Speciation analysis of gadolinium-based MRI contrast agents in blood plasma by hydrophilic interaction chromatography/electrospray mass spectrometry

The first analytical method for simultaneous speciation analysis of five of the most important gadolinium-based magnetic resonance imaging (MRI) contrast agents in blood plasma samples was developed. Gd-DTPA (Magnevist), Gd-BT-DO3A (Gadovist), Gd-DOTA (Dotarem), Gd-DTPA-BMA (Omniscan), and Gd-BOPTA (Multihance) were separated by hydrophilic interaction liquid chromatography (HILIC) and detected with electrospray mass spectrometry (ESI-MS). Spiking experiments of blank plasma with Magnevist and Gadovist were performed to determine the analytical figures of merit and the recovery rates. The limits of detection ranged from 1 x 10 (-7) to 1 x 10 (-6) mol/L depending on the ionization properties of the individual compounds, and limits of quantification ranged from 5 x 10 (-7) to 5 x 10 (-6) mol/L. The linear concentration range comprised 2 orders of magnitude. With application of this method, blood plasma samples of 10 healthy volunteers, with Magnevist or Gadovist medication, were analyzed for Gd-DTPA and Gd-BT-DO3A, respectively. The obtained results were successfully validated with inductively coupled plasma-optical emission spectroscopy (ICP-OES).     

Liquid chromatography with on-line electrochemical derivatization and fluorescence detection for the determination of phenols

A new methodological approach for the determination of monosubstituted phenols is described. After liquid chromatographic separation of the analytes, an on-line electrochemical derivatization is carried out and the reaction products are detected fluorometrically. Phenols are oxidized in the electrochemical cell to form fluorescent dimers and higher oligomers, which were identified by on-line electrochemistry/mass spectrometry. Major advantages of the proposed method include enhanced selectivity and sensitivity. Without prior enrichment of the analytes, limits of detection down to 2 x 10(-9) M (20 fmol) may be reached for selected phenols, e.g., for 4-octylphenol, 4-ethylphenol, and 4-(1-indanyl)phenol. Only readily available instrumentation is required for these measurements.     

Photothermal deflection determination of iron(II) with ferrozine with sorption preconcentration on Silufol plates

Photothermal deflection spectroscopy was applied to the selective detection of iron(II) chelate with ferrozine by its sorption preconcentration on Silufol plates. The linearity range was 1 x 10(-11) - 6 x 10(-8) mol cm(-2) of chelate at the plate surface, which corresponded to 1 x 10(-9) -4 x 10(-6) M of chelate in solution. The limits of detection and quantification are 8 x 10(-12) and 2.5 x 10(-11) mol cm(-2) at the plate from 15 microL of test solution (0.5 nM and 1.5 nM in solution, respectively), and the absolute detection limit is 8 fmol in the whole spot applied to a plate. Characteristics and features of photothermal deflection detection are discussed.     

Quantitative counting of single fluorescent molecules by combined electrochemical adsorption accumulation and total internal reflection fluorescence microscopy

We developed an ultrasensitive quantitative single-molecule imaging method for fluorescent molecules using a combination of electrochemical adsorption accumulation and total internal reflection fluorescence microscopy (TIRFM). We chose rhodamine 6G (R6G, fluorescence dye) or goat anti-rat IgG(H+L) (IgG(H+L)-488), a protein labeled by Alexa Fluor 488 or DNA labeled by 6- CR6G (DNA-R6G) as the model molecules. The fluorescent molecules were accumulated on a light transparent indium tin oxide (ITO) conductive microscope coverslip using electrochemical adsorption in a stirred solution. Then, images of the fluorescent molecules accumulated on the ITO coverslip sized 40 x 40 microm were acquired using an objective-type TIRFM instrument coupled with a high-sensitivity electron multiplying charge-coupled device. One hundred images of the fluorescent molecules accumulated on the coverslip were taken consecutively, one by one, by moving the coverslip with the aid of a three-dimensional positioner. Finally, we counted the number of fluorescent spots corresponding to single fluorescent molecules on the images. The linear relationships between the number of fluorescent molecules and the concentration were obtained in the range of 5 x 10(-15) to 5 x 10(-12) mol/L for R6G, 3 x 10(-15) to 2 x 10(-12) mol/L for IgG(H+L)-488, and 3 x 10(-15) to 2 x 10(-12) mol/L for DNA-R6G.     

