Negative Polarity Mode Research Articles

Identification of canine mammary tumor-associated metabolites using untargeted metabolomics

The canine mammary tumor is the most common tumor type in female dogs and seriously threatens their life. Currently, no effective treatments are available for this condition. Hence, it is essential to identify biomarkers that positively influence the early diagnosis and treatment and prognosis of this disease. To provide a basis for early diagnosis of canine breast tumors, in this study, 23 dogs with mammary tumors were identified via histopathological examination combined with ancillary diagnoses via blood examinations and diagnostic imaging. The canine mammary tumor and tumor-adjacent healthy tissues were collected, and their metabolites were identified utilizing a UHPLC-qTOF-MS-based untargeted metabolomics approach. The metabolic results revealed a total of 979 ion features in the positive polarity mode and 371 ion features in the negative polarity mode in the tissues of two groups; among them, 536 differential metabolites (385 in the positive and 151 in the negative polarity mode) were analyzed by PCA and PLS-DA. Subsequently, the enrichment pathways purine metabolism, alpha-linolenic acid metabolism, vitamin B6 metabolism, and fatty acid biosynthesis were analyzed using Metaboanalyst 4.0, which suggested that these pathways were valuable diagnostic and therapeutic targets. Moreover, the receiver operating characteristic curves further confirmed 13Z,16Z-docosadienoic acid, 23-nordeoxycholic acid, and (±)12(13)-DiHOME as expected candidate biomarkers of canine mammary tumors. In conclusion, the discovery of tumor biomarkers based on untargeted metabolomics is informative for pathological mechanism studies and facilitates the early diagnosis of canine mammary tumors.

Combination of metabolome and proteome analyses provides insights into the mechanism underlying growth differences in Acipenser dabryanus

To analyze the differences between different-sized Acipenser dabryanus, we randomly selected 600 3-month-old A. dabryanus juveniles. Four months later, the blood and white muscle of these fish were analyzed. The results showed no significant difference in the length-weight relationship (LWR) b value between the large and small A. dabryanus. The levels of serum growth hormone (gh) and insulin-like growth factor 1 (igf1) in the large A. dabryanus were significantly lower than those in the small, whereas the activity levels of Total superoxide dismutase (T-sod) and catalase (cat) were opposite to the results of gh and igf1. A total of 212 and 245 metabolites showed significant changes in the positive and negative polarity mode, respectively. Among 3,308 proteins identified, 69 proteins showed upregulated expression, and 185 proteins showed downregulated expression. These results indicated that the growth advantage of A. dabryanus was closely related to glycolysis, protein synthesis, and antioxidant function.

Identification of Canine Pyometra-Associated Metabolites Using Untargeted Metabolomics.

Canine pyometra frequently occurs in middle-aged to older intact bitches, which seriously affects the life of dogs and brings an economic loss to their owners. Hence, finding a key metabolite is very important for the diagnosis and development of a new safe and effective therapy for the disease. In this study, dogs with pyometra were identified by blood examinations, laboratory analyses and diagnostic imaging, and fifteen endometrium tissues of sick dogs with pyometra and fifteen controls were collected and their metabolites were identified utilizing a UHPLC-qTOF-MS-based untargeted metabolomics approach. The results indicated that the elevated inflammatory cells were observed in dogs with pyometra, suggesting that sick dogs suffered systemic inflammation. In the untargeted metabolic profile, 705 ion features in the positive polarity mode and 414 ion features in the negative polarity mode were obtained in endometrium tissues of sick dogs with pyometra, with a total of 275 differential metabolites (173 in positive and 102 in negative polarity modes). Moreover, the multivariate statistical analyses such as PCA and PLS-DA also showed that the metabolites were significantly different between the two groups. Then, these differential metabolites were subjected to pathway analysis using Metaboanalyst 4.0, and Galactose metabolism, cAMP signaling pathway and Glycerophospholipid metabolism were enriched, proving some insights into the metabolic changes during pyometra. Moreover, the receiver operating characteristic curves further confirmed kynurenic acid was expected to be a candidate biomarker of canine pyometra. In conclusion, this study provided a new idea for exploring early diagnosis methods and a safe and effective therapy for canine pyometra.

MALDI Matrices for the Analysis of Low Molecular Weight Compounds: Rational Design, Challenges and Perspectives.

