High lateral resolution vs molecular preservation in near-IR fs-laser desorption postionization mass spectrometry.

Ultrashort pulse length lasers operating in the near-infrared region show promise for submicrometer lateral resolution by laser desorption-based mass spectrometry (MS) imaging. However, these experiments must balance lateral resolution and molecular fragmentation since abundant atomic ions are observed at the high laser irradiances that can be generated by tightly focused ultrashort pulse laser beams. It is shown here that combining ultrashort pulse laser desorption with laser postionization (fs-LDPI) allows for a considerable increase of molecular ion signal while operating with lower laser irradiances, yielding the added benefit of reduced molecular fragmentation. This Letter presents several experimental results in support of the fs-LDPI approach for MS imaging. First, the lateral resolution for MS imaging of molecular species desorbed by ∼75 fs, 800 nm laser pulses was determined to be <2 µm for a simulated organic electronic device under vacuum. Next, the dependence of precursor ion survival on both desorption laser fluence and delay between desorption and photoionization laser pulses was observed for a small molecule desorbed from an organic multilayer that was originally devised as a model of a bacterial biofilm. When considered in light of recent results in the literature (Milasinovic et al. J. Phys. Chem. C 2014, DOI: 10.1021/jp504062u), these experiments demonstrate the potential for submicrometer spatial resolution MS imaging by fs-LDPI.

Near infrared (NIR) strong field ionization and imaging of C60 sputtered molecules: overcoming matrix effects and improving sensitivity.

Strong field ionization (SFI) was applied for the secondary neutral mass spectrometry (SNMS) of patterned rubrene films, mouse brain sections, and Botryococcus braunii algal cell colonies. Molecular ions of rubrene, cholesterol, C31 diene/triene, and three wax monoesters were detected, representing some of the largest organic molecules ever ionized intact by a laser post-ionization experiment. In rubrene, the SFI SNMS molecular ion signal was ~4 times higher than in the corresponding secondary-ion mass spectroscopy (SIMS) analysis. In the biological samples, the achieved signal improvements varied among molecules and sampling locations, with SFI SNMS, in some cases, revealing analytes made completely undetectable by the influence of matrix effects in SIMS.

A tetraene aldehyde as the major sex pheromone component of the promethea moth (Callosamia promethea (Drury)).

The promethea moth Callosamia promethea is one of three species of silkmoths from the genus Callosamia that occur in North America. Cross attraction of males to heterospecific calling females has been observed in the field, and hybrid progeny have been produced by pairing heterospecifics in captivity. These observations suggest that all three species share or have considerable overlap in the sex attractant pheromones produced by females, so that other prezygotic isolating mechanisms, such as diel differences in reproductive activity, limit hybridization in the field. Coupled gas chromatography-electroantennogram detection and gas chromatography- mass-spectrometry analyses of extracts of volatiles collected from female promethea moths supported the identification of (4E,6E,11Z,13Z)-hexadeca-4,6,11,13-tetraenal [(4E,6E,11Z,13Z)-16:Ald] as the compound in extracts that elicited the largest responses from antennae of males. The identification was confirmed by non-selective synthesis of several isomers as analytical standards, and stereoselective synthesis of (4E,6E,11Z,13Z)-16:Ald for testing in field trials. Male moths were strongly attracted to synthetic (4E,6E,11Z,13Z)-16:Ald, suggesting that this compound is the major and possibly the only component of the sex pheromone of these large saturniid moths. Based on the cross-attraction of heterospecifics, it is likely that this is also a major pheromone component of the other two North American Callosamia species as well.

Antitumour compounds from a saline soil isolate, Streptomyces griseoincarnatus CTF15.

A new actinomycete strain designated as Streptomyces sp. CTF15 was isolated from a saline soil using casein-KNO(3) agar medium. The strain Streptomyces sp. CTF15 exhibited promising antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Streptomyces viridochromogens Tu57 and high cytotoxicity (91.2% mortality) against Artimia salina in biological screening. The cultivation of this strain in a 50 L lab fermenter and subsequent isolation and purification by a series of chromatographic techniques and structure elucidation by MS and NMR analysis of the active metabolites revealed that it is a highly stable producer of resistomycin (1), tetracenomycin D (2) and actinomycin D (3), even under non-optimised culture conditions. The morphological, microscopic, biochemical and physiological characterisation suggested that the strain CTF15 belongs to the genus Streptomyces. A partial 16S rRNA gene sequence (1429 bp) from the strain CTF15 was determined and found to have high identity (99%) with Streptomyces griseoincarnatus. As such, this is the first report of a strain of S. griseoincarnatus capable of producing these three bioactive compounds simultaneously.

