Liquid chromatography
In response to the growing concerns regarding pharmaceutical contamination of our aquatic systems, targeted actions are being implemented to align with the recommendations of the European Commission. However, a challenge lies in finding effective, accurate, and green chemistry-compliant methods for analyzing these compounds in complex matrices. This study introduces a highly sensitive and sustainable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneously determining carbamazepine, caffeine, and ibuprofen in water and wastewater. This method exhibits impressive advantages: exceptional sensitivity, high selectivity, and an economical sample preparation strategy resulting from the absence of an evaporation step after solid-phase extraction (SPE), as well as a short analysis time (10 min). Following the International Council for Harmonization (ICH) guidelines Q2(R2), the developed and validated method proved to be specific, linear (correlation coefficients ≥ 0.999), precise (RSD < 5.0%), and accurate (recovery rates ranging from 77 to 160%). The limits of detection were 300 ng/L for caffeine, 200 ng/L for ibuprofen, and 100 ng/L for carbamazepine, respectively. The limits of quantification (LOQs) were 1000 ng/L for caffeine, 600 ng/L for ibuprofen, and 300 ng/L for carbamazepine. The advanced UHPLC-MS/MS method presented in this article constitutes a green and blue analytical technique for the precise detection and quantification of trace levels of pharmaceutical contaminants in aquatic environments. This method has been validated and exemplified using a case study from the Kraków area, highlighting its high efficiency, reliability, and minimal environmental impact. This approach aligns with the concept of sustainable analytics, combining ecological aspects with high-quality results. This study is therefore crucial for the effective monitoring of pollutants, the assessment of environmental and health risks, and ensuring water quality.
Green Analytical Chemistry (GAC) and quality standards in monitoring represent primary aspects of contemporary scientific inquiry to establish effective, safe, and environmentally benign analytical methodologies. The synergistic integration of these components creates an innovative approach for modern monitoring practices, a development of particular salience given the escalating global issue of pharmaceutical presence in aqueous environments. GAC focuses on techniques that limit the consumption of substances, energy, and waste, while maintaining precision, which in monitoring translates into ecological and effective solutions in long-term surveillance programs. Conversely, quality standards in monitoring assure the reliability and accuracy of obtained data, which are indispensable for informed decision-making in environmental stewardship, public health initiatives, and industrial operations. The integration of these two aspects enables the development of robust and sustainable analytical methods that are technically effective and consistent with the principles of sustainable development 1.
In recent decades, the global issue of environmental contamination by pharmaceuticals has become a subject of intense scientific scrutiny and growing public concern. These compounds, often referred to as “emerging contaminants”, encompass a broad spectrum of substances, from analgesics and antibiotics to hormones and psychoactive drugs, that were not traditionally monitored in the environment. The escalating global consumption of pharmaceuticals, both by humans and in veterinary applications, results in their continuous and significant introduction into the aquatic environment through municipal and industrial wastewaters. Conventional wastewater treatment plants often demonstrate insufficient efficacy in eliminating these compounds, leading to their pervasive presence in surface and groundwaters2,3,4. The estimated average annual consumption of pharmaceuticals is approximately 15 g per capita, translating to about 1.2 × 105 tons per year globally, considering a world population of 8,119 million people2,5. Particularly concerning are data indicating that a substantial fraction (10–20%) of ingested pharmaceuticals is excreted largely unchanged into wastewater6,7, and their metabolites can transform into active compounds in ecosystems8,9,10,11. Additional significant sources of pharmaceutical pollution include industrial discharges, household disposal, landfill leachate, agricultural practices, and aquaculture feed additives12,13,14. Despite relatively low individual discharge quantities, the persistent release of these substances leads to their accumulation in aquatic ecosystems15. Globally, high concentrations of active pharmaceutical ingredients are often associated with arid climates, inadequate sanitation infrastructure, and direct contact between surface waters and municipal landfills, while low levels are characteristic of limited anthropogenic impact, advanced wastewater treatment, and high river flow rates7,16,17,18.
The presence of pharmaceuticals in the environment poses a serious ecological threat. Even at trace concentrations, they can exert toxic effects on aquatic organisms, lead to endocrine disruption (e.g., feminization of fish), and contribute to the development of antibiotic resistance, representing a global public health challenge. Furthermore, long-term exposure to these substances in drinking water raises potential risks to human health, underscoring the urgent need for effective monitoring.
