Novel comprehensive multidimensional liquid chromatography approach for elucidation of the microbosphere of shikimate-producing Escherichia coli SP1.1/pKD15.071 strain Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2017-11-22 Francesco Cacciola, Domenica Mangraviti, Francesca Rigano, Paola Donato, Paola Dugo, Luigi Mondello, Hernan J. Cortes
Shikimic acid is a intermediate of aromatic amino acid biosynthesis and the preferred starting material for production of the most commonly prescribed anti-influenza drug, Tamiflu. Its six-membered carbocyclic ring is adorned with several chiral centers and various functionalities, making shikimic acid a valuable chiral synthon. When microbially-produced, in addition to shikimic acid, numerous other metabolites are exported out of the cytoplasm and accumulate in the culture medium. This extracellular matrix of metabolites is referred to as the microbosphere. Due to the high sample complexity, in this study, the microbosphere of shikimate-producing Escherichia coli SP1.1/pKD15.071 was analyzed by liquid chromatography and comprehensive two-dimensional liquid chromatography coupled to photodiode array and mass spectrometry detection. GC analysis of the trimethylsilyl derivatives was also carried out in order to support the elucidation of the selected metabolites in the microbosphere. The elucidation of the metabolic fraction of this bacterial strain might be of valid aid for improving, through genetic changes, the concentration and yield of shikimic acid synthesized from glucose.
Peptidomic strategy for purification and identification of potential ACE-inhibitory and antioxidant peptides in Tetradesmus obliquus microalgae Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-02-23 Carmela Maria Montone, Anna Laura Capriotti, Chiara Cavaliere, Giorgia La Barbera, Susy Piovesana, Riccardo Zenezini Chiozzi, Aldo Laganà
Microalgae are unicellular marine organisms that have promoted complex biochemical pathways to survive in greatly competitive marine environments. They could contain significant amounts of high-quality proteins which, because of their structural diversity, contain a range of yet undiscovered novel bioactive peptides. In this work, a peptidomic platform was developed for the separation and identification of bioactive peptides in protein hydrolysates. In this work, a peptidomic platform was developed for the extraction, separation, and identification of bioactive peptides in protein hydrolysates. Indeed, extraction of proteins from recalcitrant tissues is still a challenge due to their strong cell walls and high levels of non-protein interfering compounds. Therefore, seven different protein extraction protocols, based on mechanical and chemical methods, were tested in order to produce high-quality protein extracts. Proteins obtained by means of the best protocol, consisting of milling the recalcitrant tissue with glass beads, were subjected to enzymatic digestion with Alcalase® and subsequently the hydrolysate was purified by two-dimensional semi-preparative reversed phase liquid chromatography. Fractions were assayed for antioxidant and antihypertensive activities and only the most active ones were finally analyzed by RP nanoHPLC-MS/MS. Around 500 peptide sequences were identified in these fractions. The identified peptides were subjected to an in silico analysis by PeptideRanker algorithm in order to assign a score of bioactivity probability. Twenty-five sequenced peptides were found with potential antioxidant and angiotensin-converting-enzyme-inhibitory activities. Four of these peptides, WPRGYFL, GPDRPKFLGPF, WYGPDRPKFL, SDWDRF, were selected for synthesis and in vitro tested for specific bioactivity, exhibiting good values of antioxidant and ACE-inhibitory activity.
Recent trends and analytical challenges in plant bioactive peptide separation, identification and validation Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-01-20 Susy Piovesana, Anna Laura Capriotti, Chiara Cavaliere, Giorgia La Barbera, Carmela Maria Montone, Riccardo Zenezini Chiozzi, Aldo Laganà
Interest in research into bioactive peptides (BPs) is growing because of their health-promoting ability. Several bioactivities have been ascribed to peptides, including antioxidant, antihypertensive and antimicrobial properties. As they can be produced from precursor proteins, the investigation of BPs in foods is becoming increasingly popular. For the same reason, production of BPs from by-products has also emerged as a possible means of reducing waste and recovering value-added compounds suitable for functional food production and supplements. Milk, meat and fish are the most investigated sources of BPs, but vegetable-derived peptides are also of interest. Vegetables are commonly consumed, and agro-industrial wastes constitute a cheap, large and lower environmental impact source of proteins. The use of advanced analytical techniques for separation and identification of peptides would greatly benefit the discovery of new BPs. In this context, this review provides an overview of the most recent applications in BP investigations for vegetable food and by-products. The most important issues regarding peptide isolation and separation, by single or multiple chromatographic techniques, are discussed. Additionally, problems connected with peptide identification in plants and non-model plants are discussed regarding the particular case of BP identification. Finally, the issue of peptide validation to confirm sequence and bioactivity is presented.
