Sample Treatment Procedures Research Articles

Green and miniaturized ultrasonic-assisted extraction using natural deep eutectic solvents to extract phenolic compounds from tea samples

Natural deep eutectic solvents (NADES) have emerged as promising green solvents, particularly useful in sample treatment procedures. For the first time, different types of NADES were prepared and evaluated as potential phenolic extraction solvents from tea samples (Camellia sinensis). The NADES, composed of lactic acid:glycerol:water in a molar ratio of 1:1:3, provided the best results for the simultaneous extraction of the target phenolic compounds. This NADES was also characterized by using nuclear magnetic resonance (NMR), and the presence of nuclear Overhauser enhancement effects confirmed its supramolecular structure. The experimental conditions affecting the miniaturized ultrasonic-assisted extraction (UAE) were optimized to maximize extraction efficiency by performing a design of experiments. The proposed sample treatment was combined with ultra-high performance liquid-chromatography coupled to tandem mass spectrometry detection (UHPLC-MS/MS) to determine 21 phenolic compounds including catechin derivatives and other minor compounds. The UAE-UHPLC-MS/MS method was successfully characterized and applied to the analysis of various commercial green, black, white and red tea samples. In all cases, 18 phenolic compounds were determined with concentrations ranging from 0.3 to 29,125 mg kg−1. The proposed sample treatment is a high-throughput, easy, fast and robust alternative to conventional procedures, which reduces organic solvent consumption and costs, aligned with green sample preparation principles.

Development of a fast and low-cost aqueous based-extraction protocol for the simultaneous extraction and characterization of SiO2 and TiO2 (nano)particles in confectionary products

BackgroundThe determination of (nano)particulate content from food additives has been a long-standing concern for authorities since it is of vital importance for ensuring food safety, regulatory adherence, and transparent consumer information. Nonetheless, a critical step in these determinations is the refinement of a careful and quantitative extraction process for particles that may be found within complex matrices such as confectionery products. The development of new technologies and analysis methods for nanoparticles is ongoing. Whereas new technologies and analysis methods for nanoparticles are being developed, the extraction of (nano)particles of different nature has not been adequately addressed in the literature. ResultsA simple aqueous extraction procedure was found to be suitable for the simultaneous extraction of TiO2 and SiO2 (nano)particles from five confectionery products. Neither the extraction agents (water, lipase, pancreatin and Tris-HCl solutions) nor the methods (manual shaking, ultrasonic bath, ultrasonic probe and ultrafiltration) altered the size, morphology, or aggregation state of either type of particle, as revealed by the micrographs obtained by Transmission Electron Microscopy (TEM). Single-particle ICP-MS (spICP-MS) determined that the optimal conditions for extracting both types of particles involve manual shaking using water as the solvent. Furthermore, the use of enzymes seemed to hinder the determination of both types of particles by spICP-MS. (Nano)particles of TiO2 and SiO2 were detected in all the confectionaries, even though the E171 additive was only labeled in one of them. The average percentage of nanoparticulate TiO2 material in the evaluated products was 30 %, while no nanometer-sized particles of SiO2 were detected. SignificanceEnsuring food safety, regulatory compliance and transparent consumer information relies on getting reliable results that connect with the application of sample treatment procedures for detecting unaltered nanoparticles in food products. The presented research introduces an economical, swift, user-friendly, environmentally responsible, and harmonious extraction method for the concurrent analysis of TiO2 and SiO2 particles in confectionery samples. Furthermore, particles from additives not included in the labeling have been detected, characterized, and quantified in the confectionary products.

