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Characterization of Adeno-Associated Virus (AAV) Gene Therapy Products

Recent Advances and Applications of Passive Sampling Devices

Trends in Sample Preparation

Case Studies Dealing with Cannabis sativa L.

From “Green” to “Sustainable” Sample Preparation in Omics Studies in the Natural Product Field

Onsite Environmental Extraction Based on Portable and Affordable Stirred Devices

A team of researchers recently set out to explore the potential and limitations of reversed hydrophilic interaction liquid chromatography (revHILIC) compared to other chromatographic techniques for the analysis of small molecules and peptides. The team published their findings online in the May 2023 edition of 

(1). The authors — Davy Guillarme from the University of Geneva, Switzerland with Soraya Chapel, Florent Rouviere and Sabine Heinisch from the University of Lyon, France — defined RevHILIC as a technique where a very polar stationary phase, such as bare silica, is used under gradient elution conditions with the mobile phase increasing from a low concentration of acetonitrile (1-5%) to a higher concentration (40%).

The primary aim of this research was to compare the capabilities of revHILIC with reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC). The team initially evaluated the retention behavior of revHILIC against RPLC and HILIC using mixtures of peptides and pharmaceutical compounds. Results indicated that selectivity for RPLC was greater than revHILIC, which was greater than HILIC for both sample types.

The researcher then explored the possibilities of incorporating revHILIC into two-dimensional liquid chromatography (2D-LC) configurations by combining RPLC, revHILIC, or HILIC with RPLC in an on-line comprehensive (LC × LC) mode. The researchers reported that revHILIC × RPLC combination exhibited remarkable performance in terms of peak capacity and sensitivity, providing not only impressive results but also complementary selectivity compared to the more conventional RPLC × RPLC and HILIC × RPLC setups. The elution order and retention time range varied significantly between the three techniques, affirming the potential of revHILIC as a viable option for 2D-LC analysis of small molecules and peptides, according to the researchers.

For 2D-LC analyses, the researchers investigated RPLC, HILIC, or revHILIC in the first dimension, coupled with RPLC in the second dimension. The revHILIC × RPLC configuration demonstrated superior performance with increased peak capacity and sensitivity compared to HILIC × RPLC. The effective peak capacity of revHILIC × RPLC and RPLC × RPLC exhibited a substantial enhancement, showcasing a potential increase of around 70% and 160%, respectively. Additionally, sensitivity saw a notable improvement of 6 to 8 times in revHILIC × RPLC compared to HILIC × RPLC.

The researchers concluded that revHILIC, under gradient conditions, is a potentially useful strategy for the analysis of small drugs and peptides and could be considered for on-line LC × LC applications for applications in life science analysis.

This article was written with the help of artificial intelligence and has been edited to ensure accuracy and clarity. You can read more about our

Chapel S.; Rouviere F.; Guillarme D.; Heinische S. Reversed HILIC Gradient: A Powerful Strategy for On-Line Comprehensive 2D-LC. 

, 28(9), 3907. DOI: 10.3390/molecules28093907

Events

A recently published study explores and potential and limitations of reversed hydrophilic interaction liquid chromatography. 

revHILIC: A New 2D-LC Tool for Life Science Analysis?

A group of researchers, led by Fred E. Regnier of Purdue University, recently a published a manuscript in 

 exploring the benefits of a mobile affinity selection chromatography (MASC) luminon assay method to assess the quality of therapeutic monoclonal antibodies (mAbs) (1). The researchers concluded that this MASC approach overcomes limitations associated with the traditional two-step quality assessment process and offers numerous important advantages.

Therapeutic monoclonal antibodies (mAbs) are subject to stringent quality verification by regulatory agencies. The traditional two-step approach involves protein A affinity chromatography for host-cell proteome and metabolome removal, followed by liquid chromatography–mass spectrometry (LC–MS) for mAb quality assessment. However, this process faces challenges including a time-consuming sample preparation step and a lack of higher-order structure analyses. The published research explored the potential of using a mobile affinity selection chromatography (MASC) luminon assay—which integrates sample preparation and molecular-recognition-based analyses in a single isocratically eluted column —to assess mAbs.

