Recent advances in the chromatographic analysis of endocannabinoids and phytocannabinoids in biological samples.

Endocannabinoid system, including endocannabinoid neurotransmitters (eCBs), has gained much attention over the last years due to its involvement with the pathophysiology of diseases and the potential use of Cannabis sativa (marijuana). The identification of eCBs and phytocannabinoids in biological samples for forensic, clinical, or therapeutic drug monitoring purposes constitutes a still significant challenge. In this scoping review, the recent advantages, and limitations of the eCBs and phytocannabinoids quantification in biological samples are described. Published studies from 2018-2023 were searched in 8 databases, and after screening and exclusions, the selected 38 articles had their data tabulated, summarized, and analyzed. The main characteristics of the eCBs and phytocannabinoids analyzed and the potential use of each biological sample were described, indicating gaps in the literature that still need to be explored. Well-established and innovative sample preparation protocols, and chromatographic separations, such as GC, HPLC, and UHPLC, are reviewed highlighting their respective advantages, drawbacks, and challenges. Lastly, future approaches, challenges, and tendencies in the quantification analysis of cannabinoids are discussed.

Water as a green solvent for sustainable sample preparation: single drop microextraction of N-nitrosamines from losartan tablets.

Water, renowned for its sustainability and minimal toxicity, is an ideal candidate for environmentally friendly solvent-based microextraction. However, its potential as an extractant solvent in miniaturized sample preparation remains largely unexplored. This paper pioneers using water as the extraction solvent in headspace single-drop microextraction (HS-SDME) for N-nitrosamines from losartan tablets. Autonomous HS-SDME is executed by an Arduino-controlled, lab-made Cartesian robot, using water for the online preconcentration of enriched extracts through direct injection into a column-switching system. Critical experimental parameters influencing HS-SDME performance are systematically explored through univariate and multivariate experiments. While most previously reported methods for determining N-nitrosamines in pharmaceutical formulations rely on highly selective mass spectrometry detection techniques to handle the strong matrix effects typical of pharmaceutical samples, the water-based HS-SDME method efficiently eliminates the interfering effects of a large amount of the pharmaceutical active ingredient and tablet excipients, allowing straightforward analysis using high-performance liquid chromatography with ultraviolet detection (HPLC-UV-Vis). Under optimized conditions, the developed method exhibits linear responses from 100 to 2400 ng g-1, demonstrating appropriate detectability, precision, and accuracy for the proposed application. Additionally, the environmental sustainability of the method is assessed using the AGREEprep methodology, positioning it as an outstanding green alternative for determining hazardous contaminants in pharmaceutical products.

Advances and Applications of Hybrid Graphene-Based Materials as Sorbents for Solid Phase Microextraction Techniques.

The advancement of traditional sample preparation techniques has brought about miniaturization systems designed to scale down conventional methods and advocate for environmentally friendly analytical approaches. Although often referred to as green analytical strategies, the effectiveness of these methods is intricately linked to the properties of the sorbent utilized. Moreover, to fully embrace implementing these methods, it is crucial to innovate and develop new sorbent or solid phases that enhance the adaptability of miniaturized techniques across various matrices and analytes. Graphene-based materials exhibit remarkable versatility and modification potential, making them ideal sorbents for miniaturized strategies due to their high surface area and functional groups. Their notable adsorption capability and alignment with green synthesis approaches, such as bio-based graphene materials, enable the use of less sorbent and the creation of biodegradable materials, enhancing their eco-friendly aspects towards green analytical practices. Therefore, this study provides an overview of different types of hybrid graphene-based materials as well as their applications in crucial miniaturized techniques, focusing on offline methodologies such as stir bar sorptive extraction (SBSE), microextraction by packed sorbent (MEPS), pipette-tip solid-phase extraction (PT-SPE), disposable pipette extraction (DPX), dispersive micro-solid-phase extraction (d-µ-SPE), and magnetic solid-phase extraction (MSPE).

Advances on multiclass pesticide residue determination in citrus fruits and citrus-derived products - A critical review.