Tandem laser-induced fluorescence and mass spectrometry detection for high-performance liquid chromatography analysis of the in vitro metabolism of doxorubicin.

Structural characterization, identification, and quantification of xenobiotics and their metabolic products commonly require the use of at least two different techniques. This has been the case in the analysis of metabolic products of doxorubicin, a widely used fluorescent anthracycline for the treatment of tumors and leukemia. In this work, we combine high-performance liquid chromatography (HPLC) with a tandem laser-induced fluorescence (LIF) and mass spectrometry (MS) detection scheme for the characterization of doxorubicin and its metabolites produced in the postmitochondrial fraction prepared from Fischer 344 rat liver. LIF detection allowed quantification of the metabolic compounds while MS detection aided in the identification of the metabolites. Using this HPLC-LIF-MS methodology, the disappearance of doxorubicin and the appearance of 7-deoxydoxorubicinone and 7-deoxydoxorubicinolone were monitored over the course of 40 min. This application demonstrates the potential of the tandem LIF-MS detection scheme in quantification and characterization of biotransformations of fluorescent xenobiotics of biomedical and environmental relevance. Furthermore, this detection scheme would be particularly relevant in the analysis of fluorescent analytes in complex samples and in validation of methods for the analysis of such samples that typically rely only on LIF detectors.

The effect of laser wavelength on the Raman Spectra of phenanthrene, chrysene, and tetracene: implications for extra-terrestrial detection of polyaromatic hydrocarbons.

Raman spectroscopy, with visible laser (514 and 633 nm) and near infrared (785 and 1064 nm) excitation, has been used to obtain high quality spectra of phenanthrene, chrysene, and tetracene. Samples with dimensions from a minimum size of 10 microm have been analyzed utilizing a Raman microprobe fitted with a charge-coupled device (CCD) array detector and a FT-Raman instrument. Fluorescence is observed for samples using visible 514, 633 and near infrared 785 nm excitation but most of the samples can be measured with a near infrared 1064 nm Nd:YAG laser.

Singlet oxygen quenching effects of phosphatidylcholine in emulsion containing sunflower oil.

Effects of phosphatidylcholine (PC) on the oxidation of oil by singlet oxygen in a W/O microemulsion and an emulsion food model containing tocopherol-stripped sunflower oil (TSSO) have been studied. The W/O microemulsion consisted of methylene chloride, butanol, and sodium dodecyl sulfate with PC (0, 250, 1000 ppm) and TSSO (0, 3.3, 16.5, 33 mg/mL). Production of singlet oxygen in the microemulsion was done chemically with hydrogen peroxide in the presence of sodium molybdate, and indirectly evaluated by rubrene oxidation at A529. The emulsion food model consisted of TSSO, distilled water, and xanthan gum with addition of 250 ppm PC and 4 ppm chlorophyll b, and was placed at 25 degrees C under fluorescent lights (1700 lux) for 24 h. The oxidation of TSSO was determined by thin-layer chromatography and values of conjugated dienoic acid (CDA) and peroxides (POV). PC significantly decreased the oxidation of rubrene and TSSO in the W/O microemulsion, but its content was decreased to approximately one-half by a 20-min reaction, indicating its degradation. This clearly shows that PC acted as an antioxidant via chemical quenching of singlet oxygen in the W/O microemulsion. A possible synergism between PC and TSSO was observed in singlet oxygen quenching in the microemulsion. PC also significantly decreased the chlorophyll-photosensitized oxidation of TSSO in the emulsion food model, possibly by singlet oxygen quenching. This study clearly suggested that PC be used as an antioxidant to improve the lipid oxidative stability of an emulsion food containing chlorophyll under light.

Determination of doxorubicin and metabolites in murine specimens by high-performance liquid chromatography.

