Improvement of chemical analysis of antibiotics: XXIII. Identification of residual tetracyclines in bovine tissues by electrospray high-performance liquid chromatography–tandem mass spectrometry

To reliably identify the residual tetracycline antibiotics (TCs), oxytetracycline (OTC), tetracycline, chlortetracycline (CTC) and doxycycline (DC), in bovine tissues, we have established a confirmation method using electrospray ionization liquid chromatography–tandem mass spectrometry (ESI LC–MS–MS) with daughter ion scan. All TCs gave [M+H−NH₃]⁺ and [M+H−NH₃−H₂O]⁺ as the product ions, except for DC when [M+H]⁺ was selected as the precursor ion. The combination of C₁₈ cartridge clean-up and the present ESI LC–MS–MS method can reliably identify TCs fortified at a concentration of 0.1 ppm in bovine tissues, including liver, kidney and muscle, and has been successfully applied to the identification of residual OTC in bovine liver and residual CTC in bovine muscle samples previously found at concentrations of 0.58 ppm and 0.38 ppm by LC, respectively.     

A fragmentation study of isoflavones by IT-TOF-MS using biosynthesized isotopes

To aid in the identification and quantification of biologically and agriculturally significant natural products, tandem mass spectrometry can provide accurate structural information with high selectivity and sensitivity. In this study, diagnostic fragmentation patterns of isoflavonoids were examined by liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS). The fragmentation scheme for [M+H?2CO]⁺ ions derived from isoflavones and [M+H?B-ring?CO]⁺ ions derived from 5-hydroxyisoflavones, were investigated using different isotopically labeled isoflavones, specifically [1′,2′,3′,4′,5′,6′,2,3,4-¹³C₉] and [2′,3′,5′,6′,2-D₅] isoflavones. Specific isotopically labeled isoflavones were prepared through the biosynthetic incorporation of pharmacologically applied ¹³C- and D-labelled L-phenylalanine precursors in soybean plants following the application of insect elicitors. Using this approach, we empirically demonstrate that the [M+H?2CO]⁺ ion is generated by an intramolecular proton rearrangement during fragmentation. Furthermore, [M+H?B-ring?CO]⁺ ion is demonstrated to contain a C₂H moiety derived from C-ring of 5-hydroxyisoflavones. A mechanistic understanding of characteristic isoflavone fragmentation patterns contributes to the efficacy and confidence in identifying related isoflavones by LC-MSⁿ.     

Evaluation of automated electrospray-TOF mass spectrometryfor metabolic fingerprinting of the plant metabolome

Metabolic fingerprinting is increasingly employed in microbial and plant metabolomics. Identification and evaluation of analytical factors that influence mass spectra produced with automated electrospray time of flight mass spectrometry to support metabolic fingerprinting are described. Instrument resolution of 4000 (FWHM) at mass 200 Da provided detection of ions of the same nominal mass but different monoisotopic masses. Complex mass spectra were obtained from polar extracts of tomato fruit in positive and negative ion mode. These spectra consist of metabolite ions (molecular, adduct and fragment) and those derived from the extraction medium, largely in the form of [M+H]⁺, [M–H]^–, [M+Na]⁺, [M+K]⁺, [2M+H]⁺, [M+Cl]^– and [2M–H]^–. Ionisation suppression reduced sensitivity, although its effect was consistent for a wide range of metabolite concentrations. Variability in ion signal intensity was lower in analytical (2.2–30.1%) compared to biological (within fruit 9.6–27.6%; between-fruit 13.2–34.4%) replicates. The method is applicable to high throughput metabolic fingerprinting and, with accurate mass measurements, is able to provide reductions in data complexity and preliminary identification of metabolites.     

