A dual-column HPLC method for the simultaneous determination of DHPG (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine) and its mono and diesters in biological samples

A convenient method for the simultaneous determination of various DHPG species present in biological samples is presented. This method utilizes a cation exchange column (25 cm X 4.6 mm i.d.) coupled in series to a short reversed-phase column (5 cm X 4.6 mm i.d.). The mobile phase consists of methanol:0.005M ammonium phosphate buffer, pH 2.5. There is a large polarity difference between DHPG and its esters due to the non-polar side chain of the ester moiety. The simultaneous determination of the diesters, monoesters, and DHPG in these samples using only the cation exchange or the reversed-phase column is not possible without time-consuming gradient elution. In the reversed-phase mode alone, the esters are highly retained relative to DHPG, whereas the esters are only slightly retained on a cation exchange column and are insensitive to changes in pH and ionic strength of the mobile phase. However, a combination of these two columns provides interesting selectivity for these compounds and offers a unique way of controlling the retention times of these species relative to each other. The retention time of esters can be selectively altered (with respect to DHPG) by changing the composition of methanol in the mobile phase. In contrast, the retention time of DHPG is controlled by changing the buffer strength and pH of the mobile phase.     

On the direct determination of metals in lubricating oils by ICP

New equipment and procedures are evaluated for the direct analysis of metals in lubricating oils without the need for sample pretreatment or dilution. A modified Babington principle nebulizer equipped with a sample heater is shown to be capable of producing aerosols from undiluted oils, which are suitable for introduction into an inductively coupled plasma atomic emission spectrophotometer. Heating the samples immediately prior to nebulization greatly increases the output of aerosol and reduces output variations in emission intensity due to differences in oil manufacture and viscosity. The type of organometallic complex used in the preparation of standards is shown to be unimportant if the plasma observation region is properly chosen. Performance of a conventional plasma geometry and an inverted torch geometry on analysis of field collected oil samples is presented.     

A Lock-and-Kill Anticancer Photoactivated Chemotherapy Agent†

Photosubstitutionally active ruthenium complexes show high potential as prodrugs for the photoactivated chemotherapy (PACT) treatment of tumors. One of the problems in PACT is that the localization of the ruthenium compound is hard to trace. Here, a ruthenium PACT prodrug, [Ru(3)(biq)(STF-31)](PF₆)₂ (where 3 = 3-(([2,2′:6′,2″-ter- pyridin]-4′-yloxy)propyl-4-(pyren-1-yl)butanoate) and biq = 2,2′-biquinoline), has been prepared, in which a pyrene tracker is attached via an ester bond. The proximity between the fluorophore and the ruthenium center leads to fluorescence quenching. Upon intracellular hydrolysis of the ester linkage, however, the fluorescence of the pyrene moiety is recovered, thus demonstrating prodrug cellular uptake. Further light irradiation of this molecule liberates by photosubstitution STF-31, a known cytotoxic nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, as well as singlet oxygen via excitation of the free pyrene chromophore. The dark and light cytotoxicity of the prodrug, embedded in liposomes, as well as the appearance of blue emission upon uptake, were evaluated in A375 human skin melanoma cells. The cytotoxicity of the liposome-embedded prodrug was indeed increased by light irradiation. This work realizes an in vitro proof-of-concept of the lock-and-kill principle, which may ultimately be used to design strategies aimed at knowing where and when light irradiation should be realized in vivo.     

Cyclic esters of aliphatic diacids with pyrocatechol and hydroquinone

Cyclic esters of adipic acid, suberic acid and sebacic acid were prepared by reaction of the acid dichlorides and pyrocatechol or hydroquinone in benzene under high dilution conditions. While only the cyclic dimers could be obtained from hydroquinone, pyrocatechol formed cyclic monomers as well as cyclic dimers (and also a cyclic dimer with succinic acid). The structure of all compounds was confirmed by¹H-NMR- and mass spectra. The crystal structures of the pyrocatechol esters were determined by single crystal X-ray analysis.

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Cyclische Ester aliphatischer Dicarbonsäuren mit Brenzcatechin und Hydrochinon

Zusammenfassung Es wurden cyclische Ester von Adipinsäure, Korksäure und Sebacinsäure durch Reaktion der entsprechenden Säure-Dichloride mit Brenzcatechin oder Hydrochinon bei hoher Verdünnung hergestellt. Während mit Hydrochinon lediglich die cyclischen Dimeren erhalten werden konnten, wurden mit Brenzcatechin sowohl cyclische Monomere als auch cyclische Dimere erhalten (ebenfalls das cyclische Dimer mit Bernsteinsäure). Die Strukturen wurden mittels¹H-NMR und Massenspektren gesichert. Die Kristallstrukturen der Brenzcatechin-Ester wurden mittels Röntgenstrukturanalyse bestimmt.