The MASC luminon assay allowed the rapid assessment of mAb titer and aggregate content within 10 min, eliminating the need for the protein A step with the potential to improve quality evaluation at all stages of mAb development and manufacturing process, according to the researchers.

The MASC luminon assay was found to be a swift and accurate method for identifying and quantifying two critical mAb quality features: titer and percent aggregate content. In comparison to the traditional online two dimensional liquid chromatography (2D-LC) protein A-SEC assays, MASC exhibits similar speed but offers improved accuracy and reproducibility. The elimination of the protein A purification step addresses various sources of variability, ensuring consistent results. This novel MASC luminon assay used in process analytical technology (PAT) enabled cellular extract preparation, analyte-specific fluorescent coding, and proteoform separation in a single isocratically eluted column.

A significant advantage of the MASC luminon assay is its applicability to diverse therapeutic mAb subclasses without the need for preliminary removal of host-cell impurities. By targeting the fragment crytallizable (Fc) region with fluorescent coding, the assay facilitates rapid quantification of mAb titer and aggregate content, crucial in assessing toxicity and immunogenicity risks. The assay's ability to establish a chronological pattern of monomer to aggregate ratios provides early warnings of mAb quality drift to aid fast validation and remediation efforts, according to the researchers.

Although the MASC luminon assay does not provide structural information beyond the Fc region, it does evaluate the mAb function and higher-order structure. This means it is useful as a rapid screening tool for identifying protein quality issues, which can lead the way for subsequent in-depth analysis using liquid chromatography tandem mass spectrometry (LC–MS) methods. The simplicity and speed of this MASC luminon assay enabled data-dependent decision-making—which is important in therapeutic mAb quality management during process development and manufacturing. Additionally, the research highlighted that compatibility with multiple IgG subclasses and allotypes of human or humanized antibodies positions make this MASC approach useful as a versatile and powerful addition to process analytical technology (PAT).

The researchers concluded that the MASC luminon assay's speed and versatility make it a useful tool for mAb quality at all stages of the discovery, process R&D, and manufacturing of mAbs.

Narsimhan M.L; Kim J.; Morris N.A.; Bower M.A; Gunawardena H.P.; Bowen E.; Regnier F.E. Mobile Affinity Selection Chromatography Analysis of Therapeutic Monoclonal Antibodies. 

https://doi.org/10.1021/acs.analchem.3c02180

Researchers explore the benefits of a mobile affinity selection chromatography (MASC) luminon assay method to assess the quality of therapeutic monoclonal antibodies.

Mobile Affinity Selection Chromatography Advances Quality Assessment of Monoclonal Antibodies 

Regulators are focused on making sure that animal-derived foodstuffs do not contain pharmaceutical residues, which could negatively impact human health.

A recent study conducted by Veena Jain and Somendu Kumar Roy from CSIR-Indian Institute of Toxicology Research and the Academy of Scientific and Innovative Research explored a new way to detect pharmaceutical residues in foodstuffs (1). Their study, published in the journal 

, showcased a novel and improved method to detect coccidiostat residues in chicken liver (1).

Their published study highlighted the use of a chromatographic technique called ionic liquid-dispersive liquid‒liquid microextraction (IL-DLLME) coupled with ultrahigh-pressure liquid chromatography with photodiode array detection (UHPLC-PDA). Using this technique allowed the researchers to identify and quantify five coccidiostats present in chicken liver samples (1).

Developing the method requires a multifaceted optimization process. The researchers employed a screening design, response surface methodology (RSM), and desirability function (1). Through this, the team achieved optimal extraction conditions that allowed for a robust analysis. The resulting method displayed linearity, with a coefficient of determination (

) ≥ 0.999 across concentrations ranging from 39 to 1250 ng/mL (1).

