High-resolution Accurate Mass Spectrometry Research Articles

Identification of unexpected chemical contaminants in baby food coming from plastic packaging migration by high resolution accurate mass spectrometry

Plastic multilayers are widely used for baby food packaging. However, it is important to consider that migration of food contact materials (FCM) into the baby food can occur. The comprehensive identification of potential migrants, including intentionally added substances (IAS) and non-intentionally added substances (NIAS), is required to assess the safety of these packaging materials. In this study, high resolution accurate mass spectrometry (HRAMS) with a data-independent acquisition method of sequential mass windows enables the detection of substances with corresponding deconvoluted fragment mass spectra. The identification of unexpected migrants present in the food simulants and in real baby food was facilitated by filtering strategies and by an in-house library. This approach has allowed the identification of 42 migrants, including eight NIAS detected for the first time. Two oligomers were quantified by means of reference standard materials at concentration levels above 0.010 mg/kg, exceeding the maximum residue levels for baby food.

MON-487 Simultaneous Monitoring of 13 IGF-1 Variants in a Clinical Mass Spectrometry Assay

In the clinical laboratory, identification and quantification of intact proteins by high resolution accurate mass (HRAM) mass spectrometry (MS) is a highly specific and robust technique. However, protein variants with mass-to-charge ratios (m/z) differing from the wild type are not measured. In addition, variants may not be resolved from the wild type or from each other. HRAM can enable resolution of IGF-1 and its variants at the level of isotopic masses. However, the choice of which isotope to monitor is critically important as intact IGF-I produces a wide isotopic envelope of up to 13 detectable peaks. For this purpose, we employ the concept of Peak Isotopic Index (PII) wherein peaks in the isotopic clusters are designated by a subscript. We designate IGF-10 for the monoisotopic peak, and IGF-11, IGF-12, etc., for heavier isotopic peaks. The same applies to IGF-1 variants. Unfortunately, most of the variant isotopic peaks are not resolved. Consequently, simultaneous monitoring of multiple variants is challenging. In the current study, our objective was to simultaneous monitor 13 IGF-1 variants in a routine clinical analysis employing HRAM MS. For the routine IGF-1 assay we used a Thermo Q Exactive™ Focus Hybrid Quadrupole-Orbitrap™ MS coupled to a multiplex high performance liquid chromatography (HPLC) system. To efficiently monitor all the variants, they were split into 4 groups, each corresponding to a single m/z for that group of variants: Group 1 (P66A4, R36Q6) monitored at m/z 1089.8050; Group 2 (A67S4, T29I8) monitored at m/z 1095.8065; Group 3 (S34N2, A70P3, A38V1, M59R4) monitored at m/z 1097.0960; and Group 4 (V17M4, V44M4, T4M6, A67T6, A70T6) monitored at m/z 1098.0969. All specimens that were suspected to contain IGF-1 variants were submitted for DNA sequencing for identification and/or confirmation. We analyzed 77,231 samples and identified 302 patients (0.4%) with IGF-1 variants. Most (290, 96.0%) were Group 4 variants, compared to 8 (2.7%) from Group 3, 3 (1.0%) from Group 2, and 1 (0.3%) from Group 1. Of the samples analyzed, 299 corresponded to patients heterozygous for a particular IGF-1 variant; the amount of wild type IGF-1 reported for those patients was a half of the total IGF-1 present in the sample. We also identified 3 homozygous patients (all variants belonging to Group 4), in which no wild type IGF-1 was detected and all of the IGF-1 was expressed in a form of a variant. In this study we demonstrated that 13 IGF-1 variants can be routinely screened by HRAM MS assay using only 4 m/z ratios. To our knowledge, this is the largest number of IGF-1 variants monitored in a clinical setting. While the clinical significance of these variants may be presently unknown, knowledge that the variants are present may help avoid misdiagnosis on the basis of low wild type IGF-1 levels

Study on heterogeneous photocatalytic ozonation degradation of ciprofloxacin by TiO2/carbon dots: Kinetic, mechanism and pathway investigation

