Ultra-high Performance Liquid Chromatography Research Articles

Development and Validation of a UHPLC-MS/MS Method for the Simultaneous Quantification of Candesartan and Bisoprolol Together with Other 16 Antihypertensive Drugs in Plasma Samples.

Antihypertensive pharmacological therapy is often characterized by a coadministration of different classes of drugs. Therefore, analytical methods allowing the simultaneous quantification of many drugs are needed for therapeutic drug monitoring (TDM) purposes in this context. In particular, TDM represents a useful tool to discriminate poor adherence from real cases of resistant hypertension. For this reason, the aim of this study is to validate, following the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) guidelines, an ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous quantification of 18 antihypertensive drugs in human plasma. A LX-50 coupled with a QSight 220 UHPLC-MS/MS system with electrospray ionization and multiple reaction monitoring mode was used, after a binary gradient separation (13 min) on a reverse-phase Acquity UPLC HSS T3 [1.8 μm, 2.1 mm × 150 mm] column. Method validation showed a stable and acceptable matrix effect, recovery, high accuracy, and precision, assessing the eligibility of this method for routine use in the clinical context.

Quantitative analysis of cannabinoids and metabolites in oral fluid by volumetric absorptive microsampling combined with UHPLC-HRMS.

With recent evolution of cannabis legalization around the world and multiplication of cannabis derived products, identifying and qualifying cannabis consumption has a proven interest. Although blood, plasma, and urine are common matrices widely used in toxicology laboratories, oral fluid presents specific advantages. In the context of doping tests, addiction consultation or roadside checks, where other matrices are impractical to collect or can be adulterated, oral fluid is a promising matrix that allows a non-invasive, rapid, and monitored self-sampling. However, available devices required a consequent volume of oral fluid,more than 250 µL, sometimes difficult to collect. We present here a fully optimized quantitative method for seven cannabinoids, including four metabolites, in oral fluid, Δ9-tetrahydrocannabinol, 11-hydroxy-Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol; cannabidiol, 7-hydroxy and 7-carboxycannabidiol; and cannabinol. After self-collection of 20 µL using an accurate and precise volumetric absorptive microsampling device (VAMS®), cannabinoids were derivatized with 2-fluoro-1-methylpyridinium p-toluenesulfonate to increase sensitivity. The successive steps of the proposed method, including biosampling, 1 h sample preparation with derivatization, and acquisition by ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry, were fully optimized. A limit of quantification of 0.5 ng/mL (≈10 pg per sampling) was thus targeted, adapted to the legal threshold required by the authorities and to clinical monitoring. Applied to six cannabis consumers, the proposed method made it possible to quantify in 20 µL oral fluid samples, Δ9-tetrahydrocannabinol ranging from 0.5 to 6236 ng/mL, cannabidiol from 0.6 to 190 ng/mL and cannabinol from 0.5 to 118 ng/mL.

Simultaneous quantification of 14 glucosinolates in rapeseeds by ultra high performance liquid chromatography – Tandem mass spectrometry

Glucosinolates' species and content serve as crucial indicators for Brassicaceae plants and their products. However, endogenous enzymes in Brassicaceae can hydrolyze glucosinolates, influencing their quantitative detection. Our study utilized rapeseed, a vital Brassicaceae oil crop, to establish a novel UHPLC-MS/MS method for glucosinolate determination, incorporating a pretreatment approach with enzyme inhibition. This enabled intact glucosinolate determination at room temperature. The straightforward process involved homogenizing samples with saturated sodium chloride and methanol, followed by vortex extraction, centrifugation, and filtration. The method exhibited a strong linear relationship (R2 > 0.990), with LODs and LOQs ranging from 0.1 to 1.1 mg/kg and 0.3–3.7 mg/kg, respectively. Intra-day precision (7 repetitions) ranged from 3.21 to 10.47 %, while inter-day precision (3 days) ranged from 3.13 to 15.97 %.

