Krisztina Ludányi

GlycoMiner: a new software tool to elucidate glycopeptide composition

New computer software, GlycoMiner, has been developed to automatically identify tandem (MS/MS) spectra obtained in liquid chromatography/mass spectrometry (LC/MS) runs which correspond to N-glycopeptides. The program complements conventional proteomics analysis, and can be used in a high-throughput environment. The program interprets the spectra and determines the structure of the corresponding glycopeptides. GlycoMiner runs under Windows, can process spectra obtained on various instruments, and can be downloaded from our website (w3.chemres.hu/ms/glycominer). The algorithm works similarly to a human expert; evaluates the low mass oxonium ions; deduces oligosaccharide losses from the protonated molecule; and identifies the mass of the peptide residue. The program has been tested on tryptic digests of two glycopeptides: AGP (which has five different N-glycosylation sites) and transferrin (with two N-glycosylation sites). Results have been evaluated both manually and by GlycoMiner. Out of 3132 MS/MS spectra 338 were found to correspond to glycopeptides; identification by GlycoMiner showed a 0.1% false positive and 0.1% false negative rate. From these it was possible to identify 196 glycan structures manually; GlycoMiner correctly identified all of these, with no false positives. The rest were low quality spectra, not suitable for structure assignment.

Aqueous solvent system for solubilization of azole compounds

Azoles have a wide spectrum antimycotic activity, but due to some derivatives exhibiting poor water solubility their parenteral administration is limited. The influence of solubilizers on the aqueous solubility of the itraconazole, ketoconazole and miconazole was investigated in order to enhance their solubility for a possible parenteral dosage form. The solubilizer effect of acetate, phosphate and gluconate solutions were studied, along with ethanol, glycerol, macrogol 400, propylene glycol and surfactants, such as polysorbate 20, 60, 80 and sodium taurocholeate. The solubilizing effect of these excipients in binary or ternary combinations was also studied. An HPLC method was used for the solubility assay of the azoles. All of the assessed excipients showed considerable solubility enhancement characteristics, moreover the binary and ternary combinations showed synergistic effects solubilizing more miconazole than what they solubilized separately. Ternary combinations were capable of solubilizing more than 30 mg/ml miconazole, and more than 135 mg/ml of ketoconazole, which in both cases is a very substantial increase in solubility compared to their water solubility. The amount of solubilized drugs may well be used therapeutically, and the formulated solvent system can serve as a base for parenteral solutions.

In vitro food-drug interaction study: Which milk component has a decreasing effect on the bioavailability of ciprofloxacin?

The purpose of the present work was developing an in vitro dissolution test to highlight the possible molecular background causing ciprofloxacin (CPFX)-milk interaction. The in vitro dissolution of CPFX from film-coated tablets (Ciprinol) 500mg) was examined at different pH values, simulating certain parts of the gastrointestinal tract, in the presence of water, low-fat milk, casein- or calcium enriched water. In order to determine the amount of dissolved CPFX, solid phase extraction sample preparation followed by high performance liquid chromatography coupled with mass spectrometry was applied. Comparing the dissolution efficiency values in various media, it can be concluded, that casein has a more pronounced effect on the absorbable amount of the antibiotic at each pH value studied, than calcium. In the case of concomitant intake of CPFX film-coated tablet and milk or other dairy products not only the complexation with calcium, but also the adsorption of CPFX on the surface of proteins decreases the absorbable amount of CPFX.

Comparative evaluation of the effect of cyclodextrins and pH on aqueous solubility of apigenin

The aqueous solubility of a flavonoid, apigenin, was studied in the presence of first generation cyclodextrins (α-CyD, β-CyD, γ-CyD), ionic and nonionic synthetic derivatives of β-CyD, namely SBE-β-CyD, HP-β-CyD and RM-β-CyD at various physiological pH. The order of solubility enhancement was as follows: RM-β-CyD>SBE-β-CyD>γ-CyD>HP-β-CyD>β-CyD>α-CyD. The phase solubility diagrams of HP-β-CyD and SBE-β-CyD indicated Higuchi AL subtype behavior, suggesting 1:1 stoichiometry of the complex. In contrast, AP subtype, so higher order complex formation can be assumed in the case of RM-β-CyD and γ-CyD. The formation of inclusion complexes has been confirmed by absorption and fluorescence spectroscopic measurements. Increased antioxidant activity was observed due to the inclusion complexes. These results prove that synthetic derivatives of β-CyD will be potentially useful excipients in the development of drug delivery systems for healthcare products containing flavonoids.

Analysis of pyridinium aldoximes - a chromatographic approach.

Pyridinium aldoximes are used as antidotes to organophosphorus cholinesterase inhibitors. All pyridinium aldoximes (oximes) are highly polar quaternary ammonium compounds showing low to minimal blood-brain-barrier (BBB) penetration. Oximes are separated using reversed-phase (RP) HPLC methods and/or thin-layer chromatography (TLC). The chemical structures, elementary compositions, molecular sizes and the calculated logP values of several mono- and bis-pyridinium aldoximes are given. Chromatographic and electrophoretic analyses of oximes are detailed, including the stationary and mobile phase composition and the mode of detection. Degradation pathways and products are also discussed. To characterize oximes lipophilicity/hydrophilicity an in silico method was used and expanded as to describe organophosphorus compound adducts with several pyridinium aldoximes.

