Environmental impact of RP-HPLC strategy for detection of selected antibiotics residues in wastewater: Evaluating of quality tools

1 Introduction

Public health risks are associated with antibiotic residues in wastewater, as well as environmental effects. Dispersal of these residues can produce antibiotic-resistant bacteria in water systems, causing antibiotic-resistant bacteria proliferation in humans [1,2]. Investigation has indicated that seasonal variations exist in the amount of antibiotic residues found in environmental samples. For example, elevated concentrations of specific antibiotics, like enrofloxacin, have been observed in meat samples throughout the spring. Sewage from agricultural sources may contain residues of cephradine (CEP), oxytetracycline (OXY), and tetracycline (TET) in varying quantities due to seasonal changes [3]. For public health and environmental reasons, detecting OXY, TET, and CEP residues in poultry slaughterhouse wastewater is crucial. Analyzing residues using advanced detection methods is possible through chromatography, capillary electrophoresis, immunological techniques, and biosensors. In veterinary and human medicine, CEP’s broad spectrum activity against bacteria makes it one of the most widely prescribed first-generation cephalosporin antibiotics. As a result of its frequent use, it is present in wastewater systems. Due to its use to treat bacterial infections, it is often detected in hospital effluents and municipal wastewater. TET and OXY are two antibiotics in the TET class extensively used in human and animal medicine. Since they are widely used in agriculture and aquaculture, they are common environmental contaminants [4]. Agricultural runoff and wastewater from livestock farming contain these compounds and are used to promote growth and treat animals. Antibiotics like these are listed on the Watch List of the European Union’s Water Framework Directive because they may pose a safety risk. To effectively assess treatment effectiveness and mitigate environmental impact, it is essential to detect and quantify them in wastewater [5,6,7,8]. To mitigate risks related to antibiotic resistance, it is necessary to monitor antibiotic contamination continuously and implement effective waste management practices. Livestock use antibiotics to prevent sickness and encourage growth. Nevertheless, excessive use leaves residues in animal feces, whose wastewater can then be released into the environment. Public health may be at risk due to this contamination’s potential to foster antibiotic-resistant bacteria [9].

During the last two decades, sustainable chemistry has gained significant attention. Chemical reactions can be made more environmentally friendly and safer by using renewable resources. As chemistry moves towards green and sustainable practices, developing sustainable chemical processes and managing them effectively is imperative [10]. An innovative approach to chemical research and engineering emphasizes green and white chemistry principles. The method’s integrated approach ensures unmatched protection against dangerous substances when it comes to chemical processes and products [11]. Using green and white chemistry, scientists and engineers can design environmentally friendly, long-lasting, and sustainable products and processes [12]. It is essential to evaluate HPLC procedures ecologically in order to determine whether or not they are eco-friendly [13]. Several tools were used to assist this investigation, including Blue Applicability Grade Index (BAGI) and RGB. Several other environmental sustainability analyses have also been created, including the complementary green analytical procedure index (ComplexGAPI), analytical GREEnness (AGREE), analytical method greenness score (AMGS), green analytical procedure index (GAPI), high-performance liquid chromatography environmental assessment tool (HPLC-EAT), analytical greenness metric for sample preparation (AGREEprep), analytical method volume intensity (AMVI), and the analytical Eco-Scale (ESA) [14].

In the pharmaceutical industry, Lean Six Sigma has become one of the most commonly discussed topics, so manufacturers will implement it to reduce operational costs, reduce waste, predict errors during every stage of the process, eliminate defects, and ensure that production processes will still be capable during the process [15,16].

OXY and TET are commonly used antibiotics in the production of chickens and rabbits (Figure 1a and b). Compared to TET, CEP, a cephalosporin antibiotic, is less frequently examined in environmental samples (Figure 1c). Nonetheless, only a few techniques were available for identifying veterinary antimicrobial medicines in wastewater using LC-MS/MS. One such method involved the analysis of 92 compounds belonging to eight different classes [17]. A single appropriate HPLC-MS/MS technique for identifying sulfachloropyridazine, enrofloxacin, and OXY residues in lettuce plants [18]. An HPLC-DAD procedure has been described for the quantification of OXY, TET, chlorotetracycline, doxycycline, enrofloxacin, ciprofloxacin, sarafloxacin, and flumequine residues in animal drinking water [19]. Furthermore, a study reported the identification of OXY and chloramphenicol in untreated wastewater effluents using RP-HPLC technology [20]. Hassouna and Mohamed developed a highly effective HPLC technique to detect cephalosporin residues on stainless-steel plates and human plasma that had been intentionally contaminated [21].