Deuterium effect on ionization and fragmentation patterns of monosaccharides ionized by atmospheric pressure chemical ionization

Glucose, galactose, and mannose in H₂O and D₂O were ionized by an atmospheric pressure chemical ionization (APCI) method. Isotope effects on fragmentation patterns of the monosaccharides were examined by deuterium replacement of the -OH groups to distinguish the isomers with a single mass spectrometer. The most abundant ions were the [M+H₂O]⁺ and [M_D5+D+D₂O]⁺ for using H₂O and D₂O as solvent and eluent, respectively. Major fragment ions were the [M−OH]⁺ and [M−OH−H₂O]⁺ in H₂O, while those in D₂O were the [M_D5+D−D₂O]⁺ and [M_D5+D−2D₂O]⁺. The differences in the product ions generated in H₂O and D₂O were due to enhancement of the strength of hydrogen bonding by the deuterium replacement. Variations of the ion intensity ratios of the [M−OH]⁺/[M−OH−H₂O]⁺ and [M_D5−OD]⁺/[M_D5−OD−D₂O]⁺ with the fragmentor voltage showed different trends depending on the kind of monosaccharides. By comparing the ion intensity ratios of the [M+H₂O]⁺/M⁺, [M_D5+D+D₂O]⁺/[M_D5+D]⁺, [M−OH]⁺/[M−OH−H₂O]⁺, and [M_D5+D−D₂O]⁺/[M_D5+D−2D₂O]⁺, it was possible to distinguish the isomers of monosaccharides.     

Improvement of chemical analysis of antibiotics: XXIII. Identification of residual tetracyclines in bovine tissues by electrospray high-performance liquid chromatography–tandem mass spectrometry

To reliably identify the residual tetracycline antibiotics (TCs), oxytetracycline (OTC), tetracycline, chlortetracycline (CTC) and doxycycline (DC), in bovine tissues, we have established a confirmation method using electrospray ionization liquid chromatography–tandem mass spectrometry (ESI LC–MS–MS) with daughter ion scan. All TCs gave [M+H−NH₃]⁺ and [M+H−NH₃−H₂O]⁺ as the product ions, except for DC when [M+H]⁺ was selected as the precursor ion. The combination of C₁₈ cartridge clean-up and the present ESI LC–MS–MS method can reliably identify TCs fortified at a concentration of 0.1 ppm in bovine tissues, including liver, kidney and muscle, and has been successfully applied to the identification of residual OTC in bovine liver and residual CTC in bovine muscle samples previously found at concentrations of 0.58 ppm and 0.38 ppm by LC, respectively.     

Structure identification of natural rhamnolipid mixtures by fast atom bombardment tandem mass spectrometry

Two rhamnobiose-lipid preparations have been studied by fast atom bombardment (FAB) tandem mass spectrometry. The principal rhanobiose-lipids contain the -hydroxydecanoyl--hydroxydecanoate Rha-Rha-C₁₀-C₁₀ and the -hydroxytetradecanoyl--hydroxytetradecanoate Rha-Rha-C₁₄-C₁₄. Both preparations contain minor components which are heterogenous in -hydroxy fatty acid composition. FAB ionization of rhamnobiose-lipids in the presence of Na⁺ shows the formation of both [M + Na]⁺, [M + 2Na - H]⁺, [M + 3Na - 2H]⁺ and [M - H]^– ions. Tandem mass spectrometry of the [M + 2Na - H]⁺ and [M - H]^– ions give information about the sequence of the building blocks. Particularly, heterogeneity in -hydroxy fatty acid composition is determined for the principal components and all the minor components present in the preparations.     

Negative and positive ion chemical ionization mass spectrometry of dioximes and their nickel complexes

The negative ion mass spectra of Ni(LH)₂ (where LH₂ is glyoxime, methylglyoxime, dimethylglyoxime and diphenylglyoxime), in the presence of ammonia or methane at 0.5 torr, are reported and compared with the spectra of the free ligands. In each case, the base peak of the complex is the molecular negative ion and the extent of fragmentation was found to decrease gradually going from the glyoximato to the diphenylglyoximato derivative. In the chemical ionization mass spectra of the free ligands, the [M]⁻ ion is absent in all cases. The base peak is [M  H]⁻ for methylglyoximine, dimethylglyoxime and diphenylglyoxime and [M  H  H₂]⁻ for glyoxime. The fragmentation occurs largely by loss of H, OH, H₂O and NO species. The positive ion chemical ionization mass spectra of the same complexes show very abundant [M + H]⁺ and [M]⁺ ions and weak fragments, whilst a rather high fragmentation is observed for the corresponding free ligands.     

