirements depend on the use of these principles.

3.Green Assessment Tools for Analytical Methods

3.1.GAC

Green chemistry (GC), which is based on the 12 principles put forward by Anastas and Warner, has become more important worldwide for encouraging sustainable practices in labs and businesses. Green analytical chemistry (GAC), which aims to lessen the effects of analytical operations on the environment, human health, and safety, developed from this basis. To make analytical processes safer and more sustainable, GAC places a strong focus on the use of less hazardous or solvent-free extraction methods, downsized sample processing, and eco-friendly detection tools. Maintaining excellent analytical performance and accuracy while protecting the environment is a significant problem for GAC. The 12 principles of GAC, which offer organized guidelines for greener analytical approaches, were created by revising the original GC principles by adding eight new concepts and choosing four important ones. In order to assess and enhance the sustainability of sample preparation methods, ten green sample preparation (GSP) principles were also established. Assessing and reducing the environmental effect of analytical techniques is crucial since not all of them can fully adhere to GAC standards. Due to the shortcomings of traditional evaluation techniques, a number of greenness assessment measures were created. NEMI, Analytical Eco-Scale, GAPI, AGREE, RGB models, BAGI, HEXAGON, and other tools are often used; some are specifically made for certain tests, while others are generally applicable. In addition to addressing contemporary requirements and future prospects in the field of green analytical chemistry, this paper examines 15 prominent GAC metrics and discusses their concepts, benefits, drawbacks, and real-world applications^(13,14,15).

3.2NEMI

One of the first measures in green analytical chemistry (GAC) is the National Environmental Methods Index (NEMI), which was created in 2002 by the Methodologies and Data Comparability Board. It employs a straightforward circular pictogram with four portions, each of which represents a distinct environmental criterion, and serves as a searchable database. A section is colored green if the analytical method meets the requirement: chemicals must not be listed as persistent, bioaccumulative, and toxic (PBT); Sample pH must be between 2 and 12 to prevent corrosive effects; waste generation cannot be more than 50 g; and solvents cannot be on toxic waste lists (D, F, P, and U). NEMI is appreciated for its ease of use and capacity to deliver rapid visual data on the environmental effect of an analytical technique. Nevertheless, it might be time-consuming to operate and only provides generic and qualitative information. It was used to assess three analytical techniques: a liquid-liquid extraction UPLC-MS/MS approach, an HPLC-UV assay for spiked milk samples, and a UV spectroscopic method without chromatographic separation. Among these, the third method showed better greenness due to higher throughput and shorter run time. To overcome NEMI’s limitations, advanced NEMI was introduced, incorporating a green–yellow–red colour scale to provide semi-quantitative evaluation. Further improvement led to the development of the Analytical Green Profile (AGP), which assesses safety, health, energy, waste, and environmental impact using NFPA-based scoring and colour coding, offering a more comprehensive evaluation of analytical procedures^(16,17,18).

3.3.ECO-SCALE

A popular green analytical chemistry (GAC) measure that assesses the greenness of analytical techniques quantitatively is the Analytical Eco-Scale, which was first proposed in 2012. Penalty points are deducted based on solvent and reagent dangers, amounts utilized, energy consumption, occupational risks, and waste creation. The highest score for a perfect green analysis is 100. Although relatively few techniques completely satisfy these requirements, a "ideal green analysis" calls for non-hazardous chemicals, energy usage of less than 0.1 kWh per sample, and zero waste creation. Penalty points are calculated considering the type and amount of chemicals, following the Globally Harmonized System (GHS), which uses hazard pictograms and signal words such as “danger” and “warning.” The number of pictograms and the signal word determine hazard severity, and these values are multiplied by quantity-based penalty factors. Additional penalties are assigned for higher energy use, vapour release, waste amount, and waste treatment methods. The number of points for penalties are subtracted from 100 to determine the ultimate Eco-Scale score, with scores above 75 indicating excellent greenness, 50–75 acceptable greenness, and below 50 inadequate performance. The Analytical Eco-Scale is valued for its simplicity, quantitative capability, and ease of comparison between methods. However, it does not provide detailed insight into the specific sources of environmental impact. To address this limitation, the Green Certificate Modified Eco-Scale was later developed, categorizing Eco-Scale values into seven colour-coded levels for clearer interpretation and improved evaluation of analytical methods^(19,20).

