High-resolution Quadrupole Research Articles

Determination of N-Nitrosamines by Gas Chromatography Coupled to Quadrupole–Time-of-Flight Mass Spectrometry in Water Samples

An analytical method based on high-resolution quadrupole–time-of-flight (QToF) mass spectrometry has been developed as an alternative to the classical method, using a low-resolution ion trap (IT) analyzer to reduce interferences in N-nitrosamines determination. Extraction of the targeted compounds was performed by solid-phase extraction (SPE) following the United States Environmental Protection Agency (USEPA) -521 method. First, both electron impact (EI) and positive chemical ionization (PCI) using methane as ionization gas were compared, along with IT and QToF detection. Then, parameters such as limits of detection (LOD) and quantification (LOQ), linearity, and repeatability were assessed. The results showed that the QToF mass analyzer combined with PCI was the best system for the determination of the N-nitrosamines, with instrumental LOD and LOQ in the ranges of 0.2–4 and 0.6–11 ng mL−1, respectively, which translated into method LOD and LOQ in the ranges of 0.2–1.3 and 0.6–3.9 ng L−1, respectively. The analysis of real samples showed the presence of 6 of the N-nitrosamines in influent, effluent, and tap water. N-nitrosodimethylamine (NDMA) was quantified in all the analyzed samples at concentrations between 1 and 27 ng L−1. Moreover, four additional nitrosamines were found in tap and wastewater samples.

Study on the Cure Behavior of a Novel Photocurable Material Using UPLC-Q-TOF-MS

An UPLC-Q-TOF-MS method is developed for cure degree measurement and cure behavior analysis on a novel photocurable adhesive material which is composed of specially designed acrylate oligomers, acrylate monomers, photo-initiators and additives such as ultra-violet absorbent, antioxidant stabilizer, optical stabilizer, etc. The photocurable adhesive material, in both cured and uncured state, were separated by Ultra-Performance Liquid Chromatography (UPLC) and the low molecular weight components were detected and determined quantitatively by high resolution Quadrupole Time-Of-Flight mass spectrometry (Q-TOF-MS) under Atmosphere Pressure Chemical Ionization (APCI) mode. Cure behaviors of all photo-reactive components in the photocurable adhesive material such as acrylate monomers and photo-initiators were studied by quantitatively measuring the amount of each reactive components in different stages of curing. Both the conversion of each acrylate monomers and photo-initiators at different curing energy conditions were calculated and discussed. Nearly full cure was obtained at cure energy of 200 mJ/cm2 for 4-hydroxybutyl acrylate and acryloyl morphine, as well as the two bifunctional monomers, 1,6-hexandiol diacrylate and dimethylol tricyclodecane diacrylate. Only 42.7% and 85.0% conversion were achieved for benzyl acrylate and isobornyl acrylate, respectively while consumption of TPO, a photo-initiator, was 38.0% at this cure energy. The results showed that a minimum 2000 mJ/cm2 energy condition is needed to achieve full cure of all acrylate monomers and enough decomposition of photo-initiator. This study indicated that UPLC-Q-TOF-MS is an effective and precise analytical method for cure degree measurement and cure behavior analysis on the photocurable materials.

Chemotyping and identification of protected Dalbergiatimber using gas chromatography quadrupole time of flight mass spectrometry

