Mass Spectrometric Method Research Articles

Chiral analysis of ostarine in commercially available products and doping control urine samples

Various substances classified as prohibited in sport by the World Anti-Doping Agency (WADA) comprise one or more chiral centers. One such substance is enobosarm ((2S)-3-(4-cyanophenoxy)-N-[4-cyano-3-(trifluoromethyl)phenyl]-2‑hydroxy-2-methylpropanamide, S-ostarine, S-22, MK-2866, GTx-024, VERU-024). Although ostarine is not an approved drug, it has been readily available via Internet-based suppliers, and to date it is unknown to what extent R-ostarine, S-ostarine, or a mixture of both is used in these products. Likewise, such information is not available with regards to urine samples that returned an adverse analytical finding in the context of sports drug testing programs and, hence, this pilot-study aimed at establishing a test method to provide first insights into the presence of R- and S-ostarine in commercially available products and in authentic doping control urine samples.Here, a chiral liquid chromatographic - mass spectrometric method, employing a teicoplanin-containing (chiral) stationary phase that allows the differentiation of R- and S-enantiomers of ostarine, is presented. Ostarine products were extracted and diluted prior to analysis, while urine samples were prepared for analysis by enzymatic hydrolysis followed by liquid-liquid extraction.Seven products declaring to contain ostarine were investigated, five of which were ordered via the Internet and two were confiscated and made available by a customs authority. Only 2 of the 5 purchased products contained ostarine. In these two products and the confiscated products, only the ostarine S-enantiomer was found. Further, five authentic doping control urine samples containing the target analyte were subjected to chiral analysis, also confirming the sole presence of the S-ostarine enantiomer.The developed approach proved suitable for the separation of ostarine enantiomers, which adds further information e.g. in the context of illicit drug production, trafficking, and doping controls, especially when additional details are desirable that might assist in determining the origin of a drug or drug residue.

A screening method for heavy metals in consumer goods using reactive desorption electrospray ionization mass spectrometry.

Contamination of everyday goods with heavy metals such as nickel, cadmium, and lead known to be hazardous to the health of customers is an ongoing problem. Here, a mass spectrometric screening method based on reactive desorption electrospray ionization (DESI) is presented for the analysis of metals in consumer goods such as jewelry, tableware, and paintings. The method detects oxidized species of lead, nickel, cadmium, copper, and iron from the surface of objects without sample preparation. Positively charged metal ions form singly and doubly negatively charged complexes with ethylenediaminetetraacetic acid added to the DESI solvent, which are analyzed by a mass spectrometer. Qualitative and quantitative performance of the method was elucidated with metal salt standards. Subsequently, authentic samples were analyzed qualitatively. Reactive DESI-MS was able to detect lead and cadmium in eight out of nine consumer goods. For tableware, these heavy metals were found to be localized in the print as determined by reactive DESI imaging. In addition, mockup paintings generated from modern and historical pigments of Pb, Cu, Cd, and Fe in various media (acrylic binder, egg tempera, and linseed oil) were measured to show the suitability of the method for art authentication and conservation. The developed method expands the range of analytes accessible by DESI-MS to metal ions. Hence, DESI becomes a suitable ionization technique for an increasing number of analyte classes, which are of interest in chemical screening of consumer goods.

Carboxylate Bridged Binuclear Cobalt(II) Complexes Catalyze the Hydrolysis of Thiolates to Alcohols

AbstractA binuclear Co(II) complex, [Co2(PhBIMP)(μ‐O2CPh)(MeCN)]2+ (1) (PhBIMP is the anion of 2,6‐bis[(bis((N‐1‐methyl‐4,5‐diphenylimidazoylmethyl) amino)methyl]‐4‐methylphenol) catalyzes the hydrolysis of thiolates to the corresponding alcohols/phenols at room temperature. Hydrolysis of 11 different thiolates has been investigated with a catalyst loading of 4–6.7 mol% and with the highest TON values reaching 22. Furthermore, the effects of electron donating and electron withdrawing groups on the bridging benzoate of 1 have been investigated. The intermediates, products, and fate of the catalyst have been identified by mass spectrometric, gas chromatographic and gravimetric methods, and based on that, a mechanism for the catalytic cycle has been proposed.

