Analytical and Bioanalytical Chemistry (v.406, #20)

Statistical filtering in fluorescence microscopy and fluorescence correlation spectroscopy by Radek Macháň; Peter Kapusta; Martin Hof (4797-4813).
We review the principles and applications of statistical filtering in multichannel fluorescence microscopy. This alternative approach to separation of signals from individual fluorophore populations has many important advantages, especially when spectral and/or temporal overlap, or the complicated nature of those signals, makes their discrimination or sorting impossible by means of hardware. This situation is typically encountered for biological samples. This review of well established statistical filtering techniques and of emerging, very promising new methods of analysis reveals remarkable progress in bioanalytical applications of fluorescence microscopy.
Keywords: Fluorescence lifetime correlation spectroscopy; Fluorescence spectral correlation spectroscopy; Filtered fluorescence correlation spectroscopy; Raster lifetime image correlation spectroscopy; Time-correlated single photon counting

USP compendial methods for analysis of heparin: chromatographic determination of molecular weight distributions for heparin sodium by Barbara Mulloy; Alan Heath; Zachary Shriver; Fabian Jameison; Ali Al Hakim; Tina S. Morris; Anita Y. Szajek (4815-4823).
worked at the National Institute for Biological Standards and Control for many years, specialising in the physicochemical characterization of biological medicines. She is now Visiting Professor at the Institute of Pharmaceutical Science, King’s College London, and has honorary appointments also at Imperial College and Royal Holloway University of London. Barbara is a member of the United States Pharmacopeial Convention’s expert panels on unfractionated heparin and low molecular weight heparin. has recently retired from National Institute for Biological Standards and Control, where he worked for 26 years, and was head of the biostatistics group. Alan specializes in the statistical analysis of assays for biological medicines, and multicentre studies to establish biological and other reference materials. is currently a research affiliate at MIT and Vice President of Research at Visterra. He received his Ph.D. degree in applied biosciences from MIT, in the area of sequencing complex polysaccharides through integrating analytical techniques and bioinformatics. is a reference standards scientist at the United States Pharmacopeial Convention. He is responsible for the development and establishment of reference standards for compendial use. He manages a diverse portfolio of reference standards including small molecules, complex actives and biologicals. joined the Food and Drug Administration in 1994 as a review chemist in the division of gastrointestinal and coagulation drug products. He became Heparin Expert in 2001 and was promoted to Chemistry, Manufacturing and Control Lead in 2005. He is currently a branch chief in the Office of New Drug Quality Assessment (Branch II, Division I). is Vice President, Biologics and Biotechnology, USP-NF at the United States Pharmacopeial Convention. She coordinates all standard-setting activities in the division related to biologics and biotechnology for the US-based compendia and manages the scientific staff responsible for the relevant expert committees, the development of biological reference materials, and the biologics laboratory at the United States Pharmacopeial Convention headquarters. Before she joined the United States Pharmacopeial Convention, her industrial experience included major biotechnology companies in the areas of analytical development, especially mass spectrometry, and recombinant protein characterization. is a principal scientific liaison in biologics and biotechnology in the Department of Global Science and Standards Division at the United States Pharmacopeial Convention. She is the scientific liaison for the USP Unfractionated Heparin Expert Panel, the USP Low Molecular Weight Heparins Expert Panel, the USP Therapeutic Peptides Expert Panel, and the USP Monographs-B&B 1 Expert Committee as well as other expert panels. Since the 2007 heparin contamination crisis, she has been working on revising the United States Pharmacopeia ‘Heparin sodium’ monograph to include new and improved tests to control quality and impurities. Broad standard tableFood and Drug AdministrationHeparin sodium drug productNumber-average molecular weightPeak molecular weightWeight-average molecular weightMolecular weightRelative standard deviationStandard deviationSize-exclusion chromatography United States Pharmacopeia

Model system for targeted drug release triggered by immune-specific signals by Shay Mailloux; Nataliia Guz; Maria Gamella Carballo; José M. Pingarrón; Evgeny Katz (4825-4829).
A new sense-and-act system was realized by integrating a biocatalytic/bioaffinity electrode responding to immune signals represented by an antibody and a polymer-modified electrode loaded with drug-mimicking species. The release of the drug-mimicking species was achieved specifically in response to a signal antibody, thus demonstrating for the first time an immune-induced drug-releasing process. The present approach promises new options for future applications in controlled drug release and personalized medicine. Figure Electrochemical immune-sensing system was integrated with the substance-releasing modified electrode to demonstrate the immune-triggered drug release process
Keywords: Bioanalytical methods; Drug-mimicking release; Immune sensing; Bioelectrocatalysis; Modified electrode

