Liquid Chromatography-quadrupole Time Of Flight Research Articles

The Effects of Encapsulation on the In Vitro Anti-Clostridial Activity of Olive Mill Wastewater Polyphenolic Extracts: A Promising Strategy to Limit Microbial Growth in Food Systems.

Despite the technologies applied to food production, microbial contamination and chemical deterioration are still matters of great concern. In order to limit these phenomena, new natural approaches should be applied. In this context, the present study aimed to assess the antioxidant and anti-Clostridial effects of two different polyphenolic extracts derived from olive mill vegetation water, one liquid (LE) and one encapsulated (EE). The extracts have been preliminary characterized using Liquid Chromatography Quadrupole Time-Of Flight spectrometry. The Oxygen Radical Absorbance Capacity method was used to determine the antioxidant capacity, registering a higher value for EE compared to that for LE (3256 ± 85 and 2446 ± 13 µgTE/g, respectively). The antibacterial activity against C. perfringens, C. botulinum and C. difficile was studied by the agar well diffusion method, MIC and MBC determination and a time-kill test. The results confirm that EE and LE are able to limit microbial growth, albeit with minor effects when the phenolic compounds are encapsulated. Further studies are needed to evaluate the possible application of these extracts in food systems.

Characterization of Polyhexamethylene Guanidine Oligomers in Solutions and Aerosols Emitted during Humidifier Use

The behavior of polyhexamethylene guanidine (PHMG), the causative agent of many humidifier-induced lung diseases, is not well known because of its various oligomer structures and analytical difficulties. The aim of this study was to identify different PHMG oligomer types both in solution and aerosols and to estimate the airborne concentration of oligomers during humidifier use. Three products containing PHMG as the main component were diluted to the manufacturer’s recommended concentration (6.5 ppm) or the worst-case concentration (65 ppm or 125 ppm). Samples were qualitatively and quantitatively analyzed with liquid chromatography-quadrupole time-of-flight (LC-qToF) mass spectrometry in the diluted solution and in the air at 0.5 m and 1 m. The LC-qToF data were processed using UNIFI software to characterize the PHMG structure. For all products in both the humidifier solution and air, the linear type was predominant over the branched/cyclic structure, but each product had different characteristics. The linear structure in the Oxy product, the main product of lung diseases, accounted for 90.6%, while that of the Scunder and BOC Sciences’ products accounted for 78.6% and 75.8%, respectively. The concentration of the oligomer in air for the Oxy product was estimated to be 35.89 and 390.96 μg/m3 at 6.5 and 65 ppm, respectively. Most of the oligomers in the solution were found in air at a short distance (0.5 m), with a negligible concentration beyond 1 m. Oligomers with 1–7 monomer units were identified in the humidifier solution, whereas mainly monomers, dimers, and trimers were identified in the air. The results of this study will facilitate further investigations of the mechanisms of lung disease by identifying the behaviors and forms of PHMG in the air, along with previously revealed toxicity results.

Multi-Omics Reveals the Effects of Cannabidiol on Gut Microbiota and Metabolic Phenotypes.

Introduction: Cannabidiol (CBD) has important pharmacological activity, which includes antispasmodic, antioxidant, antithrombotic, and antianxiety properties. CBD has been applied as a health supplement to atherosclerosis. However, CBDs effect on gut microbiota and metabolic phenotype is unclear. Materials and Methods: We constructed a high production of cardiovascular risk factors, such as trimethylamine-N-oxide (TMAO) and phenylacetylglutamine (PAGln), in a mouse model using Clostridium sporogenes colonization. We used 16S ribosomal RNA (rRNA) gene sequencing and ultra-high performance liquid chromatography-quadrupole time-of flight mass spectrometry-based metabolomics to evaluate the effect of CBD on gut microbiota and plasma metabolites. Results: CBD decreased the levels of creatine kinase (CK), alanine transaminase (ALT), and low-density lipoprotein cholesterol and markedly increased high-density lipoprotein cholesterol. Furthermore, CBD treatment increased the abundance of beneficial bacteria, which include Lachnospiraceae_NK4A136 and Blautia in the gut, but it decreased the levels of TMAO and PAGln in the plasma. Conclusion: CBD might have beneficial effects for cardiovascular protection.

