Liquid Chromatography-fluorescence Detection Research Articles

Fabric phase sorptive extraction increases the accuracy of liquid chromatography-fluorescence detection of zearalenone in food samples

ABSTRACT The accuracy of mycotoxins detection in the food samples has always been controversial and more sensitive methods help better regulatory activities and, therefore, the quality of final products. Zearalenone is a toxic substance from mycotoxins mainly produced by Fusarium fungi that has proven endocrine effects and is potentially carcinogenic. This research aimed to apply the fabric phase sorptive extraction (FPSE) technique to the liquid chromatography- fluorescence detection method to increase the sensitivity and accuracy of zearalenone extraction and determination in food samples. Applying graphene nanoparticles in the sol-gel process modifies the method depending on the amount of nanoparticles, volume and type of the back-extraction solvent, sample pH, ionic strength, and back-extraction time, which are all considered and investigated. At analytical conditions, the limit of quantification (LOQ) 16.57 and detection (LOD) of 4.97 ng/mL were observed. The method’s accuracy was determined by three spiking levels of 100, 150, and 200 ng/mL in the real samples. The mean recovery percentage in wheat were 61.69, 70.56, and 97.95%, and in the rice, 58.13, 66.00, and 102.59, respectively. The average percentages of the relative standard deviation of the instrument for intra-and inter-laboratory repeatability were less than 1.7%. This work provided a superior analytical condition to the conventional detection procedures in food safety laboratories.

High-Performance Liquid Chromatography-Fluorescence Detection Method for Ochratoxin A Quantification in Small Mice Sample Volumes: Versatile Application across Diverse Matrices Relevant for Neurodegeneration Research.

Ochratoxin A (OTA) is a mycotoxin commonly found in various food products, which poses potential health risks to humans and animals. Recently, more attention has been directed towards its potential neurodegenerative effects. However, there are currently no fully validated HPLC analytical methods established for its quantification in mice, the primary animal model in this field, that include pivotal tissues in this area of research, such as the intestine and brain. To address this gap, we developed and validated a highly sensitive, rapid, and simple method using HPLC-FLD for OTA determination in mice tissues (kidney, liver, brain, and intestine) as well as plasma samples. The method was rigorously validated for selectivity, linearity, accuracy, precision, recovery, dilution integrity, carry-over effect, stability, and robustness, meeting the validation criteria outlined by FDA and EMA guidelines. Furthermore, the described method enables the quantification of OTA in each individual sample using minimal tissue mass while maintaining excellent recovery values. The applicability of the method was demonstrated in a repeated low-dose OTA study in Balb/c mice, which, together with the inclusion of relevant and less common tissues in the validation process, underscore its suitability for neurodegeneration-related research.

Establishment of the HPLC fluorescence detection method for plasma trace ivermectin and its pharmacokinetics in Bactrian camel.

Ivermectin (IVM), a widely used veterinary anthelmintic, lacks recommended doses for Bactrian camels. This study aims to establish its pharmacokinetics in Bactrian camels, comparing with other livestock. A method for high-performance liquid chromatography fluorescence detection of IVM in plasma was developed. IVM exhibited linear scaling (y=0.6946x+0.0088, R2=0.9988) within 0.025-5ng/mL, with a lower limit of quantification of 25.00pg/mL, high recovery (>70%) and low RSD (<7%). In Bactrian camels, IVM injection showed a low Cmax, extended Tmax and apparent secondary absorption compared to cattle and sheep. Slow absorption and widespread distribution were observed, with peak concentration and area under the curve correlating positively with the dose. This study provides insights into IVM pharmacokinetics in Bactrian camels, informing dose determination and highlighting potential metabolic differences compared to other livestock.