Implementation of quality by design and green chemistry principles to validate the stability indicating method on UHPLC for the simultaneous quantification of Glycopyrronium, Fluticasone, Vilanterol in inhaled formulation

The combination of Glycopyrronium, Fluticasone, and Vilanterol is utilized to manage chronic obstructive pulmonary disease (COPD) and mitigate asthma symptoms. Glycopyrronium functions as an anticholinergic compound, Fluticasone serves as a corticosteroid that mitigates inflammation, and Vilanterol operates as a long-acting beta-agonist that facilitates the relaxation of the bronchial passages. The simultaneous quantification of Glycopyrronium, Vilanterol, and Fluticasone was accomplished through the utilization of Ultra-High-Performance Liquid Chromatography (UHPLC) employing a Symmetry Shield C18 column (4.6 mm x 100 mm ID, 2.6µm particle size), with a flow rate of 0.6 mL/min, and a gradient elution employing pH 3.0 Ammonium Formate as mobile phase A and ethanol as mobile phase B. By the guidelines delineated by the International Conference on Harmonization (ICH) Q2, method validation encompassed specificity (with the purity angle for the three chromatographic peaks exceeding the purity threshold under all degradation conditions), linearity (exhibiting an R² value greater than 0.999 from the lower limit of quantification to 200%), accuracy (with recovery rates ranging from 95% to 105%), robustness (the influence of variations in mobile phase buffer pH, column temperature, and flow rate was scrutinized utilizing Analytical Quality by Design principles), and assessments of method precision. The Green Analytical Procedure Index (GAPI), Analytical Greenness (AGREE), and analytical eco-scale evaluations substantiated the method's advantageous characteristics regarding environmental sustainability.

Exploring the Differences of Chemical Components of Citri Reticulatae Pericarpium Powder Decoction and Pieces Decoction Based on UHPLC-Q-Exactive Orbitrap MS/MS and Network Pharmacology.

Taking Citri Reticulatae Pericarpium (CRP) as an example, it is proved that there are differences between the powder decoction and pieces decoction of traditional Chinese medicine (TCM). In this study, an ultra-high performance liquid chromatography quadrupole exactive orbitrap MS/MS (UHPLC-Q-Exactive Orbitrap MS/MS) method was established to characterize 80 chemical components of CRP. The content of components was compared based on extraction rate, alcohol solubility rate, and mass spectrum peak area. The result showed that the content in CRP powder decoction was generally higher than that in CRP pieces decoction. The principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used to distinguish between the two decoctions. In addition, cardiovascular diseases (CVDs) were used as a model to investigate whether the increased content of components has practical significance. Network pharmacology screened five core targets of CRP in CVDs. The results of molecular docking indicated that the binding energies were all ≤ -5.0kcal/mol between effective compounds (M34, M57, M80, etc.) and key targets (Akt1, SRC, ESR1, EGFR, and PTGS2) with good affinity. These results provide an important reference for further development and use of CRP powder decoction.

In vitro screening of UGT2B10 in silico prioritized putative ligands from drugs used in the pediatric hematopoietic stem cell transplantation setting.

UGT2B10 is a phase II drug metabolizing enzyme with limited information on its role in the metabolism of drugs, especially in the pediatric hematopoietic stem cell transplantation setting. Previously, we investigated UGT2B10's role through in silico analyses and prioritized acetaminophen (APAP), lorazepam (LOR), mycophenolic acid (MPA), and voriconazole N-oxide (VCZ N-oxide) for invitro investigations. In this report, we present invitro screening of these candidates and of voriconazole (VCZ) to assess their potential to be substrates and/or inhibitors of UGT2B10. Enzyme kinetics experiments included recombinant UGT2B10 and analytical methods based on ultra high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS). To determine potential substrates, candidates were incubated at various therapeutically observed concentrations with recombinant UGT2B10 to identify the corresponding glucuronide metabolite. Inhibition capacity was tested using the selective probe cotinine for its glucuronidation to cotinine N-ß-d-glucuronide. IC50 was determined for compounds exhibiting inhibition. Among the tested compounds, LOR (IC50 = 0.01 μM, R2 = 0.9257) and MPA (IC50 = 0.38 mM, R2 = 0.9212) exhibited inhibition potential for UGT2B10. None of the other tested compounds featured inhibition potential and none of the compounds tested exhibited metabolism through UGT2B10. Further exploration on the clinical relevance of this inhibition using modeling strategies, overlapping nature with other UGT isoforms, and screening other molecules for their inhibition potential on UGT2B10 is warranted.