The application of agrochemicals in citrus fruits is widely used to improve the quality of crops, increase production yields, and prolong post-harvest life. However, these substances are potentially toxic for humans and the ecosystem due to their widespread use, high stability, and bioaccumulation. Conventional techniques for determining pesticide residues in citrus fruits are chromatographic methods coupled with different detectors. However, in recent years, the need for analytical strategies that are less polluting for the environment has encouraged the appearance of new alternatives, such as sensors and biosensors, which allow selective and sensitive detection of pesticide residues in real time. A comprehensive overview of the analytical platforms used to determine pesticide residues in citrus fruits and citrus-derived products is presented herein. The review focuses on the evolution of these methods since 2015, their limitations, and possible future perspectives for improving pesticide residue determination and reducing environmental contamination.

Liquid-liquid and solid-liquid extractions with low-temperature partitioning - A review.

The paper represents the first review of solvent extraction techniques utilizing the low-temperature partitioning/purification (LTP) approach. Initially conceived in the 1960s to purify extracts from fatty matrices, it wasn't until the 2000s that this approach received increasing attention for its efficacy in extracting organic compounds from diverse samples, often without additional cleanup steps. This review covers a brief history and proposes a mechanism for LTP-based solvent extraction. Furthermore, the principal practical issues of the technique are spotlighted, elucidating the factors influencing extraction efficiency. The advantages, limitations, and potential combinations with other extraction techniques of the LTP-based solvent extractions are analyzed. The versatility of the LTP approach is demonstrated by its applications in extracting various compounds from food, environmental, and biological samples, emphasizing its potential for rapid sample preparation with minimal steps, few chemicals, and minimal analyst intervention.

Robust assessment of sample preparation protocols for proteomics of cells and tissues.

In proteomic studies, the reliability and reproducibility of results hinge on well-executed protein extraction and digestion protocols. Here, we systematically compared three established digestion methods for macrophages, namely filter-assisted sample preparation (FASP), in-solution, and in-gel digestion protocols. We also compared lyophilization and manual lysis for liver tissue protein extraction, each of them tested using either sodium deoxycholate (SDC)- or RIPA-based lysis buffer. For the macrophage cell line, FASP using passivated filter units outperformed the other tested methods regarding the number of identified peptides and proteins. However, a careful standardization has shown that all three methods can yield robust results across a wide range of starting material (even starting with 1 µg of proteins). Importantly, inter and intra-day coefficients of variance (CVs) were determined for all sample preparation protocols. Thus, the median inter-day CVs for in-solution, in-gel and FASP protocols were respectively 10, 8 and 9%, very similar to the median CVs obtained for the intra-day analysis (9, 8 and 8%, respectively). Moreover, FASP digestion presented 80% of proteins with a CV lower than 25%, followed closely by in-gel digestion (78%) and in-solution sample preparation (72%) protocols. For tissue proteomics, both manual lysis and lyophilization presented similar proteome coverage and reproducibility, but the efficiency of protein extraction depended on the lysis buffer used, with RIPA buffer showing better results. In conclusion, although each sample preparation method has its own particularity, they are all suited for successful proteomic experiments if a careful standardization of the sample preparation workflow is carried out.

Microwave-induced combustion for further determination of potentially toxic elements in honey by ICP-MS.

Microwave-induced combustion (MIC) was proposed in this study for honey decomposition aiming for As, Cd, Hg, and Pb determination by inductively coupled plasma mass spectrometry (ICP-MS). Sample mass (up to 1.0 g), absorbing solution (0.5 to 14.4 mol L-1 HNO3, and H2O), heating program, and combustion aids were evaluated. The Eurachem guidelines were used for method validation. The proposed method enabled combustion of a high sample mass (0.8 g of honey, with 0.4 g of microcrystalline cellulose and 100 µL of 6 mol L-1 NH4NO3) using 6 mL of an absorbing solution consisting of 1 mol L-1 HNO3, which resulted in low residual carbon in solution (< 25 mg L-1). Honey samples from different geographical origins were analyzed. Results showed no significant difference in comparison to other two microwave decomposition methods, based on microwave-assisted wet digestion with single reaction chamber (MAWD-SRC) and microwave-assisted wet digestion (MAWD). Standard addition experiments resulted in recoveries higher than 98%. The limits of detection ranged from 1.10 (As) to 4.60 ng g-1 (Pb). In addition to using only diluted reagents and resulting in digests virtually free of interferences, the proposed method was faster (< 30 min) than most of those presented in the literature.

Layer-by-layer assembly of PDDA/MWCNTs thin films as an efficient strategy for extraction of organic compounds from complex samples.

