Liquid Chromatography Orbitrap Mass Spectrometry Research Articles

Targeted Offline Two-Dimensional HPLC and UHPLC-Orbitrap-MS Combined with Molecular Networking Reveal the Effect of Processing on Chemical Constituents of Xuetong (the Stem of Kadsura heteroclita)

Xuetong, the dried stem of Kadsura heteroclita (Roxb.) Craib, is a traditional Tujia medicine extensively used to treat rheumatoid arthritis (RA). All traditional Chinese medicines (TCMs) necessitate a processing stage called “Paozhi” before clinical application. However, there is a dearth of research concerning the processing methods employed for Xuetong. To investigate the impact of vinegar and wine processing on the chemical constituents of Xuetong, this study devised a targeted offline two-dimensional (2D) high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography-orbitrap mass spectrometry (UHPLC-orbitrap-MS) method. By incorporating various MS data-processing techniques, such as molecular networking technology, fragment-ion similarity searching (FISh), online and offline database matching, and fragmentation pattern analysis, a total of 158 components were identified in Xuetong. Among them, 14 were verified by comparison with the reference standards. Notably, aside from triterpenoids and lignans, catechin derivatives were found to be the predominant constituents of Xuetong, and their levels exhibited a significant decrease following processing. This method significantly improved peak capacity and resolution, overcoming the limitations of 1D LC in simultaneously analyzing highly polar catechin derivatives and less polar triterpenoids and lignans. Moreover, the developed method shows promise for Xuetong’s quality control.

Insight into the Mechanism Underlying the Reduction of Digestibility and IgG/IgE Binding Ability in Ovalbumin during Different High-Temperature Conduction Modes-Induced Glycation.

Effects of different high-temperature conduction modes [high-temperature air conduction (HAC), high-temperature contact conduction (HCC), high-temperature steam conduction (HSC)]-induced glycation on the digestibility and IgG/IgE-binding ability of ovalbumin (OVA) were studied and the mechanisms were investigated. The conformation in OVA-HSC showed minimal structural changes based on circular dichroism, fluorescence, and ultraviolet spectroscopy. The degree of hydrolysis analysis indicated that glycated OVA was more resistant to digestive enzymes. Liquid chromatography-Orbitrap mass spectrometry identified 11, 14, and 15 glycation sites in OVA-HAC, OVA-HCC, and OVA-HSC, respectively. The IgG/IgE-binding ability of OVA was reduced during glycation and digestion, and the interactions among glycation, allergenicity, and digestibility were further investigated. Glycation sites masked the IgG/IgE epitopes resulting in a reduction in allergenicity. Digestion enzymes destroyed the IgG/IgE epitopes thus reducing allergenicity. Meanwhile, the glycation site in proximity to the digestion site of pepsin was observed to cause a reduction in digestibility.

MATLAB language assisted data acquisition and processing in liquid chromatography Orbitrap mass spectrometry: Application to the identification and differentiation of Radix Bupleuri from its adulterants

Comprehensive and rapid analysis of secondary metabolites like saponins remains challenging. This study aimed to establish a semi-automated workflow for filtration, identification, and characterization of saikosaponins in six Bupleurum species. Radix Bupleuri, a high-sales herbal medicine, is often adulterated, restricting its quality control and applications. Two authentic Radix Bupleuri species and four major adulterants were analyzed through UHPLC-LTQ-Orbitrap-MS for targeted saikosaponin analysis. To reveal trace saikosaponins and obtain quality fragment data, a MATLAB-based process automatically enumerating “sugar chain + aglycone + side chain” combinations and deduplicating generated a predicted saikosaponin database covering all possible saikosaponins as a precursor ion list for comprehensive targeted acquisition. To focus on informative ions and reduce MS analysis workload, we utilized MATLAB to automatically filtrate the false positive ions by MS1 and MS2 spectrometry. The newly established MATLAB-assisted data acquisition approach exhibited 50 % improvement in characterization of targeted saikosaponins. Furthermore, positive and negative ionization workflows were designed for accurate saikosaponins characterization based on fragmentation rules. In total, 707 saikosaponins were characterized, including over 500 potential new compounds and previously unreported C29 aglycones. We identified 25 saikosaponins present in both authentic species but absent in adulterants as potential markers. This unprecedented comprehensive multi-origin species differentiation demonstrates the promise of MATLAB-assisted acquisition and processing to advance saponin identification and standardize the Radix Bupleuri market.

