Assigning precursor–product ion relationships in indiscriminant MS/MS data from non-targeted metabolite profiling studies

Tandem mass spectrometry using precursor ion selection (MS/MS) is an invaluable tool for structural elucidation of small molecules. In non-targeted metabolite profiling studies, instrument duty cycle limitations and experimental costs have driven efforts towards alternate approaches. Recently, researchers have begun to explore methods for collecting indiscriminant MS/MS (idMS/MS) data in which the fragmentation process does not involve precursor ion isolation. While this approach has many advantages, importantly speed, sensitivity and coverage, confident assignment of precursor–product ion relationships is challenging, which has inhibited broad adoption of the technique. Here, we present an approach that uses open source software to improve the assignment of precursor–product relationships in idMS/MS data by appending a dataset-wide correlational analysis to existing tools. The utility of the approach was demonstrated using a dataset of standard compounds spiked into a malt-barley background, as well as unspiked human serum. The workflow was able to recreate idMS/MS spectra which are highly similar to standard MS/MS spectra of authentic standards, even in the presence of a complex matrix background. The application of this approach has the potential to generate high quality idMS/MS spectra for each detectable molecular feature, which will streamline the identification process for non-targeted metabolite profiling studies.     

Reference intervals of urinary steroid metabolites using gas chromatography-mass spectrometry in Chinese adults

BACKGROUND: Urinary steroid profiling by GC or GC-MS are established clinical tools to complement other biochemical tests in the diagnosis and investigation of a wide range of adrenocortical disorders, but normative data on adults using the more specific GC-MS are lacking. Our objective was to set up the reference intervals of commonly detected urinary steroid metabolites as well as marker metabolites seen in disease states. METHOD: Apparently healthy adult Chinese males and females were recruited by completing health questionnaires. A 24-h urine specimen was collected from all the participants for urinary steroid profiling by GC-MS in cyclic scan mode. The analyzer was calibrated by using authentic steroid standards. Statistical methods recommended by the National Committee for Clinical Laboratory Standards were followed for setting up the reference intervals of various steroid metabolites. After outliers were excluded, the data were tested for the necessity to partition into sex-, menopausal status- and age-specific reference intervals. RESULTS: 83 males and 89 females were recruited for the study. Necessity to partition into sex-specific reference intervals was demonstrated for almost all steroid metabolites. Menopausal status and age also had a significant impact on steroid metabolite excretion, making separate reference intervals necessary. CONCLUSIONS: We have set up the normative data on the levels of urinary steroid metabolite excretion in Chinese adults for future reference in patient management and research in steroid metabolism.     

Identification of novel lignans in the whole grain rye bran by non-targeted LC–MS metabolite profiling

Rye (Secale cereale) is among the richest dietary sources of lignan phytochemicals. Lignans are one of the suggested metabolite groups to contribute to the beneficial health effects of whole grain products evidenced in epidemiological studies. So far, the complete repertoire of lignan derivatives in rye, especially in the bran, has not been fully described. In this study, ten novel oligomeric sesqui- and dilignans were identified in rye bran by the use of high resolution LC?CMS analysis (i.e., UPLC-qTOF-MS/MS). Putative identification of lignan components in the bran was performed by combining: (i) detailed inspection of the fragmentation behavior of available standard compounds belonging to different lignan types, (ii) interpretation of MS/MS data obtained from unknown metabolites in the samples. This combined analysis, particularly detailed MS/MS characterization, is most valuable for non-targeted assays in metabolite-rich matrices such as plant extracts, in which the verification of identity with authentic standards for each detected metabolite is normally not possible. Metabolomics analysis will increasingly aid in deciphering the active compounds in dietary products as part of studies aiming at elucidating the link between human health and nutrition.     