Detection of Virulence Genes in Pseudomonas aeruginosa Isolates by Polymerase Chain Reaction and Investigation of High-Risk Clones by Matrix Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometer Method

Pseudomonas aeruginosa is a non-fermentative gram-negative bacillus. Many virulence factors play a role in the pathogenesis of P.aeruginosa. The aim of this study was to early detection of ST111, ST175, ST235, ST253, ST395 which are named high-risk clones with increased epidemic potential due to multidrug resistance in P.aeruginosa isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method and to evaluate the relationship between high-risk clones and the presence of P.aeruginosa virulence factors and carbapenemase production genes.P.aeruginosa isolates (n= 100) found to be resistant to at least imipenem or meropenem antibiotics isolated from the various clinical samples in the medical microbiology laboratory between 01.01.2021 and 07.06.2022 were included in the study. For the detection of virulence genes uniplex polymerase chain reaction (PCR) for toxA and multiplex PCR for algD, plcN, lasB, plcH were performed in P.aeruginosa isolates. In the detection of carbapenemase genes, two separate multiplex PCRs used for blaKPC , blaNDM , blaVIM , blaOXA-48 and for blaIMP , blaSPM , blaSIM , blaGIM , blaGES . Investigation of the peaks specific to high-risk clones was performed by using VITEK®-MS (bioMérieux, France) system. P.aeruginosa isolates were mostly isolated from intensive care units (45%) and respiratory tract samples (46%). The antibiotic to which the isolates were found to be most susceptible was amikacin, while highest resistance was detected for piperacillin. In PCR results, toxA, lasB, plcH, plcN and algD were detected as 89%, 99%, 98%, 100%, 100%, respectively. When the presence of characteristic peaks belonging to high-risk clones was evaluated with MALDI-TOF MS, ST253 (7%) and ST175 (2%) were detected. The peaks specific to ST235 and ST395 clones were not detected in our study. blaVIM was detected in two isolates and blaGES-5 carbapenemase was detected in two isolates. Virulence factors were detected at high rates in both high-risk clones and other strains and no significant relationship was found between high-risk clones and virulence factors. Early detection of high-risk clones, identification of antimicrobial resistance mechanisms will help to develop strategic treatment options and prevent their worldwide spread.

Aza-Prilezhaev Aziridination-Enabled Multidimensional Analysis of Isomeric Lipids via High-Resolution U-Shaped Mobility Analyzer-Mass Spectrometry.

Unsaturated lipids constitute a significant portion of the lipidome, serving as players of multifaceted functions involving cellular signaling, membrane structure, and bioenergetics. While derivatization-assisted liquid chromatography tandem mass spectrometry (LC-MS/MS) remains the gold standard technique in lipidome, it mainly faces challenges in efficiently labeling the carbon-carbon double bond (C═C) and differentiating isomeric lipids in full dimension. This presents a need for new orthogonal methodologies. Herein, a metal- and additive-free aza-Prilezhaev aziridination (APA)-enabled ion mobility mass spectrometric method is developed for probing multiple levels of unsaturated lipid isomerization with high sensitivity. Both unsaturated polar and nonpolar lipids can be efficiently labeled in the form of N-H aziridine without significant side reactions. The signal intensity can be increased by up to 3 orders of magnitude, achieving the nM detection limit. Abundant site-specific fragmentation ions indicate C═C location and sn-position in MS/MS spectra. Better yet, a stable monoaziridination product is dominant, simplifying the spectrum for lipids with multiple double bonds. Coupled with a U-shaped mobility analyzer, identification of geometric isomers and separation of different lipid classes can be achieved. Additionally, a unique pseudo MS3 mode with UMA-QTOF MS boosts the sensitivity for generating diagnostic fragments. Overall, the current method provides a comprehensive solution for deep-profiling lipidomics, which is valuable for lipid marker discovery in disease monitoring and diagnosis.

Liquid chromatography with tandem mass spectrometric method for determination of 52 antibiotics in human whole blood and urine and application to forensic cases.