Identification of antidiabetic constituents in Polygonatum odoratum (Mill.) Druce by UPLC-Orbitrap-MS, network pharmacology and molecular docking

Polygonatum odoratum has been known as having therapeutic effects on diabetes. However, due to its complex composition, it is difficult to elucidate the molecular mechanisms of its functions. In this study, we investigated the material basis and molecular mechanisms underlying the antidiabetic activities of methanol extract of P. odoratum (Mill.) Druce by ultra-performance liquid chromatography orbitrap mass spectrometry (UPLC-Orbitrap-MS) and network pharmacology. In specificity, we first constructed a compound-protein interaction network for type 2 diabetes mellitus (T2DM) to identify potential drug targets. Disease Ontology (DO), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were then conducted to discover the pathways involved in the antidiabetic activities of P. odoratum. Among the 334 compounds detected in P. odoratum, 277 active constituents and 897 corresponding targets were identified to be associated with antidiabetic activities. Five compounds and five targets were consequently obtained by analyzing the compound-target-pathway network. The five compounds were 9-Aminocamptothecin, 9-Methoxycamptothecin, 5-Hydroxy-1-tetralone, 5,7,3′-Trihydroxy-6,4′,5′-trimethoxyflavone, and Diacerein, while the five targets were ACTB, JUN, STAT3, HIF1A, and MMP9. In addition, 30 antidiabetic-related pathways were recognized by using KEGG pathway analysis. Moreover, molecular docking was performed with the five essential compounds and the top five targets based on degree ranking. Our study confirms that active components in P. odoratum can exert antidiabetic activities via multi-component, multi-target, and multi-pathway mechanisms of action, which serves as a practical guidance for further experimental investigations into the biological functions of the methanol extract of P. odoratum in the treatment of T2DM.

Development of Chemical Isotope Labeling Liquid Chromatography Orbitrap Mass Spectrometry for Comprehensive Analysis of Dipeptides.

Dipeptides have recently attracted considerable attention due to their newly found biological functions and potential biomarkers of diseases. Global analysis of dipeptides (400 common dipeptides in total number) in samples of complex matrices would enable functional studies of dipeptides and biomarker discovery. In this work, we report a method for high-coverage detection and accurate relative quantification of dipeptides. This method is based on differential chemical isotope labeling (CIL) of dipeptides with dansylation and liquid chromatography Orbitrap tandem mass spectrometry (LC-Orbitrap-MS). An optimized LC gradient ensured the separation of dansyl-dipeptides, including positional isomers (e.g., leucine- and isoleucine-containing dipeptides). MS/MS collision energy in Orbitrap MS was optimized to provide characteristic fragment ion information to sequence dansyl-dipeptides. Using the optimized conditions, a CIL standard library consisting of retention time, MS, and MS/MS information of a whole set of 400 dansyl-dipeptides was constructed to facilitate rapid dipeptide identification. For qualitative analysis of dipeptides in real samples, IsoMS data processing software's parameters were tuned to improve the coverage of dipeptide annotation. Data-dependent acquisition was also carried out to improve the reliability of dipeptide identification. As examples of applications, we successfully identified a total of 321 dipeptides in rice wines and 105 dipeptides in human serum samples. For quantitative analysis, we demonstrated that the intensity ratios of the peak pairs from 96% of the dansyl-dipeptides detectable in a 1:1 mixture of 12C- and 13C-labeled rice wine samples were within ±20% of an expected value of 1.0. More than 90% of dipeptides were detected with a relative standard deviation of less than 10%, showing good performance of relative quantification.