Moreover, the refined technique had a relative standard deviation (%RSD) ≥ 0.30–6.53% and significant recovery rates within the range of 61.57–107.49% (1). The limits of detection (LODs) and limits of quantitation (LOQs) for the coccidiostats ranged from 3.1 to 8.7 ng/mL and 10 to 29 ng/mL, respectively, ensuring the method's sensitivity in detecting trace amounts of these substances (1).

Using this method allowed the researchers to detect coccidiostat residues in chicken liver samples. Their findings were then confirmed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. As a result, LC–MS/MS analysis helped confirm that the novel method the researchers developed proved to be a reproducible means of detecting these residues, shedding light on potential risks associated with poultry products and human consumption (1).

(1) Jain, V., Roy, S.K. Multiple Response Optimization of an Ionic Liquid-DLLME and UHPLC-DAD Analysis of Five Coccidiostats in the Chicken Liver with LC–MS/MS Confirmation. 

In a recent study conducted by two researchers from the CSIR-Indian Institute of Toxicology Research and the Academy of Scientific and Innovative Research, an analytical method was used to reveal coccidiostat residues in poultry.

Advancements in Analytical Techniques Reveal Coccidiostat Residues in Poultry

scientists from South Dakota State University and the University of Colorado School of Medicine developed a method to detect traces of cyanide in blood samples using liquid chromatography–tandem mass spectrometry (LC–MS/MS) (1).

Intense cluttering of blood cells | Image Credit: © Argus - stock.adobe.com

Cyanide (CN) can be a dangerous poison (cyanide anion [CN

] or hydrogen cyanide [HCN]), but it is also a common chemical component in different natural and anthropogenic substances. Acute CN toxicity happens by blocking terminal electron transfer by inhibiting cytochrome c oxidase, which plays an important role in cellular respiration (2). The blockage is caused by cyanide poisoning the mitochondrial electron transport chain within cells, making the cells unable to derive energy from oxygen. This can result in cellular hypoxia, cytotoxic anoxia, and potential death. As such, it is critical to check for blood CN concentrations, along with the major metabolite, thiocyanate (SCN

However, simultaneously detecting CN and SCN

 in blood can be a complicated process. As part of this study, the scientists created a method to detect CN and SCN

 from human ante- and postmortem blood using LC–MS/MS analysis. Sample preparation for CN involved active microdiffusion with subsequent chemical modification using naphthalene-2,3-dicarboxaldehyde (NDA) and taurine (i.e., the capture solution). SCN

 preparation was conducted with protein precipitation and monobromobimane (MBB) modification.

After the experiment, it was revealed that the method had good sensitivity for CN, with antemortem limit of detection (LODs) of 219 nM and 605 nM for CN and SCN

, respectively, and postmortem LODs of 352 nM and 509 nM. Additionally, the method’s dynamic rangers were 5–500 µM for antemortem blood and 10–500 µM for postmortem blood. The method produced good accuracy (100 ± 15%) and precision (≤ 15.2% relative standard deviation), all while being able to detect elevated levels of CN and SCN

(1) Alluhayb, A. H.; Severance, C.; Hendry-Hofer, T.; Bebarta, V. S.; Logue, B. A. Concurrent determination of cyanide and thiocyanate in human and swine antemortem and postmortem blood by high-performance liquid chromatography–tandem mass spectrometry. 

https://doi.org/10.1007/s00216-023-04939-6

https://chem.libretexts.org/Courses/Saint_Marys_College_Notre_Dame_IN/CHEM_342%3A_Bio-inorganic_Chemistry/Readings/Metals_in_Biological_Systems_(Saint_Mary%27s_College)/Cytochrome_Oxidase

https://centerforhealthsecurity.org/sites/default/files/2023-02/cyanide.pdf

Articles

Scientists from South Dakota State University and the University of Colorado School of Medicine created a system to detect traces of cyanide in blood samples using liquid chromatography–tandem mass spectrometry (LC–MS/MS).