A method for the fabrication of low-noise carbon fiber nanoelectrodes

A new and facile method has been developed for the fabrication of low-noise carbon fiber microelectrodes (CFMEs) and carbon fiber nanoelectrodes (CFNEs). The carbon fiber was flame-fuse sealed in the tip of the glass capillary. The CFMEs were made by cutting the protruding carbon fiber to the desired length, and the CFNEs were achieved by etching the protruding carbon on the flame to form a nanometer-scale tip. The tip of CFNEs can be controlled within the range from 100 to 300 nm. Thus, no epoxy wax was involved in the CFMEs and CFNEs. The experimental results of inspecting CFMEs and CFNEs by scanning electron microscopy demonstrated that the surface of the electrodes and the glass/fiber interface are very smooth. Therefore, the noise caused by the glass/fiber of these electrodes is much lower than that of the electrodes fabricated conventionlly. The electrodes were characterized by ferricyanide, catecholamine (dopamine,DA), norepinephrine (NE), and epinephrine (E)) and 5-hydroxytryptamine (5-HT) neurotransmitters using CV, LSV, DPV, and FSCV. The results showed that the CFMEs and CFNEs have very excellent electrochemical behavior and high sensitivity. The CV and DPV detection limits of DA, NE, and E are 7.6 x 10(-8), 7.0 x 10(-8), and 5.0 x 10(-8) mol/L, and the DPV detection limits of DA, NE, and E are 4.0 x 10(-8), 1.0 x 10(-7), and 2.2 x 10(-7) mol/L, respectively. This experiment offers a new and facile method for the fabrication of CFMEs and CFNEs of very high sensitivity and low noise.     

Measurement of 18 perfluorinated organic acids and amides in human serum using on-line solid-phase extraction

We have developed an on-line solid-phase extraction (SPE) method coupled to high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) for measuring trace levels of 18 perfluorinated chemicals (3 perfluorosulfonates, 8 perfluorocarboxylates, 7 perfluorosulfonamides) in serum. Without protein precipitation, only dilution with 0.1 M formic acid, one aliquot of 100 microL of serum was injected into a commercial column switching system that allowed for concurrent SPE and HPLC-MS/MS acquisition. First, the analytes were concentrated on a C18 SPE column. Then, this column was placed automatically in front of a C8 analytic HPLC column for chromatographic separation of the analytes. Detection and quantification were done using negative-ion TurboIonSpray ionization, a variant of electrospray ionization, MS/MS. Excellent recovery was achieved for all analytes including the volatile sulfonamide derivatives that could not be determined before using traditional off-line SPE methods. The high throughput and low limits of detection (0.05-0.8 ng/mL) using a small sample volume (100 microL of serum) and isotope dilution quantification make this method suitable for large-scale epidemiologic studies.     

Generation and identification of reactive metabolites by electrochemistry and immobilized enzymes coupled on-line to liquid chromatography/mass spectrometry

The detection of reactive metabolites using conventional in vivo and in vitro techniques is hampered because the intermediately formed reactive species are prone to covalent binding to cellular macromolecules. Therefore, the application of improved methods is required. The on-line coupling of an electrochemical reactor and horseradish peroxidase immobilized on magnetic microparticles with liquid chromatography/mass spectrometry (EC/LC/MS or HRP/LC/MS) allows the direct detection of reactive metabolites of the model compounds amodiaquine, amsacrine, and mitoxantrone, which are all known for readily binding to cellular macromolecules after metabolization by cytochrome P450. EC/LC/MS and HRP/LC/MS experiments were compared to rat liver microsome incubations and proved to be valuable complementary methods since reactive quinone, quinone imine, and quinone diimine species could be detected directly and not only after trapping with glutathione. Furthermore, N-dealkylation and N-oxidation of amodiaquine were successfully simulated by electrochemical oxidation reactions, as well as the formation of an aldehyde. Therefore, EC/LC/MS and HRP/LC/MS are promising tools for the identification of both reactive and stable metabolites in drug development.     

