Electrospray[+] tandem quadrupole mass spectrometry in the elucidation of ergot alkaloids chromatographed by HPLC: screening of grass or forage samples for novel toxic compounds

Ergot alkaloids are mycotoxins generated by grass and grain pathogens such as Claviceps, for example. Ergot alkaloid-poisoning syndromes, such as tall fescue toxicosis from endophyte-infected tall fescue grass, are important veterinary problems for cattle, horses, sheep, pigs and chickens, with consequent impact on food, meat and dairy industries. Damage to livestock is of the order of a billion dollars a year in the United States alone. HPLC with UV and fluorescence detection are the predominant means of ergot alkaloid determination, with focus on quantitation of the marker compound ergovaline, although ELISA methods are undergoing investigation. These techniques are excellent for rapid detection, but of poor specificity in defining new or poorly characterized ergot alkaloids and related compounds. This paper demonstrates the facility of using electrospray(+) mass spectrometry with multiple reaction monitoring (MRM) detection during chromatographic examination of ergot alkaloid standards of lysergic acid, lysergol, ergonovine, ergovaline, ergotamine, ergocornine, ergocryptine and ergocrystine by HPLC. Ergoline-8 position epimers could be separated on the gradient HPLC system for ergocornine, ergocrystine and ergonovine and appeared as shoulders for ergotamine and ergovaline; epimers generally showed different patterns of relative intensity for specific MRM transitions. There was reasonable correspondence between retention of standards on the 2-mm ESI(+)MS phenyl-hexyl-based reverse phase column and those on the 4-mm C18-based column. Since up to 10% of clinical cases involving toxin exposure display unidentified chromatographic peaks, 11 samples of feed components associated with such cases were studied with developed MRM methods to attempt elucidation of crucial components if possible. Ergotamine appeared in all, ergovaline appeared in five and ergocornine appeared in six; ergonovine, ergocryptine, ergocrystine and lysergol also appeared in several. In addition, molecular weights of compounds newly revealed by mass spectrometry suggested ergosine, ergostine and ergoptine in four samples, for which standards were not available. Dehydrated products of ergotamine, ergocrystine and ergocornine were discovered, along with dihydrogenated ergocrystine and ergocryptine in seven of the samples, and the issue was raised as to whether dehydration was strictly an instrument-derived artifact. Finally, five of the samples, along with fescue seed standard, evidenced one or more of 14 new ergot alkaloids ranging in size from 381 to 611 molecular weight and with key mass spectral characteristics of ergot alkaloids, specifically the pair of peaks m/z 223 and 208, corresponding to the ergoline ring system and its demethylated variant, respectively. It is anticipated that findings such as these will provide impetus to future development of analytical methodology for these heretofore relatively rare ergot alkaloid species.     

Synthesis of 2/6-(polychloromethyl)pyridine-carboxylates

Due to their relationship to certain bio-active 2,6-(polyfluoromethyl)-3,5-pyridinedicarboxylates 12 , methodology was developed to prepare new, but analogous 2/6 (polychloromethyl)pyridinecarboxylates 6, 9, 10, 13, 18 and 20. Successful methods to prepare these materials include several chlorination procedures, mixed Hantzsch sequences, and aluminum chloride interchanges with 12 and 16.     

The Effect of Electron-Donating and Electron-Withdrawing Substituents on 1H-and 13C-NMR chemical shifts of novel 7′-aryl-substituted 7′-apo-β-carotenes

The synthesis of 7′-aryl-7′-apo-β-carotenes, where aryl (Ar) is Ph, 4-NO₂C₆H₄, 4-MeOC₆H₄, 4-(MeO₂C)C₆H₄, C₆F₅, and 2,4,6-Me₃C₆H₂, is described. NMR Chemical shifts of all H- and C-atoms are presented, together with specific examples of the spectra. In contrast to ¹H chemical shifts which, except for H? C(8′) and H? C(7′), did not differ greatly from those of β,β-carotene, considerable variations in ¹³C chemical shifts were observed. Signals of the C(α) atoms of the polyene chain [C(β)? C(α)] +_n Ar were shielded, those of the C(β) atoms were deshielded, with some exceptions when n = 1; the effects decreased with increasing n.     

A concise synthesis of the naphthalene portion of purpuromycin

A concise synthesis of naphthalene compounds for incorporation into a synthetic sequence for the rubromycin family of natural products is presented. These highly substituted naphthalenes are generated in seven and nine steps, respectively, from 2,4,5-trimethoxybenzaldehyde. Three ring-forming methods were explored and the controlled oxygenation of different positions was investigated to yield differentially substituted/protected systems. Key steps to the final products include a Stobbe condensation to form the ring system and a novel series of regioselective oxidations to introduce the required oxygen functionality. These naphthalene products incorporate orthogonal protecting groups and are suitable for combination with a variety of coupling partners.     

Connection digraphs and second-order line digraphs

The primary result of this paper gives a set of necessary and sufficient conditions for a digraph to be second-order line digraph of some digraph. A directed analogue of the concept of an intersection graph, defined for collections of ordered pairs of sets and called the connection digraph, is used to achieve this result.