3.4.GAPI

A semi-quantitative statistic called GAPI (Green Analytical Procedure Index) was developed by P?otka-Wasylka to assess how environmentally friendly each step of an analytical process is. It employs a three-color scale—green for low impact, yellow for medium impact, and red for high impact—to show the consequences on the environment and human health. The pictogram is made up of five pentagrams, each of which represents a separate stage of the analytical process. The first pentagram assesses sampling aspects such as collection method, preservation, transportation, and storage conditions. The second focuses on the type of analytical method and the need for sample processing, with an internal circle indicating whether the method supports both qualitative and quantitative analysis. The third pentagram evaluates sample preparation , such as extraction scale (nano, micro, or macro), solvent type (solvent-free, green, or non-green), and extra preparation procedures. Based on NFPA ratings, the fourth looks at solvent amount and reagent health and safety risks. The fifth evaluates waste creation, waste treatment techniques, energy usage, and occupational dangers. GAPI integrates qualitative and semi-quantitative evaluations and offers a thorough visual summary of an analytical method's environmental effect. It is easy to use and useful for comparing approaches, but its classification ranges might be imprecise because methods with very different amounts of waste could get the same grade. All things considered, GAPI is a useful but imperfect method for assessing analytical greenness.

3.5.AGREE

 AGREE is a comprehensive and contemporary green analytical chemistry (GAC) metric that was developed by Pena-Pereira et al. to assess the greenness of analytical techniques in an easy-to-understand manner. It is based on the 12 principles of GAC, which are converted into a 0–1 scoring scale, and the final greenness score is calculated from the combined assessment of these principles. With 12 colored segments grouped around a center circle, each of which represents a GAC principle, the AGREE pictogram graphically depicts the evaluation. Each segment's weight is reflected in its breadth, and its hue varies from red (the least green) to dark green (the most green). Both visual and quantitative data are provided by the pictogram's center, which shows the overall color and number score. AGREE is valued for its thoroughness, adaptability, and user-friendliness, which enable transparent comparison of analytical techniques. Nevertheless, it does not thoroughly assess sample preparation and does not take into account chemicals, solvents, energy consumption, or trash produced during pre-extraction activities. The 10 principles of green sample preparation (GSP) served as the foundation for the development of AGREE prep in order to close this gap. Using a 0–1 scale and color-coded pictogram, AGREE prep assesses factors such solvent safety, material sustainability, waste reduction, automation, decreased sample volume, energy efficiency, and operator safety. While AGREE prep offers a comprehensive evaluation of sample preparation, it ignores sample storage, transportation, and financial considerations.

3.6RGB

Red, green, and blue are the three major colors used in the RGB model, commonly referred to as "white" analytical chemistry, which is a complete green analytical chemistry (GAC) metric. In this paradigm, output and operational efficiency are represented by blue, safety and sustainability by green, and analytical performance by red. The overall compliance of an analytical method with these criteria is expressed quantitatively through a Color Score (CS), ranging from 0% to 100%. Based on the CS value, methods are classified as satisfactory (66.6–100%), tolerable (33.3–66.6%), or unsatisfactory (0–33.3%). The combined color outcome visually represents the balance of performance, safety, and efficiency. The RGB evaluation is typically performed using Excel and considers method safety, reproducibility, and analytical quality. Although it provides a holistic assessment, the evaluation process can be complex and time-consuming.

3.7.Analytical Method Greenness Score or AMGS
 

AMGS is a newly created GAC measure that uses a single numerical score to represent an analytical method's greenness, with lower values denoting better environmental performance. Solvent use, waste production, energy consumption of both instruments and solvents, and safety, health, and environmental (SHE) aspects of solvents are all taken into account when calculating the score. It incorporates parameters such as solvent mass used in sample preparation and mobile phase, cumulative energy demand (CED) for solvent production and disposal, and instrument energy usage. The final AMGS value is derived from a weighted combination of solvent consumption, SHE factors, and energy demand. Unlike pictogram-based tools, AMGS provides only a numerical result without color visualization. While it offers a concise and focused assessment, the calculation procedure is relatively detailed and time-intensive                                           