In-line RNA-based microreactor direct mass spectrometry for ultrasensitive and rapid assay of terminal deoxynucleotidyl transferase activity

Terminal deoxynucleotidyl transferase (TdT), a unique template-independent DNA polymerase, plays a crucial role in the human adaptive immune system and is considered a promising biomarker for the diagnosis of various forms of acute or chronic leukemia. The accurate and sensitive detection of trace TdT is of pivotal importance to fulfill the significant medical interest in understanding its pathological functions and diagnosing TdT-related diseases. We hereby present an in-line RNA-based microreactor direct mass spectrometry (MS) method and its application for ultrasensitive, accurate, and rapid analysis of trace TdT activity in leukemic cell samples. A specially designed RNA-based microreactor is fabricated by immobilizing short RNA sequence via covalent Au–S bond on the inner surface of a capillary pre-modified with three-dimensional porous layer (PL) and Au nanoparticles (AuNPs). Utilizing this PL@Au@RNA microreactor, the signal of target TdT is conversed into reporter molecules (adenine), which exhibit a strong MS response. This conversion process enables efficient signal amplification and enhances detection sensitivity. The outlet end of the PL@Au@RNA microreactor is deliberately crafted into a porous tip, serving as an electrospray ionization (ESI) interface to directly couple to ESI-MS in-line. This design facilitates the direct transmission of the generated signaling molecules into the MS system, eliminating the need for laborious sample treatment procedures. By implementing this RNA-based microreactor in direct MS analysis, we have achieved remarkable sensitivity in detecting TdT activity with the limit-of-detection of 4 × 10−9 U, surpassing other reported methods in literature by three to four orders of magnitude. Furthermore, each assay requires a minimal sample volume of merely 10 nL. This method has successfully demonstrated its application in accurately and efficiently detecting TdT activity in leukemia cells, and its detection results are consistent with those obtained by ELISA kits.

Ecofriendly UPLC-MS/MS method for simultaneous assay of veterinary drugs residues; erythromycin, sulfadiazine and trimethoprim in edible chicken tissues, evaluation of method greenness and whiteness

Antibiotics are the key veterinary drugs used in animal food production. The uncontrolled widespread use of antibiotics may lead to the accumulation of their residues in foodstuff such as milk, eggs, meat and muscles. An Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method was developed for simultaneous determination of triple combination of erythromycin thiocyanate (ERY), sulfadiazine sodium (SFD) and trimethoprim (TMP) in their dosage form and in different chicken tissues including muscles and liver samples. A Simple extraction and cleaning up procedure was applied using 0.1 N EDTA and methanol to remove the interfering substances and hence increase the efficiency of chromatographic separation. Moreover, separation was performed within 1.2 min on Agilent Poroshell 123EC-C18 column using a mobile phase mixture of methanol: water (90:10, v/v), at a flow rate of 0.5 mL/min using quadrupole tandem mass spectrometer detector. The method was validated according to Food and Drug Administration (FDA) guidelines and linearity was achieved in the range of 2.5–1000 ng/mL for ERY and TMP and in the range of for 5–2000 ng/mL for SFD using tamsulosin (TAM) as an internal standard. All the validation results were within the acceptable ranges. Moreover, Three green metric systems including Green Analytical Procedure Index (GAPI), Analytical Eco-Scale and Analytical GREEnness (AGREE) were used to assess the greenness of the developed method. As well as, the novel blue applicability grade index (BAGI) tool was applied to evaluate the practicality of the method. Results of these tools proved the low environmental impact of the method regarding solvent consumption, analysis time, amount of sample needed, sample treatment procedure, and waste product. The developed method has the privilege of high sensitivity and selectivity as well as short analysis time; hence it can be used to test the presence of the residues of studied drugs in various marketed meat samples to assure human safety and maintain public health. Moreover, it is the first developed method for their analysis in liver samples.

Filtered Saliva for Rapid and Accurate Analyte Detection for POC Diagnostics.