In this study, the objective was mainly focusing on the mechanism investigation of ciprofloxacin (CIP) degradation by photocatalytic ozonation process which carried out by ozone and TiO2 with a low content of carbon-dots (CDs) under simulated sunlight irradiation. The physicochemical properties of the prepared photocatalysts were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscope (SEM) X-ray photoelectron spectroscopy (XPS) and zeta potential. Comprehensive investigation has proven the process to be efficient in the removal of CIP with high yield of reactive species (OH, O2—, h+, etc.). Kinetic model on pH investigation found out a repulsive force between the photocatalysts and CIP intensified with the increasing pH, so did the production rate of hydroxyl radicals (OH), while eventually reached a balance and achieved a maximum degradation rate. The results indicated that the enhancement mechanism was triggered by the photoexcited electron accumulated on CDs and transferred by ozone, resulting in the continuous generation of h+, O3— and O2—. Possible photocatalytic ozonation degradation pathways of CIP were proposed according to the identifications of intermediates using high-resolution accurate-mass spectrometry (HRAM) LC-MS/MS.

Integrated liquid chromatography-mass spectrometry and nuclear magnetic resonance spectra for the comprehensive characterization of various components in the Shuxuening injection

Shuxuening injection (SXNI) is made from the extract of Ginkgo biloba, and used for curing coronary heart disease, stenocardia and cerebral vasospasm. However, the flavonoids and terpene trilactones (TTLs) only account for about 30% of the dry weight of SXNI according to the current quality control standard. Thus, systematic investigation of the chemical composition of SXNI is needed to guarantee the drug safety. In this study, the integral approach of ultra high performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high resolution accurate mass spectrometry (UHPLC-Q-Orbitrap HRMS) and nuclear magnetic resonance (NMR) techniques, combining with the solvent partition, were used for identifying the phytochemical compounds in SXNI. The NMR analysis revealed the possible structure types based on the characteristic chemical shifts, coupling constants, and peak shape, which gave the clues for the further UHPLC-Q-Orbitrap HRMS analysis of previously unknown or uncatalogued compounds. The characteristic ions and structural network of flavonoids and TTLs were generated and used in the UHPLC-Q-Orbitrap HRMS based structure identification. Only 72 compounds could be identified by the direct UHPLC-Q-Orbitrap HRMS analysis of SXNI, while this systematic strategy led to the identification of 133 compounds, which included 73 flavonoids, 5 biflavone, 6 proanthocyanidin, 14 TTLs, 6 fatty acids, 5 fatty amides, 12 organic acids, 1 amino acid, 3 excipients, 2 saccharides and 6 other components. In addition, the presence of steroids and triglycerides were also suggested by NMR. The absence of toxic components in SXNI was also confirmed. In addition, the NMR spectrum also revealed some unknown signals, which need to be further identified. This approach was proved to be useful for the rapid and effective identification of various compounds in the TCM preparations.

High-throughput quantification of carboxymethyl lysine in serum and plasma using high-resolution accurate mass Orbitrap mass spectrometry.

BackgroundCarboxymethyl lysine is an advanced glycation end product of interest as a potential biomarker of cardiovascular and other diseases. Available methods involve ELISA, with potential interference, or isotope dilution mass spectrometry (IDMS), with low-throughput sample preparation.MethodsA high-throughput sample preparation method based on 96-well plates was developed. Protein-bound carboxymethyl lysine and lysine were quantified by IDMS using reversed phase chromatography coupled to a high-resolution accurate mass Orbitrap Exactive mass spectrometer. The carboxymethyl lysine concentration (normalized to lysine concentration) was measured in 1714 plasma samples from the British Regional Heart Study (BRHS).ResultsFor carboxymethyl lysine, the lower limit of quantification (LLOQ) was estimated at 0.16 μM and the assay was linear between 0.25 and 10 μM. For lysine, the LLOQ was estimated at 3.79 mM, and the assay was linear between 2.5 and 100 mM. The intra-assay coefficient of variation was 17.2% for carboxymethyl lysine, 9.3% for lysine and 10.5% for normalized carboxymethyl lysine. The inter-assay coefficient of variation was 18.1% for carboxymethyl lysine, 14.8 for lysine and 16.2% for normalized carboxymethyl lysine. The median and inter-quartile range of all study samples in each batch were monitored. A mean carboxymethyl lysine concentration of 2.7 μM (IQR 2.0–3.2 μM, range 0.2–17.4 μM) and a mean normalized carboxymethyl lysine concentration of 69 μM/M lysine (IQR 54–76 μM/M, range 19–453 μM/M) were measured in the BRHS.ConclusionThis high-throughput sample preparation method makes it possible to analyse large cohorts required to determine the potential of carboxymethyl lysine as a biomarker.