Differentiating ultra-high temperature milk and reconstituted milk using an untargeted peptidomic approach with chemometrics

An untargeted peptide profiling based on ultra-performance liquid chromatography quadrupole time-of flight mass spectrometry with chemometrics was performed to differentiate ultra-high temperature processed milk and reconstituted milk. Thirty-three marker peptides were identified, primarily released from the C- or N-terminal of β-casein and αs1-casein. These peptides were produced by heating and protease hydrolysis. Additional heating and storage time experiments showed that the level of 18 marker peptides increased with heat load and storage time, whereas 15 peptides were solely influenced by heat load. The peptides from β-casein showed higher sensitivity to thermal stress compared to those from αs1-casein. Additionally, eight modified peptides of casein were identified as indicators of milk thermal processing. The identified marker peptides can distinguish ultra-high temperature processed milk and reconstituted milk, and are suitable for monitoring heating processes and storage of milk.

Antimicrobial residue assessment in 5,357 commercialized meat samples from the Spain-France cross-border area: A new approach for effective monitoring

Although antimicrobials are valuable allies in animal production, their extended use has led to unexpected threats associated with the emergence and propagation of antimicrobial resistance. Moreover, when withdrawal periods in food-producing animals are not observed, antimicrobial residues can access the food chain, causing direct toxicity, allergies, and/or intestinal microbiota dysbiosis in consumers.Given that Spain and France are the largest meat producers in the EU and also count among the top consumers of meat, our study's aim was to investigate the presence of antimicrobials in commercialized meat purchased in the Spain-France cross-border area (POCTEFA region). 5,357 meat samples were collected from different animal species and a variety of different retailer types in Spain (Zaragoza, Bilbao, and Logroño) as well as in France (Toulouse and Perpignan). Meat samples were analysed by a screening method (Explorer®+QuinoScan®), yielding 194 positive samples, which were further evaluated by UPLC-QTOF (Ultra Performance Liquid Chromatography-Quadrupole Time of Flight) for confirmation. Chromatographic analyses found antimicrobial residues in 30 samples, although only 5 of them (0.093% of initial samples) were non-compliant according to the current legislation. Further studies suggested that this mismatch between screening and confirmatory analyses might be due to the presence of biologically active metabolites derived from degradation of antimicrobials that were not identified by the targeted UPLC-QTOF method, but which might play a decisive role in the inhibition of the biological Explorer® test. Although chromatographic techniques detect the marker compounds determined by European and national regulations, and although they are the methods selected for official control of antimicrobials in food, certain unknown metabolites might escape their monitoring. This thus suggests that biological tests are the most adequate ones in terms of ideal consumer health protection.

Metabolic consequences for mice lacking Endosialin: LC\u2013MS/MS-based metabolic phenotyping of serum from C56Bl/6J Control and CD248 knock\u2010out mice

IntroductionThe Endosialin/CD248/TEM1 protein is expressed in adipose tissue and its expression increases with obesity. Recently, genetic deletion of CD248 has been shown to protect mice against atherosclerosis on a high fat diet.ObjectivesWe investigated the effect of high fat diet feeding on visceral fat pads and circulating lipid profiles in CD248 knockout mice compared to controls.MethodsFrom 10 weeks old, CD248−/− and +/+ mice were fed either chow (normal) diet or a high fat diet for 13 weeks. After 13 weeks the metabolic profiles and relative quantities of circulating lipid species were assessed using ultra high performance liquid chromatography-quadrupole time-of flight mass spectrometry (UHPLC–MS) with high resolution accurate mass (HRAM) capability.ResultsWe demonstrate a specific reduction in the size of the perirenal fat pad in CD248−/− mice compared to CD248+/+, despite similar food intake. More strikingly, we identify significant, diet-dependent differences in the serum metabolic phenotypes of CD248 null compared to age and sex-matched wildtype control mice. Generalised protection from HFD-induced lipid accumulation was observed in CD248 null mice compared to wildtype, with particular reduction noted in the lysophosphatidylcholines, phosphatidylcholines, cholesterol and carnitine.ConclusionsOverall these results show a clear and protective metabolic consequence of CD248 deletion in mice, implicating CD248 in lipid metabolism or trafficking and opening new avenues for further investigation using anti-CD248 targeting agents.