An integrated method for rapid extraction of fluorescent whitening agents from selected cosmetics by in situ formation of deep eutectic solvent

A novel extraction methodology was developed based on deep eutectic solvent (DES) in situ formation. The solvent was synthesized from a binary mixture of L-cysteine, functioning as a hydrogen bond acceptor, and 2-methylphenol, serving as a hydrogen bond donor. The DES’s inherent molecular structures and extensive hydrogen bonding networks were characterized using the Fourier-transform infrared spectroscopy (FT-IR), while its hydrophobic nature was assessed by determining molecular polarity. Its extraction efficiency was demonstrated through the extraction of fluorescent whitening agents (FWAs) in cosmetic products, acting as a model analytical application. This was accomplished using vortex-assisted emulsification solid–liquid extraction (SLE) method, followed by high-performance liquid chromatography-fluorescence detection. Response surface methodology was employed to optimize extraction conditions in a multivariate experimental design. This approach streamlined the multi-operation extraction process via ingeniously integrating extraction and clean-up into a single step, resulting in significant reductions in extraction time required and high sample throughputs of approximately 40 samples per hour. The methodology was validated and exhibited excellent linearity in concentration ranges of 0.01–24 mg/L for five FWAs and 0.01–40 mg/L for other three FWAs, with correlation coefficients (r) ranging from 0.9985 to 1.000. The limits of detection and quantification were in the range of 0.03–0.26 mg kg−1 and 0.10–0.87 mg kg−1, respectively. The reliability and practicality of the proposed method were affirmed by quantifying eight FWAs in ninety-four commercially available facial masks and whitening creams, yielding recoveries values between 90.0 % and 106.0 % with relative standard deviations (RSDs) of 1.0–4.3 %. Inorganic ions and organic compounds present in the samples had minimal impact on FWAs detection following sample pre-treatment. This procedure showcased exceptional extraction capabilities towards a diverse array of FWAs with the aid of multiple interactions, suggesting significant potential for increasing sample throughput while reducing organic solvent and material consumptions compared with conventional SLE methods. The low-toxicity DES is an excellent alternative to traditional extractants typically applied to SLE techniques.

Determination of ochratoxin A in brown rice through a rapid, straightforward strategy based on FaTEx coupled with high-performance liquid chromatography–fluorescence detection

Ochratoxin A is a mycotoxin commonly found in cereals. In this study, a rapid and straightforward method was developed for measuring trace concentrations of ochratoxin A in brown rice; the method combines the fast toxin extraction (FaTEx) technique with high-performance liquid chromatography–fluorescence detection. The proposed method was validated and exhibited high performance and satisfactory accuracy (91.6%–99.2%), precision (relative standard deviation < 3%), linearity (R2 > 0.9999), sensitivity (limit of quantification = 1.0 ng/g), and matrix effects (magnitude = −2%). The proposed method is simple, accurate, inexpensive, reliable, and environmental friendly and requires minimal sample preparation. It was successfully used to analyze brown rice samples from markets and rice shops in Taiwan. To our knowledge, no study has reported the use of FaTEx for the extraction of ochratoxin A from cereals. The proposed method overcomes the shortcomings of traditional column cleanup and satisfies current food safety and analysis demands.

Determination of bisphenols in beeswax based on sugaring out–assisted liquid–liquid extraction: Method development and application in survey, recycling and degradation studies