The withdrawal time of enrofloxacin, sulfachloropyrazine sodium, and doxycycline as well as the in vitro binding interaction with melanin in black-feathered silky fowl

The problem of veterinary drug residues in black-feathered silky fowl (BFS) has garnered significant attention. This study investigated the residue elimination patterns and causes of enrofloxacin (ENR), sulfachloropyrazine sodium (SPZ), and doxycycline (DOX) in BFS using ultra-high performance liquid chromatography mass spectrometry (UPLC-MS), along with the drug adsorption and melanin binding tests. The findings indicated that ENR and SPZ were metabolized slowly, DOX was metabolized faster in BFS, with optimum withdrawal time was 87.8, 37.26, and 24.7 days, respectively. The adsorption capacity of skin + fat to drugs and the binding capacity of melanin to the three drugs in BFS were in the order of ENR > SPZ > DOX, confirming that melanin's binding ability influences the drug residues in BFS. This study is highly significant for addressing the issue of veterinary drug residues in BFS.

Associations between volatile organic compounds exposure and multiple oxidative damage biomarkers: Method development, human exposure, and application for e-waste pollution prediction

Atmospheric monitoring studies reveal substantial health risks from exposure to volatile organic compounds (VOCs) for workers and nearby children in e-waste recycling areas (ERA), yet internal exposure risks are seldom examined. To address this, we developed a method to simultaneously analyze 12 urinary VOC metabolites (mVOCs) and oxidative damage biomarkers (ODBs) in workers and children from ERA and general adults from control areas using ultrahigh performance liquid chromatography coupled with quadrupole/orbitrap high-resolution mass spectrometry (UPLC-Orbitrap-HRMS). The results showed that e-waste workers exhibited significantly higher levels of VOC exposure and ODBs than e-waste children and control adults. Exceeding 91.1 %, 69.1 %, 20.8 %, 19.7 %, and 3.26 % of e-waste workers faced non-carcinogenic risk from exposure to acrolein, acrylonitrile, acrylamide, 1,3-butadiene, and 1,2-dichloroethane, respectively. The weighted quantile sum, quantile g-computation, and Bayesian kernel machine regression models consistently indicated significant positive associations between these VOC mixtures and cholesterol ODB levels (i.e., glycocholic acid, cholic acid, and glycochenodeoxycholic acid), highlighting the necessity for improved protective measures for occupational workers. Interestingly, cholesterol ODBs significantly mediated the association between VOCs exposure and nucleic acid ODBs, accounting for 12.0–26.0 % of the association with 8-hydroxy-2′-deoxyguanosine (an oxidative DNA damage biomarker) and 25.4–53.4 % with 8-hydroxyguanosine (an oxidative RNA damage biomarker). This suggests that cholesterol ODBs potentially serve as better indicators of health risks from VOC exposure than nucleic acid ODBs. Additionally, the combination of mVOCs and ODBs (Mean AUC: 0.906, ACC: 0.821) as exposure fingerprints outperformed either mVOCs (Mean AUC: 0.878, ACC: 0.802) or ODBs (Mean AUC: 0.843, ACC: 0.768) alone in predicting the presence of e-waste pollution, underscoring the importance of integrating exposure and effect biomarker fingerprints to accurately capture e-waste pollution characteristic. Our findings offer a novel approach for screening e-waste pollution in unknow e-waste recycling sites and provide a foundation for developing high-precision prediction models for other polluting industries.

Integration of ion mobility-quadrupole time-of-flight mass spectrometry and triple quadrupole-ion trap mass spectrometry coupled with ultra-high performance liquid chromatography for characterizing and quantitatively assay the saponins of Panax quinquefolius flower

Panax quinquefolius (American ginseng) is globally popular serving as herbal medicine or tonifying agent. Compared with the root, studies on the flower of Panax quinquefolius (PQF) in respect of the multicomponent characterization and quality evaluation is scarce. This work was designed, by feat of two liquid chromatography–mass spectrometry platforms, to characterize and quantitatively assay the saponins of PQF. Ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) combined with data-independent high-definition MSE (HDMSE) was developed to acquire the structure information of PQF saponins. Consequently, efficient and more accurate identification (higher identification accuracy and much lower false positives rate) of the involved saponins were accomplished by the intelligent peak annotation workflows of the UNIFI bioinformatics platform incorporated with a “Multi-dimensional Information Library of Ginsenosides” (GinMIL). Totally 128 ginsenosides were identified or tentatively characterized, and 45 thereof were identified by comparing with the reference standards. Additionally, an efficient UHPLC/QTrap-MS (triple quadrupole-ion trap mass spectrometry) method enabling quantitative assay of 21 ginsenosides in PQF was established and validated by scheduled multiple reaction monitoring (sMRM). It gave low limit of detection (0.011–0.65 pg) and limit of quantification (0.053–4.1 pg), good linearity (R2 > 0.99), intra-/inter-day precision (RSD < 13.6 %), stability (RSD < 3.5 %), repeatability (RSD < 7.5 %), and recovery (RSD < 13.5 %). It application to quantify 21 ginsenosides in 14 batches of PQF samples revealed differences in their saponin content. Conclusively, systematic multi-component characterization and multi-indicator quantitation of PQF were achieved, thus providing the methodological reference for the quality investigation of functional herbs.