Saliva has shown considerable promise as a diagnostic medium for point-of-care (POC) and over-the-counter (OTC) diagnostic devices due to the non-invasive nature of its collection. However, a significant limitation of saliva-based detection is undesirable interference in a sensor's readout caused by interfering components in saliva. In this study, we develop standardized sample treatment procedures to eliminate bubbles and interfering molecules while preserving the sample's target molecules such as spike (S) protein and glucose. We then test the compatibility of the pretreatment system with our previously designed SARS-CoV-2 and glucose diagnostic biosensing systems for detecting S protein and glucose in subject saliva. Ultimately, the effectiveness of each filter in enhancing biomarker sensitivity is assessed. The results show that a 20 mg nylon wool (NW) filter shows an 80% change in viscosity reduction with only a 6% reduction in protein content, making it an appropriate filter for the salivary S protein diagnostic system. Meanwhile, a 30 mg cotton wool (CW) filter is identified as the optimal choice for salivary glucose detection, achieving a 90% change in viscosity reduction and a 60.7% reduction in protein content with a minimal 4.3% reduction in glucose content. The NW pretreatment filtration significantly improves the limit of detection (LOD) for salivary S protein detection by five times (from 0.5 nM to 0.1 nM) and it reduces the relative standard deviation (RSD) two times compared to unfiltered saliva. Conversely, the CW filter used for salivary glucose detection demonstrated improved linearity with an R2 of 0.99 and a sensitivity of 36.6 μA/mM·cm2, over twice as high as unfiltered saliva. This unique filtration process can be extended to any POC diagnostic system and optimized for any biomarker detection, making electrochemical POC diagnostics more viable in the current market.

Enhancing Anaerobic Digestion Process: A Comprehensive Optimization Study

Anaerobic digestion constitutes a series of chemical reactions facilitated by anaerobic microorganisms, wherein organic matter is converted into biofuels. We did a bibliometric study to comprehend the factors influencing the optimization of this process. This investigation underscored crucial parameters affecting methane and biogas production: temperature, pH, hydraulic retention time (HRT), carbon-to-nitrogen ratio, and sample preparation and treatment procedures. We recommend an average of parameters as T = 35°C, pH = 6, HRT = 21 days, and total chemical oxygen demand > 8.5 COD/m³.d to attain optimal outcomes, it is recommended to maintain. By adhering to these guidelines, the process can be enhanced, thereby fostering greater efficiency in biogas production.

Determination of norfloxacin, cipfloxacin, levofloxacin and moxifloxacin in urine by HPLC with a two-channel fluorescence detector

A sensitive HPLC-FLD method with two-channel fluorescence detection was established and validated for quantification of four fluoroquinolones (norfloxacin, ciprofloxacin, levofloxacin and moxifloxacin) in urine samples. The fluorescent detector was set at two-channel excitation and emission wavelengths: 280/445 nm (for NOR, CIP) and 290/500 nm (for LEVO, MOXI). The measurement protocol was in compatible with both of the sample treatment procedures including the liquid-liquid extraction (LLE) and solid-phase extraction (SPE). The method was well-validated with the limit of quantification: 0.027; 0.034; 0.024; 0.020 µg/mL (for LLE), and 0.028; 0.033; 0.024; 0.020 µg/mL (for SPE) for NOR, CIP, LEVO, MOXI, respectively. The method has been proved to be precise and accurate, with the relative standard deviation lower than 3% and the recovery ranging from 98.0%¸102.0%, for all fluoroquinolones. The proposed HPLC-FLD method provides an alternative approach for the simultaneous analysis of fluoroquinolones in urine. ® 2023 Journal of Science and Technology - NTTU

Critical evaluation of energy dispersive X-ray fluorescence spectrometry for multielemental analysis of coffee samples: Sample preparation, quantification and chemometric approaches

In the present contribution, a critical evaluation of the use of EDXRF (Energy dispersive X-ray fluorescence spectrometry) for elemental composition determination (K, Ca, Mn, Fe, Cu, Zn, Sr and Rb) of coffee samples is presented. Firstly, several sample treatment procedures (pressed pellets and loose powder) and quantification strategies (fundamental parameters and empirical calibration) were evaluated.In addition to quality and nutritional purposes, information of elemental content in coffee samples can be also useful for coffee traceability and provenance studies. In view of that, a comparison of different tools such as explorative principal component analysis (PCA) and classification by partial least square regression (PLS-DA) were also tested as discriminating tools of different types of coffee samples (i.e., pure, instant and coffee with additives). Chemometric analysis was performed using coffee elemental concentrations determined by the developed EDXRF method and, for the first time, using raw EDXRF spectra obtained, without the need of a fitting and quantification procedure.