High-resolution chemical profiling and antiparasitic potential of the tropical shrub Dillenia suffruticosa

The aquaculture industry is growing rapidly throughout the world, but due to intensification, fish hatcheries are often faced with infestations of parasites, which can lead to economic loss. Among these parasites, the leech Zeylanicobdella arugamensis (Hirudinea: Piscicolidae) has been reported to impact hybrid groupers and other hosts. The objective of this study was to test the antiparasitic potential of chromatographic fractions of a crude methanolic extract of the tropical shrub Dillenia suffruticosa. The phytochemical composition of the shrub was determined using high-resolution liquid chromatography (LC)–quadrupole time-of-flight (QTOF)–mass spectrometry (MS) to narrow down the metabolites responsible for its antiparasitic properties. Seven fractions of a methanolic extract of D. suffruticosa were obtained through flash column chromatography. Various concentrations of the fractions were prepared and tested against Z. arugamensis. In the bioassay conducted with fresh leeches, significant mortality was induced by fraction 6 at 31.66 ± 4.88 min, followed by fraction 5 (39.58 ± 2.94 min), fraction 3 (63.75 ± 6.61 min) and fraction 4 (65.25 ± 4.98 min). Chemical profiling using LC–QTOF–MS identified 17 secondary metabolites comprising triterpenoids, sterols, flavones, a glycoside, a non-flavone phenolid, a pyrrolizine, a fatty acid and a fatty amide. Thus, our study indicated that the D. suffruticosa fractions contained potent bioactive compounds with antiparasitic potential.

White poplar: Targeted isolation of pancreatic lipase inhibitors

In order to discover new anti-obesity agents, twenty two natural extracts from medicinal plants and agricultural wastes were screened by pancreatic lipase inhibition assay. The methanolic extract of poplar wood (Populus alba L.), an important raw material for paper industry, showed promising inhibitory activity, its IC50 value was 7.15 μg/mL. Through bio-guided fractionation, the ethyl acetate fraction (E) was found to be the major contributor to the activity, IC50 = 4.7 μg/mL. Hence, it was subjected to bio-guided isolation and nine compounds (1–9) were identified from the active sub-fractions. The isolates were identified by means of one- and two-dimensional nuclear magnetic spectroscopy (1D, 2D-NMR), as well as high-performance liquid chromatography quadrupole time-of flight mass spectrometry (HPLC-QTOF-MS). (+)-Taxifolin (5) and (+)-ampelopsin (4) showed the highest activity, their IC50 values were 23.2 and 46.2 μM, respectively. The results indicated that flavonoids and phenolic acids in poplar wood could serve as potential candidates for development of new anti-obesity agents.

Synergistic multiple active species for the degradation of sulfamethoxazole by peroxymonosulfate in the presence of CuO@FeOx@Fe0

