On-line Electrospray Ionization Research Articles

Short and medium chain acylcarnitines as markers of outcome in diabetic and non-diabetic subjects with acute coronary syndromes

BackgroundCarnitine metabolism produces numerous molecular species of short-, medium-, and long-chain acylcarnitines, which play important roles in energy homeostasis and fatty acid transport in the myocardium. Given that disturbances in the carnitine metabolism are linked to cardiometabolic disease, we studied the relationship of circulating acylcarnitines with outcomes in patients with acute coronary syndromes (ACS) and evaluated differences in circulating levels of these metabolites between diabetic and non-diabetic patients. MethodsHarnessing a prospective multicentre cohort study (SPUM-ACS; NCT01000701), we measured plasma levels of acylcarnitines, carnitine, and carnitine metabolites to assess their relationship with adjudicated major adverse cardiac events (MACE), defined as composite of myocardial infarction, stroke, clinically indicated revascularization, or death of any cause. The SPUM-ACS study enrolled patients presenting with ACS to Swiss University Hospitals between 2009 and 2012. Acetylcarnitine, octanoylcarnitine, proprionylcarnitine, butyrylcarnitine, pentanoylcarnitine, hexanoylcarnitine, carnitine, γ-butyrobetaine, and trimethylamine N-oxide were measured in plasma using stable isotope dilution high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry. ResultsA total of 1683 patients with ACS were included in the study. All measured metabolites except γ-butyrobetaine and carnitine were higher in diabetic subject (n = 294) than in non-diabetic subjects (n = 1389). On univariate analysis, all metabolites, apart from octenoylcarnitine, were significantly associated with MACE at 1 year. After multivariable adjustment for established risk factors, acetylcarnitine remained an independent predictor of MACE at 1-year (quartile 4 vs. quartile 1, adjusted hazard ratio 2.06; 95% confidence interval 1.12–3.80, P = 0.020). ConclusionCirculating levels of acetylcarnitine independently predict residual cardiovascular risk in patients with ACS.

Online Sequential Determination of Organic/Inorganic Lead Speciation in PM2.5 Using Electrochemical Mass Spectrometry.

The information regarding the occurrence and abundance of lead (Pb) in PM2.5 is useful for the evaluation of air pollution status and tracing the pollution source. Herein, electrochemical mass spectrometry (EC-MS) for sequential determination of Pb species in PM2.5 samples without sample pretreatment has been developed using the combination of online sequential extraction with mass spectrometry (MS) detection. Four kinds of Pb species including water-soluble Pb compounds, fat-soluble Pb compounds, water/fat-insoluble Pb compounds, and a water/fat-insoluble Pb element were sequentially extracted from PM2.5 samples, in which water-soluble Pb compounds, fat-soluble Pb compounds, and water/fat-insoluble Pb compounds were extracted sequentially by elution using H2O, CH3OH, and EDTA-2Na as the eluent respectively, while the water/fat-insoluble Pb element was extracted by electrolysis using EDTA-2Na as the electrolyte. The extracted water-soluble Pb compounds, water/fat-insoluble Pb compounds, and water/fat-insoluble Pb element were transformed into EDTA-Pb in real time for online electrospray ionization mass spectrometry analysis, while the extracted fat-soluble Pb compounds were directly detected by electrospray ionization mass spectrometry. The advantages of the reported method include the obviation of sample pretreatment, high speed of analysis (<60 min/sample), low detection limit (0.16 pg), low sample consumption (30 μg), and high accuracy (>90%), which indicates the potential of this method for the rapid quantitative species detection of metals in environmental particulate matter samples.

Oxidation of Catechols at the Air-Water Interface by Nitrate Radicals.