A sensitive and selective reversed-phase high-performance liquid chromatographic method for the quantification of doxorubicin and its metabolites doxorubicinol, 7-deoxydoxorubicinone and 7-deoxydoxorubicinolone was developed and validated for a variety of murine specimens. Daunorubicin was used as internal standard. Sample pretreatment involved liquid-liquid extraction of 200 microl sample with 1 ml of chloroform-1-propanol (4:1, v/v). Chromatographic separation was achieved isocratically on a LiChrosorb RP-8 analytical column at ambient temperature. The mobile phase consisted of acidified water (pH 2.05)-acetonitrile-tetrahydrofuran (80:30:1, v/v/v). The column effluent was monitored fluorimetrically at an excitation wavelength of 460 nm and an emission wavelength of 550 nm. The lower limits of quantitation were in the range 1.8-2.4 nM. Spiked murine specimens and samples from treated mice were subjected to stability studies. The results demonstrated the importance of validation in all relevant specimens, since the accuracy and precision were highly matrix-dependent. Accuracies and precisions of measured drug concentrations in liver, spleen, muscle, gastrointestinal tissues, diluted bile, feces and urine were lower than in the other matrices. Doxorubicin was unstable in diluted bile, but not in the other specimens. The method is suitable for studying the pharmacokinetics of doxorubicin and its metabolites in mice.

A sensitive procedure for the quantitation of free and N-(2-hydroxypropyl) methacrylamide polymer-bound doxorubicin (PK1) and some of its metabolites, 13-dihydrodoxorubicin, 13-dihydrodoxorubicinone and doxorubicinone, in human plasma and urine by reversed-phase HPLC with fluorimetric detection.

A high-performance liquid chromatographic assay has been developed and validated for the determination in plasma and urine of doxorubicin (DXR) and some of its metabolites released in vivo from an N-(2-hydroxypropyl)methacrylamide (HPMA) polymer containing DXR linked through its aminosugar moiety to the polymer via an oligopeptide spacer (PK1). The method also allows measurement of the DXR still bound to the polymer. Following addition of two internal standards, the free compounds were extracted twice with isopropanol-chloroform (25:75, v/v). The first extraction was performed at physiological pH and the second after buffering at pH 8.4, in order to extract the aglycones and the glycosides, respectively. Determination of total DXR (polymer-bound plus free DXR) was performed, after quantitative acid hydrolysis to release doxorubicinone from free or polymer-bound DXR, by extraction with the same solvent mixture at pH 7.4. In both cases the organic phase was evaporated to dryness; the compounds were then separated by reversed-phase high-performance liquid chromatography (HPLC) under isocratic conditions and quantitated by fluorimetric detection. In the chromatograms all the analytes appeared to be separated at the baseline and no interference from blank human plasma and urine was observed. The suitability of the method for in vivo samples was checked by the analysis of plasma and urine samples obtained from a cancer patient who had received a single intravenous dose of the test compound.

Fast atom bombardment mass spectrometric analysis of anthracyclines and anthracyclinones.

A mass spectral characterization of a set of anthracyclines and anthracyclinones comprised of daunorubicin, adriamycin and their modified analogs was carried out by using negative and positive fast atom bombardment (FAB) ionization techniques. Addition of more than one hydrogen to the molecular ions of the anthracyclines was observed. The choice of the FAB matrix played an important role in the characterization of these compounds. The dominant ions in the molecular ion region were M-. (or M+.) and MH- (or MH+.2) when sulfolane and glycerol, respectively, were employed as the FAB solvents. The major fragmentation was cleavage of the glycosidic bond with the charge retention mainly on the aglycone moiety. Aromatization of the tetracyclic ring promoted further fragmentation of the aglycone moiety. The anthracyclinones could be characterized only by negative FAB ionization using sulfolane as the FAB matrix. The assigned fragmentation pathways were confirmed by acquiring metastable ion spectra using B/E linked-field scans.

Alpha-actin synthesis changes in cultured cardiac myocytes: relationship to anthracycline structure.