     

Quantitative determination of tertatolol in biological fluids by gas chromatography-mass spectrometry

Quantitative determination of tertatolol concentrations in plasma and urine was performed by gas chromatography-mass spectrometry in the chemical-ionization mode with ammonia after successive extractions of the beta-blocking drug in alkaline, acid and final alkaline medium. [2H9]Tertatolol, isotopically stable under the operating conditions employed, was used as an internal standard, thus allowing quantities of 1 ng/ml to be specifically determined. Overall analytical error was less than 10%. Prior to isothermal chromatography at 240 degrees C on a column packed with 3% SE-30, both compounds were silylated with bis(trimethylsilyl)trifluoroacetamide. Detection was performed by monitoring the quasimolecular ions of tertatolol, m/z 368 and m/z 377, for the [2H9]tertatolol in the chemical-ionization mode with ammonia. The calibration curves obtained had linear characteristics for the concentration range 1-1125 ng/ml.     

PHOTOCHEMICAL RING-OPENING IN meso-CHLORINATED CHLOROPHYLLS

Abstract— Irradiation of 20-chloro-chlorophylls of the a-type with visible light produces long-wavelength shifted photoproducts, which transform in the dark to linear tetrapyrroles (bile pigments). The possible significance for chlorophyll degradation is discussed.     

Quantitative analysis by high-performance liquid chromatography atmospheric pressure chemical ionization mass spectrometry: The determination of the renin inhibitor CP-80,794 in human serum

A specific method was developed for the quantitative determination of the renin inhibitor CP-BO,794. Serum extracts containing the drug and an internal standard were injected into a standard reverse-phase high-performance liquid chromatography (HPLC) column. The mobile phase, methanol/water (8/2), flowed at 1 ml/min through the column and then via a heated nebulizer interface into a corona discharge atmospheric pressure chemical ionization source. The assay minimum limit of quantification was 50 pg/mL. It exhibits satisfactory accuracy and precision over the range 50 pg/ml, to 10 ng/mL. A minor modification of the HPLC mobile phase was necessary to attain extremely low detection limits. The addition of a structural analogue contributed to enhancing the precision of the assay.     

Determination of 4-hydroxyandrostenedione in plasma and urine by extractive alkylation and electron-capture gas chromatography

A sensitive and specific quantitative assay has been developed for the determination of 4-hydroxyandrostenedione (4-OHA), a potent aromatase inhibitor used in the treatment of estrogen-dependent breast cancer. This steroid has a high first-pass metabolism and is extensively metabolized, mainly by glucuronidation. Plasma levels of unchanged 4-OHA are very low, even after high peroral doses. The analytical method is based on the addition of 17 alpha-ethinylestradiol (internal standard), liquid-liquid extraction from biological material followed by extractive alkylation with pentafluorobenzyl bromide and quantitation by gas chromatography. The method has been validated for sensitivity, accuracy and precision and was found to be suitable for application to pharmacokinetic and bioavailability studies of peroral formulations of 4-OHA.     

Context-based identification of protein-protein interfaces and "hot-spot" residues

Reliable determination of protein-protein interaction sites is of critical importance for structure-based design of small molecules modulating protein function through macromolecular interfaces. We present an alignment-free computational method for prediction of protein-protein interface residues. The method ("iPred") is based on a knowledge-based scoring function adapted from the field of protein folding and small molecule docking. Based on a training set of 394 hetero-dimeric proteins iPred achieves sustained accuracy on an external unbound test set. Prediction robustness was assessed from more than 1500 diverse complexes containing homo- and hetero-dimers. The technique does not rely on sequence conservation, so that rapid interface identification is possible even for proteins for which homologs are unknown or lack conserved residue patterns in interface region. Functional "hot-spot" residues are enriched among the predicted interface residues, rendering the method predestined for macromolecular binding site identification and drug design studies aiming at modulating protein-protein interaction that might influence protein function. For a comparative structural model of peptidase HtrA from Helicobacter pylori, we performed mutation studies for predicted hot-spot residues, which were confirmed as functionally relevant for HtrA activity or oligomerization.