Electrospray Ionization Technique Research Articles

Simultaneous determination of five main active bufadienolides of Chan Su in rat plasma by liquid chromatography tandem mass spectrometry

To study the pharmacokinetics of Chan Su, a sensitive and selective method was developed and validated for the determination of five main bufadienolides (cinobufagin, resibufogenin, bufalin, bufotalin and arenobufagin) in rat plasma. The analytes were extracted by liquid–liquid extraction with ethyl acetate after internal standard (IS, caudatin) spiked. The separation was performed by a ZORBAX SB-C 18 column (3.5 μm, 2.1 mm × 100 mm) and a C 18 guard column (5 μm, 4.0 mm × 2.0 mm) with an isocratic mobile phase consisted of acetonitrile–water–formic acid (50:50:0.05, v/v/v) at a flow rate of 0.3 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple reaction monitoring mode (MRM) using the electrospray ionization technique in positive mode. The nominal retention times for cinobufagin, resibufogenin, bufalin, bufotalin, arenobufagin and caudatin were 3.07, 3.55, 2.30, 1.62, 1.22 and 3.43 min, respectively. All analytes showed good linearity in a wide concentration range ( r > 0.995) and their lower limits of quantification (LLOQ) were all 1.0 ng/mL. The method was linear for all analytes with correlation coefficients >0.995 for all analytes. The average extract recoveries of the five analytes from rat plasma were all over 85%, the precisions and accuracies determined were all within 15%. This method has been successfully applied to pharmacokinetic study of Chan Su in rats following oral administration.

Propagation of structural deviations of poly(amidoamine) fan-shape dendrimers (generations 0–3) characterized by MALDI and electrospray mass spectrometry

Fan-shape PAMAM dendrimers, from generations 0 to 3, were analyzed by mass spectrometry, using both MALDI and electrospray ionization techniques, to identify any structural deviations present in each sample. First, it could be concluded that all detected molecules were present in the samples as they were detected in MALDI as well as in electrospray mass spectra. Apart from commonly reported dendrimer defects (“missing arm” and “molecular loop”), new impurities were found to arise from propagation of these defects during the synthesis of upper generations. These assignments were based on both compound molecular weight and, when ions were detected with sufficient abundance, deviations from perfect structure behaviour during MS/MS experiments. Since new impurities could be created, either from perfect or defective molecules, during each new generation dendrimer synthesis, models were built to predict the molecular weight of a compound as a function of its synthesis history and efficiently guide mass spectral interpretation.

LC–MS/MS method for the simultaneous determination of icariin and its major metabolites in rat plasma

A rapid and sensitive method using liquid chromatography–tandem mass spectroscopy (LC–MS/MS) was developed and validated for the simultaneous quantitative determination of icariin and its two major metabolites, icariside I and icariside II in rat plasma. The analytes were extracted by liquid–liquid extraction with ethyl acetate after internal standard (daidzein) spiked. The separation was performed by a ZORBAX SB-C 18 column (3.5 μm, 2.1 mm × 100 mm) and a C 18 guard column (5 μm, 4.0 mm × 2.0 mm) with an isocratic mobile phase consisting of acetonitrile–water–formic acid (50:50:0.05, v/v/v) at a flow rate of 0.25 mL/min. The Agilent G6410A triple quadrupole LC–MS system was operated under the multiple reaction monitoring (MRM) mode using the electrospray ionization technique in positive mode. The nominal retention times for icariin, icariside I, icariside II and daidzein were 1.21, 1.88, 2.34 and 1.35 min, respectively. The lower limits of quantification (LLOQ) of icariin, icariside I and icariside II of the method were 1.0, 0.5 and 0.5 ng/mL, respectively. The method was linear for icariin and its metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 12.5%. This method has been applied successfully to a pharmacokinetic study involving the intragastric administration of icariin to rats.

