1 Introduction

Paclitaxel (PTX) is a widely used chemotherapeutic agent for treating various cancers, including breast, ovarian, and non-small cell lung cancer [1]. It functions by stabilizing microtubules, disrupting the cell cycle, and inducing apoptosis, making it a key component of cancer therapy [2]. Additionally, PTX has demonstrated effectiveness in overcoming drug resistance in certain cancer types, further enhancing its clinical relevance [3]. High-performance liquid chromatography (HPLC) is a standard technique for PTX quantification in pharmaceutical formulations and biological samples due to its accuracy and reliability [4]. However, conventional HPLC methods involve the extensive use of organic solvents and reagents, leading to significant environmental concerns. Despite the widespread adoption of HPLC for PTX analysis, limited studies have assessed its environmental impact, highlighting the need for more sustainable analytical approaches.

White Analytical Chemistry (WAC) offers an advanced alternative to Green Analytical Chemistry (GAC) by integrating sustainability with analytical efficiency. Unlike GAC, which primarily focuses on minimizing environmental impact through solvent reduction and waste management, WAC ensures that method performance, precision, and robustness remain uncompromised. Recent studies have demonstrated WAC’s practical application in analytical method development. For example, a stability-indicating HPTLC method incorporating WAC-driven modifications improved both analytical performance and environmental sustainability [5]. Similarly, GAC principles have been successfully applied in chromatographic optimization to enhance efficiency while reducing solvent waste.

For PTX quantification, WAC facilitates the creation of optimized analytical methods that maintain high sensitivity while minimizing hazardous solvent use and energy consumption [6]. By incorporating systematic approaches such as design of experiments (DoE) and analytical quality by design (AQbD), WAC enhances reproducibility and efficiency. Additionally, assessment models like the RGB model play a crucial role in quantifying the balance between sustainability and analytical performance, ensuring that greener modifications do not compromise method effectiveness [7]. Despite its benefits, WAC requires extensive validation and optimization, which may pose challenges for routine pharmaceutical applications. However, its ability to integrate sustainability with high analytical performance makes it a promising approach for enhancing environmentally responsible pharmaceutical analysis [8, 9].

As eco-friendliness becomes more important, this study uses seven well-known tools to compare the greenness of different HPLC-based methods for PTX analysis. The tools are NEMI, Complex NEMI, Analytical Eco-Scale, SPMS, ChlorTox, RGBfast, and BAGI. By using these evaluation models, this study hopes to give a complete plan for improving analytical methods in a way that is responsible to the environment without lowering the quality of the analysis.

2 Greenness tools

2.1 National Environmental Methods Index (NEMI)

The National Environmental Methods Index (NEMI), created in 2002 by the Multiagency Methods and Data Comparability Board (MDCB), serves as a key resource for evaluating the environmental sustainability of analytical methods. This tool assesses methods based on four primary factors: the presence of persistent, bio-accumulative, and toxic (PBT) substances, the use of hazardous chemicals, the inclusion of corrosive materials, and the amount of waste generated [10]. For a method to align with sustainability goals, it must exclude PBT substances, avoid hazardous and corrosive chemicals (2 ≤ pH ≤ 12), and minimize waste up to 50 g. These criteria are visually represented using a circular diagram divided into four quadrants, where green indicates compliance and blank sections highlight areas for improvement [11]. Each quadrant addresses a specific criterion: the first excludes PBT substances as listed in the EPA’s Toxic Release Inventory (TRI); the second targets hazardous waste based on the EPA's Resource Conservation and Recovery Act (RCRA) categories (e.g., F, K, P, U Lists); the third ensures chemicals fall within a safe pH range; and the fourth emphasizes waste reduction to under 50 g [12, 13]. Table 1 summarizes these evaluation criteria.

Table 1 Evaluation criteria for the National Environmental Methods Index (NEMI)
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While NEMI is effective for promoting sustainable practices in analytical chemistry, it primarily provides a qualitative evaluation and does not address all the 12 GAC principles, such as energy use in instrumentation or sampling techniques. NEMI remains an effective and widely used tool for promoting sustainable practices in analytical science, based on criteria [14] presented in Table 1.