In this study, bimetallic particles (CuO@FeOx@Fe0) were prepared by displacement plating and calcination to activate peroxymonosulfate (PMS) for the degradation of sulfamethoxazole (SMX). First, the key preparation parameters (i.e., theoretical Cu mass loading (TMLCu) and calcination temperature) of CuO@FeOx@Fe0 preparation were investigated by single-factor experiments. Then the properties of CuO@FeOx@Fe0 were analyzed by scanning electron microscope energy dispersive spectrometry (SEM-EDS), energy dispersive spectrometry mapping (EDS-mapping), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). Subsequently, effects of PMS dosage, CuO@FeOx@Fe0 dosage and initial pH on the degradation of sulfamethoxazole were studied respectively. Meanwhile, the results of control experiments indicated that the initial SMX degradation efficiency of CuO@FeOx@Fe0 system was slower, but the final degradation rate was higher than that of Fe0/PMS system, which was due to the sustained release effect of Fe2+. In addition, the quantitative experiment of SO4− indicated that SO4− generated continuously in CuO@FeOx@Fe0/PMS system. Furthermore, the detection results of quenching tests and electron paramagnetic resonance (EPR) revealed the generation of multiple reactive oxygen species (i.e., SO4−, HO, O2−, 1O2) in CuO@FeOx@Fe0/PMS system, which degraded SMX synergistically. Finally, the SMX degradation pathways were proposed based on the intermediates detected by Liquid Chromatography-Quadrupole time of flight – Mass Spectrum (LC-QTOF-MS/MS) and the activation mechanism of CuO@FeOx@Fe0/PMS system was analyzed in detail. This study provided a new insight on the application of CuO@FeOx@Fe0 on organic pollutant degradation.

Metabolic profiling of Solanum villosum Mill subsp. miniatum (bernh. ex willd.): Hepatoprotective and antifibrotic activity in a rat model of liver fibrosis

Aim/Background: To assess the liver antifibrotic action of ethanolic extract of aerial parts of Solanum villosum Mill subsp. miniatum (Bernh. ex Willd.) (SVE) and correlate this activity with its high performance liquid chromatography-quadrupole time of flight (HPLC-qTOF) Electrospray Ionisation Mass Spectrometry (ESIMS) phytochemical profile. Materials and Methods: The median lethal dose of SVE was determined, and a rat model of carbon tetrachloride (CCl4)-induced liver fibrosis was used to evaluate its antifibrotic activity. Markers for hepatotoxicity, fibrosis, and oxidative stress were assessed, and histopathological features of liver tissues were examined. Metabolite profiling of SVE was achieved via HPLC-qTOF-ESIMS coupled with Photodiode array (PDA). Liver fibrosis was induced in rats by oral administration of CCl4for 6 weeks. Silymarin (positive control) and SVE (100 and 250 mg/kg) were orally administered daily for 6 weeks. Results: Compared to CCl4-intoxicated group, administration of SVE obviously ameliorated fibrosis of the hepatic capsule associated with aggregation of multiple focal fat cells formation. Both silymarin and SVE ameliorated the rise in serum markers of hepatotoxicity (alanine transaminase, aspartate transaminase, and alkaline phosphatase), markedly attenuated CCl4-induced oxidative stress. The antifibrotic activity of SVE was evidenced by inhibiting the rise in hepatic hydroxyproline content and accumulation of collagen. This was confirmed by the ability of SVE to inhibit alterations in expression of the fibrosis-related genes Collagen Iα, matrixmetalloproteinase-2 (MMP-2), tissue inhibitor matrix metalloproteinase-2, and transforming growth factor beta 1. HPLC-qTOF-ESIMS analysis of SVE revealed the presence of 47 metabolites, among which 33 were tentatively identified. Conclusion: SVE exhibited hepatoprotective and antifibrotic activities in rats by enhancing the antioxidant capacity and regulating expression of fibrogenic mediators.

Untargeted metabolomics reveals transformation pathways and metabolic response of the earthworm Perionyx excavatus after exposure to triphenyl phosphate