Figure 1

Chemical structure of (a) OXY, (b) TET, and (c) CEP.

No documented techniques for detecting OXY, TET, and CEP residues in wastewater from chicken slaughterhouses, rabbit farms, and pharmaceutical factories are available. A primary objective of this study is to resolve this inconsistency by developing a cutting-edge, effective, and environmentally friendly HPLC method that utilizes principles of white and green chemistry to rapidly and accurately identify traces of OXY, TET, and CEP in wastewater originating from poultry slaughterhouses, rabbit farms, and pharmaceutical factories. We have employed eight dependable evaluation tools to assess the ecological sustainability of our processes: GAPI, ESA, AMGS, AGREE, HPLC-EAT, AGREEprep, ComplexGAPI, and AMVI. In addition to widely recognized environmentally friendly initiatives, the White analytical chemistry metric BAGI serves as a valuable complement. The results confirm our method’s ecological sustainability. The method’s accuracy was assessed utilizing an exclusive Six Sigma approach. This demonstrated that the proposed HPLC method provides a fast, sensitive, and dependable approach.

2 Materials and methods

3 Results

The study selected HPLC as the method for tracking three antibiotics (OXY, TET, and CEP) in pharmaceutical wastewater, chicken slaughter, and rabbit farms.

3.1 Methods development and optimization

A novel RP-HPLC method was developed to assess the targeted analytes in wastewater from poultry slaughter, rabbit farms, and pharmaceutical factories by utilizing distinct mobile phase compositions. Preliminary experiments were carried out to ascertain the optimal parameters for establishing an efficient methodology for pharmaceutical analysis. Modifications were implemented in the detector wavelength, column temperature, and mobile phase composition to optimize separation and precision. Multiple studies were conducted to change the composition of the mobile phase experimentally. The experiment commenced with a volumetric ratio of water to methanol of 50:50. The concentration of the organic modifier (methanol) was systematically changed between 45 and 65% using neutral and acidic water. The mobile phase was observed to have a significant impact on the separation process. Higher methanol levels led to longer retention durations, while increasing the amount of water eventually caused the three components to combine. The longer duration of drug retention while using larger concentrations of organic modifiers may be attributed to the methanolysis of medicines at elevated methanol levels.

Additionally, because of the ionization of chemicals, basic pH was not effective at resolving components. A high degree of dependability exists between analytes and their ionization states in the presence of a low pH mobile phase. The ionization of many analytes is increased in alkaline (basic) conditions, particularly for those with acidic functional groups. As a result of this ionization, they are less likely to interact with the hydrophobic stationary phase (e.g., C18), leading to poor retention and resolution. In contrast, analytes that have basic functional groups remain protonated under acidic conditions, enhancing their retention on stationary phases. Based on these results, basic pH was ineffective for resolving components because ionization disrupted the interaction of the analytes with the stationary phase and contributed to co-elution.

Analytes are more likely to be retained and separated when they are protonated, which can be achieved by using phosphate buffer solution (modified with formic acid) in a mobile phase. By increasing the concentration of formic acid, stronger interactions with the stationary phase and enhanced protonation increased the retention of analytes. While maintaining a pH within a stable range to ensure stable ionization states, separation efficiency remained unaffected.

A 55% methanol and 45% phosphate buffer solution pH 4.2 produced a good separation in a reasonable amount of time. The retention increased as formic acid’s potency increased, although separation was unaffected. The mobile phase combining 55% methanol with 45% phosphate buffer solution pH 4.2 (modified with formic acid) allowed a good separation to be achieved in a reasonable timeframe. Analytes interacted more efficiently with the stationary phase when formic acid was added, as it promoted protonation of the analytes. The experiment also made use of packaging materials and columns of varying lengths. The columns ranged in length from 50 to 250 mm and contained C8, phenyl, and cyano. It was found that the C18 stationary phase had the best hydrophobic interaction with the analytes, which resulted in better retention and resolution as compared to the other columns investigated. Despite being similar, the C8 column demonstrated weaker retention, while the phenyl and cyano columns showed different selectivity patterns that were less effective. The Agilent Zorbax C18 column (25 × 0.46 cm, 5 μm) was the best choice. The column was heated to a temperature of 20–40°C. An important factor in drug separation is elevated temperatures. Our method optimization included testing buffer solutions at different pH values (e.g., pH 4, 6, and 8) to assess their impact on extraction efficiency. Based on the results, pH 4.2 provided the highest recovery rates and reproducibility. Maintaining the samples at 4°C during analysis ensured the stability of the analytes. Obtaining reproducible retention times and peak shapes requires a consistent column temperature. As a result of our optimization studies, 25°C provided the highest separation efficiency for the target analytes. Chromatographic performance can be improved by higher temperatures for some compounds, but sensitive analytes may be degraded by high temperatures. By keeping the column at 25°C, the antibiotics remained stable during analysis.