Abundant substituted catechols are emitted to, and created in, the atmosphere during wildfires and anthropogenic combustion and agro-industrial processes. While ozone (O3) and hydroxyl radicals (HO•) efficiently react in a 1 μs contact time with catechols at the air-water interface, the nighttime reactivity dominated by nitrate radicals (NO3) remains unexplored. Herein, online electrospray ionization mass spectrometry (OESI-MS) is used to explore the reaction of NO3(g) with a series of representative catechols (catechol, pyrogallol, 3-methylcatechol, 4-methylcatechol, and 3-methoxycatechol) on the surface of aqueous microdroplets. The work detects the ultrafast generation of nitrocatechol (aromatic) compounds, which are major constituents of atmospheric brown carbon. Two mechanisms are proposed to produce nitrocatechols, one (equivalent to H atom abstraction) following fast electron transfer from the catechols (QH2) to NO3, forming NO3- and QH2•+ that quickly deprotonates into a semiquinone radical (QH•). The second mechanism proceeds via cyclohexadienyl radical intermediates from NO3 attack to the ring. Experiments in the pH range from 4 to 8 showed that the production of nitrocatechols was favored under the most acidic conditions. Mechanistically, the results explain the interfacial production of chromophoric nitrocatechols that modify the absorption properties of tropospheric particles, making them more susceptible to photooxidation, and alter the Earth's radiative forcing.

Identification of Exhaled Metabolites in Children with Cystic Fibrosis.

The early detection of inflammation and infection is important to prevent irreversible lung damage in cystic fibrosis. Novel and non-invasive monitoring tools would be of high benefit for the quality of life of patients. Our group previously detected over 100 exhaled mass-to-charge (m/z) features, using on-line secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS), which distinguish children with cystic fibrosis from healthy controls. The aim of this study was to annotate as many m/z features as possible with putative chemical structures. Compound identification was performed by applying a rigorous workflow, which included the analysis of on-line MS2 spectra and a literature comparison. A total of 49 discriminatory exhaled compounds were putatively identified. A group of compounds including glycolic acid, glyceric acid and xanthine were elevated in the cystic fibrosis group. A large group of acylcarnitines and aldehydes were found to be decreased in cystic fibrosis. The proposed compound identification workflow was used to identify signatures of volatile organic compounds that discriminate children with cystic fibrosis from healthy controls, which is the first step for future non-invasive and personalized applications.

Serum trimethylamine-N-oxide is associated with incident type 2 diabetes in middle-aged and older adults: a prospective cohort study

BackgroundThe role of trimethylamine-N-oxide (TMAO) in the development of diabetes remains controversial, and prospective data are few. We aimed to investigate the association between serum TMAO and incident type 2 diabetes in middle-aged and older adults.MethodsThis study was based on the Guangzhou Nutrition and Health Study (GNHS), a community-based prospective cohort study in China. A total of 2088 diabetes-free participants aged 40–75 years were included from 2008 to 2010. Incident type 2 diabetes was ascertained during follow-up visits. Baseline serum TMAO was measured by high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry. Hazard ratios (HRs) and 95% confidence intervals (95% CIs) for diabetes across tertiles of serum TMAO were calculated using Cox proportional hazard models. Prospective associations of serum TMAO with changes in glycemic traits (fasting glucose, HbA1c, insulin, HOMA-IR) over time were estimated using linear mixed-effects models (LMEMs).ResultsWe ascertained 254 incident type 2 diabetes cases during a median follow-up of 8.9 years. The median (interquartile range) of serum TMAO was 1.54 (0.86–2.91) μmol/L. From the first to the third tertile of serum TMAO, the multivariable-adjusted HRs for diabetes were 1.00 (reference), 1.17 (95% CI: 0.84–1.61), and 1.42 (95% CI: 1.03–1.96) (P-trend = 0.031). LMEMs showed that the estimated yearly change in fasting glucose was 0.011 (0.001–0.022) mmol/L/y in the highest tertile of serum TMAO, compared with the lowest tertile (P-interaction = 0.044). Serum TMAO was not associated with longitudinal changes in HbA1c, insulin or HOMA-IR.ConclusionsOur findings suggested that higher serum TMAO was associated with a higher risk of type 2 diabetes and an increase in fasting glucose among middle-aged and older Chinese adults.Trial registration: NCT03179657. https://clinicaltrials.gov/ct2/show/NCT03179657?term=NCT03179657&draw=2&rank=1