Discovery, biosynthesis, and structure elucidation of new metabolites of norandrostenedione using in vitro systems

The aim of our study was to demonstrate the positive impact that in vitro systems could have on the synthesis and characterization of unknown metabolites of banned doping agents. Using norandrostenedione (estr-4-en-3,17-dione), we were able to identify and characterize by GC/MS and LC/UV/MS several new hydroxylated metabolites formed in human hepatocyte incubations. The site of hydroxylation of M1, M2, M3, and M5 was demonstrated to be at C-6beta position by incubating estr-4-en-6beta-ol-3,17-dione (M4), which is the direct 6beta-hydroxylated metabolite of norandrostenedione. The structure of M5 was confirmed to be estr-4-en-6beta,17beta-diol-3-one (6beta-hydroxynortestosterone) using a commercially available authentic standard. For the other metabolites, M1, M2, and M3, no standards were available. Due to limited access to fresh human liver tissues, in vitro incubation conditions in rat liver subcellular fractions and hepatocytes were optimized as an alternative to produce sufficient quantities of the unknown metabolites for MS and/or NMR characterization. The structure of M1 was assigned to 5alpha-estran-3alpha,6beta-diol-17-one (6beta-hydroxynorandrosterone) and M3 to 5alpha-estran-3beta,6beta-diol-17-one (6beta-hydroxynorepiandrosterone) based on NMR data. M2 is proposed to be 5beta-estran-3alpha,6beta-diol-17-one (6beta-hydroxynoretiocholanolone) based on GC/MS fragmentation of the TMS-enol bis-TMS-ether derivative. The in vitro approach reported here, in addition to urinary excretion studies in humans, could contribute significantly to the discovery, the synthesis, and structure elucidation of new markers of doping agents.     

Microdetermination of the 6,15-diketo-13,14-dihydroprostaglandin F1alpha in human plasma using gas chromatography/selected ion monitoring with [18O]6,15-diketo-13,14-dihydro-prostaglandin F1alpha as an internal standard

We devised a simple and effective purification method for the microdetermination of 6,15-diketo-13,14-dihydro-prostaglandin F1alpha (DK), a metabolite of prostacyclin (PGI2). [18O]DK was synthesized from the repeated base-catalyzed hydrolysis of methyl ester derivatives in [18O] water to obtain an internal standard for the gas chromatography/selected ion monitoring (GC/SIM) of DK. The methyl ester-methoxime-tert-butyldimethylsilyl ether derivative was prepared, then gas chromatography/selected ion monitoring was carried out by monitoring the ion at m/z 613.4 for DK and at m/z 617.4 for an internal standard. A good linear response over the range of 10 pg to 10 ng was demonstrated. We detected DK to a level of about 297.8 pg/ml in human plasma. This method can be used to determine DK in biological samples.     

Detection and quantitation of 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one, the major urinary metabolite of methenolone acetate (Primobolan) by isotope dilution--mass spectrometry

A highly accurate method has been developed for detection and quantitation of 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one, the major urinary metabolite of methenolone acetate (Primobolan) in man. Unlabelled as well as 2H-labelled 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one were synthesized from 1-methylen-5 alpha-androstane-3,17-dione. A fixed amount of the internal standard was added to a fixed amount of urine and the mixture was treated with Helix pomatia for 24 h. After extraction and purification by t.l.c., the mixture was converted into methoxime--trimethylsilyl derivative and analyzed by combined GC--MS. Unlabelled 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one could be quantitated from the ratio between the tracings of the ions at m/z 372 and m/z 375 (corresponding to the M-31 ions). In alternative procedures, the ions at m/z 403 and m/z 406 (molecular ions) as well as m/z 282 and m/z 285 (M-90-31 ions) could be used. Under the conditions employed, the metabolite could be identified and quantitated in concentrations exceeding 10 ng/ml. Significant amounts of the metabolite could be detected in urine during 5 days after a single oral ingestion of 10 mg of Primobolan. The method has been successfully used for analyses of urine samples obtained from athletes involved in competition.     

