Ultra-high Performance Liquid Chromatography Research Articles

Identification of volatile and non-volatile migrants released during sous vide cooking by UPLC-IMS-QTOF and DI-SPME-GC-MS using a design of experiments approach

The rise of new culinary advances combined with science has made it possible to find new optimum methods of cooking, such as vacuum cooking (Sous Vide), that allow achieving pleasant textures, preserving the genuine nature of the food and also its original flavour. Sous Vide involves the direct contact of plastic materials with food during cooking, causing the possible release of chemical compounds that may alter the properties of the food and pose a risk to human health. In this work, the migration kinetics that takes place during this cooking process have been estimated thanks to a design of experiments (DoE). DoE has been applied to 4 samples of vacuum cooking bags containing 3 food simulants (ethanol 10%, acetic acid 3% and isooctane) under typical cooking conditions of the culinary technique. The study also allowed the identification of the main volatile and non-volatile migrants that can be transferred from this particular packaging. Twenty-nine non-volatile species were identified by ultra-high performance liquid chromatography coupled to a quadrupole-time-of-flight ion-mobility separation mass spectrometer (UPLC-IMS-QTOF) and sixty-four volatile species were identified by solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). Furthermore, a comprehensive literature analysis was conducted to explore the applications, origins, and potential toxicity of these compounds.

Cilentan Cichorium intybus L. organs: UHPLC-QqTOF-MS/MS analysis for new antioxidant scenario, exploitable locally and beyond

In the framework aimed at evaluating the close link between the chemical composition of poorly explored organs of local edible medicinal plants in the Cilento region (Southern Italy) and their recovery for new health products, Cichorium intybus L. was of interest. Ultra-High Performance Liquid Chromatography Quadrupole-Time-Of-Flight tandem Mass Spectrometry (UHPLC-QqTOF-MS/MS) investigation revealed that sesquiterpene lactones characterized roots and stems, while hydroxycinnamic derivatives accumulated in florets, bracts, and leaves. Florets diverged for anthocyanin derivatives, while roots differed by low flavonoid content. Despite shared phytochemical constituents, their ratio was responsible for different antioxidant responses. Notably, extracts, particularly rich in hydroxycinnamoyl derivatives, showed protective effects against oxidative stress induced by hydrogen peroxide in SH-SY5Y and HaCaT cell lines. The wound healing assay revealed marked inhibition of SH-SY5Y cell migration, avoiding wound closure. Overall, the research encourages the use of different organs from Cilentan wild chicory to obtain antioxidant safe and consumer-guaranteed products.

Evaluation of pesticide residues in rice marketed in Cape Verde by QuEChERS-LC/GC-MS analysis

In this work, a multiresidue pesticide analysis using both gas chromatography and ultra-high performance liquid chromatography coupled to mass spectrometry systems has been conducted to determine 151 pesticides in rice samples consumed by the population of Cape Verde. The extraction and clean-up procedures were carried out by the QuEChERS method. The validation of both methodologies achieved determination coefficients higher than 0.9902 in matrix-matched calibration, relative recovery values in the range 58–120% with relative standard deviation lower than 20%, and limits of quantification of the method in the range 0.008–0.017 mg/kg, which agreed with the legal limits established by both EU regulations and the Codex Alimentarius guidelines. Among the total of 98 rice samples analysed, 91 samples were contaminated by pesticides at concentrations up to 18.3 mg/kg, with pirimiphos-methyl, propiconazole, tebuconazole, and imidacloprid being the most frequently detected. Following EU regulations, 14 non-compliances were found in 13 samples, whereas only one non-compliance was identified according to the Codex. Besides, a risk assessment based on the dietary exposure was performed for the pesticides found in the analysed rice samples. The results showed the need to carry out stricter controls to ensure the security of food agricultural products to protect consumers’ health.

Image analysis and polyphenol profiling unveil red-flesh apple phenotype complexity.