Association of Trimethylamine N-Oxide and Metabolites With Mortality in Older Adults

Little is known about the association of trimethylamine N-oxide (TMAO), a novel plasma metabolite derived from L-carnitine and phosphatidylcholine, and related metabolites (ie, choline, betaine, carnitine, and butyrobetaine) with risk of death among older adults in the general population. To investigate the associations of serial measures of plasma TMAO and related metabolites with risk of total and cause-specific death (ie, deaths from cardiovascular diseases [CVDs] and non-CVDs) among older adults in the US. This prospective cohort study involved 5333 participants from the Cardiovascular Health Study-a community-based longitudinal cohort of adults aged 65 years or older-who were followed up from June 1, 1989, to December 31, 2015. Participants were from 4 communities in the US (Forsyth County, North Carolina; Sacramento County, California; Washington County, Maryland; and Allegheny County, Pennsylvania). Data were analyzed from March 17 to June 23, 2021. Plasma TMAO, choline, betaine, carnitine, and butyrobetaine levels were measured using stored samples from baseline (June 1, 1989, to May 31, 1990, or November 1, 1992, to June 31, 1993) and follow-up examination (June 1, 1996, to May 31, 1997). Measurements were performed through stable-isotope dilution liquid chromatography with tandem mass spectrometry using high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry. Deaths (total and cause specific) were adjudicated by a centralized Cardiovascular Health Study events committee based on information from medical records, laboratory and diagnostic reports, death certificates, and/or interviews with next of kin. The associations of each metabolite with mortality were assessed using Cox proportional hazards regression models. Among 5333 participants in the analytic sample, the mean (SD) age was 73 (6) years; 2149 participants (40.3%) were male, 3184 (59.7%) were female, 848 (15.9%) were African American, 4450 (83.4%) were White, and 35 (0.01%) were of other races (12 were American Indian or Alaska Native, 4 were Asian or Pacific Islander, and 19 were of other races or ethnicities). During a median follow-up of 13.2 years (range, 0-26.9 years), 4791 deaths occurred. After adjustment for potential confounders, the hazard ratios for death from any cause (ie, total mortality) comparing extreme quintiles (fifth vs first) of plasma concentrations were 1.30 (95% CI, 1.17-1.44) for TMAO, 1.19 (95% CI, 1.08-1.32) for choline, 1.26 (95% CI, 1.15-1.40) for carnitine, and 1.26 (95% CI, 1.13-1.40) for butyrobetaine. Plasma betaine was not associated with risk of death. The extent of risk estimates was similar for CVD and non-CVD mortality. In this cohort study, plasma concentrations of TMAO and related metabolites were positively associated with risk of death. These findings suggest that circulating TMAO is an important novel risk factor associated with death among older adults.

Trimethylamine N-oxide predicts stroke severity in diabetic patients with acute ischaemic stroke and is related to glycemic variability.

The present study analyzed the relationship between circulating trimethylamine N-oxide (TMAO) levels and stroke severity in diabetic patients with acute ischaemic stroke. A further aim was to investigate whether higher TMAO levels were associated with platelet aggregation and glycemic variability. This was a cross-sectional analysis of 108 patients with type 2 diabetes mellitus (DM) undergoing acute ischaemic stroke and 60 healthy controls. Fasting plasma TMAO was measured using high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry. Plasma TMAO levels of patients with acute ischaemic stroke were significantly higher than those of healthy controls. Amongst stroke patients, 50 were defined as undergoing mild stroke, and their plasma TMAO levels were lower compared to those with moderate to severe stroke. Platelet aggregation and mean amplitude of glycemic excursions were both correlated with plasma TMAO levels and these relationships remained significant in multiple linear regression analyses. Moreover, in streptozotocin-induced diabetic rats fed a diet enriched with choline to increase TMAO synthesis, platelet aggregation was significantly increased in the DM+choline and fluctuating DM (FDM) + choline groups compared to the control group. This increase was abolished in rats receiving oral antibiotics, which markedly reduced plasma TMAO levels. Importantly, compared with the DM+choline group, the FDM+choline group displayed significantly elevated TMAO levels and higher platelet aggregation. Our results demonstrated that higher plasma TMAO levels were associated with stroke severity and suggested a novel link between plasma TMAO levels and glycemic variability in diabetic patients with acute ischaemic stroke.