On-line electrochemistry/thermospray/tandem mass spectrometry as a new approach to the study of redox reactions: the oxidation of uric acid

The electrochemical oxidation pathway of uric acid was determined by on-line electrochemistry/thermospray/tandem mass spectrometry. Intermediates and products formed as a result of electrooxidation were monitored as the electrode potential was varied. Several reaction intermediates have been identified and characterized by tandem mass spectrometry. The tandem mass spectrometric results provide convincing evidence that the primary intermediate produced during the electrooxidation of uric acid has a quinonoid diimine structure. The results indicate that once formed via electrooxidation, the primary intermediate can follow three distinct reaction pathways to produce the identified final products. The final electrochemical oxidation products observed in these studies were urea, CO2, alloxan, alloxan monohydrate, allantoin, 5-hydroxyhydantoin-5-carboxamide, and parabanic acid. The solution reactions that follow the initial electron transfer at the electrode are affected by the vaporizer tip temperature of the thermospray probe. In particular, it was found that at different tip temperatures either hydrolysis or ammonolysis reactions of the initial electrochemical oxidation products can occur. Most importantly, the results show that the on-line combination of electrochemistry with thermospray/tandem mass spectrometry provides otherwise difficult to obtain information about redox and associated chemical reactions of biological molecules such as the structure of reaction intermediates and products, as well as providing insight into reaction pathways.     

Monocrystalline diamond paste-based electrodes and their applications for the determination of Fe(II) in vitamins

A new class of electrochemical sensors, namely, electrodes based on diamond paste, was designed using monocrystalline diamond (natural diamond 1 microm and synthetic diamond, 50 microm (synthetic-1) and 1 microm (synthetic-2)) powder and paraffin oil. The characterization of the electrodes was performed using cyclic voltammetry and differential pulse voltammetry. Fe(II) was determined by differential pulse voltammetry (DPV) at 75 mV (vs Ag/AgCl) using all diamond paste-based electrodes. The linear concentration range was between 10(-8) and 10(-4) mol/L for both the natural diamond and synthetic-2 with detection limits of 10(-10) and 10(-9) mol/L, respectively, whereas the linear concentration range for synthetic-1 was between 10(-7) and 10(-3) mol/L with a detection limit of 10(-8) mol/L Fe(II) was determined successfully from four types of pharmaceutical products. The recovery values of Fe(II) in the pharmaceutical products were higher than 98.00% with relative standard deviation values < 5%.     

On-line probe for fast electrochemistry/electrospray mass spectrometry. Investigation of polycyclic aromatic hydrocarbons

A newly invented probe accessory for fast electrochemistry/electrospray mass spectrometry (EC/ESMS) is presented and evaluated. The device features a low-volume, three-electrode electrochemical cell which has been designed with a minimum distance between the working electrode and the "Taylor cone" inherent to the electrospray process. This configuration limits the time between electrochemical generation of ions and mass spectrometric analysis to an absolute minimum. A fused-silica layer insulates the microcylinder working electrode from the sample solution until immediately prior to the electrospray region, postponing electrode processes until the last moment. The same fused-silica layer insulates the working electrode from the surrounding auxiliary electrode, a stainless steel capillary that also serves as the electrospray capillary. The performance and capabilities of the novel electrochemistry/electrospray mass spectrometry system have been evaluated using polycyclic aromatic hydrocarbons (PAHs) as test analytes. In the positive ion EC/ESMS mode, oxidized forms (one-electron removal) of PAHs are produced in high yield. The ability to analyze reaction products appearing subsequent to the initial oxidation is also demonstrated.     

