Liquid Chromatography-fluorescence Research Articles

Development and validation of a DLLME-HPLC-FLD method for determination of aflatoxins in Chrysanthemum morifolium based on quality by design principles.

Aflatoxins, potent carcinogens produced by Aspergillus species, present significant health risks and commonly contaminate herbal products such as Chrysanthemum morifolium. Detecting these toxins in C. morifolium proves challenging due to the complex nature of the herbal matrix and the fluctuating levels of toxins found in different samples. This study aimed to develop and optimize a novel method for the detection of aflatoxins in C. morifolium using dispersive liquid-liquid microextraction combined with high-performance liquid chromatography-fluorescence detection based on quality by design principles. The method involved determining critical method attributes and parameters through the Plackett-Burman design, followed by optimization using the Box-Behnken design. Monte Carlo simulation was employed to establish a design space, which was experimentally verified. Method validation was performed to confirm accuracy, precision, and stability. The developed method exhibited excellent linearity (R2 > 0.9991) for aflatoxins B1, B2, G1, and G2 across a range of concentrations, with recovery rates between 85.52% and 102.01%. The validated method effectively quantified aflatoxins in C. morifolium under different storage conditions, highlighting the impact of temperature and storage time on aflatoxin production. This study successfully established a reliable and effective method for the detection of aflatoxins in C. morifolium, highlighting the importance of strict storage conditions to reduce aflatoxin contamination. Using a quality by design framework, the method demonstrated robustness and high analytical performance, making it suitable for routine quality control of herbal products.

Development of Magnetic Porous Polymer Composite for Magnetic Solid Phase Extraction of Three Fluoroquinolones in Milk.

In this study, a magnetic porous polymer composite with both hydrophilic and hydrophobic groups was synthesized for magnetic solid phase extraction (MSPE) of milk substrates. Optimization was conducted on various parameters, including adsorption dose, solution pH, adsorption time, and some elution conditions. Coupled with a high-performance liquid chromatography fluorescence detector, a novel MSPE method for determination of norfloxacin (NFX), ciprofloxacin (CIP), and enrofloxacin (ENR) in milk was developed based on magnetic metal organic framework polystyrene polymer (Fe3O4@MOF@PLS) as adsorbent. The Fe3O4@MOF@PLS exhibited significantly improved adsorption performance compared to MOF and PLS. Under optimized experimental conditions, the method exhibited good linearity for the three fluoroquinolones (FQs) in the range of 0.5-1000 μg/kg, with limit of detections (LODs) ranging from 0.21 to 1.33 μg/kg, and limit of quantitations (LOQs) from 0.71 to 4.42 μg/kg. The relative standard deviation (RSD) for the three FQs were 3.4-8.8%. The recoveries of three FQs in milk samples ranged from 84.2% to 106.2%. This method was successfully applied to the detection of three FQs in 20 types of milk, demonstrating its simplicity, speed, and effectiveness in analyte enrichment and separation. The method presented advantages in adsorbent dosage, adsorption time, LODs, and LOQs, making it valuable for the analysis and detection of FQs in milk.

One-pot derivatization/magnetic solid-phase extraction combined with high-performance liquid chromatography-fluorescence detection for rapid analysis of biogenic amines in alcoholic beverages

The determination of biogenic amines (BAs) in alcoholic beverages is crucial for assessing their health impact, ensuring beverage quality, and guaranteeing safety. Herein, a rapid one-pot derivatization/magnetic solid-phase extraction (OPD/MSPE) method was proposed using 6-aminoquinolinyl-N-hydroxysuccinimide carbamate as the derivatization reagent and magnetic hydroxyl-functionalized multi-walled carbon nanotubes as the extraction material. Integration of derivatization and extraction steps simplifies the sample preparation process, taking only three minutes and eliminating the need for centrifugation by utilizing magnetic sorbent. The resulting desorption solution was directly analyzed by high-performance liquid chromatography-fluorescence detection (HPLC-FLD) without any evaporation or reconstitution steps. The integrated OPD/MSPE-HPLC-FLD method demonstrates excellent linearity (R2 > 0.992), accuracy (relative recoveries: 85.1–109.2%), precision (RSDs≤9.7%) and detection limits (limits of detection: 0.3–2 ng/mL). It has been successfully applied to determine free BAs in various alcoholic beverages, including red wine, Baijiu, Huangjiu, and beer. This method enables rapid, sensitive and precise analysis of BAs in alcoholic beverages.