Microsampling with a Solid-Phase Extraction Cartridge: Storage and Online Mass Spectrometry Analysis.

This study aims to introduce the concept of utilizing a solid-phase extraction (SPE) cartridge for remote biofluid collection, followed by direct sample analysis at a later time. For this, a dried matrix spot was prepared in a syringe, in the form of SPE cartridge for the first time to enable small biofluid collection (microsampling), storage, shipment, and online electrospray ionization (ESI) mass spectrometry (MS) analysis of the stored dried samples. The SPE sorbents were packed into an ESI syringe and the resultant cartridge was used for sampling small volumes (<20 μL) of different complex biological fluids including blood, plasma, serum, and urine. The collected sample was stored in the dry state within the confinement of the SPE sorbent at room temperature, and analyte stability (e.g., diazepam) was maintained for more than a year. Direct coupling of the SPE cartridge to MS provides excellent accuracy, precision, and sensitivity for analyzing illicit drugs present in the biofluid. The corresponding mechanism of wrong-way positive ion generation from highly basic elution solvents was explored. Without chromatography, our direct SPE-ESI-MS analysis technique afforded detection limits as low as 26 and 140 pg/mL for raw urine and untreated plasma, respectively. These promising results proved that the new syringe-based SPE cartridge can serve as a good alternative to conventional microsampling techniques in terms of analyte stability, ease of operation and versatility, and analytical sensitivity and speed.

One-step hexaplex immunoassays by on-line paper substrate-based electrospray ionization mass spectrometry for combined cancer biomarker screening.

Mass spectrometry (MS) is attractive as a multiplexed immunoassay readout benefiting from its high sensitivity, speed and mass resolution. Here, a simple paper-based hexaplex immunoassay with an on-line MS readout was proposed, using functionalized paper as the immune substrates, along with rhodamine-based mass tags assembled on gold nanoparticles prepared as the mass probes (MPs). Simultaneous immune capture and labeling were conducted in one step on paper substrates in 96-well plates with a high throughput within 30 minutes, and the on-line efficient dissociation of the mass tags highly facilitated the hexaplex readout of the immune signals by a newly established on-line paper substrate-based electrospray ionization-MS setup. Six MPs were synthesized for the simultaneous quantification of six important cancer protein markers (cancer antigen 15-3, cancer antigen 19-9, carcinoma embryonic antigen, cancer antigen 125, human epididymis protein 4, and alpha fetoprotein) using only 10 μL serum, presenting satisfactory sensitivity, accuracy and specificity. This platform was further tested in screening for the six biomarkers in serum samples of patients with breast, liver and gastric cancers, showing its high potential for sensitive and specific early cancer diagnosis.

Serum choline is associated with hepatocellular carcinoma survival: a prospective cohort study

