Electrospray Interface Research Articles

Effects of inorganic seeds on secondary organic aerosol formation from photochemical oxidation of acetone in a chamber

Photochemical oxidations of acetone were studied under different inorganic seed (NaCl, (NH4)2SO4 and NaNO3) conditions in a self-made chamber. The results show that no secondary organic aerosol (SOA) can be formed in the experiments either in the absence of artificially added seed particles or in the presence of solid status of the added particles. Liquid water content is the key factor for the formation of SOA in the experiments with seeds. The amount of SOA was only about 4–7 μg m−3 in the experiments with the initial acetone of ∼15 ppm under different seed conditions. The analysis of SOA compositions by Exactive-Orbitrap mass spectrometer equipped with electro-spray interface (ESI-MS) shows that chlorine-containing and sulfur-containing compounds were detected in SOA formed from the experiments with NaCl and (NH4)2SO4 seeds, respectively, which were not identified in SOA from those with NaNO3. The compositions of SOA were mainly esters, organonitrates, hydroperoxides, etc. It is concluded that inorganic seed particles participated into the formation of SOA. Acetone SOA was mainly formed in the aqueous phase in which dissolved SOA precursors underwent further oxidation reactions, esterification reactions and/or radical-radical reactions. Our experiments further demonstrate that low-molecular-weight VOCs, such as acetone, can form SOA under certain conditions in the atmosphere, although their contributions to SOA may not be large.

Determination of the bioavailability of selected pharmaceutical residues in fish plasma using liquid chromatography coupled to tandem mass spectrometry

Aquatic systems near major urban centers are constantly contaminated with effluent from wastewater treatment plants. Pharmaceuticals are part of the contamination and several classes of drugs have been detected in surface waters in the last decade. To better understand the impact of those pharmaceuticals in ecosystems, the exposure to aquatic species needs to be investigated. This study presents a new simple and rugged quantitative method for the determination of several classes of drugs using 100μL of plasma from fish environmentally exposed to a major but highly diluted urban effluent. Six common drugs (i.e., diclofenac, ibuprofen, naproxen, salbutamol, sulfamethoxazole and trimethoprim) and one major metabolite (2-hydroxy-ibuprofen), present in significant amount in impacted waterways have been selected for the development and validation of the method. First, all drugs were extracted using cation exchange solid phase extraction (SPE) and eluted with two solvent mixtures. Then, the extracts were analyzed using a reverse-phase analytical column Waters® CORTECS C18+ (150×2.1mm, 2.7μm) within 14min. MS/MS was performed with an electrospray (ESI) interface in positive ion mode, with multiple reaction monitoring (MRM) experiment acquiring two product ions per drugs. Quantification has been made with standard curves for each analyte using isotopically labeled internal standards. This method has high sensitivity with limits of quantification of 1ngmL−1 for each drug, except for ibuprofen and its metabolite 2-hydroxy-ibuprofen at 2ngmL−1. The precision of the method was below 11%, the accuracy between 94 and 105% and overall recovery between 94 and 111% for all drugs, with high selectivity. Application of the method to plasma samples from wild northern pike inhabiting the St. Lawrence River collected over a three-year period showed the presence of naproxen, diclofenac, trimethoprim and salbutamol at very low concentrations (around 1ngmL−1).

A sensitive UHPLC-MS/MS method for the simultaneous quantification of three lignans in human plasma and its application to a pharmacokinetic study.

The aim of this study was to develop an analytical method to simultaneously analyze schizandrin, schizandrol B, and gomisin N lignans in human plasma using ultra high performance liquid chromatography with tandem mass spectrometry. The three lignans were separated using a mobile phase of water and acetonitrile containing 0.02% acetic acid equipped with a Kinetex C18 column (2.1mm×50mm, 1.7μm). This analysis was achieved by multiple reaction monitoring mode in an electrospray interface. The mass transitions were m/z 433.1→384.0 for schizandrin, 398.8→367.8 for schizandrol B, and 400.6→299.8 for gomisin N. Liquid-liquid extraction with methyl tert-butyl ether was used to obtain the three lignans. The chromatograms showed high resolution, sensitivity, and selectivity with no interference with plasma constituents. The calibration curves for the three lignans in human plasma were 0.05-50ng/mL and displayed excellent linearity with correlation coefficients greater than 0.99. Precision for all three lignans was within 11.23%. The accuracy was 88.3-99.0% for schizandrin, 90.6-103.4% for schizandrol B, and 90.2-103.5% for gomisin N. The developed simultaneous analytical method satisfied the criteria of international guidance and could be successfully applied to the pharmacokinetic study of three lignans after oral administration of Schisandrae Fructus extract powder to humans.