studied food chemistry in Braunschweig, Germany, and is currently a Ph.D. student in the Department of Analytical Chemistry at the Helmholtz Centre for Environmental Research–UFZ in Leipzig, Germany. His research focuses on the uptake of environmental contaminates in zebrafish embryos and combines analytical and ecotoxicological issues. studied chemistry in Leipzig, Germany, and received his Ph.D. degree in 2011 from the University of Regensburg, Germany. From 2011 to 2013, he was a postdoctoral researcher at the Helmholtz Centre for Environmental Research—UFZ in Leipzig, Germany. Currently, he works as a research scientist at MVZ Labordiagnostik Dessau in Dessau, Germany. is a research scientist and workgroup leader in the Department Bioanalytical Ecotoxicology at the Helmholtz Centre for Environmental Research—UFZ in Leipzig, Germany. His main research interest is the evaluation of fish embryos as alternatives to fish tests. His recent work focuses on the toxicokinetics of pollutants in zebrafish embryos and reactions to toxins at the zebrafish embryo proteome level. is Head of the Department of Analytical Chemistry at the Helmholtz Centre of Environmental Research—UFZ in Leipzig, Germany, and Professor for Analytical Chemistry at the University of Leipzig. He has been active for several years in developing analytical methods for organic trace analytes from environmental matrices, and in applying these methods to study the occurrence and fate of contaminants, in cooperation with engineers, (micro)biologists and ecotoxicologists.An analytical method using high-performance liquid chromatography–tandem mass spectrometry was developed to determine internal concentrations of 34 test compounds such as pharmaceuticals and pesticides in zebrafish embryos (ZFE), among them, cimetidine, 2,4-dichlorophenoxyacetic acid, metoprolol, atropine and phenytoin. For qualification and quantification, multiple reaction monitoring mode was used. The linear range extends from 0.075 ng/mL for thiacloprid and metazachlor and 7.5 ng/mL for coniine and clofibrate to 250 ng/mL for many of the test compounds. Matrix effects were strongest for nicotine, but never exceeded ±20 % for any of the developmental stages of the ZFE. Method recoveries ranged from 90 to 110 % from an analysis of nine pooled ZFE. These findings together with the simple sample preparation mean this approach is suitable for the determination of internal concentrations from only nine individual ZFE in all life stages up to 96 h post-fertilization. Exemplarily, the time course of the internal concentrations of clofibric acid, metribuzin and benzocaine in ZFE was studied over 96 h, and three different patterns were distinguished, on the basis of the speed and extent of uptake and whether or not a steady state was reached. Decreasing internal concentrations may be due to metabolism in the ZFE. Figure Quantification of polar pollutants in different life stages of the zebrafish embryo by LC-MS/MS
Keywords: Bioconcentration; Toxicodynamic; Absorption, distribution, metabolism and excretion; Larvae; Electrospray; Toxicity

Multi-analyte homogenous immunoassay based on quenching of quantum dots by functionalized graphene by L. Anfossi; P. Calza; F. Sordello; C. Giovannoli; F. Di Nardo; C. Passini; M. Cerruti; I. Y. Goryacheva; E. S. Speranskaya; C. Baggiani (4841-4849).
We propose a homogenous multi-analyte immunoassay based on the quenching of quantum dot (QD) fluorescence by means of graphene. Two QDs with emission maxima at 636 and 607 nm were bound to antibodies selective for mouse or chicken immunoglobulins, respectively, and graphene functionalized with carboxylic moieties was employed to covalently link the respective antigen. The antibody-antigen interaction led graphene close enough to QDs to quench the QD fluorescence by resonance energy transfer. The addition of free antigens that competed with those linked to graphene acted as a “turn-on” effect on QD fluorescence. Fluorescence emitted by the two QDs could be recorded simultaneously since the QDs emitted light at different wavelengths while being excited at the same wavelength and proved to be linearly correlated with free antigen concentration. The developed assay allows measuring both antigens over 2–3 orders of magnitude and showed estimated limits of detection in the nanomolar range. This approach is thus a promising universal strategy to develop homogenous immunoassays for diverse antigens (cells, proteins, low-molecular-mass analytes) in a multi-analyte configuration.
Keywords: Immunoassay; Antibody; Homogeneous assay; Quantum dot; Graphene; Resonance energy transfer; Fluorescence quenching

Multiplexed microfluidic enzyme assays for simultaneous detection of lipolysis products from adipocytes by Colleen E. Dugan; William P. Cawthorn; Ormond A. MacDougald; Robert T. Kennedy (4851-4859).
Microfluidics has enabled new cell biology experiments. Incorporating chemical monitoring of cellular secretion into chips offers the potential to increase information content and utility of such systems. In this work, an integrated, multilayer polydimethylsiloxane microfluidic chip was developed to simultaneously measure fatty acids and glycerol secreted from cultured adipocytes on chip in near real time. Approximately 48,000 adipocytes were loaded into a cell chamber in a reversibly sealed chip. Cells were perfused at 0.75 μL/min. Cell perfusate was split and directed to separate, continuously operating fluorescent enzyme assay channel networks. The fluorescent assay products were detected simultaneously near the outlet of the chip. The fatty acid and glycerol assays had linear dynamic ranges of 150 and 110 μM and limit of detection (LOD) of 6 and 5 μM, respectively. Surface modifications including pretreatment with sodium dodecyl sulfate were utilized to prevent adsorption of fatty acids to the chip surface. Using the chip, basal fatty acid and glycerol concentrations ranged from 0.18 to 0.7 nmol × 106 cell−1 min−1 and from 0.23 to 0.85 nmol × 106 cell−1 min−1, respectively. Using valves built into the chip, the perfusion solution was switched to add 20 μM isoproterenol, a β-adrenergic agonist, which stimulates the release of glycerol and fatty acids in adipocytes. This manipulation resulted in a rapid and stable 1.5- to 6.0-fold increase of non-esterified fatty acid (NEFA) and glycerol. The ratio of NEFA:glycerol released increased with adipocyte age. These experiments illustrate the potential for performing multiple real-time assays on cells in culture using microfluidic devices.
Keywords: Adipocytes; Enzyme assay; Integration; Microfluidics; Multiplex