Screening for anti-proliferative and anti-inflammatory components from Rhamnus davurica Pall. using bio-affinity ultrafiltration with multiple drug targets Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-02-24 Guilin Chen, Jianlin Wu, Na Li, Mingquan Guo
Rhamnus davurica Pall. (R. davurica) has been used as a traditional medicine for many years in China and abroad and shown a wide spectrum of biological activities. Previously, we reported the phytochemical fingerprinting profile of R. davurica, its distinct anti-proliferative activities against HT-29 and SGC-7901 cell lines, and the topoisomerase I (Top I) ligands based on bio-affinity ultrafiltration and HPLC-MS (UF-HPLC-MS). Nevertheless, among the 32 peaks detected in the fingerprinting profile, the common bioactive constituents responsible for the anti-inflammatory and anti-proliferative activities in the extracts remain elusive. To further explore the specific responsible components for their diversified activities and their potential action targets/mechanisms, the method based on bio-affinity UF-HPLC-MS using therapeutic targets like Top I and cyclooxygenase 2 (COX-2) was established to rapidly screen and identify the ligands binding to these known target enzymes. As a result, 12 components were revealed as potential Top I ligands along with 11 components as potential COX-2 ligands, where several components were revealed to possess both activities. Further validations of these bioactive components have also been conducted and confirmed their highlighted activities. This integrated method of UF-HPLC-MS exhibits high efficiency in rapidly screening for multi-target bioactive components responsible for multiple pharmacological effects from the complex natural products and could be very useful to explain the complex action mechanisms of herb medicines in a complex multi-component and multi-target mode at the molecular level.
Facile derivatization of ultratrace carboxylic acids in saliva for quantification by HPLC–MS/MS Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-11 Chao Guo, Dongmei Li, Cuimei Liu, Zhenpeng Guo, Yi Chen
It remains an issue to directly quantify trace biologically important carboxyl compounds in body fluids. Herein we propose an innovative method to determine α-lipoic acid, 2-(β-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman, prostaglandin E2, cholic acid, and chenodeoxycholic acid in saliva. The method consists of two successive steps: fast and direct labeling of the target analytes with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide followed by ultrahigh-performance liquid chromatography–tandem mass spectrometry analysis. The method exhibited a wide linear range from 2.5 to 2500 pg/mL, with linear coefficients greater than 0.9963 and limits of detection and quantification as low as 0.10 and 0.33 pg/mL, respectively. The method precision was evaluated, with relative standard deviations ranging from 2.12% to 10.63% for intraday assays and from 2.98% to 12.88% for interday assays. The recoveries were measured by our spiking saliva samples with standards at three different levels, and ranged from 72.5% to 98.0%. Real applicability was validated by direct quantification of trace target analytes in human saliva, with simple pretreatment, use of a small sample volume, and a short analysis time.
Multiclass screening of >200 pharmaceutical and other residues in aquatic foods by ultrahigh-performance liquid chromatography–quadrupole-Orbitrap mass spectrometry Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-11 Cong Kong, Yang Wang, Yuanfei Huang, Huijuan Yu
A quick screening method of more than 200 pharmaceutical and other residues in aquatic foods based on ultrahigh-performance liquid chromatography–quadrupole-Orbitrap mass spectrometry (UHPLC-Q/Orbitrap MS) was established. In this method, after the addition of 200 μL of 1 M EDTA-Na2, 2 g of each sample homogenate was extracted successively with 10 mL of acetonitrile and 10 mL of ethyl acetate. The extracts were combined, dried under nitrogen flow, and redissolved in 0.1% formic acid in acetonitrile/water (4:6, v/v) for analysis. The prepared samples were analyzed by UHPLC- Q/Orbitrap MS system in Full MS/ddMS2 (full-scan data-dependent MS/MS) mode. Compound identification was performed through comparison of the sample data with the database for standard chemicals, including the retention time, precursor ion, product ions, and isotope pattern for all 206 compounds. Five different aquatic food matrices (carp, shrimp, crab, eel, and mussel) spiked with the analytes at 1, 10, and 50 ng/g were evaluated to assess recoveries, precision, matrix effects, stability, and detection limits using the method. UHPLC analyses required 25 min, and 178–200 analytes met identification criteria at 50 ng/g depending on the matrix. Furthermore, practical application of this method for real samples displayed strong screening capability.