Sensitive Analysis of Nitroguanidine in Aqueous and Soil Matrices by LC-MS.

Nitroguanidine, a widely used nitramine explosive, is an environmental contaminant that is refractory, persistent, highly mobile in soils and aquifers, and yet under-researched. Nitroguanidine determination in water and soil poses an analytical challenge due its high hydrophilicity, low volatility, charge neutrality over a wide pH range, and low proton affinity which results in low electrospray interface (ESI)-MS sensitivity. A sensitive method for the determination of nitroguanidine in aqueous and soil matrices was developed. The method is based on reduction by zinc in acidic solution, hydrophobization by derivatization, preconcentration on C18 cartridge, and LC-MS quantification. The demonstrated limit of detection (LOD) reaches 5 ng/L and 22 ng/g in water and soil, respectively. Analysis of a contaminated site demonstrates that it is possible to map a contamination plume that extends over 1 km from the source of the contamination.

Use of LC-HRMS in full scan-XIC mode for multi-analyte urine drug testing - a step towards a 'black-box' solution?

The influx of new psychoactive substances (NPS) has created a need for improved methods for drug testing in toxicology laboratories. The aim of this work was to design, validate and apply a multi-analyte liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for screening of 148 target analytes belonging to the NPS class, plant alkaloids and new psychoactive therapeutic drugs. The analytical method used a fivefold dilution of urine with nine deuterated internal standards and injection of 2μl. The LC system involved a 2.0μm 100×2.0mm YMC-UltraHT Hydrosphere-C18 column and gradient elution with a flow rate of 0.5 ml/min and a total analysis time of 6.0min. Solvent A consisted of 10mmol/l ammonium formate and 0.005% formic acid, pH 4.8, and Solvent B was methanol with 10mmol/l ammonium formate and 0.005% formic acid. The HRMS (Q Exactive, Thermo Scientific) used a heated electrospray interface and was operated in positive mode with 70 000 resolution. The scan range was 100-650 Da, and data for extracted ion chromatograms used ± 10ppm tolerance. Product ion monitoring was applied for confirmation analysis and for some selected analytes also for screening. Method validation demonstrated limited influence from urine matrix, linear response within the measuring range (typically 0.1-1.0 μg/ml) and acceptable imprecision in quantification (CV <15%). A few analytes were found to be unstable in urine upon storage. The method was successfully applied for routine drug testing of 17 936 unknown samples, of which 2715 (15%) contained 52 of the 148 analytes. It is concluded that the method design based on simple dilution of urine and using LC-HRMS in extracted ion chromatogram mode may offer an analytical system for urine drug testing that fulfils the requirement of a 'black box' solution and can replace immunochemical screening applied on autoanalyzers. Copyright © 2017 John Wiley & Sons, Ltd.