The genetic basis of colour development in red-flesh apples (Malus domestica Borkh) has been widely characterised; however, current models do not explain the observed variations in red pigmentation intensity and distribution. Available methods to evaluate the red-flesh trait rely on the estimation of an average overall colour using a discrete class notation index. However, colour variations among red-flesh cultivars are continuous while development of red colour is non-homogeneous and genotype-dependent. A robust estimation of red-flesh colour intensity and distribution is essential to fully capture the diversity among genotypes and provide a basis to enable identification of loci influencing the red-flesh trait. In this study, we developed a multivariable approach to evaluate the red-flesh trait in apple. This method was implemented to study the phenotypic diversity in a segregating hybrid F1 family (91 genotypes). We developed a Python pipeline based on image and colour analysis to quantitatively dissect the red-flesh pigmentation from RGB (Red Green Blue) images and compared the efficiency of RGB and CIEL*a*b* colour spaces in discriminating genotypes previously classified with a visual notation. Chemical destructive methods, including targeted-metabolite analysis using ultra-high performance liquid chromatography with ultraviolet detection (UPLC-UV), were performed to quantify major phenolic compounds in fruits' flesh, as well as pH and water contents. Multivariate analyses were performed to study covariations of biochemical factors in relation to colour expression in CIEL*a*b* colour space. Our results indicate that anthocyanin, flavonol and flavanol concentrations, as well as pH, are closely related to flesh pigmentation in apple. Extraction of colour descriptors combined to chemical analyses helped in discriminating genotypes in relation to their flesh colour. These results suggest that the red-flesh trait in apple is a complex trait associated with several biochemical factors.

Efficient Quality Control of Peptide Pools by UHPLC and Simultaneous UV and HRMS Detection

Peptide pools consist of short amino acid sequences and have proven to be versatile tools in various research areas in immunology and clinical applications. They are commercially available in many different compositions and variants. However, unlike other reagents that consist of only one or a few compounds, peptide pools are highly complex products which makes their quality control a major challenge. Quantitative peptide analysis usually requires sophisticated methods, in most cases isotope-labeled standards and reference materials. Usually, this would be prohibitively laborious and expensive. Therefore, an approach is needed to provide a practical and feasible method for quality control of peptide pools. With insufficient quality control, the use of such products could lead to incorrect experimental results, worsening the well-known reproducibility crisis in the biomedical sciences. Here we propose the use of ultra-high performance liquid chromatography (UHPLC) with two detectors, a standard UV detector at 214 nm for quantitative analysis and a high-resolution mass spectrometer (HRMS) for identity confirmation. To be cost-efficient and fast, quantification and identification are performed in one chromatographic run. An optimized protocol is shown, and different peak integration methods are compared and discussed. This work was performed using a peptide pool known as CEF advanced, which consists of 32 peptides derived from cytomegalovirus (CMV), Epstein–Barr virus (EBV) and influenza virus, ranging from 8 to 12 amino acids in length.

Development and validation of stability indicating ultra‐high‐performance liquid chromatographic method for assay and impurities of mexiletine hydrochloride and mexiletine hydrochloride capsules

AbstractMexiletine (MEX) is an antiarrhythmic drug used to treat acute and chronic ventricular arrhythmias. A simple isocratic, rapid, specific, stability‐indicating, and sensitive reverse‐phase ultra‐high‐performance liquid chromatography (UHPLC) method was developed and successfully validated to quantify the assay and impurities of MEX hydrochloride. The new UHPLC method showed the optimum separation of MEX and its seven impurities in 20 min by using sodium acetate buffer (0.14 mM) with 0.3% of glacial acetic acid (pH 4.8) and methanol in the ratio of 40:60 (v/v) as mobile phase. The method used an Acquity HSS T3 (100 × 3.0 mm, 1.8 µm) chromatographic column at a 0.2 mL/min flow rate. The injection volume is 2 µL, and 254 nm is the detection wavelength. Note that, 25°C is the column oven temperature. The method is linear over the concentration range of the quantification limit (0.03%) to 150% (0.3%) of the specification level (0.2%) for the impurities and 50%–150% for the assay method. The quality control lab of bulk drugs and finished dosage form successfully applied the new UHPLC method for quantification of assay and impurities of MEX hydrochloride.