Occurrence of aflatoxin M1 in cheese products commonly available in Sri Lankan market

Aflatoxins (AFs) are a group of mycotoxins produced by certain fungi of Aspergillus spp. AFs of major concern are B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2), and M1 (AFM1). AFM1 is a hydroxylated metabolite of AFB1 formed inside the animal's body which is excreted into milk of cows that consumed AFB1 contaminated feed. Consumption of AFM1-contaminated milk and subsequent dairy products causes negative health effects in consumers. This study was conducted to determine the occurrence and levels of AFM1 in cheese products available in the Sri Lankan market where AFM1 is not regularly monitored in milk while having an outdated regulatory limit of 1 ppb established for dairy products. Processed cheese (n = 28), hard cheese (n = 14), semi-hard cheese (n = 5), and soft cheese (n = 3) representing seven popular brands were collected. The samples were analyzed by Ultra High-Performance Liquid Chromatography Fluorescence Detection. AFM1 was detected in 40 samples (80 %), while 17 (34 %) and 37 (74 %) of the samples had AFM1 levels exceeding the maximum permitted limit set by Codex Alimentarius Commission (0.5 ppb) and the Netherlands (0.2 ppb). Further, 10 samples violated the Sri Lankan maximum limit of 1 ppb. Thirteen out of the 14 hard cheese (92.9 %, 0.11–14.43 ppb) and all semi-hard cheese samples (100 %, 0.29–0.65 ppb) contained AFM1. Most of the soft (66.7 %, 0.35–0.45 ppb) and processed (71.4 %, 0.11–1.35 ppb) cheese samples had AFM1. Most of the locally manufactured cheese products in Sri Lankan market may pose health risks to consumers. The results highlight the significance of regular monitoring of AFM1 in dairy products and the importance of updating regulations on par with international standards concurrently to ensure consumer safety.

Precise method to measure fungal and bacterial necromass using high pressure liquid chromatography with fluorescence detector adjusted to inorganic, organic and peat soils

Soil organic matter is the dominant pool of carbon (C) in terrestrial ecosystems. Recent advances in understanding of the mechanisms of C stabilization in the soil emphasize microbes as the main drivers. Special attention is placed on the accumulation of bacterial and fungal necromasses. This calls for development of fast and reliable methods to estimate microbial necromass in a various type of soils, including peat soils. Here we provide precise method to measure fungal and bacterial necromasses with high-pressure liquid chromatography-fluorescence detector (HPLC-FLD) and its comparison with gas chromatography method. Purity of the chromatographic peaks was confirmed with mass spectrometry. The HPLC-FLD method provides reliable results for mineral, organic and highly organic peat soils.

Honeycomb-like triazine-based conjugated microporous polymers exhibiting simultaneous and ultrasensitive detection of fifteen polycyclic aromatic hydrocarbons using solid-phase microextraction technique

BackgroundSince the severe hazard to the ecosystem and widespread distribution through biological and man-made ways of polycyclic aromatic hydrocarbons (PAHs), it is very urgent to establish the ultrasensitive analytical method to quantitatively and directly monitor PAHs in real samples. However, because of the complicated environmental matrix and their trace concentration, the pre-concentration process is a necessary step to analyze of these compounds. In this study, solid phase microextraction (SPME) technique was proposed to separate and enrich fifteen trace PAHs from environmental samples. ResultsIn this work, a honeycomb-like triazine-based conjugated microporous polymers (T-CMPs) were prepared by Yamamoto reaction and firstly used as SPME coating material for the ultrasensitive direct-immersion-SPME of PAHs prior to high performance liquid chromatography-fluorescence detector (HPLC-FLD). The synthesized T-CMPs was characterized using various spectroscopy and electron microscopy techniques. The unique porous network of T-CMPs might deliver abundant adsorption sites for PAHs. Orthogonal experimental design (OED) was used to investigate the influence of four experimental parameters on the enrichment ability. Under optimal situation, a wide linear range (which lasted from 0.003 to 1000 μg L−1) with the coefficients of determination (R2) varying 0.9981 to 0.9993 was obtained. The limits of detection (LODs) for the analytes varied from 0.001 to 1.650 μg L−1, and the limits of quantification (LOQs) were between 0.003 and 4.960 μg L−1. The proposed method was effectively employed to the simultaneous and ultrasensitive detection of fifteen PAHs in industrial wastewaters. The relative recoveries for PAHs analysis varied from 74.6 % to 105 % with the relative standard deviations (RSD) of 0.1 %–7.5 % in real water samples. SignificanceThe prepared SPME coating material exhibited a simultaneous, high extraction and adsorption capacity for fifteen PAHs due to its honeycomb-like porous structure, ultra-large specific surface area, strong π-π stacking, and hydrophobic interactions. The present research developed a novel strategy for the construction of SPME fiber coating composites and demonstrated great application potential in the field of sample pretreatment and environmental analytical chemistry.