Leaf cuticle-assisted phototransformation of isoproturon

Isoproturon is a pre- and post-emergence herbicide used generally to manage Phalaris minor and Avena ludoviciana in wheat. The importance of post-emergence and foliarly applied pesticides has been increasing. The first reaction environment of these herbicides is the leaf surface made up of polymerised esters of higher fatty acids. In the presence of sunlight, the fatty substances on leaf surfaces generate reactive radicals, or ions which may interact with herbicide molecules leading towards degradation resulting in the loss of bioefficacy of herbicides and the formation of metabolites of unknown toxicity. The present experiment was conducted with the objective to know the photochemical behaviour of isoproturon on different leaf-extracted epicuticular cutin surfaces. Epicuticular waxes, i.e. cutins were extracted from P. minor and A. ludoviciana leaf surfaces using dichloromethane as a solvent and further purified with activated charcoal. On the cutin surface under UV-light (365 nm), the transformation of the herbicide is very slow as it is evident from its higher half-life values, 75 min on P. minor cutin surface and 115 min on A. ludoviciana cutin, as compared to that on the glass surface (half-life 53 min). A similar pattern was also observed under the sunlight condition. It may be due to the quenching effect imparted by the cutin material or simply screening effect of it on the herbicide. The sunlight-irradiated extracts of isoproturon and its degradation products were analysed by LC–MS/MS using electrospray interfacing technique and the structures of six different photoproducts were characterised by their respective spectra. The degradation pathways involved mainly demethylation and ring oxidation processes forming hydroxylated compounds. The mixture of these photoproducts were found to be non-toxic to A. ludoviciana and P. minor.

Capillary electrophoresis coupled to mass spectrometry employing hexafluoro-2-propanol for the determination of nucleosides and nucleotide mono-, di- and tri-phosphates in baby foods

The present work describes a method for the simultaneous determination of unmodified nucleosides and nucleotide mono-, di- and tri-phosphates by capillary electrophoresis coupled to mass spectrometry (CE-MS). The use of hexafluoro-2-propanol (HFIP) in the separation medium, and as an additive to the sheath liquid of the electrospray interface (ESI), generated a highly efficient and sensitive method. Instrumental limits of detection in the range of 14–53ngmL−1 for nucleosides and 7–23, 20–49 and 64–124ngmL−1 for nucleotide mono-, di-, and tri-phosphates, respectively, were found. Sample treatment involved diluting an aliquot of baby food with ultra-high quality water and applying centrifugation-assisted ultrafiltration (CUF). The proposed method was validated and used to analyse a variety of baby food samples (16 in total) such as fish, meat, fruits, and baby dairy desserts that may endogenously contain these analytes.

Phototransformation of isoproturon in soil

The present experiment was conducted with an objective to reveal the photochemical behaviour of isoproturon on different soil surfaces, viz. red, black and alluvial soil under sunlight. The half-life values of isoproturon on glass surface was found as 25.38 days. But on soil surfaces, the rate of photolysis was changed with half-life values of 20.76, 27.38 and 28.02 days under sunlight for red, black and alluvial soil, respectively. The slower reaction rate on the surfaces of black and alluvial soil was due to the quenching effect imparted by humic substances, which were absent on glass surface and less in red soil. The sunlight-irradiated extracts of isoproturon and its degradation products were analysed by LC-MS/MS using electrospray interfacing technique and the structures of six different photoproducts were characterised by their respective spectra as 3-(4-isopropyl-2/3-hydroxyphenyl)-1-methyl-1-hydroxymethylurea (I), 3-(4-isopropyl-2/3-hydroxyphenyl)-1-methyl-1-(N, N-dimethylcarbamoyl)urea (II), 3-(4-isopropyl-2/3-hydroxyphenyl)-1-methyl-1-(N-methylcarbamoyl)urea (III), 3-(4-isopropyl-2/3-hydroxyphenyl)-1-methyl-1-carbamoyl urea (IV), N-dimethyl-N-methyl urea (V), N-dimethyl-N-dimethyl urea (VI). The products were mainly formed through demethylation, ring oxidation and rearrangement. Thus, sunlight induced photodegradation may contribute in the dissipation of isoproturon in soil minimising the load of environmental hazards.