3.8.BAGI

 BAGI is a newly created green analytical chemistry (GAC) measure that evaluates the practical usability and greenness of analytical techniques based on white analytical chemistry concepts. It presents evaluation findings in an easy-to-understand manner by combining a pictogram, color scale, and numerical score. . The BAGI score ranges from 25 to 100, with higher scores indicating better environmental performance and practicality. This metric evaluates analytical methods using 10 specific criteria, each associated with defined score values and color hues, allowing rapid and straightforward assessment. The greenness score can be calculated through its dedicated online platform, making it user-friendly and accessible. Although BAGI is simple, efficient, and useful as a complementary tool alongside metrics such as AGREE prep and Complex GAPI, it has certain limitations. Better environmental performance and practicality are indicated by higher BAGI scores, which range from 25 to 100. This measure allows for quick and simple evaluation by evaluating analytical techniques using ten distinct criteria, each of which is linked to predetermined score values and color colors. Its specialized web platform makes it easy to compute the greenness score, making it accessible and user-friendly. BAGI has certain drawbacks while being straightforward, effective, and helpful when used in conjunction with metrics like Complex GAPI and AGREE prep^.(21,22)

4.EMERGING GREEN STRATEGIES IN EFAVIRENZ ANALYSIS:

4.1.HYDROTROPIC SOLUBLIZATION

One of the most frequent challenges in the screening of novel chemical compounds and the creation of therapeutic formulations is the solubilization of medications with limited solubility.1,2 There are several medications in the US and Indian pharmacopoeias that are either insoluble or weakly soluble in water. Because poor solubility can lead to poor bioavailability, this poses a significant issue for medication development. One of the most crucial physicochemical characteristics for drug development is solubility.3 A lot of recently created medications are poorly soluble. These medications are frequently dissolved using organic solvents such methanol, chloroform, dimethyl formamide, and acetonitrile, however these solvents have disadvantages like high cost, volatility, pollution, and toxicity. A safe, economical, and environmentally beneficial substitute for solubilizing poorly soluble medications is provided by hydrotropic agents.

Neuberg described hydrotropy for the first time in 1916. It describes the phenomena where another solute becomes more soluble in water when a substantial quantity of another substance is added. Concentrated aqueous solutions of sodium benzoate, sodium salicylate, urea, nicotinamide, sodium citrate, and sodium acetate are a few examples of hydrotropic agents.6 An anionic group and a hydrotropic aromatic ring or ring system are the two fundamental components of hydrotropic agents, which are often anionic organic salts. The anionic group is responsible for lity, which could have a little impact on the phenomena.7The increase in a substance's solubility in water caused by a high concentration of additives has been referred to as hydrotropy. Many weakly water-soluble medications have been shown to become more soluble in water when concentrated aqueous hydrotropic solution of sodium benzoate, urea, nicotinamide, sodium salicylate, sodium ascorbate, and sodium glycinate are used. In the current study, the weakly water-soluble antiretroviral medication efavirenz was dissolved using the hydrotropic solubilization approach⁽²³⁾.

4.2.MINIATURIZATION AND MICROEXTRACTION

A paradigm shift toward sustainable pharmaceutical analysis is represented by the development of deep eutectic solvents (DESs) and ionic liquids (ILs) as effective green solvents for microextraction, providing attractive substitutes for traditional organic solvents for efavirenz determination. These task-specific solvents are especially appealing for sample preparation in antiretroviral medication monitoring because of their lower volatility, adjustable physicochemical characteristics, and lower toxicity when compared to conventional solvents. Hydrogen bonding between a hydrogen bond donor (HBD) and acceptor (HBA), such as choline chloride mixed with urea or carboxylic acids, creates DESs, which are low-melting mixtures with enhanced solubilization power that are perfect for extracting moderately lipophilic compounds like efavirenz (log P ≈ 4.5). On the other hand, ILs are salts made up of anions and organic cations (pyridinium, ammonium, or imidazolium) whose structural diversity allows for the optimization of pharmacological microextraction. Both solvent types are appropriate substitutes for hazardous organic solvents in efavirenz analysis due to their high solvation capabilities, low vapor pressure, and great thermal stability.

There are important differences between DESs and ILs that affect their green credentials, even if they share traits like tunability, broad liquid ranges, and dual organic/inorganic dissolving capability. While ILs are made up of discrete anion-cation pairs, DESs are formed by HBA-HBD interactions, and DES synthesis is more scalable and economical. DESs are often favored from a biodegradability and sustainability standpoint since they are thought to be less dangerous and more ecologically friendly than ILs. A useful framework for choosing the best solvent systems for specific pharmaceutical applications has been provided by comprehensive classification systems that have divided DESs into oxygen-containing solvents (alcohol-, polyalcohol-, organic acid-based), nitrogen-containing variants (urea-based, amine-based, ammonium salts-based), and phosphorus- or sulfur-containing systems.