FACTORS INFLUENCING THE EFFLUX OF [3H]GAMMAAMINOBUTYRIC ACID FROM SATELLITE GLIAL CELLS IN RAT SENSORY GANGLIA

The spontaneous efflux of [³H]GABA from the satellite glial cells of rat dorsal root ganglia and the efflux evoked by 64 mM-K⁺ were studied in the presence of 10^-5M-amino-oxyacetic acid and found not to be affected by 10^-4M-D 600 or by elevated (9.6mM) Ca²⁺ in the absence of Mg²⁺. [³H]GABA efflux was increased by replacing sodium ions in the washing medium by choline ions and 64 mM-K⁺ failed to increase the efflux further. The drugs veratridine (10^-6 and 10^-4M) and batrachotoxin (10^-8 and 10^-6 M) failed to alter the spontaneous efflux of [³H]GABA from the glial cells. A variety of compounds, including amino acids, a GABA analogue and a GABA antagonist were tested for their ability to affect [³H]GABA efflux. The results indicated that compounds which inhibit GABA uptake into glial cells were also able to stimulate [³H]GABA efflux from these cells. The results are discussed with reference to possible mechanisms involved in the release of GABA from glial cells.     

Analysis of sesterterpenoids from Aspergillus terreus using ESI‐QTOF and ESI‐IT

Introduction – Biosynthesis of terretonin was studied due to the interesting skeleton of this series of sesterterpenoids. Very recently, López‐Gresa reported two new sesterterpenoids (terretonins E and F) which are inhibitors of the mammalian mitochondrial respiratory chain. Mass spectrometry (MS), especially tandem mass spectrometry, has been one of the most important physicochemical methods for the identification of trace natural products due to it rapidity, sensitivity and low levels of sample consumption. The potential application prospect and unique skeleton prompted us to study structural characterisation using MS. Objective – To obtain sufficient information for rapid structural elucidation of this class of compounds using MS. Methodology – The elemental composition of the product ions was confirmed by low‐energy ESI‐CID‐QTOF‐MS/MS analyses. The fragmentation pathways were postulated on the basis of ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ spectra. Common features and major differences between ESI‐QTOF‐MS/MS and IT‐MSⁿ spectra were compared. For ESI‐QTOF‐MS/MS/MS experiments, capillary exit voltage was raised to induce in‐source dissociation. Ammonium acetate or acetic acid were added into solutions to improve the intensity of [M + H]⁺. The collision energy was optimised to achieve sufficient fragmentation. Some fragmentation pathways were unambiguously proposed by the variety of abundance of fragment ions at different collision energies even without MSⁿ spectra. Results – Fragmentation pathways of five representative sesterterpenoids were elucidated using ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ in both positive‐ and negative‐ion mode. The key group of characterising fragmentation profiles was ring B, and these fragmentation patterns are helpful to identify different types of sestertepenoids. Conclusion – Complementary information obtained from fragmentation experiments of [M + H]⁺ (or [M + NH₄]⁺) and [M ? H]^? precursor ions is especially valuable for rapid identification of this kind of sesterterpenoid.     

Sodium-Dependent Efflux of [3H]GABA from Synaptosomes Probably Related to Mitochondrial Calcium Mobilization