The analysis of low molecular weight (LMW) compounds is of great interest to detect small pharmaceutical drugs rapidly and sensitively, or to trace and understand metabolic pathways. Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI MS) plays a central role in the analysis of high molecular weight (bio)molecules. However, its application for LMW compounds is restricted by spectral interferences in the low m/z region, which are produced by conventional organic matrices. Several strategies regarding sample preparation have been investigated to overcome this problem. A different rationale is centred on developing new matrices which not only meet the fundamental requirements of good absorption and high ionization efficiency, but are also vacuum stable and “MALDI silent”, i. e., do not give matrix‐related signals in the LMW area. This review gives an overview on the rational design strategies used to develop matrix systems for the analysis of LMW compounds, focusing on (i) the modification of well‐known matrices, (ii) the search for high molecular weight matrices, (iii) the development of binary, hybrid and nanomaterial‐based matrices, (iv) the advance of reactive matrices and (v) the progress made regarding matrices for negative or dual polarity mode.

Studying dynamic aroma release by headspace-solid phase microextraction-gas chromatography-ion mobility spectrometry (HS-SPME-GC-IMS): method optimization, validation, and application.

To understand aroma perception from complex food matrices' determination of dynamic aroma release during simulated oral processing is necessary. In this study optimization, validation and application of a novel method coupling headspace-solidphase microextraction (HS-SPME) with gas chromatography-ion mobility spectrometry (GC-IMS) is presented. Thirteen character impact compounds imparting different chemical properties are studied to understand capabilities and limitations of the method. It was shown for the first time that the temperature of the IMS sample inlet can be increased up to 200°C without instrumental constraints. Linear calibration was possible for eleven of the thirteen compounds with one decade dynamic range. The limit of detection and quantitation were 2.1-63.0ppb and 7.2-210.1ppb, respectively. Diacetyl could be detected in negative polarity mode of IMS, however with lower precision compared to the compounds detected in positive mode. Limitations of the method were short HS-SPME extraction time, which in the case of caproic acid was not sufficient for reliable quantification. Additionally, δ-decalactone could not be detected due to maximum GC temperature of 200°C. Application of the method to determine dynamic aroma release from a dairy matrix was successfully shown for nine compounds. Analysis of complex food matrix was performed with similar precision compared to analysis in aqueous solution, thus proving high robustness of the method towards matrix effects.

Time-of-flight secondary ion mass spectrometry and X-ray photoelectron spectroscopy study of 2-phenylimidazole on brass.

This work presents the first surface analysis investigation of 2-phenylimidazole (PhI) as a corrosion inhibitor for brass in a 3 wt.% NaCl solution using time-of-flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy (XPS). A time-of-flight secondary ion mass spectrometer was used to describe the elemental and molecular specific signals on the brass surface. Gas cluster/monoatomic ion beam depth profiling and two-dimensional (2D) imaging showed the surface properties of the PhI surface layer. In addition, detailed XPS was used to describe the element-specific signals on the brass surface. Furthermore, principal component analysis demonstrated the distribution of the different phases of the surface. Finally, the applicability of PhI in hot corrosion studies was suggested by a thermal stability experiment. TOF-SIMS measurements showed an intense positive-ion TOF-SIMS signal for protonated PhI, i.e. C9 H9 N2 + at m/z 145.07. Moreover, there was an intense negative-ion TOF-SIMS signal for deprotonated PhI, i.e. C9 H7 N2 ¯ at m/z 143.06. Gas cluster ion beam sputtering associated with the analysis of the XPS-excited Cu L3 M4,5 M4,5 revealed a connection between Cu(I) and PhI to form organometallic complexes. The formation of the organometallic complexes with Cu and Zn metal atoms/ions was identified using TOF-SIMS in positive and negative polarity mode: PhI-Cu, PhI-Cu2 , PhI-Zn, and PhI-Zn2 complexes were present on the brass surface. The TOF-SIMS measurements were supported by XPS measurements.