Effects of relative humidity on atmospheric organosulfur species derived from photooxidation and nocturnal chemistry in a forest environment

The molecular composition of organic aerosols in ambient PM2.5 was investigated at the northern foothills of Qinling Mountain region in central China during the summer of 2022. The molecular characteristics of organic matter were analyzed using ultrahigh performance liquid chromatography coupled with high-resolution Orbitrap mass spectrometry. The number of molecular formula assignments was dominated by organosulfur species (OrgS, on average 28–47% in total). Both the number and peak area of OrgS increased significantly with higher relative humidity (RH), while those of CHO and CHON species decreased, indicating the different impacts of RH on individual organic groups. The entire study period was divided into two distinct stages, including a low RH and a high RH period (LRH and HRH). The enhanced formation of CHOS with O>7 in HRH was primarily attributed to photochemical oxidation, whereas the increase in CHONS with O>7 was driven by enhanced NO3 radical-initiated oxidation under elevated RH conditions. Nearly 50% of OrgS presented only in HRH had aliphatic structures with C≥9. Although isoprene-derived organosulfates dominated the peak area across the entire period, the monoterpenes and long-chain alkanes-derived organosulfates largely increased in HRH. The strong correlations between aerosol liquid water content and OrgS containing high oxygen content (O>7) indicate that aqueous-phase reactions played a crucial role in multigenerational oxidation of OrgS. This study highlights the important effects of RH on the formation of OrgS, particularly for organosulfates derived from monoterpenes and long-chain alkanes.

Comparative Analysis of N-Lactoyl-phenylalanine and 3-Phenyllactic Acid Production in Lactic Acid Bacteria from Kimchi: Metabolic Insights and Influencing Factors.

N-Lactoyl-phenylalanine (Lac-Phe) is a metabolite known for its appetite-suppressing and antiobesity properties, while phenyllactic acid (PLA) is recognized for its antibacterial activity. Both metabolites are derived from phenylalanine and lactic acid metabolism through peptidase and dehydrogenase activities. The aim of this study was to investigate the production of Lac-Phe and PLA in kimchi, focusing on the role of lactic acid bacteria (LAB). Ultrahigh performance liquid chromatography coupled with time-of-flight mass spectrometry was used to quantify these metabolites in homemade and commercial kimchi. Lac-Phe and PLA were detected in both kimchi sample types. Various genera, including Lactobacillus, Leuconostoc, and Weissella, were evaluated for Lac-Phe and PLA production. LAB strains exhibiting high Lac-Phe production generally showed lower PLA production, indicating an inverse relationship between these two metabolites. Analysis of dipeptidase sequences revealed that the presence of carnosine dipeptidase 2 (CNDP2)-like M20 metallopeptidase is crucial for Lac-Phe production in LAB. Additionally, phenylalanine was identified as a major factor for both Lac-Phe and PLA production, whereas lactic acid supplementation did not significantly affect their production levels.

Decelerated skin aging effect of rubber (Hevea brasiliensis) seed oil in cell culture assays

Rubber seeds, the abundant by-products of rubber tree (Hevea brasiliensis), have been studied for sustainable utilization. Nevertheless, there is no information available regarding activity against skin aging. The study aimed to prepare rubber seed oil (RSO) and evaluate fatty acid compositions by gas chromatography - mass spectrometry (GC/MS), linamarin contamination by ultra-high performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS). Additionally, cytotoxicity assay and anti-skin aging activities, including cell proliferating stimulation, cellular antioxidant, collagen stimulation, and matrix metalloproteinase-2 (MMP-2) inhibition, were analyzed in immortalized human skin keratinocytes (HaCaT cells) and human dermal fibroblasts. RSO was pale-yellow oily liquid with an extraction yield of 35.79 ± 0.52%. Principal fatty acids were comprised of oleic (43.37 ± 0.76%), linoleic (38.49 ± 0.81%), palmitic (11.47 ± 0.12%), and stearic (6.66 ± 0.05%) acids. Linamarin contamination was not detected in 100 µg/mL RSO, demonstrating the absence of a cyanogenic glucoside. Non-cytotoxic concentrations of RSO in both cells were in the range of 0.0001–0.1 mg/mL. Activities of RSO against skin aging included the cell proliferating stimulation, the antioxidant activity, the collagen stimulation, and the MMP-2 suppression at mRNA expression level and enzymatic activity. Study results have suggested that rubber seeds can probably be employed as a promising ingredient in the preparations designed for deceleration of skin aging.