Quantitative Comparison of Tandem Mass Spectra Obtained on Various Instruments

AbstractThe similarity between two tandem mass spectra, which were measured on different instruments, was compared quantitatively using the similarity index (SI), defined as the dot product of the square root of peak intensities in the respective spectra. This function was found to be useful for comparing energy-dependent tandem mass spectra obtained on various instruments. Spectral comparisons show the similarity index in a 2D “heat map”, indicating which collision energy combinations result in similar spectra, and how good this agreement is. The results and methodology can be used in the pharma industry to design experiments and equipment well suited for good reproducibility. We suggest that to get good long-term reproducibility, it is best to adjust the collision energy to yield a spectrum very similar to a reference spectrum. It is likely to yield better results than using the same tuning file, which, for example, does not take into account that contamination of the ion source due to extended use may influence instrument tuning. The methodology may be used to characterize energy dependence on various instrument types, to optimize instrumentation, and to study the influence or correlation between various experimental parameters. Graphical Abstractᅟ

New steroid-fused P-heterocycles: Part II. Synthesis and conformational study of oxazaphosphorino[16,17-e]estrone derivatives

16Beta-aminomethyl-17beta-hydroxyestrone 3-methyl ether 6 and its N-propyl (17), N-benzyl (18) and N-arylmethyl derivatives (19-22) were subjected to ring closure reactions with phenylphosphonic dichloride in order to synthetize P-epimeric oxazaphosphorinanes 23a, 24-29 in which the hetero ring is condensed to ring D of the sterane skeleton. The stereostructures of the products were evaluated by 1H, 13C and 31P NMR spectroscopy. The geometry was optimized by utilizing the B3LYP DFT method. The NMR spectral data and the results of the ab initio calculations demonstrated that the stereostructure of the hetero ring was strongly affected by the rigid sterane framework condensed to it, and the phosphoramidate ring proved to adopt predominantly a distorted-boat conformation, regardless of the P-configuration.

BIOTRANSFORMATION OF DERAMCICLANE IN PRIMARY HEPATOCYTES OF RAT, MOUSE, RABBIT, DOG, AND HUMAN

The metabolic fate of deramciclane [(1R,2S,4R)-(–)-2-phenyl-2-(2′-dimethylamino-ethoxy)-1,7,7-trimethyl-bicyclo[2.2.1]heptane], a new anxiolytic drug candidate, has been determined in rat, mouse, rabbit, dog, and human hepatocytes. Rat and rabbit cells were the most active, whereas the rate of metabolism was quite slow in human hepatocytes. During biotransformation, deramciclane underwent side chain modification and oxidation at several positions of the molecule. The side chain modification led to the formation of N-desmethyl deramciclane and phenylborneol. The oxidation of deramciclane resulted in several hydroxy-, carboxy-, and N-oxide derivatives. The hydroxylation took place at primary or secondary carbons of the camphor ring as well as at the side chain; furthermore, dihydroxylated derivatives were also found. The side chain-modified metabolites were also oxidized to hydroxy- or carboxy-derivatives. Conjugation of phase I metabolites, as a route of elimination, was also observed in rat, rabbit, and dog hepatocytes. Although there were some species differences in biotransformation of deramciclane, it was concluded that phase I metabolism in human liver cells seemed to be similar to the metabolism in the hepatocytes isolated from rat. With careful approach, the rat model may be considered to be predictive for human metabolism of deramciclane.

Fully on-line hyphenation of an experimental OPLC separation unit with diode-array detection and mass-spectrometry (OPLC-DAD-MS) for analysis of xanthine compounds

The newly developed experimental OPLC separation unit 100 (OSU 100) has been used for fully on-line multiple hyphenation using the one-channel flowing eluent wall (FEW) arrangement at the inlet. OSU 100-UV and OSU 100-DAD-ESI-MS with manual injector or autosampler were used to test the connected systems (OPLC-UV; OPLC-DAD-ESI-MS), using xanthine standards (caffeine, theophylline, theobromine) and green tea leaf extract as model compounds. The mobile phase chloroform-trifluoroacetic acid-acetonitrile-methanol, 76:4:6.67:13.33 (v/v) is suitable for rapid and selective separation, DAD, and ESI-MS of xanthines. The detection sensitivity of OPLC-UV or OPLC-DAD was increased by hyphenation with ESI-MS coupled in series. In contrast with DAD, the extracted ion chromatogram (m/z = 181.1 Da) after background subtraction yields readily measurable peaks from the small amounts of theophylline and theobromine in tea leaf extract. Analysis of xanthines was achieved within 10 min with less than 2 mL mobile phase. Owing to the openable adsorbent layer in OPLC the bed conditions can, moreover, be checked off-line after single or multiple fully online separations.

Metabolism of Moexipril to Moexiprilat: Determination of In Vitro Metabolism Using HPLC-ES-MS

Moexipril is a long-acting, non-sulfhydryl angiotensine-converting enzyme inhibitor. It is used for treatment of arterial hypertension. Moexipril is the prodrug, yielding moexiprilat by hydrolysis of an ethyl ester group. Moexiprilat is the metabolite responsible for the pharmacological effect after moexipril administration. Samples of rat and human microsomal preparations exposed to moexipril treatment were analyzed by HPLC using octyl silica stationary phase and isocratic elution. To detect moexipril and moexiprilat the separation was monitored by both ultraviolet and mass specific detection. The rat liver microsomal preparation was more effective to in producing moexiprilat than the similar one derived from human liver cell lines. While additional potential metabolites of moexipril were suggested by computer-modeling, moexiprilat was the sole metabolite detected after microsomal treatment.