Optimization and validation of a label-free MRM method for the quantification of cytochrome P450 isoforms in biological samples by Ahmad Al Ali; David Touboul; Jean-Pierre Le Caër; Isabelle Schmitz-Afonso; Jean-Pierre Flinois; Catherine Marchetti; Isabelle De Waziers; Alain Brunelle; Olivier Laprévote; Philippe Beaune (4861-4874).
Cytochromes P450 (CYPs) play critical roles in oxidative metabolism of many endogenous and exogenous compounds. Protein expression levels of CYPs in liver provide relevant information for a better understanding of the importance of CYPs in pharmacology and toxicology. This work aimed at establishing a simple method to quantify six CYPs (CYP3A4, CYP3A5, CYP1A2, CYP2D6, CYP2C9, and CYP2J2) in various biological samples without isotopic labeling. The biological matrix was spiked with the standard peptides prior to the digestion step to realize a label-free quantification by mass spectrometry. The method was validated and applied to quantify these six isoforms in both human liver microsomes and mitochondria, but also in recombinant expression systems such as baculosomes and the HepG2 cell line. The results showed intra-assay and interassay accuracy and precision within 16 % and 5 %, respectively, at the low quality control level, and demonstrated the advantages of the method in terms of reproducibility and cost. Figure Calibration curve in complex matrix for CYPs quantification
Keywords: Cytochrome P450; Mass spectrometry; Multiple reaction monitoring; Label-free quantification; Liver microsomes; Baculosomes

Phenotyping of CYP450 in human liver microsomes using the cocktail approach by Dany Spaggiari; Laurent Geiser; Youssef Daali; Serge Rudaz (4875-4887).
The cocktail approach is an advantageous strategy used to monitor the activities of several cytochromes P450 (CYPs) in a single test to increase the throughput of in vitro phenotyping studies. In this study, a cocktail mixture was developed with eight CYP-specific probe substrates to simultaneously evaluate the activity of the most important CYPs, namely, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and the CYP3A subfamily. After cocktail incubation in the presence of human liver microsomes (HLMs), the eight selected substrates and their specific metabolites were analyzed by ultra-high-pressure liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry. Qualitative and quantitative data were simultaneously acquired to produce an overview of the extended phase I biotransformation routes for each probe substrate in the HLMs and to generate phenotypic profiles of various HLMs. A comparison of the cocktail strategy with an individual substrate assay for each CYP produced similar results. Moreover, the cocktail was tested on HLMs with different allelic variants and/or in the presence of selective inhibitors. The results were in agreement with the genetic polymorphisms of the CYPs and the expected effect of the alterations. All of these experiments confirmed the reliability of this cocktail assay for phenotyping of the microsomal CYPs.
Keywords: Cocktail approach; Ultra-high-pressure liquid chromatography–quadrupole time-of-flight mass spectrometry; Cytochrome P450 profile; Phenotyping; Genetic polymorphism; Human liver microsomes

A fluorescence lifetime-based binding assay for acetylpolyamine amidohydrolases from Pseudomonas aeruginosa using a [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ligand probe by Christian Meyners; Robert Wawrzinek; Andreas Krämer; Steffen Hinz; Pablo Wessig; Franz-Josef Meyer-Almes (4889-4897).
High-throughput assays for drug screening applications have to fulfill particular specifications. Besides the capability to identify even compounds with low potency, one of the major issues is to minimize the number of false-positive hits in a screening campaign in order to reduce the logistic effort for the subsequent cherry picking and confirmation procedure. In this respect, fluorescence lifetime (FLT) appears as an ideal readout parameter that is supposed to be robust against autofluorescent and light-absorbing compounds, the most common source of systematic false positives. The extraordinary fluorescence features of the recently discovered [1,3]dioxolo[4,5-f][1,3] benzodioxole dyes were exploited to develop an FLT-based binding assay with exceptionally robust readout. The assay setup was comprehensively validated and shown to comply not only with all requirements for a powerful high-throughput screening assay but also to be suitable to determine accurate binding constants for inhibitors against enzymes of the histone deacetylase family. Using the described binding assay, the first inhibitors against three members of this enzyme family from Pseudomonas aeruginosa were identified. The compounds were characterized in terms of potency and selectivity profile. The novel ligand probe should also be applicable to other homologues of the histone deacetylase family that are inhibited by N-hydroxy-N′-phenyloctandiamide. Figure ᅟ
Keywords: Histone deacetylases; Acetylpolyamine amidohydrolases; Fluorescence life time; Binding assay; Pseudomonas aeruginosa