The identification and quantification of three new 6alpha-hydroxylated corticosteroids in human neonatal urine

The 24 hours urines of six two days old fullterm newborn infants were investigated for polar corticosteroids. 6alpha-hydroxy-tetrahydrocortisone, 6alpha-hydroxy-20alpha-cortolone and 6alpha-hydroxy-20beta-cortolone were identified by gas chromatographic-mass spectrometric comparison of the urinary steroids to compounds synthesized previously. These 6alpha-hydroxylated corticosteroids as well as seven other polar corticosteroids were quantified by gas chromatography or mass fragmentography. It was shown that the newly identified steroids constituted a quantitatively important part of the neonatal urinary corticosteroids. The unconjugated- and glucuronic acid conjugated steroids were quantified separately. It was found that the extent of glucuronoconjugation decreased with increasing polarity of the steroid moiety.     

Direct determination of anabolic steroid conjugates in human urine by combined high-performance liquid chromatography and tandem mass spectrometry

A novel screening procedure for the sulfate and glucuronide conjugates of testosterone (T) and epitestosterone (E) in human urine was developed based on liquid-solid extraction and microbore high-performance liquid chromatography combined on-line with ion-spray tandem mass spectrometry. Confirmation of the sulfate and glucuronide conjugates of testosterone and epitestosterone isolated frrm normal human urine was acheived by selected reaction monitoring of characteristic product ions of the parent compounds. Endogenous levels of the steroid conjugates are detected in normal male urine and an increase is observed when the sample is fortified with authentic analytical standards of the conjugates. Calibration curves of all steroid conjugates in urine are linear over a range of twenty. Deuterated internal standards of testosterone glucuronide and epitestosterone sulfate were used for quantitation of the endogenous conjugates. T/E ratios were determined based on the glucuronide fractions of six replicates from a normal male and were shown to be statistically reproducible and below the accepted T/E threshold of 6:1. Sulfate conjugates were shown to be present at significantly lower levels in the urine. The method has potential as an alternative for monitoring anabolic steroid conjugates in human urin.     

Characterization of linoleic acid nitration in human blood plasma by mass spectrometry

Nitric oxide (*NO) is a pervasive free radical species that concentrates in lipophilic compartments to serve as a potent inhibitor of lipid and low-density lipoprotein oxidation processes. In this study, we synthesized, characterized, and detected nitrated derivatives of linoleic acid (18:2) in human blood plasma using high-pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry. While the reaction of nitronium tetrafluoroborate with 18:2 presented ions with a mass/charge (m/z) ratio of 324 in the negative ion mode, characteristic of nitrolinoleate (LNO(2)), the reaction of nitrite (NO(2)(-)) with linoleic acid hydroperoxide yielded nitrohydroxylinoleate (LNO(2)OH, m/z 340). Further analysis by MS/MS gave a major fragment at m/z 46, characteristic of a nitro group (-NO(2)) present in the parent ion. This was confirmed by using [(15)N]O(2), which gave products of m/z 325 and 341, that after fragmentation yielded a daughter ion at m/z 47. Moreover, a C-NO(2) structure was also demonstrated in LNO(2)OH by nuclear magnetic resonance spectroscopy ((15)N NMR, delta 375.9), as well as by infrared analysis in both LNO(2)OH (nu(max) = 3427, 1553, and 1374 cm(-1)) and LNO(2) (nu(max) = 1552 and 1373 cm(-1)). Stable products with m/z of 324 and 340, which possessed the same chromatographic characteristics and fragmentation pattern as synthesized standards, were found in human plasma of normolipidemic and hyperlipidemic donors. The presence of these novel nitrogen-containing oxidized lipid adducts in human plasma could represent "footprints" of the antioxidant action of *NO on lipid oxidation and/or a pro-oxidant and nitrating action of *NO-derived species.     