Detecting Cyanide in Blood Using LC–MS/MS

Scientists from multiple universities in China used laser desorption ionization mass spectrometry (LDI-MS) to detect toxic alkaloids in traditional Chinese medicines (TCM), publishing their findings in the journal of 

 (1). LDI-MS is a technique that ionizes molecules using a ultraviolet laser.

Chinese herbs | Image Credit: © ft2010 - stock.adobe.com

Alkaloids are amines that occur naturally in plants, mainly produced as defense mechanisms to protect against herbivores (2–3). Different toxic alkaloids can affect the 

, digestive processes, reproduction, and the immune system, among other systems (2). To best protect consumers, it is essential for scientists to identify which alkaloids are in different substances to minimize the risk of poisoning consumers.

In this study, the team studied various extracts from TCM including Fuzi (

), which were all found to contain alkaloids. The scientists investigated twelve nanomaterials (NMs) as matrices, finding that molybdenum disulfide (MoS2) and defect-rich-tungsten trioxide (D-WO3) were the most effective at detecting alkaloids). They also found use in converting these materials into 3D nanomaterial plates, where the NMs are doped into resin and formed via 3D printing.

The researchers obtained good alkaloid quantification using a chemothermal compound as an internal standard. This approach enabled the creation of a LDI-MS approach that achieves clean backgrounds for alkaloid detection, while the 3D NM plates facilitated mass spectrometry imaging of alkaloids in TCMs. This method still needs further development, but the scientists believe it to have great potential in medicine and food safety.

(1) Wang, C.; Qin, L-Y.; Li, D-M.; Hu, L-G.; Xue, J-J.; Zhai, X-P.; Wang, Q.; Guo, L; Tang, L.; Xie, J-W. Doped nanomaterial facilitates 3D printing target plate for rapid detection of alkaloids in laser desorption/ionization mass spectrometry. 

https://doi.org/10.1007/s00216-023-04961-8

https://www.sciencedirect.com/topics/food-science/alkaloid

Topical Collection “Toxicity of Natural Alkaloids”. 

https://www.mdpi.com/journal/toxins/topical_collections/natural-alkaloids

Scientists recently used laser desorption ionization mass spectrometry (LDI-MS) to detect toxic alkaloids in traditional Chinese medicines (TCM).

Toxic Alkaloids Detected in Chinese Medicines Using Laser Desorption Ionization

A group of scientists from Uppsala University and the Karolinska Institute in Sweden analyzed the metabolomics of individual cells using a pneumatically assisted (PA) nanospray desorption electrospray ionization (nano-DESI) probe of their own creation. Their work was published in the 

Journal of the American Society of Mass Spectrometry

Cell Cross Section | Image Credit: © alswart - stock.adobe.com

Cellular heterogeneity is necessary to mitigate stress and improve a biological system’s resilience; however, to achieve this, scientists must be able to chemically analyze individual cells. One means of studying single-cell processes is metabolomics, which can analyze changes in seconds to minutes. This process can be challenging, partly due to rapid metabolite turnover rates, high complexity of metabolomes, and wide concentration ranges within a cell volume.

For this research, the scientists used a pneumatically assisted (PA) nanospray desorption electrospray ionization (nano-DESI), with the nano-DESI system that was previously used by the scientists for cheek cell metabolomics. DESI is a system that uses an electrospray source of highly charged aqueous spray droplets to desorb and ionize analyte molecules from a sample’s surface. This system allows a sample to be analyzed in its native state (2). The combination was also combined with tapered capillaries to show cellular imaging of IMR-90 cells and global metabolomics of individual INS-1 cells, while also showing a throughput of three cells per minute. In total, the scientists showed metabolomics optimization and exposure conditions for 479 unique INS-1 cells.

Using the PA nano-DESI probe, scientists were able to sample six lines from a large senescent IMR-90 cell, which allowed for a comprehensive assessment of metabolite distribution across the cell area. Different lipid species were evenly distributed over the large cell, which contrasts with other amino acids, which typically show heterogeneous distributions over a cell area.