74 THE BOVINE EMBRYO INFLUENCES THE PROTEOME OF THE OVIDUCTAL FLUID

It has been shown that the equine embryo is able to modulate the proteome of the oviduct, increasing the presence of certain proteins involved in the embryo-maternal communication (Smits et al. 2016 Reprod. Fertil. Dev. doi: 10.1071/RD15481). In cattle, the presence of a single embryo did not affect the transcriptome of the oviduct, whereas multiple embryos induced changes (Maillo et al. 2015 Biol. Reprod. 92, 144). The aim of the present study was to examine the effect of the presence of an embryo on the oviduct fluid proteome. Cross-bred beef heifers were synchronized, and those in standing oestrus were randomly allocated to cyclic, not bred (n = 7), or pregnant, artificially inseminated (n = 11), groups. All heifers were slaughtered on Day 3 after oestrus. The oviducts from each animal were isolated, straightened, and cut, separating ampulla and isthmus. Each portion was flushed with 500 μL of PBS free of protein to confirm the presence of an oocyte/embryo. Recovered unfertilized oocytes (cyclic group) and embryos (pregnant group) were located in the isthmus of the oviduct ipsilateral to the corpus luteum. Flushings from 4 cyclic and 4 pregnant heifers (8-cell embryos) were used for proteomic analysis, including blank controls of PBS. Comparison of ipsilateral ampulla and isthmus in pregnant and cyclic heifers did not reveal any differences. Therefore, samples from ipsilateral oviducts were compared between cyclic and pregnant heifers. Total protein (150 μg) from cyclic and pregnant samples was labelled with different cyanine fluorescent probes and separated according to the isoelectric point using immobilized pH gradient strips (pH 3–10, 17 cm, Protean® IEF cell system, Bio Rad, Hercules, CA, USA). Second dimension was performed in a polyacrylamide gel (12%) in the presence of SDS using a Protean II XL system (Bio Rad). The images were obtained with the Typhoon 9410 scanner and analysed with the Progenesis SameSpots software v. 4.0. The results of image analysis showed 42 different spots between the 2 groups (ANOVA P &lt; 0.01 and fold difference &gt;1.5), the majority (38) of which were more abundant in pregnant heifers. Gels were stained with Coomassie blue to detect the spots. The differential spots were digested with trypsin and analysed with Agilent Ion-Trap XCT Plus mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray interface. Twenty spots were highly abundant in pregnant heifers. From these, 3 different proteins were identified with score &gt;5.0 and % SPI &gt;76.0 by mass spectrometry, corresponding to SERPINA1, serum albumin, and serum transferrin. These proteins are abundant in plasma, suggesting that the embryo may be able to activate pathways to increase the permeability of the vascular endothelium in the oviduct. The same proteins were also identified in the equine oviducal proteome in the presence of an embryo, suggesting a possible conserved mechanism between species. In conclusion, presence of an embryo in the bovine oviduct increases the abundance of some proteins that may be related with its early development. This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL-2012–37510 and AGL-2015–70140-R) and Science Foundation Ireland (13/IA/1983).

Simultaneous Determination of Nine Sulphonamides by LC-MS for Routine Control of Raw Honey Samples

A liquid chromatography-mass spectrometry method for the simultaneous determination of nine sulphonamides (sulphadiazine, sulphathiazole, sulphapyridine, sulphamerazine, sulphamethizole, sulphasoxazole, sulphamethoxypyridazine, sulphamethoxazole and sulphadimethoxine) in honey samples was developed. Sample pretreatment included acidic hydrolysis, followed by solid-phase extraction on a Sep-Pack® C18. Separation is carried out in a C18 column and mobile phases consisting of acetic acid/methanol mixtures. Detection is performed by mass spectrometry with ion trap, using electrospray interface in the positive ionization mode. Method validation has been performed according to European Commission Decision 2002/657/EC criteria, in terms of linearity, trueness, precision, specificity, decision limit (CCα) and detection capability (CCβ). Linearity was obtained with an average coefficient of determination (R2) higher than 0.9995 in matrix. Typical recoveries in fortified honey samples ranged between 89 and 118 % at all levels tested (5, 10, 50 and 100 μg/kg). The method demonstrated good intra- and interbatch precision and accuracy. No interferences with the peaks of interest were observed throughout the chromatographic run. The method has been successfully applied to the analysis of raw honey samples. Forty-five (21 %) of 215 analysed were positive (range 6.7–140.8 μg/kg) for sulphonamides.