Food safety is a major concern for suppliers in the food chain to ensure the safety of their products. The identification procedure requested by norms is tedious, and it often requires systematic controls and qualified staff to perform the necessary analyses. Raman spectroscopy offers new opportunities to rapidly and efficiently ascertain the presence of pathogens in samples. Nevertheless, this technique requires a standardized procedure to be applied in the industrial context. Our study shows that the variability between spectral fingerprints is related to the physiological state of the microbial species and the growth phase of the bacteria plays a crucial role in its identification by Raman spectroscopy. To improve the discrimination between closely related bacterial species, a procedure based on the selection of bacterial spectra in the exponential growth phase was proposed. Different ways to introduce Raman spectroscopy in the ISO 6579:2002 standards are also proposed from the entire process to a shorter protocol. In the latter case, the identification of bacterial colonies after the selective enrichment step was proposed with the advantages of this path in terms of simplicity and rapidity (analysis time is reduced up to 50 h from the 100 h required by the standard). The protocol validated using six food categories from industrial partners have presented a good correlation by confirmation with other laboratory classical methods. In the future, this procedure could be introduced to the control system of the food production chain with a reliable database for various microorganisms encountered in this field.
Keywords: Raman spectroscopy; Identification; Bacteria; Food

Quantitative monitoring of yeast fermentation using Raman spectroscopy by Jens A. Iversen; Rolf W. Berg; Birgitte K. Ahring (4911-4919).
Compared to traditional IR methods, Raman spectroscopy has the advantage of only minimal interference from water when measuring aqueous samples, which makes this method potentially useful for in situ monitoring of important industrial bioprocesses. This study demonstrates real-time monitoring of a Saccharomyces cerevisiae fermentation process using a Raman spectroscopy instrument equipped with a robust sapphire ball probe. A method was developed to correct the Raman signal for the attenuation caused by light scattering cell particulate, hence enabling quantification of reaction components and possibly measurement of yeast cell concentrations. Extinction of Raman intensities to more than 50 % during fermentation was normalized with approximated extinction expressions using Raman signal of water around 1,627 cm−1 as internal standard to correct for the effect of scattering. Complicated standard multi-variant chemometric techniques, such as PLS, were avoided in the quantification model, as an attempt to keep the monitoring method as simple as possible and still get satisfactory estimations. Instead, estimations were made with a two-step approach, where initial scattering correction of attenuated signals was followed by linear regression. In situ quantification measurements of the fermentation resulted in root mean square errors of prediction (RMSEP) of 2.357, 1.611, and 0.633 g/L for glucose, ethanol, and yeast concentrations, respectively.
Keywords: Raman spectroscopy; Sapphire ball probe; On-line monitoring; Yeast fermentation; Quantitative analysis; Scattering correction

Enzymatic hydrolysis-based absolute quantification of triacylglycerols in plant oil by use of a single marker by Ting Tan; Chang-jiang-sheng Lai; Su-ling Zeng; E-Hu Liu; Ping Li (4921-4929).
Absolute quantification of triacylglycerols (TAGs) in plant oils is a challenge for analysts, because most of the necessary chemical standards are unavailable. In this study, a new method for absolute quantification analysis of multi-components by use of a single marker (AQAMS), using two crucial technologies, evaluation of the collection recovery without chemical standards and enzymatic hydrolysis, was used for determining the absolute content of TAGs in brucea javanica oil (BJO), using glycerol as the marker. The TAGs in BJO were initially characterized using ultrafast liquid chromatography tandem atmospheric-pressure-chemical-ionization mass spectrometry. Then the TAGs in BJO were individually collected, by target-fraction collection via high-performance liquid chromatography coupled with an evaporative-light-scattering detector (HPLC-ELSD), and their recoveries were calculated by use of a novel non-standard evaluated recovery strategy (NSER). The results revealed that the collection procedure was feasible and reliable. Finally, modified commercial TAG assay kits using glycerol as the marker were used to determine the absolute abundance of individual TAGs in the plant oils. Comparing the result with that obtained by HPLC-ELSD analysis using triolein standard, the content of triolein determined by AQAMS was closely matched. The proposed strategy is a practical measure for solving the problem of the lack of chemical standards, and provides a new method for absolute quantification in natural products of multi-components with the same backbone. Figure Enzymatic hydrolysis-based absolute quantification of triacylglycerols
Keywords: Absolute quantification; Triacylglycerols; Brucea javanica oil; Non-standard evaluated recovery strategy; Colorimetric assay kits

