between maps, which further complicates the analysis. In this work, we have
implemented two significant improvements to the PF2D chromatofusing step. First,
we used a novel prototypic electrode (p/n A48657) that has been designed not only to
accurately measure changes in pH, but also to give accurate pH measurements
Materials and methods
Experiments were carried out on the commercially available GCxGC-TOFMS Pegasus 4D ® system (Leco Corp., St Joseph, MI, USA). The modulation was obtained from a ‘quad jet’ system (dual-stage modulator) based on the use of a dual cryogenic (liquid nitrogen cooled) cold jet and a dual hot jet installed on an Agilent 6890 GC unit.
manuka (Leptospermum scoparium), rewarewa (Knightia excelsa), kamahi (Weinmannia racemosa), and tawari (Ixerba brexioides), acquired in a local market in New Zealand.
Derivatization Procedure. Derivatization (oximation plus trimethyl- silylation) of disaccharides was carried out according to Sanz et al. (7). Standard mixtures were separately prepared by diluting 10 mg of different saccharides into 10 mL of ethanol/water (70:30, v/v). One milliliter of each solution was mixed with 0.5 mL of internal standard (phenyl- β- D -gluco- side, 1 mg mL -1 ). The mixture was evaporated to dryness under vacuum. Oximes were formed by the addition of 350 μL of 2.5% hydroxylamine chloride in pyridine and heating for 30 min at 75 °C. Silylation of these derivatives was further performed by adding 350 μL of hexamethyldisi- lazane and 35 μL of trifluoroacetic acid and heating for 30 min at 45 °C. After reaction, samples were centrifuged at 8000 rpm for 10 min, and supernatant was taken for analysis. To eliminate the excess of derivatiza- tion reagents, supernatant was submitted to a liquid -liquid extraction using hexane/water (1:1, v/v). After vortex mixing and separation of phases, the organic phase was used for analysis.
which is heated by a resistive film painted onto the capil- lary surface and cooled by ambient air. This modulator is difficult to operate and has only a short lifetime. A similar thermal modulator using a wire, instead of the painted film, has higher durability, but sluggish thermal response (de Geus et al., 1997). A more robust thermal modulator using a rotat- ing heated sweeper showed good performance; however, the operation temperature of the GC oven must be about 100 ◦ C lower than the maximum allowed temperature of the station- ary phase in the modulation capillary (Phillips et al., 1999). Instead of using heating, Kinghorn and Marriott (1998b) de- veloped a modulator using cooling. This modulator regu- larly traps and releases solutes from the first column by mov- ing a cryogenic trap back and forth along the second col- umn. While achieving good performance, the prototype of this modulator showed problems with ice build-up, which were overcome in modified designs (Kinghorn et al., 2000; Beens et al., 2001b). Both the heated sweeper technique and the moving cryogenic trap technique have a common draw- back, i.e. frequent breakage of capillaries by moving parts in the system. More recent development of the modulation technique is the jet-cooled modulator, which uses no mov- ing parts. Adapted from the jet-cooled thermal modulator (Ledford and Billesbach, 2000), a jet-cooled/heated modula- tor was reported by Ledford (2000). The modulator employs two cold and two hot nitrogen jets that are pulsed to alter- nately cool and heat two spots at the front end of the second column for focusing and remobilizing analytes eluting from the first column. While this type of modulator allows excel- lent modulation of compounds even as volatile as methane, use of liquid nitrogen is limited by its availability and re- quires bulky facilities for storage and insulation. A practi- cal solution has already been demonstrated by Beens et al. (2001a), who used a CO 2 -cooled jet modulator and obtained
Because of the use of a regular GC oven and liquid nitrogen as the cryogenic ﬂuid for modulation, our approach did not allow us to consider a signiﬁcant part of the MTS-VP VOCs that comprise highly volatile compounds (below butane). The use of lower trapping tem- peratures during TD sampling and trapping, as well as a dedicated negative temperature GC oven ﬁtted with a ﬂow modulator would be of interest to extend the present work to low boiling compounds. Nevertheless, analytes listed in Table 2 signiﬁcantly enlarge the list of the few previous reports on MTS-VP components and offer a more comprehensive overview as the majority of the previous studies relied on target analysis and were limited in terms of the description of the VOC proﬁle. Only one study previously reported 92 tentatively identiﬁed compounds in MTS-VP from 1R4F refer- ence cigarette material using a dedicated GC–MS technique capable to isolate molecular weights in the range from 28 to 136 (C1–C10)  . Our approach was limited to the range from 69 to 218 (C4–C14) but we attempted to make a fair comparison with the study of Dong et al.  by considering the retention time window from 3-methyl-
cryogenic-free thermal modulator and flow-based systems (3). The development of the liquid nitrogen-based modulator by Zoex ® represents the first robust commercial modulator system. Unfortunately, even if very efficient, the large consumption of cryo-fluids has discouraged many potential industrial users. The development of flow modulators certainly permitted to compensate that and opened the door to a lower running cost GC×GC solution. This was however accompanied by more complex method development needs and limitations regarding the coupling to vacuum outlet MS systems due to the required high career gas flow of such flow modulators. INTRODUCTION