Modern methods for regulation nicotine containing products

Issues of assessing the content of carbonyl compounds in the aerosol of innovative nicotine-containing products: nicotine delivery systems of various designs (LUXLITE, VON ERL My, iKuu i200) and electric tobacco heating system iQOS, by high-performance liquid chromatography/mass spectrometry. At the international level (CORESTA, ISO/TC 126), a lot of work is being done to create methods for quality control of nicotine-containing products. Requirements for nicotine-containing products are not regulated within the EAEU. The monitoring of world studies to determine the content of toxic and conditionally toxic components of aerosol NSP, carried out using the method of machine Smoking. Vniitti IS developing a methodology for comprehensive evaluation of innovative nicotine-containing products on the basis of modern methods of product quality control. Of particular interest is the determination of the content of carbonyl compounds in the aerosol generated by SDN and ESNT, which started in the laboratory of tobacco production technology within the Eurasian Economic Union. The results of machine Smoking according to the canadian intensive method, ISO method, VNIITTI method, standard and experimental CORESTA methods are analyzed. Developed testing methodology and collection of aerosol-nicotine produce using a laboratory five-CERULEAN Smoking machines SM 405. The technique of quantitative determination of carbonyl compounds using high-performance liquid chromatomass spectrometry LCMS/MS by detection at an electrospray source in the negative polarity mode on analytical equipment was validated: chromatograph Thermo Scientific Dionex UltiMate 3000 and mass spectrometer TSQ Quantiva. Validation of methods for collecting and determining the content of carbonyl compounds (formaldehyde, acetaldehyde and acrolein) in the aerosol of nicotine-containing products of various types allowed to obtain reliable data to establish the potential of innovative products to reduce the risk compared with traditional cigarettes. Experimental data on the quantitative determination of carbonyl compounds in the NSP aerosol were obtained. Further research will focus on the development of evidence-based safety requirements for innovative types of nicotine-containing products.

Study of Scale Formation on Al2O3-Scale Forming Alloys by Time-of-Flight Secondary Ion Mass Spectrometry (TOF–SIMS)

Abstract Al2O3-scale formation on high temperature Ni-base and Fe-base structural alloys is a subject of significant technological and scientific interest. Such scale formation involves either (or both) inward oxygen grain boundary diffusion and new oxide growth at the scale/metal interface, or outward aluminum grain boundary diffusion and new oxide growth at the scale/gas interface. The details of scale growth have been studied using a commercial Kanthal A1 alloy and so-called double oxidation experiments—growth of an Al2O3 scale in 16O2, followed by further oxidation for twice the 16O2 duration in 18O2—at temperatures between 1000 and 1400°C. A time-of-flight secondary ion mass spectrometer (TOF-SIMS) with isotopic imaging resolution of ≈100 nm was used for determination of the 16O and 18O distributions in the scale. A focused primary Ga+ ion beam was used for imaging the oxygen isotopes in the negative polarity mode, and a de-focused Xe+ beam was used for sputtering. One novel aspect of this work was the use of taper sections to compensate for the sub-micrometer scale thickness of this oxidation-resistant alloy.

Negative Polarity Helium Charge Transfer Dissociation Tandem Mass Spectrometry: Radical-Initiated Fragmentation of Complex Polysulfated Anions.

This work provides the first use of helium charge transfer dissociation (He-CTD) tandem mass spectrometry (MS/MS) in negative polarity mode. Three sulfated oligosaccharides of natural origin were chosen as representative structures that are difficult to solve by conventional MS/MS approaches. Negative polarity He-CTD provided a full set of structurally informative fragments, which permitted the unambiguous determination of the complete structures of these molecules, including the characterization of labile sulfated functional groups. Despite close structural features, the three molecules led to distinct fragmentation patterns depending on the position of the sulfate group in the heterocycle. The observed fragments showed a consistent radical-initiated mechanism of dissociation, which shares similarities with fragment types produced in electron detachment dissociation (EDD), negative electron transfer dissociation (NETD), or extreme UV photodissociation (XUV-PD). Short times of data collection and the fact that the technique can be affordably implementable in any standard laboratory and with a classical ion trap mass spectrometer were other remarkable characteristics of negative polarity He-CTD.

Online preconcentration by electrokinetic supercharging for separation of endocrine disrupting chemical and phenolic pollutants in water samples.

Online preconcentration using electrokinetic supercharging (EKS) was proposed to enhance the sensitivity of separation for endocrine disrupting chemical (methylparaben (MP)) and phenolic pollutants (2-nitrophenol (NP) and 4-chlorophenol (CP)) in water sample. Important EKS and separation conditions such as the concentration of BGE; the choice of terminating electrolyte (TE); and the injection time of leading electrolyte (LE), sample, and TE were optimized. The optimum EKS-CE conditions were as follows: BGE comprising of 12 mM sodium tetraborate pH 10.1, 100 mM sodium chloride as LE hydrodynamically injected at 50 mbar for 30 s, electrokinetic injection (EKI) of sample at -3 kV for 200 s, and 100 mM CHES as TE hydrodynamically injected at 50 mbar for 40s. The separation was conducted at negative polarity mode and UV detection at 214 nm. Under these conditions, the sensitivity of analytes was enhanced from 100- to 737-fold as compared to normal CZE with hydrodynamic injection, giving LOD of 4.89, 5.29, and 53μg/L for MP, NP and CP, respectively. The LODs were adequate for the analysis of NP and CP in environmental water sample having concentration at or lower than their maximum admissible concentration limit (240 and 2000 μg/L for NP and CP). The LOD of MP can be suitable for the analysis of MP exists at mid-microgram per liter level, even though the LOD was slightly higher than the concentration usually found in water samples (from ng/L to 1 μg/L). The method repeatabilities (%RSD) were in the range of 1.07-2.39% (migration time) and 8.28-14.0% (peak area).