A rapid and reliable method was developed and validated for the simultaneous analysis of 52 antibiotics (cephalosporins, penicillins, carbapenems, lincosamides, quinolones, nitroimidazoles, macrolides, sulfonamides, tetracyclines, glycopeptide) in urine and whole blood by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Analytes were extracted by dilution or protein precipitation and analyzed on an Agilent 1260 HPLC system coupled to an Agilent 6470 Triple Quadrupole Mass Spectrometer. The method attended method validation criteria. The limits of detection were equal or lower than 2.0ng/mL, whereas the limits of quantification ranged from 0.1 to 10.0ng/mL, from 0.1 to 5.0ng/mL, in urine and whole blood, respectively. For all analytes, the bias and intra- and inter-day precision values were less than 14.7%. The ranges of recovery values of all antibiotics were 76.5-124.5% in whole blood and 76.3-121.8% in urine, values of the effects were lower than 25% in two matrices. No evidence of carryover was observed. The study of sample stability showed that almost all analytes were stable at 24°C for 24h, all analytes were stable at -20°C for 14 days and at -80°C for 30 days. Freeze-thaw cycles stability showed that antibiotics were stable except for imipenem. Autosampler stability study showed that all analytes were stable for 24h, except for imipenem and amoxicillin. Applicability was proven by analyzing authentic whole blood (n = 86) and urine (n = 79) samples from patients under antibiotics treatment. Therefore, this method was applied to the analysis 3 forensic allergy cases, which were positive for at least one analyte. A simple, sensitive and high-throughput method for the simultaneous determination of different classes of antibiotics in urine and whole blood samples was developed and applied. This sensitive method was successfully applied to forensic cases.

Development and validation of high-performance liquid chromatography-Orbitrap mass spectrometric method for quantification of NDMA in ranitidine drug products and evaluation of antioxidants as inhibitors of classical nitrosation reaction.

N-Nitroso dimethylamine (NDMA) is a mutagenic impurity detected in several ranitidine products. The amino functional group of ranitidine is a risk factor for classical nitrosation-induced NDMA formation in ranitidine drug products during storage conditions. The United States Food and Drug Administration (US FDA) recommended the use of antioxidants to control NDMA in drug products. Considering the need for sensitive analytics, a liquid chromatography/high-resolution mass spectrometry (LC-HRMS) method was developed and validated to detect NDMA in this pilot study to demonstrate the antioxidants as inhibitors of nitrosation reactions. The method, utilizing an EC-C18 column and tuned to atmospheric pressure chemical ionization/selected ion monitoring (APCI/SIM) mode, separated NDMA (m/z: 75.0553; tR: 3.71 min) and ranitidine (m/z: 315.1485; tR: 8.61 min). APCI mode exhibited four times higher sensitivity to NDMA than electrospray ionization (ESI) mode. Classical nitrosation of the dimethyl amino group of ranitidine was studied with sodium nitrite in solid pellets. Antioxidants (alpha-tocopherol, ascorbic acid, and trolox) were evaluated as NDMA attenuators in ranitidine pellets under vulnerable storage conditions. The developed method quantified NDMA levels in samples, extracted with methanol through vortex shaking for 45 min. The method achieved a limit of detection (LOD) and limit of quantitation (LOQ) of 0.01 and 0.05 ng/mL, respectively, with linearity within 1-5000 ng/mL (R1: 0.9995). It demonstrated good intra-day and inter-day precision (% RSD [relative standard deviation]: <2) and accuracy (96.83%-101.72%). Nitrosation of ranitidine induced by nitrite was significant (p < 0.001; R2 = 0.9579) at various sodium nitrite levels. All antioxidants efficiently attenuated NDMA formation during ranitidine nitrosation. Ascorbic acid exhibited the highest NDMA attenuation (96.98%), followed by trolox (90.58%). This study recommends 1% ascorbic acid and trolox as potent NDMA attenuators in ranitidine drug products. This study compared the effectiveness of antioxidants as NDMA attenuators in ranitidine under storage conditions susceptible to NDMA generation. The study concluded that ascorbic acid and trolox are potent inhibitors of NDMA formation and nitrosation attenuators in ranitidine drug products.