Metabolome alterations in INS-1 cells were monitored in low and high glucose concentrations, using 93 and 97 cells respectively. Additionally, using a touch-down mode with an analysis of 3 cells per minute, there was higher throughput for metabolomics for cells spotted and analyzed in an array.

(1) Marques, C.; Friedrich, F.; Liu, L.; Castoldi, F.; Pietrocola, F.; Lanekoff, I. Global and Spatial Metabolomics of Individual Cells Using a Tapered Pneumatically Assisted nano-DESI Probe.

https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/desorption-electrospray-ionization

A group of scientists recently analyzed the metabolomics of individual cells using a pneumatically assisted (PA) nanospray desorption electrospray ionization (nano-DESI) probe of their own creation.

Pneumatically Assisted Nano-DESI Probe for Metabolomics

Patrick Lavery is an Editor for the MJH Life Sciences brands LCGC and Spectroscopy, and their respective websites, 

. He previously spent nearly a decade as a news anchor, reporter, and producer at New Jersey 101.5 FM.

A recent report by Vantage Market Research

 estimates that the value of the global proteomics market will reach $107.66 billion by 2030, the result of a predicted 15% exponential growth in the next six-plus years. Market size in 2022 was estimated at $35.2 billion.

DNA and proteomics. DNA, background Generative AI | Image Credit: © Катерина Євтехова - stock.adobe.com

The demand for proteomics and its associated techniques have increased over the last several years, according to Vantage, because of its role in disease diagnosis, particularly the early detection of cancer. The research firm said that the biomarkers identified by proteomics help to discover therapeutic targets in biospecimens that drive market demand.

As of 2022, Vantage said, the proteomics market was dominated by North America, with the continent’s 42.3% share of revenue attributed to generally advanced healthcare infrastructure. But trend-watchers say governments primarily in the Asia/Pacific region, like those of China and India, but also in other corners of the world such as Brazil, are now investing heavily in the research and development of new potential drugs as proteomics research and technology continues to transform the pharmaceutical industry. Those are the regions which are forecast to experience the maximum market growth by 2030.

Where instrumentation technologies are concerned, Vantage predicts chromatography to claim the fastest market share growth. Chromatography is also predicted to be the area in which reagent growth most impacts the industry. Vantage lists nine companies as major players in the global proteomics market scene currently: Illumina, Agilent, Bio-Rad, GE Healthcare, Thermo Fisher, Hoffman-La Roche, Promega, Merck, and Danaher.

Proteomics-based solutions, according to the report, reach farther than just cancer detection and treatment. As the frequency of other chronic illnesses such as diabetes and heart disease increase worldwide, the need for customizable medicines that detect protein biomarkers is expected to intensify.

Spectroscopy, electrophoresis, and protein microarrays and fractionation are some of the other instrumentation technologies that can be anticipated to undergo market growth between now and the end of the 2020s.

Chromatography is predicted to be a major player among instrument technologies, but spectroscopy and electrophoresis may also increase their market shares.

Proteomics Market to Reach $107B Valuation by 2030

Beijing, China, is known for its poor air quality. Because of a combination of being the world’s largest greenhouse gas emitter and having many coal plants in its metropolitan area, Beijing is one of the most polluted cities, with hazardous PM2.5 particles more than 20 times higher than the guidelines established by the World Health Organization (WHO) (1).

As a result of this issue, scientists in China are searching for ways to test air quality in Beijing and across the globe. A recent study published in 

 explores a novel process involving ultrasound-assisted extraction (UAE), online solid phase extraction (online SPE), and liquid chromatography–tandem mass spectrometry (LC–MS/MS) (2).

