Systematic UPLC-ESI-MS/MS study on the occurrence of staurosporine and derivatives in associated marine microorganisms from Eudistoma vannamei

Former bioactivity-guided analysis of the marine invertebrate Eudistoma vannamei led to the isolation of staurosporine derivatives, which revealed strong cytotoxic activity against several human cancer cell lines. The occurrence of such alkaloids in E. vannamei may be correlated to the presence of associated biota, such as Streptomyces bacteria. In agreement to this hypothesis, marine microorganisms associated with E. vannamei were recovered and cultured, leading to a total of 84 isolated bacterial strains. Gas phase fragmentation reactions of staurosporine and derivatives were systematically studied and the analyzed results further supported by computational chemistry studies. The resulting fragment patterns were used to search for the presence of different derivatives in extracts of isolated microorganisms, thereby using LC-MS/MS analysis in MRM mode. These results evidenced that one isolated Streptomyces sp. was able to generate staurosporine, while none of the hydroxy-7-oxo derivatives were detected. Finally, significant cytotoxic activity against human cancer lines was observed for one of the extracts.

Messa a punto e validazione di metodiche analitiche per la determinazione di micotossine in matrici biologiche mediante UPLC-MS/MS

La presenza di micotossine nelle materie prime desta grande preoccupazione a causa delle importanti implicazioni nella sicurezza di alimenti e mangimi. Lo scopo di questo lavoro è stato quello di mettere a punto e validare una metodica analitica rapida e semplice, in cromatografia liquida ad ultra prestazione accoppiata a spettrometria di massa-tandem (UPLC-MS/MS), per la determinazione simultanea di differenti micotossine: aflatossine (B1, B2, G1, G2), ocratossina A, fumonisine (B1, B2), deossinivalenolo e zearalenone in matrici biologiche. Il metodo sviluppato per l’analisi di campioni di mangime secco per cani ha mostrato prestazioni adeguate ed è stato applicato a 49 campioni reperibili in commercio, al fine di valutare la sua efficacia e di ottenere alcuni dati preliminari sulla contaminazione da micotossine in alimenti per cani disponibili sul mercato italiano. Lo studio ha evidenziato una percentuale alta di campioni positivi, contenenti principalmente fumonisine, deossinivalenolo e ocratossina A; tutti i tenori si sono dimostrati inferiori al limite di legge previsto (Racc. CE 576/2006). Una seconda metodica è stata messa a punto e validata per l’identificazione e la quantificazione micotossine in campioni di formaggio; per questa matrice è stata inserita anche l’aflatossina M1, specifica dei prodotti lattiero - caseari. Le differenti proprietà chimico-fisiche degli analiti e la complessità della matrice hanno implicato alcune difficoltà nello sviluppo della metodica. Tuttavia, il metodo validato si è mostrato rapido, semplice ed affidabile ed è stato applicato a diversi tipi di formaggi per verificarne la versatilità. I risultati preliminari hanno mostrato l’assenza di contaminazione da parte delle micotossine in oggetto. Entrambi i metodi si sono dimostrati utili per il monitoraggio di contaminanti in matrici complesse ad oggi ancora poco studiate.

Comparative metabolism of cyclophosphamide and ifosfamide in the mouse using UPLC-ESI-QTOFMS-based metabolomics

Ifosfamide (IF) and cyclophosphamide (CP) are common chemotherapeutic agents. Interestingly, while the two drugs are isomers, only IF treatment is known to cause nephrotoxicity and neurotoxicity. Therefore, it was anticipated that a comparison of IF and CP drug metabolites in the mouse would reveal reasons for this selective toxicity. Drug metabolites were profiled by ultra-performance liquid chromatography-linked electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS), and the results analyzed by multivariate data analysis. Of the total 23 drug metabolites identified by UPLC-ESI-QTOFMS for both IF and CP, five were found to be novel. Ifosfamide preferentially underwent N-dechloroethylation, the pathway yielding 2-chloroacetaldehyde, while cyclophosphamide preferentially underwent ring-opening, the pathway yielding acrolein (AC). Additionally, S-carboxymethylcysteine and thiodiglycolic acid, two downstream IF and CP metabolites, were produced similarly in both IF- and CP-treated mice. This may suggest that other metabolites, perhaps precursors of thiodiglycolic acid, may be responsible for IF encephalopathy and nephropathy.