Impact of CYP2D6*2A, CYP2D6*4 and CYP3A5*3 genetic polymorphisms on Bisoprolol peak concentration and clinical response in acute coronary syndrome patients.

Acute coronary syndrome (ACS) represents a major cause of death. Bisoprolol is commonly used in the management of ACS. This study aims to investigate the impact of CYP2D6*2A, CYP2D6*4 and CYP3A5*3 genetic polymorphisms on pharmacokinetics and clinical response of bisoprolol in ACS patients. This is an open-label cohort study that included 127 ACS patients and studied the effect of CYP3A5*3, CYP2D6*2A and CYP2D6*4 genotyping using real-time polymerase chain reaction on steady state bisoprolol plasma peak concentration analysed by high performance liquid chromatography-fluorescence detector. Regarding CYP3A5*3, the mean peak bisoprolol concentration for CC, CT and TT genotypes were 4.25 ± 1.20, 3.93 ± 1.10 and 1.79 ± 0.69 ng/mL, respectively (P < .001). Higher systolic (126 ± 5.47 mmHg), diastolic blood pressure (82 ± 2.73 mmHg) and heart rate (97.80 ± 3.03 beats/min) were also observed in CYP3A5*3 TT carriers (P < .05). In CYP2D6*2A, the peak concentration of bisoprolol was lower in CC carriers (3.54 ± 1 ng/mL) compared to GG (4.38 ± 1.25 ng/mL) and GC carriers (4.07 ± 1.29 ng/mL, P = .019). In CYP2D6*4, the mean bisoprolol peak concentration in CC carriers was 3.98 ± 1.31 ng/mL, which was lower than T allele carriers (4.5 ± 0.8, P = .02). No differences in heart rate, systolic, diastolic blood pressure or bisoprolol dose were observed among CYP2D6*2A or CYP2D6*4 variants. Smokers exhibited lower bisoprolol peak concentration (3.96 ± 1.2 ng/mL) compared to nonsmokers (4.55 ± 1.34 ng/mL, P = .037). There is an association between CYP3A5*3, CYP2D6*4, CYP2D6*2A variants and bisoprolol peak concentration, which may serve as a guide in the future in choosing the optimum dose of bisoprolol in ACS patients.

Assay for okadaic acid O-acyl transferase using HPLC-FLD.

Dinophysistoxin 1 (DTX1, 1) and okadaic acid (OA, 2), produced by the dinoflagellates Dinophysis spp. and Prorocentrum spp., are primary diarrhetic shellfish toxins (DSTs), which may cause gastric illness in people consuming such as bivalves. Both compounds convert to dinophysistoxin 3 (DTX3, 3; generic name for 1 and 2 with fatty acids conjugated at 7-OH) in bivalves. The enzyme okadaic acid O-acyl transferase (OOAT) is a membrane protein found in the microsomes of the digestive glands of bivalves. In this study, we established an in vitro enzymatic conversion reaction using 4-nitro-2,1,3-benzoxadiazole (NBD)-OA (4), an OA derivative conjugated with (R)-(-)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (NBD-APy) on 1-CO2H, as a substrate. We detected the enzymatically produced 3, NBD-7-O-palmitoyl-OA (NBD-Pal-OA), using high-performance liquid chromatography-fluorescence detection. We believe that an OOAT assay using 4 will facilitate the fractionation and isolation of OOAT in the future.

Novel ferrofluid based on hydrophobic deep eutectic solvents for separation and analysis of trace estrogens in environmental water and urine samples.

A novel ferrofluid prepared from ahydrophobic deep eutectic solvent (DES) and Fe3O4@graphite composite materials was introduced as a green microextraction medium for the separation and enrichment of trace estrogens in real samples. It was found that the ferrofluid greatly improved the capacity and selectivity of target analytes, benefiting from the enrichment of both DES and Fe3O4@graphite composite materials. Using a combination of high-performance liquid chromatography-fluorescence detection (HPLC-FLD) and vortex-assisted liquid-liquid microextraction (VALLME), a new method was established for simultaneous rapid processing and accurate determination of three estrogens (estradiol [E2], estriol [E3], and ethinyl estradiol [EE2]) in environmental water and urine samples. Key parameters affecting the extraction efficiency were optimized using a single-factor approach and response surface methodology. Under optimal conditions, this method yielded a low limit of detection (1.01 ng L-1, 3.03 ng L-1, and 25.0 ng L-1 for EE2, E2, and E3, respectively), wide linear range (3-200,000ng L-1), high enrichment factors (9.81-47.2), and satisfactory recovery (73.8-129.0%). Compared with traditional analytical techniques, this method avoids the use of volatile toxic organic extraction solvents and cumbersome phase separation operations.

