Mass spectral characterization of doxylamine and its rhesus monkey urinary metabolites

This study describes the use of mass spectrometry (MS), high-performance liquid chromatography (HPLC) and chemical derivatization techniques for the identification of doxylamine and five rhesus monkey urinary metabolites. The analyses were performed using chemical ionization mass spectrometry with either methane or ammonia as the reagent gas. The confirmation of the structures of two of these urinary metabolites was aided by the synthesis of doxylamine N-oxide and desmethyldoxylamine and by the use of methylation and acetylation derivatization techniques. Doxylamine N-oxide, desmethyldoxylamine, didesmethyldoxylamine, and two metabolites which resulted from the cleavage of the aliphatic tertiary nitrogen side chain to the subsequent 2-[1-phenyl-1-(2-pyridinyl)ethoxy]acetic acid or 2-[1-phenyl-1-(2-pyridinyl)ethoxy]methanol compounds were isolated and identified from rhesus monkey urine. Additional data concerning the mass spectral analysis of derivatization or reaction products from the three chloroformate reactions with doxylamine, and the synthesis and separation techniques which afforded mass spectral identification of the urinary metabolites are also presented.     

Mass spectrometric identification of 13C-labeled metabolites during anaerobic propanoic acid oxidation

Biowaste digestion is a possibility to gain biogas as a renewable fuel source. However, the anaerobic food chain may be disrupted by, e.g., substrate overload or by inhibitors, leading to the accumulation of volatile fatty acids (VFAs), predominantly of propanoic acid (PA). VFA Accumulation may cause a rapid pH decrease, less biogas production, or even a total inhibition. To maintain high biogas productivity or to prevent a collapse of methanogenesis, metabolic properties of the degrading microorganisms must be elucidated, e.g., by investigation of the established pathways for degradation of VFAs. A Dani 3950 headspace system (HS), a Varian 431 gas chromatograph (GC), and a Varian 210 mass spectrometer (MS) have been combined to quantify and specifically identify metabolites of PA oxidation. The use of [1-(13)C]-labeled PA as a carbon source for microorganisms allows differentiation between the methyl-malonyl-CoA or the C(6)-dismutation pathway, both resulting in AcOH production. Appearance of the (13)C-moiety either in the COO or Me group of AcO can easily be detected by MS. The methyl-malonyl-CoA pathway was successfully identified as the only pathway of PA degradation by organisms in a lab-scale anaerobic digester. A similar approach can be applied to any degradation pathway involving VFAs.     

Clinical chemistry of serotonin and metabolites

Analyses of serotonin and other 5-hydroxyindoles, such as its precursor 5-hydroxytryptophan and major metabolite 5-hydroxyindoleacetic acid (5-HIAA), are indispensable for the elucidation of their (patho)physiological roles. In clinical chemistry attention is mainly focused on the diagnosis and follow-up of carcinoid tumours. For this most laboratories routinely measure urinary 5-HIAA. More recently, measurements of serotonin in platelets and urine have been advocated. Platelet serotonin may be the most sensitive indole marker for the detection of carcinoid tumours that secrete only small amounts of serotonin and/or its precursor 5-hydroxytryptophan. Although several chromatographic techniques have emerged for the analysis of tryptophan-related indoles, HPLC with either electrochemical or fluorometric detection have become the methods of choice for their quantification. HPLC-based methods combine selectivity, sensitivity and high precision, and enable the simultaneous investigation of several metabolically related indoles. This review aims to place the analysis of indoles in biological matrices in a biochemical, physiological and clinical perspective and highlights several important steps in their chromatographic analysis and quantification.     

Metabolism of 4-hydroxyandrostenedione and 4-hydroxytestosterone: Mass spectrometric identification of urinary metabolites