Chemical characterization and comparative analysis of different parts of Cocculus orbiculatus through UHPLC-Q-TOF-MS.

Cocculus orbiculatus (L.) DC. (C. orbiculatus) is a medicinal herb valued for its dried roots with anti-inflammatory, analgesic, diuretic, and other therapeutic properties. Despite its traditional applications, chemical investigations into C. orbiculatus remain limited, focusing predominantly on alkaloids and flavonoids. Furthermore, the therapeutic use of C. orbiculatus predominantly focuses on the roots, leaving the stems, a significant portion of the plant, underutilized. This study employed ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) with in-house and online databases for comprehensive identification of components in various plant parts. Subsequently, untargeted metabolomics was employed to analyze differences in components across different harvest periods and plant sections of C. orbiculatus, aiming to screen for distinct components in different parts of the plant. Finally, metabolomic analysis of the roots and stems, which contribute significantly to the plant's weight, was conducted using chemometrics, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA), and heatmaps. A total of 113 components, including alkaloids, flavonoids, and organic acids, were annotated across the root, stem, leaf, flower, and fruit, along with numerous previously unreported compounds. Metabolomic analyses revealed substantial differences in components between the root and stem compared to the leaf, flower, and fruit during the same harvest period. PLS-DA and OPLS-DA annotated 10 differentiating components (VIP > 1.5, P < 0.05, FC > 2 or FC < 0.67), with 5 unique to the root and stem, exhibiting lower mass spectrometric responses. This study provided the first characterization of 113 chemical constituents in different parts of C. orbiculatus, laying the groundwork for pharmacological research and advocating for the enhanced utilization of its stem.

Quality markers of Polygala fallax Hemsl decoction based on qualitative and quantitative analysis combined with network pharmacology and chemometric analysis.

Polygala fallax Hemsl (PFH) is a widely used herbal medicine in Guangxi, China. At present, research on PFH mainly focuses on extraction technology and cultivation, lacking quality control standards for systematic evaluation. The study aimed to assess the quality of PFH from different sources and to predict markers that would help assess quality. Fingerprinting of 15 batches of PFH samples was performed by ultra-high performance liquid chromatography (UPLC) and similarity was assessed using hierarchical cluster analysis (HCA), principal component analysis (PCA), and orthogonal partial least squares discrimination (OPLS-DA). Differential components were screened by mathematical analysis, and a "component-target-pathway" network map was constructed in combination with network pharmacology, quality markers (Q-markers) of PFH were predicted, and quantitative analysis was performed. Fifteen batches were fingerprinted for PFH, with 11 common peaks, and peak 5 was identified as 4-hydroxybenzoic acid, which was generally consistent with the results of HCA, PCA, and OPLS-DA. Network pharmacology screened 18 potential compounds, 45 core targets, and 20 key pathways, integrating fingerprinting, pattern recognition, and network pharmacology methods. One of the potential Q-markers that can identify the principle of testability, efficacy, and specificity is 4-hydroxybenzoic acid, whose content ranges from 0.0188 to 1.4517 mg/g. The potential Q-markers of PFH were predicted by integrating fingerprinting, pattern recognition, and network pharmacological analysis, which provided a scientific basis for the overall control and evaluation of the quality of PFH and a theoretical reference for the study of the quality standard of multi-base traditional Chinese medicine.

Wine- and stir-frying processing of Cuscutae Semen enhance its ability to alleviate oxidative stress and apoptosis via the Keap 1-Nrf2/HO-1 and PI3K/AKT pathways in H2O2-challenged KGN human granulosa cell line