Polyalanine – a practical polypeptide mass calibration standard for matrix‐assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry in positive and negative mode

Mass calibration in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is currently obtained using mixtures of individual peptides. This procedure has several drawbacks, including laborious preparation, limited shelf life and unequal calibration mass spacing. Polyalanine, a simple to prepare polydisperse molar mass standard, alleviates these problems. Polyalanine is prepared by the typical protocols for biological sample analytics. Polyalanine is the first polymeric standard providing abundant signals in both, positive and negative polarity mode with the typical matrices DHB and HCCA. Facile MS as well as MS/MS calibration is thus enabled for the first time in both polarity modes by employing this polydisperse standard.

Liquid chromatography/negative electrospray ionization ion trap MS2 mass spectrometry application for the determination of microcystins occurrence in Southern Portugal water reservoirs

Microcystins (MCs) are toxins produced by cyanobacteria which are common organisms in the phytoplankton of eutrophic lakes, rivers and freshwater reservoirs. In the present work, a novel method of liquid chromatography–electrospray ion trap tandem mass spectrometry (LC/ESI/Ion trap–MS/MS), operated in the negative ionization mode, was developed for the analysis of these cyanotoxins. The method was applied to determine the amounts of total microcystins-LR, -YR and -RR in two water reservoirs in Southern Portugal, namely Alqueva and Beliche. A total of 30 water samples were analysed along 2011. Solid phase extraction (SPE) was used for sample cleaning-up and analyte enrichment. The extracted toxins were separated on a C18 column with a gradient of acetonitrile/water with 0.1% formic acid. Detection of microcystins was carried out using multiple reaction monitoring (MRM) in the negative polarity mode, as this method gave a higher selectivity. The MC-RR, YR and LR quantification limits were 17.9, 31.7 and 15.8 ng/L, respectively; quite below the limits recommended by WHO guidelines for drinking water (1 μg/L). Total MC highest concentrations were found in the warm months of June, July and September in Alqueva sampling sites, with concentrations of MC LR and RR ranging 17–344 and 25–212 ng/L, respectively, showing comparable results for MC-RR and LR and slightly lower concentration of MC-YR. Detected values for Beliche reservoir were below quantification limits.

Metabolism schemes of chlorogenic, quinic and caffeic acid derivatives in rats deduced by molecular fragments detected with optimized UPLC–APCI-MS/MS conditions

The synthesized chlorogenic, quinic, and caffeic acid derivatives with antifungal activity were investigated on their metabolism and biotransformation properties in rats detected by optimized UPLC–APCI-MS/MS conditions. The metabolic scheme was deduced from the molecular fragments detected in biotransformed samples in vivo. The ion sources of APCI and ESI under positive or negative ion polarity mode for detection molecular fragments were compared to create the optimized UPLC–MS/MS conditions. The plasma and biliary samples from rats after oral or intravenous administration of the compounds were analyzed for their biotransformation in vivo. The absorption, distribution, hepatic metabolism, and disposal from systemic circulation of the substrates, and isomerization of their metabolites would be finished in 4h after oral administration. The detection of the metabolites after HCHO, H2N-orn-4-(octyloxy) aniline, and CH4 elimination, and isomerization of their metabolites illustrated for the metabolic pathway of chlorogenic and quinic acid derivates in vivo. Caffeic acid was found to dissociate easily from chlorogenic and caffeic acid derivatives in vivo, whereas quinic acid derivative seemed more stable in the systemic circulation of rats but had the tendency to isomerize in hepatic metabolism. In addition, in vitro incubation of the 3 substrates with pooled rat liver microsomes confirmed that CYP450 played the role in catalysis for biotransformation of these compounds.