Four-Dimensional Lipidomic Analysis Using Comprehensive Online UHPLC × UHPSFC/Tandem Mass Spectrometry.

Multidimensional chromatography offers enhanced chromatographic resolution and peak capacity, which are crucial for analyzing complex samples. This study presents a novel comprehensive online multidimensional chromatography method for the lipidomic analysis of biological samples, combining lipid class and lipid species separation approaches. The method combines optimized reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) in the first dimension, utilizing a 150 mm long C18 column, with ultrahigh-performance supercritical fluid chromatography (UHPSFC) in the second dimension, using a 10 mm long silica column, both with sub-2 μm particles. A key advantage of employing UHPSFC in the second dimension is its ability to perform ultrafast analysis using gradient elution with a sampling time of 0.55 min. This approach offers a significant increase in the peak capacity. Compared to our routinely used 1D methods, the peak capacity of the 4D system is 10 times higher than RP-UHPLC and 18 times higher than UHPSFC. The entire chromatographic system is coupled with a high-resolution quadrupole-time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) in both full-scan and tandem mass spectrometry (MS/MS) and with positive- and negative-ion polarities, enabling the detailed characterization of the lipidome. The confident identification of lipid species is achieved through characteristic ions in both polarity modes, information from MS elevated energy (MSE) and fast data-dependent analysis scans, and mass accuracy below 5 ppm. This analytical method has been used to characterize the lipidomic profile of the total lipid extract from human plasma, which has led to the identification of 298 lipid species from 16 lipid subclasses.

Impact of Cooking on Tuber Color, Texture, and Metabolites in Different Potato Varieties

Potatoes are a globally important crop with high nutritional value. Different potato varieties display notable variations in color, texture, and nutrient composition. However, the influence of cooking on tuber color, texture, and metabolites has not been comprehensively explored. This study evaluated the color and texture of five potato varieties before and after cooking. Cooking significantly altered tuber color, decreased hardness and adhesiveness, and increased springiness, particularly after steaming. The metabolomic analysis of Zhongshu 49 (ZS49) and Shishu 3 (SH3) tubers was conducted using gas chromatography–mass spectrometry (GC-MS) and ultra-high performance liquid chromatography (UHPLC)-MS/MS. GC-MS identified 122 volatile metabolites, with 42 significantly varying between cooking treatments, while UHPLC-MS/MS detected 755 nonvolatile metabolites, 445 of which showed significant differences. Compared to ZS49, SH3 exhibited a marked increase in umami- and flavor-related metabolites, especially after cooking. This study provides new insights into how cooking affects the quality, texture, and metabolite profiles of potato tubers.

Serum neurotransmitter analysis of motor and non-motor symptoms in Parkinson’s patients

Clinical symptoms of Parkinson’s disease (PD) are classified into motor and non-motor symptoms. Mental disorders, especially depression, are one of the major non-motor manifestations of PD. However, the underlying mechanisms remain poorly understood. In the present study, 21 neurotransmitters associated with mental disorders were measured in serum samples from patients and controls using the ultra-high performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) assay. Additionally, five clinical scales—the MDS Unified Parkinson’s Disease Rating Scale (UPDRS), the Non-Motor Symptoms Scale (NMSS), the Mini-Mental State Examination (MMSE), the Hamilton Anxiety Scale (HAMA), and the Hamilton Depression Scale (HAMD)—were used to evaluate the severity of both motor and non-motor symptoms in PD patients. Analysis of neurotransmitter metabolism revealed significant changes in the tryptophan (Trp) metabolic pathway in PD patients. Specifically, levels of Trp, kynurenine (KYN), kynurenic acid (KA), nicotinamide (NAM), and 5-methoxyltryptamine (MeOTA) were substantially decreased. Additionally, three other excitation/inhibiting amino acids—glutamic acid (Glu), 4-aminobutyric acid (GABA), and aspartic acid (Asp)—also declined. Moreover, neurotransmitter conversion ratios, such as KA/KYN, nicotinamide/niacin (NAM/NA), 5-hydroxytryptophan/tryptophan (5-HTP/Trp), and quinolinic acid/kynurenic acid (QA/KA), provided more dynamic insights into disrupted neurotransmitter metabolism. Correlation analyses between scale scores and neurotransmitter levels showed that concentrations of xanthurenic acid (XA) and the turnover rate of 3-hydroxykynurenine (3-HK) were negatively correlated with UPDRS scores, while 5-hydroxytryptamine (5-HT) and GABA levels were negatively correlated with non-motor symptoms in PD patients. In summary, this study elucidates, for the first time, the potential association and dynamics between altered neurotransmitter metabolism and the etiology of PD in terms of motor and non-motor functions. These findings offer novel biomarkers and therapeutic targets for the treatment of PD.