Accurate Determination of Pesticide Residues in Milk by Sonication-QuEChERS Extraction and LC-LTQ/Orbitrap Mass Spectrometry

A modified, quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction procedure combined with sonication and Ultra-High Performance Liquid Chromatography–Orbitrap-Mass Spectrometry (UHPLC–Orbitrap-MS) was developed as a sensitive and reliable methodology for the determination of multiclass pesticides in full-fat milk. Different amounts of EMR-lipid sorbent were assayed for the cleanup step in order to achieve both acceptably high recoveries and low co-extractives in the final extracts. Accurate mass measurements of the analyte’s pseudo-molecular ions and tandem MS fragmentation were used to quantify and identify the target pesticides. Analytical performance characteristics of the method, such as linearity, recovery, precision, the limit of detection (LOD) and quantification (LOQ), matrix effects (ME), and expanded uncertainty, have been determined for method validation fulfilling all criteria for its use as a validated routine method. The method was successfully applied to real samples (by local farms and commercial), revealing the presence of carbendazim in one milk sample at a concentration level below the maximum residue limits.

Biomarkers related to cognitive dysfunction in APP/PS1 mice based on non-targeted metabonomics and intervention mechanism of Huanglian Jiedu Decoction

Through the non-targeted metabonomics study on endogenous substances in APP/PS1 transgenic mice, this paper aimed to discover biomarkers related to APP/PS1 mice with cognitive dysfunction, and find targets of Huanglian Jiedu Decoction(HLJDD) in the treatment of Alzheimer's disease(AD) and its mechanism. The brain tissue and serum metabolic mass spectrometry of mice were analyzed by ultra-high performance liquid chromatography-Orbitrap mass spectrometry(UPLC-Orbitrap MS). Through partial least squares-discriminant analysis(PLS-DA) and orthogonal partial least squares-discriminant analysis(OPLS-DA), the metabolic data of the normal group, the model group, the high-dose and low-dose HLJDD groups, and the berberine group were compared and analyzed to screen out potential biomarkers, and the relevant metabolic pathways were constructed with the help of the Kyoto Encyclopedia of Genes and Genomes(KEGG) database. Forty-five potential endogenous metabolites were identified, including 13 in brain and 35 in serum, among which leukotriene B4, tyrosine, and adenosine were expected to be differential metabolites related to cognitive function. HLJDD recalled 22 differential metabolites, and the pathways mainly involved in aminoacyl-tRNA biosynthesis, valine, leucine and isoleucine biosynthesis, pantothenic acid and coenzyme A biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, and arachidonic acid metabolism. These pathways suggested that the main mechanism of HLJDD in the intervention of AD was to inhibit central and peripheral inflammation, and regulate energy metabolism, fatty acid metabolism, and amino acid metabolism. HLJDD has a certain effect on the improvement of cognitive dysfunction, and regulates relative pathways by recalling endogenous differential metabolites, which helps to further discover the biomarkers of AD and clarify the intervention mechanism of HLJDD in the treatment of AD.

In vitro approaches for the identification and characterization of urinary metabolite biomarkers of synthetic cannabinoid ADB-BUTINACA