Selective labeling for the identification and semi-quantification of lipid aldehydes in food products Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-10 Boudewijn Hollebrands, Eftychia Varvaki, Sonja Kaal, Hans-Gerd Janssen
Lipid oxidation reactions in foods rich in healthy unsaturated fatty acids result in the formation of a wide range of oxidation products that can have adverse effects on food quality and safety. To improve the understanding of oxidation reactions and methods for their inhibition, detailed information on the type and levels of the oxidation products formed is required. Accurate measurement of lipid oxidation products, especially of the non-volatile aldehyde products, has so far been a challenge due to the low sensitivity and limited specificity of most analytical methods. Here, a novel normal-phase LC method that uses selective labeling of aldehydes and epoxides with 7-(diethylamino)coumarin-3-carbohydrazide (CHH) is described. Labeling of alkanals is quantitative within 10 h. For alkenals, conversion is only around 50% at 24 h reaction time. Detailed MS identification of all aldehydes and epoxides is possible by using high-resolution MS and data-dependent MS2 acquisition. Fluorescence detection was successfully used to quantify groups of oxidation products. Sensitivity was high enough to allow accurate quantification even in fresh mayonnaises, where levels of around only 0.3 g total aldehydes/kg oil were found. Individual species can be quantified by MS if suitable reference standards are available. If no standards can be used, semi-quantification using an average response factor is an option. Clearly, the novel derivatization method is suitable for monitoring secondary lipid oxidation products in the early stages of shelf life. This makes it an important tool for developing improved food products.
Mussel-inspired 3D fiber scaffolds for heart-on-a-chip toxicity studies of engineered nanomaterials Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-10 Seungkuk Ahn, Herdeline Ann M. Ardoña, Johan U. Lind, Feyisayo Eweje, Sean L. Kim, Grant M. Gonzalez, Qihan Liu, John F. Zimmerman, Georgios Pyrgiotakis, Zhenyuan Zhang, Juan Beltran-Huarac, Paul Carpinone, Brij M. Moudgil, Philip Demokritou, Kevin Kit Parker
Due to the unique physicochemical properties exhibited by materials with nanoscale dimensions, there is currently a continuous increase in the number of engineered nanomaterials (ENMs) used in consumer goods. However, several reports associate ENM exposure to negative health outcomes such as cardiovascular diseases. Therefore, understanding the pathological consequences of ENM exposure represents an important challenge, requiring model systems that can provide mechanistic insights across different levels of ENM-based toxicity. To achieve this, we developed a mussel-inspired 3D microphysiological system (MPS) to measure cardiac contractility in the presence of ENMs. While multiple cardiac MPS have been reported as alternatives to in vivo testing, most systems only partially recapitulate the native extracellular matrix (ECM) structure. Here, we show how adhesive and aligned polydopamine (PDA)/polycaprolactone (PCL) nanofiber can be used to emulate the 3D native ECM environment of the myocardium. Such nanofiber scaffolds can support the formation of anisotropic and contractile muscular tissues. By integrating these fibers in a cardiac MPS, we assessed the effects of TiO2 and Ag nanoparticles on the contractile function of cardiac tissues. We found that these ENMs decrease the contractile function of cardiac tissues through structural damage to tissue architecture. Furthermore, the MPS with embedded sensors herein presents a way to non-invasively monitor the effects of ENM on cardiac tissue contractility at different time points. These results demonstrate the utility of our MPS as an analytical platform for understanding the functional impacts of ENMs while providing a biomimetic microenvironment to in vitro cardiac tissue samples.
