Sample handling and contamination encountered when coupling offline normal phase high performance liquid chromatography fraction collection of petroleum samples to Fourier transform ion cyclotron resonance mass spectrometry

Normal phase high performance liquid chromatography (HPLC) is used to separate a gas oil petroleum sample, and the fractions are collected offline and analyzed on a high resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICR MS). The separation prior to MS analysis dilutes the sample significantly; therefore the fractions need to be prepared properly to achieve the best signal possible. The methods used to prepare the HPLC fractions for MS analysis are described, with emphasis placed on increasing the concentration of analyte species. The dilution effect also means that contamination in the MS spectra needs to be minimized. The contamination from molecular sieves, plastics, soap, etc. and interferences encountered during the offline fraction collection process are described and eliminated. A previously unreported MS contamination of iron formate clusters with a 0.8 mass defect in positive mode electrospray is also described. This interference resulted from the stainless steel tubing in the HPLC system. Contamination resulting from what has tentatively been assigned as palmitoylglycerol and stearoylglycerol was also observed; these compounds have not previously been reported as contaminant peaks.     

Fluorescent properties and resonance energy transfer of 3,4-bis(2,4-difluorophenyl)-maleimide

A fluorescent compound 3,4-bis(2,4-difluorophenyl)-maleimide from the 3,4-diaryl-substituted maleimides was synthesized and determined to have a Stokes shift of 140 nm (λ(abs) 341 nm, λ(em) 481 nm), a high fluorescent quantum yield (Φ(fl) 0.61) and an extinction coefficient ε((340)) of 48?400 M(-1) cm(-1) in dichloromethane. For the first time we demonstrated the successful implementation of a 3,4-diaryl-substituted maleimide molecule as a donor component in FRET experiments.     

A neurosteroid analogue photolabeling reagent labels the colchicine-binding site on tubulin: a mass spectrometric analysis

Previous studies have shown that the neurosteroid analogue, 6-Azi-pregnanolone (6-AziP), photolabels voltage-dependent anion channels and proteins of approximately 55 kDa in rat brain membranes. The present study used two-dimensional electrophoresis and nanoelectrospray ionization ion-trap mass spectrometry (nano-ESI-MS) to identify the 55 kDa proteins (isoelectric point 4.8) as isoforms of β-tubulin. This identification was confirmed by immunoblot and immunoprecipitation of photolabeled protein with anti-β-tubulin antibody and by the demonstration that 6-AziP photolabels purified bovine brain tubulin in a concentration-dependent pattern. To identify the photolabeling sites, purified bovine brain tubulin was photolabeled with 6-AziP, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization MS (MALDI). A 6-AziP adduct of TAVCDIPPR(m/z?= 1287.77), a β-tubulin specific peptide, was detected by MALDI. High-resolution liquid chromatography-MS/MS analysis identified that 6-AziP was covalently bound to cysteine 354 (Cys-354), previously identified as a colchicine-binding site. 6-AziP photolabeling was inhibited by 2-methoxyestradiol, an endogenous derivative of estradiol thought to bind to the colchicine site. Structural modeling predicted that neurosteroids could dock in this colchicine site at the interface between α- and β-tubulin with the photolabeling group of 6-AziP positioned proximate to Cys-354.     

Musical acoustics of orchestral water crotales

An experimental investigation of orchestral crotale vibrational modes in water is presented, along with a qualitative virtual mass model describing the observed effects. Changes in frequency, overtone ratio, and mode splitting as a function of water depth are reported for a C(6) crotale using electronic speckle-pattern interferometry. These data are related to perceived changes in pitch and timbre, along with the creation of audible beats at particular water depths. It is also shown that the suspension method used by musicians when dipping crotales into water leads to the creation of additional acoustically significant modes that are not excited when the crotales are mounted in the standard manner.     

Synthesis and Evaluation of a Macrocyclic Actinium-225 Chelator,Quality Control and In Vivo Evaluation of 225Ac-crown-αMSH Peptide

Targeted alpha-therapy (TAT) has great potential for treating a broad range of late-stage cancers by delivering a focused and lethal radiation dose to tumors. Actinium-225 (²²⁵Ac) is an emerging alpha emitter suitable for TAT; however, the availability of chelators for Ac remains limited to a small number of examples (DOTA and macropa). Herein, we report a new Ac macrocyclic chelator named ‘ crown’ , which binds quantitatively and rapidly (<10 min) to Ac at ambient temperature. We synthesized ²²⁵Ac- crown -αMSH, a peptide targeting the melanocortin 1 receptor (MC1R), specifically expressed in primary and metastatic melanoma. Biodistribution of ²²⁵Ac- crown -αMSH showed favorable tumor-to-background ratios at 2 h post injection in a preclinical model. In addition, we demonstrated dramatically different biodistrubution patterns of ²²⁵Ac- crown -αMSH when subjected to different latency times before injection. A combined quality control methodology involving HPLC, gamma spectroscopy and radioTLC is recommended.     

A Netform System for Resource Planning in the U.S. Bureau of Land Management

We report an effective model and solution procedure for a major resource management problem of the U.S. Bureau of Land Management (B.L.M.), enabling large practical applications to be solved 20-300 times faster than previously possible. The resulting system has proved a useful analysis tool, providing model interaction capabilities that are employed routinely by the B.L.M. an average of 700 times a month throughout the U.S.     

Intelligent automation of high-performance liquid chromatography method development by means of a real-time knowledge-based approach

We describe the development, attributes and capabilities of a novel type of artificial intelligence system, called LabExpert, for automation of HPLC method development. Unlike other computerised method development systems, LabExpert operates in real-time, using an artificial intelligence system and design engine to provide experimental decision outcomes relevant to the optimisation of complex separations as well as the control of the instrumentation, column selection, mobile phase choice and other experimental parameters. LabExpert manages every input parameter to a HPLC data station and evaluates each output parameter of the HPLC data station in real-time as part of its decision process. Based on a combination of inherent and user-defined evaluation criteria, the artificial intelligence system programs use a reasoning process, applying chromatographic principles and acquired experimental observations to iteratively provide a regime for a priori development of an acceptable HPLC separation method. Because remote monitoring and control are also functions of LabExpert, the system allows full-time utilisation of analytical instrumentation and associated laboratory resources. Based on our experience with LabExpert with a wide range of analyte mixtures, this artificial intelligence system consistently identified in a similar or faster time-frame preferred sets of analytical conditions that are equal in resolution, efficiency and throughput to those empirically determined by highly experienced chromatographic scientists. An illustrative example, demonstrating the potential of LabExpert in the process of method development of drug substances, is provided.