BackgroundCuscutae Semen (CS) has been prescribed in traditional Chinese medicine (TCM) for millennia as an aging inhibitor, an anti-inflammatory agent, a pain reliever, and an aphrodisiac. Its three main forms include crude Cuscutae Semen (CCS), wine-processed CS (WCS), and stir-frying-processed CS (SFCS). Premature ovarian insufficiency (POI) is a globally occurring medical condition. The present work sought a highly efficacious multi-target therapeutic approach against POI with minimal side effects. Finally, it analyzed the relative differences among CCS, WCS and SFCS in terms of their therapeutic efficacy and modes of action against H2O2-challenged KGN human granulosa cell line.MethodsIn this study, ultrahigh-performance liquid chromatography (UPLC)-Q-ExactiveTM Orbitrap-mass spectrometry (MS), oxidative stress indices, reactive oxygen species (ROS), Mitochondrial membrane potential (MMP), real-time PCR, Western blotting, and molecular docking were used to investigate the protective effect of CCS, WCS and SFCS on KGN cells oxidative stress and apoptosis mechanisms.ResultsThe results confirmed that pretreatment with CCS, WCS and SFCS reduced H2O2-induced oxidative damage, accompanied by declining ROS levels and malondialdehyde (MDA) accumulation in the KGN cells. CCS, WCS and SFCS upregulated the expression of antioxidative levels (GSH, GSH/GSSG ratio, SOD, T-AOC),mitochondrial membrane potential (MMP) and the relative mRNA(Nrf2, Keap1, NQO-1, HO-1, SOD-1, CAT). They inhibited apoptosis by upregulating Bcl-2, downregulating Bax, cleaved caspase-9, and cleaved caspase-3, and lowering the Bax/Bcl-2 ratio. They also exerted antioxidant efficacy by partially activating the PI3K/Akt and Keap1-Nrf2/HO-1 signaling pathways.ConclusionsThe results of the present work demonstrated the inhibitory efficacy of CCS, WCS and SFCS against H2O2-induced oxidative stress and apoptosis in KGN cells and showed that the associated mechanisms included Keap1-Nrf2/HO-1 activation, P-PI3K upregulation, and P-Akt-mediated PI3K-Akt pathway induction.

Comprehensive Toxicological Assessment of Halobenzoquinones in Drinking Water at Environmentally Relevant Concentration.

Halobenzoquinones (HBQs), an emerging unregulated category of disinfection byproduct (DBP) in drinking water, have aroused an increasing concern over their potential health risks. However, the chronic toxicity of HBQs at environmentally relevant concentrations remains largely unknown. Here, the occurrence and concentrations of 13 HBQs in drinking water from a northern megacity in China were examined using ultrahigh performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-MS/MS). Four HBQs, including 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), 2,6-dibromo-1,4-benzoquinone (2,6-DBBQ), 2,3,6-trichloro-1,4-benzoquinone (TriCBQ), and 2,5-dibromo-1,4-benzoquinone (2,5-DBBQ), were detected beyond 50% occurrence frequency and at median concentrations from 4 to 50 ng/L. The chronic toxicity of these four HBQs to normal human colon and liver cells (FHC and THLE-2) was investigated at these concentrations. After 90 days of exposure, 2,5-DBBQ and 2,6-DCBQ induced the highest levels of oxidative stress and deoxyribonucleic acid (DNA) damage in colon and liver cells, respectively. Moreover, 2,5-DBBQ and 2,6-DCBQ were also found to induce epithelial-mesenchymal transition (EMT) in normal human liver cells via the extracellular signal regulated kinase (ERK) signaling pathway. Importantly, heating to 100 °C (boiling) was found to efficiently reduce the levels of these four HBQs in drinking water. These results suggested that environmentally relevant concentrations of HBQs could induce cytotoxicity and genotoxicity in normal human cells, and boiling is a highly efficient way of detoxification for HBQs.

Coptidis rhizoma extract alleviates oropharyngeal candidiasis by gC1qR-EGFR/ERK/c-fos axis-induced endocytosis of oral epithelial cells