Transforming growth factor-β3/Smad2/Smad3 signaling pathway inhibition and autophagy by the Yunpi-Xiefei-Huatan decoction ameliorated airway inflammation and mucus hypersecretion in asthmatic rats

Ethnopharmacological relevanceThe Yunpi-Xiefei-Huatan decoction (YXHD) is a traditional Chinese medicine that can improve asthma-related symptoms, including cough, phlegm in the throat, and shortness of breath. However, the YXHD mechanism on asthma has not yet been elucidated. Study aimThe aim of this study is to investigate the effect of YXHD on airway inflammation, mucus hypersecretion, and autophagy modulation in asthma. Materials and methodsThe YXHD chemical constituents were observed and analyzed using high-performance liquid chromatography-mass spectrometry. Ovalbumin sensitization and stimulation were used to establish an asthma rat model. A total of 80 Sprague-Dawley (SD) rats were segmented into eight groups at random: a Normal (NC) group, a Model (Mod) group, a YXHD low-dose group (10 g/kg/d), a YXHD moderate-dose group (20 g/kg/d), a YXHD high-dose group (40 g/kg/d), a Rapamycin group (4 mg/kg/d), a 3-methyladenine (3-MA) group (15 mg/kg/d), and a Dexamethasone (DEX) group (0.5 mg/kg/d). Whole-body plethysmography (WBP) detection was used to evaluate airway hyperresponsiveness. An enzyme-linked immunosorbent assay (ELISA) was used to detect inflammatory factors in the peripheral blood. Inflammatory cells in the bronchoalveolar lavage fluid (BALF) were also counted. Pathological changes in the lung tissues were marked using hematoxylin and eosin (H&E) staining and periodic acid-Schiff (PAS) staining. The localization of MUC5AC and the co-localization of LC3B + MUC5AC were observed using immunofluorescence. The expressions of autophagy and the TGF-β3/Smad2/Smad3 pathway in the lung tissues were detected using a Western blot assay (WB) and qPCR, and the autophagosomes in the lung tissues were detected using a transmission electron microscope (TEM). ResultsTwenty signal peaks were identified using ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) technology. The TGF-β3/Smad2/Smad3 signal pathway activation was induced using ovalbumin (OVA) exposure in the rats. The upregulated expression of autophagy, enhanced MUC5AC fluorescence and LC3B fluorescence, and their co-localized expression in the airway epithelium indicated inflammatory cell infiltration and excessive mucus secretion in the lungs. This resulted in airway hyper-responsiveness. The YXHD inhibited the activation of the TGF-β3/Smad2/Smad3 signaling pathway, and autophagy expression reduced inflammatory factors, abnormal mucus secretion, and airway hyperresponsiveness. ConclusionThe YXHD improved lung function, relieved lung inflammation, and inhibited airway mucus secretions in asthmatic rat models. Its mechanism may have been related to the blockage of the TGF-β3/Smad2/Smad3 signaling pathway and autophagy downregulation.