Additionally impacted are the crucial processes of drug separation and degradation. Consequently, the suggested procedures determined that the ideal separation temperatures for the sample and column oven were 4°C and 25°C, respectively. We investigated wavelengths in the 200–400 nm range using spectrophotometry. The highest sensitivity and exceptional intensity at 257 nm, along with the biggest absorbances at 254 and 280 nm, led to the selection of OXY, TET, and CEP. For the RP-HPLC process, acidic water and methanol were mixed in a 45:55 (v/v) ratio. A pH of 4.2 was set for the mixture. Seven minutes were spent performing the operation with an injection volume of 10 μL, a flow rate of 1.0 mL·min⁻¹, and UV detection at 257 nm. Figure 2 demonstrates the enhanced efficiency and speed in resolving these problems by adopting the chromatographic condition with the shortest duration of less than six minutes.

Figure 2

HPLC chromatograms of antibiotic residues of (a) ternary mixture, (b) OXY and TET in poultry slaughter wastewater, (c) rabbit farms wastewater, and (d) pharmaceutical factory wastewater.

There are essential factors for choosing acetonitrile, magnesium stearate, and sodium chloride in the drug extraction technique; its excellent solvent properties make acetonitrile useful for drug extraction, especially because it dissolves a wide variety of compounds, including both polar and nonpolar compounds. Furthermore, it can be analyzed using many analytical techniques, including HPLC and MS [39]. An extraction solvent such as magnesium stearate protects the extraction apparatus from sticking to the surface by acting as a lubricant. By doing so, the extraction process becomes more efficient, resulting in higher yields for target compounds [40]. A process known as “salting out” utilizes sodium chloride to enhance extraction efficiency. By increasing ionic strength in the aqueous phase, this technique reduces the drug’s solubility in water and promotes its transfer into organic matter. Several extraction cycles were performed to ensure complete drug extraction, and the drug concentration in the extract was measured by HPLC to ensure high extraction efficiency [41]. Several extractions were conducted without noticeable increases in concentration. Additionally, a known standard was used to check the recovery rate of our extraction method.

3.2 Evaluation of the quality tools

The accuracy findings are expressed as the percentage recovery of the suggested technique using the Cpk method. As shown in Tables 1–3, X-bar and R charts, measured by quality tools, demonstrate that all data points fall within the control limits, as indicated by the Cpk report. According to the latest plot of the last 20 subgroups, the data are distributed randomly and uniformly around the processing center. Histograms, average probabilities, and capability plots indicate that the procedure is roughly centered around the goal. The Cpk values for OXY, TET, and CEP in the proposed approach exceed 1.33. Nevertheless, the suggested approach demonstrates higher accuracy, as indicated by the Cpk values of 1.36 and 1.64 for OXY and TET (Figure 3a and b), in contrast to the Cpk values of 1.76 for CEP (Figure 3c), respectively. In summary, these results indicate that the suggested approach can greatly improve the exactness and correctness of future studies.

Table 1

Descriptive statistics of accuracy outcomes of the demonstrated HPLC process for OXY

Variable	N	N*	Mean	SE mean	StDev	Variance	CoefVar	Sum	Sum of squares
OXY	100	0	99.580	0.112	1.118	1.249	1.12	9,958.000	991,741.280

Variable	Minimum	Q1	Median	Q3	Maximum	Range	IQR	Mode	N for mode	Skewness
OXY	96.800	98.800	99.600	100.500	101.800	5.000	1.700	100	12	−0.19

Variable	Kurtosis
OXY	−0.62

Table 2

Descriptive statistics of accuracy outcomes of the demonstrated HPLC process for TET

