Offline pentafluorophenyl (PFP)-RP prefractionation as an alternative to high-pH RP for comprehensive LC-MS/MS proteomics and phosphoproteomics.

Technological advances in liquid chromatography and tandem mass spectrometry (LC-MS/MS) have enabled comprehensive analyses of proteins and their post-translational modifications from cell culture and tissue samples. However, sample complexity necessitates offline prefractionation via a chromatographic method that is orthogonal to online reversed-phase high-performance liquid chromatography (RP-HPLC). This additional fractionation step improves target identification rates by reducing the complexity of the sample as it is introduced to the instrument. A commonly employed offline prefractionation method is high pH reversed-phase (Hi-pH RP) chromatography. Though highly orthogonal to online RP-HPLC, Hi-pH RP relies on buffers that interfere with electrospray ionization. Thus, samples that are prefractionated using Hi-pH RP are typically desalted prior to LC-MS/MS. In the present work, we evaluate an alternative offline prefractionation method, pentafluorophenyl (PFP)-based reversed-phase chromatography. Importantly, PFP prefractionation results in samples that are dried prior to analysis by LC-MS/MS. This reduction in sample handling relative to Hi-pH RP results in time savings and could facilitate higher target identification rates. Here, we have compared the performances of PFP and Hi-pH RP in offline prefractionation of peptides and phosphopeptides that have been isolated from human cervical carcinoma (HeLa) cells. Given the prevalence of isobaric mass tags for peptide quantification, we evaluated PFP chromatography of peptides labeled with tandem mass tags. Our results suggest that PFP is a viable alternative to Hi-pH RP for both peptide and phosphopeptide offline prefractionation.

Fabrication and evaluation of a fluorophilic adsorbent for multiple monolithic fiber solid-phase microextraction of fluorobenzenes.

A new type of highly fluorinated monolith (HFM) was fabricated and used as adsorbent of multiple monolithic fiber solid-phase microextraction (MMF-SPME). To prepare the HFM, a fluorinated monomer, 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptyl acrylate was in situ copolymerized with dual cross-linkers (divinylbenzene and ethylenedimethacrylate). The fabrication parameters including the content of monomer and porogenic solvent in the polymerization mixture were optimized to obtain expected extraction performance and life span. The physicochemical properties of the HFM were systematically investigated with elemental analysis, infrared spectroscopy, scanning electron microscopy and mercury intrusion porosimetry. The effective extraction of six fluorobenzenes was selected as a paradigm to demonstrate the fluorophilic characteristic of HFM/MMF-SPME. At the same time, a convenient and effective method for the determination of trace fluorobenzenes in environmental water samples was developed by coupling HFM/MMF-SPME with high performance liquid chromatography/diode array detection (HFM/MMF-SPME-HPLC/DAD). Results indicated that the limits of detection (S/N=3) for targeted compounds were in the range of 1.09-5.88µg/L. The intra-day and inter-day precision (relative standard deviations, n=4, %) at two spiked concentrations were 4.2-10.6% and 6.1-10.8%, respectively. Finally, the developed method was successfully applied to the analysis of fluorobenzenes in spiked real water samples with satisfactory recoveries and repeatability.

Bacteria detection based on the evolution of enzyme-generated volatile organic compounds: determination of Listeria monocytogenes in milk samples.

The rapid detection of Listeria monocytogenes contamination in food is essential to prevent food-borne illness in humans. The aim of this study was to differentiate non-contaminated milk from milk contaminated with L. monocytogenes using enzyme substrates coupled with the analysis of volatile organic compounds (VOCs). The method is based on the activity of ß-glucosidase and hippuricase enzymes and the detection of a specific VOC i.e. 2-nitrophenol and 3-fluoroaniline, respectively. VOCs were extracted, separated and detected by headspace-solid phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME GC-MS). This approach required the inclusion of the selective agent's cycloheximide, nalidixic acid and acriflavine HCl in the growth medium to inhibit interfering bacteria. The VOCs were liberated by L. monocytogenes provided that samples contained at least 1-1.5×10(2) CFU ml(-1) of milk prior to overnight incubation. This approach shows potential for future development as a rapid method for the detection of L. monocytogenes contaminated milk.

Determination of rosuvastatin in urine by spectrofluorimetry after liquid-liquid extraction and derivatization in acidic medium.

Statins are a class of drugs mostly used for treating hyperlipidemia, and rosuvastatin is the newest drug in the market belonging to this class. In this present work, a method was developed based on the molecular fluorescence technique, with the objective to quantify rosuvastatin in urine samples. For this purpose, the study of several parameters was made to achieve the maximum analytical signal (under reaction with sulfuric acid during 40 min). Also, a previous step to avoid matrix interference was carried out (liquid-liquid extraction). The limit of detection (LOD) and the limit of quantification (LOQ) were 0.38 and 1.28 mg L(-1), respectively. Linear relationship between rosuvastatin concentration and it's fluorescence intensity was found until 5.0 mg L(-1). The proposed method was tested in several samples spiked with rosuvastatin and recovery was found in the range of 90 ± 10%.