Assessment of Erythrocyte Transketolase, Whole Blood Thiamine Diphosphate, and Human Milk Thiamine Concentrations to Identify Infants and Young Children Responding Favorably to Therapeutic Thiamine Administration: Findings from the Lao Thiamine Study, a Prospective Cohort Study

BackgroundThere is limited information on relationships among biomarkers of thiamine status (whole blood thiamine diphosphate [ThDP], erythrocyte transketolase activity coefficient [ETKac], and human milk thiamine [MTh]) and clinical manifestations of thiamine deficiency. ObjectivesThis study aimed to explore correlations among these biomarkers and thiamine responsive disorders (TRDs), a diagnosis based on favorable clinical response to thiamine. MethodsHospitalized infants and young children (aged 21 d to <18 mo) with respiratory, cardiac, and/or neurological symptoms suggestive of thiamine deficiency were treated with parenteral thiamine (100 mg daily) for ≥3 d alongside other treatments and re-examined systematically. Clinical case reports were reviewed by 3 pediatricians, who determined TRD or non-TRD status. Children in a community comparison group were matched by age, sex, and residence. Venous whole blood ThDP and MTh were determined by high-performance liquid chromatography fluorescence detection and ETKac in washed erythrocytes by ultraviolet spectrophotometry. Associations between biomarkers were assessed using Spearman correlations, and biomarker cutoffs predictive of TRD and ETKac >1.25 were explored using area under the receiver operating characteristic curve framework. ResultsThiamine biomarkers were available for 287 hospitalized children and 228 community children (mean age 4.7 mo; 59.4% male). Median (interquartile range [IQR]) ThDP and ETKac were 66.9 nmol/L (IQR: 41.4, 96.9 nmol/L) and 1.25 nmol/L (IQR: 1.11, 1.48 nmol/L), respectively, among hospitalized children, and 64.1 nmol/L (IQR: 50.0, 85.3 nmol/L) and 1.22 nmol/L (IQR: 1.12, 1.37 nmol/L) among 228 community children (P > 0.05 for both). Forty-five percent of breastfeeding mothers of infants <6 mo had MTh <90 μg/L. ThDP and ETKac, but not MTh, were significantly different between 152 children with TRD and 122 without TRD, but overlapping distributions undermined prediction of individual responses to thiamine. ConclusionsAlthough ETKac, ThDP, and MTh are useful biomarkers of population thiamine status, none of the biomarkers reliably identified individual children with TRD. ThDP is more practical for population assessment because preparing washed erythrocytes is not required.This trial was registered at clinicaltrials.gov as NCT03626337.

MOF@N–doped carbon dots–based dispersive solid phase extraction combined dispersive liquid–liquid microextraction of several aflatoxins from raw cow’s milk samples prior to HPLC–FLD

A dispersive solid-phase extraction method based on metal–organic framework and nitrogen–doped carbon dot nanocomposite combined with magnetic ionic liquid-based dispersive liquid–liquid microextraction was established for the extraction of aflatoxins from milk samples. For this purpose, the proteins in the sample were precipitated using a zinc sulfate solution. After vortexing (for 5 min) and centrifuging, 35 mg of the sorbent were added into the aqueous phase obtained from previous step, and the mixture was vortexed for five minutes. In the following, the sorbent particles were separated and eluted with methanol in the presence of vortex agitation (4 min). After centrifuging, the eluent phase was mixed with magnetic ionic liquid (1- octyl-3-methylimidazolium tetrachloroferrate) and rapidly injected into deionized water to perform the dispersive liquid–liquid microextraction procedure. Finally, the magnetic solvent was separated in the presence of a magnet and injected into a high-performance liquid chromatography-fluorescence detector after diluting with the mobile phase. Referring to the results, the limits of detection and quantification of the studied aflatoxins were in the ranges of 0.69–1.09 and 2.31–3.63 ng/L, respectively. Extraction recoveries were in the range of 61 to 73 %. At the end, the suggested approach was employed in the analysis of the aflatoxins in the raw cow’s milk samples collected from different regions of East Azarbaijan and AFM1 was found in several samples (34 out of 60 samples). Also, AFB1 was found in three samples.