The gas chromatographic separation of fatty acid methyl esters (FAMEs) on ionic liquid stationary phases was investigated. Seven commercially available ionic liquid columns were tested using a test mixture containing 37 fatty acid methyl esters. The influence of column temperature on the elution order was studied using five different temperature programs. Retention times were highly reproducible. Similar retention behavior was observed for the IL59, IL60, and IL61 columns. The peak pair C18:1 cis/trans was not baseline resolved on these columns, whose stationary phases are highly similar. C18:2 cis/trans, C18:3 n6/n3, and C20:3 n6/n3 were baseline separated on all columns. Baseline separation of the complete test mix was only obtained on the IL82 column using a heating rate of 5 K/min. In general, retention times decreased with increasing column polarity but unsaturated FAMEs were retained stronger compared to their saturated counterparts. Except for the IL59 column, retention crossover was observed when the temperature program was changed.
Keywords: Ionic liquid columns; Gas chromatography; Fatty acid methyl esters; Retention crossover

Mixed-mode liquid chromatography coupled to tandem mass spectrometry for the analysis of aminoglycosides in meat by Élida Alechaga; Encarnación Moyano; M. Teresa Galceran (4941-4953).
A novel LC-MS/MS method has been developed for the determination of 13 aminoglycoside antibiotics in meat products. Among the chromatographic columns tested, the mixed-mode Obelisc R provided the best performance. Electrospray has been used for the coupling of the LC and the effect of temperature on the ionization has been evaluated. The mass spectra of AGs have been studied in order to select the most adequate precursor and product ions for quantitation and confirmation in SRM mode, showing that the single charged [M+H]+ provided better precisions than the double charged [M+2H]2+. Accurate mass measurements have been performed in order to confirm the molecular composition of the product ions, allowing the establishment of a new mechanism for some product ions of STR and DHSTR. A sample treatment based on an extraction and a SPE clean-up has been applied to a wide variety of meat products such as frankfurters; sausages; and minced meat of pork, veal, and chicken. Method limits of quantitation in the low microgram per kilogram level (1–50 μg kg−1), precisions %RSD below 15 % and accuracies expressed as relative errors below 23 % have been obtained, making the proposed method suitable for routine analysis.
Keywords: Mixed-mode chromatography; Aminoglycosides; LC-MS/MS; Mass spectrometry; Meat products

A novel ionic liquid-modified organic-polymer monolith as the sorbent for in-tube solid-phase microextraction of acidic food additives by Ting-Ting Wang; Yi-Hui Chen; Jun-Feng Ma; Min-Jie Hu; Ying Li; Jiang-Hua Fang; Hao-Qi Gao (4955-4963).
A novel ionic liquid-modified organic-polymer monolithic capillary column was prepared and used for in-tube solid-phase microextraction (SPME) of acidic food additives. The primary amino group of 1-aminopropyl-3-methylimidazolium chloride was reacted with the epoxide group of glycidyl methacrylate. The as-prepared new monomer was then copolymerized in situ with acrylamide and N,N’-methylenebisacrylamide in the presence of polyethylene glycol (PEG)-8000 and PEG-10,000 as porogens. The extraction performance of the developed monolithic sorbent was evaluated for benzoic acid, 3-hydroxybenzoic acid, cinnamic acid, 2,4-dichlorophenoxyacetic acid, and 3-(trifluoromethyl)-cinnamic acid. Such a sorbent, bearing hydrophobic and anion-exchange groups, had high extraction efficiency towards the test compounds. The adsorption capacities for the analytes dissolved in water ranged from 0.18 to 1.74 μg cm−1. Good linear calibration curves (R 2 > 0.99) were obtained, and the limits of detection (S/N = 3) for the analytes were found to be in the range 1.2–13.5 ng mL−1. The recoveries of five acidic food additives spiked in Coca-Cola beverage samples ranged from 85.4 % to 98.3 %, with RSD less than 6.9 %. The excellent applicability of the ionic liquid (IL)-modified monolithic column was further tested by the determination of benzoic acid content in Sprite samples, further illustrating its good potential for analyzing food additives in complex samples. Graphical abstract ᅟ
Keywords: Ionic liquid; Food additive; Monolithic column; In-tube solid-phase microextraction