This article presents the assembly and characterization of poly(diallyldimethylammonium chloride)/multi-walled carbon nanotubes (PDDA/MWCNTs) thin films on borosilicate bottles using a layer-by-layer (LBL) approach. The thin films, consisting of 10 bilayers of coating materials, were thoroughly characterized using UV-VIS spectroscopy, scanning electron microscopy (SEM), and zeta potential measurements. The modified bottles were then utilized for the extraction of analytes with diverse acid-base characteristics, including drugs, illicit drugs, and pesticides, from saliva, urine, and surface water samples. The studied analytes can be adsorbed on the surface of the LBL film mainly through hydrogen bonding and/or hydrophobic interactions. Remarkably high extraction percentages of up to 92 % were achieved, accompanied by an impressive enhancement in the analytical signal of up to 12 times when the sample volume was increased from 0.7 to 10 mL. These results highlight the outstanding extraction and sorption capabilities of the developed material. Additionally, the (PDDA/MWCNTs)10 films exhibited notable resistance to extraction and desorption processes, enabling their reuse for at least 5 cycles. The straightforward and cost-effective fabrication of these sorbent materials using the LBL technique, combined with the ability to extract target compounds during sample transportation and/or storage, renders this sample preparation method a promising alternative.

Low cost sample preparation method using ultrasound for the determination of environmentally critical elements in seaweed.

In this study, ultrasound (US) was evaluated for As, Cd, Pb, Mn, Sr and V extraction from seaweed samples. The following parameters of ultrasound-assisted extraction (UAE) using an US bath were: frequency (25 to 130 kHz), amplitude (30 to 100%), temperature (30 to 80 °C), sample mass (50 to 200 mg), extractant concentration (1 to 3 mol L-1 of HNO3) and treatment time (5 to 30 min). Acoustic density and power density distribution were calculated using the calorimetric method and mapping of the acoustic pressure distribution was also evaluated. The optimized UAE conditions were 200 mg of sample in 10 mL of 2 mol L-1 HNO3 and 30 min of sonication in a 25 kHz US bath (37.2 ± 4.0 W L-1) at 70% of amplitude and 70 °C. Analytes were quantified using inductively coupled plasma mass spectrometry and results were compared with values obtained using "silent" conditions (magnetic or mechanical stirring at 500 rpm, and without stirring), and a reference method based on microwave-assisted wet digestion (MAWD). The UAE method demonstrated the best extraction efficiency (higher than 95%) for all analytes, especially for As, Cd and V, with lower standard deviations (up to 5%) and lower blank values in comparison with the silent conditions. The proposed UAE method was more advantageous than the reference method, being faster, simpler, safer, more environmentally friendly, and with higher detectability (lower limits of quantification, from 0.0033 to 1.34 µg g-1). In addition, negligible blank values were obtained for UAE and no interference were observed in the determination step. Furthermore, the optimized UAE method was applied for Antarctic seaweed samples and comparison with results obtained by MAWD was satisfactory. In this sense, UAE is demonstrated to be a suitable option for sample preparation of seaweed samples and further determination of environmentally critical elements avoiding the use of concentrated reagents as in the MAWD reference method.

Pyrohydrolysis as a sample preparation method for the subsequent halogen determination: A review.

The use of pyrohydrolysis as a sample preparation method for further halogen determination is extensively discussed in this review, covering studies published in the last 30 years. This method is compatible with both organic (such as cellulose, fossil fuels, carbon nanotubes, and graphite) and inorganic (such as rocks, silicates, alumina, and nuclear fuels) matrices. It has also been used for samples with different organic matter content, such as coal, mineral supplements, and soil. Sample masses vary greatly and are dependent on organic matter content in the samples, ranging from 50 mg to up to 500 mg for organic samples, and up to 4 g to inorganic samples. Different additives, such as V2O5 and cellulose, or flame retardants, such as silica, could also be used to improve analyte recovery using pyrohydrolysis. Dilute alkaline solutions or even water have been used as absorbing solutions, with mainly NaOH, NH4OH, and mixtures of NaHCO3 and Na2CO3 being applied. Furthermore, pyrohydrolysis is compatible with detection techniques such as ion chromatography, inductively coupled plasma mass spectrometry, ion selective electrode, inductively coupled plasma optical emission spectrometry, energy-dispersive X-ray fluorescence spectrometry, spectrophotometry, and isotope ratio mass spectrometry. Other advantages usually related to this method are the low residual carbon concentration of digests and the low residue generation. A critical comparison with alkaline extraction, alkaline fusion, Schöniger oxygen flask combustion, combustion bomb and microwave-induced combustion is also provided.