Metabolism of arachidonic acid by human nasal and bronchial epithelial cells

Nasal and bronchial epithelium from normal human nasal turbinates was isolated from surgical specimens and used to study arachidonic acid metabolism. High-performance liquid chromatography analysis of cell incubations in the presence of calcium ionophore, A23187, showed the formation of 15-lipoxygenase products. The major arachidonic acid metabolite with bronchial and nasal tissue was 15-HETE identified by uv spectroscopy, coelution with the authentic standards by HPLC, and GC-mass spectrometry. The second major metabolite, formed from either arachidonic acid or 15-HPETE, was identified as 13-hydroxy-14,15-epoxy-5,8,11-eicosatetraenoic acid (15-alpha-HEPA) by uv spectroscopy, coelution with the authentic standard, and GC-mass spectrometry. In addition, two 8,15-diHETEs and two 8,15-LTs were identified by uv spectroscopy and coelution with the authentic standards by HPLC on both reverse-phase and normal-phase HPLC. Also isolated and identified were 14,15-diHETEs, and 12-HETE. Nasal epithelial cells appear to be more active than nasal bronchial cells in oxidizing arachidonic acid. However, the profile of metabolites from these normal tissue preparations was similar. The addition of 15-lipoxygenase products to nasal epithelium weakly stimulated Cl- ion secretion. These studies indicate that human pulmonary epithelial cells selectively oxidize arachidonic acid to 15-lipoxygenase metabolites.     

Determination of Cloretazine (VNP40101M) and its active metabolite (VNP4090CE) in human plasma by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS)

A sensitive method for the determination of Cloretazine (VNP40101M) and its metabolite (VNP4090CE) with an internal standard (ISTD) in human plasma was developed using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Acidified plasma samples (500 microL) were prepared using solid phase extraction (SPE) columns, and 25 microL of the reconstituted sample was injected onto an Ascentis C18 HPLC column (3 microm, 5 cmx2.1 mm) with an isocratic mobile phase. Analytes were detected with an API-3000 LC-MS/MS System at unit (Q1) and low (Q3) resolution in negative multiple reaction monitoring mode: m/z 249.0 (precursor ion) to m/z 114.9 (product ion) for both Cloretazine (at 3.64 min) and VNP4090CE (at 2.91 min), and m/z 253.0 (precursor ion) to m/z 116.9 (product ion) for the ISTD. The mean recovery for Cloretazine (VNP40101M) and its metabolite (VNP4090CE) was greater than 87% with a lower limit of quantification of 1.0 ng/mL for Cloretazine (S/N=9.7, CV相似文献   

Rapid determination of chloramphenicol and its glucuronide in food products by liquid chromatography-electrospray negative ionization tandem mass spectrometry

Chloramphenicol (CAP) is subjected to monitoring in food products, with a minimum required performance level set at 0.3 ng/g. CAP was isolated from chicken meat and seafood by very simple solvent extraction procedure. For honey, a fast SPE procedure was applied. CAP-D5 was used as internal standard. HPLC separation was done on RP18 123 mm x 3 mm column in acetonitrile-ammonium formate 10 mM, pH 3.0 (40:60) at flow rate of 0.3 ml/min. A TSQ Quantum instrument with ESI source has been used in negative ionization mode. A MRM procedure has been applied and following transitions were monitored: m/z 321 > 152 (quantifier), 321 > 194, 321 > 257(qualifiers), 326 > 157 (IS). CAP peak was eluted at around 5 min; the total run time was 7 min. LOD was around 0.1 ng/g meat or 0.05 ng/g honey. Matrix effects were studied for all materials used, involving injection of blank extracts with post-column infusion of CAP, as well as checking the influence of the co-injected blank extracts on the signal intensity of CAP. No influence of matrix on the results of CAP determination were observed. The method allows analyzing up to 30 duplicate samples per day, including all calibration standards. Additionally, the method for determination of CAP glucuronide (CAP-G) was established, using urine from rats that were given this drug as a source of the metabolite. Full validation of the metabolite was not possible, due to the unavailability of reference standard.     