A Question of Opioid Diversion or Compliance

A 60-year-old female patient with a history of chronic pain and headache presented to the Pain Management Clinic for follow-up assessment of her prescribed medications, as well as to determine the patient's suitability for enrollment in the medical cannabis program. The patient's chronic pain and headache were secondary to traumatic brain injury sustained in a motor vehicle accident approximately 2 years before her appointment. The patient also was afflicted by chronic pain in her right arm due to nerve damage associated with surgery for de Quervain tendonitis. Following the motor vehicle accident, the patient had been evaluated at a multidisciplinary traumatic brain injury clinic and had been prescribed Tylenol #4 (acetaminophen 300 mg/codeine 60 mg) up to a maximum of 4 times daily for pain, and lorazepam (1 mg every 8 h as needed) for anxiety and insomnia. To determine the patient's suitability for the medical cannabis program, the patient was required to submit a urine sample for drugs of abuse testing. Analysis of the sample was performed by a combination of immunoassay (amphetamines, barbiturates, benzodiazepines, benzoylecgonine, carboxy-tetrahydrocannabinol, and phencyclidine) and high-resolution accurate mass spectrometry (opioids). Following review of the patient's urine test results the clinician contacted the laboratory for assistance with the interpretation of the opioid results. A review of the immunoassay screens only demonstrated presumptive positive results for the amphetamine class; however, reflexive confirmatory testing via liquid chromatography–tandem mass spectrometry for amphetamines (amphetamine, methamphetamine, phentermine, methylenedioxyamphetamine, methylenedioxymethamphetamine, pseudoephedrine/ephedrine) was negative (Table 1). View this table: Table 1 Laboratory test results. The high-resolution accurate mass spectrometry results were more complex and demonstrated the presence of codeine, codeine-6-β-glucuronide, morphine, and morphine-6-β-glucuronide, which were consistent with the use of codeine (Table 1) (1, 2). However, the analysis also unexpectedly revealed the presence of hydrocodone, norhydrocodone, dihydrocodeine, and hydromorphone-3-β-glucuronide (Table 1). ### QUESTIONS TO CONSIDER 1. What are the …

A single run, rapid polarity switching method for determination of 30 pharmaceuticals and personal care products in waste water using Q-Exactive Orbitrap high resolution accurate mass spectrometry

The analytical capability of the UPLC-Q Exactive™ Orbitrap MS was exploited for simultaneous determination of 30 acidic and basic PPCPs in a single run, using rapid polarity switching of the electrospray ionisation source. Full scan MS mode at resolution of 35,000 FWHM, Automatic gain control (AGC) target of 1E6 ions at injection time of 50 ms provided the optimum parameters for high sensitivity, together with sufficient data points per peak (≥15) for improved reproducibility. In addition to chromatographic retention times, method selectivity was achieved via applying high resolution accurate mass with low mass tolerance filter (<5 ppm) for identification of each target compound. Six-point linear calibration curves (R2 > 0.95) were established for all target analytes over a concentration range of 1–1500 ng/ml. Good results were obtained for method accuracy (% recovery = 76–104%), inter- and intra-day precision (relative standard deviation <15%) at 3 concentration levels. Instrumental detection and quantification limits ranged from (0.02–1.21 ng/ml) and (0.07–4.05 ng/ml), respectively. While optimised MS/MS analysis through parallel reaction monitoring (PRM) mode provided slightly higher sensitivity, Full scan MS mode allowed for higher mass resolution (selectivity), more data points per peak (reproducibility) and more importantly, the potential for post-acquisition screening of non-target compounds. Following solid phase extraction (SPE) of target analytes, the method was successfully applied to provide first data on PPCPs occurrence in effluent and surface water samples (n = 10) from Egypt. Moreover, screening for non-target compounds revealed the presence of bisphenol A, which was further confirmed via matching with an authentic standard. Overall, this study provides first insight into the high analytical capabilities of the Q-Exactive™ Orbitrap platform for both targeted/non-targeted analysis of PPCPs in environmental matrices.

Reconsidering mass spectrometric fragmentation in electron ionization mass spectrometry - new insights from recent instrumentation and isotopic labelling exemplified by ketoprofen and related compounds.