Surface desorption atmospheric pressure chemical ionization mass spectrometry for direct ambient sample analysis without toxic chemical contamination

Ambient mass spectrometry, pioneered with desorption electrospray ionization (DESI) technique, is of increasing interest in recent years. In this study, a corona discharge ionization source is adapted for direct surface desorption chemical ionization of compounds on various surfaces at atmospheric pressure. Ambient air, with about 60% relative humidity, is used as a reagent to generate primary ions such as H(3)O(+), which is then directed to impact the sample surface for desorption and ionization. Under experimental conditions, protonated or deprotonated molecules of analytes present on various samples are observed using positive or negative corona discharge. Fast detection of trace amounts of analytes present in pharmaceutical preparations, viz foods, skins and clothes has been demonstrated without any sample pretreatment. Taking the advantage of the gasless setup, powder samples such as amino acids and mixtures of pharmaceutical preparations are rapidly analyzed. Impurities such as sudan dyes in tomato sauce are detected semiquantitatively. Molecular markers (e.g. putrescine) for meat spoilage are successfully identified from an artificially spoiled fish sample. Chemical warfare agent stimulants, explosives and herbicides are directly detected from the skin samples and clothing exposed to these compounds. This provides a detection limit of sub-pg (S/N > or = 3) range in MS2. Metabolites and consumed chemicals such as glucose are detected successfully from human skins. Conclusively, surface desorption atmospheric pressure chemical ionization (DAPCI) mass spectrometry, without toxic chemical contamination, detects various compounds in complex matrices, showing promising applications for analyses of human related samples.

Liquid chromatography tandem mass spectrometry method for simultaneous determination of antidiabetic drugs metformin and glyburide in human plasma

A simple and rapid liquid chromatography/tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the simultaneous quantitation of antidiabetic drugs metformin and glyburide in human plasma using glimepiride as internal standard (IS). After acidic acetonitrile-induced protein precipitation of the plasma samples, metformin, glyburide and IS were chromatographed on reverse phase C18 (50mm×4.6mm i.d., 5μm) analytical column. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique and operating in multiple reaction monitoring (MRM) and positive ion mode. The total chromatographic run time was 3.5min and calibration curves were linear over the concentration range of 20–2500ng/ml for metformin and 5–500ng/ml for glyburide. The method was validated for selectivity, sensitivity, recovery, linearity, accuracy and precision, dilution integrity and stability studies. The recoveries obtained for the analytes and IS (≥69%) were consistent and reproducible. Inter-batch and intra-batch coefficient of variation across four validation runs (LLOQ, LQC, MQC and HQC) was less than 8%. The accuracy determined at these levels was within ±8% in terms of relative error (RE). The method was applied to a bioequivalence study of 500mg metformin and 5mg of glyburide tablet after oral administration to 28 healthy human subjects under condition of fasting.

PO9-216 AGE-DEPENDENT EFFECT ON RESISTIN BLOOD LEVEL AND ITS ROLE IN THE INFLAMMATION AND CALCIFYING PROCESS OF THE AORTIC VALVE