A simple, fast and sensitive screening LC-ESI-MS/MS method for antibiotics in fish

The objective of this study was to develop and validate a fast, sensitive and simple liquid chromatography–electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the screening of six classes of antibiotics (aminoglycosides, beta-lactams, macrolides, quinolones, sulfonamides and tetracyclines) in fish. Samples were extracted with trichloroacetic acid. LC separation was achieved on a Zorbax Eclipse XDB C18 column and gradient elution using 0.1% heptafluorobutyric acid in water and acetonitrile as mobile phase. Analysis was carried out in multiple reaction monitoring mode via electrospray interface operated in the positive ionization mode, with sulfaphenazole as internal standard. The method was suitable for routine screening purposes of 40 antibiotics, according to EC Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines, taking into consideration threshold value, cut-off factor, detection capability, limit of detection, sensitivity and specificity. Real fish samples (n=193) from aquaculture were analyzed and 15% were positive for enrofloxacin (quinolone), one of them at a higher concentration than the level of interest (50µgkg−1), suggesting possible contamination or illegal use of that antibiotic.

Preparation and application of immunoaffinity column coupled with dcELISA detection for aflatoxins in eight grain foods

This paper described a reliable and simple analytical method for the determination of aflatoxins (AFB1, AFB2, AFG1, AFG2) in cereals, peanuts, vegetable oils and fermented foods like beer, soybean sauce and soybean paste based on immunoaffinity column (IAC) cleanup coupled with direct competitive enzyme-linked immunosorbent assay (dcELISA) detection and confirmed by ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Under the optimized conditions, the home-made IAC capacity was 293, 60, 220 and 45 ng/mL gel for AFB1, AFB2, AFG1 and AFG2, respectively. Eight grain foods were pretreated with IAC, and then the extracts were detected by the dcELISA. The results showed that the recoveries of IAC-dcELISA ranged from 71.5% to 119.8% (relative standard deviations ≤ 16.67%) that were higher than dcELISA without IAC cleanup procedure by at least 30% on average, indicating that the matrix interference of different samples could be alleviated via the pretreatment of IAC, especially for fermented foods. The developed method was also confirmed by UHPLC-MS/MS with electrospray interface in positive mode using multiple reaction monitoring. In conclusion, this assay could be used as an effective analytical method for the determination of aflatoxins in complex grain foods.

Liquid chromatography-tandem mass spectrometric assay for the T790M mutant EGFR inhibitor osimertinib (AZD9291) in human plasma.

A method for the quantitative analysis by ultra-performance liquid chromatography-tandem mass spectrometry of the highly selective irreversible covalent inhibitor of EGFR-TK, osimertinib in human plasma was developed and validated, using pazopanib as an internal standard. The validation was performed in a range from 1 to 1000ng/ml, with the lowest level corresponding to the lower limit of quantitation. Gradient elution was performed on a 1.8μm particle trifunctional bonded C18 column by 1% (v/v) formic acid in water, and acetonitrile as mobile phase. The analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer after positive ionization with the heated electrospray interface. Within-day precisions ranged from 3.4 to 10.3%, and between-day precisions from 3.8 to 10.4%, accuracies were 95.5-102.8%. Plasma (either lithium heparin or sodium EDTA) pretreatment was performed by salting-out assisted liquid-liquid extraction using acetonitrile and magnesium sulfate. This method was used to analyze the osimertinib blood plasma levels of five adult patients with metastatic T790M mutated non-small cellular lung carcinoma for therapeutic drug monitoring purposes.

Development of high-throughput multi-residue method for non-steroidal anti-inflammatory drugs monitoring in swine muscle by LC-MS/MS