In various fields of chemical analyses, structurally unknown analytes are considered. Proper structure confirmation may be challenged by the low amounts of analytes that are available, e.g. in early stage drug development, in metabolism studies, in toxicology or in environmental analyses. In these cases, mass spectrometric techniques are often used to build up structure proposals for these unknowns. Fragmentation reactions in mass spectrometry are known to follow definite pathways that may help to assign structural elements by fragment ion recognition. This work illustrates an investigation of fragmentation reactions for gas chromatography/electron ionization mass spectrometric characterization of benzophenone derivatives using the analgesic drug ketoprofen and seven of its related compounds as model compounds. Deuteration and 18 O-labelling experiments along with high-resolution accurate mass and tandem mass spectrometry (MS/MS) were used to further elucidate fragmentation pathways and to substantiate rationales for structure assignments. Low-energy ionization was investigated to increase confidence in the identity of the molecular ion. The high-resolution mass analyses yielded unexpected differences that led to reconsideration of the proposals. Site-specific isotopic labelling helped to directly trace back fragment ions to their respective structural elements. The proposed fragmentation pathways were substantiated by MS/MS experiments. The described method may offer a perspective to increase the level of confidence in unknown analyses, where reference material is not (yet) available.

Gas-Phase Chemistry in the GC Orbitrap Mass Spectrometer.

Gas-phase reactions of temporally stored ions play a significant role in trapped ion mass spectrometry. Especially highly labile ion species generated through electron ionization (EI) are prone to undergo gas-phase reactions after relaxation to a low vibrational state. Here, we show that in the C-Trap of the Q Exactive GC Orbitrap mass spectrometer, gaseous water reacts with radical cations of various compound classes. High-resolution accurate mass spectrometry of the resulting ions provides a key to the mechanistic understanding of the chemistry of high energetic species generated during EI. We systematically addressed water adduct formation by use of H2O and D218O in the C-Trap. Mass spectra of halogen cyanides XCN (X=Cl, Br, I) showed the formation of HXCN+ species, indicating hydrogen atomic transfer reactions. Relative ratios of HXCN+/XCN+• increased as the electronegativity of the halide increased. The common internal calibrant perfluorotributylamine forms oxygenated products from water reactive fragment ions. These can be explained by the addition of water to an initial cation followed by elimination of two HF molecules. This addition/elimination chemistry can also explain [M+2]+ and [M+3]+ ions that commonly occur in mass spectra of silylated analytes. High-resolution accurate mass spectra of trimethylsilyl (TMS) derivatives revealed these as [M-CH3•+H2O]+ and [M-CH4+H2O]•+, respectively. This study explains common fragment ions in ion trap mass spectrometry. It also opens up perspectives for the systematic mechanistic and kinetic investigation of high-energy ion reactivity. Graphical Abstract.

The effect of freeze-dried Carica papaya leaf juice treatment on NS1 and viremia levels in dengue fever mice model

BackgroundCarica papaya leaf juice (CPLJ) was well known for its thrombocytosis activity in rodents and dengue patients. However, the effect of CPLJ treatment on other parameters that could contribute to dengue pathogenesis such as nonstructural protein 1 (NS1) production and viremia level have never been highlighted in any clinical and in vivo studies. The aim of this study is to investigate the effect of freeze-dried CPLJ treatment on NS1 and viremia levels of dengue fever mouse model.MethodsThe dengue infection in mouse model was established by inoculation of non-mouse adapted New Guinea C strain dengue virus (DEN-2) in AG129 mice. The freeze-dried CPLJ compounds were identified by Ultra-High Performance Liquid Chromatography High Resolution Accurate Mass Spectrometry analysis. The infected AG129 mice were orally treated with 500 mg/kg/day and 1000 mg/kg/day of freeze-dried CPLJ, starting on day 1 post infection for 3 consecutive days. The blood samples were collected from submandibular vein for plasma NS1 assay and quantitation of viral RNA level by quantitative reverse transcription PCR.ResultsThe AG129 mice infected with dengue virus showed marked increase in the production of plasma NS1, which was detectable on day 1 post infection, peaked on day 3 post-infection and started to decline from day 5 post infection. The infection also caused splenomegaly. Twenty-four compounds were identified in the freeze-dried CPLJ. Oral treatment with 500 mg/kg/day and 1000 mg/kg/day of freeze-dried CPLJ did not affect the plasma NS1 and dengue viral RNA levels. However, the morbidity level of infected AG129 mice were slightly decreased when treated with freeze-dried CPLJ.ConclusionOral treatment of 500 mg/kg/day and 1000 mg/kg/day of freeze-dried CPLJ at the onset of viremia did not affect the plasma NS1 and viral RNA levels in AG129 mice infected with non-mouse adapted New Guinea C strain dengue virus.