To identify the major metabolites and investigate the key disposition kinetics of synthetic cannabinoid ADB-BUTINACA so as to establish the optimal urinary biomarkers for forensic surveillance. Novel synthetic cannabinoids (SCs) continue to evolve and cause widespread intoxications globally. Despite this increasing prevalence of abuse, severe toxicities associated with SC intake coupled with uncertainties in identifying the parent drug in biological samples remain perennial problems undermining forensic management. ADB-BUTINACA is an emerging synthetic cannabinoid that was first identified in Europe in 2019 and entered Singapore's drug scene with a total of 40 and 107 occurrences in 2020 and 2021, respectively. Due to the unavailability of toxicological and metabolic data, there is a need to establish urinary metabolite biomarkers for detection of ADB-BUTINACA consumption and elucidate its biotransformation pathways for rationalizing its toxicological implications. The metabolites of ADB-BUTINACA were identified in human liver microsomes (HLM) using liquid chromatography Orbitrap mass spectrometry analysis. Enzyme-specific inhibitors and recombinant enzymes were adopted for the reaction phenotyping of ADB-BUTINACA. Recombinant enzymes were used to generate a pool of key Phase 1 metabolites in situ and the metabolic stability of these metabolites were determined. By coupling in vitro metabolism and authentic human urine analyses, a panel of urinary metabolite biomarkers for the detection of ADB-BUTINACA abuse was proposed. A total of fifteen metabolites of ADB-BUTINACA were identified with key biotransformations being hydroxylation, N-debutylation, dihydrodiol formation, and oxidative deamination. From the reaction phenotyping studies, it was established that ADB-BUTINACA was rapidly eliminated via CYP3A4-, CYP2C19-, and CYP3A5-mediated metabolism pathways. Three major monohydroxylated metabolites were generated in situ, and they demonstrated greater metabolic stability compared to the parent drug, ADB-BUTINACA. Four urinary biomarker metabolites of ADB-BUTINACA, namely 3 hydroxylated metabolites and 1 oxidative deaminated metabolite, were identified from in vitro metabolite profiling coupled with authentic urinary analyses. The 2-pronged approach of using specific enzyme inhibitors and recombinant enzymes have provided adequate evidence to ascertain the metabolic routes of ADB-BUTINACA. Findings also confirmed that cytochrome P450 (CYP450) is the main class of enzymes contributing to the elimination of ADB-BUTINACA. Through this study, a feasible approach of combining in vitro HLM metabolite profiling with authentic human urine analyses for the comprehensive identification of urinary metabolite biomarkers of ADB-BUTINACA is established. The metabolic profile of ADB-BUTINACA and its key metabolizing enzymes and biotransformation pathways had been characterized in this study. This innovative approach used to identify the biomarkers can be applied to other existing and new prevailing SCs for diagnosing abuse.

Optimizing accelerated solvent extraction combined with liquid chromatography-Orbitrap mass spectrometry for efficient lipid profile characterization of mozzarella cheese

In this study, a novel Accelerated Solvent Extraction (ASE) procedure combined with UHPLC-Q-Orbitrap-MS was developed for detailed untargeted lipid profile of mozzarella cheese. Response Surface Methodology and Pareto front, using a Central Composite Design (CCD), were employed to define the optimised combination of extraction temperature, number of extraction cycles and mix of solvents. LipidSearch™ software was used for a reliable and accurate lipid identification. A total of 13 subclasses, including ceramides, diacylglycerols, triacylglycerols, lysophosphatidylcholines, lysophosphatidylethanolamines, phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, sphingomyelins, bismethyl phosphatidic acids, cholesterol ester, zymosterol ester, hexosyl ceramides were measured. The elaboration of the CCD showed that the solvents ratio was the main factor affecting the extraction efficiency. The optimised ASE method, together with the Folch extraction, synergistically contributed to a complete characterization of lipid profile of mozzarella cheese, confirming ASE technique, associated with high resolution mass spectrometry detection, as an efficient tool for Lipidomics in food science.

Ultra-high performance liquid chromatography-orbitrap high-resolution mass spectrometry for rapid screening and identification of 32 illegally added drugs in slimming and anti-impotence health foods

建立了基于超高效液相色谱-Orbitrap高分辨质谱的快速筛查及确证减肥和壮阳类保健食品中32种非法添加药物的分析方法,并总结了数据库建立和应用的相关要点。研究对象聚焦于非法添加药物的衍生物,在对比正负离子模式下各化合物响应强度的基础上建立了高分辨质谱信息库,对提取溶剂、色谱柱温度等实验条件进行了详细探究,尽可能给出了较宽的标准曲线线性范围。使用Hypersil gold vanquish色谱柱(100 mm×2.1 mm, 1.9 μm),梯度洗脱,流量0.3 mL/min,正、负离子全扫描/数据依赖的二级扫描模式,在17 min内完成32种目标化合物的数据采集,通过TraceFinder软件进行快速定性筛查和定量。结果显示在17 min内32种化合物能得到较好分离;2种基质加标溶液中32种化合物的一级质谱离子精确质量数的实测值与理论值均在5×10-6误差之内,二级碎片离子质量数的实测值与理论值均在1×10-5误差之内;方法学验证结果表明,所有化合物均显示出优异的线性关系,相关系数(r2)均大于0.99;固体基质中除达泊西汀、羟基硫代豪莫西地那非、硫代豪莫西地那非、硫代西地那非、去甲基硫代西地那非的回收率较低外,其余27种化合物的回收率为50.5%~84.5%,相对标准偏差(RSD)为1.2%~13%,液体基质中32种化合物的回收率范围为60.4%~109.3%, RSD为0.77%~8.2%;在48份减肥及壮阳类保健食品中检出1份阳性样品,检出率2.08%。该方法操作简单,结果定性准确,可用于减肥及壮阳类保健食品中32种非法添加药物的快速筛查及确证。

Comprehensive lipidomic analysis reveals regulation of glyceride metabolism in rat visceral adipose tissue by high-altitude chronic hypoxia.