Cannabinoid Modulation of Monoamine Levels in Mouse Brain: Unveiling Neurochemical Dynamics through an Innovative High-performance Liquid Chromatography-Fluorescence Detection Bioanalysis

Homeostasis of monoaminergic pathways is compromised in aging and neurodegenerative/neurological diseases such as Alzheimer’s disease and depression. On the other hand, their modulation has also been linked to the mechanism of action of several drugs. Therefore, monitoring the levels of noradrenaline (NA), adrenaline (AD), dopamine (DA), and serotonin (5-HT), as well as those of tryptophan (TRYP), the precursor of 5-HT, and DA metabolites, namely 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), is fundamental for assessing disease severity and progression. This work aimed to develop and validate the first High-Performance Liquid Chromatography (HPLC) coupled with fluorescence detector (FLD) method that simultaneously and accurately quantifies NA, AD, DA, DOPAC, HVA, 5-HT and TRYP in mouse brain and prefrontal cortex (PFC) matrices. Previous sample preparation by protein precipitation was required to extract the compounds. Calibration curves were plotted using the background subtraction approach to reduce the interference of the endogenous analytes. Intra and inter-day accuracy and precision were within the ranges defined by ICH (The International Council for Harmonisation) guideline for bioanalytical method validation. Following validation, the impact of cannabidiol (CBD), cannabigerol (CBG), and cannabidivarin (CBDV) was explored in mice brains post-administration, revealing significant alterations in specific neurotransmitter levels upon cannabinoid exposure and shedding light on the complex modulation of neurochemical dynamics by cannabinoids. This research highlights the fit-for-purpose of the HPLC-FLD method and provides insights into potential mechanisms underlying phytocannabinoid actions in the central nervous system (CNS).

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.

In situ extraction and enrichment of multiple amino acids by integrated mechanochemical extraction and aqueous two-phase system

A highly efficient and environment-friendly extraction technology was established to extract and enrich multiple amino acids (AAs) in situ by deep eutectic solvent (DES)-assisted mechanochemical extraction (MCE) and aqueous two-phase system (ATPS). Firstly, the DES was prepared from betaine and ethylene glycol in a 1:2 M ratio. Then, a series of conditions were evaluated by single-factor and multifactor experiments to select the optimum experimental parameters. The highest extraction yields of AAs were obtained by DES-assisted MCE and ATPS enrichment, including that the amount of DES was 0.97 mL, the milling time was 4.88 min, the amount of salt was 8 g, and the solid-to-liquid ratio was 0.055 g mL−1. After extracting, the diluted AA extracts were analyzed by a high-performance liquid chromatography-fluorescence detector after pre-column derivatization with 6-aminoquinolyl-N-hydroxysccinimidyl carbamate. The methodological validation demonstrated the proposed method exhibited satisfactory limits of detection ranged from 0.11 to 0.82 ng mL−1, reliable interday precision (%RSD) of 0.014–0.067 % for retention time and 1.29–3.34 % for the peak area, acceptable reproducibility with %RSD below 4.1 %, and good %recovery between 90.3 % and 122.5 % at three spiked concentrations. The surface morphology of raw, only ground, and DES-ground sample powders was tested by scanning electron microscopy to further explore the changes in the physicochemical properties of samples. By comparing with other AAs extraction methods, the developed method is not only eco-friendly, but greatly improves the yield of free AAs extracted from chrysanthemum.

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.

Assessing in vitro stability of remdesivir (GS-5734) and conversion to GS-441524 in feline plasma and whole blood

Feline infectious peritonitis (FIP) is a potentially fatal coronavirus-driven disease of cats. Treatment with nucleoside analogue GS-441524 and or prodrug remdesivir (RDV) have produced remission in both experimentally induced and naturally occurring FIP, yet information regarding metabolism of RDV into GS-441524 in cats is scarce. This study assessed possible phase I metabolism of RDV in cats, utilising an in vitro feline microsome model with in vitro t1/2 and in vitro Clint calculated using the substrate depletion method. A previously validated high-performance liquid chromatography (HPLC) fluorescence method was utilised for detection and analysis of RDV and GS-441524. Qualitative yield of RDV and intermediate metabolite GS-441524 were determined following microsome incubation, then compared to whole blood and plasma incubations. In vitro microsome incubation resulted in rapid depletion of RDV, though it did not appear to resemble a conventional phase I-dependent reaction in cats, as it is in humans and dogs. Depletion of RDV into GS-441524 was demonstrated in whole blood in vitro, suggesting cats convert RDV to GS-441524, likely via blood esterases, as observed in mice and rats. RDV metabolism is unlikely to be impacted by impaired liver function in cats. Furthermore, as RDV depletes within minutes, whereas GS-441524 is very stable, whole blood or plasma GS-441524 concentrations, rather than plasma RDV concentrations, are more appropriate for therapeutic drug monitoring (TDM) in cats receiving RDV.

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.