BackgroundHigher choline and betaine levels have been linked to lower risk of liver cancer, whereas existing data in relation to hepatocellular carcinoma (HCC) prognosis are scarce. Our objective was to examine the associations of the serum choline and betaine with HCC survival.Methods866 newly diagnosed HCC patients were enrolled in the Guangdong Liver Cancer Cohort. Serum choline and betaine were assessed using high-performance liquid chromatography with online electro-spray ionization tandem mass spectrometry. Liver cancer-specific survival (LCSS) and overall survival (OS) were calculated. Cox proportional hazards models were used to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs).ResultsSerum choline levels were associated with better LCSS (T3 vs. T1: HR = 0.69, 95% CI: 0.51–0.94; P-trend < 0.05) and OS (T3 vs. T1: HR = 0.73, 95% CI: 0.54–0.99; P-trend < 0.05). The associations were significantly modified by C-reactive protein (CRP) levels but not by other selected prognostic factors including sex, age, etc. The favorable associations between serum choline and LCSS and OS were only existed among patients with CRP ≥3.0 mg/L. No significant associations were found between serum betaine levels and either LCSS or OS.ConclusionsThis study revealed that higher serum choline levels were associated with better HCC survival, especially in HCC patients with systemic inflammation status. No significant associations were found between serum betaine and HCC survival. Our findings suggest the benefits of choline on HCC survival.Trial registrationThe Guangdong Liver Cancer Cohort was registered at clinicaltrials.gov as NCT03297255.

Targeting a Subset of the Membrane Proteome for Top–Down Mass Spectrometry: Introducing the Proteolipidome

A subsection of integral membrane proteins partition into chloroform during a chloroform/methanol/water extraction primarily designed to extract lipids. Traditionally, these proteins were called proteolipids due to their lipid-like properties; the c-subunit of the ATP synthase integral FO component is the best known due to its abundance. In this manuscript, we investigate purification of proteolipid proteins away from lipids for high-resolution mass spectrometry. Size-exclusion chromatography on silica beads using a chloroform/methanol/aqueous formic acid (4/4/1; v/v) mobile phase allowed the separation of larger proteins (>3 kDa) from lipids (<1.5 kDa) and analysis by online electrospray ionization mass spectrometry. Fraction collection for mass spectrometry was limited by presence of plasticizers and other contaminants solubilized by chloroform. Drying down of the protein sample followed by resuspension in formic acid (70%) allowed reverse-phase chromatography on a polymeric support at elevated temperature, as described previously. Fractions collected in this way could be stored for extended periods at −80 °C without adducts or contaminants. Top–down mass spectrometry enabled the definition of PsaI as a novel proteolipid of spinach thylakoid membrane. Proteolipid preparation worked similarly when total membranes from mouse brains were extracted with chloroform. While it might be tempting to use the described extraction, we prefer to broaden the meaning of the term, whereby the proteolipidome is defined as a novel biological membrane proteome that includes the full complement of membrane proteins, their binding partners/ligands and their tightly bound structural lipids that constitute each protein–lipid complex’s functional unit; that is, a complete description of a biological membrane.

Stability of Monoterpene-Derived α-Hydroxyalkyl-Hydroperoxides in Aqueous Organic Media: Relevance to the Fate of Hydroperoxides in Aerosol Particle Phases.

The α-hydroxyalkyl-hydroperoxides [R-(H)C(-OH)(-OOH), α-HH] produced in the ozonolysis of unsaturated organic compounds may contribute to secondary organic aerosol (SOA) aging. α-HHs' inherent instability, however, hampers their detection and a positive assessment of their actual role. Here we report, for the first time, the rates and products of the decomposition of the α-HHs generated in the ozonolysis of atmospherically important monoterpenes α-pinene (α-P), d-limonene (d-L), γ-terpinene (γ-Tn), and α-terpineol (α-Tp) in water/acetonitrile (W/AN) mixtures. We detect α-HHs and multifunctional decomposition products as chloride adducts by online electrospray ionization mass spectrometry. Experiments involving D2O and H218O, instead of H216O, and an OH-radical scavenger show that α-HHs decompose into gem-diols + H2O2 rather than free radicals. α-HHs decay mono- or biexponentially depending on molecular structure and solvent composition. e-Fold times, τ1/e, in water-rich solvent mixtures range from τ1/e = 15-45 min for monoterpene-derived α-HHs to τ1/e > 103 min for the α-Tp-derived α-HH. All τ1/e's dramatically increase in <20% (v/v) water. Decay rates of the α-Tp-derived α-HH in pure water increase at lower pH (2.3 ≤ pH ≤ 3.3). The hydroperoxides detected in day-old SOA samples may reflect their increased stability in water-poor media and/or the slow decomposition of α-HHs from functionalized terpenes.