Determination of Variance of Secondary Metabolites in Lettuces Grown Under Different Light Sources by Flow Injection Mass Spectrometric (FIMS) Fingerprinting and ANOVA–PCA

Lettuce (Lactuca sativa L.) is one of the most consumed vegetables in the world and different management practice can result in considerable variability of the secondary metabolites. Flow injection mass spectrometry (FIMS) combined with analysis of variance–principle component analysis (ANOVA–PCA) was used to study differences in the secondary metabolites originating from different lighting conditions (Sunlight, white light, and florescent light) and lettuce varieties (Romaine and Lollo Rossa). Ultra-high-performance liquid chromatography–high-resolution accurate mass spectrometry was used for putative marker compound identification. Quinic acid, caffeic acid, chlorogenic acid, L-chicoric acid, and quercetin malonyl glucoside varied significantly for Romaine lettuce grown under different light conditions. The study showed that the combination of FIMS fingerprinting and ANOVA–PCA can be a useful tool for the characterization of the sources of variance in plant materials regarding to genetic, environmental, and management factors.

Metabolomics approach in lung tissue of septic rats and the interventional effects of Xuebijing injection using UHPLC-Q-Orbitrap-HRMS.

Sepsis is the dysregulated host response to an infection which leads to life-threatening organ dysfunction. Metabolomic profiling in bio-fluid or tissue is vital for elucidating the pathogenesis of sepsis and evaluating therapeutic effects of medication. In this study, an untargeted metabolomics approach was applied to study the metabolic changes in lung tissue of septic rats induced by cecal ligation and puncture (CLP) and investigate the treatment effects of Xubijing injection (XBJ). Metabolomics analyses were performed on ultra-high performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry (UHPLC-Q-Orbitrap-HRMS) together with multivariate statistical analysis. A total of 26 differential metabolites between CLP and sham-operated group were identified. The altered metabolic pathways included energy metabolism, amino metabolism, lipid metabolism, fatty acid metabolism and hormone metabolism. Among the 26-varied metabolites, 15 were significantly regulated after XBJ treatment. The metabolic pathway network of sepsis was drawn to interpret the pathological feature of lung damage caused by sepsis and the underlying regulating mechanism of XBJ on the molecular levels. Our findings display that LC-MS-based metabolomics is a useful tool for uncovering the underlying molecular mechanism of sepsis, and XBJ may exert therapeutic effect by regulating multiple metabolic pathways.

High-resolution mass spectrometry with data independent acquisition for the comprehensive non-targeted analysis of migrating chemicals coming from multilayer plastic packaging materials used for fruit purée and juice

EU legislation requires that chemicals migrating from food packaging into food do not endanger human health. A proper safety assessment must go further than simply testing for known ingredients used to make the packaging materials. The identification of all potential migrants, including non-intentionally added substances (NIAS), is required to assess the safety of these materials. In this study, the benefits of using LC-ESI-Q-TOF-MS equipment combined with the data-independent acquisition method SWATH (Sequential Windowed Acquisition of All THeoretical MS) have been evaluated for the identification of unexpected migrants from multilayer plastic packaging materials. Identification of non-target peaks followed by filtering strategies facilitated the comprehensive identification procedure of unexpected migrants present in the food simulants. MS and MS/MS data from the most abundant ions in the samples were processed using formula finding and searched against open chemical and spectral databases, such as ChemSpider and METLIN, combined with bibliographic search. This approach has allowed the identification of 26 potential migrants, of which 23 were NIAS. Cyclic oligomers, possibly coming from polyurethane adhesives, were the main migrants in the studied materials. Evaluation of the toxicity of the tentatively identified migrants was carried out, showing that most of them were classified in the higher toxicity class according to Cramer rules. Most of the compounds detected in this study were previously reported in other studies of NIAS migration from plastic packaging materials, except for five of the tentatively identified oligomers that were found for the first time in this study. This study shows the high potential of non-targeted approaches using high resolution accurate mass spectrometry (HRAMS) with SWATH acquisition for the characterization of food packaging materials aimed at food safety evaluation.

