Tímea Imre

Mass spectrometric and linear discriminant analysis of N-glycans of human serum alpha-1-acid glycoprotein in cancer patients and healthy individuals

N-glycan oligosaccharides of human serum alpha(1)-acid glycoprotein (AGP) samples isolated from 43 individuals (healthy individuals and patients with lymphoma and with ovarian tumor) were analyzed by MALDI-TOF mass spectrometry and a multivariate statistical method (linear discriminant analysis, LDA). 34 different glycan structures have been identified. From the glycosylation pattern determined by mass spectrometry fucosylation and branching indices have been calculated. These parameters show only small differences between the patient groups studied, but these differences are not sufficiently large to use as a potential biomarker. LDA analysis, on the other hand shows a very good separation between the three groups (with a classification of 88%). Cross-validation indicates that the method has predictive power: Identifying cancerous vs. healthy individuals shows 96% selectivity and 93% specificity; identification of lymphoma vs. the mixed group of healthy and ovarian tumor cases is also promising (72% selectivity and 84% specificity). The pilot study presented here demonstrates that mass spectrometry combined with linear discriminant analysis (LDA) may provide valuable data for identifying and studying the pathophysiology of malignant diseases.

Enantiomeric separation of antimalarial drugs by capillary electrophoresis using neutral and negatively charged cyclodextrins

Capillary electrophoresis (CE) methods for chiral resolution of five antimalarial drugs (primaquine, tafenoquine, mefloquine, chloroquine and quinacrine) were developed by using a wide selection of neutral and anionic cyclodextrin (CD) derivatives. The use of sulfobutyl-β-CD and carboxymethyl-β-CD (CMBCD) resulted in good resolution of quinacrine and tafenoquine, respectively. New results are presented for resolutions of chloroquine and mefloquine. Application of carboxyalkyl- and sulfobutyl-CD derivatives provided improved resolution for primaquine. The impurity in primaquine sample detected by CE was identified as quinocide by MS and NMR. CMBCD provided not only the best separation of primaquine from quinocide but also the simultaneous complete resolution of both compounds.

Diastereoselective synthesis of 1,2,3,6-tetrahydrophosphinine 1-oxides with an exocyclic P-function by a Michael type addition

The anions generated from diphenylphosphine oxide or dialkyl phosphites add easily at the α,β-double-bond of 1,2-dihydrophosphinine oxides 1 to afford a single diastereomer of 3-substituted tetrahydrophosphinine oxides 2–4 existing in a twist-boat conformation.

Induced chirality upon binding of cis-parinaric acid to bovine β-lactoglobulin: spectroscopic characterization of the complex

Binding of the polyunsaturated cis‐parinaric acid to bovine β‐lactoglobulin (BLG) was studied by circular dichroism (CD), electronic absorption spectroscopy and mass spectrometry methods. Upon protein binding, the UV absorption band of parinaric acid is red shifted by ca. 5 nm, showing hypochromism and reduced vibrational fine structure, suggesting that the ligand binds as a monomer in non‐planar geometry. In the CD spectra measured at pH 7.36 and 8.5 a strong, negative Cotton band appears centered at 310 nm (Δϵ=−25 M−1 cm−1) corresponding to the long‐wavelength absorption band of cis‐parinaric acid. The source of this induced optical activity is the helical distortion of the polyene chromophore caused by the chiral protein environment. From CD spectral data the value of the association constant was calculated to be 4.7×105 M−1 at pH 7.36. CD and mass spectrometry measurements showed that parinaric acid binds weakly to BLG in acidic solution, though small peaks at mass 18559 and 18645 can be obtained in the reconstructed electrospray mass spectrum; these correspond to the binding of parinaric acid in 1:1 stoichiometry to both monomer variants of BLG B and A. The hydrophobic interior cavity of BLG was assigned as the primary binding site of cis‐parinaric acid.

endo and exo Ring fusion in the Diels–Alder reaction of 1-(2,4,6-trialkylphenyl-)3-methylphospholes with maleic acid derivatives

Abstract Diels–Alder reaction of the title phospholes and N -phenylmaleimide afforded, surprisingly, a mixture of endo and exo fused cycloadducts with the P-aryl substituent anti to the double bond giving, after oxidation the corresponding P-oxides. The P-center of the exo ring fused P-oxides was found to be inverted under the conditions of the oxidation. The cycloaddition of triisopropylphenylphosphole with N -methylmaleimide or with maleic acid anhydride gave the corresponding endo fused phosphanorbornenes affording stable products after oxidation. Relative stability of the possible isomers was evaluated experimentally and by quantum chemical calculations.

Specific behavior of the p-aminothiophenol--silver sol system in their Ultra-Violet-Visible (UV-Visible) and Surface Enhanced Raman (SERS) spectra.