It has been suggested that mitochondria might modify transmitter release through the control of intracellular Ca²⁺levels. Treatments known to inhibit Ca²⁺retention by mitochondria lead to an increased transmitter liberation in the absence of external Ca²⁺, both at the frog neuromuscular junction and from isolated nerve endings. Sodium ions stimulate Ca²⁺efflux from mitochondria isolated from excitable tissues. In the present study, the effect of increasing internal Na⁺ levels on [³H]γ-aminobutyric acid ([³H]GABa) release from isolated nerve endings is reported. Results show that the efflux of [³H]GABA from prelabeled synaptosomes is stimulated by ouabain, veratrine, gramicidin D, and K⁺-free medium, which increase the internal sodium concentration. This effect was not observed when Na⁺ was omitted from the incubation medium and it was independent of external Ca²⁺, the experiments having been performed in a Ca²⁺-free, EGTA-containing medium. Since preincubation of synaptosomes with 2,4-diaminobutyric acid did not prevent the stimulatory effect of increased internal Na⁺ levels on [³H]GABA efflux, it appears to be unrelated to an enhanced activity of the outward carrier-mediated GABA transport. These results suggest that the augmented release of [³H]GABA may be due to an increased Ca²⁺efflux from mitochondria eiicited by the accumulation of Na⁺ at the nerve endings. Sandoval M. E. Sodium-dependent efflux of [³H]GABA from synaptosomes probably related to mitochondrial calcium mobilization. J. Neurochem. 35 , 915–921 (1980).     

Fast Atom Bombardment and Tandem Mass Spectrometry for the Identification of Nucleoside Adducts with Phenyl Glycidyl Ether

Abstract

The present study deals with the use of fast atom bombardment (FAB) in combination with constant neutral loss (CNL) scanning, high resolution mass spectrometry and tandem mass spectrometry (MS-MS) with collisionally activated decomposition (CAD), as complementary methods for the identification and structural analysis of phenyl glycidyl ether-nucleoside adducts. Selective detection of the parent ions of the modified nucleosides at the 1–10 ng level has been achieved by suitably designed CNL scans. The elemental composition of the adducts has been determined by accurate mass measurements. CAD-MS has been carried out on the [M + H]⁺ and [M - H]^? ions to derive structural data on the size and nature of the base, sugar and alkyl substituent. In some cases, information on the alkylation site has been obtained, which is very useful for distinguishing isomeric adducts.     

Atmospheric pressure photoionization mass spectrometry of per-O-methylated oligosaccharides related to D-xylans

Three d-xylan type per-O-methylated trisaccharides with various types of linkages between the d-xylopyranose units were examined by atmospheric pressure photoionization (APPI) mass spectrometry in the positive ion mode. The most interesting feature of a thermospray mass spectrum using the APPI source with UV lamp switched off, is the exclusive production of [M+Na]⁺ adduct ions. [M+Na]⁺ cationized ions are the most abundant species in the case of APPI mass spectrometry. The second ionization process has no analogy in the case of substances studied using APPI to date. This aspect involves the addition of a water molecule to the molecular ion of a per-O-methylated saccharide, giving rise to [M+H₂O]⁺ adduct ions. The [M+H₂O]⁺ species are readily detected at m/z 544, and are clearly visible for all three isomers studied. The MS/MS spectrum of [M+Na]⁺ ions contains a base peak at m/z 375, produced by a Y-type cleavage of the trisaccharide, along with a hydrogen rearrangement on the terminal interglycosidically linkage glycosidic oxygen atom. The [M+H₂O]⁺ species fragment largely give rise to ions at m/z 175, 143 and, as a result, the m/z 111 ion is unique to nonreducing terminal units.     

Vesicular and carrier-mediatied depolarization-induced release of [3H]GABA: Inhibition by amiloride and verapamil

The Ca²⁺-dependent, presumably exocytotic fraction of the [³H]GABA released by depolarization is dissected from the depolarization-induced Na⁺-dependent, carrier-mediated fraction of [³H]GABA release in mouse brain synaptosomes. GABA homoexchange is prevented by the [³H]GABA carrier blocker, DABA. The absence of external Na⁺ completely abolishes the release of the carrier-mediated, presumably cytoplasmic release of [³H]GABA induced by homoexchange and heteroexchange with GABA and DABA, respectively. The carrier-mediated, Na⁺-dependent fraction of the depolarization-induced release of [³H]GABA is resistant to tetrodotoxin (TTX) but is sensitive to amiloride and verapamil. The Ca²⁺-dependent fraction of the [³H]GABA released by high K⁺ depolarization is also completely abolished by amiloride (from 300 M) and sensitive to verapamil (30 M), but in contrast is insensitive to the absence of external Na⁺ and to DABA. On the basis of these results we conclude that amiloride and verapamil inhibit high K⁺-induced release of [³H]GABA by antagonizing the entrance of Ca²⁺ (and possibly Na⁺ when external Ca²⁺ is absent) through a population of voltage sensitive presynaptic Ca²⁺ channels activated by depolarization.Depto. de Biología Molecular Instituto de Investigaciones Biomédicas UNAM.     