The international trade in illegally logged and environmentally endangered timber has spurred enforcement agencies to seek additional technical procedures for the identification of wood species. All Dalbergia species are listed under the Convention on International Trade in Endangered Species (CITES) which is the reason this genus was chosen for study. Multiple sources of the heartwood from different Dalbergia species were extracted and chromatographic profiles collected by gas chromatography with high resolution quadrupole Time of Flight mass spectrometry (GC/QToF). The collected data was mined to select peaks and mass ions representative of the investigated Dalbergia species, and used to develop a Microsoft Excel® template offering immediate graphical representation of the results. Using wood specimens sourced from different xylaria, this graphical fingerprint proved adept at definitive identification of Dalbergia species. The CITES Appendix I species, D. nigra, was easily distinguished from D. melanoxylon and look-alike species of other genera. Similarly, a number of other Dalbergia species were differentiated using this current approach. Kernel discrimination analysis (KDA) was applied to increase the confidence of the species identification. The mislabeling of specimens appears to be common, and the emerging technique of GC/QToF in combination with other techniques, offers improved confidence in identification. GC/QToF further provides automation, the dimension of chromatography to avoid interferences, and production of reproducible electron impact positive (EI+) spectra. The prospect of building an EI+ spectral database for future wood identification is an important feature considering the limited accessibility of authenticated wood species specimens.

Molecular Level Analysis Reveals Changes in Chemical Composition of Dissolved Organic Matter From South Texas Rivers After High Flow Events

Riverine dissolved organic matter (DOM) is a major source of reduced carbon exported from land to marine environments, and the inflow of riverine organic matter greatly affects biogeochemical cycling in estuaries and bays. Thus, any change in DOM composition, such as changes caused by flood waters as a result of storms and hurricanes, can subsequently affect estuarine environments. To investigate the impact of high flow events on riverine DOM, multidimensional molecular level information of DOM from four south Texas Rivers (Aransas, Lavaca, Mission, and Nueces Rivers) was acquired using high-resolution Ion Mobility Quadrupole Time of Flight Liquid Chromatography Mass Spectrometry (IM Q-TOF LCMS). Base-flow samples were collected in May, July and October of 2016, June of 2017, and March of 2018, while high-flow samples were collected in September of 2017, and June and September of 2018. Based on the molecular formula assigned from IM Q-TOF LCMS, H/C ratio decreased during high-flow event (1.52 to 1.51 in ESI+; 1.19 to 1.07 in ESI-), while O/C ratio increased (0.31 to 0.33 in ESI-). Furthermore, DOM shifted from a protein-like and lipid-like dominated community at base flow condition, to a lignin, tannin and condensed aromatic structure dominated one during high flow event based on MS and tandem MS data. These changes in high-flow riverine DOM indicate an increase of terrestrial signal, which likely is a result of mobilization of terrestrial organic matter from the watersheds by flooding. These mobilized DOM, though refractory at high-flow condition in rivers, could be reactive in coastal regions when condition changes, and thus potentially fuel microbial activities downstream. In addition, about 3.76 – 21.8% of DOM molecules contain structural isomers among different flow conditions. This low number of isomer percentages suggests that as the products of various enzymatic biochemical reactions, the number of isomers in DOM is constrained. Taken together, our study provides insights into structural changes of riverine DOM in response to extreme climate events in subtropical regions and have implications in understanding biogeochemical changes in estuaries under a changing climate.

Quantitation of Cannabinoids in Cannabis Dried Plant Materials, Concentrates, and Oils Using Liquid Chromatography–Diode Array Detection Technique with Optional Mass Spectrometric Detection: Single-Laboratory Validation Study, First Action 2018.11

Abstract This paper describes a single-laboratory validation of a liquid chromatography–diode array detection (LC–DAD) method for quantification of 12 major cannabinoids in Cannabis dried plant materials, concentrates, and oils. The method met Standard Method Performance Requirements for quantitative analysis of cannabinoids in Cannabis concentrates and Cannabis dried plant materials. The LOQs were in the range 0.003–0.10% (w/w), depending on the analyte and matrix. Spike recoveries were between 96.7 and 101.3% with relative SDs (RSDs) ≤2.3%. Precision expressed as repeatability and intermediate precision was within 0.3–4.8 and 1.1–5.1%, respectively. The chromatographic separation conditions used in this versatile method are compatible with both DAD–UV and MS detection. During method validation, high-resolution quadrupole time-of-flight MS was employed as a secondary detector (connected in series to the LC–DAD instrument) to provide high confidence identification of target analytes and as a tool for monitoring other cannabinoids for which reference standards were not available. The obtained results demonstrate applicability of the method to quantitative analysis of important cannabinoids in dried plants, concentrates, and oils. Limited data were generated for a food matrix (Cannabis-containing cookies) using this method with LC coupled to a compact single quadrupole mass spectrometer.