2.2 Modified NEMI/Analytical Greenness Profile (AGP)

The Modified National Environmental Method Index (Modified NEMI), also referred to as the Analytical Greenness Profile (AGP) or the Raynie and Driver tool, was introduced in 2009 to advance the traditional NEMI by offering a semi-quantitative framework for assessing the environmental impact of analytical methods. Unlike the original NEMI, which uses a circular pictogram divided into four quadrants to evaluate Persistent Bio accumulative and Toxic (PBT) chemicals, hazardous waste, pH range, and waste generation [20]. The Modified NEMI employs a pentagram design. This pentagram assesses five critical parameters: health hazards, safety hazards, environmental hazards, energy consumption, and waste production, providing a more comprehensive and holistic evaluation. Each parameter is scored on a three-point scale (1 to 3) and represented visually using a color-coded system that is often used to indicate levels of risk, with green representing low risk, yellow indicating moderate risk, and red signifying high risk, allowing for a nuanced understanding of environmental impact [21].

A key advancement of the Modified NEMI is the inclusion of energy consumption and the environmental footprint of instrumentation, metrics absent in the traditional model. It is particularly effective for comparing the greenness profiles of various analytical techniques. Health and safety evaluations rely on National Fire Protection Association (NFPA) codes [22] and standards, as detailed on their official site https://www.newenv.com/resources/nfpa-chemicals/. Ensuring specificity and alignment with established safety protocols. Table 2 summarizes the criteria for the Modified NEMI tool, illustrating the evaluation of health, safety, environmental hazards, energy use, and waste production with relevant examples and their color-coded risk levels.

Table 2 Modified NEMI evaluation criteria [23]
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While transitioning from qualitative to semi-quantitative analysis, the tool utilizes NFPA-based scoring to provide detailed insights into health and safety risks. However, despite its expanded capabilities, the Modified NEMI does not detail the sources of negative environmental impacts. Its broader scope and increased complexity represent a slight departure from the simplicity of GAC principles, reflecting a trade-off between depth and ease of use.

2.3 Analytical Eco-Scale

The Analytical Eco-Scale (AES), introduced in 2012, serves as a semi-quantitative tool that evaluates the environmental impact of analytical methods by integrating both descriptive and numerical data. This approach enables researchers to reasonably estimate the greenness profile of a method [24]. The AES scoring begins with a base of 100, representing an ideal environmentally friendly method, from which penalty points are deducted for deviations from best practices. For example, Chemicals are categorized according to their hazard levels using the Globally Harmonized System (GHS) https://pubchem.ncbi.nlm.nih.gov/. With more hazardous chemicals resulting in higher penalty points. Reagents are penalized according to their volume and hazard level, while additional deductions account for energy consumption and the effectiveness of waste management strategies (e.g., recycling, degradation, or passivation) [25, 26]. Methods scoring ≥ 75 are classified as eco-friendly, those between 50 and 74 as moderately eco-friendly, and scores below 50 are non-eco-friendly. Table 3 summarizes the criteria and penalty points used in the AES evaluation framework. Table 3 provides detailed insights into the evaluation parameters; this comprehensive system ensures a balanced assessment of the environmental impact, providing a practical tool for optimizing analytical methods in alignment with green chemistry principles [27, 28].

Table 3 Analytical Eco-Scale criteria and penalty points
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2.4 Sample preparation metric of sustainability (SPMS)

The sample preparation metric of sustainability (SPMS), introduced by Raul Gonzalez-Martín in 2023, provides a framework for evaluating the sustainability of sample preparation techniques (accessible via https://doi.org/https://doi.org/10.1016/j.chroma.2023.46429 [29]). This metric is visually represented as a clock-like diagram, with a central greenness score surrounded by four key parameters: sample information, extractant information, procedural details, and energy consumption and waste. By assessing these factors, SPMS calculates a sustainability score, enabling the identification and adoption of greener, more efficient preparation methods.

As outlined in Table 4, the SPMS evaluation assigns weighted scores based on criteria such as sample quantity, type and amount of extractant used, procedural complexity, and waste management. This tool is particularly valuable for pinpointing areas for improvement in sample preparation practices. In this study, SPMS was instrumental in identifying methods with lower extractant use and minimal energy requirements, providing actionable insights for enhancing environmental sustainability [30].