Triphenyl phosphate (TPHP) is one of the most highly utilized organophosphorus flame retardants, and has been frequently detected in various environmental matrices, including soil. So far, limited information is known regarding the potential toxicity of TPHP to the earthworm-soil ecosystem. We investigated the metabolism of TPHP and the perturbation of the endogenous metabolome in the earthworm, Perionyx excavatus, using gas chromatography mass spectrometry (GC-MS) and liquid chromatography quadrupole time-of-flight (LC-QTOF)-based untargeted metabolomics approach after acute exposure to TPHP for one and two days through a filter paper contact test, as well as after chronic exposure for 28 days in a soil microcosm experiment. TPHP showed low bioaccumulation potential in the earthworm-soil ecosystem at concentrations of 10 mg/kg and 50 mg/kg. Identified phase I metabolites include diphenyl phosphate, mono-hydroxylated and di-hydroxylated TPHP. Two groups of phase II metabolites, thiol conjugates (including mercaptolactic acid, cysteine, cysteinylglycine, and mercaptoethanol conjugates) and glucoside conjugates (including glucoside, glucoside-phosphate, and C14H19O10P conjugates), were putatively identified. Only acute TPHP exposure caused significant perturbations of the endogenous metabolome in earthworms, featuring fluctuations in amino acids, glucose, inosine and phospholipids. These results reveal novel phase II metabolism and toxicity of TPHP in P. excavatus.

A Metabolomics Study on the Bone Protective Effects of a Lignan-Rich Fraction From Sambucus Williamsii Ramulus in Aged Rats.

The lignan-rich fraction (SWR) of Sambucus Williamsii Ramulus, a folk herbal medicine in China for treatment of bone diseases, has previously reported to exert protective effects on bone without exerting uterotrophic effects in ovariectomized (OVX) mice. The aim of the present study was to identify the potential metabolites and the associated metabolic pathways that contribute to the beneficial effects of SWR on bone in vivo. Aged female Sprague Dawley rats (9 months old) were either sham-operated or ovariectomized for 12 weeks, before receiving treatment for another 12 weeks with the following treatment groups (n = 12 each): vehicle (Sham), vehicle (OVX), Premarin (130 μg/kg) or low (57 mg/kg), medium (114 mg/kg), and high (228 mg/kg) doses of SWR. The results showed that SWRH significantly suppressed bone loss, improved bone micro-architecture and increased bone strength on tibia without stimulating uterus weight gain in OVX rats. Premarin exerted similar bone protective effects as SWRH but elicited uterotrophic effects in OVX rats. The metabolic profiles of serum samples were analyzed by using ultra-performance liquid chromatography quadrupole time-of flight mass spectrometry and gas chromatography time-of flight mass spectrometry, and the metabolites that were significantly altered were identified by multivariate statistical analysis. Our study indicated that SWRH effectively restored the changes of 26 metabolites induced by estrogen-deficiency in OVX rats, which related to lipids, amino acids, tryptophan metabolisms, and anti-oxidative system. A subsequent validation showed that the serum level of superoxide dismutase and catalase were indeed up-regulated, while the serotonin level in a tryptophan hydroxylase 1 (TPH1) high expressing cells (rats RBL-2H3 cells) was down regulated after treatment with SWR. The results also suggested that the gut-microbiota may play an important role on the bone protective effects of SWR. The current study provides insight for understanding the unique mechanism of actions of SWR that might be involved in achieving bone protective effects in vivo.

Phytochemical Screening of Quaking Aspen (Populus tremuloides) Extracts by UPLC-QTOF-MS and Evaluation of their Antimicrobial Activity.

The continual emergence of pathogen resistance is a recurring challenge and pushes for the development of antimicrobial compounds. Here, we investigated compounds from quaking aspen trees (Populus tremuloides) as potential antimicrobial agents. Several extractions using different solvents were realized, and corresponding antimicrobial activity was tested against eight microorganisms. Results revealed that polar extraction solvents including water, ethanol and methanol gave the best extraction yields (>15.07%). Minimal inhibition concentration (MIC) and minimal bactericidal/fungicidal concentration (MBC/MFC) demonstrated that water extracts had the best antimicrobial activity by a weak to moderate inhibition of growth of all eight tested microorganisms in addition to having a bactericidal effect on three of them. The quaking aspen methanol extract also displayed antimicrobial activity but to a lower level than the water extract. Ultra-performance liquid chromatography quadrupole time-of flight mass spectrometry (UPLC-QTOF-MS) analysis led to the identification of 92 compounds, mainly polyphenols in both extracts, with 22 molecules previously known for their antimicrobial properties. According to the relative abundance, 4-hydroxybenzaldehyde (5.44% in methanol extract) and kaempferol (5.03% in water extract) were the most abundant antimicrobial compounds. Among antimicrobial molecules identified, nine were from the flavonoid family. The results of our study demonstrate the interest of using quaking aspen as source of antimicrobial compounds.