Enhancement of ionization efficiency by electrochemical reaction products in on-line electrochemistry/electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

A miniaturized two-electrode electrochemical (EC) cell was developed and was coupled on-line with an electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer (ESI-FTICR MS). Electrochemistry on-line with mass spectrometry, EC/ESI-FTICR MS, of triphenylamine (TPA), which undergoes one-electron oxidation to form a radical cation (TPA*+), demonstrates a significant sensitivity enhancement compared to ESI-FTICR MS. The on-line EC cell configuration with a stainless steel ES needle as the working electrode produces the highest sensitivity in EC/ESI-MS. The results provide evidence that, during the ES ionization, electrolytic reactions occur mainly in the ES tip region, as previously predicted. The results demonstrate that ESI-MS signal suppression by tetrabutylammonium perchlorate electrolyte, which can be a problem, is minimized in EC/ESI-MS. TPA*+ dimer tetraphenylbenzidine (TPB) can be detected by EC/ESI-MS, together with TPA*+, as TPB*+ and TPB2+. The high mass resolving power of FTICR MS was exploited to identify TPB2+ dication in the presence of [TPA*+ - H*]+ ions of the same m/z, from their respective isotopic distributions. The dimer dication TPB2+ can be detected only in EC/ESI-MS.     

Determination of pentachlorophenol at carbon nanotubes modified electrode incorporated with beta-cyclodextrin

A facile and reliable electrochemical technique at beta-cyclodextrin incorporated carbon nanotubes modified glassy carbon electrode (beta-CD/CNTs/GCE) was proposed for determination of pentachlorophenol (PCP). The electrochemical behavior of PCP at the beta-CD/CNTs/GCE was investigated by cyclic voltammetry and linear sweep voltammetry. The beta-CD/CNTs/GCE showed good analytical performance characteristics in electrocatalytic oxidation of PCP, compared with the simple carbon nanotube modified electrode (CNTs/GCE) and bare glassy carbon electrode (GCE). After accumulation for 5 min on beta-CD/CNTs/GCE, the peak current increased linearly with the concentration of PCP in the range from 8.0 x 10(-7) to 1.04 x 10(-5) mol/L. The detection limit was 4.0 x 10(-8) mol/L at 3 sigma level. The proposed electrode presented good repeatability for the determination of PCP in artificial wastewater, and the recovery was 97%-103%. This modified electrode combined the advantages of carbon nanotubes and supramolecular cyclodextrin, leading to new capabilities for electrochemical detection of PCP.     

Analysis of perchlorate in water by reversed-phase LC/ESI-MS/MS using an internal standard technique

A new method was developed for the analysis of perchlorate in water by using reversed-phase liquid chromatograhy/electrospray ionization-mass spectrometry/mass spectrometry (LC/ESI-MS/MS) in the negative ESI mode. Selective and sensitive perchlorate detection was obtained by monitoring the 35ClO4- --> 35ClO3- and 37ClO4- --> 37ClO3- mass transitions. The 35ClO4- --> 35ClO3- transition was quantitated against the internal standard oxygen-labeled sodium perchlorate (NaCl18O4). Sample pretreatment for the removal of major common anions and dissolved metal ions along with internal standard quantitation sufficiently compensated for ion suppression caused by the matrix. The 37ClO4- --> 37ClO3- transition was examined to provide additional specificity. The method sensitivity, accuracy, and precision were investigated by analyzing fortified blank samples, field samples, and performance evaluation samples. The results (1.01-13.5 microg/L) for the proficiency evaluation samples differed from the certified values (1.04-14.1 microg/L) by 3-18%. The developed reversed-phase LC/ESI-MS/MS method was rapid, accurate, and reproducible. The calculated method detection limits were 0.007 microg/L for deionized reagent water and 0.009 microg/L for synthesized reagent water, respectively. The minimum reporting limit was conservatively set to 0.05 microg/L.     