Table 4 SPMS criteria for sample preparation sustainability
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2.5 ChlorTox

The Chloroform-Oriented Toxicity Assessment Scale (ChlorTox) was introduced by Nowak et al. in 2023 is an innovative tool developed to evaluate the chemical risk associated with analytical procedures (available at: https://ars.els-cdn.com/content/image/1-s2.0-S2772577423000174-mmc1.xlsx). This tool aligns with GAC principles by promoting the adoption of safer and more environmentally sustainable practices. ChlorTox systematically calculates the risk of a chemical by comparing its hazard potential to chloroform, which serves as the reference standard [31]. The ChlorTox value is calculated using the formula:

$${\text{ChlorTox }} = \left( {\frac{{{\text{CH}}_{sub} }}{{{\text{CH}}_{{{\text{CHCl}}3}} }}} \right) \times {\text{m sub}}$$

here CHsub is the hazard of the substance under assessment, CHCHCl3 is the hazard of chloroform, and m sub is the mass of the substance required for a single analysis. The summation of ChlorTox values for all chemicals in a method provides the Total ChlorTox score, offering an overall measure of the chemical risk associated with the procedure [32].

ChlorTox incorporates two hazard assessment models:

The weighted hazards number (WHN) is a tool designed to evaluate chemical hazards using information from safety data sheets (SDS) that follow the globally harmonized system (GHS). Hazards are divided into four categories, with Category 1 being the most hazardous and Category 4 the least. Each category is assigned a weight: 1.0 for Category 1, 0.75 for Category 2, 0.5 for Category 3, and 0.25 for Category 4. The WHN is calculated using the formula [33]:

$${\text{CH}}sub = (1 \times N_{Cat1 } ) + \, (0.{75} \times N_{Cat2 } ) + \, (0.{5} \times N_{Cat3 } ) + \, (0.{25} \times N_{Cat4} )$$

Here Ncat represents the number of hazards in each category.

For example, chloroform has a WHN value of 5.83. Chemicals with WHN values below 5.83 are less hazardous, while those with higher values pose greater risks. To ensure accurate comparisons, the latest SDS data should be used, considering the physical state (solid or liquid) of the substance.

The CHEMS-1 This model assigns scores to solvents based on their toxicity, ranging from 0 (non-toxic) to 5 (highly hazardous). Chloroform, for example, \(CHsub\) has a value of 103.8. The CHEMS-1 model evaluates factors such as biodegradability (\(HV_{BOD}\)) hydrolysis (\(HV_{HYD}\)) and bioconcentration (\(HV_{BCF}\)), alongside toxicity parameters such as oral toxicity (\(HV_{ORAL}\)), inhalation toxicity (\(HV_{INH}\)), carcinogenicity (\(HV_{CAR}\)), and other harmful effects (\(HV_{HE}\)). The formula to calculate the total hazard value (tHV) in the CHEMS-1 model is:

$${\text{t}}HV = \, [HV_{BOD} + HV_{HYD} + HV_{BCF} ] \times [HV_{ORAL} + HV_{INH} + HV_{CAR} + HV_{FA} + HV_{FC} ]$$

This calculation provides an aggregate score that reflects both the exposure risks and the toxicological hazards of a given solvent, facilitating informed decision-making in selecting safer alternatives.

2.6 RGBfast

The RGBfast, developed by Nowak et al. in 2024, is a comprehensive tool designed to evaluate the quality and sustainability of analytical techniques across four key dimensions: Greenness, Redness, Blueness, and Whiteness. Despite the growing focus on sustainability in analytical chemistry, many existing evaluation models fail to provide a multidimensional assessment that balances environmental, operational, and methodological quality. The RGBfast tool addresses this gap by offering a holistic approach that integrates these aspects into a single framework. Implemented via an Excel-based template (accessible at https://ars.els-cdn.com/content/image/1-s2.0-S2772577424000296-mmc1.xlsx), it allows users to evaluate and compare up to ten analytical methods simultaneously [34].

The"Greenness"dimension focuses on environmental sustainability, assessing factors like reagent toxicity, consumption, and energy use, while"Redness"evaluates analytical performance through validation parameters such as trueness, precision, and sensitivity. The"Blueness"dimension addresses operational efficiency, including cost and time effectiveness, ease of instrument handling, and the number of analysis stages. Finally,"Whiteness"integrates all three dimensions, providing an overall quality score that reflects both sustainability and methodological robustness [35].