Regulated and emerging mycotoxins occurrence in plant-based beverages from the Italian market

This study aimed to optimize a salting-out assisted liquid-liquid extraction (SALLE) procedure combined with ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS/MS) for the simultaneous determination of 19 mycotoxins in plant-based beverages. The targeted mycotoxins were aflatoxins (AFB1, AFB2, AFG1, AFG2), alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), fumonisins (FB1 and FB2), HT-2/T-2, ochratoxin A (OTA), zearalenone (ZEN), deoxynivalenol (DON), enniatins (ENNB, ENNB1, ENNA, ENNA1), and beauvericin (BEA). Satisfactory performance characteristics were obtained, with recoveries values > 70% and precision with RSD <20%. The methodology developed was applied to 96 commercial samples from Italy. All the samples analyzed contained at least one mycotoxin, and a particular incidence of non-regulated mycotoxins, mainly enniatins, BEA, and Alternaria toxins, was evidenced in all plant-based beverages. The contamination level ranged from 0.02 μg L−1 for BEA to 4.61 μg L−1 for DON. Overall, the present study contributes to providing comprehensive occurrence mycotoxin data in plant-based beverages, which is necessary for further evaluation of exposure and risk characterization.

Impact of easing COVID-19 restrictions on antibiotic usage in Eastern China using wastewater-based epidemiology

Coronavirus Disease 2019 (COVID-19) emerged in December 2019, prompting the implementation of a “zero-COVID” policy in Mainland China. The easing of this policy in December 2022 led to a surge in COVID cases, which was believed to significantly increase antibiotic usage, potentially due to antibiotic misuse or increased coinfections. Our study aimed to compare antibiotic consumption and patterns before and after this policy adjustment. We utilised wastewater-based epidemiology (WBE) to analyse antibiotic levels in samples collected from five wastewater treatment plants in Eastern China during January and February of 2021 and 2023. 27 antibiotics were quantified using ultra-high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UPLC-MS/MS) and analysed via WBE, with the resulting estimates compared with catchment-specific prescription data. 23 antibiotics were detected in wastewater samples, with a substantial increase in usage in 2023 (ranging from 531% to 3734%), consistent with prescription data. Here, we show a significant rise in antibiotic consumption during the COVID-19 surge and this underscores the need for further investigation into the impacts of inappropriate antibiotic use in China.

Detection and assessment of antimicrobial resistance promotion risk in the Little Akaki River wastewater.

The misuse of antimicrobial agents in healthcare and animal farming has led to their release into the environment through wastewater, contributing to the emergence of resistant bacteria. This study analyzed selected antimicrobial concentration and antimicrobial resistance (AMR) risk in the Little Akaki River wastewater. Samples collected from March to December 2019, June 2020, and August 2022 were processed using solid phase extraction with a hydrophilic-lipophilic balance (HLB) cartridge and analyzed through ultra-high-performance liquid chromatography (UHPLC) with mass spectrometry. Risk quotients (RQ) were calculated as the ratio of measured environmental concentrations (MEC) to predicted no-effect concentrations (PNEC). Ciprofloxacin concentrations ranged from 5.62 to 9.34 µg/L, cefotaxime from 1.89 to 64.79 µg/L, and sulfamethoxazole from 29.11 to 248.77 µg/L, with higher concentrations observed during the wet season. MECs for ciprofloxacin, cefotaxime, and sulfamethoxazole exceeded their PNECs (RQ > 2), indicating a high risk of resistance development. Sulfamethoxazole posed the greatest risk due to its consistently elevated levels. In contrast, erythromycin concentrations remained below the PNEC, suggesting minimal resistance risk. These findings highlight the urgent need for wastewater treatment strategies to mitigate AMR risks in aquatic environments.

Implementation of Sensitive Method for Determination of Benzophenone and Camphor UV Filters in Human Urine

The level of the human body’s burden of benzophenone and camphor ultraviolet (UV) filters can be estimated from their urinary levels. The present study describes the implementations and validation of the sensitive analytical method for the analysis of seven benzophenone and two camphor UV filters in urine. Sample preparation includes overnight enzymatic hydrolysis and ethyl acetate extraction followed by purification by dispersive solid-phase extraction using a sorbent Z-Sep. For the analysis, ultra-high performance liquid chromatography coupled with tandem mass spectrometry was used. Validation was performed using a Standard Reference Material® 3673 and an artificially contaminated urine sample. Target analyte recoveries ranged from 79–113% with repeatability expressed as a relative standard deviation of 2–15%. The limits of quantification were between 0.001 and 0.100 ng/mL in urine. This method was subsequently applied to examine the urine samples collected from Czech women. The analytes benzophenone-1 and 4-hydroxy-benzophenone were the most common analytes present in 100% of the samples, whereas benzophenone-3 was quantified in only 90% of the urine samples. The other four determined benzophenone derivatives were quantified in ≤33% of the samples. The derivatives of camphor were not detected in any samples. This method could be applied in biomonitoring studies.