4-Hydroxyandrost-4-ene-3,17-dione is a second generation, irreversible aromatase inhibitor and commonly used as anti breast cancer medication for postmenopausal women. 4-Hydroxytestosterone is advertised as anabolic steroid and does not have any therapeutic indication. Both substances are prohibited in sports by the World Anti-Doping Agency, and, due to a considerable increase of structurally related steroids with anabolic effects offered via the internet, the metabolism of two representative candidates was investigated. Excretion studies were conducted with oral applications of 100mg of 4-hydroxyandrostenedione or 200mg of 4-hydroxytestosterone to healthy male volunteers. Urine samples were analyzed for metabolic products using conventional gas chromatography-mass spectrometry approaches, and the identification of urinary metabolites was based on reference substances, which were synthesized and structurally characterized by nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. Identified phase-I as well as phase-II metabolites were identical for both substances. Regarding phase-I metabolism 4-hydroxyandrostenedione (1) and its reduction products 3beta-hydroxy-5alpha-androstane-4,17-dione (2) and 3alpha-hydroxy-5beta-androstane-4,17-dione (3) were detected. Further reductive conversion led to all possible isomers of 3xi,4xi-dihydroxy-5xi-androstan-17-one (4, 6-11) except 3alpha,4alpha-dihydroxy-5beta-androstan-17-one (5). Out of the 17beta-hydroxylated analogs 4-hydroxytestosterone (18), 3beta,17beta-dihydroxy-5alpha-androstan-4-one (19), 3alpha,17beta-dihydroxy-5beta-androstan-4-one (20), 5alpha-androstane-3beta,4beta,17beta-triol (21), 5alpha-androstane-3alpha,4beta,17beta-triol (26) and 5alpha-androstane-3alpha,4alpha,17beta-triol (28) were identified in the post administration urine specimens. Furthermore 4-hydroxyandrosta-4,6-diene-3,17-dione (29) and 4-hydroxyandrosta-1,4-diene-3,17-dione (30) were determined as oxidation products. Conjugation was diverse and included glucuronidation and sulfatation.     

Mass spectral characterization of two onion constituents with prostaglandin E-like activity as lipoxygenase metabolites of linoleic acid

Onions ($\text{Allium cepa}$ have been used in folk medicine for the treatment of hypertension and gastrointestinal ulcers, which are cases where the administration of prostaglandins is considered. Using bioassay guided fractionation and gas chromatography/mass spectrometry (GC/MS) we have isolated two biologically active fractions with PGE-like properties and have characterized these products as lipoxygenase metabolites of linoleic acid.For the isolation of the active principles, onion bulbs were homogenized in phosphate buffer and the PGE-like products were isolated using Amberlite XAD-2 absorption, silicic acid column chromatography and silicagel thin layer chromatography. The PGE-like activity was estimated in a cascade superfusion system in which the isolated rabbit coeliac artery, the rabbit mesenteric artery and the rat fundus were used as assay organs.For the GC/MS characterization, various types of volatile derivatives were prepared in order to facilitate the structure elucidation. Derivatization included hydrogenation, methyl ester formation, n-butyl boronate formation and trimethylsilylation. The active fractions yielded identical electron-impact mass spectra, indicating that they are stereoisomeric, and each fraction was identified as a mixture of two positional isomers, i.e. of 9,12,13-trihydroxy-10-octadecenoic and of 9,10,13-trihydroxy-11-octadecenoic acid. With regard to the structure elucidation of the latter isomers, the mixed hydrogenated, n-butyl boronate, methyl ester, TMS-ether derivatives were shown to be of particular value for the determination of the vicinal diol function.The isomeric trihydroxylated octadecenoic acids have been described for the first time as metabolites of linoleic acid in wheat flour incubates. In this system, the trihydroxylated octadecenoic acids were shown to be formed through a sequence of three reactions, including an initial 9- or 13-lipoxygenation yielding hydroperoxy octadecadienoic acids, followed by rearrangement into unstable allylic epoxy hydroxy octadecenoic acids, which in turn are hydrolyzed to trihydroxy octadecenoic acids. Furthermore, structurally related trihydroxy octadecadienoic acids have also recently been isolated from a plant source, more specifically from the roots of $\text{Bryonia Alba}$, which are used for the same medicinal purposes as onion bulbs.     

Mass spectral characterization of 6-oxo-OGF

The mass spectra of eleven derivatives are presented to provide structural support for the recently discovered prostaglandin, 6-oxo-PGF1alpha, which we have isolated from incubations of arachidonic acid with ram seminal vesicles or released during isolated perfusions of sensitized guinea pig lungs.     

Biotransformation of 17-alkylsteroids in the equine: gas chromatographic-mass spectral identification of ten intermediate metabolites of methyltestosterone

The metabolism of the orally active anabolic steroid methyltestosterone in the equine was investigated by administration of the drug along with a tritiated traces. In this study some of the metabolites were identified and a radio immunoassay screen and immunoaffinity chromatography gel for methyltestosterone were also evaluated. Pathway intermediates, in particular the 17-methylandrostanediols, were studied to gain an insight into the most likely stereochemistry of the major metabolites. The predominant phase I biotransformations involve reduction of the A ring 3-oxo and 4-ene groups to yield predominantly 3β-hydroxy-5-androstane products and hydroxylation of the steroid nucleus at several positions. Epimerisation of the 17-methyl group also occurred. Ten steroids could be positively identified by comparison with authentic references materials and many other triol, tetrol and pentols were also observed. Phase II metabolites and sulphate conjugates in particular, were common.     