Steroids were first analyzed by immunoassay-based methods followed by gas chromatography mass spectrometry (GC–MS or GC–MS/MS) with derivatization techniques since steroids are neutral and do not ionize at a high level using the electrospray ionization technique. We now report a validated liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantification of seven steroidal compounds, i.e., estradiol (E2), estrone (E1), testosterone (Testo), dihydrotestosterone (DHT), androst-5-ene-3β, 17β-diol (5-diol), dehydroepiandrosterone (DHEA) and androstenedione (4-dione). The system used is a UPLC–MS/MS (Qtrap 6500) system. With this method, the sample preparation is the combination of liquid–liquid extraction and a simple selective derivatization for only E1 and E2. This assay method is simple and practically eliminates potential contamination. Low quantification limits of 1 pg/mL, 4 pg/mL, 50 pg/mL, 10 pg/mL, 100 pg/mL, 500 pg/mL and 100 pg/mL have been found, respectively for the steroids mentioned above. Without derivatization, DHT sensitivity can be as low as 4 pg/mL with S/N ≥ 5. A full validation has been performed for the seven compounds in compliance with GLP and FDA guidelines for bioanalytical method development and validation. Recovery of all seven compounds in unstripped serum is similar to that in stripped serum: 72.1–84.7% for E2, 83.6–94.5% for E1, 88.2–90.3% for Testo, 82.0–90.6% for DHT, 84.9–92.0% for 5-diol, 88.1–93.8% for DHEA and 86.2–90.3% for 4-dione, respectively. A good linearity is obtained with R > 0.99 for each compound within its calibration range. Accuracies of all levels of QC are within the range of 15% for all seven compounds. The between day variation coefficients are 6.1–8.9% for the low limits of quantification of all seven compounds with 0.7–6.1% for higher levels of QCs for all seven compounds. All results of other test parameters similarly meet the acceptance criteria of EndoCeutics SOPs and FDA guidelines. By comparison of GC–MS/MS and LC–MS/MS data for six derivatized and nonderivatized free steroids, the present data show the crucial importance to use validated assays according to the FDA guidelines to increase specificity, precision and reliability of the absolute values associated with MS/MS-based assays. This method has already been applied to series of samples from clinical trials and is ready for wide clinical use.

Mass Spectrometry-Based Approaches for Structural Studies on Protein Complexes at Low-Resolution

Almost two decades after the introduction of the electrospray ionization (ESI) and the matrix-assisted laser desorption ionization (MALDI) techniques, mass spectrometry (MS) has become a key technology in the emerging field of proteomics. MS-based procedures combined with various affinity-trapping methods allowed massive identification of constitutive elements in multi-protein complexes from different organisms. Similarly, various MS-based strategies have been developed and applied to low-resolution structural studies on protein and protein complex. In fact, products generated either by limited proteolysis, selective chemical modification or radical probe reactions performed on isolated proteins have been characterized by ESI and MALDI techniques, providing information on the location of residues accessible on molecular surface. Differential experiments performed before and following complex formation allowed the identification of masked regions in each component of the complex after binding, generated as a result of macromolecular interaction. Similarly, MS analysis of protein complex cross-linking products has led to the identification of spatially closed amino acids occurring at molecular interface. Nowadays, these methodologies are used for experimental validation of insilico generated models and verification of interface regions predicted by bioinformatics computations. Accordingly, these approaches present a fundamental resource when severe limitations using high-resolution methods, such as X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy, which arise from the amount of sample required, difficulties in crystallization or solubilization, may hinder a definitive structural characterization. In this review, the combined use of chemical cross-linking, limited proteolysis, selective chemical modification or radical probe reactions with MS analysis is reviewed and discussed in the point of view of structural biology studies on protein and protein complexes. Keywords: Protein complex, protein interaction, cross-linking, limited proteolysis, selective chemical modification, radical probe, molecular modelling

Dissipation of sulfosulfuron in water – Bioaccumulation of residues in fish – LC-MS/MS-ESI identification and quantification of metabolites

Dissipation study of sulfosulfuron in natural water and its bioaccumulation in fish was conducted at 25+/-2 degrees C and at two different concentration levels 1mgl(-1) and 2mgl(-1). The dissipation data in water showed the DT50 and DT90 values 67-76 and 222-253 days and followed first order kinetics. Bioaccumulation of sulfosulfuron in fish was conducted under static conditions exposing the fish at one-tenth of sub-lethal concentration 9mgl(-1) and at double the concentration 18mgl(-1), for a period of 56 days. On different occasions fish samples were collected and analyzed. A HPLC-RF method was used for the quantification of sulfosulfuron and aminopyrimidine with the limit of quantification 0.001microg ml(-1). Results showed the accumulation of residues of sulfosulfuron in fish over the concentration range 0.009-0.496microg g(-1). Both in water and fish samples, identified the presence of metabolites aminopyrimidine, desmethyl sulfosulfuron, guanidine, sulfonamide, ethyl sulfone and rearranged amine. The formations of these metabolites are confirmed by LC-MS/MS analysis. An LC-MS/MS electro spray ionization technique was used for this purpose. One of the metabolite Aminopyrimidine was identified at higher concentration levels (0.01-0.1microg ml(-1)) when compared with other metabolites. Subsequently dissipation of aminopyrimidine in water and its bioaccumulation was also studied at the concentration level 1mgl(-1) and 2mgl(-1). The calculated DT50 and DT90 values are 66-68 days and 218-226 days, respectively. This followed first order kinetics. Three hundred days after the exposure complete demineralization was observed.