The methodology of sugaring out–assisted liquid–liquid extraction (SULLE) coupled with high-performance liquid chromatography–fluorescence detection was devised for quantifying bisphenol A (BPA) and bisphenol B (BPB) in beeswax. The effectiveness of SULLE was methodically explored and proved superior to the salting out–assisted liquid–liquid extraction approach for beeswax sample preparation. The analytical performance underwent comprehensive validation, revealing detection limits of 10 μg/kg for BPA and 20 μg/kg for BPB. The method developed was employed to analyse commercial beeswax (n = 15), beeswax foundation (n = 15) and wild-build comb wax (n = 26) samples. The analysis revealed BPA presence in four commercial beeswax samples and three beeswax foundation samples, with the highest detected residue content being 88 ± 7 μg/kg. For BPB, two beeswax foundation samples were positive, with concentrations below the limits of quantification and 85 ± 4 μg/kg, respectively. No bisphenols were detected in wild-build comb wax. Furthermore, the bisphenol removal efficacy of two recycling methods—boiling in water and methanol extraction—was assessed. The findings indicated that after four recycling cycles using water boiling, 9.6% of BPA and 29.2% of BPB remained in the beeswax. Whereas methanol extraction resulted in approximately 7% residual after one recycling process. A long-term study over 210 days revealed the slow degradation of bisphenols in comb beeswax. This degradation fitted well with a first-order model, indicating half-lives (DT50) of 139 days for BPA and 151 days for BPB, respectively. This research provides the first report on bisphenol contamination in beeswax. The low removal rate during the recycling process and the gradual degradation in beeswax underscore the significance of bisphenol contamination and migration in bee hives along with their potential risk to pollinators warranting concern. Furthermore, the developed SULLE method shows promise in preparing beeswax samples to analyse other analytes.

Development of a QuEChERS-HPLC-FLD Procedure for the Simultaneous Detection of Residues of Florfenicol, Its Metabolite Florfenicol Amine, and Three Fluoroquinolones in Eggs.

A method utilizing high-performance liquid chromatography-fluorescence detection (HPLC-FLD) has been developed and refined for the simultaneous detection of florfenicol (FF) and its metabolite florfenicol amine (FFA) along with three fluoroquinolone (ciprofloxacin (CIP), enrofloxacin (ENR), and sarafloxacin (SAR)) residues in different parts of eggs (whole egg, egg yolk, and egg albumen). The QuEChERS ("Quick, easy, cheap, effective, rugged, and safe") procedure utilized 0.1 M disodium EDTA solution, water, and acetonitrile as extractants; sodium sulfate, sodium chloride, and trisodium citrate as dehydrating salts; and N-propylethylenediamine and C18 as adsorbents. A dual-channel FLD method was utilized to analyze the target compounds using an XBridge BEH C18 chromatographic column (4.6 mm × 150 mm, 5 μm). The mobile phase was employed isocratically using a solution of 0.01 M sodium dihydrogen phosphate, 0.005 M sodium dodecyl sulfate, and 0.1% triethylamine (pH 4.8) in combination with acetonitrile at a ratio of 65:35 (V/V). The limits of detection (LOD) and quantification (LOQ) of the analytes ranged from 0.03 to 1.5 µg/kg and from 0.1 to 5.0 µg/kg, respectively. The recoveries of the analytes in the blank egg samples ranged from 71.9% to 94.8% when reference standard concentrations of the LOQ, half of the maximum residual limit (MRL), MRL, and twice the MRL were added. The parameters of the presented protocol were validated and subsequently applied to the analysis of real samples, demonstrating the applicability and reliability of the method.

Liquid Chromatography and Capillary Electrophoresis Analysis of 2AA-Labeled O-Glycans.

To reveal O-glycan structures in mucins, it is necessary to release covalently bound O-glycans from the polypeptide backbone and derivatize to a form suitable for structural analysis. Various derivatization methods can now be applied in the analysis of O-glycans following the development of O-glycan release methods. Among the many derivatization methods available, we prefer to use fluorescent labeling with 2-aminobenzoic acid (anthranilic acid, 2AA). 2AA-labeled O-glycans can be detected with high sensitivity using liquid chromatography fluorescence detection (LC-FD) analysis because of the strong fluorescence. In addition, as 2AA has a carboxyl group that carries a negative charge, 2AA-labeled O-glycans can be analyzed with high sensitivity in negative ion mode mass spectrometry. Furthermore, because the negative charge of 2AA provides a driving force for electrophoresis, 2AA-labeled O-glycans can be analyzed using capillary electrophoresis (CE) and capillary affinity electrophoresis. High detection sensitivity and versatility are key advantages of the 2AA-labeling method. Here we present our preferred LC-FD and CE analytical methods for 2AA-labeled O-glycans.