Metabonomic investigation of liver profiles of nonpolar metabolites obtained from alcohol-dosed rats and mice using high mass accuracy MSn analysis

Alcoholism is a complex disorder that, in man, appears to be genetically influenced, although the underlying genes and molecular pathways are not completely known. Here, the intragastric alcohol feeding model in rodents was used together with high mass accuracy LC-MS(n) analysis to assess the metabonomic changes in nonpolar metabolite profiles for livers from control and alcohol-treated rats and mice. Ion signals with a peak area variance of less than 30% (based on repeat analysis of a pooled quality control sample analyzed throughout the batch) were submitted to multivariate statistical analysis (using principal components analysis, PCA). PCA revealed robust differences between profiles from control and alcohol-treated animals from both species. The major metabolites seen to differ between control and alcohol-treated animals were identified using high accuracy MS(n) data and verified using external search engines ( http://www.lipidmaps.org ; http://www.hmdb.ca; http://www.genome.jp/kegg/ ) and authentic standards. The main metabolite classes to show major changes in the alcoholic liver-derived samples were fatty acyls, fatty acid ethyl esters, glycerolipids, and phosphatidylethanol homologues. Significant metabolites that were up-regulated by alcohol treatment in both rat and mouse livers included fatty acyls, metabolites such as octadecatrienoic acid and eicosapentaenoic acid, a number of fatty acid ethyl esters such as ethyl arachidonate, ethyl docosahexaenoic acid, ethyl linoleate, and ethyl oleate and phosphatidylethanol (PEth) homologues (predominantly PEth 18:0/18:2 and PEth 16:0/18:2; PEth homologues are currently considered as potential biomarkers for harmful and prolonged alcohol consumption in man). A number of glycerophospholipids resulted in both up-regulation (m/z 903.7436 [M + H](+) corresponding to a triglyceride) and down-regulation (m/z 667.5296 [M + H](+) corresponding to a diglyceride). Metabolite profiles were broadly similar in both mouse and rat models. However, there were a number of significant differences in the alcohol-treated group particularly in the marked down-regulation of retinol and free cholesterol in the mouse compared to the rat. Unique markers for alcohol treatment included ethyl docosahexaenoic acid. Metabolites were identified with high confidence using predominantly negative ion MS(n) data for the fatty acyl components to match to www.lipidmaps.org MS and MS/MS databases; interpreting positive ion data needed to take into account possible adduct ions which may confound the identification of other lipid classes. The observed changes in lipid profiles were consistent with alcohol-induced liver injury in humans.     

The comparative metabonomics of age-related changes in the urinary composition of male Wistar-derived and Zucker (fa/fa) obese rats

The global metabolite profiles of endogenous compounds excreted in urine by male Wistar-derived and Zucker (fa/fa) obese rats were investigated from 4 to 20 weeks of age using both 1H NMR spectroscopy and HPLC-TOF/MS with electrospray ionisation (ESI). Multivariate data analysis was then performed on the resulting data which showed that the composition of the samples changed with age, enabling age-related metabolic trajectories to be constructed. At 4 weeks it was possible to observe differences between the urinary metabolite profiles from the two strains, with the difference becoming more pronounced over time resulting in a marked divergence in their metabolic trajectories at 8-10 weeks. The changes in metabolite profiles detected using 1H NMR spectroscopy included increased protein and glucose combined with reduced taurine concentrations in the urine of the Zucker animals compared to the Wistar-derived strain. In the case of HPLC-MS a number of ions were found to be present at increased levels in the urine of 20 week old Zucker rats compared to Wistar-derived rats including m/z 71.0204, 111.0054, 115.0019, 133.0167 and 149.0454 (negative ion ESI) and m/z 97.0764 and 162.1147 (positive ion ESI). Conversely, ions m/z 101.026 and 173.085 (negative ion ESI) and m/z 187.144 and 215.103 (positive ion ESI) were present in decreased amounts in urine from Zucker compared to Wistar-derived rats. Metabolite identities proposed for these ions include fumarate, maleate, furoic acid, ribose, suberic acid, carnitine and pyrimidine nucleoside. The utility of applying metabonomics to understanding disease processes and the biological relevance of some of the findings are discussed.     