The UV-Visible and Surface Enhanced Raman Spectroscopy (SERS) behavior of silver sol (a typical SERS agent) were studied in the presence of different bifunctional thiols such as p-aminothiophenol, p-mercaptobenzoic acid, p-nitrothiophenol, p-aminothiophenol hydrochloride, and 2-mercaptoethylamine hydrochloride in diluted aqueous solution. Our results confirm that the p-aminothiophenol induced aggregation of citrate stabilized silver colloid originates from its electrostatic nature, as well as the azo-bridge formation cannot be the reason of the observed time dependent UV-Visible spectra. Based on our parallel SERS and electrospray ionization mass spectrometry measurements, we have concluded that certain amount of oxidized form of the probe molecule has to be present for the so-called b2-mode enhancement in the SERS spectrum of p-aminothiophenol. Our findings seem to support the idea that the azo-bridge formation is responsible for the b2-mode enhancement in the SERS spectrum of p-aminothiophenol.

Pegylated liposomal formulation of doxorubicin overcomes drug resistance in a genetically engineered mouse model of breast cancer

Abstract Success of cancer treatment is often hampered by the emergence of multidrug resistance (MDR) mediated by P‐glycoprotein (ABCB1/Pgp). Doxorubicin (DOX) is recognized by Pgp and therefore it can induce therapy resistance in breast cancer patients. In this study our aim was to evaluate the susceptibility of the pegylated liposomal formulation of doxorubicin (PLD/Doxil®/Caelyx®) to MDR. We show that cells selected to be resistant to DOX are cross‐resistant to PLD and PLD is also ineffective in an allograft model of doxorubicin‐resistant mouse B‐cell leukemia. In contrast, PLD was far more efficient than DOX as reflected by a significant increase of both relapse‐free and overall survival of Brca1−/−;p53−/− mammary tumor bearing mice. Increased survival could be explained by the delayed onset of drug resistance. Consistent with the higher Pgp levels needed to confer resistance, PLD administration was able to overcome doxorubicin insensitivity of the mouse mammary tumors. Our results indicate that the favorable pharmacokinetics achieved with PLD can effectively overcome Pgp‐mediated resistance, suggesting that PLD therapy could be a promising strategy for the treatment of therapy‐resistant breast cancer patients. Graphical abstract Figure. No Caption available.

Simultaneous determination of thirteen different steroid hormones using micro UHPLC-MS/MS with on-line SPE system

Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LOQ). Micro UHPLC coupled to sensitive tandem mass spectrometry provides state of the art solution for such analytical problems. Using on-line SPE with column switching on a micro UHPLC-MS/MS system allowed to decrease LOQ without any complex sample preparation protocol. The presented method is capable of reaching satisfactory low LOQ values for analysis of thirteen different steroid molecules from human plasma without the most commonly used off-line SPE or compound derivatization. Steroids were determined by using two simple sample preparation methods, based on lower and higher plasma steroid concentrations. In the first method, higher analyte concentrations were directly determined after protein precipitation with methanol. The organic phase obtained from the precipitation was diluted with water and directly injected into the LC-MS system. In the second method, low steroid levels were determined by concentrating the organic phase after steroid extraction. In this case, analytes were extracted with ethyl acetate and reconstituted in 90/10 water/acetonitrile following evaporation to dryness. This step provided much lower LOQs, outperforming previously published values. The method has been validated and subsequently applied to clinical laboratory measurement.

Pushing quantitation limits in micro UHPLC–MS/MS analysis of steroid hormones by sample dilution using high volume injection

Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LoQ). Micro UHPLC coupling with sensitive tandem mass spectrometry provides state of the art solutions for such analytical problems. Decreased column volume in micro LC limits the injectable sample volume. However, if analyte concentration is extremely low, it might be necessary to inject high sample volumes. This is particularly critical for strong sample solvents and weakly retained analytes, which are often the case when preparing biological samples (protein precipitation, sample extraction, etc.). In that case, high injection volumes may cause band broadening, peak distortion or even elution in dead volume. In this study, we evaluated possibilities of high volume injection onto microbore RP-LC columns, when sample solvent is diluted. The presented micro RP-LC-MS/MS method was optimized for the analysis of steroid hormones from human plasma after protein precipitation with organic solvents. A proper sample dilution procedure helps to increase the injection volume without compromising peak shapes. Finally, due to increased injection volume, the limit of quantitation can be decreased by a factor of 2-5, depending on the analytes and the experimental conditions.

Efficient Synthesis of Mixed Phosphonates by the Fragmentation‐Related Phosphorylation of Alcohols Applying a 2‐phosphabicyclo[2.2.2]octa‐5,7‐Diene 2‐Oxide Precursor

Abstract The photochemically induced phosphonylation of C1–C4 alcohols by the fragmentation of a P‐ethoxy 2‐phosphabicyclo[2.2.2]octa‐5,7‐diene 2‐oxide (1) yielded the phosphonates (3) with two different alkoxy group efficiently, without the slightest extent of transesterification.