Visual colorimetric detection of tin(II) and nitrite using a molybdenum oxide nanomaterial-based three-input logic gate Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-09 Jiayan Du, Mengxin Zhao, Wei Huang, Yuequan Deng, Yi He
We report a molybdenum oxide (MoO3) nanomaterial-based three-input logic gate that uses Sn2+, NO2−, and H+ ions as inputs. Under acidic conditions, Sn2+ is able to reduce MoO3 nanosheets, generating oxygen-vacancy-rich MoO3−x nanomaterials along with strong localized surface plasmon resonance (LSPR) and an intense blue solution as the output signal. When NO2− is introduced, the redox reaction between the MoO3 nanosheets and Sn2+ is strongly inhibited because the NO2− consumes both H+ and Sn2+. The three-input logic gate was employed for the visual colorimetric detection of Sn2+ and NO2− under different input states. The colorimetric assay’s limit of detection for Sn2+ and the lowest concentration of NO2− detectable by the assay were found to be 27.5 nM and 0.1 μM, respectively. The assay permits the visual detection of Sn2+ and NO2− down to concentrations as low as 2 μM and 25 μM, respectively. The applicability of the logic-gate-based colorimetric assay was demonstrated by using it to detect Sn2+ and NO2− in several water sources.
Synchrotron- and focal plane array-based Fourier-transform infrared spectroscopy differentiates the basalis and functionalis epithelial endometrial regions and identifies putative stem cell regions of human endometrial glands Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-09 Georgios Theophilou, Camilo L. M. Morais, Diane E. Halliwell, Kássio M. G. Lima, Josephine Drury, Pierre L. Martin-Hirsch, Helen F. Stringfellow, Dharani K. Hapangama, Francis L. Martin
The cyclical process of regeneration of the endometrium suggests that it may contain a cell population that can provide daughter cells with high proliferative potential. These cell lineages are clinically significant as they may represent clonogenic cells that may also be involved in tumourigenesis as well as endometriotic lesion development. To determine whether the putative stem cell location within human uterine tissue can be derived using vibrational spectroscopy techniques, normal endometrial tissue was interrogated by two spectroscopic techniques. Paraffin-embedded uterine tissues containing endometrial glands were sectioned to 10-μm-thick parallel tissue sections and were floated onto BaF2 slides for synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy and globar focal plane array-based FTIR spectroscopy. Different spectral characteristics were identified depending on the location of the glands examined. The resulting infrared spectra were subjected to multivariate analysis to determine associated biophysical differences along the length of longitudinal and crosscut gland sections. Comparison of the epithelial cellular layer of transverse gland sections revealed alterations indicating the presence of putative transient-amplifying-like cells in the basalis and mitotic cells in the functionalis. SR-FTIR microspectroscopy of the base of the endometrial glands identified the location where putative stem cells may reside at the same time pointing towards νsPO2− in DNA and RNA, nucleic acids and amide I and II vibrations as major discriminating factors. This study supports the view that vibration spectroscopy technologies are a powerful adjunct to our understanding of the stem cell biology of endometrial tissue.
Facile determination of sphingolipids under alkali condition using metal-free column by LC-MS/MS Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-09 Siddabasave Gowda B. Gowda, Kazutaka Ikeda, Makoto Arita
Extraction and analysis of sphingolipids from biological samples is a critical step in lipidomics, especially for minor species such as sphingoid bases and sphingosine-1-phosphate. Although several liquid chromatography-mass spectrometry methods enabling the determination of sphingolipid molecular species have been reported, they were limited in analytical sensitivity and reproducibility by causing significant peak tailing, especially by the presence of phosphate groups, and most of the extraction techniques are laborious and do not cover a broad range of sphingolipid metabolites. In this study, we developed a rapid single-phase extraction and highly sensitive analytical method for the detection and quantification of sphingolipids (including phosphates) comprehensively using liquid chromatography-triple quadruple mass spectrometry. After validating the reliability of the method, we analyzed the intestinal tissue sphingolipids of germ-free (GF) and specific pathogen-free (SPF) mice and found significantly higher levels of free sphingoid bases and sphingosine-1-phosphate in the GF condition as compared to the SPF condition. This method enables a rapid extraction and highly sensitive determination of sphingolipids comprehensively at low femtomolar ranges.