Determination of norfloxacin, cipfloxacin, levofloxacin and moxifloxacin in urine by HPLC with a two-channel fluorescence detector

A sensitive HPLC-FLD method with two-channel fluorescence detection was established and validated for quantification of four fluoroquinolones (norfloxacin, ciprofloxacin, levofloxacin and moxifloxacin) in urine samples. The fluorescent detector was set at two-channel excitation and emission wavelengths: 280/445 nm (for NOR, CIP) and 290/500 nm (for LEVO, MOXI). The measurement protocol was in compatible with both of the sample treatment procedures including the liquid-liquid extraction (LLE) and solid-phase extraction (SPE). The method was well-validated with the limit of quantification: 0.027; 0.034; 0.024; 0.020 µg/mL (for LLE), and 0.028; 0.033; 0.024; 0.020 µg/mL (for SPE) for NOR, CIP, LEVO, MOXI, respectively. The method has been proved to be precise and accurate, with the relative standard deviation lower than 3% and the recovery ranging from 98.0%¸102.0%, for all fluoroquinolones. The proposed HPLC-FLD method provides an alternative approach for the simultaneous analysis of fluoroquinolones in urine.
 ® 2023 Journal of Science and Technology - NTTU

Application of capillary electrophoresis with capacitively contactless conductivity detection for biomedical analysis.

The coupling of capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C4 D) has become convenient analytical method for determination of small molecules that do not possess chromogenic or fluorogenic group. The implementations of CE with C4 D in the determination of inorganic and organic ions and amino acids in biomedical field are demonstrated. Attention on background electrolyte composition, sample treatment procedures, and the utilize of multi-detection systems are described. A number of tables summarizing highly developed CE-C4 D methods and the figures of merit attained are involved. Lastly, concluding remarks and perspectives are argued.

Improved LC-MS/MS method for the simultaneous quantification of tacrolimus and cyclosporine A in human blood and application to therapeutic drug monitoring.

In order to facilitate therapeutic drug monitoring of tacrolimus and cyclosporine A in clinical practice, a simple, rapid, robust, sensitive and specific LC-MS/MS assay was developed and validated for the simultaneous determination of tacrolimus and cyclosporine A in human whole blood. Erythrocytes were destroyed using internal standard solution with 10% (w/v) zinc sulfate in water. The analytes were extracted from 100 μl of whole blood by protein precipitation with acetonitrile. Chromatographic separation was conducted on a Kinetex PFP column (60°C) by a gradient elution with a flow rate of 0.450 ml/min in 2.5 min. Quantitative analysis was performed using electrospray ionization and multiple reaction monitoring in positive ionization mode. The method was fully validated as per current guidelines on bioanalytical methodologies of the US Food and Drug Administration and European Medicines Agency. The method developed was applied successfully in analyzing clinical samples from patients administered tacrolimus or cyclosporine A. The sample treatment procedure was rationalized and improved to fulfill the complete target extraction. The chromatography conditions were optimized to achieve rapid and accurate quantification of both analytes. This method may be beneficial as a constructive input for the therapeutic drug monitoring of tacrolimus and cyclosporine A in obtaining individualized therapy.

Passive Sampler Technology for Viral Detection in Wastewater-Based Surveillance: Current State and Nanomaterial Opportunities.

Although wastewater-based surveillance (WBS) is an efficient community-wide surveillance tool, its implementation for pathogen surveillance remains limited by ineffective sample treatment procedures, as the complex composition of wastewater often interferes with biomarker recovery. Moreover, current sampling protocols based on grab samples are susceptible to fluctuant biomarker concentrations and may increase operative costs, often rendering such systems inaccessible to communities in low-to-middle-income countries (LMICs). As a response, passive samplers have emerged as a way to make wastewater sampling more efficient and obtain more reliable, consistent data. Therefore, this study aims to review recent developments in passive sampling technologies to provide researchers with the tools to develop novel passive sampling strategies. Although promising advances in the development of nanostructured passive samplers have been reported, optimization remains a significant area of opportunity for researchers in the area, as methods for flexible, robust adsorption and recovery of viral genetic materials would greatly improve the efficacy of WBS systems while making them more accessible for communities worldwide.