9-Fluorenylmethyl Chloroformate Labeling for O-Glycan Analysis.

Structural analysis of O-glycans from mucins and characterization of the interaction of these glycans with other biomolecules are essential for a full understanding of mucins. Various techniques have been developed for the structural and functional analysis of glycans. While 9-fluorenylmethyl chloroformate (Fmoc-Cl) is generally used to protect amino groups in peptide synthesis, it can also be used as a glycan-labeling reagent for structural analysis. Fmoc-labeled glycans are strongly fluorescent and can be analyzed with high sensitivity using liquid chromatography-fluorescence detection (LC-FD) analysis as well as being analyzed with high sensitivity by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Fmoc-labeled glycans can be easily delabeled and converted to glycosylamine-form or free (hemiacetal or aldehyde)-form glycans that can be used to fabricate glycan arrays or synthesize glycosyl dendrimers. This derivatization allows for the isolation from biological samples of glycans that are difficult to synthesize chemically, as well as the fabrication of immobilized-glycan devices. The Fmoc labeling method promises to be a tool for accelerating O-glycan structural analysis and an understanding of molecular interactions. In this chapter, we introduce the Fmoc labeling method for analysis of O-glycans and fabrication of O-glycan arrays.

A novel method for evaluating pseudoallergy based on β-hexosaminidase and its application for traditional Chinese medicine injections.

Pseudoallergy is a typical and common adverse drug reaction to injections, especially in traditional Chinese medicine injections (TCMIs). At present, the evaluation methods for pseudoallergy include cell methods in vitro and animal methods in vivo. The mast cell evaluation method based on the β-hexosaminidase (β-Hex)-catalyzed substrate, 4-nitrophenyl-β-N-acetyl-D-glucosaminide (4-NPG), is an important method for the evaluation of drug-induced pseudoallergy, but it is prone to false positive results and has insufficient sensitivity. In this study, a novel β-Hex evaluation system with rat basophilic leukemia-2H3 cells based on high-performance liquid chromatography-fluorescence detection (HPLC-FLD) was established, which effectively increased the sensitivity and avoided false positive results. Cell viabilities were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay. In addition, a method for the determination of histamine, which is another indicator in the development of pseudoallergy, was established to validate the above method. The results of this novel method indicated that two TCMIs (Shuxuening injection and Shenqi Fuzheng injection), which were considered to be pseudoallergenic using 4-NPG, were not pseudoallergenic. Overall, the novel β-Hex/HPLC-FLD evaluation system using Rat basophilic leukemia-2H3 cells established was effective and precise. It could be used for the evaluation of pseudoallergic reactions caused by TCMIs and other injections.

One-pot derivatization/magnetic solid-phase extraction coupled with liquid chromatography-fluorescence detection for the rapid determination of sulfonamide residues in honey

Consuming foods with excess sulfonamide residues threatens human health, underscoring the importance of their detection in food. This study presents an innovative one-pot derivatization/magnetic solid-phase extraction (OPD/MSPE) method for sulfonamides analysis. This approach integrates the derivatization and extraction steps into a single process. The sample solution, along with the derivatization reagent fluorescamine and the sorbent magnetic hydroxyl multi-walled carbon nanotubes, is mixed and vortexed for 3 min. This procedure simultaneously conducts derivatization and extraction, with easy phase separation using an external magnet. This streamlined sample preparation method is completed in only 5 min and, when combined with liquid chromatography-fluorescence detection (LC-FLD), demonstrates excellent linearity (R2 > 0.99) and satisfactory detection limits (0.004–0.04 ng/g) for the quantification of nine sulfonamides in honey samples. The proposed OPD/MSPE-LC-FLD method is distinguished by its simplicity, rapidity, high sensitivity, and specificity, making it an outstanding advancement in the field of food safety analysis.