Rapid Determination of Aminoglycosides in Milk by Exact Ion Masses Using Ultra-High-Performance Liquid Chromatography–High Resolution Quadrupole Time-of-Flight Mass Spectrometry

A method is proposed for the rapid determination of 10 aminoglycosides (amikacin, apramycin, gentamycin, hygromycin B, dihydrostreptomycin, kanamycin A, neomycin, paromomycin, spectinomycin, and streptomycin) in milk by exact ion masses in ultra-high-performance liquid chromatography–high resolution quadrupole time-of-flight mass spectrometry. A simple method for sample preparation is proposed. The determination of aminoglycosides is possible along with other veterinary drugs in milk under the conditions of reversed-phase chromatography with an addition of an ion-pair reagent (heptafluorobutyric acid) to the sample extract being analyzed. A significant matrix effect is observed in the determination of aminoglycosides in milk (79–96%), which is reduced by diluting the extract tenfold with deionized water. Quantitative determination was performed by the method of absolute calibration. The analytical ranges for aminoglycoside were 10–1000 ng/g. The relative standard deviation of the results of analysis did not exceed 0.17. The duration of the identification of aminoglycosides in milk was 15 min; the duration of the determination of the detected analytes was 30–40 min.

Occurrence and spatial distribution of statins, fibrates and their metabolites in aquatic environments

Due to widespread occurrence of lipid lowering drugs such as statins, fibrates and their metabolites in the aquatic environments, there is a worldwide growing concern in their role in water quality and aquatic biota. However, this concern is limited by ability to address their occurrence, distribution, fate and eco-toxicological effects. This study focuses on the quantification of the levels of statins, fibrates and their metabolites in the aquatic environments using Ultra-High Performance Liquid Chromatography coupled to high resolution quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS). The developed UHPLC–QTOF–MS based method was successfully applied to the analysis of statins, fibrates and metabolites in real water samples collected from Daspoort WWWs influent and effluent and Apies River. A series of statin compounds (mevastatin, simvastatin, pravastatin, rosuvastatin, fluvastatin, atorvastatin), fibrates (gemfibrozil, fenofibrate) and the corresponding metabolites (clofibric and fenofibric acids) were detected and quantified in the range of 0.56–19.90 µg/L in both waste and River water samples. In general, the results of the present study are an indication of pollution hazards from wastewater treatment processes and these levels poses a huge risk to the growth and reproduction of aquatic organisms. Thus, regulating the limit levels of statins, fibrates and metabolites in any type of water is paramount as it will provide the vital information on the toxic risks associated with organic pollutants of pharmaceutical origin.

Comprehensive lipidomics of mouse plasma using class-specific surrogate calibrants and SWATH acquisition for large-scale lipid quantification in untargeted analysis