To use the tool, users input data, such as trueness, precision, ChlorTox scores, sample throughput, and energy consumption, into the Excel template. Scores ranging from 0 (worst) to 100 (best) are assigned to each parameter, enabling a quantitative evaluation. Specific scoring formulas are applied to calculate weighted scores for each dimension, such as [36]:

$${\text{Redness }} = \sqrt {Score _{{R_{1} }} \times Score _{{R_{2} }} \times Score _{{R_{3} }} }$$

where R1 = trueness, R2 = precision, and R3 = LOD

$${\text{Greenness }} = \sqrt[3]{{Score _{{G_{1} 2}} \times Score _{{G_{\frac{2}{B2}} }} }}$$

where G1 = ChlorTox score and G2/B2 = Energy consumption.:

$${\text{Blueness }} = \sqrt[3]{{Score _{{B_{1} 2}} \times Score _{{G_{\frac{2}{B2}} }} }}$$

where B1 = Sample throughput and G2/B2 = Energy consumption.

Whiteness is an integrated score that combines all attributes, calculated by:

$${\text{Whiteness }} = \sqrt[9]{{Score _{{R_{1} }} \times Score _{{R_{2} }} \times Score _{{R_{3} }} \times Score _{{G_{1} 2}} \times Score _{{B_{1} 2}} \times Score _{{G_{\frac{2}{B2}} }} }}$$

2.7 BAGI

The BAGI tool, introduced by Manousi et al. in 2023, provides a comprehensive framework for assessing the applicability and effectiveness of analytical methods based on the ten principles of White Analytical Chemistry (WAC). It is accessible through the official website (mostwiedzy.pl/bagi) and features an online application at https://bagi-index.anvil.app for user convenience [37]. This tool employs a scoring system ranging from 25 to 100, where higher scores indicate greater applicability and effectiveness. The results are visually represented using a color-coded system: dark blue (10 points) signifies high compliance, blue (7.5 points) indicates medium compliance, light blue (5 points) represents low compliance, and white (2.5 points) denotes no compliance with the evaluated criteria [38]. The overall score is prominently displayed in the center of an asteroid-shaped pictogram. A score of 100 reflects excellent applicability, while 25 represents the lowest performance. A score above 60 is generally recommended for optimal results. This tool enables users to evaluate both the strengths and limitations of a method while facilitating the comparison of different methods.The ten principles assessed by BAGI are outlined in Table 5 [39].

Table 5 Criteria for BAGI tool along with scores
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2.8 Pharmacopoeial Standards for Greenness Assessment

Pharmacopoeial standards promote sustainable analytical methods by encouraging the use of eco-friendly solvents, minimizing hazardous reagents, and implementing waste reduction strategies [40]. The USP General Chapter < 467 > sets residual solvent limits based on toxicity and environmental impact, guiding laboratories toward safer alternatives [41]. Similarly, the European Pharmacopoeia (Ph. Eur.) General Chapter 5.4 recommends reducing the use of Class 1 and Class 2 solvents due to their health and environmental hazards [42]. Additionally, the ICH Q3 C (R6) guideline defines acceptable residual solvent levels to encourage the use of less toxic options in pharmaceutical analysis [43].

Efforts to minimize hazardous reagents align with regulatory recommendations such as the FDA Guidance for Industry on Residual Solvents, which classifies solvents based on toxicity and permitted daily exposure, ensuring compliance with USP General Chapter < 467 > [44]. Similarly, the European Medicines Agency (EMA) guideline establishes solvent limits to ensure patient safety while promoting sustainability in pharmaceutical processes [45].

To enhance sustainability, waste reduction and energy efficiency are key considerations in pharmacopoeial guidelines. The USP and Ph. Eur. advocate for the use of automated and miniaturized techniques, such as micro-extraction and capillary electrophoresis, to minimize solvent use and waste generation [46].

By incorporating these elements, pharmacopoeial standards create a structured framework for greener analytical methods, ensuring compliance with environmental regulations while maintaining accuracy and efficiency in pharmaceutical analysis.