Determination of intracellular dopamine by liquid chromatography–fluorescence detection with post-column derivatization using the König reaction

Dopamine is an important neurotransmitter, and the disruption of dopaminergic homeostasis causes various neurological diseases such as Parkinson’s disease. Analysis of intracellular dopamine levels is important to understand the pathology of neurological diseases. We have developed a new method for the fluorometric detection of dopamine by adopting the König reaction, which is commonly used for the detection of cyanide, thiocyanate, and selenocyanate, and demonstrated that it can be applied to the determination of intracellular dopamine levels. The present method only requires a conventional LC system with isocratic elution and post-column derivatization and is simple to perform. The LOD, LOQ, and linearity range were 10.8 nM, 32.8 nM, and 0.05–10 μM, respectively, with accuracies of 101.8–106.3 % and precisions within 5 %, which are sufficient for the quantification of intracellular dopamine. We also determined dopamine levels in PC12 cells and found that the levels increased and decreased when the cells were exposed to L-dopa and cyanide, respectively, possibly because of the conversion of L-dopa into dopamine and the depletion of intracellular dopamine by exposing cells to cyanide, respectively. These results suggest the applicability of the present method, and that this new use of the König reaction offers a reliable and useful means of quantifying intracellular dopamine.

Adaptive polarity of graphene oxide anchored silica doped with C18 for effective enrichment of aflatoxins from foodstuff

Selecting adsorbent with appropriate polarity is a practicable way to improve the enrichment efficiency for aflatoxins (AFs) during the solid phase extraction in sample pretreatment. In this study, graphene oxide anchored silica was prepared and applied to dop with C18 with suitable polarity for effective pre-concentration and purification of AFs from foodstuff. With systematic optimizing, GO anchored silica/C18 (1/1, w/w) exhibited excellent extraction performance for AFs. As the medium, a facile and efficient pipette-tip solid-phase extraction (PT-SPE) technique was developed for enrichment of AFs. Coupling with high-performance liquid chromatography fluorescence detection (HPLC-FLD), the developed analysis method illustrated the lower limits of detection of 0.075 ng/g and 0.17 ng/g for AFG2, AFB2 and AFG1, AFB1 (R2 > 0.99). The recoveries were calculated as 71.27 ∼ 94.52 % for the spiking level of 1, 5, and 20 ng/g with the intra-day and inter-day precision of 1.30 to 4.01 and 3.56 to 5.78, respectively. Hence, the developed PT-SPE-HPLC-FLD determination method was sensitive, accurate, and convenient for quantitative analysis of AFs at trace contents in foodstuff.

A novel deep eutectic solvent as an extractant for the determination of bisphenol compounds in edible oils by high-performance liquid-phase fluorescence detection

In this study, an efficient, low-cost and environmentally friendly deep eutectic solvent (DES) was prepared using methanol and betaine, which was then employed as an extractant to detect BPA and BPF in several edible oils by high performance liquid chromatography-fluorescence detection (HPLC-FLD) with good results. The electrostatic potentials of methanol, betaine, water and BPs were analyzed by quantum chemical computational techniques to explain the mechanism of DES extraction. Several important parameters affecting the extraction efficiency, composition, volume and extraction time of DES were optimized. The optimum conditions for the use of DES were the molar ratio of 1:30 (betaine: methanol), the volume of DES of 200 μL and the extraction time of 1.5 min. The results of the method evaluation showed a wide linear range (0.01–0.8 μg g−1) and a high linear correlation coefficient (R2 ≥0.994). The limit of detection (LOD) and limit of quantification (LOQ) values ranged from 1.65 to 2.92 ng g−1 and 5.45–9.64 ng g−1, respectively. The greenness of the assay was evaluated by applying the green analytical procedure index method, and the results showed that the experimental method has the advantages of simple sample pre-treatment and low amount of waste generated during the assay. The experimental method has potential applications in the field of the detection of bisphenol compounds in edible oils.

In Situ Formation of a Relatively Transparent Ion-Associate Liquid Phase from an Aqueous Phase and Its Application to Microextraction/High-Performance Liquid Chromatography-Fluorescence Detection of Bisphenol A in Water.