Lipidomics has gained rising attention in recent years. Several strategies for lipidomic profiling have been developed, with targeted analysis of selected lipid species, typically utilized for lipid quantification by low-resolution triple quadrupole MS/MS, and untargeted analysis by high-resolution MS instruments, focusing on hypothesis generation for prognostic, diagnostic and/or disease-relevant biomarker discovery. The latter methodologies generally yield relative quantification data with limited inter-assay comparability. In this work we aimed to combine untargeted analysis and absolute quantification to enhance data quality and to obtain independent results for optimum comparability to previous studies or database entries. For the lipidomic analysis of mouse plasma, RP-UHPLC hyphenated to a high-resolution quadrupole TOF mass spectrometer in comprehensive data-independent SWATH acquisition mode was employed. This way, quantifiable data on the MS and the MS/MS level were recorded, which increases assay specificity and quantitative performance. Due to the lack of an appropriate blank matrix for untargeted lipidomics, we herein established a sophisticated strategy for lipid class-specific calibration with stable isotope labeled standards (surrogate calibrants). LLOQs were in the range between 10 and 50 ng mL−1 for LPC, LPE, PI, PS, PG, SM, PC, PE, DAG) or 100–700 ng mL−1 (MAG, TAG), except for cholesterol and CE (1–20 μg mL−1). Acceptable values for accuracy and precision well below ±15% bias were reached for the majority of surrogate calibrants. However, to achieve sufficient accuracy for target lipids, response factors to corresponding surrogate calibrants are required. An approach to estimate response factors via a standard reference material (NIST SRM 1950) was therefore conducted. Furthermore, a useful workflow for post-acquisition re-calibration, involving response factor determination and iteratively built libraries, is suggested. In comparison to single-point calibration, the presented surrogate calibrant method was shown to yield results with improved accuracy that are largely in accordance with standard addition. Quantitative results of real samples (high-fat diet vs control diet) were then compared to two previously published dietary mouse plasma studies that provided absolute lipid levels and showed similar trends.

Stability study and oxidative degradation kinetics of posaconazole

Posaconazole (POS), a triazole derivative used for the treatment of systemic fungal infections, was recently found to be a potential therapeutic alternative for patients with chronic Chagas disease. It is necessary to determine the stability of POS and identify its degradation products to achieve the expected pharmacological effects and assess the risks of drug toxicity associated with this novel therapeutic application. This study aimed to evaluate POS stability under varied conditions using forced degradation tests (acidic and basic hydrolysis, thermolysis, and oxidation). A high-performance liquid chromatography (HPLC) method was developed for the separation and identification of POS and its degradation products. POS was stable to acid and basic hydrolysis and thermolysis but not to oxidation. POS and its degradation products were characterized by high-resolution positive mode electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Two degradation products with m/z values of 717 and 733 were detected. Molecular modeling was used for the calculation of the highest occupied molecular orbital (HOMO) and electrostatic potential changes. The results suggest that POS degradation begins with the breakage of its piperazine ring. This is the first study to report the kinetics of POS oxidative degradation under different oxidizing agent concentrations, temperatures, and pH conditions. The reaction followed second-order kinetics under all tested conditions. The analytical methods developed in this study were suitable to delineate the stability profile of POS. Elucidation of the degradation kinetics of POS is essential to ensure the stability of the drug in pharmaceutical formulations.

Ammonia/chlorine synergistic oxidation process applied to the removal of N, N-diethyl-3-toluamide

The ammonia/chlorination process is important for the treatment of ammonia-rich water, such as the ammonia removal, disinfection strategies, and disinfection byproducts controlling. Recently, the simultaneous removal of recalcitrant micropollutants during ammonia/chlorination has attracted attention. In this study, ammonia/chlorination was employed to remove the chlorine-resistant micropollutant N, N-diethyl-3-toluamide (DEET). Using the optimal chlorine to ammonia molar ratio (Cl/N) of 1.6, nearly 60% of DEET was removed by ammonia/chlorination within 15 min, while chlorination or chloramination alone showed minimal DEET removal. The synergistic effect obtained from the combination of ammonia and chlorine is attributed to reactive species generated from the decomposition of chloramine over Cl/N 1.0 to 1.6, being proved by the significantly inhibited performance (almost 95%) by t-butanol. The aqueous matrix was found to affect the results, and acidic/basic solutions reduced DEET removal by 70% to 79%, while the presence of natural organic matter (NOM 6 mg-C/L) suppressed the removal by 33%. The quantity of DEET removed per mole of decomposed chloramine (α) is proposed as a measure of the efficiency of this synergistic oxidation process, and the α values were determined to be 0.034, 0.022 and 0.010 in the synthetic solution, a solution containing 6 mg-C/L NOM and a wastewater effluent, respectively. Trials using high resolution quadrupole time of flight mass spectrometry and 15N isotope labelling, allowed the transformation products to be identified, and hydroxylation was found to be the dominant DEET degradation pathway.