UPLC-MS-based urine metabolomics reveals indole-3-lactic acid and phenyllactic acid as conserved biomarkers for alcohol-induced liver disease in the Ppara-null mouse model

Since the development and prognosis of alcohol-induced liver disease (ALD) vary significantly with genetic background, identification of a genetic background-independent noninvasive ALD biomarker would significantly improve screening and diagnosis. This study explored the effect of genetic background on the ALD-associated urinary metabolome using the Ppara-null mouse model on two different backgrounds, C57BL/6 (B6) and 129/SvJ (129S), along with their wild-type counterparts. Reversed-phase gradient UPLC-ESI-QTOF-MS analysis revealed that urinary excretion of a number of metabolites, such as ethylsulfate, 4-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid sulfate, adipic acid, pimelic acid, xanthurenic acid, and taurine, were background-dependent. Elevation of ethyl-β-d-glucuronide and N-acetylglycine was found to be a common signature of the metabolomic response to alcohol exposure in wild-type as well as in Ppara-null mice of both strains. However, increased excretion of indole-3-lactic acid and phenyllactic acid was found to be a conserved feature exclusively associated with the alcohol-treated Ppara-null mouse on both backgrounds that develop liver pathologies similar to the early stages of human ALD. These markers reflected the biochemical events associated with early stages of ALD pathogenesis. The results suggest that indole-3-lactic acid and phenyllactic acid are potential candidates for conserved and pathology-specific high-throughput noninvasive biomarkers for early stages of ALD.

Radiation metabolomics. 4. UPLC-ESI-QTOFMS-Based metabolomics for urinary biomarker discovery in gamma-irradiated rats

Radiation metabolomics has aided in the identification of a number of biomarkers in cells and mice by ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) and in rats by gas chromatography-coupled mass spectrometry (GCMS). These markers have been shown to be both dose- and time-dependent. Here UPLC-ESI-QTOFMS was used to analyze rat urine samples taken from 12 rats over 7 days; they were either sham-irradiated or γ-irradiated with 3 Gy after 4 days of metabolic cage acclimatization. Using multivariate data analysis, nine urinary biomarkers of γ radiation in rats were identified, including a novel mammalian metabolite, N-acetyltaurine. These upregulated urinary biomarkers were confirmed through tandem mass spectrometry and comparisons with authentic standards. They include thymidine, 2'-deoxyuridine, 2'deoxyxanthosine, N(1)-acetylspermidine, N-acetylglucosamine/galactosamine-6-sulfate, N-acetyltaurine, N-hexanoylglycine, taurine and, tentatively, isethionic acid. Of these metabolites, 2'-deoxyuridine and thymidine were previously identified in the rat by GCMS (observed as uridine and thymine) and in the mouse by UPLC-ESI-QTOFMS. 2'Deoxyxanthosine, taurine and N-hexanoylglycine were also seen in the mouse by UPLC-ESI-QTOFMS. These are now unequivocal cross-species biomarkers for ionizing radiation exposure. Downregulated biomarkers were shown to be related to food deprivation and starvation mechanisms. The UPLC-ESI-QTOFMS approach has aided in the advance for finding common biomarkers of ionizing radiation exposure.

A comprehensive understanding of thioTEPA metabolism in the mouse using UPLC-ESI-QTOFMS-based metabolomics

ThioTEPA, an alkylating agent with anti-tumor activity, has been used as an effective anticancer drug since the 1950s. However, a complete understanding of how its alkylating activity relates to clinical efficacy has not been achieved, the total urinary excretion of thioTEPA and its metabolites is not resolved, and the mechanism of formation of the potentially toxic metabolites S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA) remains unclear. In this study, the metabolism of thioTEPA in a mouse model was comprehensively investigated using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) based-metabolomics. The nine metabolites identified in mouse urine suggest that thioTEPA underwent ring-opening, N-dechloroethylation, and conjugation reactions in vivo. SCMC and TDGA, two downstream thioTEPA metabolites, were produced from thioTEPA from two novel metabolites 1,2,3-trichloroTEPA (VII) and dechloroethyltrichloroTEPA (VIII). SCMC and TDGA excretion were increased about 4-fold and 2-fold, respectively, in urine following the thioTEPA treatment. The main mouse metabolites of thioTEPA in vivo were TEPA (II), monochloroTEPA (III) and thioTEPA-mercapturate (IV). In addition, five thioTEPA metabolites were detected in serum and all shared similar disposition. Although thioTEPA has a unique chemical structure which is not maintained in the majority of its metabolites, metabolomic analysis of its biotransformation greatly contributed to the investigation of thioTEPA metabolism in vivo, and provides useful information to understand comprehensively the pharmacological activity and potential toxicity of thioTEPA in the clinic.