COMPREHENSIVE TWO-DIMENSIONAL GAS CHROMATOGRAPHY COUPLED TO TIME-OF-FLIGHT MASS SPECTROMETRY FOR THE ANALYSIS OF VAPOUR PHASE MAINSTREAM CIGARETTE SMOKE
Benjamin Savareear 1 , Radoslaw Lizak 1 , Jean-Francois Focant 1 , Michal Brokl 2 , Christopher Wright 2
Statistical Methods in Quality by Design Approach to LiquidChromatography Methods Development Hermane T. Avohou, Cédric Hubert, Benjamin Debrus, Pierre Lebrun, Serge Rudaz, Bruno Boulanger,and Philippe Hubert
Analytical quality by design (AQbD) approach is more and more advocated for the development of analytical methods. In brief, AQbD is a systematic and risk-based approach to method development and optimization that begins with predefined objectives, seeks method understanding and defines a method control strategy based on scientific knowledge and quality risk management tools. Contrary to the classical quality-by-testing (QbT) approach, which is rather unstructured and proceeds mostly by trial-and-error, AQbD approach is a structured and science-based. It enables an efficient search for optimal conditions and a deeper understanding of the underlying separation processes. As results, effective optimization of the method, robustness building and quality risks management as required by regulations may be more easily achieved. A key output of the AQbD strategy is the design space, which defines an envelope of operable region of method parameters that guarantees with a high probability acceptable method performances in routine.
is monotonously increasing (asymptoting to constant).
The first case is physically more relevant, because in most cases of interest dissipation dominates over forcing at the small scales. In this case E(y) behaves qualitatively similar as in the two examples considered in section IV. Namely, if we take the same Gaussian forcing as in these two examples, we will have E(y) which has a maximum at y = 0, smooth everywhere (including y = 0) and rapidly decaying to zero for y → ∞. However, there is a big difference from the previous examples in that now the characteristic width of function E(y), and respectively the width of the spectrum in the k y variable, is of the same order as
11] . Consequently, the number of reports and applications utilizing GC × GC has increased as illustrated in Figure S1 in the supporting information (SI). Increased separation capability, however, does not necessarily solve the general challenge in chromatography, namely coelution, or facilitate the extraction of meaningful chemical infor- mation. In fact, it demands fast detector acquisition techniques re- sulting in information rich data sets with higher order complexity and ﬁle size  especially when coupled to sophisticated detec- tion techniques. Evaluation of these datasets is considered a major challenge in GC × GC and the community agrees that growth, de- velopment and a certain degree of automation is needed [ 4 , 6 , 13– 17 ] in this particular area. It is thereby little surprising that the variety and availability of dedicated GC × GC software packages (SPs) rose within the last years.
Optimal in vitro metabolism of APAP was examined by GSH trapping experiments (data not shown). Oxidizing RLM and CYP3A4 Supersomes were each tested with either NADPH, or NADP + and a NADPH regenerating system. RLM incubation in combination with a NADPH regenerating system and 3A4 Supersomes in the presence of NADPH yielded optimal conditions for APAP in vitro metabolism. For both strategies, a combination of 1 h open tube incubation followed by 2 h closed tube was found best for oxidation. It must be noted that GST stock solutions contained reduced GSH, which also bound to NAPQI (data not shown). Denaturation and excessive buffer exchange of GSTs, to yield completely GSH-free stocks, did not permit the study of binding capabilities based on protein structure. Additionally, excessive treatment of proteins led to important sample loss, different for each protein (data not shown). Furthermore, inclusion of a His-tag during expression (for subsequent Ni-nitrilotriacetic acid purification) might alter protein folding and thus binding results ( Ledent et al., 1997 ). The use of non-mutant GSTs purified over GSH affinity combined with solvent-free elution seemed optimal in this study. These facts were considered and APAP concentrations in final incubations were adjusted accordingly to assure detectable amounts of NAPQI-GST being formed. Subsequently, protein digestion, SPE and offline fractionation were optimized for highest sequence coverage of target GST from database search results of DDA runs. Trypsin and pepsin digestion (4 and 1 h, respectively) in parallel was found optimal to yield a sequence coverage >83% (data not shown). Standard iodo-APAP-GST was used to test cysteine coverage and detection of all possible modified cysteine (see Table 2). Only the sites Cys15 and 102 were detected with a confidence <95%, based on small sequence size and low fragmentation of tryptic C 15 (iodo-APAP)AALR, and poor abundance of peptic GVEDLRC 102 (iodo-APAP)KYISL. For peptide separation, high-pH RP and fraction concatenation was used as an alternative strategy to conventional ion exchange offline fractionation ( Zhou, 2003; Yang et al., 2012 ). This enabled orthogonal separation to online (low-pH) RP chromatography without a desalting SPE step, thus reducing potential sample loss ( Wang et al., 2011; Di Palma et al., 2012 ).