3 Results and discussion

The assessment of various reported methods was conducted using seven greenness tools, following guidelines and criteria of each tool. The generated pictograms and detailed evaluations are summarized in Table 6, highlights the greenness of each method across several parameters using selected tools and details of scoring is given in Supplementary file 1.

Table 6 Assessment of greenness of HPLC methods for Paclitaxel using seven tools
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3.1 NEMI analysis

According to the NEMI tool, Method 9 was classified as non-compliant, as all four quadrant parameters—persistent, bioaccumulative, and toxic (PBT), hazardous, corrosive, and waste generation—were left blank, indicating a failure to meet green criteria. In contrast, Methods 1, 2, 3, 4, and 5 aligned with NEMI principles and were classified as green. Method 7 was found to be partially green, with only the PBT quadrant marked green, while the remaining three quadrants were left blank. This indicates the absence of PBT-listed substances based on EPA-TRI data, but the method falls short in other environmental and safety aspects.

3.2 Modified NEMI assessment

The modified NEMI tool identified Methods 3, 4, and 6 as relatively greener due to favorable ratings across five critical parameters: safety hazards, health hazards, environmental impact, waste generation, and energy consumption. Specifically, Method 3 was classified as green due to its limited use of hazardous materials (<50 g), moderate toxicity and flammability (NFPA 2–3), moderate energy consumption, and waste generation between 50 and 250 g (rated yellow). Method 4 was classified as green in two quadrants—hazardous material usage (<50 g) and waste generation (<50 g)—while the remaining quadrants were rated yellow, indicating moderate greenness. Similarly, Method 6 was rated green for PBT substances as no PBT-listed chemicals were involved; however, the other quadrants were yellow, reflecting moderate greenness.

In comparison, Methods 1, 7, and 8 were considered less green. Method 1 involved a PBT-listed chemical, resulting in a red rating. Methods 7 and 8 were rated red in the safety quadrant due to high flammability (NFPA 4), with moderate environmental and waste impacts (yellow rating).

3.3 Analytical Eco-Scale assessment

A notable contrast in environmental impact was observed between Methods 3 and 6, as reflected in their Analytical Eco-Scale (AES) scores of 82 and 85, respectively. Method 3 demonstrated a more sustainable profile due to its efficient reagent usage, minimal energy consumption, and effective waste management, making it an environmentally preferable approach. In contrast, although Method 6 achieved a relatively high score, it still incorporated hazardous solvents, excessive waste generation, and inefficient disposal practices, contributing to a higher penalty in its assessment.

Among all methods, Methods 5, 7, and 8 attained the highest AES scores (90, 90, and 88, respectively), indicating superior environmental performance. Their high scores can be attributed to optimized reagent consumption, reduced waste production, and effective solvent recovery or waste treatment strategies. These results highlight how the strategic selection of solvents, controlled reagent usage, and energy-efficient instrumentation significantly enhance sustainability in analytical methodologies.

On the other hand, Method 2 recorded the lowest AES score (34), indicating a considerable environmental burden due to excessive solvent consumption, high waste output, and inadequate waste treatment strategies. This finding suggests that Method 2 could benefit from modifications such as adopting greener solvent alternatives, minimizing solvent usage, and implementing recycling systems to enhance its environmental sustainability.

3.4 SPMS tool evaluation

The SPMS (Sustainable Process Metrics System) tool evaluated the sustainability of all selected methods and ranked Method 2 as the greenest, with a score of 9.47. This method satisfied all SPMS criteria, including sample quantity, extractant use, energy consumption, waste generation, and reusability, all rated green. In contrast, Method 8 scored 4.42, indicating lower sustainability. The extractant usage in Method 8 was categorized in the orange range (0.5 < x ≤ 1), emphasizing moderate greenness, while the use of conventionally persistent extractants received a red rating. Additionally, waste generation in Method 8 (50 < x ≤ 100) further contributed to its lower overall score.