Pharmaceuticals from various therapeutic groups commonly detected in surface water and wastewater were considered as indicators of anthropogenic contamination3: caffeine (CAF), a widely used psychoactive agent; ibuprofen (IBU), a common non-steroidal anti-inflammatory drug; and carbamazepine (CBM), an anticonvulsant (Table 1).
Their selection is dictated not only by their widespread environmental presence but also by their specific chemical and pharmacological properties, which can influence their behavior in aquatic ecosystems. Carbamazepine (CBM), a dibenzoazepine derivative with anticonvulsant properties, stands out as one of the most widely accepted and well-established indicators of environmental contamination due to its high chemical stability, widespread medical use, and poor biodegradability in wastewater treatment plants7,19. Caffeine (CAF), as one of the most widely consumed psychoactive substances globally, is present in coffee, tea, energy drinks, and numerous other products. Its immense global consumption directly translates to a continuous and significant release into sewage systems and subsequently into the aquatic environment. For this reason, caffeine is broadly recognized as an excellent marker for domestic wastewater contamination, serving as a valuable indicator of human impact on surface waters20,21. Its presence typically correlates with the influx of insufficiently treated sewage. Despite undergoing faster biodegradation than CBM, CAF is still frequently detected in surface waters, especially near urban agglomerations and wastewater discharge points. The variable presence and degradation levels of CAF provide additional insights into the self-purification processes of waters and the efficiency of wastewater treatment plants (WWTPs)22.
Ibuprofen (IBU) is one of the most frequently used over-the-counter pain relievers and anti-inflammatory drugs. Its massive global consumption leads to continuous and significant introduction into the aquatic environment23. Although IBU is partially degradable in wastewater treatment plants (WWTPs), substantial quantities still pass into receiving waters. Its presence in treated effluent and surface waters is well-documented, making it a significant “emerging contaminant”. Crucially, environmental concentrations of IBU can exert negative ecotoxicological effects on aquatic organisms, including fish and invertebrates, impacting their health, development, behavior, and hormonal balance. Therefore, monitoring its presence is vital for assessing the ecological quality of water bodies24. Ibuprofen also serves as a representative example of the broad class of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), which are among the most frequently detected pharmaceuticals in aquatic environments. Monitoring its presence allows for a general estimation of the environmental burden from these substances25.
A variety of conventional analytical techniques are utilized to monitor pharmaceutical contaminants in aquatic environments, with each method offering different levels of sensitivity, selectivity, and applicability. One of the most commonly used techniques is UV–Vis spectrophotometry, which is based on measuring the absorption of ultraviolet and visible light by the analytes. Although this method is simple and widely available, it suffers from low selectivity and a high susceptibility to interference from other light-absorbing substances, which complicates the analysis of complex environmental matrices26,27,28,29. Similar limitations are observed in spectrofluorometry, which measures the fluorescence of molecules, often requiring prior derivatization. Despite its high sensitivity for certain compounds, the technique is prone to matrix interferences and is not suitable for broad-spectrum pharmaceutical analysis30,31,32. Fourier Transform Infrared (FTIR) spectroscopy enables the identification of chemical compounds based on the characteristic vibrational bands of functional groups. However, its applicability in detecting trace concentrations of pharmaceuticals in environmental samples is limited due to its relatively low sensitivity and inability to effectively analyze complex mixtures. Potentiometry, an electrochemical method, is relatively simple and cost-effective but suffers from low sensitivity and selectivity, and it is particularly prone to ionic interferences in environmental matrices33. Capillary electrophoresis (CE) offers improved resolution by separating analytes based on their electrophoretic mobility. Despite its advantages of minimal sample consumption and good separation efficiency, CE is generally more difficult to couple with mass spectrometric detection and tends to have lower sensitivity compared to modern analytical methods34,35. High-Performance Liquid Chromatography (HPLC) is among the most widely used chromatographic techniques for pharmaceutical analysis, enabling compound separation based on retention behavior and detection via UV, DAD, or FLD detectors. However, HPLC is limited by its lower selectivity and the inability to reliably identify analytes in complex environmental matrices26,36,37,38,39,40. Gas Chromatography coupled with Mass Spectrometry (GC–MS), on the other hand, is a sensitive and selective technique for the analysis of volatile and thermally stable compounds. Nonetheless, most pharmaceuticals found in aquatic environments are non-volatile and/or thermally labile, often requiring laborious and inefficient derivatization steps, which complicate sample preparation and may result in analyte loss41,42,43.