UPLC-ESI-TOFMS-based metabolomics and gene expression dynamics inspector self-organizing metabolomic maps as tools for understanding the cellular response to ionizing radiation

Global transcriptomic and proteomic profiling platforms have yielded important insights into the complex response to ionizing radiation (IR). Nonetheless, little is known about the ways in which small cellular metabolite concentrations change in response to IR. Here, a metabolomics approach using ultraperformance liquid chromatography coupled with electrospray time-of-flight mass spectrometry was used to profile, over time, the hydrophilic metabolome of TK6 cells exposed to IR doses ranging from 0.5 to 8.0 Gy. Multivariate data analysis of the positive ions revealed dose- and time-dependent clustering of the irradiated cells and identified certain constituents of the water-soluble metabolome as being significantly depleted as early as 1 h after IR. Tandem mass spectrometry was used to confirm metabolite identity. Many of the depleted metabolites are associated with oxidative stress and DNA repair pathways. Included are reduced glutathione, adenosine monophosphate, nicotinamide adenine dinucleotide, and spermine. Similar measurements were performed with a transformed fibroblast cell line, BJ, and it was found that a subset of the identified TK6 metabolites were effective in IR dose discrimination. The GEDI (Gene Expression Dynamics Inspector) algorithm, which is based on self-organizing maps, was used to visualize dynamic global changes in the TK6 metabolome that resulted from IR. It revealed dose-dependent clustering of ions sharing the same trends in concentration change across radiation doses. "Radiation metabolomics," the application of metabolomic analysis to the field of radiobiology, promises to increase our understanding of cellular responses to stressors such as radiation.

Evaluation of an alternative spectroscopic approach for aflatoxin analysis: Comparative analysis of food and feed samples with UPLC-MS/MS

Increasing research has highlighted the effects of changing climates on the occurrence and prevalence of toxigenic Aspergillus species producing aflatoxins. There is concern of the toxicological effects to human health and animal productivity following acute and chronic exposure that may affect the future ability to provide safe and sufficient food globally. Considerable research has focused on the detection of these toxins, based on the physicochemical and biochemical properties of the aflatoxin compounds, in agricultural products for human and animal consumption. As improvements in food security continue more regulations for acceptable levels of aflatoxins have arisen globally; the most stringent in Europe. These regulations are important for developing countries as aflatoxin occurrence is high significantly effecting international trade and the economy. In developed countries analytical approaches have become highly sophisticated, capable of attaining results with high precision and accuracy, suitable for regulatory laboratories. Regrettably, many countries that are affected by aflatoxin contamination do not have resources for high tech HPLC and MS instrumentation and require more affordable, yet robust equally accurate alternatives that may be used by producers, processors and traders in emerging economies. It is especially important that those companies wishing to exploit the opportunities offered by lucrative but highly regulated markets in the developed world, have access to analytical methods that will ensure that their exports meet their customers quality and safety requirements.

This work evaluates the ToxiMet system as an alternative approach to UPLC–MS/MS for the detection and determination of aflatoxins relative to current European regulatory standards. Four commodities: rice grain, maize cracked and flour, peanut paste and dried distillers grains were analysed for natural aflatoxin contamination. For B1 and total aflatoxins determination the qualitative correlation, above or below the regulatory limit, was good for all commodities with the exception of the dried distillers grain samples for B1 for which no calibration existed. For B1 the quantitative R² correlations were 0.92, 0.92, 0.88 (<250 μg/kg) and 0.7 for rice, maize, peanuts and dried distillers grain samples respectively whereas for total aflatoxins the quantitative correlation was 0.92, 0.94, 0.88 and 0.91. The ToxiMet system could be used as an alternative for aflatoxin analysis for current legislation but some consideration should be given to aflatoxin M1 regulatory levels for these commodities considering the high levels detected in this study especially for maize and peanuts

Desenvolvimento e validação de metodologia analítica para a determinação de fármacos em amostyras de água, superficial e tratada, utilizando a cromatografia líquida de ultra performance acoplada a espectrometria de massas tandem (UPLC-MS/MS)

Dissertação (Mestrado)

Seasonal and Species Variation of the Hepatotoxin Indospicine in Australian Indigofera Legumes as Measured by UPLC−MS/MS