ABSTRACTA reliable and simple method for the detection and quantification of residues of 14 non-steroidal anti-inflammatory drugs and a metamizole metabolite in swine muscle was developed using liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). The samples were extracted with acetonitrile (ACN) in solid–liquid extraction followed by a low-temperature partitioning (LLE-LTP) process at –20 ± 2°C. After evaporation to dryness, the residue was reconstituted with hexane and a mixture of water:acetonitrile (1:1). LC separation was achieved on a reversed-phase (RP18) column with gradient elution using water (phase A) and ACN (phase B) both containing 1 mmol l–1 ammonium acetate (NH4COO) with 0.025% acetic acid. Analysis was carried out on a triple-quadrupole tandem mass spectrometer (LC-MS/MS) in multiple reaction monitoring mode using an electrospray interface in negative and positive mode in a single run. Method validation was performed according to the criteria of Commission Decision No. 2002/657/EC. The matrix effect and linearity were evaluated. Decision limit (CCα), detection capability (CCβ), accuracy and repeatability of the method are also reported. The proposed method proved to be simple, easy and adequate for high-throughput analysis and was applied to routine analysis by the Brazilian Ministry of Agriculture, Livestock and Food Supply.

Novel pilot scale continuous photocatalytic membrane reactor for removal of organic micropollutants from water

A novel, fully automated photocatalytic membrane reactor (PMR) pilot unit is presented and evaluated (at different operating process parameters and real treatment conditions) for degradation of the pharmaceutical diclofenac (DCF), a typical recalcitrant micropollutant. The PMR-pilot has a maximum system capacity of 1.2m3/d of treated water, 52W of UV-C power, and combines two modern processes; i.e. heterogeneous photocatalysis and membrane separation, employing dispersed TiO2 nanoparticles with UV-C irradiation, and submerged ultrafiltration hollow fibers, respectively. Pilot tests with two types of natural (tap and surface) water under stable, continuous operation, permitted the assessment of the PMR unit performance (for DCF degradation, TOC mineralization) in terms of hydraulic residence time (HRT), TiO2 loading, UV-C irradiation dose, and DCF feed concentration. Under the specific conditions tested, the catalyst loading and UV-C radiant power significantly affected DCF removal; however, HRT had a rather insignificant effect. Additionally, several transformation products (TPs) were identified by means of liquid chromatography–mass spectrometry using electrospray interfacing technique, thus, shedding light into possible reaction pathways leading to DCF mineralization.

(Congreso Nacional de la Sociedad Mexicana de Electroquímica Poster Winner) Capillary Electrophoresis Coupled on Line with Mass Spectrometry Via an Electrospray Interface (CE-ESI-MS) for the Characterization of Cyclic Peptides and Their Possible Aggregation into Peptide Nanotubes