Ethnopharmacological relevanceCoptidis rhizoma, first recorded in the “Shen Nong's Herbal Classic”, is one of the traditional Chinese medicine (TCM) used to treat infectious diseases, with reputed effectiveness against oropharyngeal candidiasis (OPC). Studies have demonstrated the inhibitory properties of C. rhizoma (CRE) against Candida albicans, yet there is limited information available regarding its treatment mechanism for OPC. Aim of the studyOur previous research has suggested that CRE can prevent the formation of C. albicans hyphae and their invasion of the oral mucosa, thereby exerting a therapeutic effect on OPC. Nevertheless, the precise therapeutic mechanisms remain incompletely understood. Previous studies have revealed that a receptor for globular heads of C1q (gC1qR), a crucial co-receptor of the epidermal growth factor receptor (EGFR), facilitates the EGFR-mediated internalization of C. albicans. Therefore, this study aims to investigate the potential mechanism of action of CRE and its primary component, berberine (BBR), in treating OPC by exploring their effects on the gC1qR-EGFR co-receptor. Materials and methodsTo identify the chemical components of CRE, we utilized Ultra-high performance liquid chromatography in conjunction with quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MSE), revealing the presence of at least 18 distinct components. To observe the therapeutic effects of CRE on OPC at the animal level, we employed hematoxylin and eosin staining, periodic acid-Schiff staining, scanning electron microscopy, and fungal load detection. Subsequently, we evaluated the anti-inflammatory properties of CRE and its main component, BBR, in treating OPC. This was achieved through enzyme-linked immunosorbent assay (ELISA) both at the animal and cellular levels. Additionally, we assessed the ability of C. albicans to disrupt the epithelial barrier of FaDu cells by studying the protective effects of BBR on the fusion barrier using the transwell assay. To further explore the underlying mechanisms, we analyzed the effects of BBR on the gC1qR-EGFR/extracellular signal-regulated kinase/c-Fos signaling pathway at the cellular level using qRT-PCR, western blotting, and immunofluorescence. Furthermore, we validated the effects of BBR on the gC1qR-EGFR co-receptor through ELISA, qRT-PCR, and western blotting. Finally, to confirm the outcomes observed at the cellular level, we validated the impact of CRE on the gC1qR-EGFR co-receptor in vivo using qRT-PCR, western blotting, and immunofluorescence. These comprehensive methods allowed us to gain a deeper understanding of the therapeutic mechanisms of CRE and BBR in treating OPC. ResultsOur findings indicate that CRE and its primary component, BBR, effectively alleviated the symptoms of OPC by modulating the gC1qR-EGFR co-receptor. The chemical composition of CRE and BBR was accurately identified using UPLC-Q/TOF-MSE. The gC1qR-EGFR co-receptor plays a crucial role in regulating downstream signaling pathways, emerging as a potential therapeutic target for OPC treatment. Through both in vitro and in vivo experiments, we explored the therapeutic potential of CRE and BBR in OPC. Additionally, we employed overexpression and silencing techniques to confirm that BBR can indeed influence the gC1qR-EGFR co-receptor and regulate the gC1qR-EGFR/extracellular signal-regulated kinase (ERK)/c-Fos signaling pathway, leading to improved OPC outcomes. Furthermore, the significance of CRE's effect on the gC1qR-EGFR co-receptor was validated in vivo. ConclusionOur study demonstrates that CRE and its main component, BBR, can effectively alleviate OPC symptoms by targeting the gC1qR-EGFR heterodimer receptor. This discovery offers a promising new therapeutic approach for the treatment of OPC.

Impact of extraction method on the lipids of Himalayan marmot oil with ultrahigh-performance liquid chromatography Q-Exactive Orbitrap mass spectrometry analysis.

Himalayan marmot oil (SPO) has been used for pharmaceutical purposes for centuries, but its composition is still unclear. The bioactivity of SPO highly depends on the techniques used for its processing. This study focused on the comprehensive lipidomics of SPO, especially on the ones derived from dry rendering, wet rendering, cold pressing, and ultrasound-assisted solvent extraction. We performed lipid profiling of SPO acquired by different extraction methods using ultrahigh-performance liquid chromatography Q-Exactive Orbitrap mass spectrometry, and 17 classes of lipids (2 BMPs, 12 LysoPCs, 9 LysoPEs, 41 PCs, 24 PEs, 23 Plasmenyl-PCs, 10 Plasmenyl-PEs, 10 MGs, 63 DGs, 187 TGs, 2 MGDGs, 3 Cer[NDS]s, 22 Cer[NS]s, 2 GlcCer[NS]s, 14 SMs, 14 CEs, and 6 AcylCarnitines) were characterized. Fifty-five lipids were differentially altered (VIP > 1.5, p < 0.05) between the extraction techniques, which can be used as potential biomarkers to differentiate SPO extracted by various methods. Additionally, the contents of oleic acid and arachidic acid were abundant in all samples that may suggest their medicinal values and are conducive to in-depth research. These findings reveal the alterations of lipid profile and free fatty acid composition in SPO obtained with different extraction methods, providing a theoretical foundation for investigating its important components as functional factors in medicines and cosmetics.