By reducing solvent consumption, resolving matrix interferences, and accelerating processing times, the incorporation of DESs and ILs into microextraction techniques—specifically, solid-phase microextraction (SPME) and liquid-phase microextraction (LPME)—has transformed pharmaceutical trace analysis. These methods are very effective for drug analysis in complicated matrices including biological fluids and wastewater, which is directly related to environmental fate studies and efavirenz therapeutic medication monitoring. In addition to improving sustainability, the use of DESs and ILs increases extraction selectivity and sensitivity through adjustable polarity, allowing for the best possible recovery of both hydrophilic and hydrophobic medications while reducing matrix interference. DES-based microextraction techniques have the ability to provide clean extracts appropriate for chromatographic analysis and high enrichment factors for efavirenz in particular, which is consistent with green chemistry principles while preserving the analytical rigor needed for regulatory compliance. The development and validation of DES-based microextraction procedures for efavirenz in biological and environmental matrices, as well as the methodical assessment of extraction efficiency, greenness metrics, and comparability with recognized reference techniques, should be the main goals of future research⁽²⁴⁾.

5.CHALLENGES IN DEVELOPING GREEN EVAFIRENZ METHODS

Key barriers include:

5.1Poor intrinsic aqueous solubility of efavirenz

The development of sustainable methodologies for Efavirenz (EFV) is fundamentally obstructed by its intrinsic hydrophobicity and negligible aqueous solubility. As a BCS Class II antiretroviral, EFV’s lipophilic structure necessitates the use of traditional organic solvents, which conflicts with the core tenets of Green Chemistry that prioritize aqueous or bio-based media. This 'solubility barrier' complicates both sample preparation in analytical workflows and the transition toward solvent-free synthetic routes. Recent efforts to overcome this have shifted toward the use of supramolecular hosts like cyclodextrins and the formulation of nanomicelles, which can enhance apparent solubility by several orders of magnitude. However, a significant challenge remains in balancing the 'greenness' of these additives against the energy-intensive mechanical processes, such as nanomilling or hot-melt extrusion, required to disrupt the crystalline lattice of the drug. Consequently, a truly green method for EFV must not only replace toxic solvents but also address the total environmental footprint of the solubilizing agents and the energy requirements of the enabling technologies."

5.2.Sensitivity limitations of greener techniques

Green analytical chemistry (GAC) has gained considerable attention due to its emphasis on reducing hazardous solvent consumption, minimizing waste generation, and improving operator safety. The application of aqueous media, bio-based solvents, and hydrotropic agents such as urea and sodium benzoate has been widely explored as environmentally benign alternatives to traditional organic solvents in pharmaceutical analysis. Although these greener methodologies significantly enhance the environmental profile of analytical procedures, certain sensitivity-related limitations have been reported. One of the primary challenges includes comparatively higher limits of detection (LOD) and quantification (LOQ) when water or hydrotropic systems replace strong organic solvents like methanol or acetonitrile. Method sensitivity may be impacted by lower molar absorptivity and poorer absorbance responses resulting from reduced the dissolution efficiency for poorly soluble in water medications. . Additionally, some hydrotropic agents may contribute to baseline interference in the UV region, reducing signal-to-noise ratio at low concentration levels. In comparison with advanced hyphenated techniques such as HPLC or LC–MS, green UV-spectrophotometric methods are generally less suitable for trace-level impurity detection. Nevertheless, for routine quality control applications—where extreme sensitivity is not mandatory—the environmental sustainability, safety advantages, and cost-effectiveness of green analytical techniques outweigh their moderate sensitivity limitations. Thus, current research seeks to uphold the principles of green analytical chemistry while striking a balance between ecological effect and analytical performance⁽²⁵⁾.