Adipose tissue plays a central role in energy substrate homeostasis and is a key regulator of lipid flow throughout these processes. As hypoxia affects lipid metabolism in adipose tissue, we aimed to investigate the effects of high-altitude chronic hypoxia on lipid metabolism in the adipose tissue of rats using a lipidomic analysis approach. Visceral adipose tissues from rats housed in a high-altitude hypoxia environment representing 4,300 m with 14.07% oxygen (hypoxia group) and from rats housed in a low-altitude normoxia environment representing 41 m with 20.95% oxygen (normoxia group) for 8 weeks were analyzed using an ultra-performance liquid chromatography-Orbitrap mass spectrometry system. After 8 weeks, the body weight and visceral adipose tissue weight of the hypoxia group were significantly decreased compared to those of the normoxia group (p < 0.05). The area and diameter of visceral adipose cells in the hypoxia group were significantly smaller than those of visceral adipose cells in the normoxia group (p < 0.05). The results of lipidomic analysis showed a total of 21 lipid classes and 819 lipid species. The total lipid concentration of the hypoxia group was lower than that in the normoxia group (p < 0.05). Concentrations of diacylglycerols and triacylglycerols in the hypoxia group were significantly lower than those in the normoxia group (p < 0.05). Using univariate and multivariate analyses, we identified 74 lipids that were significantly altered between the normoxia and hypoxia groups. These results demonstrate that high-altitude chronic hypoxia changes the metabolism of visceral adipose glycerides, which may potentially modulate other metabolic processes.

Alkaloid Profiling, Anti-Enzymatic and Antiproliferative Activity of the Endemic Chilean Amaryllidaceae Phycella cyrtanthoides

This research aims to identify the alkaloid profile and to evaluate the enzyme inhibitory potential and antiproliferative effects of the Amaryllidaceae plant Phycella cyrtanthoides. The alkaloid extracts from bulbs and leaves were analyzed using ultrahigh performance liquid chromatography orbitrap mass spectrometry (UHPLC-Orbitrap-MS) analysis. A total of 70 alkaloids were detected in the P. cyrtanthoides’ extracts. The enzyme inhibition potential against cholinesterases (AChE: acetylcholinesterase, and BChE butyrylcholinesterase) and tyrosinase were studied. Bulbs displayed the best IC50 values against AChE (4.29 ± 0.03 µg/mL) and BChE (18.32 ± 0.03 µg/mL). These results were consistent with docking experiments with selected major compounds in the active sites of enzymes, while no activity was observed against tyrosinase enzyme. Antiproliferative effects were investigated against human cervical (HeLa), lung (A549, SW1573), colon (WiDr), and breast (HBL-100, T-47D) tumor cell lines. Bulbs and leaves were active in all cell lines (GI50 < 2.5 µg/mL). These findings suggest that the endemic Chilean plant P. cyrtanthoides contains diverse types of bioactive alkaloids with antiproliferative activities and inhibitory effects with potential therapeutic applications for neurodegenerative diseases

High-throughput glycosylation analysis of intact monoclonal antibodies by mass spectrometry coupled with capillary electrophoresis and liquid chromatography.