The design of a simple approach enabling the detection of bisphenol A (BPA) in water samples without the need for large amounts of solvents is of utmost importance. This paper reports a simple method for the separation, concentration, and quantification of BPA in water samples using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) after its microextraction into an in situ formed organic ion-associate (IA) liquid phase (LP). Novel IA phase components without conjugated double bonds, such as benzene rings, were investigated. Ethylhexyloxypropylamine hydrochloride and sodium dodecyl sulfate solutions were added to the water samples to form IAs. The aqueous phase and ion-associate liquid phase (IALP) were separated by centrifugation. The aqueous phase was removed, and the liquid phase was recovered and measured using HPLC-FLD or HPLC-electrochemical detection (ECD). The concentrated phase (IALP) had a relatively low viscosity and could be injected directly into the chromatograph without dissolving it in organic solvents. The detection limits for BPA by HPLC-FLD and HPLC-ECD were 0.009 and 0.3 µg L-1, respectively.

High-performance liquid chromatography with fluorescence detection method for measuring colistin in human serum and its application in critically ill patients treated with colistin methanesulfonate sodium

Colistin, in the form of colistin methanesulfonate sodium (CMS), has a narrow therapeutic window, and colistin levels must be monitored during treatment. The aim of this study was to develop and validate a high-performance liquid chromatography-fluorescence detection (HPLC-FLD) method for measuring colistin in human serum. Colistin was extracted from human serum using trichloroacetic acid and methanol for protein precipitation, followed by in-solid phase extraction derivatization with 9-fluorenylmethyl chloroformate. HPLC-FLD system using a reverse-phase HPLC C18 column and a mobile phase consisting of acetonitrile, tetrahydrofuran, and water (80%:4%:16%) were used for the quantification of colistin A, colistin B, and polymyxin B (internal standard) in human serum. The extraction recoveries were between 71% and 103%. Linear calibration curves were obtained for colistin in concentrations from 0.3 to 8.0 μg/mL, with good fit (r2 = 0.9993). The lower limit of quantitation was 0.3 μg/mL. The intra- and inter-day precision of the assay was 0.5–5% and 3.5–9.4%, respectively. The accuracy ranged from 98% to 100%. This validated method was successfully applied to the analysis of serum-receiving CMS in critically ill patients. This method will be useful to assist colistin dosage adjustment based on individual blood colistin levels for optimization of colistin therapy.

Elucidating the relation between residual oxidants formation behavior and quinolone antibiotic removal efficiency on catalytic ozonation in seawater-based aquaculture wastewater

Owing to the recent developments in aquaculture, the discharge of antibiotic-laden wastewater has become a growing concern. Quinolone antibiotics, which cause central nervous system and cardiac conduction disorders, are commonly detected in aquaculture wastewater. To remove antibiotics in wastewater, the ozone process is a commonly utilized method. In the process of seawater ozone treatment, bromide ions (Br−) produce total residual oxidant, such as hypobromous acid (HOBr). However, the oxidation potential of the total residual oxidant (TRO) is lower than that of ozone, and the presence of inhibitors, such as ammonium, and dimethyl sulfide can reduce the residual oxidant concentration. Therefore, the seawater ozone process requires a higher ozone injection than freshwater. In this study, various factors affecting TRO production were evaluated to determine the optimal ozone-catalytic process using gamma-alumina and elucidate the relationship between TRO production and antibiotic removal efficiency for efficient seawater-based wastewater treatment. The removal rates of representative quinolone antibiotics, such as enrofloxacin, ciprofloxacin, and oxolinic acid, were evaluated using liquid chromatography-fluorescence detection. The addition of a catalyst enabled the reaction to maintain a higher TRO concentration and improves antibiotic removal efficiency, even in the presence of ammonium in the aquaculture wastewater with the same ozone dosage applied. The results reveal that the ozone-catalyst process contributes to increased TRO production to suppress bacterial growth and effectively remove non-degradable organic matter, such as antibiotics, in seawater-based wastewater treatment.