Quantification of suvorexant in blood using liquid chromatography-quadrupole/time of flight (LC-Q/TOF) mass spectrometry

Suvorexant is a novel drug for the treatment of insomnia that is marketed under the trade name Belsomra®. Unlike other hypnotics, suvorexant is a dual orexin receptor antagonist that is believed to have a lower abuse potential compared to other therapeutics. Although sedative hypnotics feature prominently in forensic toxicology investigations, there have been limited reports that describe the analysis of suvorexant in biological samples. Following a 10-mg oral dose, peak concentrations are typically <200 ng/mL. A highly sensitive assay is required because forensic toxicology laboratories are often required to identify a drug several hours after a single dose. A new analytical procedure for the quantification of suvorexant in whole blood was developed that will aid in the identification of this new drug in forensic toxicology casework. A simple acidic/neutral liquid-liquid extraction (LLE) was used to isolate suvorexant from whole blood followed by liquid chromatography-quadrupole/time of flight (LC-Q/TOF) mass spectrometry analysis using positive electrospray ionization (ESI). The extraction efficiencies of various solvents in blood were evaluated in addition to limit of detection, limit of quantitation, precision, accuracy and bias, calibration model, matrix effects, interferences, and carryover. The recovery of suvorexant was evaluated using four different extraction solvents (N-butyl chloride, ether/toluene (1:1), hexane/ethyl acetate (9:1), and methyl tert-butyl ether (MTBE). Although no significant differences in analytical recovery were observed, N-butyl chloride demonstrated improved reproducibility, efficiency and convenience. A weighted (1/x) quadratic calibration model was selected over a range of 2–200 ng/mL (R2 = 0.995). Using only 0.5 mL whole blood, limits of detection and quantification were 0.5 ng/mL. Intra-assay (n = 5) and inter-assay (n = 15) precision (% CV) were ≤ 13% and bias ranged from −5 to 2% at concentrations of 5, 50, and 160 ng/mL. Matrix effects were 16% (9% CV) and 15% (8% CV) for 20 ng/mL and 100 ng/mL (n = 20), respectively. No qualitative interferences or carryover were observed; however, a quantitative interference with the internal standard (estazolam-D5) could be attributed to sertraline when present at a 10-fold higher concentration. In the absence of a commercially available deuterated internal standard, the potential for quantitative interferences using LC-based methods are discussed.

Next-generation metabolic screening: targeted and untargeted metabolomics for the diagnosis of inborn errors of metabolism in individual patients

The implementation of whole-exome sequencing in clinical diagnostics has generated a need for functional evaluation of genetic variants. In the field of inborn errors of metabolism (IEM), a diverse spectrum of targeted biochemical assays is employed to analyze a limited amount of metabolites. We now present a single-platform, high-resolution liquid chromatography quadrupole time of flight (LC-QTOF) method that can be applied for holistic metabolic profiling in plasma of individual IEM-suspected patients. This method, which we termed “next-generation metabolic screening” (NGMS), can detect >10,000 features in each sample. In the NGMS workflow, features identified in patient and control samples are aligned using the “various forms of chromatography mass spectrometry (XCMS)” software package. Subsequently, all features are annotated using the Human Metabolome Database, and statistical testing is performed to identify significantly perturbed metabolite concentrations in a patient sample compared with controls. We propose three main modalities to analyze complex, untargeted metabolomics data. First, a targeted evaluation can be done based on identified genetic variants of uncertain significance in metabolic pathways. Second, we developed a panel of IEM-related metabolites to filter untargeted metabolomics data. Based on this IEM-panel approach, we provided the correct diagnosis for 42 of 46 IEMs. As a last modality, metabolomics data can be analyzed in an untargeted setting, which we term “open the metabolome” analysis. This approach identifies potential novel biomarkers in known IEMs and leads to identification of biomarkers for as yet unknown IEMs. We are convinced that NGMS is the way forward in laboratory diagnostics of IEMs.

