Novel octreotide dicarba-analogues with high affinity and different selectivity for somatostatin receptors

A limited set of novel octreotide dicarba-analogues with non-native aromatic side chains in positions 7 and/or 10 were synthesized. Their affinity toward the ssts1-5 was determined. Derivative 4 exhibited a pan-somatostatin activity, except sst4, and derivative 8 exhibited high affinity and selectivity toward sst5. Actually, compound 8 has similar sst5 affinity (IC50 4.9 nM) to SRIF-28 and octreotide. Structure-activity relationships suggest that the Z geometry of the double-bond bridge is that preferred by the receptors. The NMR study on the conformations of these compounds in SDS(-d25) micelles solution shows that all these analogues have the pharmacophore beta-turn spanning Xaa7-D-Trp8-Lys9-Yaa10 residues. Notably, the correlation between conformation families and affinity data strongly indicates that the sst5 selectivity is favored by a helical conformation involving the C-terminus triad, while a pan-SRIF mimic activity is based mainly on a conformational equilibrium between extended and folded conformational states.

Informatics for cross-sample analysis with comprehensive two-dimensional gas chromatography and high-resolution mass spectrometry (GCxGC-HRMS)

This paper describes informatics for cross-sample analysis with comprehensive two-dimensional gas chromatography (GCxGC) and high-resolution mass spectrometry (HRMS). GCxGC-HRMS analysis produces large data sets that are rich with information, but highly complex. The size of the data and volume of information requires automated processing for comprehensive cross-sample analysis, but the complexity poses a challenge for developing robust methods. The approach developed here analyzes GCxGC-HRMS data from multiple samples to extract a feature template that comprehensively captures the pattern of peaks detected in the retention-times plane. Then, for each sample chromatogram, the template is geometrically transformed to align with the detected peak pattern and generate a set of feature measurements for cross-sample analyses such as sample classification and biomarker discovery. The approach avoids the intractable problem of comprehensive peak matching by using a few reliable peaks for alignment and peak-based retention-plane windows to define comprehensive features that can be reliably matched for cross-sample analysis. The informatics are demonstrated with a set of 18 samples from breast-cancer tumors, each from different individuals, six each for Grades 1-3. The features allow classification that matches grading by a cancer pathologist with 78% success in leave-one-out cross-validation experiments. The HRMS signatures of the features of interest can be examined for determining elemental compositions and identifying compounds.

Determination of voriconazole in human serum and plasma by micellar electrokinetic chromatography

The micellar electrokinetic capillary chromatography (MEKC) separation and analysis of voriconazole and UK 115794 (internal standard) were examined and an assay for determination of voriconazole in human plasma and serum was developed. The MEKC medium comprises a 2:15 (v/v) mixture of methanol and a pH 9.3 buffer composed of 5mM Na(2)B(4)O(7), 7 mM Na(2)HPO(4) and 54 mM SDS. Sample preparation is based upon liquid/liquid extraction with ethylacetate and dichloromethane (75%/25%) at physiological pH. Using this approach with 250 microl serum or plasma and reconstitution of the dried extract into 100 microl of a buffer composed of 0.5mM Na(2)B(4)O(7) and 0.7 mM Na(2)HPO(4) (pH 9.3), the detection and quantitation limits were determined to be 0.1 and 0.2 microg/ml, respectively, a sensitivity that is suitable for therapeutic drug monitoring of voriconazole (provisional therapeutic range: 1-6 microg/ml) in human plasma and serum samples. The method was validated and compared to an HPLC method, showing excellent agreement between the two for a set of 91 samples that stemmed from patients being treated with voriconazole. The MEKC assay is also demonstrated to be suitable to explore pharmacokinetic data of voriconazole.