Identification of a novel selenium metabolite, Se-methyl-N-acetylselenohexosamine, in rat urine by high-performance liquid chromatography--inductively coupled plasma mass spectrometry and--electrospray ionization tandem mass spectrometry

The major urinary metabolite of selenium (Se) in rats was identified by HPLC-inductively coupled argon plasma mass spectrometry (ICP-MS) and--electrospray tandem mass spectrometry (ESI-MS/MS). As the urine sample was rich in matrices such as sodium chloride and urea, it was partially purified to meet the requirements for ESI-MS. The group of signals corresponding to the Se isotope ratio was detected in both the positive and negative ion modes at m/z 300 ([M+H]+) and 358 ([M+CH3COO]-) for 80Se, respectively. These results suggested that the molecular mass of the Se metabolite was 299 Da for 80Se. The Se metabolite was deduced to contain one methylselenyl group, one acetyl group and at least two hydroxyl groups from the mass spectra of the fragment ions. The spectrum of the Se metabolite was completely identical to that of the synthetic selenosugar, 2-acetamide-1,2-dideoxy-beta-D-glucopyranosyl methylselenide. However, the chromatographic behavior of the Se metabolite was slightly different from that of the synthetic selenosugar. Thus, the major urinary Se metabolite was assigned as a diastereomer of a selenosugar, Se-methyl-N-acetyl-selenohexosamine.     

Improved assays of alpha-lactalbumin and galactosyltransferase

The development and evaluation of a method for the determination of galactosyltransferase and alpha-lactalbumin activities using the addition of Dowex resin to the sample to separate substrate from products are described. For both assays galactosyltransferase activity was optimized by the addition of detergent, and relevant control incubations were included. The assay conditions were optimized for epididymal tissue and standards, and the assays were validated for accuracy and specificity with authentic bovine proteins and lactating rat mammary gland homogenates. Galactosyltransferase and alpha-lactalbumin activities in tissues were dependent on the extraction procedure used. Epididymal and testicular homogenates reduced the slopes of internal standards of galactosyltransferase but only testicular homogenates depressed slopes of internal standards of alpha-lactalbumin, necessitating the use of internal standards in the validation of the assays.     

C20 steroids: The metabolism of 3-hydroxy-19-nor[21-14C]pregna-1,3,5(10)-trien-20-one in the rabbit

1. The metabolism of 3-hydroxy-19-norpregna-1,3,5(10)-trien-20-one, a possible product of the aromatization of progesterone or pregnenolone, has been studied. 2. After oral administration of this C(20) steroid as the 21-(14)C-labelled compound to two groups of rabbits, the excretion pattern of metabolites in the urine was examined. 3. At 14 days after administration, 3.3-6.5% of the radioactivity had appeared in the urine, 71-79% in the faeces and approx. 10% remained in the gut. 4. A metabolite, isolated from urine mainly as the unconjugated steroid, was identified as 19-norpregna-1,3,5(10)-triene-3,20alpha-diol and constituted 18.5-22% of the total urinary radioactivity. 5. A minor component of the urinary unconjugated steroids was identified as 19-norpregna-1,3,5(10)-triene-3,17alpha,20alpha-triol. 6. A further 2-7.5% of the total urinary radioactivity, isolated only from the urinary sulphate fraction, was tentatively identified as an 18-oxygenated derivative of the administered steroid.     