Tissue Imaging at Atmospheric Pressure Using Desorption Electrospray Ionization (DESI) Mass Spectrometry

Images of the masses: Two-dimensional molecular imaging of intact rat brain tissue under ambient conditions was achieved by using the mass spectrometric technique of desorption electrospray ionization (DESI). The distributions of specific lipids, such as sulfatide 24:1, reveal distinctive subanatomical features of the rat brain (cc=corpus callosum; aca=anterior part of anterior commissure) with spatial resolution of less than 500 μm.

Simultaneous determination of sulforaphane and its major metabolites from biological matrices with liquid chromatography–tandem mass spectroscopy

A simple, sensitive and specific LC-MS/MS method for the simultaneous determination of sulforaphane (SFN) and its major metabolites, the glutathione (SFN-GSH) and N-acetyl cysteine conjugates (SFN-NAC) from biological matrices was developed and validated. The assay procedure involved solid-phase extratcion of all three analytes from rat intestinal perfusate using C2 extraction cartridges, whereas from rat plasma, metabolites were extracted by solid-phase extraction and SFN was extracted by liquid-liquid extraction with ethyl acetate. Chromatographic separation of SFN, SFN-GSH and SFN-NAC was achieved on a C8 reverse phase column with a mobile phase gradient (Mobile Phase A: 10mM ammonium acetate buffer, pH: 4.5 and Mobile Phase B: acetonitrile with 0.1% formic acid) at a flow rate of 0.3 mL/min. The Finnigan LCQ LC-MS/MS was operated under the selective reaction monitoring mode using the electrospray ionization technique in positive mode. The nominal retention times for SFN-GSH, SFN-NAC and SFN were 8.4, 11.0, and 28.2 min,, respectively. The method was linear for SFN and its metabolites with correlation coefficients >0.998 for all analytes. The limit of quantification was 0.01-0.1 microm depending on analyte and matrix, whereas the mean recoveries from spiked plasma and perfusate samples were approximately 90%. The method was further validated according to U.S. Food and Drug Administration guidance in terms of accuracy and precision. Stability of compounds was established in a battery of stability studies, i.e., bench top, auto-sampler and long-term storage stability as well as freeze/thaw cycles. The utility of the assay was confirmed by the analysis of intestinal perfusate and plasma samples from single-pass intestinal perfusion studies with mesenteric vein cannulation in rats.

Design, Fabrication and Study of Micro-Electrospray Chips

This paper described design, fabrication and study of micro electrospray chips. The micro electrospray chips are fabricated by using micro-electro-mechanical system (MEMS) technology. The micro electrospray techniques can be used in many applications, including high viscous fluid dispenses for 3D rapid prototyping and biological sample analysis in mass spectrometer. In this study, micro electrospray chips with different wedge angels are investigated by using microscopic visualization techniques. Four spray modes are identified at various operation voltages and currents. It is found that the voltage for stable cone-jet mode decrease as wedge angle becomes smaller. On the other hand, it is also found that stable cone-jet mode represent a constant current range in current (I)-voltage(Ф) curve. It allows us to easily identify if the micro electrospray chips operate at cone-jet mode.