Effect of Liposomes Containing Various Divalent Cations on the Release of Acetylcholine from Synaptosomes

Abstract: The effect of increasing the cytoplasmic levels of various divalent cations on the release of [³H]acetylcholine ([³H]ACh) from synaptosomes was investigated. Synaptosomes prepared from rat brain and prelabeled with [³H]choline were incubated with liposomes containing Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Sr²⁺, or Ba²⁺. This treatment allows the transfer of the aqueous contents of the liposomes to the cytoplasm of the synaptosomes. The efflux of radioactivity subsequent to this treatment was measured, and the relative proportions of [³H]ACh and [³H]choline were determined. The release of radioactivity from synaptosomes incubated with liposomes containing Mg²⁺, Mn²⁺, or Co²⁺ was not altered when compared with synaptosomes incubated either without liposomes or with liposomes containing isotonic K⁺/Na⁺. Synaptosomes incubated with liposomes containing Ca²⁺, Sr²⁺, or Ba²⁺, however, released significantly more radioactivity than did controls. Moreover, the released radioactivity consisted almost entirely of [³H]ACh. Liposomes containing either Ca²⁺ or Sr²⁺ were equally effective in promoting the release of [³H]ACh from synaptosomes, whereas liposomes containing Ba²⁺ were 2.5 times more effective in promoting the release of [³H]ACh than were liposomes containing either Ca²⁺ or Sr²⁺. Since liposomes introduce their aqueous contents into cytoplasm via a mechanism not involving plasma membrane channels, the increased release of [³H]ACh caused by liposomes containing Ca²⁺, Sr²⁺, or Ba²⁺ is attributable to an increase in the intrasynaptosomal concentration of these ions, and not to their passage through calcium channels.     

Characterization of the chromophores of pyoverdins and related siderophores by electrospray tandem mass spectrometry

Characteristic fragment ions of the various chromophores of the pyoverdin siderophore family obtained by collision activated dissociation of the [M+2H]²⁺ ions are reported allowing unambiguous identification. Tandem mass spectrometrical studies revealed the existence of the first example of a ferribactin with a succinamide side chain, and they add some information to the problem in which way a malic acid side chain is attached to the chromophore.     

Studies on the mechanism of modulation of [3H]noradrenaline release from rat hippocampal synaptosomes by GABA and benzodiazepine receptors

Release of [³H]noradrenaline from rat hippocampal synaptosomes was triggered by pulses of 25 mM K⁺, 5 μM veratridine or superfusion with the Ca²⁺ ionophore A23187. GABA with bicuculline or chlordiazepoxide depressed the release of [³H]noradrenaline evoked by depolarisation but not by the Ca²⁺ ionophore. 8 Br-cAMP with [Ca²⁺]₀ 0.3 mM had no effect on spontaneous or K⁺-evoked release of [³H]noradrenaline and completely blocked the effect of chlordiazepoxide and GABA with bicuculline. With [Ca²⁺]₀ 1 mM 8 Br-cAMP enhanced spontaneous and K⁺-evoked release of [³H]noradrenaline, and reversed the depression caused by GABA with bicuculline. GABA alone evoked Ca²⁺-dependent release of [³H]noradrenaline which was sensitive to [Cl^?]₀. The results suggest that the GABA_A-receptor mediated release of [³H]noradrenaline is due to depolarisation resulting from increased Cl^? conductance whereas the depression of depolarisation-dependent release of [³H]noradrenaline by GABA_B or benzodiazepine receptors is mediated by a cAMP-dependent decrease in the voltage-dependent Ca²⁺ conductance.     