A novel liquid chromatography Orbitrap mass spectrometry method with full scan for simultaneous determination of multiple bioactive constituents of Shenkang injection in rat tissues: Application to tissue distribution and pharmacokinetic studies.

Shenkang injection is a traditional Chinese formula with good curative effect on chronic renal failure. In this paper, a novel, rapid and sensitive ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole Orbitrap high-resolution accurate mass spectrometry was developed and validated for simultaneous determination of seven bioactive constituents of Shenkang injection in rat plasma and tissues after intravenous administration. Acetonitrile was used as a protein precipitation agent in biological samples disposal with carbamazepine as internal standard. The chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of acetonitrile and water (containing 0.1% formic acid). The MS analysis was performed in the full-scan positive and negative ion mode. The lower limits of quantification for the seven analytes in rat plasma and tissues were 0.1-10 ng/mL. The validated method was successfully applied to tissue distribution and pharmacokinetic studies of Shenkang injection after intravenous administration. The results of the tissue distribution study showed that the high concentrations of seven constituents were primarily in the kidney tract. This is the first report of the application of Q-Orbitrap with full-scan mass spectrometry in tissue distribution and pharmacokinetic studies of Shenkang injection.

Rapid analysis of fentanyls and other novel psychoactive substances in substance use disorder patient urine using paper spray mass spectrometry.

Drug overdose deaths due to fentanyls and other novel psychoactive substances (NPS) are on the rise. The higher potencies of fentanyl analogs compared with morphine require new technologies to identify and quantitate NPS. Paper spray tandem mass spectrometry (MS/MS) and high-resolution mass spectrometry were used to identify and measure fentanyl analogs as well as common drugs of abuse in urine samples from substance use disorder clinics. Ten-microliter urine samples were deposited directly on paper spray cartridges previously loaded with internal standards, dried, and analyzed with no other sample treatment. Quantitative results were obtained using MS/MS. Individual drugs were identified using high-resolution accurate mass spectrometry, and confirmed by data-dependent MS/MS. Calibration curves in urine were linear over a range of 0.5-50ng/mL with R2 of 0.99 or better for eight representative fentanyl analogs. Cartridges preloaded with internal standards demonstrated satisfactory quantitative results compared with LC/MS. Direct identification and confirmation of fentanyl analogs and other common drugs of abuse in urine using high-resolution accurate mass and MS/MS fragmentation were demonstrated at low picogram levels. Paper spray mass spectrometry can reliably identify and quantitate fentanyl analogs and other drugs of abuse in urine. Using paper spray cartridges as collection devices reduces exposure and transportation risks associated with biological fluids. Cartridges preloaded with labeled internal standards can be effective for targeted screening of fentanyl analogs and other drugs of abuse.

Microbial diversity and health-promoting properties of the traditional Bulgarian yogurt

ABSTRACTBulgarian yogurt is usually associated with good health and longevity. This study aimed to analyse the current microbial content of homemade Bulgarian yogurt. Identification by 16S rDNA sequencing revealed that out of 76 isolated strains, 53% belonged to Lactobacillus delbrueckii subsp. bulgaricus, 14% to other lactobacilli, and 32% to lactic acid cocci (Streptococcus thermophilus, Pediococcus acidilactici, Enterococcus faecium and Leuconostoc). All isolates inhibited the growth of pathogenic bacteria (10% of them by the putative action of bacteriocins); 18 isolates were able to produce extracellular exopolysaccharides (EPS), whereas 24% of them demonstrated extremely high proteolytic activity. Remarkably, 10 Lb. bulgaricus strains produced prebiotic galactooligosaccharides (GOS). High-resolution accurate mass spectrometry (HRAMS) analysis revealed production of tri- and tetrasaccharides containing atypical β(1→4) glycosidic bonds, reported for the first time for Lb. bulgaricus. Based on the beneficial features, along with good technological behaviour, we recommend several isolates as highly promising for Bulgarian yogurt starters.

Promotion of mammalian angiogenesis by neolignans derived from soybean extracellular fluids.