Degradation of matrix glycosaminoglycans by peroxynitrite/peroxynitrous acid: evidence for a hydroxyl-radical-like mechanism

The oxidant peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) is generated at sites of inflammation via reaction of O2.- with .NO. Previous studies have shown that these species can oxidize cellular targets, but few data are available on damage to extracellular matrix and its components, despite evidence for matrix modification in a number of pathologies. In the current study we show that reaction of ONOO-/ONOOH with glycosaminoglycans results in extensive polymer fragmentation. Bolus authentic ONOO-/ONOOH modifies hyaluronan, heparin, and chondroitin, dermatan, and heparan sulfates, in a concentration-dependent, but O2-independent, manner. The ONOO-/ONOOH generator 3-(4-morpholinyl)sydnoneimine produces similar time- and concentration-dependent damage. These reactions generate specific polymer fragments via cleavage at disaccharide intervals. Studies at different pH values, and in the presence of bicarbonate, are consistent with ONOOH, rather than the carbonate adduct, CO3.- or ONOO-, being the source of damage. EPR spin trapping experiments have provided evidence for the formation of carbon-centered radicals on glycosaminoglycans and related monosaccharides; the similarity of these spectra to those obtained with authentic HO. is consistent with fragmentation being induced by this oxidant. These data suggest that extracellular matrix fragmentation at sites of inflammation may be due, in part, to the formation and reactions of ONOOH.     

Identification of Putative Steroid Receptor Antagonists in Bottled Water: Combining Bioassays and High-Resolution Mass Spectrometry

Endocrine disrupting chemicals (EDCs) are man-made compounds interfering with hormone signaling and thereby adversely affecting human health. Recent reports provide evidence for the presence of EDCs in commercially available bottled water, including steroid receptor agonists and antagonists. However, since these findings are based on biological data the causative chemicals remain unidentified and, therefore, inaccessible for toxicological evaluation. Thus, the aim of this study is to assess the antiestrogenic and antiandrogenic activity of bottled water and to identify the causative steroid receptor antagonists. We evaluated the antiestrogenic and antiandrogenic activity of 18 bottled water products in reporter gene assays for human estrogen receptor alpha and androgen receptor. Using nontarget high-resolution mass spectrometry (LTQ-Orbitrap Velos), we acquired corresponding analytical data. We combined the biological and chemical information to determine the exact mass of the tentative steroid receptor antagonist. Further MSⁿ experiments elucidated the molecule’s structure and enabled its identification. We detected significant antiestrogenicity in 13 of 18 products. 16 samples were antiandrogenic inhibiting the androgen receptor by up to 90%. Nontarget chemical analysis revealed that out of 24520 candidates present in bottled water one was consistently correlated with the antagonistic activity. By combining experimental and in silico MSⁿ data we identified this compound as di(2-ethylhexyl) fumarate (DEHF). We confirmed the identity and biological activity of DEHF and additional isomers of dioctyl fumarate and maleate using authentic standards. Since DEHF is antiestrogenic but not antiandrogenic we conclude that additional, yet unidentified EDCs must contribute to the antagonistic effect of bottled water. Applying a novel approach to combine biological and chemical analysis this is the first study to identify so far unknown EDCs in bottled water. Notably, dioctyl fumarates and maleates have been overlooked by science and regulation to date. This illustrates the need to identify novel toxicologically relevant compounds to establish a more holistic picture of the human exposome.     

Gas chromatography-mass spectrometry and the measurement of vitamin D metabolites in human serum or plasma

Although methods for the measurement of vitamin D metabolites continue to be developed, few have been properly validated by comparison with methods based on gas chromatography-mass spectrometry, widely accepted as being the definitive methodology. To the best of our knowledge, only three such comparisons have been carried out (14, 42, 83), all three examining HPLC assays for 25-OH-D. This lack of proper validation leads to lack of certainty as to the specificity of many assays widely used for clinical investigation. In our view there is an obvious need for the continuing development of mass fragmentographic assays for vitamin D and its metabolites, primarily for use as reference procedures for the evaluation of less rigorous methodologies. Provided standards, both labeled and unlabeled, become more widely available, development of specific mass fragmentographic assays for any metabolite of vitamin D should be possible. For metabolites where no specific binding protein or antiserum is available, mass fragmentography may be the only alternative.