Analysis of llicit and illicit drugs in waste, surface and lake water samples using large volume direct injection high performance liquid chromatography – Electrospray tandem mass spectrometry (HPLC–MS/MS)

http://www.sciencedirect.com/science/article/pii/S0045653510008891

Development and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of nine corticosteroid residues in bovine liver samples

A liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory method for the simultaneous determination of nine corticosteroids in liver, including the four MRL compounds listed in Council Regulation 37/2010, was developed. After an enzymatic deconjugation and a solvent extraction of the liver tissue, the resulting solution was cleaned up through an SPE Oasis HLB cartridge. The analytes were then detected by liquid chromatography-negative-ion electrospray tandem mass spectrometry, using deuterium-labelled internal standards. The procedure was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/EC criteria. The results showed that the method was suitable for statutory residue testing regarding the following performance characteristics: instrumental linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit (CCα), detection capability (CCβ) and ruggedness. All the corticosteroids can be detected at a concentration around 1 μg kg(-1); the recoveries were above 62% for all the analytes. Repeatability and reproducibility (within-laboratory reproducibility) for all the analytes were below 7.65% and 15.5%, respectively.

Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery

Trypanosoma brucei and related pathogens transcribe most genes as polycistronic arrays that are subsequently processed into monocistronic mRNAs. Expression is frequently regulated post-transcriptionally by cis-acting elements in the untranslated regions (UTRs). GPEET and EP procyclins are the major surface proteins of procyclic (insect midgut) forms of T. brucei. Three regulatory elements common to the 3' UTRs of both mRNAs regulate mRNA turnover and translation. The glycerol-responsive element (GRE) is unique to the GPEET 3' UTR and regulates its expression independently from EP. A synthetic RNA encompassing the GRE showed robust sequence-specific interactions with cytoplasmic proteins in electromobility shift assays. This, combined with column chromatography, led to the identification of 3 Alba-domain proteins. RNAi against Alba3 caused a growth phenotype and reduced the levels of Alba1 and Alba2 proteins, indicative of interactions between family members. Tandem-affinity purification and co-immunoprecipitation verified these interactions and also identified Alba4 in sub-stoichiometric amounts. Alba proteins are cytoplasmic and are recruited to starvation granules together with poly(A) RNA. Concomitant depletion of all four Alba proteins by RNAi specifically reduced translation of a reporter transcript flanked by the GPEET 3' UTR. Pulldown of tagged Alba proteins confirmed interactions with poly(A) binding proteins, ribosomal protein P0 and, in the case of Alba3, the cap-binding protein eIF4E4. In addition, Alba2 and Alba3 partially cosediment with polyribosomes in sucrose gradients. Alba-domain proteins seem to have exhibited great functional plasticity in the course of evolution. First identified as DNA-binding proteins in Archaea, then in association with nuclear RNase MRP/P in yeast and mammalian cells, they were recently described as components of a translationally silent complex containing stage-regulated mRNAs in Plasmodium. Our results are also consistent with stage-specific regulation of translation in trypanosomes, but most likely in the context of initiation.

On-line SPE LC-MS/MS for the quantification of Δ9-tetrahydrocannabinol (THC) and its two major metabolites in human peripheral blood by liquid chromatography tandem mass spectrometry

A universal and robust analytical method for the determination of Δ9-tetrahydrocannabinol (THC) and two of its metabolites Δ9-(11-OH)-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-carboxy-tetrahydrocannabinol (THC-COOH) in human whole blood was developed and validated for use in forensic toxicology. Protein precipitation, integrated solid phase extraction and on-line enrichment followed by high-performance liquid chromatography separation and detection with a triple quadrupole mass spectrometer were combined. The linear ranges used for the three cannabinoids were from 0.5 to 20 ng/mL for THC and 11-OH-THC and from 2.5 to 100 ng/mL for THC-COOH, therefore covering the requirements for forensic use. Correlation coefficients of 0.9980 or better were achieved for all three analytes. No relevant hydrolysis was observed for THC-COOH glucuronide with this procedure--in contrast to our previous GC-MS procedure, which obviously lead to an artificial increase of the THC-COOH concentration due to the hydrolysis of the glucuronide-conjugate occurring at high pH during the phase-transfer catalyzed methylation step.