Led by Chao Wang from the China National Environmental Monitoring Centre, the research team deployed this highly sensitive method to determine 17 nitrated and oxygenated polycyclic aromatic hydrocarbons (NPAHs and OPAHs) in PM2.5. Using UAE, online SPE, and LC–MS/MS required significant refinement. The researcher’s methodology covered a spectrum of parameters, which include solvents and times used in UAE, extract filtering, online SPE conditions, and LC–MS settings (2). By optimizing the method for these parameters, the research team achieved isomeric separation and detection of mononitro-PAHs, dinitro-PAHs, and OPAHs. This was made possible by leveraging the proposed MS/MS fragmentation pathway in negative mode atmospheric pressure chemical ionization (2).

The research team used a method that had an online SPE advantage, which enabled the attainment of low limits of detection for NPAHs (ranging from 0.001 to 0.042 μg/L) and OPAHs (ranging from 0.01 to 0.038 μg/L) (2). Moreover, the recovery rates, spanning from 64% to 108% at three spiking levels, exhibited commendable relative standard deviations (RSDs) of 3% to 23% (2).

Notably, this study marks one of the first applications of the LC–MS methodology for the quantification of three carcinogenic dinitro-PAHs – specifically, 1,3-dinitropyrene, 1,6-dinitropyrene, and 1,8-dinitropyrene – within atmospheric PM2.5 (2).

The researchers deployed this method twice in Beijing: once in the summer, and once in the winter, with the goal being to determine the concentrations of various NPAHs and OPAHs in PM2.5 samples. The findings revealed concentrations ranging from ∑NPAH: 9.4 to 303.3 pg m

, painting a comprehensive picture of the diverse pollutants present in the capital’s air (2).

The new methodology helps us to understand and improve the monitoring of air quality in urban areas and advances environmental science applications by offering scientists another roadmap to quantify pollutants in the air.

(1) Walsh, M. China's smog problem explained. 

(Accessed 21 November 2023). Available at: 

https://phys.org/news/2023-11-china-smog-problem.html

(2) Wang, C.; Zhu, W.; Shi, Z.; Li, J. A Sensitive Online SPE-LC–APCI–MS/MS Method for Simultaneous Determination of 17 Nitrated and Oxygenated Polycyclic Aromatic Hydrocarbons in Atmospheric Particulate Matter. 

A recent study conducted in Beijing, China, presented a novel method involving LC–MS/MS, UAE, and online SPE for characterizing environmental pollutants. 

Advanced Method Unveils Diverse Pollutants in Beijing’s Air

As the field of biopharmaceuticals continues to evolve, the pursuit of efficient analytical techniques plays a pivotal role in understanding crucial quality attributes. The 

Journal of the American Society for Mass Spectrometry

 has recently published a study by research scientists Jennifer L. Lippens, Heath C. Timmons, and Tawyna G. Flick, of Amgen in Thousand Oaks, California, and Crystal Welch and Aditya Kulkarni, of 908 Devices in Boston, Massachusetts, that introduces microfluidic capillary electrophoresis mass spectrometry (CE-MS) as a rapid avenue for intact mass analysis of oligonucleotide single strands (1).

Ressearcher mix DNA, polymerase and oligonucleotides in PCR tubes on the thermal cycler | Image Credit: © HYUNGKEUN - stock.adobe.com

Oligonucleotide analysis, a domain which is crucial for biopharmaceutical advancements, traditionally relies on liquid chromatography–mass spectrometry (LC–MS). However, LC–MS methods often demand lengthy sample run times (exceeding 15 min) to achieve the necessary separation of diverse oligonucleotide species. Furthermore, the use of additives like triethylamine and 1,1,1,3,3,3-hexafluoro-2-propanol in LC methods, while effective, may not align with the preferences of mass spectrometry instrumentation.

To address these challenges, the research team leveraged the ZipChip technology within the microfluidic CE-MS platform to streamline intact mass analysis of oligonucleotide single strands. The ZipChip is a microfluidic device that combines capillary electrophoresis (CE) and electrospray ionization (ESI). The results proved to be significant, achieving baseline separation of oligonucleotides with equal lengths in less than 4 min, marking a substantial reduction in analysis time compared to traditional methods.