A reliable and repeatable isotachophoretic method for the simultaneous determination of K+, Na+, Ca2+, Mg2+, and ammonium in berries of different blue honeysuckle cultivars was developed. The usefulness of the complex-forming equilibria in determining the cations mentioned above was studied. The addition of 7.5 mM 18-crown-6 and 2 mM α-hydroxyisobutyric acid to sulfuric acid enhanced the separation capacity of the leading electrolyte. The terminating electrolyte of 10 mM TRIS buffered to pH 4.30 was chosen as most appropriate for these studies. An ultrasound-assisted procedure for the extraction of macroelements from dried fruit of Lonicera caerulea L. was investigated using response surface methodology. The Box-Behnken experimental design showed the optimal extraction conditions as follows: temperature of 36 °C, extraction time of 42.3 min and solid-to-liquid ratio of 1:800 v/w. Detection limits for the method ranged from 0.030 to 0.097 mg/l with precision expressed by RSD ranging from 1.4 % for Mg to 4.4 % for Na. The results showed that blue honeysuckle berries are a good source of potassium, from 199 to 402 mg/100 g FW, especially ‘Zielona’ and ‘Zojka’ cultivars. The average content of other analytes in these berries was 5.2 mg Na, 24.8 mg Ca, 7.5 mg Mg and 52.0 mg ammonium per 100 g FW. The berry products contained lower amounts of minerals especially Mg and K. However, a glass of freshly squeezed juice can provide almost 20 % of the RDI of potassium. The results obtained using both the developed isotachophoretic method and ion chromatographic procedure did not differ significantly. Figure ᅟ
Keywords: Electroanalytical methods; Capillary isotachophoresis; Ion chromatography; Response surface methodology; Macroelements; Blue honeysuckle berry

Xanthine oxidase (XOD) is a key oxidative enzyme to the pathogenesis of hyperuricemia and certain diseases induced by excessive reactive oxygen species. XOD inhibitors could provide an important therapeutic approach to treat such diseases. A new method using affinity selection-based two-dimensional chromatography coupled with liquid chromatography-mass spectrometry was developed for the online screening of potential XOD inhibitors from Radix Salviae Miltiorrhizae. Based on our previous study, the two-dimensional, turbulent-flow chromatography (TFC) was changed to a mixed-mode anion-exchange/reversed-phase column and one reversed-phase column. The developed method was validated to be selective and sensitive for screening XOD-binding compounds, especially weak acidic ones, in the extracts. Three salvianolic acids were screened from the Radix Salviae Miltiorrhizae extract via the developed method. The XOD inhibitory activities of salvianolic acid C and salvianolic acid A were confirmed, and their inhibitory modes were measured. Salvianolic acid C exhibited potent XOD inhibitory activity with an IC50 of 9.07 μM. This work demonstrated that the developed online, two-dimensional TFC/LC-MS method was effective in discovering the binding affinity of new compounds from natural extracts for target proteins, even at low concentrations. ᅟ
Keywords: Affinity selection-based two-dimensional chromatography; Ion exchange chromatography; Xanthine oxidase inhibitor; Radix Salviae Miltiorrhizae

Measurement of meropenem concentration in different human biological fluids by ultra-performance liquid chromatography-tandem mass spectrometry by Raül Rigo-Bonnin; Roser Juvany-Roig; Elisabet Leiva-Badosa; Joan Sabater-Riera; Xosé L. Pérez-Fernández; Paola Cárdenas-Campos; Enric Sospedra-Martínez; Helena Colom; Pedro Alía (4997-5007).
Meropenem is a broad-spectrum antibiotic, often used for the empirical treatment of infections in critically ill patients with acute kidney injury. Meropenem has clinically insignificant protein binding and, as a carbapenem antibiotic, shows time-dependent bacterial killing, meaning that the unbound or free antibiotic concentration in blood should be maintained above the minimal inhibitory concentration of the pathogen for at least 40 % of the dosing interval. We developed and validated simple chromatographic methods by ultra-performance liquid chromatography-tandem mass spectrometry to measure plasma, filtrate-dialysate, and urine concentrations of meropenem. Chromatographic separation was achieved using an Acquity® UPLC® BEHTM (2.1 × 100 mm id, 1.7 μm) reverse-phase C18 column, with a water/acetonitrile linear gradient containing 0.1 % formic acid at a 0.4-mL/min flow rate. Meropenem and its internal standard (ertapenem) were detected by electrospray ionization mass spectrometry in positive ion multiple reaction monitoring mode. The limits of quantification were 0.27, 0.24, and 1.22 mg/L, and linearity was observed between 0.27–150, 0.24–150, and 1.22–2,000 mg/L for plasma, filtrate-dialysate, and urine samples, respectively. Coefficients of variation and relative biases were less than 13.5 and 8.0 % for all biological fluids. Recovery values were greater than 68.3 %. Evaluation of the matrix effect showed ion suppression for meropenem and ertapenem. No carry-over was observed. The validated methods are useful for both therapeutic drug monitoring and pharmacokinetic studies. It could be applied to daily clinical laboratory practice to measure the concentration of meropenem in plasma, filtrate-dialysate, and urine. Figure Meropenem chemical structures
Keywords: Filtrate-dialysate; Ertapenem; Meropenem; Plasma; Therapeutic drug monitoring; UPLC-MS/MS; Urine