Limitation standard of toxic aconitines in Aconitum proprietary Chinese medicines using on-line extraction electrospray ionization mass spectrometry

Development of rapid analytical methods and establishment of toxic component limitation standards are of great importance in quality control of traditional Chinese medicine. Herein, an on-line extraction electrospray ionization mass spectrometry (oEESI-MS) coupled with a novel whole process integral quantification strategy was developed and applied to direct determination of nine key aconitine-type alkaloids in 20 Aconitum proprietary Chinese medicines (APCMs). Multi-type dosage forms (e.g., tablets, capsules, pills, granules, and liquid preparation) of APCM could be determined directly with excellent versatility. The strategy has the characteristics of high throughput, good tolerance of matrix interference, small amount of sample (∼0.5 mg) and reagent (∼240 μL) consumption, and short analysis time for single sample (<15 min). The results were proved to be credible by high performance liquid chromatography−mass spectrometry (LC–MS) and electrospray ionization mass spectrometry, respectively. Moreover, the limitation standard for the toxic aconitines in 20 APCMs was established based on the holistic weight toxicity (HWT) evaluation and the Chinese Pharmacopoeia severally, and turned out that HWT-based toxicity evaluation results were closer to the real clinical applications. Hence, a more accurate and reliable APCM toxicity limitation was established and expected to play an important guiding role in clinics. The current study extended the power of ambient MS as a method for the direct quantification of molecules in complex samples, which is commonly required in pharmaceutical analysis, food safety control, public security, and many other disciplines.

Online mass spectrometry of CE (SDS)-separated proteins by two-dimensional capillary electrophoresis.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the fundamental technique for protein separation by size. Applying this technology in capillary format, gaining high separation efficiency in a more automated way, is a key technology for size separation of proteins in the biopharmaceutical industry. However, unequivocal identification by online mass spectrometry (MS) is impossible so far, due to strong interference in the electrospray process by SDS and other components of the SDS-MW separation gel buffer. Here, a heart-cut two-dimensional electrophoretic separation system applying an electrically isolated valve with an internal loop of 20nL is presented. The peak of interest in the CE (SDS) separation is transferred to the CZE-MS, where electrospray-interfering substances of the SDS-MW gel are separated prior to online electrospray ionization mass spectrometry. An online SDS removal strategy for decomplexing the protein-SDS complex is implemented in the second dimension, consisting of the co-injection of organic solvent and cationic surfactant. This online CE (SDS)-CZE-MS system allows MS characterization of proteoforms separated in generic CE (SDS), gaining additional separation in the CZE and detailed MS information. In general, the system can be applied to all kinds of proteins separated by CE (SDS). Here, we present results of the CE (SDS)-CZE-MS system on the analysis of several biopharmaceutically relevant antibody impurities and fragments. Additionally, the versatile application spectrum of the system is demonstrated by the analysis of extracted proteins from soybean flour. The online hyphenation of CE (SDS) resolving power and MS identification capabilities will be a powerful tool for protein and mAb characterization. Graphical abstract Two-dimensional capillary electrophoresis system hyphenated with mass spectrometry for the characterization of CE (SDS)-separated proteins. As first dimension, a generic and high MS-interfering CE (SDS) separation is performed for size separation. After heart-cut transfer of the unknown CE (SDS) protein peak, via a four-port nanoliter valve to a volatile electrolyte system as second dimension, interference-free mass spectrometric data of separated mAb fragments and soybean proteins are obtained.