The analysis of monoclonal antibodies glycosylation is a crucial quality control attribute of biopharmaceutical drugs. High throughput screening approaches for antibody glycoform analysis are required in various stages of process optimization. Here, we present high throughput screening suitable mass spectrometry-based workflows for the analysis of intact antibody glycosylation out of cell supernatants. Capillary electrophoresis and liquid chromatography were coupled with quadrupole time-of-flight mass spectrometry or Orbitrap mass spectrometry. Both separation methods offer fast separation (10-15min) and the capability to prevent the separated cell supernatant matrix to enter the mass spectrometry by post-separation valving. Both mass spectrometry instruments provide comparable results and both are sufficient to determine the glycosylation pattern of the five major glycoforms of the measured antibodies. However, the Orbitrap yields higher sensitivity of 25μg/mL (CE-nanoCEasy-Orbitrap mass spectrometry) and 5μg/mL (liquid chromatography-Orbitrap mass spectrometry). Data processing was optimized for a faster processing and easier detection of low abundant glycoforms based on averaged charge-deconvoluted mass spectra. This approach combines a non-target glycoform analysis while yielding the same glycosylation pattern as the traditional approach based on extracted ion traces. The presented methods enable the high throughput screening of the glycosylation pattern of antibodies down to low μg/mL-range out of cell supernatant without any sample preparation.

Serum Metabolomics in Patients with Coexisting NAFLD and T2DM Using Liquid Chromatography-Mass Spectrometry.

Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM) frequently coexist and can act synergistically to drive adverse outcomes of one another. This study aimed to unravel the metabolomic changes in patients with NAFLD and T2DM, to identify potential noninvasive biomarkers, and to provide insights for understanding the link between NAFLD and T2DM. Three hundred participants aged 35 to 70 years who were diagnosed with NAFLD (n = 100), T2DM (n = 100), or a comorbidity of NAFLD and T2DM (n = 100) were included in this study. Anthropometrics and routine blood chemistry were assessed after overnight fast. The global serum metabolomic analysis was performed by ultra-performance liquid chromatography-Orbitrap mass spectrometry. Multivariate data analysis methods were utilized to identify the potential biomarkers. A set of serum biomarkers that could effectively separate NAFLD from NAFLD + T2DM and T2DM from NAFLD + T2DM were identified. We found that patients with coexisting NAFLD and T2DM had significantly higher levels of total protein (TP), triglycerides (TG), glucose in urine, and gamma-hydroxybutyric acid than those with NAFLD and had significant increased levels of TP, albumin, alanine aminotransferase, aspartate aminotransferase, total cholesterol, cholinesterase, TG, low-density lipoprotein, and apolipoprotein A when compared to patients with T2DM. The metabolomics results provide evidence that the comorbidity of NAFLD and T2DM considerably altered patients' metabolomics patterns compared to those of patients with only NAFLD or T2DM.

Isolation and quantification of diarylheptanoids from European hornbeam (Carpinus betulus L.) and HPLC-ESI-MS/MS characterization of its antioxidative phenolics

Detailed polyphenol profiling of European hornbeam (Carpinus betulus L.) bark, leaf, male and female catkin extracts was performed by high-performance liquid chromatography–diode array detection coupled to tandem mass spectrometry (HPLC-DAD-MS/MS). A total of 194 compounds were characterized and tentatively identified. Gallo- and ellagitannins dominated in the methanol extracts, while flavonol glycosides and methoxylated flavones prevailed in the ethyl acetate samples. In the quest for diarylheptanoids, twelve compounds were isolated by the combination of subsequent reversed-phase flash chromatographic and high-performance liquid chromatographic methods. The structural elucidation of the isolated components was performed by ultrahigh-performance liquid chromatography-Orbitrap mass spectrometry (UHPLC‐Orbitrap‐MS) as well as 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. Six known cyclic diarylheptanoids, together with a new compound were described in Carpinus betulus for the first time. The occurrence of a linear diarylheptanoid and a lignan has also been unprecedented in the genus Carpinus. Moreover, three known flavonol glycosides were isolated. Based on the identification of characteristic fragment ions, a new mass spectrometric fragmentation pathway for meta,meta-cyclophane-type diarylheptanoids was proposed. Quantities of the four major cyclic diarylheptanoids in European hornbeam were determined by a validated UHPLC-DAD method for the first time. The antioxidant properties of the extracts and the isolated compounds were assessed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Contribution of the individual constituents to the total radical scavenging activity of the samples was evaluated by an off-line DPPH-HPLC-DAD method. This allowed the identification of gallo- and ellagitannin derivatives as the constituents being primarily responsible for the antioxidant capacity of the extracts.