Stable Interstrand Cross-Links Generated from the Repair of 1,N6-Ethenoadenine in DNA by α-Ketoglutarate/Fe(II)-Dependent Dioxygenase ALKBH2.

DNA cross-links severely challenge replication and transcription in cells, promoting senescence and cell death. In this paper, we report a novel type of DNA interstrand cross-link (ICL) produced as a side product during the attempted repair of 1,N6-ethenoadenine (εA) by human α-ketoglutarate/Fe(II)-dependent enzyme ALKBH2. This stable/nonreversible ICL was characterized by denaturing polyacrylamide gel electrophoresis analysis and quantified by high-resolution LC-MS in well-matched and mismatched DNA duplexes, yielding 5.7% as the highest level for cross-link formation. The binary lesion is proposed to be generated through covalent bond formation between the epoxide intermediate of εA repair and the exocyclic N6-amino group of adenine or the N4-amino group of cytosine residues in the complementary strand under physiological conditions. The cross-links occur in diverse sequence contexts, and molecular dynamics simulations rationalize the context specificity of cross-link formation. In addition, the cross-link generated from attempted εA repair was detected in cells by highly sensitive LC-MS techniques, giving biological relevance to the cross-link adducts. Overall, a combination of biochemical, computational, and mass spectrometric methods was used to discover and characterize this new type of stable cross-link both in vitro and in human cells, thereby uniquely demonstrating the existence of a potentially harmful ICL during DNA repair by human ALKBH2.

Stochastic dynamics mass spectrometric structural analysis of poly(methyl methacrylate)

Polymers such as poly(methyl methacrylate), comprising of all-carbon backbones are regarded as ubiquitous functional materials mainly due to their low cost, unique physical properties, and impressive robustness. The latter properties are results from relative chemical inertness of the polymer backbone carbon–carbon bonds. The highlighted stability crucially challenges not only innovations to plastic recycling polymer materials, but also developments of reliable analytical protocols for determining both quantitatively and structurally polymers. Despite, the fact that there are numerous applications of mass spectrometric methods to polymer science, the analysis involves chiefly annotation of monomers of polymers and additives, owing to the fact that the additives are low molecular weight analytes. The exact structural determining of end-chain groups, particularly highlighting chemically substituted end carbon–carbon bond represents significant challenge even utilizing the superior features of the analytical mass spectrometric instrumentation. This study, in the latter context, illustrates innovative stochastic dynamics approach and model equations capable of not only determining unambiguously substituted carbon–carbon end chain of the entitled polymer but also to predict mass spectra of its derivatives: thus, extending crucially the applicability of the method to many fields of the fundamental scientific and industrial research. The latter claim is argued still at the beginning of the study with the achieved method performances showing |r|=0.99998. The study utilizes experimental and theoretical mass spectrometric data on high resolution electrospray ionization mass spectrometry; high accuracy quantum chemical static methods, molecular dynamics; and chemometrics.

Appearance of hexahydrocannabinols as recreational drugs and implications for cannabis drug testing – focus on HHC, HHC-P, HHC-O and HHC-H

This study investigated the effects of hexahydrocannabinol (HHC) and other unclassified cannabinoids, which were recently introduced to the recreational drug market, on cannabis drug testing in urine and oral fluid samples. After the appearance of HHC in Sweden in 2022, the number of posts about HHC on an online drug discussion forum increased significantly in the spring of 2023, indicating increased interest and use. In parallel, the frequency of false positive screening tests for tetrahydrocannabinol (THC) in oral fluid, and for its carboxy metabolite (THC-COOH) in urine, rose from <2% to >10%. This suggested that HHC cross-reacted with the antibodies in the immunoassay screening, which was confirmed in spiking experiments with HHC, HHC-COOH, HHC acetate (HHC-O), hexahydrocannabihexol (HHC-H), hexahydrocannabiphorol (HHC-P), and THC-P. When HHC and HHC-P were classified as narcotics in Sweden on 11 July 2023, they disappeared from the online and street shops market and were replaced by other unregulated variants (e.g. HHC-O and THC-P). In urine samples submitted for routine cannabis drug testing, HHC-COOH concentrations up to 205 (mean 60, median 27) µg/L were observed. To conclude, cannabis drug testing cannot rely on results from immunoassay screening, as it cannot distinguish between different tetra- and hexahydrocannabinols, some being classified but others unregulated. The current trend for increased use of unregulated cannabinols will likely increase the proportion of positive cannabis screening results that need to be confirmed with mass spectrometric methods. However, the observed cross-reactivity also means a way to pick up use of new cannabinoids that otherwise risk going undetected.