One-pot aqueous synthesis PbS quantum dots and their Hg2+ sensitive properties

In this study, we report the synthesis and stability of PbS quantum dots (QDs) using an aqueous route with L-Cysteine (L-Cys) as the capping molecule. The as-synthesized L-Cys-capped PbS QDs were characterized by high resolution transmission electron microscopy (HRTEM), and X-ray diffraction (XRD), the results indicated that the QDs were about 4 nm in size and dispersed well with a rock salt crystalline structure, and there was L-Cys on the surface of QDs, which was confirmed by Fourier transform infrared (FT-IR) spectrometry. The influence of various experimental variables, including amounts of capping ligand, pH value and refluxing time, on the luminescent properties of the obtained QDs have been systematically investigated. The QDs exhibited optimal PL intensity when Pb: L-Cys: S = 1:2.2:0.3. In addition, the as-prepared QDs could be used as fluorescence probes to detect Hg2+ ions in aqueous media. The response of QDs fluorescence probes was linearly proportional to the concentration of Hg2+ ions ranging from 8 x 10(-9) to 2 x 10(-6) mol x L(-1) with a limit of detection of 2 x 10(-9) mol x L(-1). Furthermore, the method was successfully applied to the determine Hg2+ ions in different real samples.     

Redox and double-layer charging of phenothiazine functionalized monolayer-protected clusters

Monolayer-protected Au clusters (MPCs) have been prepared with mixed monolayers of alkanethiolates and alkanethiolates terminally omega-functionalized with phenothiazine. The mixed monolayer MPCs can contain as many as 10 phenothiazines/MPC; these electron donors are electroactive in rapid, successive one-electron reactions. Surface adsorption of the functionalized MPCs is evident in cyclic voltammetry. Double-potential-step chronocoulometry with incremented potential steps was applied to unfunctionalized hexanethiolate-coated MPCs and to those functionalized with phenothiazine to analyze the coupling between the diffusion-controlled double-layer charging of the MPC cores and the oxidation of the phenothiazine centers. Apparent changes in ordering of the MPC alkanethiolate chains were observed with infrared spectroscopy in solutions of MPCs where alcohol, carboxylic acid, or phenothiazine moieties had been incorporated into the monolayer.     

碱性降解-Al~(3+)络合物形成荧光法测定强力霉素

本文提出了荧光法测定强力霉素(DC)的新方法。强力霉素碱性降解后与铝离子形成强荧光的络合物,并据此进行荧光测定,其荧光强度在4.0×10~(-8)～7.0×10~(-6)mol/L 范围内与强力霉素浓度成正比,共存的其它四环素类抗菌素不干扰强力霉素的测定,测定的检测限为3.0×10~(-8)mol/L。此法用于血清和尿液中强力霉素的测定,回收率分别为93～97%和91%～99%。     

Uracil in human DNA from subjects with normal and impaired folate status as determined by high-performance liquid chromatography-tandem mass spectrometry.

A sensitive and selective method for determination of the uracil content in human DNA was first developed on the basis of high-performance liquid chromatography-tandem mass spectrometry. Uracil was excised from DNA using uracil DNA glycosylase. The released uracil was derivatized with 4-bromomethyl-7-methoxycoumarin, thereby forming bis-N,N'-(4-methylene-7-methoxycoumaryl)-uracil (uracil-MMC). 15N2-Uracil was used as an internal standard. The analytes were separated on an Adsorbsphere XL ODS column. A SCIEX API III tandem mass spectrometer equipped with a turbo ion-spray interface was used as the detector. Multiple reaction monitoring using the parent --> product ion combinations of m/z 489 --> 232 and 491 --> 233 were used to detect uracil-MMC and the internal standard, respectively. The detection limit for this assay is <1.0 x 10(-10) mol/L uracil, and the linearity is from 1.0 x 10(-10) to 2.5 x 10(-6) mol/L. The method was used for determination of uracil in human DNA. Our data show that the uracil levels in human DNA isolated from peripheral white blood cells did not differ between subjects with folate deficiency and subjects with normal red cell folate levels.     