Liquid chromatography-quadrupole time of flight mass spectrometric method for targeted analysis of 111 nitrogen-based compounds in weight loss and ergogenic supplements

The use of supplements for weight loss and in sports as pre-workout (ergogenic) products is widespread. Many of these supplements were found to contain active components, which were not claimed on the products labels. A validated liquid chromatography high-resolution mass spectrometry quadrupole time-of-flight (LC-QToF-MS) method was developed for the simultaneous analysis of 111 amine-based compounds belonging to ergogenics, anorectics and other active components including phenethylamines (amphetamines, ephedrines), sibutramine or yohimbine. This method involves the detection of [M+H]+ ions and the separation was achieved using a C18 column, water/acetonitrile gradient as the mobile phase. The method was validated for linearity, repeatability, accuracy, stability, system suitability, limits of quantification (LOQ) and limits of detection (LOD). The limits of detection were in the range from 0.001-0.5 μg/mL. The validated method was applied to the analysis of twenty-seven weight loss and ergogenic dietary supplements. Two-thirds of the supplements contained compounds that were not listed on the product’s label. These include several phenethylamines (PEA) such as demelverine, hordenine, N, N-dimethyl-phenethylamine, synephrine, N-methyl-β-phenethylamine, and methylsynephrine. In addition, the PEA mimics such as dimethylamylamine, dimethylbutylamine other stimulants including fursultiamine, evodiamine, phenibut and theophylline were also observed. One or more of the ingredients listed on the labels were not detected in forty-four percent of the products analyzed. Positive identification was based on retention time, accurate mass and fragment ions in comparison with the respective reference standards. Development of such methods is anticipated to be of aid to regulatory agencies for the identification of undeclared exogenous components that are found in many dietary supplement products.

Phototransformation of three selected pharmaceuticals, naproxen, 17α-Ethinylestradiol and tetracycline in water: Identification of photoproducts and transformation pathways

Transformation products (TPs) may be formed during various processes, however, it was proved that one of the main mechanisms responsible for degradation of pharmaceuticals in natural waters is photolysis. Three compounds were selected, tetracycline (TC), 17-α-ethinylestradiol (EE2) and naproxen (NAP), for degradation experiments which were performed using a xenon lamp emitting light with a spectrum closely approximating that of natural sunlight. Identification of photodegradation products was achieved via liquid chromatography coupled with high resolution quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) providing accurate mass measurements and MS/MS experiments for structural elucidation. Results indicate that degradation of compounds under the study follows pseudo-first-order kinetics. Photolysis rate constants (k) were determined and half-lifes (t1/2) calculated to be 11, 120, and 13 min for TC, EE2 and NAP, respectively. Xenon lamp irradiation led to the formation of several photoproducts. Many of them have been identified for the first time. LC-QTOF-MS analysis allowed for the identification of seven, four, and eight TPs of TC, EE2 and NAP, respectively. Finally, the presence of parent compounds and identified TPs was investigated in various real-world water samples. Only three degradation products of 17-α-ethinylestradiol and naproxen were detected in wastewater effluent and treated water samples. Quantitative structure-activity relationship (QSAR) approach was applied in order to estimate physical-chemical properties of selected pharmaceuticals and their TPs. This allowed predicting the fate of all analytes in the aqueous environment.