Identification and quantification of synthetic cathinones in blood and urine using liquid chromatography-quadrupole/time of flight (LC-Q/TOF) mass spectrometry.

Synthetic cathinones continue to present a formidable challenge to forensic toxicology laboratories despite the fact that they are often encountered in impaired driving and death investigations. Due to limitations in immunoassay-based screening technologies, many forensic toxicology laboratories must rely on more labor intensive chromatographic-based screening approaches in order to detect these drugs in biological evidence. Solid phase extraction (SPE) and liquid chromatography-quadrupole/time of flight (LC-Q/TOF) mass spectrometry were used to identify twenty-two synthetic cathinones in urine and blood. Target drugs included methcathinone, ethcathinone, pentedrone, buphedrone, 3-fluoromethcathinone (3-FMC), 4-fluoromethcathinone (4-FMC), 4-methylethcathinone (4-MEC), 4-ethylmethcathinone (4-EMC), mephedrone, methedrone, 3,4-dimethylmethcathinone (3,4-DMMC), ethylone, butylone, pentylone, eutylone, methylone, methylenedioxypyrovalerone (MDPV), 4-methylpyrrolidinobutiophenone (MPBP), 3,4-methylenedioxypyrrolidinobutiophenone (MDPBP), α-pyrrolidinopentiphenone (α-PVP), pyrovalerone, and naphyrone. A total of nine deuterated internal standards were employed. Using traditional reversed phase chromatography all positional isomers, including 3-FMC and 4-FMC, were separated in 12min. The procedure was validated in accordance with the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation. Extraction efficiencies were 84-104% and 81-93% in urine and blood, respectively. Limits of quantitation in both matrices were 0.25-5ng/mL. Precision, bias and matrix effect were all within acceptable thresholds and the assay was free from more than fifty interferences. The validated method was used to identify cathinones in authentic urine case samples (n=20) and these results highlight important considerations for cathinone stability and the subsequent interpretation of results.

Regulation of Acyl Homoserine Lactone Synthesis in Pseudomonas putida JMQS1 Under Phenol Stress

The cell density-dependent gene expression in gram-negative bacteria is through the activity of acyl homoserine lactone signal molecules. The novel strain Pseudomonas putida JMQS1 isolated from detergent-contaminated soil exhibited quorum sensing along with its ability to degrade phenol. The response to Chromobacterium violaceum DSTS-1 mutant biosensor and luxI and luxR gene-specific amplification was used to characterize the quorum sensing property of the isolate. A regulation in the synthesis of various acyl homoserine lactone molecules, viz C6HSL in the initial stages of phenol stress, C8HSL during degradation, and 3OC12HSL on completion of degradation could be identified by liquid chromatography-quadrupole time of flight. Thin-layer chromatography, Fourier transform infrared spectroscopy, and gas chromatography mass spectrometry confirmed the complete degradation of phenol in 48–56 h. P. putida JMQS1 exhibited adaptation over phenol stress through the selective activation of the quorum sensing signal molecules depending on the changing physiological conditions. This study proposes an efficient method for enhancing the degradation of toxic organic pollutants by the supplementation of acyl homoserine lactone signal molecules.

Accurate Mass GC/LC-Quadrupole Time of Flight Mass Spectrometry Analysis of Fatty Acids and Triacylglycerols of Spicy Fruits from the Apiaceae Family.