Quality fluctuation detection of an herbal injection based on biological fingerprint combined with chemical fingerprint by Lele Zhang; Lina Ma; Wuwen Feng; Congen Zhang; Feiya Sheng; Yi Zhang; Chen Xu; Gang Dong; Xiaoping Dong; Xiaohe Xiao; Dan Yan (5009-5018).
Herbal injection is one of the most important preparations of traditional Chinese medicine. More than 130 types of herbal injections are used clinically for 400 million patients annually with total sales of over four billion US dollars per year. However, the current quality control (QC) methods relying mainly on chemical fingerprints (CF) can hardly ensure quality and safety of the herbal injections with complex chemical composition and have resulted in an increase in serious adverse drug reactions. In this study, a comprehensive approach for the QC of a controversial herbal injection Shuang-Huang-Lian lyophilized powder (SHL) was established based on the quality fluctuation detection by a combination of CF and biological fingerprint (BF). High-performance liquid chromatography and the impedance-based xCELLigence system were applied to establish the CF and BF, respectively. In addition, multivariate analysis was performed to evaluate the discriminant ability of the two methods. The results showed that being subjected to environmental influence like oxygen/air, high temperature, and extreme illumination could lead to quality fluctuation of SHL. The combination of chemical and biological fingerprint method is a more powerful tool for the QC of SHL because it can clearly discriminate different groups of abnormal samples. This method can be used for the detection of quality fluctuation of SHL and can provide reference for the quality control of other herbal injections.
Keywords: Herbal injection; Quality control; Shuang-Huang-Lian lyophilized powder; Biological fingerprint; Chemical fingerprint; Quality fluctuation

Trace levels of the veterinary antibiotic compound sulfadiazine (SDZ) can be determined in agricultural drainage water samples with this new method. Optimized sample pretreatment and solid-phase extraction was combined with liquid chromatography coupled to tandem mass spectrometry (SPE LC-MS/MS) using positive electrospray ionization. The linear dynamic range for the LC-MS/MS was assessed from 5 μg/L to 25 mg/L with a 15-point calibration curve displaying a coefficient of correlation r 2 = 0.9915. Agricultural drainage water spiked at a concentration of 25 ng/L gave recoveries between 63 and 98 % (relative standard deviation 15 %), while at 10 ng/L, it showed a lower recovery of 32 % (relative standard deviation 47 %). The final SPE LC-MS/MS method had a limit of detection (LOD)Method and a limit of quantification (LOQ)Method of 7.5 and 23 ng/L agricultural drainage water, respectively. Determination of SDZ, spiked at a realistic concentration of 50 μg/L, in artificial drainage water (ADW) containing common and high levels of phosphate (0.05, 0.5, and 5 mg/L) gave recoveries between 70 and 92 % (relative standard deviation 7.4–12.9 %). Analysis of the same realistic concentration of SDZ in ADW, spiked with common and high levels of dissolved organic carbon (2, 6, and 15 mg/L) confirmed the possible adaptation of a tandem solid-phase extraction (strong anion exchange (SAX)-hydrophilic-lipophilic balance (HLB)) followed by liquid chromatography-tandem mass spectrometry methodology. Recoveries obtained ranged from 104 to 109 % (relative standard deviation 2.8–5.2 %). The new methods enable determination of the veterinary antibiotic compound SDZ in agricultural drainage water from field experiments and monitoring schemes for phosphate- and dissolved organic carbon (DOC)-rich water samples in intensive farming areas. Figure Clean-up and up-concentration of sulfadiazine from agricultural drainage water
Keywords: SDZ; LC-MS/MS; SPE; Veterinary antibiotic; Sulfonamide

Pericellular matrix plays an active role in retention and cellular uptake of large-sized nanoparticles by Ruili Xu; Bin Xiong; Rui Zhou; Hui Shen; Edward S. Yeung; Yan He (5031-5037).
As the outmost coating of cells, the pericellular matrix (PCM) involved in various cellular functions has been exploited previously to be able to accumulate 120 nm Au nanoparticles (NPs), adjust their diffusion coefficient similar to that of membrane receptors, and enhance their uptake efficiency. In this study, the interactions between PCM and NPs with different sizes and materials were systematically investigated. We found that PCM can selectively enhance the retention and cellular uptake of NPs with diameters from 50 to 180 nm, but has no enhancement effect for 20 nm NPs. Identical behaviors of PCM was observed for both Au NPs and polystyrene NPs, indicating that this unique phenomenon is more related to the dimensions of the NPs. The study of single-particle tracking of 50–180 nm NPs on the surface of thick PCM cells revealed that PCM actively adjusts the diffusion coefficient of NPs to ∼0.1 μm2/s regardless of their sizes. By blocking the receptor-mediated endocytosis (RME) pathway with four different inhibitors, this active role of PCM can be effectively suppressed, further confirming that the trapping and retention of NPs by PCM is an inherent biological function. These findings provided new insights for better understanding of the RME pathway and may have promising NP-based applications for controlled drug delivery and therapy in biomedicine. Figure In this study, we revealed the active role of pericellular matrix (PCM) during the endocytosis of large nanoparticles (NPs). PCM can selectively enhance the retention of NPs larger than 50 nm and adjust the diffusion coefficient of large-sized NPs to ~0.1 μm2/s regardless of their sizes, which is beneficial for the binding of NPs with receptors and followed cellular endocytosis. This active role can be suppressed by endocytosis inhibitors, suggesting the trapping of PCM on NPs is an integral part of receptor-mediated endocytosis
Keywords: Pericellular matrix; Nanoparticles; Cell; Receptor-mediated endocytosis; Retention