Simultaneous Determination of D and L Enantiomers of 2-Hydroxyglutarate by UHPLC-MS/MS Method in Human Biological Fluids and its Clinical Application.

2-hydroxyglutarate has limited abundance in normal tissues but a high level under certain pathologic conditions. To clarify the diagnostic value of two chiral isomers of 2-hydroxyglutarate in plasma and urine of Chinese cancer patients, an ultra-high performance liquid chromatography-tandem mass spectrometric method was developed for simultaneous quantification of D-/L-2-hydroxyglutarate. The selected D-/L-2-hydroxyglutarate-d5 as internal standards were added to samples before the SPE on Waters Oasis® MAX 96-Well plate (30μm, 60mg). A derivatization step with (+)-O,O'-diacetyl-L-tartaric anhydride permitted the chromatography separation of D-/L-2-hydroxyglutarate on an ACQUITY UPLC-HSS T3 column (50 × 2.1mm, i.d. 1.8μm) with acetonitrile and water (containing 0.1% formic acid and 10mmol ammonium acetate) as the mobile phase. The calibration curves showed good linearity (R ≥ 0.99) over the concentration ranges of 200-5,000ng/mL and 500-20,000ng/mL for analysis of D-/L-2-hydroxyglutarate in plasma and urine samples, respectively. Intra- and inter-run precision were ≤ 12.33%, and the accuracy was within the range of -10.44 to 13.90%. This method was further successfully applied to clinical sample analysis in isocitrate dehydrogenase 1/2 mutated Chinese cancer patients.

Germ-free status but not subacute polychlorinated biphenyl (PCB) exposure altered hepatic phosphatidylcholine and ether-phosphatidylcholine levels in mice

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that pose a current ecosystem and human health concern. PCB exposure impacts the gut microbiome in animal models, suggesting a mechanistic link between PCB exposure and adverse health outcomes. The presence and absence of the microbiome and exposure to PCBs independently affect the lipid composition in the liver, which in turn affects the PCB disposition in target tissues, such as the liver. Here, we investigated microbiome × subacute PCB effects on the hepatic lipid composition of conventional and germ-free female mice exposed to 0, 6, or 30 mg/kg body weight of an environmental PCB mixture in sterile corn oil once daily for 3 consecutive days. Hepatic triacylglyceride and polar lipid levels were quantified using mass spectrometric methods following the subacute PCB exposure. The lipidomic analysis revealed no PCB effect on the hepatic levels. No microbiome effect was observed on levels of triacylglyceride and most polar lipid classes. The total hepatic levels of phosphatidylcholine (PC) and ether-phosphatidylcholine (ePC) lipids were lower in germ-free mice than the conventional mice from the same exposure group. Moreover, levels of several unsaturated PCs, such as PC(36:5) and PC(42:10), and ePCs, such as ePC(36:2) and ePC(4:2), were lower in germ-free than conventional female mice. Based on a KEGG pathway meta-analysis of RNA sequencing data, the ether lipid metabolism pathway is altered in the germ-free mouse liver. In contrast to the liver, extractable lipid levels, determined gravimetrically, differed in several tissues from naïve conventional vs. germ-free mice. Overall, microbiome × subacute PCB exposure effects on hepatic lipid composition are unlikely to affect PCB distribution into the mouse liver. Further studies are needed to assess how the different extractable lipid levels in other tissues alter PCB distribution in conventional vs. germ-free mice.

Improving predictions of compound amenability for liquid chromatography-mass spectrometry to enhance non-targeted analysis.