Neonicotinoids: An overview of the newest sample preparation procedures of environmental, biological and food matrices

An overview of the actual state-of-the-art sample treatment procedures for trace and ultra-trace determination of neonicotinoids (NQs) in environmental, food and biological samples is presented. Analytical procedures for NQs, sometimes together with their metabolites/by-products, and NQ-related new generation pesticides have been considered, focusing on sample preparation, which is essential in most cases due to both the complexity of the matrices and low concentrations of residual analytes. Different strategies have been practised for analytes extraction/preconcentration/cleanup, entailing various techniques as solid-liquid extraction (SLE), liquid-liquid extraction (LLE), offline and online solid-phase extraction (SPE), dispersive SPE (d-SPE), magnetic SPE (MSPE), solid-phase microextraction (SPME) and the quick, easy, cheap, effective, rugged and safe (QuEChERS) approach. New solid phases, including inorganic and carbon-based materials, have been prepared, also starting from “green” precursors, and successfully employed in the sample treatment step before hyphenated liquid chromatography or direct electrochemical detection. As well, new environmentally friendly solvents have been evaluated for LLE. The most recent studies (2022-present), around 50 papers, have been reviewed by matrix type, briefly described and commented concerning extraction efficiency, matrix effect, sensitivity, ease of application and sample throughput. Advantages and drawbacks were highlighted, as well as new achievements paving the way for further improvements.

Applicability of total reflection x‐ray fluorescence for heavy metal analysis in Lake Baikal sponges

AbstractOxidative stress of Lake Baikal organisms can be caused by pollution of nearshore with anionic surfactants or heavy metals. Some specific heavy metals are essential for aquatic organisms. At present, there are no data concerning heavy metal content caused by anthropogenic pollution or chemotaxonomic features of Baikal sponges. Here, we consider the applicability of total reflection x‐ray fluorescence (TXRF) to analyze heavy metals in Lubomirskia baikalensis to fill this gap. It was shown that TXRF method can be successfully applied to the fast and reliable quantification of Cu, Zn, Fe, and Mn in this object. Evaluation of different sample treatment procedures showed that the acid digestion by HNO3/H2O2 mixture is an optimal procedure for the preparation of sponges compared with suspension preparation. It helps to improve sensitivity, eliminate the effects of particle size and specimen heterogeneity. The total uncertainty of the TXRF results associated with the sample preparation, taking, depositing, and measuring of the specimen was 6%–18% for Mn, 3%–5% for Cu, 5%–12% for Fe, and 4%–5% for Zn. To validate the developed method, the TXRF results were compared with the data obtained by wavelength dispersive x‐ray fluorescence spectrometry. A good agreement of results was achieved. TXRF data show the dominance of essential heavy metals in L. baikalensis (n = 14) among other heavy metals. Correlations between Cu/Zn contents (R2 = 0.515) as well as between Fe/Mn contents (R2 = 0.972) can be explained by the contribution of them to L. baikalensis antioxidant system.

Quantitative plasma profiling by 1H NMR-based metabolomics: impact of sample treatment.

Introduction: There is evidence that sample treatment of blood-based biosamples may affect integral signals in nuclear magnetic resonance-based metabolomics. The presence of macromolecules in plasma/serum samples makes investigating low-molecular-weight metabolites challenging. It is particularly relevant in the targeted approach, in which absolute concentrations of selected metabolites are often quantified based on the area of integral signals. Since there are a few treatments of plasma/serum samples for quantitative analysis without a universally accepted method, this topic remains of interest for future research. Methods: In this work, targeted metabolomic profiling of 43 metabolites was performed on pooled plasma to compare four methodologies consisting of Carr-Purcell-Meiboom-Gill (CPMG) editing, ultrafiltration, protein precipitation with methanol, and glycerophospholipid solid-phase extraction (g-SPE) for phospholipid removal; prior to NMR metabolomics analysis. The effect of the sample treatments on the metabolite concentrations was evaluated using a permutation test of multiclass and pairwise Fisher scores. Results: Results showed that methanol precipitation and ultrafiltration had a higher number of metabolites with coefficient of variation (CV) values above 20%. G-SPE and CPMG editing demonstrated better precision for most of the metabolites analyzed. However, differential quantification performance between procedures were metabolite-dependent. For example, pairwise comparisons showed that methanol precipitation and CPMG editing were suitable for quantifying citrate, while g-SPE showed better results for 2-hydroxybutyrate and tryptophan. Discussion: There are alterations in the absolute concentration of various metabolites that are dependent on the procedure. Considering these alterations is essential before proceeding with the quantification of treatment-sensitive metabolites in biological samples for improving biomarker discovery and biological interpretations. The study demonstrated that g-SPE and CPMG editing are effective methods for removing proteins and phospholipids from plasma samples for quantitative NMR analysis of metabolites. However, careful consideration should be given to the specific metabolites of interest and their susceptibility to the sample treatment procedures. These findings contribute to the development of optimized sample preparation protocols for metabolomics studies using NMR spectroscopy.