Self-reported caffeine consumption miss-matched consumption measured by plasma levels of caffeine and its metabolites: results from two population-based studies.

Self-reported caffeine consumption has been widely used in research while it may be subject to bias. We sought to investigate the associations between self-reported caffeine consumption and plasma levels of caffeine and its two main metabolites (paraxanthine and theophylline) in the community. Data from two population-based studies (SKIPOGH1 and 2 (N = 1246) and CoLaus|PsyCoLaus (N = 4461)) conducted in Switzerland were used. Self-reported caffeine consumption was assessed using questionnaires. Plasma levels of caffeine and its metabolites were quantified by ultra-high performance liquid chromatography coupled to a tandem quadrupole mass spectrometer. In both studies, mean log plasma levels of caffeine and its two metabolites were over 6.48 (plasma levels = 652 ng/ml) when no caffeine consumption was reported. Subsequently, nonlinear associations between log plasma levels and self-reported caffeine consumption were observed in SKIPOGH, with a change of the slope at 3-5 cups of espresso per day in SKIPOGH1 but not SKIPOGH2. In CoLaus|PsyCoLaus, increased daily consumption of caffeinated beverages was associated with increased log plasma levels with a change of the slope at 3 cups. In both studies, declared caffeine consumption higher than 3-5 cups per day was not associated with higher plasma levels of caffeine and its metabolites. Self-reports of no or low caffeine consumption and consumption of more than 3-5 cups of coffee should be interpreted with caution, with possible under- or over-estimation. Quantifying plasma levels of caffeine and its metabolites may contribute to a better estimation of caffeine intake.

Investigation of oligomeric proanthocyanidins extracted from Rhodiolae Crenulatae Radix et Rhizomes using deep eutectic solvents and identified via data-dependent-acquisition mass-spectroscopy

In this study, 34 deep eutectic solvents were successfully prepared for the extraction of proanthocyanidin from Rhodiolae Crenulatae Radix et Rhizomes. The extraction process was optimized using single factor exploration and Box-Behnken design-response surface analysis. The extraction rate was significantly improved when the molar ratio of choline chloride to 1,3-propanediol was 1:3.5 and the water content was 30% (V/V) in deep eutectic solvents. AB-8 macroporous resin and ethyl acetate were used for separation and refining, and the oligomer-rich proanthocyanidin components were eventually obtained. The ultraviolet (UV) and infrared spectrometer (IR) spectra showed that the proanthocyanidins were mainly composed of catechin and epicatechin. To further clarify the chemical composition of proanthocyanidin, an ion scan list containing 156 proanthocyanidins precursors was obtained by constructing a proanthocyanidins structural library and mass defect filtering algorithm, combined with the Full mass spectrometry (MS)/dd-MS2 scan mode that turns on the “if idle pick others” function. By using Ultra high performance liquid chromatography & High-resolution mass spectrometer (UHPLC/Orbitrap MS), the analysis used both targeted and non-targeted methods to detect proanthocyanidins. Finally, 50 oligomeric proanthocyanidin compounds were identified, including 7 monomers, 22 dimers, 20 trimers, and 1 tetramer, most of which were procyanidins of proanthocyanidins (84%), and a small amount of prodelphinidin (14%) and other types of proanthocyanidins (2%), which enabled the systematic characterization of proanthocyanidin components from Rhodiolae Crenulatae Radix et Rhizomes. Meanwhile, the comparison with the grape seeds oligomeric proanthocyanidins standard (United States Pharmacopoeia) revealed that the proanthocyanidins in Rhodiolae Crenulatae Radix et Rhizomes were more abundant, suggesting that the proanthocyanidins in Rhodiolae Crenulatae Radix et Rhizomes has promising applications.