A new ultrasound-assisted microextraction for elemental impurities determination in tablets by ICP-MS according to USP chapters 232 and 233.

Along with the United States Pharmacopeia (USP) chapters 232 and 233 regarding elemental impurities in pharmaceutical products, new challenges have been imposed in terms of sample preparation procedures prior to inductively coupled plasma mass spectrometry analysis, considering the matrix complexities. As so, a new microextraction procedure assisted by ultrasound using a cup-horn sonoreactor, minimal reactants, and sample was proposed and validated according to USP. The procedure was optimized with samples of milled tablets and 3 different acid mixtures (HNO3, 3HNO3:1HCl, and 9HNO3:1HF) and it was compared with microwave-assisted acid digestion. In the validation step, recoveries ranging from 85 to 120 % and RSD below 10 % were obtained for 22 analytes (except Ag and Pt) with satisfactory linearity and good sensitivity. The method was then applied for 37 samples of antidepressants, which presented trace levels of As, Ba, Cd, Co, Cr, Cu, Ni, Pb, Pd, Sn, and V.

Polyphenol analysis in human milk by a rapid, cost-effective, and accurate micromethod: translational development / Análise de polifenóis no leite humano por meio de um micrométodo rápido, econômico e preciso: desenvolvimento translacional

ABSTRACT Objective: To develop a rapid method for analysing polyphenols, which are potentially active antioxidants against neonatal oxidative stress, from small human milk (HM) volumes. Methods: Acid and alkaline extractions were compared using two dyes: Folin-Ciocalteu and Fast Blue BB. Linearity, sensitivity, recovery percentage, polyphenol content, precision, and stability were assessed in 14 HM samples and compared using the Kruskal-Wallis H test (p<0.05). The best technique was applied to 284 HM samples to determine their polyphenolic content and its association with maternal diet by multifactorial linear regression. Results: Acidic extraction successfully recovered the gallic acid reference standard, whereas alkaline extraction overestimated it. Calibration curves for all methods were linear (R2>0.96) up to 500 mg/L. All bicarbonate-based Folin-Ciocalteu methods assayed were stable and repeatable, whereas Fast Blue BB-based variants were not. HM polyphenols (mean=94.68 mg/L) positively correlated to the dietary intake of hydroxycinnamic acids, the most consumed polyphenolic family in this population. Conclusions: A bicarbonate-based Folin-Ciocalteu micromethod allowed the accurate determination of polyphenols in HM, which might be useful for translational research settings and HM banks.

RESUMO Objetivo: Desenvolver um método rápido para analisar polifenóis, que são antioxidantes potencialmente ativos contra o estresse oxidativo neonatal, em pequenos volumes de leite humano (LH). Métodos: Foram comparadas extrações ácidas e alcalinas usando dois corantes: Folin-Ciocalteu e Fast Blue BB. Foram avaliadas variáveis como linearidade, sensibilidade, percentagem de recuperação, teor de polifenóis, precisão e estabilidade em 14 amostras de LH, comparadas usando o teste de Kruskal-Wallis H (p<0,05). A melhor técnica foi aplicada a 284 amostras de LH para determinar seu teor polifenólico e sua associação com a dieta materna por regressão linear multifatorial. Resultados: A extração ácida recuperou com sucesso o padrão de referência do ácido gálico, enquanto a extração alcalina o superestimou. As curvas de calibração para todos os métodos foram lineares (R2>0,96) até os 500 mg/L. Todos os métodos testados baseados em Folin-Ciocalteu com bicarbonato foram estáveis e repetíveis, enquanto as variantes baseadas em Fast Blue BB não. Os polifenóis do HM (média=94,68 mg/L) correlacionaram-se positivamente com a ingestão dietética de ácidos hidroxicinâmicos, a família de polifenóis mais consumida nesta população. Conclusões: Um micrométodo baseado em bicarbonato de Folin-Ciocalteu permitiu a determinação precisa de polifenóis no HM, o que pode ser útil para configurações de pesquisa translacional e bancos de HM.