Emerging Trends in Pharmacotoxicological and Forensic Sample Treatment Procedures

One of the most discussed topics concerns the sample preparation before the analysis and, therefore, all the operations necessary to eliminate the interferents, clean up the specimens, and extract the analytes of interest, reducing the matrix effect. This review highlights the fundamental steps in the treatment of postmortem samples used in forensic analysis. Through critical literature research, it was possible to choose among the countless works that could provide a general overview of the state-of-the-art in this field. Different biological matrices have been considered; blood and urine (the traditional biological fluids) are used to investigate the presence of substances that may have caused death, whilst other body fluids, such as bile and oral fluids, are still under discussion for their usability (and suitability). In the second part of the review, all the solid matrices obtained after autopsy were further divided into conventional and unconventional matrices to facilitate proper understanding. The choice of literature was also made according to the most widely used pretreatment techniques and the most representative innovative techniques.

Dispersive Liquid - Liquid Microextraction for the Determination of Sulfonamide Residues in Egg Samples

A dispersive liquid-liquid microextraction combined with high-performance liquid chromatography-diode array detection was developed for the extraction and determination of 15 sulfonamide residues in the egg matrix. The effects of various parameters such as the type, volume, and composition of extraction solvent for sample treatment procedure, the type and volume of disperser solvent, centrifugation time, salting-out effect and solution pH were studied, and optimum conditions were established. Linearity was found in the range of 5.4 – 1 000 μg kg-1 with regression coefficients ranging from 0.9918 - 0.9987. The limit of detection (LOD) and limit of quantification (LOQ) values of the proposed method were in the range of 4.3 - 8.0 μg kg-1 and 12.9 - 24.0 μg kg-1, respectively. Satisfactory intra-day and inter-day precision results in the range of 6.3 - 17.5% and 4.8 - 16.8%, respectively, were achieved. The accuracy of the method was acceptable with percentage recovery in the range of 73 - 108% and %RSD values in the range of 1.1 - 16.5%. The proposed method was applied in chicken egg samples obtained from supermarkets, and findings confirmed that the method is feasible to be used for extraction and determination sulfonamide residues in egg and related complex biological matrices.

New Challenges in (Bio)Analytical Sample Treatment Procedures for Clinical Applications

The primary cause of poor and ambiguous results obtained from the bioanalytical process is the sample pre-treatment, especially in clinical analysis because it involves dealing with complex sample matrices, such as whole blood, urine, saliva, serum, and plasma. So, the aim of this review is to focus attention on the classical and new techniques of pre-treatment for biological samples used in the bioanalytical process. We discussed the methods generally used for these types of complex samples. Undoubtedly, it is a daunting task to deal with biological samples because the analyst may encounter a substantial loss of the analytes of interest, or the overall analysis may be too time-consuming. Nowadays, we are inclined to use green solvents for the environment, but without sacrificing analytical performance and selectivity. All the characteristics mentioned above should be added to the difficulty of the withdrawal of samples like blood because it can be an invasive practice. For these reasons, now we can also find in the literature the use of saliva as alternative biological samples and new techniques that do not require substantial sample pre-treatment, such as fabric phase sorptive extraction (FPSE). The text has been divided into the following two distinct parts: firstly, we described clinical applications under different subsections, such as anticancer drugs, antibiotics, vitamins, antivirals, non-steroidal anti-inflammatory drugs, statin, imidazoles, and triazoles. The second part is dedicated to sample preparation techniques for diagnostic purposes and is divided into the following different sample preparation techniques: solid-phase microextraction (SPME), microextraction by packed sorbent (MEPS), dispersive liquid–liquid microextraction (DDLME), and fabric phase sorptive extraction (FPSE).