Simultaneous quantification of seven B vitamins from wheat grains using UHPLC-MS/MS

B-group vitamins are important micronutrients for maintaining human health; nevertheless, B vitamin deficiency is a globally widespread issue. Thus, it is relevant to accurately assess the B-vitamin content in staple crop products such as wheat grains. Here, we developed a multi-enzyme extraction method allowing accurate quantification of seven B vitamins in wheat using ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Free forms of thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), biotin (B7) and folates (B9) were determined with recoveries ranging from 81 to 118% and accuracy below 15% bias. The precision was below 20% relative standard deviation and the internal standards adequately compensated for matrix effects. The method was applied to determine the B vitamin stabilities in wheat grains stored at different temperatures and periods. The results provide an important basis in future studies aiming at understanding nutritional availability of B vitamins.

A novel enrichment and sensitive method for rapid determination of 4 sulfonamide antibiotics residues in fish

Sulfonamide antibiotics with superior performance are widely used in aquaculture. However, the residues of sulfonamide antibiotics in aquatic products may pose a threat to human health. In this work, a novel type of magnetic molecularly imprinted polymer (mMIP) was synthesized and coupled with ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) for the selective separation and determination of 4 sulfonamide antibiotics residues in tilapia. The structure and adsorption properties of the polymers were characterized and evaluated. The key mMIP variables were investigated to determine the optimal magnetic solid phase extraction conditions. Results showed that the mMIPs had saturated adsorption capacities of 137.4, 78.7, 65.2, and 58.4 mg/g for trimethoprim, sulfadiazine, sulfamethazine, and sulfamethoxazole, respectively. Under optimized conditions, the linear range of the method for the detection of the 4 sulfonamides was 0.1–100 ng mL−1 (R > 0.991), the detection limit was 0.015 μg kg−1 (S/N = 3), the recovery rate was 78.6–101%, and the relative standard deviation (RSD%) was 4.2–7.8%. This new rapid and ultra-sensitive pretreatment method was successfully applied to the monitoring of trace sulfonamide antibiotics residues in fish samples.

Magnetic-stirring-enhanced mechanical amorphous dispersion extraction for the hydrophobic phytochemical constituents using an aqueous solution from a medicinal plant

A method involving chitosan-assisted magnetic-stirring-enhanced mechanical amorphous dispersion extraction was developed and utilized to extract hydrophobic anthraquinones from Rhei Radix et Rhizoma prior to ultrahigh performance liquid chromatography analysis. Incorporating natural chitosan as a dispersant facilitated the extraction of hydrophobic anthraquinones using purified water, considerably enhancing the eco-friendliness of the extraction methodology. To optimize extraction efficiency, an extensive evaluation of the crucial parameters influencing rhubarb yield was conducted. Furthermore, a response surface methodology was applied to optimize the extraction conditions. Under these optimized conditions, the method exhibited linearity ranges of 0.1–100 µg/mL, with correlation coefficients between 0.9990 and 0.9998. The method’s intraday (n = 6) and interday (n = 6) precision levels were maintained at ≤3.58%, which was considered to be within acceptable limits. The computed detection and quantification limits were 16.54–24.60 and 54.91–82.04 ng/mL, respectively. Consequently, this optimized method was effectively employed to extract five specific compounds (aloe-emodin, emodin, rhein, chrysophanol, and physcion) from Rhei Radix et Rhizoma, achieving recoveries ranging from 86.43% to 102.75%.