Rapid analysis of controlled substances using desorption electrospray ionization mass spectrometry

The recently developed technique of desorption electrospray ionization (DESI) has been applied to the rapid analysis of controlled substances. Experiments have been performed using a commercial ThermoFinnigan LCQ Advantage MAX ion-trap mass spectrometer with limited modifications. Results from the ambient sampling of licit and illicit tablets demonstrate the ability of the DESI technique to detect the main active ingredient(s) or controlled substance(s), even in the presence of other higher-concentration components. Full-scan mass spectrometry data provide preliminary identification by molecular weight determination, while rapid analysis using the tandem mass spectrometry (MS/MS) mode provides fragmentation data which, when compared to the laboratory-generated ESI-MS/MS spectral library, provide structural information and final identification of the active ingredient(s). The consecutive analysis of tablets containing different active components indicates there is no cross-contamination or interference from tablet to tablet, demonstrating the reliability of the DESI technique for rapid sampling (one tablet/min or better). Active ingredients have been detected for tablets in which the active component represents less than 1% of the total tablet weight, demonstrating the sensitivity of the technique. The real-time sampling of cannabis plant material is also presented.

Characterization of Various Analytes Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and 2-[(2E)-3-(4-tert-Butylphenyl)-2-methylprop-2- enylidene]malononitrile Matrix

2-[(2E)-3-(4-tert-Butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB) is a nonpolar, aprotic matrix and was used in the analysis of a variety of compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The classes of compounds include coordination compounds, organometallics, conjugated organic compounds (including porphyrins and phthalocyanines), carbohydrates, calixarenes, and macrocycles. For some samples, comparisons are made with spectra acquired with the use of 1,8,9-trihydroxyanthracene (dithranol), 2,5-dihydroxybenzoic acid, and 2,4,6-trihydroxyacetophenone matrixes. Traditionally, the majority of these compounds would have been analyzed by fast-atom bombardment (FAB), liquid secondary ion mass spectrometry (LSIMS), or electrospray techniques, but this work shows that MALDI-TOFMS using DCTB has advantages over these techniques, particularly FAB and LSIMS. Certain limitations of DCTB are noted, for example, in the analysis of water-soluble compounds such as peptides, proteins, and oligonucleotides, and good working practices for the use of the matrix are also outlined.

Development of Mass Spectrometric Method for Analysis of Cyclic Nitramine Explosives DTIW and HNIW

Mass spectrometric (MS) methods are used for the analysis of two novel nitramine explosives-hexanitrohexaazaisowurzitane (HNIW) and 4,10-dinitro-2,6,8,12-tetraoxa-4,10-diazaisowurzitane (DTIW). The methods include electrospray (ESI) and atmospheric pressure chemical ionization techniques for liquid chromatography/MS (LC/MS), chemical ionization for direct introduction (DCI), and gas chromatography/MS (CI-GC/MS). It is found that HNIW (438 Da) is detectable using both positive and negative modes of DCI and in the negative mode ESI-MS. Several anions were found to complex with HNIW, e.g., CF3CO2-, Cl-, Br-, I-, NO3-, and NO2-. On the other hand, DTIW could only be detected using positive DCI and CI-GC/MS, where an MH+ ion (m/z 263) was formed. The fragmentation pathways of the two nitramines were further studied by MS2 experiments. Apparently, the main fragmentation pathway of the MH+ ion of DTIW involves the loss of nitrous acid. Several anion adducts of HNIW that were studied dissociate to afford neutral HNIW and the added anions. However, Cl-, Br-, I-, and NO2- afford a series of fragments that resulted from the dissociation of the isowurzitane structure. For these anions, limit of detection was also found. To understand some of the HNIW fragmentation pathways, DFT calculations were used.