Mass spectrometric identification of urinary and plasma metabolites of 9-hydroxy-19,20-bis-nor-prostanoic acid (rosaprostol)

9-Hydroxy-19,20-bis-nor-prostanoic acid (Rosaprostol) is an antiulcer compound with antisecretory and cytoprotective action. We studied the metabolites of Rosaprostol found in human plasma and in human and rat urine. Sixteen different metabolites were tentatively identified on the basis of their mass spectra. Two presumed metabolites were synthesized. To clarify the identities of some of them, deuterated Rosaprostol was administered to rats and mass spectra of the deuterated and protonated metabolites were examined. Rosaprostol is metabolized following three metabolic pathways leading, combined, to oxidized compounds with a lower number of carbons than the parent drug.     

Mass spectral identification of the blocked N-terminal tryptic peptide of the ATPase inhibitor from beef heart mitochondria

The endogenous cellular oncogene products, pp60c-src, exhibits a protein kinase activity, but is itself a phosphoprotein. Based on the assumption that pp60c-src might play a role in the control of cell proliferation, we have studied its behaviour as a substrate for phosphorylation known to occur when quiescent, serum-deprived cells are stimulated to enter cell cycle following addition of either serum, platelet-derived growth factor or the phorbol ester derivative, 12-O-tetradecanoyl-phorbol-13-acetate. For this purpose a partial purification of pp60c-src on DEAE ion-exchange chromatography was combined with immune precipitation. A 2-4-fold increase in serine phosphorylation of pp60c-src was consistently observed after stimulation of quiescent cells to growth.     

Mass spectral fragmentation of 5alpha-hydroxysteroids.

The mass spectra of a number of C-4alpha- and C-4beta-alkylated cholestan-3beta, 5alpha-diols have been found to contain an intense ion at m/e 332. the corresponding 6beta-alkylated (R) cholestan-3beta, 5alpha-diols exhibit an abundant ion at m/e 331 + R. The mass spectra of cholestan-5alpha-ol and cholestan-3beta, 5alpha-diol also show an ion at m/e 332 which, however, is of relatively low abundance. The ion in question appears to arise from fragmentation processes which are characteristics of 5alpha-hydroxysteroids. A similar fragmentation has been found to occur in cases of C-4 and C-6beta-alkylated 5alpha-hysroxy-cholestan-3-ones. The results of isotopic labeling, high resolution and metastable ion defocusing studies are discussed in terms of the origin of several of the ions in the spectra of the various compounds.     

Mass spectral compatibility of four proteomics stains

With the recent introduction of new fluorescence stains to the proteomics market, there is now more choice available. SYPRO Ruby, LavaPurple, Flamingo, and Krypton total protein stains were compared for ease of use, image quality, and compatibility with protein identification by peptide mass fingerprinting (PMF) (MALDI-TOF). All four stains produced good images but with slightly different staining patterns. SYPRO was found to inhibit identification of cysteine and tryptophan containing peptides, which reduced protein identification.     

Mass spectral characterization of a protein-nucleic acid photocrosslink

A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130-138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155.     

Mass spectral analysis of murine epidermal growth factor

Fast atom bombardment mass spectrometry has been used to characterize epidermal growth factor isolated from mouse submaxillary glands. The preparation is found to consist of two peptides, one of which has the average molecular weight predicted for the familiar gene product. The molecular weight of the second component is found to be reduced by the mass of one asparagine residue. These observations are discussed in light of previous reports of heterogeneity.     

Mass spectral analysis of peptides containing nitrobenzyl moieties

Summary By means of MALDI-TOF analysis of peptides containing a nitrobenzyl moiety, we have observed that the predominant signal often corresponds to ions that have eliminated multiple oxygen atoms, [M–O_n+H]⁺, and not to the protonated molecular ion, [M+H]⁺. Matrix selection for handling these types of molecules appears to be important. The MALDI-TOF matrices, such as sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid) and -cyano-4-hydroxycinnamic acid, resulted in the appearance of significant levels of the photodeoxygenated species, whereas 4-hydroxyazobenzene-2-carboxylic acid appeared to moderately and in some cases completely suppress the photodeoxygenation reaction. This phenomenon was independent of laser intensity. Photodeoxygenation was not observed with electrospray or fast atom bombardment ionization techniques. Therefore, use of MALDI should be avoided with peptides containing a nitrobenzyl moiety and similar moieties that are prone to reactive photochemistry.