3.5 ChlorTox analysis

The ChlorTox evaluation highlights significant differences in the chemical hazards of various analytical methods, focusing on Green Analytical Chemistry (GAC) principles. Method 1 stands out with the lowest hazard profile, having a WHN score of 0 and a CHEMS-1 score of 0.0000237109, making it the safest and most environmentally friendly choice. In contrast, Method 5 had much higher scores (WHN: 0.001620926, CHEMS-1: 6.994), indicating greater risks and reduced alignment with GAC. Method 6 showed moderate hazards (WHN: 0.0017753, CHEMS-1: 0.0000915222), placing it between Methods 1 and 5. This analysis emphasizes the importance of selecting low-hazard methods, such as Method 1, to ensure safer and greener laboratory practices.

3.6 RGBfast analysis

The RGBfast analysis assessed several analytical methods for drug analysis, focusing on factors such as efficiency, simplicity, and cost-effectiveness. Method 1 performed the best, with a whiteness score of 67, a greenness score of 63.9, and a redness score of 61.9, making it the most favorable choice. In contrast, Method 9 scored much lower, with a whiteness score of 42 and RGB values of 39.5 for redness, 46.0 for greenness, and 39.7 for blueness. These results indicate that Method 9 has lower whiteness, greenness, and overall environmental friendliness compared to Method 1. This highlights the importance of selecting methods that are both effective and environmentally sustainable for drug analysis.

3.7 BAGI Assessment

The BAGI analysis revealed that Method 3 outperformed Method 2, with a score of 72.5 compared to 52.5, highlighting its superior environmental sustainability and efficiency. Method 3’s ability to process large sample volumes with minimal resource use aligns with the principles of White Analytical Chemistry (WAC), making it a more sustainable choice. In contrast, Method 2, with a lower score, indicates higher resource consumption and lower efficiency, suggesting a greater environmental impact. This comparison underscores the importance of selecting analytical methods that optimize both performance and environmental responsibility.

3.8 Applications of white analytical chemistry in analytical method development

Recent studies have demonstrated successful integration of WAC principles in analytical method development. A stability-indicating HPTLC method along with WAC principles were utilized to optimiz solvent composition while reducing hazardous reagent use, maintaining high sensitivity and selectivity [56, 57]. Similarly, WAC-driven chromatographic optimizations using design of experiments (DoE) and analytical quality by design (AQbD) have led to enhanced efficiency and reduced chemical waste [58]. These advancements align with proposed study of evaluation of greenness of HPLC methods for paclitaxel, demonstrating how WAC-based modifications can improve sustainability without compromising analytical robustness.

In summary, the comprehensive evaluation using multiple greenness tools highlights methods 1, 2, and 3 as the most favorable in terms of greenness, sustainability, and performance across various parameters.

4 Conclusion

The evaluation of the reported methods based on GAC and WAC principles highlights their overall greenness, sustainability, and performance. Among the analyzed methods, Methods 1, 2, 3, and 5 emerged as the most favorable, demonstrating strong compliance with GAC principles. Method 2 achieved the highest sustainability, scoring 9.47 in the SPMS tool due to its minimal energy consumption, low extractant usage, and reduced waste generation during sample preparation. Method 3 demonstrated outstanding performance across various assessment tools, including Modified NEMI and BAGI, earning high ratings for its safety, efficiency, and minimal environmental impact. Similarly, Method 5, with an Analytical Eco-Scale (AES) score of 90, exhibited excellent environmental sustainability, reflecting its optimized reagent use and effective waste management. Additionally, Method 1 excelled in the RGBfast analysis, achieving the highest visual greenness and aligning closely with the principles of the NEMI framework, further reinforcing its environmentally friendly profile.

In contrast, Methods 7, 8, and 9 exhibited notable limitations, including hazardous material usage, flammability, and lower sustainability, as reflected in their lower scores across SPMS, ChlorTox, and RGBfast tools. The integration of GAC and WAC tools provided a robust and systematic framework for assessing the environmental impact and performance of these methods.

The incorporation of WAC principles in method development enhances not only sustainability but also analytical performance and cost-effectiveness. Recent studies illustrate the potential of WAC in optimizing chromatographic methods, reducing hazardous solvent usage, and maintaining high analytical precision. These findings reinforce WAC's role in advancing sustainable pharmaceutical analysis. By prioritizing environmentally friendly, resource-efficient analytical approaches that adhere to GAC principles, analytical scientists can contribute significantly to sustainable development goals (SDGs) while ensuring high-quality and reliable analytical outcomes.