Variable	N	N*	Mean	SE mean	StDev	Variance	CoefVar	Sum	Sum of squares
TET	100	0	99.291%	0.0915	0.915	0.837	0.92	9,929.100	985,953.130

Variable	Minimum	Q1	Median	Q3	Maximum	Range	IQR	Mode	N for mode	Skewness
TET	97.100%	98.625%	99.250%	100.000%	101.300%	4.200	1.375	99%	15	−0.02

Variable	Kurtosis
TET	−0.50

Table 3

Descriptive statistics of accuracy outcomes of the demonstrated HPLC process for CEP

Variable	N	N*	Mean	SE mean	StDev	Variance	CoefVar	Sum	Sum of squares
CEP	100	0	99.269%	0.0885	0.885	0.782	0.89	9,926.900	985,510.890

Variable	Minimum	Q1	Median	Q3	Maximum	Range	IQR	Mode	N for mode	Skewness
CEP	97.200	98.700%	99.300%	99.975%	101.200%	4.000	1.275	99%	14	0.03

Variable	Kurtosis
CEP	−0.14

Figure 3

Process capability six-pack report of accuracy results for (a) OXY, (b) TET, and (c) CEP.

3.3 Evaluation of the whiteness, blueness, and greenness of the recommended methods

3.3.1 AGREE tool

The 12 components of the Green Appraisal Guidelines form the basis of AGREE, a modern method for evaluating green profiles (Figure 4a). Figure S1a shows the results of applying this method to generate a pictogram representing a good environmental method. In the center, the HPLC method displays an AGREE score of 0.62 and several intensifying green color combinations.

Figure 4

An assessment was conducted to evaluate the sustainability of an HPLC technique using the following graphs: (a) AGREE, (b) AGREEprep, (c) GAPI, (d) ComplexGAPI, (e) BAGI, (f) AMGS, and (g) HPLC-EAT.

3.3.2 AGREEprep tool

AGREEprep is a novel indicator of environmental impact. Efficient and eco-friendly sample preparation procedures simplify the evaluation procedure. To arrive at a score between zero and one, the AGREEprep method employs ten separate procedures. A score of one indicates an ideal performance. Figure 4b shows a pictogram for every sector. The existing methodologies’ ecological sustainability score is 0.55 for the HPLC approach, as shown in Figure S1b.

3.3.3 GAPI tool

A reliable method such as GAPI is required for analyzing the degree of greenness in analytical operations. Figure 4c details this method in fifteen sections, each explaining a different aspect of the approach. Because it takes into account all important parts of the analytical process, GAPI is a helpful tool for comparing different analytical procedures. Figure S2a shows the results, which prove that the procedure is effective while being environmentally benign.

3.3.4 ComplexGAPI tool

The ComplexGAPI measure is one way to evaluate analytical methods that make use of GAC characteristics. To account for pre-analysis activities, the GAPI diagram and the standard GAPI measure are both hexagonally enlarged (Figure 4d). These treatments cover a broad variety of individuals, as shown in Figure S2b.

3.3.5 BAGI tool

The ten standards, as well as the usefulness and efficacy of a technique, can be better understood with the help of BAGI’s illustrations and scores. When using HPLC, dark blue, light blue, and dark blue represent the ultimate HPLC scores. A solution’s color indicates how well it meets the requirements. A satisfactory analytical technique is defined as one with a score of 80, as shown in Figure S2c. In Figure 4e, we can see the BAGI index pictograms.

3.3.6 AMVI

AMVI can accurately determine solvent consumption using liquid chromatography. Supplementing the methods above with liquid chromatography techniques is possible. State-of-the-art laboratory equipment allows for the execution of analytical procedures. According to the HPLC result, AMVI has an environmentally friendly score of 51.7, as shown in Table 4.

Table 4

The AMVI examination of the suggested strategy

Criteria	Suggested HPLC strategy
Solvent consumption HPLC (mL)=	105
Flow rate	1.0
Run time	7
Number of injections for one complete analysis	15
Number of analytes	3
Solvent consumption sample prep (mL)=	50
Volume (mL) for standard preparation	10
Standard preparations numbers	1
Volume (mL) for test preparation	10
Test preparations numbers	3
Volume (mL) for system suitability	10
System suitability numbers	1
Total method solvent consumption=	155
Analytical method volume intensity	51.7
% Consumption HPLC	67.74%
% Consumption preparations	32.26%

The bold values appear to highlight key summary metrics or final results that are particularly important for interpreting the data.