Unambiguous identification of γ-aminobutyric acid adducts as novel plant biomarkers and their ultra-sensitive detection by UPLC–MS/MS for retrospective analyses of nitrogen mustards exposure

Plants are widely existing in the environments and have been considered as potential sentinel species of toxic chemicals′ exposure. In this study, the deadly toxic chemicals of three nitrogen mustards (NMs, including NH1, NH2 and NH3) were selected as the investigated targets. First, the reactivities of common endogenous plant components with NMs were examined in vitro. Then, the model plant Nicotiana benthamiana Domin was exposed to NMs. Three γ-aminobutyric acid–nitrogen mustard adducts (GABA–NMs) were identified in the living plant by high resolution mass spectrometry and comparison with the synthesized references. A sensitive detection method with the limits of quantification of 0.0500ngmL-1 was developed using ultrahigh performance liquid chromatography–triple quadrupole mass spectrometry. The GABA–NMs could be detected after 120 days of the exposure and even in the dead leaves without obvious decrease. Furthermore, 20 different plant species grown in diverse climate zones were exposed to HN1, and the adduct of GABA–HN1 was identified in all the leaves. The results showed the good universality and specificity of GABA–NMs as plant biomarkers for NMs exposure. This work provides a new approach for the pollution investigation of toxic chemicals through analysing biomarkers in plant materials. Environmental implicationNitrogen mustards (NMs) are lethal blistering agents, which are listed as Schedule 1 toxic chemicals by the Organisation for the Prohibition of Chemical Weapons (OPCW). OPCW has established the worldwide network of laboratories for environmental analysis for the abuse surveillance of the lethal chemicals. NMs entering the environment will cause great harms to organisms, including cytotoxicity and genotoxicity. In this work, γ-aminobutyric acid adducts in plants after exposure to NMs were identified as biomarkers to trace the attack or leakage in the environment. The work provides a novel promising approach to evaluate environmental hazards from toxic chemicals.

Optimization and validation of metabolomics methods for feline urine and serum towards application in veterinary medicine

BackgroundMetabolomics is an emerging and powerful technology that offers a comprehensive view of an organism's physiological status. Although widely applied in human medicine, it is only recently making its introduction in veterinary medicine. As a result, validated metabolomics protocols in feline medicine are lacking at the moment. Since biological interpretation of metabolomics data can be misled by the extraction method used, species and matrix-specific optimized and validated metabolomic protocols are sorely needed. ResultsSystematic optimization was performed using fractional factorial experiments for both serum (n=57) and urine (n=24), evaluating dilution for both matrices, and aliquot and solvent volume, protein precipitation time and temperature for serum. For the targeted (n=76) and untargeted (n=1,949) validation of serum respectively, excellent instrumental, intra-assay and inter-day precision were observed (CV≤ 15% or 30%, respectively). Linearity deemed sufficient both targeted and untargeted (R2≥0.99 or 0.90, respectively). An appropriate targeted recovery between 70 and 130% was achieved. For the targeted (n=69) and untargeted (n=2,348) validation of the urinary protocol, excellent instrumental and intra-assay precision were obtained (CV≤ 15% or 30%, respectively). Subsequently, the discriminative ability of our metabolomics methods was confirmed for feline chronic kidney disease (CKD) by univariate statistics (n=41 significant metabolites for serum, and n=55 for urine, p-value<0.05) and validated OPLS-DA models (R2(Y)>0.95, Q2(Y)>0.65, p-value<0.001 for both matrices). SignificanceThis study is the first to present an optimized and validated wholistic metabolomics methods for feline serum and urine using ultra-high performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry. This robust methodology opens avenues for biomarker panel selection and a deeper understanding of feline CKD pathophysiology and other feline applications.

Trace analysis of 20 antihistamines in milk by ultrahigh performance liquid chromatography coupled with high field quadrupole orbitrap high resolution mass spectrometry followed dispersive micro solid phase extraction

Ultrahigh-performance liquid chromatography coupled with high-field quadrupole Orbitrap high resolution mass spectrometry was used for the separation and determination of 20 antihistamines, and a dispersive micro solid-phase extraction procedure using high-performance absorbing material was developed as a sample preparation strategy for extracting 20 antihistamines from milk. Instrument conditions and key parameters influencing extraction efficiency were investigated to obtain an optimized method. The limit of detection for 20 antihistamines in milk using this method is 0.05 µg/L to 1.0 μg/L. Recoveries are between 80.7% and 108.3%, and the relative standard deviation is less than 15%. It is suitable for confirmatory monitoring and quantitative analysis of 20 antihistamines in milk. The results show that antihistamines in milk may be noteworthy issues for human health and environmental pollution.