Excessive or insufficient angiogenesis is associated with major classes of chronic disease. Although less studied, small molecules which can promote angiogenesis are being sought as potential therapeutics for cardiovascular and peripheral arterial disease and stroke. Here we describe a bioassay-directed discovery approach utilising size exclusion and liquid chromatography to purify components of soybean xylem sap that have pro-angiogenic activity. Using high resolution accurate mass spectrometry and nuclear magnetic resonance spectroscopy, the structure of two pro-angiogenic molecules (FK1 and FK2) were identified as erythro-guaiacylglycerol-8-O-4'-(coniferyl alcohol) ether (eGGCE), and threo-guaiacylglycerol-8-O-4'-(coniferyl alcohol) ether (tGGCE). These two molecules, which are coniferyl neolignan stereoisomers, promoted in vitro angiogenesis in the μM to nM range. Independently sourced samples of eGGCE and tGGCE exhibited comparable pro-angiogenic activity to the soybean derived molecules. The cellular mode of action of these molecules was investigated by studying their effect on endothelial cell proliferation, migration, tube formation and adhesion to the extracellular matrix (ECM) components, fibronectin and vitronectin. They were found to enhance endothelial cell proliferation and endothelial cell tube formation on Matrigel, but did not affect endothelial cell migration or adhesion to fibronectin and vitronectin. Thus, this study has identified two coniferyl neolignan stereoisomers, eGGCE and tGGCE, as pro-angiogenic molecules, with eGGCE being less active than tGGCE.

A non-targeted metabolomic approach to identify food markers to support discrimination between organic and conventional tomato crops

In the last decade, the consumption trend of organic food has increased dramatically worldwide. However, the lack of reliable chemical markers to discriminate between organic and conventional products makes this market susceptible to food fraud in products labeled as “organic”. Metabolomic fingerprinting approach has been demonstrated as the best option for a full characterization of metabolome occurring in plants, since their pattern may reflect the impact of both endogenous and exogenous factors. In the present study, advanced technologies based on high performance liquid chromatography-high-resolution accurate mass spectrometry (HPLC-HRAMS) has been used for marker search in organic and conventional tomatoes grown in greenhouse under controlled agronomic conditions. The screening of unknown compounds comprised the retrospective analysis of all tomato samples throughout the studied period and data processing using databases (mzCloud, ChemSpider and PubChem). In addition, stable nitrogen isotope analysis (δ15N) was assessed as a possible indicator to support discrimination between both production systems using crop/fertilizer correlations. Pesticide residue analyses were also applied as a well-established way to evaluate the organic production. Finally, the evaluation by combined chemometric analysis of high-resolution accurate mass spectrometry (HRAMS) and δ15N data provided a robust classification model in accordance with the agricultural practices. Principal component analysis (PCA) showed a sample clustering according to farming systems and significant differences in the sample profile was observed for six bioactive components (L-tyrosyl-L-isoleucyl-L-threonyl-L-threonine, trilobatin, phloridzin, tomatine, phloretin and echinenone).

Prediction of collision cross section and retention time for broad scope screening in gradient reversed-phase liquid chromatography-ion mobility-high resolution accurate mass spectrometry

Exact mass, retention time (RT), and collision cross section (CCS) are used as identification parameters in liquid chromatography coupled to ion mobility high resolution accurate mass spectrometry (LC-IM-HRMS). Targeted screening analyses are now more flexible and can be expanded for suspect and non-targeted screening. These allow for tentative identification of new compounds, and in-silico predicted reference values are used for improving confidence and filtering false-positive identifications. In this work, predictions of both RT and CCS values are performed with machine learning using artificial neural networks (ANNs). Prediction was based on molecular descriptors, 827 RTs, and 357 CCS values from pharmaceuticals, drugs of abuse, and their metabolites. ANN models for the prediction of RT or CCS separately were examined, and the potential to predict both from a single model was investigated for the first time. The optimized combined RT-CCS model was a four-layered multi-layer perceptron ANN, and the 95th prediction error percentiles were within 2 min RT error and 5% relative CCS error for the external validation set (n = 36) and the full RT-CCS dataset (n = 357). 88.6% (n = 733) of predicted RTs were within 2 min error for the full dataset. Overall, when using 2 min RT error and 5% relative CCS error, 91.9% (n = 328) of compounds were retained, while 99.4% (n = 355) were retained when using at least one of these thresholds. This combined prediction approach can therefore be useful for rapid suspect/non-targeted screening involving HRMS, and will support current workflows.

Improvements in identification and quantitation of pesticide residues in food by LC-QTOF using sequential mass window acquisition (SWATH®)

The efficiency of a data independent acquisition by the sequential window acquisition of all theoretical fragment ions (SWATH®) in high resolution accurate mass spectrometry was evaluated for pesticide residue analysis.