Evaluation of different analytical approaches using total reflection X-ray fluorescence systems for multielemental analysis of human tissues with different adipose content

Elemental content plays an important role in biological processes, and so, the multielemental analysis of human tissue samples is required in biomedical research. Still, the small amount of available biological samples and the adipose content of the samples can be major setbacks for the accurate determination of elemental content.In this study, we explored the potential of several analytical approaches combined with total reflection X-ray fluorescence spectrometry (TXRF) for multielemental analysis of human tissues with different adipose content (colon, heart, liver, lung, muscle, intestine, skin, stomach, uterus, bladder and aorta). The capabilities and limitations of different sample treatment procedures (suspension and acidic digestion) and two TXRF systems with different anode configurations (Mo and W X-ray tubes) have been evaluated for such purpose. Results showed that for tissues with a higher fat content (e.g., skin, and intestine) the best strategy was the acidic digestion of the sample before TXRF analysis. However, for other tissues, acceptable results were obtained by suspending 20 mg of powdered material in 1 mL of 2 M nitric acid. A further enhancement of the limits of detection and accuracy of the results was achieved if using Mo-TXRF systems, especially for the determination of low Z elements (e.g., K, and Ca) and of elements present at low concentrations (e.g., Cu) in the human tissues.Finally, results by TXRF analysis were compared with those obtained with μ-EDXRF and ICP-OES, and a good agreement was obtained.

Sampling and laboratory protocols to study the effects of water-level management on the littoral invertebrate fauna in deep and large temperate lakes

An integrated multidisciplinary protocol on monitoring, sampling, and laboratory procedures was developed and proposed as part of the Project "Parks Verbano Ticino" in the framework of the INTERREG V-A Italy-Switzerland 2014-2020 Cooperation Program. The project's overall goal is to evaluate the effects of water-level management (hydro-morphological stress) on both macro- and meio-fauna along the shores of Lake Maggiore, a large and deep temperate lake in northwest Italy. Because of their importance in the aquatic food web, determining how this stress affects macro- and meio-faunal assemblages is difficult. The protocol developed thus includes the evaluation of hydro-morphological impacts via the Lake Habitat Survey method, which entails monitoring of human-induced impacts and related infrastructures, followed by an in-depth evaluation of the ecological health of lake habitats via chemical analyses. The protocol then describes the sampling methods for shallow lake waters (i.e., <1.5 m depths) of deep lakes, but it also provides guidance on the best time to sample, how to select sampling sites, and how to allocate sample replicates along transects. A detailed step-by-step laboratory procedure for sample treatment was provided in order to assess the structure of macro- and meio-fauna assemblages, as well as morpho-functional traits (e.g., body shape and size, biomass estimate) in response to water-level management. For the first time, a set of morphological and functional characteristics of macro- and meio-faunal taxa are proposed for comparison. The protocol for standardized trait measurement is intended to be widely used. We also proposed chironomid species-specific length-mass regression models for biomass estimation, which is important for determining growth rate and secondary production of these taxa in temperate zone lakes. Length-mass equations could shed light on the role of specific species in the flow of energy through aquatic ecosystems. The proposed protocol was evaluated by team members to ensure common utility, accuracy, and repeatability of the procedures in order for researchers and stakeholders involved in water management of lakes with similar physical characteristics to use it. The protocol, which has been adapted or simply developed to meet the needs of the Italian context, could be successfully applied to other Alpine and Mediterranean temperate, deep lakes, reservoirs, and other glacial, volcanic, and morainic lakes, as well as to a broader European context. Graphical Abstract