Four anthracyclines (AAs) were examined comparatively to determine effects on actin isoform synthesis in vitro. Cultured cardiac myocytes (CMCs) were incubated for 24 hours with 35 microCi sulfur 35-labeled methionine and 10(-10) to 10(-5) mol/L doxorubicin hydrochloride (Adriamycin) (ADR), daunomycin (DM), 5-iminodaunorubicin IDR), or 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin (MRA-CN). CMCs were harvested in buffered Triton X-100 and homogenized. Proteins in the extracts were fractionated by centrifugation. Equal protein quantities were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, to two-dimensional electrophoresis, and to autoradiography. AAs caused a dose-dependent decrease in radiolabeling of CMC proteins in Triton-soluble and -insoluble fractions of the extracts. ADR and DM (10(-6) mol/L each) and IDR (10(-5) mol/L) decreased radiolabeling of CMC polypeptides including alpha-actin. In polypeptides extracted in the Triton X-100-soluble pool, the effect on CMC alpha-actin synthesis was greater than the effect on beta- or gamma-actin. CMC alpha-actin synthesis was susceptible to ADR and to DM in a dose-dependent fashion. Contrastingly, alpha-actin radiolabeling was not altered by exposing CMCs to 10(-5) mol/L MRA-CN. Decreased sarcomeric actin isoform synthesis in vitro may reflect forms of subcellular damage in this model of anthracycline cardiomyopathy.

In-beam light absorption measurement of anthracyclines in cells with a flow-through system.

A flow-through cuvette in which cells attach as a monolayer to a quartz plate was developed for measurement of the light absorbance of anthracyclines in cells. Despite the drawback of a short path-length (of the order of the cell diameter), a dynamic flow-through set-up and baseline storage made it possible to measure intracellular absorbance and obtain spectral data for daunomycin and carminomycin. Stopping the flow and allowing the drug to equilibrate between medium and cells led to a 20% decrease of molar light absorption of cellular anthracycline, which permitted measurement of the total cellular concentration. Furthermore, accumulation and efflux kinetics were determined for H35 rat hepatoma cells. On the basis of the reported formation constant of the iron-complex of carminomycin, which is of the order of 10(34), we expected to find this complex within the cells. However, the spectrum of cellular drug did not show absorbance bands characteristic of the complex. A red shift and hypochromism were found in the daunomycin spectrum after intracellular binding, which corresponds with the spectral change observed after intercalation of daunomycin into DNA.

[Effect of the molecular structure of natural anthracycline antibiotics on their relative hydrophobicity]. / Vliianie molekuliarnoi struktury prirodnykh antratsiklinovykh antibiotikov na ikh otnositel'nuiu gidrofobnost'.

Relative hydrophobicities of anthracycline antibiotics, adriamycin, rubomycin and carminomycin, have been measured by the two-phase distribution method. Two different biphasic systems were used for this purpose. Possible reasons of discrepancies between results obtained and other authors, data are discussed. It was established that the relative hydrophobicities of the compounds investigated contradict the theory of increment additivity. The results are compared with quantum-chemical calculations.

[Kafamycin--a new pyrrol ether antibiotic]. / Kafamitsin--novyi pirroléfirnyi antibiotik.

Cafamycin is a novel polyether antibiotic active against gram-positive bacteria. It was isolated from the culture fluid of Streptomyces sp., an organism producing the anthracycline antibiotic galtamycin. The structure of cafamycin was assessed by spectral analysis: UV, PMR, mass spectroscopy and CD. Physico-chemical and biological properties of the antibiotic are described. Cafamycin was shown to be an analog of indanomycin (X-14547A), a pyrrol ether antibiotic.

[Pharmacokinetic study of aclarubicin. The distribution of the preparation and its biologically active metabolites in rat tissues]. / Farmakokineticheskoe izuchenie aklarubitsina. Raspredelenie preparata i ego biologicheski aktivnykh metabolitov v tkaniakh krys.

Tissue pharmacokinetics of aclarubicin and its active metabolites was studied with high performance liquid chromatography. The drug was administered to rats intravenously in single doses of 5 and 10 mg/kg and orally in a single dose of 10 mg/kg. With both the administration routes the highest concentrations of the drug and its metabolites were attained in the lymph nodes. Then followed the spleen and lungs. The lowest content of the drug was detected in the heart. The total values of the areas under the concentration/time curves for aclarubicin and its metabolites in the tissues of the heart, lungs, lymph nodes and spleen after oral administration were respectively 2, 3, 4 and 7 times lower than those after the drug intravenous administration in the same dose. The concentrations of the active metabolites MA144N1 and MA144T1 exceeded those of aclarubicin and were detected in the tissues within a longer period as compared to the unchanged drug. With repeated administration preferential accumulation of the metabolites in the tissues and their increased contribution to the aclarubicin antitumor effect could be suspected.