Metabolomic analysis of Lavandula dentata L. and Lavandula stoechas L. extracts by LC-QTOF/MS experiments and multivariate analysis techniques as a chemotaxonomical tool

Lavandula stoechas L. and Lavandula dentata L. have been widely used as popular herbal medicines in the Mediterranean region to treat digestive disorders and kidney diseases. Both species have quite a similar morphology, which makes them too frequently share the same popular name and be often used indistinctly in traditional medicine although they have different properties. The aim of this study was to use metabolomics approaches, followed by multivariate analyses to facilitate the taxonomical classifications. A non-targeted metabolomic strategy based on high-performance liquid chromatography coupled with high-resolution quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) has been applied to two hydroalcoholic extracts from Lavandula species (L. dentata L. and L. stoechas L.) with the aim of elucidating the major discriminatory metabolites. The principal component analysis of the LC-QTOF/MS data showed a clear discrimination between both species, using the first two principal components. The key compounds responsible for the discrimination were tentatively identified as sucrose and glycosylated flavonoids by comparison with spectral data bases and previous studies. These results clearly show that both plant species have a different expression pattern for these marker metabolites that might be potentially used as a novel chemotaxonomical tool, in addition to classical approaches.

In situ calibration of Direct Analysis in Real Time-mass spectrometry for direct quantification: Urine excretion rate index creatinine as an example

Ambient ionization in open environment brings a capability of a coupled mass spectrometry to detect target molecules in situ. However, it is limited to qualitative and semi-quantitative analysis. By coupling of an ambient ionization-based Direct Analysis in Real Time (DART) with high-resolution quadrupole time-of-flight mass spectrometry (QTOF/MS), we observe that, in one-chemical system, the target molecule displays a non-linear response in MS signal vs concentration, accompanying with large variation in MS signal, suggesting two obstacles for quantification to be overcome. Surprisingly, in a two-chemical system, we observe an apparent suppression effect. We prove that, due to this observed suppression effect, a fluctuant response in the MS signal of the stable isotope-labeled analogue can immediately reflect the change in the analyte concentration and ionization efficiency. For example, by taking advantage of this effect, even the analyte of different concentrations despairingly displayed similar signals would be accurately calibrated through the suppression of the internal stable isotope standard. This puts an important foundation on accurate and linear quantitation of analytes in complex matrix using DART-MS assay. Moreover, we for the first time demonstrate an application of in situ calibration of DART-MS for direct and accurate quantification of target molecule (creatinine) in highly complex samples (human urine) without any pre-separation. The quantification is also validated using HPLC-UV analysis (n = 38). At last, we show that stable isotope-labeled-creatinine (m/z 117.0850 amu) can be used for simultaneous in situ calibration of some other urinary metabolites with a mass/charge ratio varying from 120.069 amu to 333.125 amu.

Reduced CuFe2O4 for catalytic oxidation of methyl orange by activation of persulfate: performances and mechanisms

Bimetallic Fe0-based catalysts have been shown to be effective heterogeneous catalysts for elimination of organic pollutants by activating persulfate (PS). In this study, copper ferrite (CuFe2O4) was isothermally treated under a reducing atmosphere in order to load Fe0/Cu0 bimetallic nanoparticles on the surface of CuFe2O4. The reduced CuFe2O4 was then used as a magnetically separable catalyst for PS activation to remove methyl orange (MO) from aqueous solution. The experimental data followed well the pseudo-first-order kinetic model, and the reaction rate of the reduced CuFe2O4/PS system was 5.6 times greater than that of the CuFe2O4/PS system. The key operating parameters such as PS concentration, catalyst dosage and solution pH were investigated. Catalyst stability of reduced CuFe2O4 was also tested by consecutive reuse cycles. Quenching experiments and electron spin resonance (ESR) spectroscopy revealed that the main reactive species was sulfate radical (SO 4 ·− ). Intermediate products of MO degradation were determined by high performance liquid chromatography coupled with a high-resolution hybrid quadrupole time-of-flight mass spectrometer (LC-Q-TOF–MS) and the MO degradation pathways were proposed. Based on the analysis of catalytic activity and surface characteristics of reduced CuFe2O4, the enhanced reactivity of reduced CuFe2O4 was mainly attributed to rapid corrosion of Fe0/Cu0 bimetallic nanoparticles by PS. These findings illustrated that the reduced CuFe2O4/PS system may be an efficient technology for MO removal from wastewaters.