In comparison to these techniques, Ultra-High-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (UHPLC-MS/MS) offers several critical advantages. It provides substantially higher sensitivity and selectivity, allowing for the detection and quantification of pharmaceuticals at ng/L levels or even lower. Through the use of Multiple Reaction Monitoring (MRM), this method enables unambiguous identification of compounds based on their molecular mass and specific fragmentation patterns, minimizing matrix interferences. Unlike methods such as HPLC or GC–MS, UHPLC-MS/MS typically does not require derivatization, simplifying sample preparation and reducing the risk of analyte loss. Moreover, the use of UHPLC significantly shortens analysis time and improves resolution. Due to these attributes, UHPLC-MS/MS is currently regarded as the gold standard in the analysis of pharmaceuticals in aquatic environments, offering unmatched performance under real-world environmental monitoring conditions. Based on the above, precise, sensitive, and efficient monitoring of pharmaceuticals in the aquatic environment is essential. It enables risk assessment for ecosystems and humans, provides crucial data for informing regulatory policy, and supports the development of effective pollution management strategies. However, the determination of trace concentrations of pharmaceuticals in complex environmental matrices is associated with numerous analytical challenges. These primarily include: the low concentrations of target analytes (often at ng/L levels), the presence of numerous matrix interferences that can affect the analytical signal, and the fundamental need for high selectivity and sensitivity in analytical methods44.
In response to these challenges, this work presents the development and validation of a UHPLC-MS/MS method that not only exhibits high analytical performance but also possesses significant “green” and “blue” analytical attributes. A key innovation of presented method is the omission of the energy- and solvent-intensive evaporation step following solid-phase extraction. This directly contributes to reducing solvent consumption and waste generation, aligning with sustainability principles. The developed method is simultaneously highly sensitive and selective, enabling the detection of pharmaceuticals at ng/L levels, which is crucial for reliable ecological and human health risk assessment. It is also efficient and rapid, significantly reducing per-sample analysis time, a vital aspect for effectively conducting routine monitoring campaigns and promptly responding to changing environmental conditions. Furthermore, the method is economical, potentially lowering reagent and equipment operation costs, which is highly significant for laboratories with limited budgets. Lastly, it is more environmentally sustainable (“green”/“blue”) due to a substantial reduction in solvent consumption and waste generation, fully aligning with increasing regulatory requirements and global trends in environmental analytical chemistry45. In conclusion, analytical methods with these characteristics are indispensable for regulatory bodies, environmental agencies, and water management entities to effectively implement monitoring programs, assess the efficacy of remediation efforts, and better manage the problem of pharmaceutical contamination in the environment.
The objective of this study was to develop and validate a fast, sensitive, and simple Ultra-High-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (UHPLC-MS/MS) method for the quantification of three pharmaceuticals from different therapeutic classes (stimulants, anticonvulsants, and antipyretics) in river and wastewater matrices. The selection of the three analytes: carbamazepine (CBM), caffeine (CAF), and ibuprofen (IBU), is based on their complementarity as markers. CBM serves as an indicator of persistent pharmaceutical contamination due to its high stability and poor biodegradability. CAF, given its widespread consumption and recognizability, acts as a direct indicator of domestic wastewater contamination. IBU was chosen as a representative of widely used pharmaceuticals with proven negative impacts on aquatic ecosystems and incomplete elimination in WWTPs. Together, these compounds provide a comprehensive picture of anthropogenic contamination in aquatic environments.
Additionally, to thoroughly evaluate both the environmental footprint and the practical utility of the newly developed analytical method, a comprehensive assessment was conducted using the Analytical Greenness metric (AGREE) and the Blue Applicability Grade Index (BAGI)45. Furthermore, to assess the applicability of the developed and validated method under real conditions and to identify potential, unforeseen issues related to the sample matrix, preparation procedures, or instrumental analysis, the method was subjected to verification on real environmental water samples collected from the Kraków area. The Kraków case study is an integral part of assessing the effectiveness and usefulness of the UHPLC-MS/MS method under real environmental monitoring conditions in a region strongly affected by industrial and urban activities. The data obtained provides specific information on the level of pollution and allows for the assessment of whether the developed method is a suitable tool for long-term monitoring of water quality and assessment of the effectiveness of pharmaceutical pollution reduction strategies.
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