Rapid simultaneous identification and determination of the multiple compounds in crude Fructus Corni and its processed products by HPLC–MS/MS with multiple reaction monitoring mode

Context: Fructus Corni, a traditional Chinese medicines, is derived from the dry ripe sarcocarp of Cornus officinalis Sieb. et Zucc (Cornaceae). Gallic acid, 5-hydroxymethylfurfural, morroniside, sweroside, loganin, cornin, 7-O-methyl-morroniside and cornuside are the active constituents of Fructus Corni used in many traditional Chinese medicines. This paper describes a sensitive and specific assay for the determination of the eight bioactive compounds in crude and processed Fructus Corni extracts.Materials and methods: In this paper, the eight components were determined by high performance liquid chromatography coupled with triple quadrupole mass spectrometry (MS/MS). Quantization was based on multiple reaction monitoring using the precursor production combination for determination of the eight analytes. The analysis was performed on an Agilent Zorbax Extend C18 column (100 mm × 3.0 mm, 3.5 μm), and an electrospray ionization (ESI)-tandem interface in the positive and negative ion polarity mode was employed prior to mass spectrometric detection.Results: With the optimized conditions, the eight bioactive compounds were detected properly within 10 min. The developed method showed good precision and reproducibility with the limits of detection ranged from 0.0042 to 12.7875 ng/mL and the average recoveries ranged from 97.08 to 103.7%.Discussion and conclusion: This newly established method is validated as simple, reliable and accurate. It can be used as a valid analytical method for intrinsic quality control of crude and processed Fructus Corni.

Aquifex aeolicus tRNA (N2,N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA

Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m(2)(2)G26) in tRNA. In the reaction, N2-guanine at position 26 (m(2)G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNA(Cys) has an m(2)(2)G26m(2)G27 or m(2)(2)G26m(2)(2)G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m(2)G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme.

Crystal Structure of Soluble Domain of Malaria Sporozoite Protein UIS3 in Complex with Lipid

Malaria parasite UIS3 (up-regulated in infective sporozoites gene 3) is essential for sporozoite development in infected hepatocytes. UIS3 encodes for a membrane protein that is localized to the parasite parasitophorous vacuolar membrane in infected hepatocytes. We describe here 2.5-Å resolution crystal structure of Plasmodium falciparum UIS3 soluble domain (PfUIS3130-229) in complex with the lipid phosphatidylethanolamine (PE). PfUIS3130-229 is a novel, compact, and all α-helical structure bound to one molecule of PE. The PfUIS3130-229-PE complex structure reveals a novel binding site with specific interactions between PfUIS3130-229 and the PE head group. One acyl chain of PE wraps around part of PfUIS3130-229 and docks onto a hydrophobic channel. We additionally provide new structural and biochemical evidence of PfUIS3130-229 interactions with lipids (phosphatidylethanolamine), with phospholipid liposomes, and with the human liver fatty acid-binding protein. The direct interaction of PfUIS3130-229 with liver fatty acid-binding protein most likely provides the parasite with a conduit for importing essential fatty acids/lipids. Therefore, our analyses have implications for lipid transport into the parasite during the rapid growth phases of sporozoites. Given that PfUIS3 is essential for establishment of liver stage infection by P. falciparum, our data provide a new target for abrogating parasite development within liver cells before typical symptoms of malaria can manifest.

Determination of ethyl sulfate – a marker for recent ethanol consumption – in human urine by CE with indirect UV detection

A CE method for the determination of the ethanol consumption marker ethyl sulfate (EtS) in human urine was developed. Analysis was performed in negative polarity mode with a background electrolyte composed of 15 mM maleic acid, 1 mM phthalic acid, and 0.05 mM cetyltrimethylammonium bromide (CTAB) at pH 2.5 and indirect UV detection at 220 nm (300 nm reference wavelength). This buffer system provided selective separation conditions for EtS and vinylsulfonic acid, employed as internal standard, from urine matrix components. Sample pretreatment of urine was minimized to a 1:5 dilution with water. The optimized CE method was validated in the range of 5-700 mg/L using seven lots of urine. Intra- and inter-day precision and accuracy values, determined at 5, 60, and 700 mg/L with each lot of urine, fulfilled the requirements according to common guidelines for bioanalytical method validation. The application to forensic urine samples collected at autopsies as well as a successful cross-validation with a LC-MS/MS-based method confirmed the overall validity and real-world suitability of the developed expeditious CE assay (sample throughput 130 per day).