Advances in offline approaches for trace measurements of complex organic compound mixtures via soft ionization and high-resolution tandem mass spectrometry

Complex airborne mixtures of organic compounds can contain 10,000′s of diverse compounds at trace concentrations. Here, we incorporate high-resolution mass spectrometry into our integrated offline sampling-to-analysis measurement system for routine molecular-level speciation of complex mixtures in gas- or particle-phase samples with detection limits of 2–20 pg L−1 (i.e. 0.2–1.9 ppt in 6 L samples). Analytes desorbed from custom adsorbent tubes (or filter extracts) were separated via gas chromatography (GC) and simultaneously analyzed by an electron ionization quadrupole mass spectrometer (EI-MS), and by atmospheric pressure chemical ionization (APCI) combined with a high-resolution quadrupole time-of-flight mass spectrometer (Q-TOF) with a resolution of 25,000–40,000 M/ΔM in HR-TOF and MS/MS modes. We demonstrated our system with simple standards, a Macondo crude oil standard as a reference for complex mixtures of common airborne compounds, and ambient samples using GC-TOF and GC–MS/MS. We speciated complex mixtures at mass accuracy error (i.e. mass tolerance) down to 8 ± 2 ppm (e.g. resolving analytes of mass 270.000 u with 0.003 u accuracy) using a targeted approach with 3000 molecular formulas, including hydrocarbons and functionalized analytes containing oxygen, sulfur, nitrogen, or phosphorous. This extended from compounds with 10 to 32 carbon atoms and up to 16 hydrocarbon formulas per carbon number, and a similar range for functionalized compound classes. We also demonstrated our MS/MS capabilities to differentiate structural isomers and determine the presence of specific functional groups; and our direct-TOF capability, which bypasses high-temperature chromatographic separation to preserve functionalized analytes.

Factorial design-assisted supercritical carbon-dioxide extraction of cytotoxic active principles from Carica papaya leaf juice

The aims of this study are to investigate the selective cytotoxic activity of supercritical carbon dioxide (scCO2)-extracted freeze-dried leaf juice (FDLJ) of Carica papaya on squamous cell carcinoma (SCC25) cells, and to delineate the best small scale extraction parameters allowing maximal extract activity. Using scCO2 as a solvent, six operating parameters were studied and the supercritical fluid extraction (SFE) process investigated using a factorial design 26-2. The processing values promoting cytotoxic activity towards SCC-25 are: high pressure (250 bar), low temperature (35 °C), extended processing time (180 minutes), as well as a large amount of starting material (5 g). The factorial experimental design successfully identified the key parameters controlling the SFE of molecules cytotoxic to SCC cells from C. papaya juice. This study also validated the extraction method and showed that the SFE yield was reproducible. The chromatographic and mass spectrometric profiles of the scCO2 extract acquired with high-resolution quadrupole time-of-flight mass spectrometry (LC-QToF-MS) were used to tentatively identify the bioactive compounds using comparative analysis. The principal compounds were likely to be mainly vitamins and phytosterols, some of which are documented to be cytotoxic to cancer cells.