3.3.7 ESA

The ecological pattern is the benchmark of excellence for evaluating sustainable development programs. The ESA metric uses penalty points to appraise the synchronous technique. The method’s strong eco-scores (87 in HPLC) demonstrate its environmental sustainability. The reader can find all the details concerning the calculated penalty points in Table 5.

Table 5

Using an approved method for calculating the analytical eco-scale score incurs penalty points

	Analytical eco-scale	Penalty points
		HPLC method
Reagents	Methanol	6
	Ultra purified water	0
	Potassium dihydrogen phosphate	0
	Acetonitrile	4
	Magnesium sulfate	0
	Sodium chloride	0
	Silica gel	0
Instruments	Energy for HPLC ≤ 1.5 kW h/sample	1
	Occupational hazard	0
	Ultrasonic	1
	Waste	1
	Total penalty points	13
	Eco-scale total score	87

The bold values appear to highlight key summary metrics or final results that are particularly important for interpreting the data.

3.3.8 AMGS

With AMGS, it is simple to compare and evaluate solutions based on their potential environmental impact. Figure 4f shows the steps involved in component analysis, instrumental energy use, solvent waste formation, and waste disposal. Figure S3a demonstrates that AMGS inspections employ a perspective-based approach.

3.3.9 HPLC-EAT

With HPLC-EAT, a reliable and efficient evaluation tool for liquid chromatography processes, we may look at the characteristics in Figure S3b. To ensure that chromatographic solvents are safe and pure, this method provides a useful means of assessing their effects. Using Figure 4g, we demonstrate that it is feasible to assess how each process solvent impacts public health, the environment, and safety.

4 Discussion

A variety of factors can influence whether specific antibiotics are detected in wastewater samples, including source of contamination, since antibiotics enter wastewater from a variety of sources, including hospitals, pharmaceutical industries, and residential areas. Certain antibiotics could be detected in these sources according to their usage patterns. Hospital effluents, for instance, may contain higher concentrations of antibiotics commonly used in hospitals [43]. As antibiotics are unstable and degrade at different rates in the environment, they have varying degrees of degradation and persistence. Depending on the pH, temperature, and microbial activity of the environment, some antibiotics can degrade more rapidly, causing their absence from samples [44]. There is a wide range of effectiveness in the removal of antibiotics from wastewater due to the different wastewater treatment processes. Treatment of wastewater with conventional methods may lead to antibiotic resistance [45]. Water treatment is affected by the chemical properties of antibiotics, such as their solubility and adsorption potential. In contrast, antibiotics with a high adsorption potential can bind to sludge during treatment and remain in the aqueous phase.

Based on our study, the detected antibiotic may have been more prevalent because of its increased use in the source area or its greater resistance to degradation and treatment. However, it is also possible that the absence of antibiotics may have resulted in a faster degrading process or a more efficient removal during treatment.

It has been well explored in the past few decades that antibiotic residues can be detected in environmental samples using various methods, such as LC-MS/MS [17,18] and HPLC-DAD [20,21]. As analytical chemistry methods are evaluated, green metrics such as GAPI and AGREE are increasingly incorporated. There has been no previous study combining ecological impact assessments with RP-HPLC methods to analyze wastewater from poultry slaughters, rabbit farms, and pharmaceutical factories for the detection of OXY, TET, and CEP. However, whereas other methods have detected similar antibiotics in environmental samples, the incorporation of several green metrics and the focus on wastewater from three distinct sources are novel aspects of the manuscript, which adds value to the field by introducing methodological and sustainability innovations.