Capillary electrophoresis hyphenated to inductively coupled plasma-sector field-mass spectrometry for the detection of chromium species after incubation of chromium in simulated sweat

The presence of chromium in chromium-tanned leather represents a considerable health problem since it can lead to chronic allergic contact dermatitis. Apart from trivalent chromium (Cr(III)), which is used for tanning, leather often contains hexavalent chromium (Cr(VI)), resulting from the oxidation of Cr(III) during the tanning process. This study deals with the chromium compounds in simulated sweat when brought into contact with Cr(III) or Cr(VI) and with chromium-tanned leathers. A capillary electrophoresis (CE) method was developed, with inductively coupled plasma-sector field-mass spectrometry (ICP-SF-MS) for element-specific detection. Two different electrophoretic runs, applying once the positive and once the negative polarity mode, were necessary for the detection of positively and negatively charged chromium species. Although sometimes described in the literature, a pre-run derivatization of the chromium-species was not performed here to prevent species transformation. 50 mmol.L(-1) sodium phosphate at a pH of 2.5 was used as CE separation buffer and as make-up liquid for the CE-ICP-SF-MS interface. When applied to simulated sweat samples incubated with Cr(VI), this method showed that methionine is responsible for the reduction of Cr(VI) into Cr(III), which, at its turn, forms a complex with lactic acid. In the case of sweat plus Cr(III), the latter step was also seen. Applied to simulated sweat in contact with leather samples, the method developed showed the presence of the former species among a much more complex pattern.

Ion trap liquid chromatography/tandem mass spectrometry analysis of leukotriene B4 in exhaled breath condensate

The objective of this study is the measurement of leukotriene B7 (LTB4), a potent inflammatory mediator, in exhaled breath condensate by using liquid chromatography/mass spectrometry (LC/MS and LC/MS/MS). Condensation of exhaled breath is a non-invasive method to collect airway secretions. Deuterated (d4)-LTB4 was used as internal standard. The MS and MS/MS behavior of LTB4 and LTB4-d4 was studied by electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in both positive and negative ion polarity mode. Preliminary results show that monitoring negative ions in ESI mode has the best sensitivity for both LTB4 and LTB4-d4. Therefore, negative ESI was chosen, and the [M-H]- ions at m/z 335 and 339 were selected for quantification. The lower limit of quantification for LTB4, expressed as the lowest point of the calibration curve, was 100 pg/mL. Using this technique, we measured LTB4 in exhaled breath condensate in two healthy subjects, four asthmatic patients on anti-inflammatory treatment, and four asthmatic patients who were not on anti-inflammatory drugs. Exhaled LTB4 concentrations were detected only in asthmatic patients who were not on anti-inflammatory therapy. This method is potentially useful for non-invasive assessment of airway inflammation, but the sensitivity of the technique needs to be improved.

Chiral anion exchangers applied to capillary electrochromatography enantioseparation of oppositely charged chiral analytes: investigation of stationary and mobile phase parameters

Weak anion-exchange (WAX) type chiral stationary phases (CSPs) based on tert.-butyl carbamoyl quinine as chiral selector (SO) and different types of silica particles (porous and non-porous) as chromatographic support are evaluated in packed capillary electrochromatography (CEC). Their ability to resolve the enantiomers of negatively charged chiral analytes, e.g., N-derivatized amino acids, in the anion-exchange mode and their electrochromatographic characteristics are described in dependence of several mobile phase parameters (pH, buffer type and concentration, organic modifier type and concentration) and other experimental variables (electric field strength, capillary temperature). The inherent “zwitterionic” surface character of such silica-based WAX type CSPs (positively charged SO and negatively charged residual silanols) allows the reversal of the electroosmotic flow (EOF) towards the anode at pH values below the isoelectric point (p I) of the modified surface, whereas a cathodic EOF results at pH values above the p I. Since for negatively charged analytes also an electrophoretic transport increment has to be considered, which can be either in or against the EOF direction, several distinct modes of elution have been observed under different stationary phase and mobile phase conditions: (i) co-electrophoretic elution of the negatively charged solutes with the anodic EOF in the negative polarity mode, (ii) counter-electrophoretic elution with the cathodic EOF in the positive polarity mode, and (iii) electrophoretically dominated elution in the negative polarity mode with a cathodic EOF directed to the injection end of the capillary. Useful enantioseparations of chiral acids have been obtained with all three modes. Enantioselectivity values as high as under pressure-driven conditions and theoretical plate numbers up to 120 000 per meter could be achieved under electrically driven conditions. A repeatability study yielded RSD values below 2% for retention times and RSD values in the range of 5–10% for theoretical plate numbers and resolution, thus clearly establishing the reliability of the investigated anion-exchange type CEC enantioseparation methods.