Innovation in analytical methodologies is of high impact for biomedical and environmental applications. In this context, electrokinetic methods represent powerful tools allowing selective and ultrasensitive separations. They are easy to run, versatile, of high efficiency and require very small sample amounts. One way to improve separations is to develop new nanosupports that can be integrated within the capillaries or microchannel, so as to increase separation selectivity and to generate specific interactions with some analytes[1 ]. Thereon, we aim at developing Peptide Nanotubes (PNTs) as new nanosupports, as their structure can offer high diversity and surface functionalization, they are biocompatible and stable in different media. Their cylindrical structure could be used as sieving tools to separate small compounds, amplifying possible applications. The synthesis of PNTs depends on the cyclic peptide (CP) monomers structure and concentration. It is therefore crucial to realize the physicochemical characterization of these CPs. Then, we developed a new capillary electrophoretic method coupled on line with mass spectrometry via an electrospray interface (CE-ESI-MS) to characterize the monomers, and the study of their possible aggregation. Metodology We synthesized 8 CPs (Table 1) of three internal diameters via 8, 10 and 12 D, L alternated amino acids by Fmoc-solid phase peptide synthesis technique[2]. They were purified by HPLC and the purity was determined by the development of CE-ESI-MS methodology, which allowed identifying their corresponding molecular ions, isotopes and some adducts with NH4 + and Na+. CE analysis were performed in zone mode (λ=200nm) at 25°C, with separation at 20kV along pressure application (50mbar). BGE used was 10mM ammonium carbonate (pH8.0)/EtOH (50/50, v/v), and 10mM ammonium formiate (pH3.0)/EtOH (50/50, v/v). The neutral marker was a mixture of dimethylformamide (UV detection) and glucose (MS detection). Fused silica capillaries were of 75µm internal diameter, 80cm total length and 20cm detection length. They were previously activated by successive flushes of NaOH 1M, NaOH 0.1M, H2O and BGE (934mbar) during 15 min each. Sample concentration was 3mM (dissolved in 0.1mM BGE). MS measurements (SCAN and SIM) were done with a sheath liquid composed of H2O/EtOH (10/90, v/v) containing 2.0% AcOH (0.6µL/min), a nebulizing gas (N2,g) at 10psi pressure, and a drying gas (N2,g) at 250°C (6L/min). Fragmentor voltage was fixed at 70V, and acquisition was performed in positive mode. Results The yields of synthesized CPs were between 42.0-90.0%, which are higher values than literature[3]. These compounds are soluble in basic media (pH8.0), and dissolved in acidic media (pH3.0), a protonation is expected as well as an aggregation of monomers into PNTs. It was shown that their electrophoretic mobility (µep) was in accordance with their global charge and mass, but unexpected behaviors could be due to specific cyclic configurations of peptides, due to their sequence nature. Furthermore, MS evidenced the absence of corresponding linear, protected/partially deprotected peptides, and assemblies, in the analysis conditions, proving their purity. The effect of dissolution media on the µep (calculated according to[4]) was studied for CP-8 aaSerie3. This dissolved in basic conditions presents a µep of -4.31±0.11x10-5cm2.V-1.s-1, in agreement with negatively charged compounds. When modifying analysis conditions to acidic media, a shift in mobility was observed, leading 1.11±0.02x10-5cm2.V-1.s-1, indicating a protonation of monomers and possible self-assembly. While, CP dissolved in acidic conditions, the µep is positive (2.35±0.01x10-5cm2V-1s-1). When modifying analysis conditions to basic media, a negative value was determined (-2.94±0.3x10-5cm2V-1s-1). These results seem to indicate a self-assembly between CPs in acidic media, with different stages, as µep values are different according to pH modification pathway. To further in the interpretation, the MS signal of CP dissolved in acidic media was analyzed, showing a m/z value corresponding to twice molecular mass of monomer. This indicates a minima dimerization of CPs, and probably formation of PNTs. Conclusions We designed and synthesized eight new CPs that could provide PNTs of various properties. In view of aggregation as PNTs, their structure and purity had to be verified. Therefore, a new methodology based on CE-ESI-MS was set up. After optimizing various parameters, this methodology was demonstrated to be an innovative tool for easy and quick characterization of CP. When dissolved in acidic media, µep were drastically modified possibly due to aggregation phenomenon. These pure CPs will thus be interesting potential precursors for PNTs that could be employed as nanoplatforms in diagnostics or pseudo-sieving tools for separative purposes. [1] M. Girardot, et al., Anal.Biochem, 435, 150, (2013). [2] W. C. Chan, P. D. White, Fmoc solid phase peptide synthesis, (2000). [3] S. F. Brady, et al., JOC, 44, 3101, (1979) [4] A. Varenne, et. al., Anal.Biochem., 315, 152 (2003). Figure 1

An electrodynamic ion funnel for electrospray ionization source based time-of-flight mass spectrometry

An electrodynamic ion funnel has been developed for improving the sensitivity of electrospray ionization sources widely used in the mass spectrometric study of proteins and other biological macromolecules. The ion funnel consists of 52 electrodes and works under the combined influence of RF and DC voltages in the pressure range of 0.1 to 5 mbar. A novel feature of this ion funnel is the specific shape of the exit electrode that improves transmission of lower mass ions by reducing the depth of effective trapping potentials. In this paper, we report on the optimization of the ion funnel design using ion trajectory simulation software SIMION 8.0 especially in the mass range 500–5000 amu, followed by experimental observations of the ion transmission from the electrospray interface. It is seen that the electrospray-ion funnel combination greatly enhances the transmission when compared with an electrospray-skimmer interface. Ion currents > 1 nA could be obtained at the exit of the ion funnel for dilute Streptomycin Sulphate (∼ 1500 amu) solution with the ion funnel operating in the 500–900 kHz frequency range, amplitude of 70 Vp−p, under a DC gradient of about 20 Volts/cm at a background pressure of 0.3 mbar. Details of the construction of the ion funnel along with the experimental results are presented.