Improvements in LC/Electrospray Ion Trap Mass Spectrometry Performance Using an Off-Axis Nebulizer

Charged residues from the electrospray process have been hypothesized to limit the sensitivity and dynamic range of an ion trap mass spectrometry operation. Incorporation of an off-axis nebulizer (positioned 90-95° from the sampling orifice) was found to drastically reduce the detrimental effects caused by the charged particles or droplets compared to typical on-axis nebulization configurations (spraying 10-20° from sampling orifices). The off-axis nebulizer reduced total ion currents that enter the ion trap (through the reduction of charged residues) by a factor of 5-7 while resulting in an increase of analyte [M + H](+) signal by a factor of 6 compared to an on-axis sprayer at flow rates of 20 μL/min. At higher flow rates (e.g., 800 μL/min) these enhancements are more evident. At flows greater than 200 μL/min, off-axis nebulization reduced total ion current that enters the ion trap by a factor of 30 and resulted in a factor of more than 20 increase in [M + H](+) signal relative to on-axis nebulization. Incorporation of the off-axis nebulizer improved the detection limit and precision for determination of dihydroxyvitamin D(3) in plasma compared to on-axis nebulization. The LC/MS/MS detection limits obtained for the off-axis nebulizer on the ion trap was within a factor of 2 from the detection limit determined by the triple quadrupole. The relative standard deviation of the dihydroxyvitamin D(3) determination was less than 8% for both off-axis ion trap and triple-quadrupole determinations.     

Analysis of perchlorate in water and soil by electrospray LC/MS/MS

A method has been developed for the determination of perchlorate in water and soil matrixes using electrospray liquid chromatography/mass spectrometry/mass spectrometry. Perchlorate is quantitated by monitoring the ion signal from mass 83, which is formed by a loss of an oxygen atom from the perchlorate molecular ion. The method was developed to be effective and economical in production laboratory analysis of perchlorate in environmental water and soil samples. Data were gathered to define method sensitivity, performance, selectivity, and robustness. Analyte stability, method susceptibility to interferences, and the reliability of the chlorine isotope ratio as an identification tool were examined. The aqueous method detection limit (MDL) is 0.05 microg/L and was determined using an actual groundwater matrix. The soil MDL is 0.5 microg/kg and was determined using Ottawa sand. The stability study was performed by spiking water samples at 0.25, 10, and 20 microg/L and analyzing them 50 days later. Acceptable recoveries were obtained for all samples. The relative standard deviation (RSD) for the replicate analyses in the stability study indicates that the method is capable of RSD values less than 5% in a relatively clean groundwater matrix. The ionization suppression study was performed by spiking water samples containing 1000 mg/L carbonate, chloride, and sulfate with 0.05 and 0.5 microg/L perchlorate and then measuring the recovery of the spike. The results indicate that the procedure does not have significant suppression effects at the high salt levels tested. Calibration, quality control sample, field sample, and suppression study data were combined to examine isotope ratio reliability. The results of that work show that chlorine isotope ratios can be used to define statistical process control limits for use as an additional analyte identification tool.     

Interaction of malachite green with bovine serum albumin: determination of the binding mechanism and binding site by spectroscopic methods

The interaction between malachite green (MG) and bovine serum albumin (BSA) under simulative physiological conditions was investigated by the methods of fluorescence spectroscopy, UV-vis absorption and circular dichroism (CD) spectroscopy. Fluorescence data showed that the fluorescence quenching of BSA by MG was the result of the formation of the MG-BSA complex. According to the modified Stern-Volmer equation, the effective quenching constants (K(a)) between MG and BSA at four different temperatures were obtained to be 3.734 x 10(4), 3.264 x 10(4), 2.718 x 10(4), and 2.164 x 10(4)L mol(-1), respectively. The enthalpy change (Delta H) and entropy change (DeltaS) were calculated to be -27.25 kJ mol(-1) and -11.23 J mol(-1)K(-1), indicating that van der Waals force and hydrogen bonds were the dominant intermolecular force in stabilizing the complex. Site marker competitive experiments indicated that the binding of MG to BSA primarily took place in sub-domain IIA. The binding distance (r) between MG and the tryptophan residue of BSA was obtained to be 4.79 nm according to F?rster theory of non-radioactive energy transfer. The conformational investigation showed that the presence of MG decreased the alpha-helical content of BSA (from 62.6% to 55.6%) and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of BSA molecules.