5.3.Regulatory approval for new techniques or solvents

In pharmaceutical quality control, the regulatory adoption of innovative green solvents and ecologically friendly analytical techniques is still crucial. To guarantee accuracy, precision, specificity, linearity, robustness, and reproducibility, regulatory bodies like the European Medicines Agency (EMA), the United States Food and Drug Administration (US FDA), and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) require thorough method validation. When conventional organic solvents like methanol or acetonitrile are replaced with greener alternatives such as aqueous systems, ethanol, or hydrotropic agents, regulatory bodies require strong scientific justification and comparative validation data to demonstrate equivalent or improved analytical performance. Challenges may arise due to limited historical data, potential variations in impurity profiling, or concerns regarding long-term stability and method ruggedness. Therefore, successful regulatory approval of green analytical methods depends on rigorous validation studies, risk-based assessment, and clear documentation aligned with established pharmaceutical guidelines, ensuring that environmental sustainability does not compromise product quality or patient safety⁽²⁶⁾.

5.4.Balancing analytical robustness with environmental sustainability

Balancing Analytical Robustness with Environmental Sustainability

One of the main obstacles to the development of Green Analytical Chemistry (GAC) is striking a balance between environmental sustainability and analytical robustness. According to the validation principles described by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH Q2 guidelines), analytical robustness necessitates a technique that consistently yields accurate, precise, and repeatable results under variable experimental conditions. On the other hand, in line with the green chemistry concepts put forward by Paul Anastas and John C. Warner, environmental sustainability places a strong emphasis on reducing harmful solvents, energy use, and chemical waste. The replacement of traditional organic solvents such as methanol or acetonitrile with greener alternatives (e.g., water, ethanol, or hydrotropic systems) can influence solubility, selectivity, sensitivity, and analyte stability, potentially affecting robustness. Therefore, method optimization must incorporate solvent selection strategies based on toxicity profiles (ICH Q3C classification), lifecycle assessment, and performance equivalence studies. Emerging core concepts such as analytical miniaturization, solvent-free extraction techniques, chemometric-assisted optimization, and energy-efficient instrumentation further support the integration of sustainability without compromising data quality. Furthermore, quantitative measurement of environmental effect together with validation criteria is made possible by structured greenness assessment methods such as Analytical Eco-Scale, GAPI (Green Analytical Procedure Index), and AGREE metrics. Achieving an optimal balance thus requires interdisciplinary integration of analytical chemistry, environmental toxicology, pharmaceutical regulation, and process engineering to ensure that sustainability improvements do not undermine analytical reliability or regulatory compliance.

Addressing Challenges through Interdisciplinary Innovation and Systematic Greenness Evaluation

The transition toward sustainable analytical methodologies requires not only solvent replacement but also comprehensive interdisciplinary innovation integrating analytical chemistry, pharmaceutical sciences, environmental toxicology, materials science, and regulatory science. In order to improve analytical performance and lessen environmental impact, Green Analytical Chemistry (GAC) encourages technique redesign through downsizing, automation, in-line monitoring, solvent-free sample preparation, chemometric optimization, and energy-efficient instrumentation. However, sustainability claims must be supported by systematic and transparent greenness evaluation frameworks. Reagent toxicity, energy consumption, waste production, occupational risks, and technique throughput may all be evaluated semi-quantitatively and quantitatively using tools like the Analytical Eco-Scale, Green Analytical Procedure Index (GAPI), and AGREE (Analytical GREEnness) meter. While guaranteeing adherence to the validation guidelines established by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH), these evaluation platforms provide an impartial comparison between traditional and environmentally friendly approaches. Additionally, quality-by-design (QbD) principles, risk-based validation, solvent selection guidelines, and lifecycle thinking improve the dependability and regulatory acceptability of environmentally friendly processes. By integrating methodological innovation with standardized greenness metrics and regulatory alignment, the analytical community can overcome technical and compliance-related barriers, ensuring that sustainability improvements are scientifically justified, reproducible, and compatible with pharmaceutical quality requirements.

6.Future Perspectives

Future research directions may include:

6.1.Deep eutectic and bio-based solvents for chromatography

The exploration of deep eutectic solvents (DES) and bio?based solvents represents a pivotal advancement toward fully sustainable analytical methodologies for efavirenz. In line with the fundamental ideas of Green and White Analytical Chemistry, these substitutes present a convincing alternative to chromatographic analysis's long-standing dependence on dangerous organic solvents. Future research must prioritize the systematic evaluation of DES formulations—particularly natural deep eutectic solvents—as mobile phase modifiers to reduce acetonitrile and methanol consumption while maintaining separation efficiency and peak symmetry. Concurrently, bio?based solvents such as ethanol, glycerol, and ethyl lactate present viable, readily implementable alternatives, with established precedents for antiviral drug separations demonstrating their chromatographic competence. However, widespread adoption hinges on addressing critical challenges, including high viscosity, aqueous stability, UV transparency limitations, and ensuring batch?to?batch reproducibility to meet regulatory standards. Advancing these green solvent systems through rigorous validation and greenness metrics will not only minimize the environmental footprint of efavirenz analysis but also establish a transferable framework for sustainable pharmaceutical quality control.