The ZipChip technology, a key player in this study, facilitated the swift separation of oligonucleotide full-length products (FLPs) and their impurities, showcasing its potential as a versatile tool in oligonucleotide analysis. The rapid analysis offered by CE-MS not only accelerates the research process but also mitigates the need for mobile phase additives that might not be ideal for mass spectrometry instrumentation.

This innovative approach holds promise for the biopharmaceutical industry, where timely insights into oligonucleotide characteristics are crucial for ensuring product quality. The streamlined CE-MS technique presents itself as an efficient alternative to the time-consuming LC–MS methods, demonstrating the feasibility of rapid and reliable oligonucleotide analysis.

The study sheds light on the transformative potential of microfluidic CE-MS in the realm of oligonucleotide characterization. The ZipChip technology's ability to achieve baseline separation in a fraction of the time opens new doors for expedited analysis, offering researchers a valuable tool for gaining swift and precise insights into the intricate world of oligonucleotides.

(1) Lippens, J. L.; Timmons, H. C.; Welch, C.; Kulkarni, A.; Flick, T. G. Rapid Intact Mass Analysis and Evaluation of the Separation Potential of Microfluidic Capillary Electrophoresis Mass Spectrometry for Oligonucleotides. 

Microfluidic capillary electrophoresis mass spectrometry (CE-MS) using ZipChip technology provides intact mass evaluations and efficient separation of oligonucleotides.

Accelerating Oligonucleotide Characterization: Microfluidic Capillary Electrophoresis Mass Spectrometry Unveils Swift Analysis

, researchers proposed a new method for the determination of antibiotics and eight different metabolites, using techniques like ultrasound-assisted extraction and liquid chromatography–tandem mass spectrometry (LC–MS/MS) (1).

After antibiotics are given to a patient, they are excreted from the body as parent compounds and metabolites. This means that these substances can enter the environment as effluents or sewage sludge from wastewater treatment plants (WWTP). In 2020, the World Health Organization (WHO) warned that antibiotic resistance is increasing, making many diseases harder to treat.

In this study, the researchers developed a method for routine control of multiclass high-concern antibiotics and their main metabolites. The method was based on three different techniques: ultrasound-assisted extraction, dispersive solid-phase extraction clean-up, and analytical determination by LC–MS/MS. Extraction solvent volume, extraction time, and the number of extraction cycles affecting each stage of the method were optimized via a Box–Behnken design. The method was used on different treatment stages of sewage sludge including non-treated sludge: primary and secondary sludge; and treated sludge: digested sludge and compost and to agricultural soil.

Following the experiment, most of the compounds displayed limits of quantification in the range of 0.03–7.50 ng g−1 dry weight (dw). Accuracy values ranged from 70–102%, while precision remained below 17%. Macrolides and fluoroquinolones held the highest concentrations. The lowest antibiotic concentrations were measured in both compost (highest concentration: 27 ng g−1 dw, corresponding to norfloxacin) and soil samples (highest concentration: 93 ng g−1 dw, corresponding to a metabolite of clarithromycin).

The scientists claim this method as the first to be developed for the determination of multiclass antibiotics and their main metabolites in sludge from different treatment stages. Furthermore, it can prove useful in obtaining antibiotic information prior to sewage sludge being applied to agricultural soils, as well as those within agricultural soils.

Mejías, C.; Santos, J. L.; Martín, J.; Aparicio, I.; Alonso, E. Multiresidue method for the determination of critically and highly important classes of antibiotics and their metabolites in agricultural soils and sewage sludge. 

https://doi.org/10.1007/s00216-023-04982-3

A new study proposed a method for the determination of antibiotics and eight different metabolites, utilizing techniques like ultrasound-assisted extraction and LC–MS/MS.

Featured Content

Characterization of Adeno-Associated Virus (AAV) Gene Therapy Products

Recent Advances and Applications of Passive Sampling Devices

Trends in Sample Preparation

From “Green” to “Sustainable” Sample Preparation in Omics Studies in the Natural Product Field

Onsite Environmental Extraction Based on Portable and Affordable Stirred Devices