Should we think about green or white analytical chemistry? Case study: Accelerated sample preparation using an ultrasonic bath for the simultaneous determination of Mn and Fe in beef.

Green Analytical Chemistry (GAC) metrics include a variety of criteria, such as the regent amounts and toxicity, energy consumption, generated waste, among others. The analytical greenness metric (AGREE) and its variant for sample preparation (AGREEprep) cover different aspects that contribute to the environmental sustainability of sample preparation. White Analytical Chemistry (WAC) considers not only environmental aspects but also analytical and practical aspects with a holistic vision based on a Red-Green-Blue color model. A case study is presented to assess the green and white profile of a method based on ultrasound-assisted extraction and determination of Mn and Fe in beef using microwave-induced plasma atomic emission spectroscopy (MP AES). The method was validated and resulted simple, fast without external heating using diluted acids. It was concluded that we should think in green sample preparation with the AGREEprep tool, as well as in white holistic assessments (WAC) as both constitute complementary tools.

Sample Preparation in Microbial Metabolomics: Advances and Challenges.

Microbial metabolomics has gained significant interest as it reflects the physiological state of microorganisms. Due to the great variability of biological organisms, in terms of physicochemical characteristics and variable range of concentration of metabolites, the choice of sample preparation methods is a crucial step in the metabolomics workflow and will reflect on the quality and reliability of the results generated. The procedures applied to the preparation of microbial samples will vary according to the type of microorganism studied, the metabolomics approach (untargeted or targeted), and the analytical platform of choice. This chapter aims to provide an overview of the sample preparation workflow for microbial metabolomics, highlighting the pre-analytical factors associated with cultivation, harvesting, metabolic quenching, and extraction. Discussions focus on obtaining intracellular and extracellular metabolites. Finally, we introduced advanced sample preparation methods based on automated systems.

An open-source smart fraction collector for isocratic preparative liquid chromatography.

Preparative liquid chromatography is a technique for separating complex samples or isolating pure compounds from complex extracts. It involves eluting samples through a packed column and selectively collecting or isolating the separated bands in a sequence of fractions. Depending on the column length and the sample complexity, a large number of fractions may be obtained, making fraction collection a laborious and time-consuming process. Manual fraction collection is also tedious, error-prone, less reproducible, and susceptible to contamination. Several commercial and lab-made solutions are available for automated fraction collection, but most systems do not synchronize with the instrument detector and collect fractions at fixed volumes or time intervals. We have assembled a low-cost Arduino-based smart fraction collector that can record the signal from the UV-vis detector of the chromatography instrument and enable the automated selective collection of the targeted bands. The system consists of a robot equipped with position sensors and a 3-way solenoid valve that switches the column effluent between the waste or collection positions. By proper programming, an Arduino board records the detector response and actuates the solenoid valve, the position sensors, and the stepper motors to collect the target chromatographic bands.

Automated microextraction by packed sorbent of endocrine disruptors in wastewater using a high-throughput robotic platform followed by liquid chromatography-tandem mass spectrometry.

An automated microextraction by packed sorbent followed by liquid chromatography-tandem mass spectrometry (MEPS-LC-MS/MS) method was developed for the determination of four endocrine disruptors-parabens, benzophenones, and synthetic phenolic antioxidants-in wastewater samples. The method utilizes a lab-made repackable MEPS device and a multi-syringe robotic platform that provides flexibility to test small quantities (2 mg) of multiple extraction phases and enables high-throughput capabilities for efficient method development. The overall performance of the MEPS procedure, including the investigation of influencing variables and the optimization of operational parameters for the robotic platform, was comprehensively studied through univariate and multivariate experiments. Under optimized conditions, the target analytes were effectively extracted from a small sample volume of 1.5 mL, with competitive detectability and analytical confidence. The limits of detection ranged from 0.15 to 0.30 ng L-1, and the intra-day and inter-day relative standard deviations were between 3 and 21%. The method's applicability was successfully demonstrated by determining methylparaben, propylparaben, butylated hydroxyanisole, and oxybenzone in wastewater samples collected from the São Carlos (SP, Brazil) river. Overall, the developed method proved to be a fast, sensitive, reliable, and environmentally friendly analytical tool for water quality monitoring.

Polyaniline-silica doped with oxalic acid as a novel extractor phase in thin film solid-phase microextraction for determination of hormones in urine.