Characterization of peptides resulting from digestion of human skin elastin with elastase

Several pathological disorders are associated with abnormalities in elastic fibers, which are mainly composed of elastin. Understanding the biochemical basis of such disorders requires information about the primary structure of elastin. Since the acquisition of structural information for elastin is hampered by its extreme insolubility in water or any organic solvent, in this study, human skin elastin was digested with elastase to produce water-soluble peptides. Tandem mass spectrometry (MS/MS) experiments were performed using conventional electrospray ionization (ESI) and nano-ESI techniques coupled with ion trap and quadrupole time-of-flight (qTOF) mass analyzers, respectively. The peptides were identified from the fragment spectra using database searching and/or de novo sequencing. The cleavage sites of the enzyme and, for the first time, the extent and location of proline hydroxylation in human skin elastin were determined. A total of 117 peptides were identified with sequence coverage of 58.8%. It has been observed that 25% of proline residues in the sequenced region are hydroxylated. Elastase cleaves predominantly at the C-terminals of the amino acids Gly, Val, Leu, Ala, and Ile, and to a lesser extent at Phe, Pro, Glu, and Arg. Our results confirm a previous report that human skin elastin lacks amino acid sequences expressed by exon 26A.

Exploring and modelling the responses of electrospray and atmospheric pressure chemical ionization techniques based on molecular descriptors

Mass spectroscopy with two different ionization techniques, electrospray ionization and atmospheric pressure chemical ionization, optimized for cocaine, was applied to study 170 molecules. For each of the molecules, 429 molecular descriptors were calculated. The aim of this work is, on one hand, to determine whether it is possible to identify which is the better ionization technique, electrospray ionization or atmospheric pressure chemical ionization, to analyse a given molecule, based on the calculated descriptors, and on the other hand to correlate the response of each of the techniques individually with these descriptors. For each molecule, the ratio between the corrected responses of electrospray ionization and atmospheric pressure chemical ionization was used as criterion to decide which ionization technique was the better for the analysis. The chemometrical methods used to explore and model the data were Principal Component Analysis, Projection Pursuit, Classification and Regression Trees, Partial Least Squares and Stepwise Multiple Linear Regression. The results obtained did not allow establishing, based on the calculated molecular descriptors, which is the better ionization technique to analyse a certain molecule for the studied data.

Scission of the Lactyl Ether Bond of N-Acetylmuramic Acid by Escherichia coli “Etherase”

The ubiquitous bacterial cell wall sugar N-acetylmuramic acid (MurNAc) carries a unique D-lactyl ether substituent at the C3 position. Recently, we proposed an etherase capable of cleaving this lactyl ether to be part of the novel bacterial MurNAc dissimilation pathway (Dahl, U., Jaeger, T., Nguyen, B. T., Sattler, J. M., Mayer, C. (2004) J. Bacteriol. 186, 2385-2392). Here, we report the identification of the first known MurNAc etherase. The encoding gene murQ is located at 55 min on the Escherichia coli chromosome adjacent to murP, the MurNAc-specific phosphotransferase system. A murQ deletion mutant could not grow on MurNAc as the sole source of carbon and energy but could be complemented by expressing murQ from a plasmid. The mutant had no obvious phenotype when grown on different carbon sources but accumulated MurNAc 6-phosphate at millimolar concentrations from externally supplied MurNAc. Purified MurQ-His6 fusion protein and extracts of cells expressing murQ both catalyze the cleavage of MurNAc 6-phosphate, with GlcNAc 6-phosphate and D-lactate being the primary products. The 18O label from enriched water is incorporated into the sugar molecule, showing that the C3-O bond is cleaved and reformed by the enzyme. Moreover, an intermediate was detected and identified as an unsaturated sugar molecule. Based on this observation, we suggested a lyase-type mechanism (beta-elimination/hydration) for the cleavage of the lactyl ether bond of MurNAc 6-phosphate. Close homologs of murQ were found on the chromosome of several bacteria, and amino acid sequence similarity with the N-terminal domain of human glucokinase-regulatory protein (GckR or GKRP) was recognized.