Molecular characterization of organic aerosol in winter from Beijing using UHPLC-Orbitrap MS

The urban organic aerosol (OA) may pose a serious threat to human health and ecological environment. In order to understand the molecular characteristics of organic compounds in aerosols, atmospheric PM2.5 samples were collected in Beijing and the extracts were analyzed by liquid chromatography-Orbitrap mass spectrometry combined with negative-ion electrospray ionization, positive-ion electrospray ionization, and positive-ion atmospheric pressure photoionization sources. The combination of multiple ionization sources realized the comprehensive molecular characterization of organic compounds in OA, and 1976 (+APPI), 3038 (−ESI), and 4376 (+ESI) molecular formulas were identified in this study. Significant differences in the species, abundance, and number of subgroups (CHO, CHN, CHON, CHONS, CHOS, and CH compounds) were clarified. Chemical fingerprinting of organics in the PM2.5 extract were investigated by high-throughput non-target compound analytical methods. Structure induction of organic compounds was realized through fragmentation prediction of MS/MS spectra with Sirius software. Furthermore, a total of 50 nitroaromatic formulas, 285 organosulfates (OS) formulas, 57 nitrooxy-OS formulas, 228 CHO- formulas with carboxyl groups, and 36 monoketone formulas were determined based on diagnostic fragmentation filtering. Our results provide important insights into the molecular composition and structural characteristics of OA, and establish foundation for exploring the interaction between composition and physicochemical properties.

Urinary Metabolite Biomarkers for the Detection of Synthetic Cannabinoid ADB-BUTINACA Abuse.

(S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-butyl-1H-indazole-3carboxamide (ADB-BUTINACA) is an emerging synthetic cannabinoid that was first identified in Europe in 2019 and entered Singapore's drug scene in January 2020. Due to the unavailable toxicological and metabolic data, there is a need to establish urinary metabolite biomarkers for detection of ADB-BUTINACA consumption and elucidate its biotransformation pathways for rationalizing its toxicological implications. We characterized the metabolites of ADB-BUTINACA in human liver microsomes using liquid chromatography Orbitrap mass spectrometry analysis. Enzyme-specific inhibitors and recombinant enzymes were adopted for the reaction phenotyping of ADB-BUTINACA. We further used recombinant enzymes to generate a pool of key metabolites in situ and determined their metabolic stability. By coupling in vitro metabolism and authentic urine analyses, a panel of urinary metabolite biomarkers of ADB-BUTINACA was curated. Fifteen metabolites of ADB-BUTINACA were identified with key biotransformations being hydroxylation, N-debutylation, dihydrodiol formation, and oxidative deamination. Reaction phenotyping established that ADB-BUTINACA was rapidly eliminated via CYP2C19-, CYP3A4-, and CYP3A5-mediated metabolism. Three major monohydroxylated metabolites (M6, M12, and M14) were generated in situ, which demonstrated greater metabolic stability compared to ADB-BUTINACA. Coupling metabolite profiling with urinary analysis, we identified four urinary biomarker metabolites of ADB-BUTINACA: 3 hydroxylated metabolites (M6, M11, and M14) and 1 oxidative deaminated metabolite (M15). Our data support a panel of four urinary metabolite biomarkers for diagnosing the consumption of ADB-BUTINACA.

Abundance of organosulfates derived from biogenic volatile organic compounds: Seasonal and spatial contrasts at four sites in China