Mass-spectrometry-based non-targeted analysis (NTA), in which mass spectrometric signals are assigned chemical identities based on a systematic collation of evidence, is a growing area of interest for toxicological risk assessment. Successful NTA results in better identification of potentially hazardous pollutants within the environment, facilitating the development of targeted analytical strategies to best characterize risks to human and ecological health. A supporting component of the NTA process involves assessing whether suspected chemicals are amenable to the mass spectrometric method, which is necessary in order to assign an observed signal to the chemical structure. Prior work from this group involved the development of a random forest model for predicting the amenability of 5517 unique chemical structures to liquid chromatography-mass spectrometry (LC-MS). This work improves the interpretability of the group's prior model of the same endpoint, as well as integrating 1348 more data points across negative and positive ionization modes. We enhance interpretability by feature engineering, a machine learning practice that reduces the input dimensionality while attempting to preserve performance statistics. We emphasize the importance of interpretable machine learning models within the context of building confidence in NTA identification. The novel data were curated by the labeling of compounds as amenable or unamenable by expert curators, resulting in an enhanced set of chemical compounds to expand the applicability domain of the prior model. The balanced accuracy benchmark of the newly developed model is comparable to performance previously reported (mean CV BA is 0.84 vs. 0.82 in positive mode, and 0.85 vs. 0.82 in negative mode), while on a novel external set, derived from this work's data, the Matthews correlation coefficients (MCC) for the novel models are 0.66 and 0.68 for positive and negative mode, respectively. Our group's prior published models scored MCC of 0.55 and 0.54 on the same external sets. This demonstrates appreciable improvement over the chemical space captured by the expanded dataset. This work forms part of our ongoing efforts to develop models with higher interpretability and higher performance to support NTA efforts.

Simplified processing and rapid quantification of buprenorphine, norbuprenorphine, and their conjugated metabolites in human plasma using UPLC-MS/MS: Assessment of buprenorphine exposure during opioid use disorder treatment.

Opioid use disorder (OUD) is a chronic neurobehavioral ailment and is prevalent in pregnancy. OUD is commonly treated with methadone or buprenorphine (BUP). Pregnancy is known to alter the pharmacokinetics of drugs and may lead to changes in drug exposure and response. A simple, specific, and sensitive analytical method for measuring the parent drug and its metabolites is valuable for assessing the impact of pregnancy on drug exposure. A new liquid chromatography-tandem mass spectrometric method that utilized a simple protein precipitation procedure for sample preparation and four deuterated internal standards for quantification was developed and validated for BUP and its major metabolites (norbuprenorphine [NBUP], buprenorphine-glucuronide [BUP-G], and norbuprenorphine-glucuronide [NBUP-G]) in human plasma. The standard curve was linear over the concentration range of 0.05-100 ng/mL for BUP and NBUP, and 0.1-200 ng/mL for BUP-G and NBUP-G. Intra- and inter-day bias and precision were within ±15% of nominal values for all the analytes. Quality controls assessed at four levels showed high recovery consistently for all the analytes with minimal matrix effect. Adequate analyte stability was observed at various laboratory conditions tested. Overall, the developed method is simple, sensitive, accurate and reproducible, and was successfully applied for the quantification of BUP and its metabolites in plasma samples collected from pregnant women in a clinical study assessing BUP exposure during OUD treatment.

Investigation of Rosa species by an optimized LC-QTOF-MS/MS method using targeted and non-targeted screening strategies combined with multivariate chemometrics.

Plants of the Rosa genus are renowned for their pronounced and pleasant aroma and colors. The aim of this work was to develop a novel liquid chromatographic triple quadrupole time-of-flight tandem mass spectrometric (LC-QTOF-MS/MS) method for the investigation of the bioactive fingerprint of petals of different genotypes belonging to Rosa damascena and Rosa centifolia species. Central composite design (CCD) of response surface methodology (RSM) was used for the optimization of the LC-QTOF-MS/MS method. The method was validated and target, suspect, and non-target screening workflows were applied. Statistical analysis and chemometric tools were utilized to explore the metabolic fingerprint of the Rosa species. RSM revealed that the optimal extraction parameters involved mixing 11 mg of sample with 1mL of MeOH:H2O (70:30, v/v). Target analysis confirmed the presence of 11 analytes, all of which demonstrated low limits of quantification (LOQs; as low as 0.048 ng mg-1) and sufficient recoveries (RE: 85%-107%). In total, 28 compounds were tentatively identified through suspect analysis. Non-target analysis enabled the generation of robust OPLS-DA and HCA models that classified the samples according to their species with 100% accuracy. A novel LC-QTOF-MS/MS method was developed and applied in the analysis of 47 R.centifolia and R.damascena flowers belonging to different genotypes.