6.2.Fully aqueous LC-MS compatible systems

The development of fully aqueous LC-MS compatible systems represents a transformative frontier in greening efavirenz analysis, aiming to eliminate organic solvents entirely from mobile phases. Superheated water chromatography (also termed high-temperature liquid chromatography) has demonstrated feasibility for pharmaceutical analysis by using elevated temperatures to reduce water's polarity and enable separation of moderately hydrophobic compounds without organic modifiers. For LC-MS applications, the challenge lies in maintaining robust electrospray ionization efficiency and chromatographic resolution while operating under purely aqueous conditions. Recent advances in column chemistry, particularly the development of thermally stable stationary phases such as zirconia-based and ethylene-bridged hybrid (BEH) materials, have expanded the operational window for aqueous mobile phases . However, successful implementation of efavirenz requires systematic optimization of temperature, pH, and buffer systems to achieve adequate retention of this moderately lipophilic compound (log P approximately 4.5) while ensuring MS-compatible volatility. Future research should explore hybrid approaches combining subcritical water with low percentages of bio-based solvents as a transitional strategy, alongside the investigation of novel stationary phases designed specifically for aqueous separations. The ultimate realization of organic solvent-free LC-MS methods would represent the pinnacle of green analytical chemistry for efavirenz, eliminating solvent toxicity, reducing operational costs, and aligning with the pharmaceutical industry's net-zero commitments.

6.3.AI-guided optimisation of green analytical conditions

The integration of artificial intelligence (AI) for optimizing green analytical conditions represents a paradigm-shifting opportunity in efavirenz method development, transitioning from traditional trial-and-error approaches to data-driven, sustainable workflows. Machine learning algorithms and in silico modelling platforms can rapidly predict retention behaviour, optimal mobile phase composition, and separation outcomes from limited experimental inputs, dramatically reducing solvent consumption and analyst time during method development. Recent advances demonstrate that AI can simultaneously optimize for both chromatographic performance and environmental impact, enabling researchers to map the entire separation landscape and identify conditions that minimize the Analytical Method Greenness Score (AMGS) while maintaining resolution and efficiency. For efavirenz specifically, artificial neural networks have already been successfully applied to model degradation behaviour under various conditions, establishing a precedent for AI-guided optimization in antiretroviral drug analysis. Furthermore, AI-powered tools are emerging for rapid evaluation of method greenness using established metrics such as AGREE, Complex GAPI, and the RGB12 whiteness model, automating sustainability assessment and enabling real-time comparison of alternative approaches. However, critical challenges must be addressed, including the current limitations of black-box models lacking interpretability for regulatory acceptance, the demonstrated need for human refinement of AI-generated methods to achieve practical greenness goals, and the requirement for diverse, high-quality training datasets specific to pharmaceutical separations. Future research should focus on developing hybrid frameworks that combine mechanistic modelling with machine learning, creating explainable AI systems that can justify their optimization decisions to meet regulatory expectations in GxP environments. The ultimate realization of AI-guided optimization for efavirenz analysis promises not only accelerated method development and reduced environmental footprint but also the establishment of intelligent, adaptive analytical platforms capable of continuously improving sustainability without compromising data quality or regulatory compliance.