- the simulated two-dimensional fields look realistic, they moreover
have coherent statistical properties (cumulative rain rate distribution, power spectrum and structure function) with observed one.
- The proposed simulation processes is very general and can be adapted to any climatic area
Due to increasing insights in the diverse roles of peptides in physiological processes, peptide-based pharmaceuticals are expanding as a relatively novel class of strategies for treatment. Indeed, peptides represent an attractive point for the design of novel therapeutics. Their specificity has been observed to translate into excellent safety, tolerability, and efficacy profiles in humans. The biological functions of peptides are multiple, including carrying signals between glands and cells, structural and mechanical roles, controlling body functions, acting as chemical transporters, process regulators and many others . They are also used as drugs for the treatment of diseases, typical examples being insulin, growth hormones, and therapeutic antibodies . The unprecedented number of marketing approvals in 2012 for peptide therapeutics could be a harbinger for the innovative peptide-based drugs . New synthetic strategies for limiting metabolism and alternative routes of administration have emerged in recent years and resulted in a large number of peptide-based drugs that are now being marketed . In parallel, the increasing importance of biopharmaceuticals necessitates performance improvements in bioanalytical techniques. In particular, their quantification in complex biological samples such as human synovial fluid (HSF) requires acute sensitivity and selectivity, as all biological matrices contain a countless number of proteins, all made of the same 20 amino acids (AA) precursors. In the present study, we focused on two peptides of interest for the treatment of osteoarthritis, namely; BQ123 (Cyclo(-D-Trp-D-Asp-L-Pro-DVal- L-Leu) and R-954 (AcOrn[Oic 2 ,( αMe)Phe 5 , DβNal 7 , Ile 8 ]desArg 9 -bradykinin). Osteoarthritis is the most common form of arthritis, affecting millions of people in the United States and around the world. It is a complex disease whose etiology bridges biomechanics and biochemistry . No curative treatment exists currently and only antiinflammatory drugs are routinely prescribed for patient relief. Recently, the therapeutic effects of peptides on arthritis have been associated with reduction of the autoimmune and inflammatory responses [6, 7]. In that context, both BQ123 and R-954 have shown promising activities. BQ123 is an endothelin receptor A (ETA) antagonist  which blocks the ETA receptor, resulting in a decrease of lipid peroxidation products , increase of the reduced glutathione (GSH) level, and enhanced super oxidase dismutase (SOD) activity . Moreover, the blockage of the ETA receptor alleviates the lipopolysaccharide (LPS)- induced oxidative stress . LPS is the main causative agent inducing sepsis, stimulating macrophages to excrete large
Fig. 1: Comparison between recoveries obtained by OCLLE and classical LLE.
The Polaris C18-A column is used to be dedicated to drug and drug metabolite discovery [26,27]. The silica phase of this HPLC column is bonded to octadecyl chain with a polar group maximizing polar retention and selectivity, and eliminating silanol residues. This allowed to cover a broad range of chemically different compounds. LC gradient has been optimized to distinguish the 17 pesticides keeping in mind that coeluted compounds showing different masses could be separated by the mass spectrometer using multiple reaction monitoring (MRM) mode. In order to achieve the best compromise between time analysis and sensitivity, the number of transitions in a single window has been limited to 12. As for each precursor compound, two product ions have been recorded; this represented a maximum of six pesticides monitored by acquisition window. An example of chromatogram is presented in Fig. 2b for a methanolic standard solution showing pesticide
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Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
(Received 5 October 2011; published 21 October 2011)
We use fifth-order two-dimensional electronic spectroscopy to measure coherent four-particle dynamics in a semiconductor nanostructure. By using optical polarization control in two-quantum measurements enabled by the COLBERT spectrometer, we separate coherent signals due to bound biexcitons and unbound two-exciton correlations. The rephasing nature of the measurement allows us to separate homogeneous from inhomogeneous contributions to the two-quantum line shapes. We find that, unlike the bound biexciton state, the energy of the unbound pair and its homogeneous linewidth depend on the laser fluence. Simulations using an extended phenomenological model help determine the primary interaction mechanism that leads to the formation of the unbound exciton pair; the model also indicates that seventh-order interactions contribute to the measured spectra under high pulse fluences.