Simultaneous quantitation of nicotinamide riboside and nicotinamide in dietary supplements via HPTLC–UV with confirmation by online HPTLC–ESI–MS

The identification and quantitation of nicotinamide riboside (NAR) and its main related compound (nicotinamide) were achieved using high-performance thin-layer chromatography (HPTLC)–ultraviolet (UV) densitometry with confirmation by online electrospray ionization (ESI)–mass spectrometry (MS). As the stationary phase, HPTLC Si 60 F254 glass plates were employed; the mobile phase was ethanol–1 M ammonium acetate–formic acid (7:1:0.1, v/v/v). No derivatization was applied, and UV densitometry was performed in the absorbance mode (270 nm). The method was validated by specificity, linearity, accuracy, precision, and robustness.

Automated solid phase extraction and electrospray chip based on programmatic pneumatic micro-valves

Microfluidic chips coupled with electrospray ionization (ESI) mass spectrometry (MS) is an analytical platform with high detection throughputs and low sample consumptions. However, its applications in probing real samples are limited by the absence of reliable on-chip sample pretreatments, which are indispensable due to both the low contents of analytes and the poor salt tolerance of ESI MS. Herein, we propose an automated extraction and ESI chip (AEEC), consisting of a solid phase extraction (SPE) zone, seven on-chip pneumatic micro-valves, a monolithic ESI nozzle, and other components, to implement on-chip SPE prior to on-line ESI for analytes. In the SPE zone, magnetic silica beads were immobilized by magnets, and used as the SPE sorbent. Additionally, open and closed statuses of all micro-valves were simultaneously controlled by turning off and on applied pneumatic pressures, thus regulating the direction of various flows in AEEC. Further, the SPE in AEEC, consisting of sample introduction and extraction, elution introduction, and analytes elution, was automatically implemented by regulating the direction of both sample and elution flows in AEEC. After these steps, the analytes elution was on-line introduced to the monolithic ESI nozzle, at which the ESI for analytes was achieved. After optimizations, the AEEC-MS method was used to investigate two standard chemicals and ten pesticides, achieving a limit of detection and an enrichment ratio between 0.10 and 0.75 ng μL−1 and 2.1–6.2, respectively. In addition, linear calibration curves with and without the SPE beads were compared, demonstrating the effect of the automated SPE in improving reliability and sensitivity of the AEEC method. Finally, we applied AEEC to quantify a new herbicide, quinotrione, in sorghum plant as 0.176 mg kg−1 (RSD=5.70%), presenting an improved error compared with related reports. With the AEEC method, a SPE and ESI MS investigation for analytes could be automatically implemented within 300 s, effectively reducing random error and analytical time compared with manual strategies.

Trimethylamine N-oxide, a gut microbiota-dependent metabolite of choline, is positively associated with the risk of primary liver cancer: a case-control study

BackgroundEvidence has suggested a potential link exists between trimethylamine-N-oxide (TMAO), a choline-derived metabolite produced by gut microbiota, and some cancers, but little is known for primary liver cancer (PLC).MethodsA case-control study was designed including 671 newly diagnosed PLC patients and 671 control subjects frequency-matched by age (±5 years) and sex, in Guangdong province, China. High-performance liquid chromatography with online electrospray ionization tandem mass spectrometry (HPLC-MS/MS) was used to measure serum TMAO and choline. The associations between these biomarkers and PLC risk were evaluated using logistic regression models.ResultsSerum TMAO concentrations were greater in the PLC group than the control group (P = 0.002). Logistic regression analysis showed that the sex- and age-adjusted odds ratio (OR) and (95% confidence interval [CI]) was 3.43 (2.42–4.86) when comparing the top and bottom quartiles (Q4 vs Q1). After further adjusting for more selected confounders, the OR (95% CI) remained significant but was attenuated to 2.85 (1.59–5.11) (Q4 vs Q1). The multivariable-adjusted ORs (95% CIs) across quartiles of choline were 0.35–0.15 (P-trend < 0.001).ConclusionHigher serum levels of TMAO were associated with increased PLC risk. The association was stronger in those with lower serum levels of choline. Additional large prospective studies are required to confirm these findings.Trial registrationThis study was registered at clinicaltrials.gov as NCT 03297255.

Mechanistic Analysis of the C-H Amination Reaction of Menthol by CuBr2 and Selectfluor.