In this study, different polyanilines were synthesized and evaluated for the determination of three hormones, including 17-ß-estradiol, 17-α-ethinylestradiol, and estrone, in urine using a novel methodology based on thin film solid-phase microextraction technique, employing the sampling well plate system. The extractor phases, designated as polyaniline doped with hydrochloric acid, polyaniline doped with oxalic acid, polyaniline-silica doped with hydrochloric acid, and polyaniline-silica doped with oxalic acid, were characterized by electrical conductivity measurements, scanning electron microscopy, and Fourier transform infrared spectroscopy. The optimized extraction conditions were composed of 1.5 mL of urine and pH adjusted to 10, with no need to dilute sample and the desorption step, 300 µL of acetonitrile was used. The calibration curves were performed in the sample matrix, with detection and quantification limits ranged from 0.30 to 3.03 µg L-1 and from 1.0 to 10.0 µg L-1 , respectively, with r ≥ 0.9969. The relative recoveries ranged from 71% to 115%, and intraday precision showed values ≤12% and interday ≤20%. The applicability of the method was successfully evaluated, and six urine samples from female volunteers were analyzed. The analytes were not detected or were below the limits of quantification in these samples.

Recent Trends in Graphene-Based Sorbents for LC Analysis of Food and Environmental Water Samples.

This review provides an overview of recent advancements in applying graphene-based materials as sorbents for liquid chromatography (LC) analysis. Graphene-based materials are promising for analytical chemistry, including applications as sorbents in liquid chromatography. These sorbents can be functionalized to produce unique extraction or stationary phases. Additionally, graphene-based sorbents can be supported in various materials and have consequently been applied to produce various devices for sample preparation. Graphene-based sorbents are employed in diverse applications, including food and environmental LC analysis. This review summarizes the application of graphene-based materials in food and environmental water analysis in the last five years (2019 to 2023). Offline and online sample preparation methods, such as dispersive solid phase microextraction, stir bar sorptive extraction, pipette tip solid phase extraction, in-tube solid-phase microextraction, and others, are reviewed. The review also summarizes the application of the columns produced with graphene-based materials in separating food and water components and contaminants. Graphene-based materials have been reported as stationary phases for LC columns. Graphene-based stationary phases have been reported in packed, monolithic, and open tubular columns and have been used in LC and capillary electrochromatography modes.

A simple, low cost and fast sample preparation method for fluorine determination by ISE in leguminous seeds and oilseeds.

This work presents the first report of the application of microwave-induced combustion in disposable vessels (MIC-DV) as a simple and fast sample preparation step for fluoride determination by ion-selective electrode (ISE) in leguminous seeds and oilseeds samples. Several experimental conditions of MIC-DV were evaluated, such as sample mass, absorbing solution, volume of combustion igniter, purging time with oxygen, and the use of successive combustion cycles. The accuracy of MIC-DV/ISE method was evaluated by comparing the results obtained by microwave-induced combustion and determination by ion chromatography, with agreements ranged from 94 to 106%. The method allowed to achieve relatively low limits of quantification (8.3 to 16 µg g-1) comparing to AOAC method (40 µg g-1), besides the use of small amounts of reagents, less handling of digests, making the method greener and with portable features comparing to other methods.

Sample pooling and incurred samples improve analytical throughput and quality control of lipophilic phycotoxins screening in bivalve mollusks.

Lipophilic marine biotoxins (LMBs) are one of the main risks associated with the consumption of mussels and oysters. Sanitary and analytical control programs are developed to detect the occurrence of these toxins in seafood before they reach toxic levels. To ensure quick results, methods must be easy and fast to perform. In this work, we demonstrated that incurred samples were a viable alternative to validation and internal quality control studies for the analysis of LMBs in bivalve mollusks. These samples were used to optimize, validate, and monitor a simple and fast ultrasound-assisted extraction (UAE) procedure. An internal quality control material containing okadaic acid (227 ± 46 µg kg-1) was produced and characterized. This material had its homogeneity and stability verified and was included as a quality control in all batches of analytical routine. Besides, a sample pooling protocol for extracts analysis was developed, based on tests for COVID-19. Up to 10 samples could be analyzed simultaneously, reducing the instrumental time of analysis by up to 80%. The UAE and sample pooling approaches were then applied to more than 450 samples, of which at least 100 were positive for the okadaic acid group of toxins.