P159 MECHANISM OF DIET-DERIVED METABOLITES: A BEDSIDE TO BENCH INTERVENTION IN ULCERATIVE COLITIS

Ulcerative colitis (UC) is a chronic inflammatory bowel disease. Growing evidence demonstrates that the diet, gut microbiota and metabolites have a significant impact on UC. A high fat diet with less fiber have been linked to an increased risk of UC development. However, these complex interactions on UC are not well understood. We are performing a diet intervention study in which UC patients in remission or mildly active are sequentially given a high fat or low fat diet (4 weeks of each, 2 week wash out in between). The cross-over design and catering minimize variation. In a limited sample size of 8 patients, quality of life (QoL) was significantly improved based on low fat diet. We tested the hypothesis that this diet intervention led to a change in the microbiome (16s rRNA sequencing) and the fecal metabolites. 16S rRNA sequencing (Illumina MiSeq platform) was performed at Second Genome and used QIIME2 for microbiome analysis. The metabolites were analyzed (targeted and untargeted) by high resolution ultra-performance liquid chromatography (UPLC) and quadrupole time-of-flight (QTOF) mass spectrometry system in Dr. Chen’s lab. Stool samples were collected at 4 time points. 16S rRNA microbial profiling did not reveal any significant differences in this small sample size. We did find significant differences, however, in fecal metabolites. The untargeted analysis showed strong separation between the LFD and HFD groups. A few groups of fecal metabolites, including fatty acids (C12-C18), N-acylamino acids (N-oleoyl phenylalanine, N-palmitoyltaurine) and dehydrolithocholic acid were positively correlated with a HFD, while multiple amino acids (glycine, alanine), and AA metabolites (phenyllactic acid) were positively correlated with a LFD. Among these diet-responsive metabolites, lauric acid was increased by HFD even though no exogenous dietary intake of lauric acid (i.e. coconut or palm oil) was in either of the diets. This pilot study highlights new strategies to formulate precision medicine approaches for increasing the efficacy of dietary interventions in UC treatment.

Selectivity enhancement using sequential mass isolation window acquisition with hybrid quadrupole time-of-flight mass spectrometry for pesticide residues

The introduction of sequential mass isolation window acquisition mode in high-resolution quadrupole time-of-flight analysers undoubtedly represents an important improvement in the MS/MS spectra obtained when working in non-target analysis. However, the advantages and limitations of this approach have not been sufficiently defined and evaluated. The present work seeks to fill this gap by considering its application in non-target multiresidue pesticide analysis. This work focuses on the called SWATH® method, which combines both MS and MS/MS acquisition, dividing the entire mass range into smaller segments for the MS/MS mode. The effect of the number of mass isolation windows, the total cycle-time lapsed, the sensitivity obtained, the MS/MS spectra quality, the ion ratio stability as well as the identification and quantification capabilities has been evaluated. The use of ten mass isolation windows for data acquisition was selected as a compromise between the required points per chromatographic peak and the reduction in interferences achieved. An identification study was carried out on 141 pesticides in 20 vegetable matrices to check the false positives and false identifications found automatically, in accordance with the criteria set out in Document No. SANTE/11945/2015. Furthermore, special attention was given to certain issues that can make correct identification difficult, such as low fragment abundance due using of a generic collision energy, the matrix influence on the collision cell, the effect of the concentration level as well as deconvolution failure and mass window width. Finally, to verify the efficiency of the optimum parameters proposed, two proficiency samples were analysed, obtaining good results. This proved the benefits in terms of identification and quantification purposes.

Lipid profiling and analytical discrimination of seven cereals using high temperature gas chromatography coupled to high resolution quadrupole time-of-flight mass spectrometry

Minor lipids in cereals (such as phytosterols and alkylresorcinols) can be important for human nutrition and/or be used as biomarkers for cereal intake. However, the analysis of cereal lipids is very challenging due to the complex lipidome comprising several hundred individual compounds present over a wide range of concentrations.Here we present a method for the profiling of cereal lipids using high temperature gas chromatography coupled to high resolution mass spectrometry (GC/Q-TOF MS). The method was used to investigate the lipid profiles of 77 samples of bread wheat, spelt, einkorn, emmer, barley, rye and oats.Distinct differences in the patterns of alkylresorcinols, free and conjugated sterols and tocopherols between the cereals could be observed. Furthermore, traces of tocomonoenols and diunsaturated and methyl-alkylresorcinols (not previously reported in cereals) could be detected. Finally, the lipid patterns in the cereals could be used to separate the cereals by Principal Component Analysis.