The mechanism of the Ritter-type C-H amination reaction of menthol with acetonitrile using CuBr2, Selectfluor, and Zn(OTf)2, first disclosed by Baran and coworkers in 2012, was studied using a combination of online electrospray ionization mass spectrometry, continuous UV/vis spectrometric monitoring, and density functional theory calculations. In addition to corroborating Baran's original mechanistic proposal, these studies uncovered a second pathway to product formation, which likely only occurs in microdroplets. DFT calculations show that neither pathway has a barrier that is greater than 6.8 kcal/mol, suggesting that both mechanisms are potentially operative under ambient conditions.

Ammonium hydroxide enhancing electrospray response and boosting sensitivity of bisphenol A and its analogs

As one of the most important analytical techniques for accurate quantification of bisphenol compounds, liquid chromatography coupled to online electrospray ionization (ESI) tandem mass spectrometry exhibits relative weak ESI response in negative mode, which greatly reduces their sensitivity and limits their detection at trace levels, especially in complex samples such as blood. Based on the facilitated deprotonation of bisphenol molecule under alkaline condition, post-column injection of ammonium hydroxide (NH3·H2O) to mass spectrometer was explored to enhance the ionization efficiency of BPA and its eight analogs and improve their analytical sensitivity. Parameters effecting response intensity and stability were investigated, including mass concentration and flow rate of NH3·H2O. Under the optimal conditions with the addition of 2% (w/w) of NH3·H2O at 4 μL min−1, the instrument detection limits for bisphenol compounds were greatly lowered to 0.001–0.04 ng mL−1, which were 2–28.6 times lower than the result obtained without injecting NH3·H2O, except TBBPA (0.03 ng mL−1 in either case). The relative standard deviations (RSDs) for instrument repeatability of BPA and its analogs at three different concentration levels were in a range of 1.2–20%. Furthermore, a decreased matrix effects (90–111%) for bisphenols (except TBBPA) analysis in serum extracts were found compared with the result obtained without NH3·H2O injection (43–111%). The results demonstrated that the improved instrumental method coupled with suitable pretreatment techniques is more feasible to analyze bisphenol compounds in complex bio-samples.

PH-Dependent Singlet O2 Oxidation Kinetics of Guanine and 9-Methylguanine: An Online Mass Spectrometry and Spectroscopy Study Combined with Theoretical Exploration.

We report a kinetic and mechanistic study on the title reactions, in which 1O2 was generated by the reaction of H2O2 with Cl2 and bubbled into an aqueous solution of guanine and 9-methylguanine (9MG) at different pH values. Oxidation kinetics and product branching ratios were measured using online electrospray ionization mass spectrometry coupled with absorption and emission spectrophotometry, and product structures were determined by collision-induced dissociation (CID) tandem mass spectrometry. Experiments revealed strong pH dependence of the reactions. The oxidation of guanine is noticeable only in basic solution, while the oxidation of 9MG is weak in acidic solution, increases in neutral solution, and becomes intensive in basic solution. 5-Guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) were detected as the major oxidation products of guanine and 9MG, and Sp became dominant in basic solution. A reaction intermediate was captured in mass spectra, and assigned to gem-diol on the basis of CID measurements. This intermediate served as the precursor for the formation of Gh. After taking into account solution compositions at each pH, first-order oxidation rate constants were extracted for individual species: that is, 3.2-3.6 × 107 M-1 s-1 for deprotonated guanine, and 1.2 × 106 and 4.6-4.9 × 107 M-1 s-1 for neutral and deprotonated 9MG, respectively. Guided by approximately spin-projected density-functional-theory-calculated reaction potential energy surfaces, the kinetics for the initial 1O2 addition to guanine and 9MG was evaluated using transition state theory (TST). The comparison between TST modeling and experiment confirms that 1O2 addition is rate-limiting for oxidation, which forms endoperoxide and peroxide intermediates as determined in previous measurements of the same systems in the gas phase.