Hydrodefluorination and hydrogenation of fluorobenzene under mild aqueous conditions.

Fluorinated organic compounds are increasingly used in many applications, and their release to the environment is expected. It is therefore important to find suitable methods for degradation of fluorinated compounds under environmentally relevant conditions. In this study, a simple heterogeneous rhodium-based catalytic system (Rh/Al(2)O(3) and H(2)) for hydrodefluorination and hydrogenation of fluorobenzene under mild aqueous conditions (1 atm of H(2), ambient temperature) was developed and the underlying reaction mechanism was investigated. Fluorobenzene degraded rapidly (t(1/2) ≈ 0.2 h) to form cyclohexane and fluoride (F(-)) as the stable end products, with benzene and cyclohexene observed as intermediates. Cyclohexadiene intermediates were not observed but were expected to form during the hydrogenation of benzene. Three postulated but unobserved fluorinated intermediates were subjected to the catalytic reaction conditions, and it was concluded that they most likely do not form during the fluorobenzene degradation reaction. Isotope labeling experiments showed that the unsaturated intermediates undergo rapid and reversible hydrogenation/dehydrogenation under the reaction conditions and also that fully saturated compounds are unreactive in the catalytic system. Both molecular hydrogen and water were sources of hydrogen in the final cyclohexane product. Kinetic fitting indicated that sorption/desorption of fluorobenzene onto the catalyst surface plays an important role in the mechanism.

Occurrence and fate of rosuvastatin, rosuvastatin lactone, and atorvastatin in Canadian sewage and surface water samples.

Rosuvastatin (RST) and atorvastatin (ATO) are prescription drugs and members in the statin family used for the treatment of elevated cholesterol levels. A method using solid-phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of ATO, RST and its metabolite rosuvastatin lactone (RSTL) in sewage and surface water samples has been developed. In the influent and effluent samples collected from 11 sewage treatment plants located in Ontario, Canada, ATO, RST, and RSTL were detected in all samples with median concentrations of 166 ng L(-1) (influent) and 77 ng L(-1) (effluent) for ATO, 448 ng L(-1) (influent) and 324 ng L(-1) (effluent) for RST, as well as 158 ng L(-1) (influent) and 41 ng L(-1) (effluent) for RSTL. Due to the inter-conversion between RST and RSTL, the total concentration of RST and RSTL in a sewage sample should be reported. The median removal rate by wastewater treatment was 66% for ATO and 22% for RST and RSTL combined. These statins were quite persistent in sewage. After a storage period of 21 and 62 days, there was only a slight decrease in ATO concentration and no change in the total RST concentrations. These three compounds were also detected in a number of surface water samples at low ng L(-1) concentrations. This is the first reported occurrence and fate of RST and RSTL in the Canadian aquatic environment.

A new Met inhibitory-scaffold identified by a focused forward chemical biological screen.

The receptor tyrosine kinase Met is crucial for the genetic program causing cancer progression and metastasis. Its nodal function during aggressiveness and resistance acquisition poses Met inhibition as an obligatory step in anti-cancer targeted therapy. Here, we applied a "Met-focussed" forward chemical biological screen to discover new agents antagonizing Met-triggered biological functions. The identified new scaffold, JLK1360, has a dual mechanism of action towards Met: it impairs Met signalling and also prevents its restoration after degradation. Docking and molecular dynamics provide evidences on the interacting mode of JLK1360 within the Met ATP-binding pocket. Moreover, computational and biochemical studies also highlighted that JLK1360 has a good degree of selectivity towards Met than other RTKs tested. Altogether, these findings demonstrate that the approach we have applied is a powerful strategy to identify compounds with combined properties towards a chosen target. Our studies show how integration of chemistry, biology and computational analysis can provide robust strategies to identify new inhibitory scaffolds suitable for further development of anti-cancer targeted therapies.

Determination of the enantiomer of a cholesterol-lowering drug by cyclodextrin-modified micellar electrokinetic chromatography.

The development, optimization and application of a chiral CE (capillary electrophoresis) method for the determination of the enantiomer content of a new cholesterol-lowering drug (BMS-180431-09) is discussed. The chiral CE technique, cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC), was employed with hydroxypropyl beta-cyclodextrin as the chiral selector in the run buffer. The detection limit of the unwanted enantiomer was about 0.06% w/w. The effect of various parameters on the separation, validation data and examples of the application of the chiral CE method are included.