Atmospheric organosulfates (OSs) derived from biogenic volatile organic compounds (BVOCs) encode chemical interaction strength between anthroposphere and biosphere. We report BVOC-derived OSs in the summer of 2016 and the winter of 2017 at four locations in China (i.e., Hong Kong (HK), Guangzhou (GZ), Shanghai (SH), and Beijing (BJ)). The spatial coverage of three climatic zones from the south to the north in China is accompanied with a wide range of aerosol inorganic sulfate (4.9–13.8 μg/m3). We employed a combined targeted and untargeted approach using high-performance liquid chromatography-Orbitrap mass spectrometry to quantify/semi-quantify ~200 OSs and nitrooxy OSs derived from four types of precursors, namely C2-C3 oxygenated VOCs, isoprene, monoterpenes (MT), and sesquiterpenes (ST). The seasonal averages of the total quantified OSs across the four sites are in the range of 201–545 (summer) and 123–234 ng/m3 (winter), with the isoprene-derived OSs accounting for more than 80% (summer) and 57% (winter). The C2–3 OSs and isoprene-derived OSs share the same seasonality (summer >winter) and the same south-north spatial gradient as those of isoprene emissions. In contrast, the MT- and ST-derived OSs are of either comparable abundance or slightly higher abundance in winter at the four sites. The spatial contrasts for MT- and ST-derived OSs are not clearly discernable among GZ, SH, and BJ. HK is noted to have invariably lower abundances of all groups of OSs, in line with its aerosol inorganic sulfate being the lowest. These results indicate that BVOC emissions are the driving factor regulating the formation of C2–3 OSs and isoprene-derived OSs. Other factors, such as sulfate abundance, however, play a more important role in the formation of MT- and ST-derived OSs. This in turn suggests that the formation kinetics and/or pathways differ between these two sub-groups of BVOCs-derived OSs.

Simultaneous determination of skimmin, apiosylskimmin, 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide in rat plasma by liquid chromatography-Orbitrap mass spectrometry and its application to pharmacokinetics.

The effective fraction of coumarin glycosides from Hydrangea paniculata Sieb (HP) has been under development for the treatment of chronic kidney disease for years. Skimmin and apiosylskimmin are the main coumarin glycosides of HP, and the major metabolites in rats are 7-hydroxycoumarin (7-HC) and 7-hydroxycoumarin glucuronide (7-HCG). In this study, a sensitive and reliable liquid chromatography-Orbitrap mass spectrometry method was developed for the simultaneous determination of skimmin, apiosylskimmin, 7-HC and 7-HCG in rat plasma. The chromatographic separation was performed on a Zobax SB C18 column (2.1 × 100 mm, 3.5μm) at a flow rate of 0.3ml/min with a gradient mobile phase of water and acetonitrile containing 0.2% formic acid. Skimmin, apiosylskimmin and 7-HCG were detected in targeted-selected-ion-monitoring mode at positive ions m/z of 325.0911, 457.1331 and 339.0703, respectively. 7-HC and the internal standard were detected in parallel-reaction-monitoring mode at m/z 163.0387 → 119.0492 and 260.1641 → 116.1071 to overcome the carryover of 7-HC. Linearity was obtained for the analytes within the ranges 20-2,000 ng/ml for skimmin, 5-500 ng/ml for apiosylskimmin and 7-HC and 100-10,000 ng/ml for 7-HCG. Validation parameters were all in line with the criteria of international guidance. The method has been applied to the pharmacokinetic study of HP in rats.

A Deep Dive into the Complex Chemical Mixture and Toxicity of Tire Wear Particle Leachate in Fathead Minnow.

The ecological impact of tire wear particles in aquatic ecosystems is a growing environmental concern. We combined toxicity testing, using fathead minnow (Pimephales promelas) embryos, with nontarget high‐resolution liquid chromatography Orbitrap mass spectrometry to characterize the toxicity and chemical mixture of organic chemicals associated with tire particle leachates. We assessed: 1) exposure to tire particle leachates after leaching for 1‐, 3‐, and 10‐d; and 2) the effect of the presence and absence of small tire particulates in the leachates. We observed a decrease in embryonic heart rates, hatching success, and lengths, as well as an increase in the number of embryos with severe deformities and diminished eye and body pigmentation, after exposure to the leachates. Overall, there was a pattern whereby we observed more toxicity in the 10‐d leachates, and greater toxicity in unfiltered leachates. Redundancy analysis showed that several benzothiazoles and aryl‐amines were correlated with the toxic effects observed in the embryos. These included benzothiazole, 2‐aminobenzothiazole, 2‐mercaptobenzothiazole, N,N′‐diphenylguanidine, and N,N′‐diphenylurea. However, many other chemicals characterized as unknowns are likely to also play a key role in the adverse effects observed. Our study provides insight into the types of chemicals likely to be important toxicological drivers in tire leachates, and improves our understanding of the ecotoxicological impacts of tire wear particles. Environ Toxicol Chem 2022;41:1144–1153. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.