Liquid chromatography-tandem mass spectrometric assay for the tyrosine kinase inhibitor afatinib in mouse plasma using salting-out liquid-liquid extraction.

A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for afatinib, an irreversible inhibitor of the ErbB (erythroblastic leukemia viral oncogene homolog) tyrosine kinase family, was developed and validated. Plasma samples were pre-treated using salting-out assisted liquid-liquid extraction (SALLE) with acetonitrile, magnesium chloride and a stable isotopically labeled internal standard. After dilution, the extract was directly injected into the reversed-phase liquid chromatographic system. The eluate was transferred into the electrospray interface with positive ionization and compounds were detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was completely validated for plasma in a 0.5-500ng/ml calibration range with r(2)=0.995±0.002 (n=6) using linear regression with the inverse square of the concentration as the weighting factor for the calibration. Within-run precisions (n=18) were 2.7-11.7% and between-run (3 runs; n=18) precisions 3.0-14.5%. Accuracies were between 96-109% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully applied to determine plasma drug levels and study pharmacokinetics after oral administration of afatinib to female FVB mice.

Simultaneous quantitation of polymyxin B1, polymyxin B2 and polymyxin B1-1 in human plasma and treated human urine using solid phase extraction and liquid chromatography-tandem mass spectrometry.

Two liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods have been developed and validated for the quantitative determination of polymyxin B1, polymyxin B2 and polymyxin B1-1 concentrations in human plasma and treated urine. During method development, technical challenges such as the separation of structural isomers polymyxin B1and polymyxin B1-1 and nonspecific binding in urine samples were encountered and overcome. Two automated solid phase extraction methods were used to extract plasma samples (100μL) and urine samples (200μL) and the resulting extracts were analyzed using reversed phase LC-MS/MS with an electrospray (ESI) interface and selected reaction monitoring (SRM) in the positive ionization mode. Both methods were validated over a calibration curve range of 5.00-2000ng/mL with a linear regression and 1/x(2) weighting. The between-run relative standard deviation (%RSD) ranged from 4.5 to 9.5% for the plasma assay and from 1.1 to 7.1% for the urine assay. For the plasma assay, the between-run accuracy ranged from 100.5 to 115.2% of nominal at all QC concentrations including the LLOQ. For the urine assay, the between-run accuracy ranged from 92.0 to 106% of nominal at all QC concentrations including the LLOQ. The extraction recoveries for all polymyxins in both assays were between 54.0 and 64.2%. Long term matrix storage stability for all polymyxins was established at both -20°C and -70°C for up to 85 days in human plasma and for up to 55 days in treated human urine. Both assays were used for the measurement of polymyxin B1, polymyxin B2 and polymyxin B1-1 concentrations in human plasma and treated urine for the determination of bioequivalence and toxicokinetic parameters in clinical studies.

Liquid chromatography–tandem mass spectrometric assay for the simultaneous determination of the irreversible BTK inhibitor ibrutinib and its dihydrodiol-metabolite in plasma and its application in mouse pharmacokinetic studies

A validated simple, fast and sensitive bio-analytical assay for ibrutinib and its dihydrodiol metabolite in human and mouse plasma was set up. Sample preparation was performed by protein precipitation, and addition of the respective deuterated internal standards, followed by LC–MS/MS analysis. Separation was performed on a 3.5μm particle-size, bridged ethylene hybrid column with gradient elution by 0.1% v/v formic acid and acetonitrile. The full eluate was transferred to an electrospray interface in positive ionization mode, and subsequently analyzed by a triple quadrupole mass spectrometer by selected reaction monitoring. The assay was validated in a 5–5000 ng/ml calibration range. Both ibrutinib and dihydrodiol-ibrutinib were deemed stable under refrigerated or frozen storage conditions. At room temperature, ibrutinib showed a not earlier described instability, and revealed rapid degradation at 37°C. Finally, the assay was used for a pharmacokinetic study of plasma levels in treated FVB mice.