Livestock industries have maintained a keen interest in pasture legumes because of the high protein content and nutritive value. Leguminous Indigofera plant species have been considered as having high feeding values to be utilized as pasture, but the occurrence of the toxic constituent indospicine in some species has restricted this utility. Indospicine has caused both primary and secondary hepatotoxicosis and also reproductive losses, but has only previously been determined in a small number of Indigofera species. This paper validates a high throughput ultra-performance liquid chromatography−tandem mass spectrometry (UPLC−MS/MS) method to determine indospicine content of various Indigofera species found in Australian pasture. Twelve species of Indigofera together with Indigastrum parviflorum plants were collected and analysed. Out of the 84 samples analyzed, *I. spicata contained the highest indospicine level (1003 ± 328 mg/kg DM, n = 4) followed by I. linnaei (755 ± 490 mg/kg DM, n = 51). Indospicine was not detected in 9 of the remaining 11 species, and at only low levels (<10 mg/kg DM) in 2 out of 8 I. colutea specimens and in 1 out of 5 I. linifolia specimens. Indospicine concentrations were below quantitation levels for other Indigofera spp. (I. adesmiifolia, I. georgei, I. hirsuta, I. leucotricha,* I. oblongifolia, I. australis and I. trita) and Indigastrum parviflorum. One of the more significant findings to emerge from this study is that the indospicine content of I. linnaei is highly variable (159 to 2128 mg/kg DM, n = 51), and differs across both regions and seasons. Its first re-growth after spring rain has a higher (p < 0.01) indospicine content than growth following more substantial summer rain. The species collected include the predominant Indigofera in Australia pasture, and of these, only *I. spicata and I. linnaei contain high enough levels of indospicine to pose a potential toxic threat to grazing herbivores.

Método multiresíduo para determinação de pesticidas em amostras de alface por QuEChERS e UPLC-ESI-MS/MS.

2009

Método multirresíduo de pesticidas em camarão por QuEChERS e UPLC-ESI-MS/MS.

2009

Multiresidue determination of pesticides in surface water by SPE-HPLC-ESI and SPE-UPLC-ESI tandem mass spectrometry (MS/MS- triple quadrupole analyser).

2009

Molecularly Imprinted Solid Phase Extraction Of Fluconazole From Pharmaceutical Formulations.

This work encompasses a direct and coherent strategy to synthesise a molecularly imprinted polymer (MIP) capable of extracting fluconazole from its sample. The MIP was successfully prepared from methacrylic acid (functional monomer), ethyleneglycoldimethacrylate (crosslinker) and acetonitrile (porogenic solvent) in the presence of fluconazole as the template molecule through a non-covalent approach. The non-imprinted polymer (NIP) was prepared following the same synthetic scheme, but in the absence of the template. The data obtained from scanning electronic microscopy, infrared spectroscopy, thermogravimetric and nitrogen Brunauer-Emmett-Teller plot helped to elucidate the structural as well as the morphological characteristics of the MIP and NIP. The application of MIP as a sorbent was demonstrated by packing it in solid phase extraction cartridges to extract fluconazole from commercial capsule samples through an offline analytical procedure. The quantification of fluconazole was accomplished through UPLC-MS, which resulted in LOD≤1.63×10(-10) mM. Furthermore, a high percentage recovery of 91±10% (n=9) was obtained. The ability of the MIP for selective recognition of fluconazole was evaluated by comparison with the structural analogues, miconazole, tioconazole and secnidazole, resulting in percentage recoveries of 51, 35 and 32%, respectively.

Identification of new ozonation disinfection byproducts of 17 beta-estradiol and estrone in water

Estrogens are a class of micro-pollutants found in water at low concentrations (in the ng L(-1) range), but often sufficient to exert estrogenic effects due to their high estrogenic potency. Disinfection of waters containing estrogens through oxidative processes has been shown to lead to the formation of disinfection byproducts, which may also be estrogenic. The present work investigates the formation of disinfection byproducts of 17 beta-estradiol (E2) and estrone (E1) in the treatment of water with ozone. Experiments have been carried out at two different concentrations of the estrogens in ground water (100 ng L(-1) and 100 mu g L(-1)) and at varying ozone dosages (0-30 mg L(-1)). Detection of the estrogens and their disinfection byproducts in the water samples has been performed by means of ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with a triple quadrupole (QqQ) and a quadrupole-time of flight (QqTOF) instrument. Both E2 and El have been found to form two main byproducts, with molecular mass (MM) 288 and 278 in the case of E2, and 286 and 276 in the case of El, following presumably the same reaction pathways. The E2 byproduct with MM 288 has been identified as 10epsilon-17beta-dihydroxy-1,4-estradieno-3-one (DEO), in agreement with previously published results. The molecular structures and the formation pathways of the other three newly identified byproducts have been suggested. These byproducts have been found to be formed at both high and low concentrations of the estrogens and to be persistent even after application of high ozone dosages. (C) 2011 Elsevier Ltd. All rights reserved.