Simultaneous determination of rosuvastatin and fenofibric acid in human plasma by LC–MS/MS with electrospray ionization: Assay development, validation and application to a clinical study

A simple, sensitive and specific LC–MS/MS method for simultaneous determination of rosuvastatin (RST) and fenofibric acid (FFA) was developed and validated with 500 μL human plasma using carbamazepine as an internal standard (IS). The assay procedure involved a simple one-step liquid/liquid extraction of RST and FFA and IS from plasma into ethyl acetate. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 °C. The residue was reconstituted in the mobile phase and injected onto X-Terra MS C-18 column (4.6 mm × 50 mm, 5.0 μm). Separation of RST, FFA and IS was achieved with a mobile phase consisting of 0.05 M formic acid:acetonitrile (45:55, v/v) at a flow rate of 0.40 ml/min. The API-3000 LC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Positive ion acquisition chromatographic run was used in the present method. Nominal retention times of RST, FFA and IS were 2.35, 4.70 and 2.32 min, respectively. Absolute recovery of RST, FFA and IS was 74, 61 and 69%, respectively. The lower limit of quantification (LLOQ) of RST and FFA was 1.00 ng/ml and 0.50 μg/ml, respectively. Response function was established for the range of concentrations 1.00–50.0 ng/ml and 0.50–20.0 μg/ml for RST and FFA, respectively, with a coefficient of determination ( r 2) of 0.999 for both the compounds. The inter- and intra-day precision in the measurement of RST quality control (QC) samples 5, 15, 400 and 800 ng/ml, were in the range 8.93–9.37% relative standard deviation (R.S.D.) and 1.74–16.1% R.S.D., respectively. Similarly, the inter- and intra-day precision in the measurement of FFA quality control (QC) samples 0.5, 1.5, 8.0 and 15.0 μg/ml, were in the range 9.78–11.6% relative standard deviation (R.S.D.) and 0.22–17.4% R.S.D., respectively. Accuracy in the measurement of QC samples for RST and FFA were in the range 88.1–108 and 87–115%, respectively, of the nominal values. RST and FFA were stable in the battery of stability studies, viz., bench-top, auto-sampler and freeze/thaw cycles. Stability of RST and FFA was established for 1 month at −80 °C. The application of the assay to a clinical study confirmed the utility of the assay.

Discovering new invertebrate neuropeptides using mass spectrometry

Neuropeptides are a complex set of messenger molecules controlling a wide array of regulatory functions and behaviors within an organism. These neuromodulators are cleaved from longer protein molecules and often experience numerous post-translational modifications to achieve their bioactive form. As a result of this complexity, sensitive and versatile analysis schemes are needed to characterize neuropeptides. Mass spectrometry (MS) through a variety of approaches has fueled the discovery of hundreds of neuropeptides in invertebrate species in the last decade. Particularly successful are direct tissue and single neuron analyses by matrix-assisted laser desorption/ionization (MALDI) MS, which has been used to elucidate approximately 440 neuropeptides, and examination of neuronal homogenates by electrospray ionization techniques (ESI), also leading to the characterization of over 450 peptides. Additional MS methods with great promise for the discovery of neuropeptides are MS imaging and large-scale peptidomics studies in combination with a sequenced genome.

Complexation and Pd-Catalyzed Asymmetric Allylation with Participation of Chiral Ferrocenyliminophosphites

Chiral ferrocene-containing iminoarylphosphite ligands based on 4,4′-dimethoxy-6,6′-di-tert-butyl-biphenyldiol-2,2′ and their chelate complexes with Rh(I) and Pd(II) were synthesized for the first time. They were shown to be perspective reagents in the reactions of asymmetric allyl substitution. The Pd-catalyzed alkylation of 1,3-diphenylallylacetate with dimethyl malonate results in a 87% enantiomer excess (ee), while in sulfonylation of 1,3-diphenylallylacetate with sodium para-toluenesulfinite, this figure amounts to 67%. The results obtained were compared with coordination and catalytic efficiency of a less sterically hindered ferro-cenyliminophosphite based on pyrocatechol. The compositions and structures of new compounds were determined by the 1H, 13C, 31P NMR, IR, mass spectrometry (EI, FAB, and electrospray techniques), and elemental analysis.