6.4.Real-time, in-line monitoring technologies

The integration of real-time, in-line monitoring technologies through Process Analytical Technology (PAT) represents a transformative frontier for greening efavirenz analysis, shifting the paradigm from discrete laboratory sampling to continuous, non-destructive process oversight. Spectroscopic tools such as near-infrared (NIR), Raman, and mid-infrared (MIR) probes can be deployed directly in manufacturing lines to monitor critical quality attributes—including content uniformity and impurity profiles—without solvent consumption or sample preparation. This approach not only eliminates the environmental burden of conventional off-line testing but also enables real-time release testing (RTRT), reducing energy use, inventory costs, and analytical waste. Recent PAT applications in pharmaceutical manufacturing demonstrate that in-line NIR monitoring can achieve near-complete solvent recovery, significantly lowering CO? emissions and operational costs. For efavirenz specifically, future research should focus on developing and validating chemometric models that correlate real-time spectroscopic data with reference methods, enabling predictive process control while maintaining ICH-compliant accuracy. The convergence of PAT with artificial intelligence for data interpretation further promises autonomous optimization of manufacturing conditions, minimizing environmental impact while ensuring product quality. Ultimately, embedding real-time monitoring into efavirenz production workflows offers the dual advantage of enhanced process efficiency and substantial reduction in the analytical carbon footprint, aligning pharmaceutical quality control with circular economy principles.

6.5.Integration of sustainability metrics into regulatory guidelines

The integration of sustainability metrics into regulatory guidelines represents a transformative step toward institutionalizing green chemistry in pharmaceutical analysis, shifting from voluntary initiatives to mandated compliance. Recent landmark developments, including the world's first global standard for pharmaceutical environmental impact assessment (PAS 2090:2025), establish harmonized frameworks for measuring and reporting the lifecycle environmental footprint of pharmaceutical products. This standard, developed through multi-stakeholder consensus involving regulators, industry, and academia, provides a consistent methodology for evaluating analytical methods alongside manufacturing and supply chain impacts. Concurrently, guidance from organizations such as the ISPE emphasizes embedding sustainability into quality management systems and regulatory processes, recognizing that environmental considerations must parallel traditional quality attributes. For efavirenz analysis, the future lies in aligning method development with these emerging regulatory expectations, utilizing established green metrics—AGREE, GAPI, ComplexGAPI, and the RGB whiteness model—as standard validation parameters rather than optional adjuncts. The demonstrated application of comprehensive sustainability assessments for antiretroviral drugs, including atazanavir and ritonavir, establishes a precedent for efavirenz. However, realizing this vision requires harmonization of diverse metric systems, regulatory acceptance of novel green alternatives, and integration of sustainability performance into pharmacopoeial monographs and inspection frameworks. Ultimately, embedding sustainability metrics into regulatory guidelines will transform environmental responsibility from an academic ideal into an enforceable standard, ensuring that future efavirenz methods are judged not only by accuracy and precision but by their ecological footprint and contribution to a sustainable healthcare future.

Such advancements could transform efavirenz analysis into an environmentally responsible analytical model.

The convergence of deep eutectic and bio?based solvents, fully aqueous LC?MS systems, AI?guided optimization, and integrated sustainability metrics collectively positions efavirenz analysis as a potential vanguard for environmentally responsible pharmaceutical quality control. Such advancements could transform efavirenz analysis into an environmentally responsible analytical model, demonstrating that antiretroviral drug monitoring can achieve exceptional analytical performance while approaching zero environmental impact. This paradigm shift aligns with the broader pharmaceutical industry's commitment to net?zero emissions and sustainable supply chains, as exemplified by recent global standards for environmental footprint assessment. The methodologies developed for efavirenz—ranging from NADES?based mobile phases to machine learning?optimized green conditions—establish transferable frameworks applicable to other antiretroviral agents and pharmaceutical compounds, amplifying their sustainability impact. Furthermore, the integration of white analytical chemistry principles ensures that greenness does not compromise analytical quality, safety, or practicality, creating a holistic benchmark for method development. As regulatory bodies increasingly mandate sustainability considerations through instruments like PAS 2090:2025, efavirenz analysis can serve as a compelling case study demonstrating that rigorous pharmaceutical analysis and environmental stewardship are not mutually exclusive but synergistic objectives. Ultimately, establishing efavirenz as a green analytical model would catalyze broader adoption across pharmaceutical quality control, inspiring innovation, informing regulatory evolution, and contributing meaningfully to healthcare sustainability without compromising patient safety or therapeutic efficacy.

CONCLUSION

Existing analytical methods for efavirenz provide high sensitivity and reliability but often fall short in environmental sustainability. Incorporation of hydrotropic solubilization, green solvents, miniaturized techniques, and solvent-free detection strategies represents a crucial pathway toward greener pharmaceutical analysis. Continued research, validation, and regulatory acceptance of eco-friendly analytical methodologies will be essential to achieving sustainable quality control of efavirenz and other poorly soluble drugs.

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