Development of analytical methods for some penicillins in bovine milk by ion-paired chromatography and confirmation by thermospray mass spectrometry

Analytical methods for the determination of cloxacillin, ampicillin/hetacillin, and amoxicillin in bovine milk were developed. The methods involved ultrafiltration of milk diluted with methanol, acetonitrile, and water on a 10 000-dalton cut-off filter. Separation of penicillins from other milk components was accomplished by ion-paired chromatography using a microbore column. The penicillins were detected using ultraviolet photodiode array (UV-PDA) detection and confirmed by thermospray liquid chromatography—mass spectrometry (LC—MS). The thermospray spectra of these compounds exhibited [M + H]⁺ and [M + Na]⁺ ions along with several fragment ions. The limits of detection for these antibiotics were estimated to be 50 to 100 ppb for LC with UV-PDA detection and 100–200 ppb for thermospray LC—MS detection.     

Modulation of concanavalin A stimulation of hamster lymphoid cells by lithium chloride.

Addition of LiCl (1–25 mM) to serum-free cultures of MHA hamster thymocytes, lymph node cells, or splenocytes stimulated with concanavalin A had a biphasic effect on [³H]thymidine incorporation. These concentrations of LiCl enhanced stimulation of [³H]thymidine incorporation by suboptimal levels of concanavalin A but inhibited stimulation of optimal and supraoptimal concentrations of concanavalin A. This effect was specific for Li⁺ since it was not observed when similar concentrations of Na⁺, K⁺, or Mg²⁺ were added to cultures stimulated by concanavalin A. The inhibitory effect of LiCl on concanavalin A stimulation was not reversed by addition of Na⁺, Ca²⁺, Mg²⁺, or Ca²⁺ + Mg²⁺ to the cultures. Significant reversal of LiCl inhibition of stimulation was observed when KCl was added to the cultures. However none of the ions tested blocked the Li-induced enhancement of [³H]thymidine incorporation in the presence of suboptimal concentrations of concanavalin A.     

Opiate binding to membrane preparations of neuroblastoma x glioma hybrid cells NG108-15: effects of ions and nucleotides.

Interaction of a number of opiate agonists with the opiate receptors in NG108-15 cell membranes is influenced by ions, as well as certain nucleotides. Steady state binding of [³H]leu-enkephalin is increased by Mg⁺⁺ and decreased by Na⁺, GMP-P(NH)P, GTP, GDP, ITP and IMP-P(NH)P. Half-maximal inhibition produced by GMP-P(NH)P occurred at 4.6 μM. The dissociation of [³H]leu- and [³H]met-enkephalin, as well as [³H]etorphine, from these opiate receptors was also shown to be altered by both ions and nucleotides.     

Qualitative and semi-quantitative analyses of cytokinins using LC/APCI-MS in combination with ELISA

Application of liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) for the analysis of cytokinins was examined. The fragmentation of cytokinins was studied using authentic trans-zeatin (t-Z), trans-zeatin riboside (t-ZR), isopentenyl adenine (i⁶Ade), isopentenyl adenosine (i⁶Ado), benzyl adenine (BAde), benzyl adenosine (BAdo), and kinetin. These cytokinins were effectively ionized by APCI in aqueous acetonitrile. t-Z, i⁶Ade, BAde, and kinetin showed prominent quasi-molecular ions of [M + H]⁺, and ribosylcytokinins clearly showed both [M + H]⁺ and a characteristic fragment ion ([M + H-ribose]⁺), giving some information about their structures. The qualitative and semi-quantitative analyses of cytokinins by LC/APCI-MS were validated in combination with enzyme-linked immunosorbent assay (ELISA) through the analysis of t-ZR in the teratoma of Nicotiana tobacum. t-ZR was conclusively identified and a semi-quantitative estimate of its endogenous levels were provided by the combination of LC/APCI-MS and ELISA. The quantified values obtained by LC/ APCI-MS (single ion detection) and ELISA are in close agreement.