Measurement of mercury species in human blood using triple spike isotope dilution with SPME-GC-ICP-DRC-MS by Yuliya L. Sommer; Carl P. Verdon; Mark R. Fresquez; Cynthia D. Ward; Elliott B. Wood; Yi Pan; Kathleen L. Caldwell; Robert L. Jones (5039-5047).
The measurement of different mercury compounds in human blood can provide valuable information about the type of mercury exposure. To this end, our laboratory developed a biomonitoring method for the quantification of inorganic (iHg), methyl (MeHg), and ethyl (EtHg) mercury in whole blood using a triple-spike isotope dilution (TSID) quantification method employing capillary gas chromatography (GC) and inductively coupled dynamic reaction cell mass spectrometry (ICP-DRC-MS). We used a robotic CombiPAL® sample handling station featuring twin fiber-based solid-phase microextraction (SPME) injector heads. The use of two SPME fibers significantly reduces sample analysis cycle times making this method very suitable for high sample throughput, which is a requirement for large public health biomonitoring studies. Our sample preparation procedure involved solubilization of blood samples with tetramethylammonium hydroxide (TMAH) followed by the derivatization with sodium tetra(n-propyl)borate (NaBPr4) to promote volatility of mercury species. We thoroughly investigated mercury species stability in the blood matrix during the course of sample treatment and analysis. The method accuracy for quantifying iHg, MeHg, and EtHg was validated using NIST standard reference materials (SRM 955c level 3) and the Centre de Toxicologie du Québec (CTQ) proficiency testing (PT) samples. The limit of detection (LOD) for iHg, MeHg, and EtHg in human blood was determined to be 0.27, 0.12, and 0.16 μg/L, respectively. Figure ᅟ
Keywords: Speciation; Mercury; Blood; Biomonitoring; Isotope dilution; SPME; GC; ICP-MS

Biomonitoring of infant exposure to phenolic endocrine disruptors using urine expressed from disposable gel diapers by Liangpo Liu; Tongwei Xia; Xueqin Zhang; Dana Boyd Barr; Ambreen Alamdar; Jie Zhang; Meiping Tian; Qingyu Huang; Heqing Shen (5049-5054).
Infant exposure to endocrine disruptors (EDs) may cause adverse health effects because of their fast growth and development during this life stage. However, collecting urine from infants for exposure assessment using biological monitoring is not an easy task. For this purpose, we evaluated the feasibility of using urine expressed from disposable gel absorbent diapers (GADs) as a matrix for biomonitoring selected phenolic EDs. GADs urine was expressed with the assistance of CaCl2 and was collected using a device fabricated in our laboratory. The analytes were extracted and concentrated using a liquid-liquid method and their hydroxyl groups were modified by dansyl chloride to enhance their chromatography and detection. Finally, the analytes were measured by high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). The target chemicals were bisphenol A, triclosan, 17 α-ethynylestradiol, the natural hormone estrone, and 17 β-estradiol. The ratio of the CaCl2 to the urine-wetted gel absorbent, variation of the inter-urination volume, and analyte deposition bias in the diaper were assessed. Analyte blank values in the diapers, the sample storage stabilities, and recoveries of the analytes were also evaluated. The results showed that 70–80 % of the urine could be expressed from the diaper with the assistance of CaCl2 and 70.5–124 % of the spiked analytes can be recovered in the expressed urine. The limits of detections (LODs) were 0.02–0.27 ng/mL, well within the range for detection in human populations. Our pilot data suggest that infants are widely exposed to the selected EDs.
Keywords: Disposable gel absorbent diaper; Biomonitoring; Endocrine disruptors; Infant; Minipuberty

An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS) method was developed and validated for the quantification of serotonin (5-HT) in lamb plasma using [2d4]-serotonin ([2d4]-5-HT) as an internal standard. Charcoal-stripped human plasma was used as the blank matrix during validation, and 5-HT was quantitated using selected reaction monitoring. The UHPLC/MS/MS system consisted of an Agilent 1290 Infinity ultrahigh-performance liquid chromatograph coupled with an AB SCIEX QTRAP® 5500 hybrid linear ion trap triple quadrupole mass spectrometer. The method was validated for accuracy, precision, linearity, lower limit of quantification (LLOQ), selectivity, and other parameters. The LLOQ was 1.0 ng/mL, requiring 100 μL of sample. The method was applied to monitor the 5-HT levels in lamb plasma after the administration of fluoxetine. Tandem mass spectrometry cubed (MS3) experiments were also performed to investigate the fragmentation pattern of 5-HT and [2d4]-5-HT. A liquid chromatography-MS3 (LC/MS3) method was developed, and the UHPLC/MS/MS and the LC/MS3 methods were compared for performance. Figure ᅟ
Keywords: Serotonin; LC/MS/MS; LC/MS3 ; Method validation; Lamb plasma