Construction of magnetic hydroxyl group-enriched hyper cross-linked polymers with functional triazine as the core for efficient enrichment of plant growth regulators

The determination of trace plant growth regulators (PGRs) residues in water and food samples make it crucial to develop novel sample pretreatment methods for the enrichment of PGRs. Herein, a novel magnetic hyper cross-linked polymer (M-CTT-9OH-HCP) was constructed and served as a magnetic adsorbent for the efficient extraction of some PGRs from water, watermelon, tomatoes, and milk samples for the first time. Combined with high performance liquid chromatography-fluorescence detection (HPLC-FLD), the established method presented a good linearity (0.03–60.0 ng g−1 (ng mL−1), (r) ≥ 0.9973), satisfactory accuracy with method recoveries (83.0%-119%) and acceptable precision with the intra-day and inter-day variations (expressed as the relative standard deviations (RSDs) ≤ 9.8%). The limits of detection (LODs) and limits of quantitation (LOQs) were in the range of 0.01–1.50 and 0.03–5.00 ng g−1/ ng mL−1. The results show that the established method is sensitive and efficient for the determination of PGRs in real samples.

Study on the effectiveness of a multi-toxin immunoaffinity clean-up for reliable cost-effective HPLC-FLD analysis of mycotoxins in corn-based foods

A different, reliable, and cost-effective strategy for the analysis of aflatoxins, ochratoxin A, and zearalenone in corn-based foods was proposed, including one multi-toxin immunoaffinity column (IAC) sample preparation and three different high-performance liquid chromatography fluorescence detection methods. The analytical procedures were tested and verified, keeping in mind their occurrence at trace levels in corn-based foods. With the validation of the proposed multi-toxin IAC methodology and comparison of the performance characteristics with methods using a single-toxin IAC, we confirmed the reliability of the multi-toxin IAC procedure versus the single-toxin IAC. The methods were validated by revealing satisfactory performance characteristics; for example, the obtained values of limit of detection were significantly lower than the maximum limits for all mycotoxins of concern. In addition, the recovery values were between 70.9 % and 106.1 % for all mycotoxins of interest, with precision values lower than 10.5 %.

Miniaturized method for the quantification of persistent organic pollutants and their metabolites in HepG2 cells: assessment of their biotransformation

Biotransformation can greatly influence the accumulation and, subsequently, toxicity of substances in living beings. Although traditionally these studies to quantify metabolization of a compound have been carried out with in vivo species, currently, in vitro test methods with very different cell lines are being developed for their evaluation. However, this is still a very limited field due to multiple variables of a very diverse nature. So, an increasing number of analytical chemists are working with cells or other similar biological samples of very small size. This makes it necessary to address the development of analytical methods that allow determining their concentration both inside the cells and in their exposure medium. The aim of this study is to develop a set of analytical methodologies for the quantification of polycyclic aromatic hydrocarbons, PAHs (phenanthrene, PHE), and polybrominated diphenyl ethers, PBDEs (2,2′,4,4′-tetrabromodiphenyl ether, BDE-47), and their major metabolites in cells and their exposure medium. Analytical methodologies, based on miniaturized ultrasound probe-assisted extraction, gas chromatography–mass spectrometry–microelectron capture detector (GC–MS-µECD), and liquid chromatography–fluorescence detector (LC-FL) determination techniques, have been optimized and then applied to a biotransformation study in HepG2 at 48 h of exposure. Significant concentrations of the major metabolites of PHE (1-OH, 2-OH, 3-OH, 4-OH-, and 9-OH-PHE) and BDE-47 (5-MeO-, 5-OH-, and 3-OH-BDE-47) were detected and quantified inside the cells and in the exposure medium. These results provide a new method for determination and improve information on the metabolization ratios for a better knowledge of the metabolic pathways and their toxicity.Graphical abstract