The triacylglycerol (TAG) structure and the regio-stereospecific distribution of fatty acids (FA) of seed oils from most of the Apiaceae family are not well documented. The TAG structure ultimately determines the final physical properties of the oils and the position of FAs in the TAG molecule affects the digestion; absorption and metabolism; and physical and technological properties of TAGs. Fixed oils from the fruits of dill (Anethum graveolens), caraway (Carum carvi), cumin (Cuminum cyminum), coriander (Coriandrum sativum), anise (Pimpinella anisum), carrot (Daucus carota), celery (Apium graveolens), fennel (Foeniculum vulgare), and Khella (Ammi visnaga), all from the Apiaceae family, were extracted at room temperature in chloroform/methanol (2:1 v/v) using percolators. Crude lipids were fractionated by solid phase extraction to separate neutral triacylglycerols (TAGs) from other lipids components. Neutral TAGs were subjected to transesterification process to convert them to their corresponding fatty acids methyl esters (FAMES) using 1% boron trifluoride (BF3) in methanol. FAMES were analyzed by gas chromatography-quadrupole time of flight (GC-QTOF) mass spectrometry. Triglycerides were analyzed using high performance liquid chromatography-quadrupole time of flight (LC-QTOF) mass spectrometry. Petroselinic acid was the major fatty acid in all samples ranging from 57% of the total fatty acids in caraway up to 82% in fennel. All samples contained palmitic (16:0), palmitoleic (C16:1n-9), stearic (C18:0), petroselinic (C18:1n-12), linoleic (C18:2n-6), linolinic (18:3n-3), and arachidic (C20:0) acids. TAG were analyzed using LC-QTOF for accurate mass identification and mass spectrometry/mass spectrometry (MS/MS) techniques for regiospesific elucidation of the identified TAGs. Five major TAGs were detected in all samples but with different relative concentrations in all of the tested samples. Several other TAGs were detected as minor components and were present in some samples and absence in the others. Regiospecific analysis showed a non-random fatty acids distribution. Petroselinic acid was predominantly located at the sn-1 and sn-3 positions.

Tentative Structural Assignment of a Glucuronide Metabolite of Methyltestosterone in Tilapia Bile by Liquid Chromatography–Quadrupole-Time-of-Flight Mass Spectrometry

Methyltestosterone (MT), a strong androgenic steroid, is not approved for use in fish aquaculture in the United States. It is used in the U.S. under an investigational new animal drug exemption (INAD) only during the early life stages of fish. There is a possibility that farmers feed fish with MT to enhance production for economic gains. Therefore, there is a need to develop methods for the detection of MT and its metabolite residues in fish tissue for monitoring purposes. Previously, our laboratory developed a liquid chromatography-quadrupole time-of-flight (LC-QTOF) method for characterization of 17-O-glucuronide metabolite (MT-glu) in bile of tilapia dosed with MT. The system used was an Agilent 6530 Q-TOF equipped with electrospray jet stream technology, operating in positive ion mode. Retrospective analysis of the data generated in that experiment by a feature-finding algorithm, combined with a search against an in-house library of possible MT-metabolites, resulted in the discovery of a major glucuronide metabolite of MT in the bile extracts. Preliminary data indicate it to be a glucuronide of a hydroxylated MT (OHMT-glu) which persists in tilapia bile for at least 2 weeks after dosing. We present the tentative structural assignment of the OHMT-glu in tilapia bile and time course of development. This glucuronide can serve as a marker to monitor illegal use of MT in tilapia culture.

Metabonomics study of lung cancer cells based on liquid chromatography-mass spectrometry

The metabolic profiles of the polar metabolites and the non-polar metabolites in lung tumor cell lines H358, A549, HCC827, H1299, Calu-3, Calu-l, PC-9 and normal cell line MRC-5 were analyzed separately using high performance liquid chromatography-quadrupole time-of flight mass spectrometry (HPLC-Q-TOF/MS). Partial least square discriminant analysis ( PLS-DA) was used to process the metabolic data. The results showed that the metabolites of the lung cancer cell lines and normal cell line have significant differences. Further, 10 polar metabolites and 21 non-polar metabolites which had a significant contribution to classification were selected and preliminarily identified due to the accurate mass. Comparing with the normal cell line, the lung tumor cell lines present an abnormal metabolism in protein, fatty acid, and phospholipids. These results may provide important information for the early diagnosis of lung cancer.