Analyses of plasma metabolites using a high performance four-channel CIL LC-MS method and identification of metabolites associated with enteric methane emissions in beef cattle.

Reducing enteric methane (one greenhouse gas) emissions from beef cattle not only can be beneficial in reducing global warming, but also improve efficiency of nutrient utilization in the production system. However, direct measurement of enteric methane emissions on individual cattle is difficult and expensive. The objective of this study was to detect plasma metabolites that are associated with enteric methane emissions in beef cattle. Average enteric methane emissions (CH4) per day (AVG_DAILYCH4) for each individual cattle were measured using the GreenFeed emission monitoring (GEM) unit system, and beef cattle with divergent AVG_DAILYCH4 from Angus (n = 10 for the low CH4 group and 9 for the high CH4 group), Charolais (n = 10 for low and 10 for = high), and Kinsella Composite (n = 10 for low and 10 for high) populations were used for plasma metabolite quantification and metabolite-CH4 association analyses. Blood samples of these cattle were collected near the end of the GEM system tests and a high performance four-channel chemical isotope labeling (CIL) liquid chromatography (LC) mass spectrometer (MS) method was applied to identify and quantify concentrations of metabolites. The four-channel CIL LC-MS method detected 4235 metabolites, of which 1105 were found to be significantly associated with AVG_DAILYCH4 by a t-test, while 1305 were significantly associated with AVG_DAILYCH4 by a regression analysis at p<0.05. Both the results of the t-test and regression analysis revealed that metabolites that were associated with enteric methane emissions in beef cattle were largely breed-specific whereas 4.29% to 6.39% CH4 associated metabolites were common across the three breed populations and 11.07% to 19.08% were common between two breed populations. Pathway analyses of the CH4 associated metabolites identified top enriched molecular processes for each breed population, including arginine and proline metabolism, arginine biosynthesis, butanoate metabolism, and glutathione metabolism for Angus; beta-alanine metabolism, pyruvate metabolism, glycolysis / gluconeogenesis, and citrate cycle (TCA cycle) for Charolais; phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, arginine biosynthesis, and arginine and proline metabolism for Kinsella Composite. The detected CH4 associated metabolites and enriched molecular processes will help understand biological mechanisms of enteric methane emissions in beef cattle. The detected CH4 associated plasma metabolites will also provide valuable resources to further characterize the metabolites and verify their utility as biomarkers for selection of cattle with reduced methane emissions.

Comparing ELISA with a targeted mass spectrometric method to monitor the Mal d 1 content in apple samples

Comparing ELISA with a targeted mass spectrometric method to monitor the Mal d 1 content in apple samples

Simultaneous determination of ethyl glucuronide, cocaine, cocaethylene, and benzoylecgonine in hair by using LC–MS/MS