There are several benefits to the HPLC method, including its cost-effectiveness, accessibility, and high separation efficiency [46]. The LC-MS/MS has the advantage of being easier to operate and maintain as well. As a result, RP-HPLC is less suitable for detecting low antibiotic concentrations than LC-MS/MS due to its lack of sensitivity and specificity. In addition to offering high sensitivity and specificity, LC-MS/MS is capable of simultaneously identifying and quantifying multiple compounds [17,18]. Analytes in low concentrations as well as complex matrices are particularly well suited for this method. The LC-MS/MS method, on the other hand, requires more equipment, requires more expertise, and is more expensive. Using BAGI is an excellent way to select analytical methods that balance performance and environmental responsibility. It is especially valuable for laboratories looking to adhere to white chemistry practices. However, BAGI may have limited application due to data availability on biological activity and environmental impact for certain reagents/processes. As a result of antibiotic residues detected in wastewater from poultry slaughterhouses, rabbit farms, and pharmaceutical factories, improved wastewater treatment technologies are necessary to mitigate contamination of the environment. We developed an RP-HPLC method that provides an environmentally friendly method for detecting antibiotic residues in wastewater. Such a method is more robust than more sophisticated methods, including LC-MS/MS. As opposed to two stages of mass spectrometry in LC-MS/MS, our approach achieves comparable accuracy and sensitivity, so it is accessible without specialized equipment for a wider range of applications. Our findings may assist in developing stricter regulations related to antibiotic discharge limits and encourage the adoption of monitoring programs for antibiotic residues in wastewater. As a result of the presence of antibiotics in agricultural wastewater, livestock farming must be managed better to reduce antibiotic use and keep antibiotic resistance at bay.

The literature review indicates that out of eight environmental metrics, none of the HPLC methods are appropriate for detecting OXY, TET, and CEP residues in wastewater from rabbit farms, pharmaceutical enterprises, and poultry slaughterhouses. To evaluate the ecological sustainability of the proposed approach, the study employed a variety of green criteria such as AGREE, HPLC-EAT, ESA, ComplexGAPI, AMVI, AMGS, and AGREEprep, in addition to the BAGI metric of white chemistry. Our state-of-the-art quality tools and Lean Six-Sigma procedures are the most effective and efficient ways to adhere to sustainable and environmentally friendly chemistry principles. This infographic Figure 5 and Table 8 illustrates traces of OXY, TET, and CEP in wastewater from a pharmaceutical company, a rabbit farm, and a chicken abattoir. The data shown in Table 5 provide details on the number of samples analyzed (n = 20) and the time period during which sampling was performed (February to May 2024). The bar graph displays the residual amounts of three antibiotics in wastewater from three distinct sources. Compared to the rabbit farm, the wastewater from the poultry slaughter industry had greater TET and OXY residue levels; these residues are not present in the pharmaceutical factory. On the other hand, pharmaceutical plants identified small concentrations of CEP, which was absent from rabbit farms and poultry slaughterhouses.

Figure 5

An infographic illustrating the identification of OXY, TET, and CEP traces in wastewater originating from a poultry slaughterhouse, rabbit farms, and pharmaceutical plant.

5 Conclusion

This study established a connection between two significant and potential fields by creating a chromatographic technique for measuring targeted analytes in actual samples of wastewater. Assessing the presence of antibiotic residues (OXY, TET, and CEP) in wastewater from chicken slaughter, rabbit farms, and medicines using eight ecologically friendly metrics and the specific BAGI associated with sustainable chemistry is not feasible with HPLC techniques. Due to this rationale, analysts have effectively devised and validated an innovative approach. The researchers employed several software and technologies, such as AGREE, AGREEprep, GAPI, ComplexGAPI, ESA, AMVI, AMGS, and HPLC-EAT, to evaluate the environmental sustainability of the proposed strategy based on eight green criteria. The integration of the lean Six Sigma strategy confirmed the suggested method’s quality further, as evidenced by the attainment of Cpk values greater than 1.3. Poultry slaughterhouses, rabbit farms, and pharmaceutical manufacturers can all use the created technology to analyze wastewater for antimicrobial medicines. It is useful for gathering data on the prevalence and environmental behavior of antimicrobial medications.

6 Future plan and study limitations

The method used in this study provides a robust method for detecting antibiotic residues in wastewater. Despite this, several limitations must be acknowledged. There are numerous factors that may affect the accuracy and precision of the results due to the complexity of the wastewater matrix, despite the cleanup steps taken. Furthermore, the method focuses only on a limited number of antibiotics, potentially ignoring other compounds or metabolites. A greener alternative is also needed because organic solvents like methanol are used in the mobile phase, which raises environmental and safety concerns. It is also possible that the method’s performance will vary based on the type of wastewater used, which limits its generalizability. To address these limitations, future work will focus on broadening the scope of analytes to include a broader range of antibiotics and emerging contaminants. Besides exploring greener solvents, we will also explore alternative techniques that reduce the method’s environmental impact. We will investigate automation and high-throughput analysis to facilitate large-scale monitoring and improve reproducibility. In addition, we plan to conduct long-term monitoring studies of antibiotic residues in wastewater to evaluate trends and risks.