Alcohol and cocaine (COC) are commonly co-used drugs that cause addiction and have harmful effects. Their abuse may threaten the health of the abuser and public safety by causing serious accidents or crimes. The recidivism rate of drug-related crimes closely correlates with alcoholism. Several incidences of alcohol consumption in combination with drug abuse have been reported. Here, liquid chromatography tandem mass spectrometric method was developed to simultaneously analyze ethyl glucuronide (EtG), a metabolite of ethanol; COC; cocaethylene (CE), an alcohol-derived metabolite of COC; and benzoylecgonine (BZE), a major metabolite of COC, to determine the concurrent use of alcohol with COC. For pre-treatment, ultracentrifugation (5 min, 50,000 g) and mixed-mode anion exchange solid-phase extraction were used to increase the recovery of target compounds and minimize the matrix effect of hair. The lower limits of quantification were: 7 pg/mg (EtG), 2 pg/mg (COC), 10 pg/mg (CE), and 1 pg/mg (BZE). The correlation coefficient (r) of the calibration curve within the quantified range of target compounds was ≥ 0.9978. The intra- and inter-day accuracies were − 6.1–9.7% and − 9.3–8.3%, and intra- and inter-day precisions were 0.5–10.3% and 0.6–14.4%, respectively. The recovery, matrix effect, process efficiency, and autosampler stability were 89.2–104.8%, 81.6–105.4%, 81.5–107.1%, and 96.6–109.7%, respectively. The novel analytical method was validated with hair samples from individuals suspected of alcohol and COC use, and the method could distinguish between independent and concurrent use. Based on the findings, the analytical approach developed in this study is anticipated to be valuable in drug and alcohol dependence tests that require the simultaneous detection of alcohol and COC abuse.

Mass spectrometric methods for evaluation of voriconazole avian pharmacokinetics and the inhibition of its cytochrome P450-induced metabolism

Invasive fungal aspergillosis is a leading cause of morbidity and mortality in many species including avian species such as common ravens (Corvus corax). Methods were developed for mass spectral determination of voriconazole in raven plasma as a means of determining pharmacokinetics of this antifungal agent. Without further development, GC/MS/MS (gas chromatography-tandem quadrupole mass spectrometry) proved to be inferior to LC/MS/MS (liquid chromatography-tandem quadrupole mass spectrometry) for measurement of voriconazole levels in treated raven plasma owing to numerous heat-induced breakdown products despite protection of voriconazole functional groups with trimethylsilyl moieties. LC/MS/MS measurement revealed in multi-dosing experiments that the ravens were capable of rapid or ultrarapid metabolism of voriconazole. This accounted for the animals’ inability to raise the drug into the therapeutic range regardless of dosing regimen unless cytochrome P450 (CYP) inhibitors were included. Strategic selection of CYP inhibitors showed that of four selected compounds including cimetidine, enrofloxacin and omeprazole, only ciprofloxacin (Cipro) was able to maintain voriconazole levels in the therapeutic range until the end of the dosing period. The optimal method of administration involved maintenance doses of voriconazole at 6 mg/kg and ciprofloxacin at 20 mg/kg. Higher doses of voriconazole such as 18 mg/kg were also tenable without apparent induction of toxicity. Although most species employ CYP2C19 to metabolize voriconazole, it was necessary to speculate that voriconazole might be subject to metabolism by CYP1A2 in the ravens to explain the utility of ciprofloxacin, a previously unknown enzymatic route. Finally, despite its widespread catalog of CYP inhibitions including CYP1A2 and CYP2C19, cimetidine may be inadequate at enhancing voriconazole levels owing to its known effects on raising gastric pH, a result that may limit voriconazole solubility.

Dynamic accelerated stress test and coupled on-line analysis program to elucidate aging processes in proton exchange membrane fuel cells

The application of hydrogen proton exchange membrane fuel cells (PEMFC) in greenhouse gas emission free heavy-duty vehicles requires extremely durable PEMFC components with service lives in the range of 30,000 h. Hence suitable test and analysis methods are required that reflect realistic operation scenarios, but significantly accelerate aging. For this purpose, a dynamic accelerated stress test was developed, which is coupled with a comprehensive in-depth in-situ and ex-situ analysis program to determine the aging processes of a PEMFC membrane electrode assembly (MEA). The test comprehends dynamic cycling between low, moderate and high load, different temperature and humidity conditions as well as recovery sequences to distinguish between reversible and irreversible failure modes. All phases of the PEMFC system (i.e. solid, liquid and gaseous) are monitored on-line during aging by sophisticated electrochemical, mass spectrometric and ion chromatographic analytical methods. The structural and elemental composition of the MEA before and after the aging program (post-mortem) are investigated by X-ray fluorescence, scanning and transmission electron microscopy. This program was able to age a commercial PEMFC to end-of-life in 1000 h, while providing an accurate picture of the aging processes involved.