Method development and validation for total haloxyfop analysis in infant formulas and related ingredient matrices using liquid chromatography-tandem mass spectrometry Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-08 Urairat Koesukwiwat, Lukas Vaclavik, Katerina Mastovska
According to the European Commission directive 2006/141/EC, haloxyfop residue levels should not exceed 0.003 mg/kg in ready-to-feed infant formula, and the residue definition includes sum of haloxyfop, its esters, salts, and conjugates expressed as haloxyfop. A simple method for total haloxyfop analysis in infant formula and related ingredient matrices was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The sample preparation consisted of an alkaline hydrolysis with methanolic sodium hydroxide to release haloxyfop (parent acid) from its bound forms prior to the extraction with acetonitrile. A mixture of magnesium sulfate (MgSO4) and sodium chloride (NaCl) (4:1, w/w) was added to the extract to induce phase separation and force the analyte into the upper acetonitrile-methanol layer and then a 1-mL aliquot was subsequently cleaned up by dispersive solid phase extraction with 150 mg of MgSO4 and 50 mg of octadecyl (C18) sorbent. The analytical procedure was developed and carefully optimized to enable low-level, total haloxyfop analysis in a variety of challenging matrices, including infant formulas and their important high-carbohydrate, high-protein, high-fat, and emulsifier ingredients. The final method was validated in two different laboratories by fortifying samples with haloxyfop and haloxyfop-methyl, which was used as a model compound simulating bound forms of the analyte. Mean recoveries of haloxyfop across all fortification levels and evaluated matrices ranged between 92.2 and 114% with repeatability, within-lab reproducibility, and reproducibility RSDs ≤ 14%. Based on the validation results, this method was capable to convert the haloxyfop ester into the parent acid in a wide range of sample types and to reliably identify and quantify total haloxyfop at the target 0.003 mg/kg level in infant formulas (both powdered and ready-to-feed liquid forms).
A novel sample preparation strategy for shotgun lipidomics of phospholipids employing multilamellar vesicles Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-08 Melissa Frick, Tommy Hofmann, Caroline Haupt, Carla Schmidt
The identification of lipids in biological samples is gaining importance. The advent of mass spectrometry-based lipidomics accelerated the field allowing nowadays for identification and quantification of complete lipidomes. However, due to solubility difficulties and varying properties of different lipid classes, sample preparation for lipidomics is still an issue. Of the many lipid classes, phospholipids are the major components of biological membranes. In solution, they spontaneously form lipid vesicles of various structures such as liposomes. They are therefore often used as membrane mimics when studying biological membranes and membrane proteins. Here, we present a novel sample preparation strategy for shotgun lipidomics employing liposomes prepared from lipid standards or lipid mixtures allowing the analysis of phospholipids directly from lipid bilayers. We validated our strategy for lipid identification by tandem mass spectrometry in positive or negative ion mode using different phospholipid species from various classes. We further tested our strategy for relative quantification by mixing different ratios of phospholipid species as well as determining the distribution of lipid species in a natural lipid extract.
Preparation of a molecularly imprinted sensor based on quartz crystal microbalance for specific recognition of sialic acid in human urine Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-08 Xiuzhen Qiu, Xian-Yan Xu, Xuncai Chen, Yiyong Wu, Huishi Guo
A novel molecularly imprinted quartz crystal microbalance (QCM) sensor was successfully prepared for selective determination of sialic acid (SA) in human urine samples. To obtain the QCM sensor, we first modified the gold surface of the QCM chip by self-assembling of allylmercaptane to introduce polymerizable double bonds on the chip surface. Then, SA molecularly imprinted polymer (MIP) nanofilm was attached to the modified QCM chip surface. For comparison, we have also characterized the nonmodified and improved surfaces of the QCM sensor by using atomic force microscopy (AFM) and Fourier transform infrared (FTIR) spectroscopy. We then tested the selectivity and detection limit of the imprinted QCM sensor via a series of adsorption experiments. The results show a linear response in the range of 0.025–0.50 μmol L−1 for sialic acid. Moreover, the limit of detection (LOD) of the prepared imprinted QCM sensor was found to be 1.0 nmol L−1 for sialic acid, and high recovery values range from 87.6 to 108.5% with RSD < 8.7 (n = 5) for the spiked urine sample obtained. Overall, this work presents how a novel QCM sensor was developed and used to detect sialic acid in human urine samples.
Current status of water environment and their microbial biosensor techniques – Part II: Recent trends in microbial biosensor development Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-08 Hideaki Nakamura
In Part I of the present review series, I presented the current state of the water environment by focusing on Japanese cases and discussed the need to further develop microbial biosensor technologies for the actual water environment. I comprehensively present trends after approximately 2010 in microbial biosensor development for the water environment. In the first section, after briefly summarizing historical studies, recent studies on microbial biosensor principles are introduced. In the second section, recent application studies for the water environment are also introduced. Finally, I conclude the present review series by describing the need to further develop microbial biosensor technologies.
Affinity capillary electrophoresis for identification of active drug candidates in myotonic dystrophy type 1 Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-08 Ioan O. Neaga, Stephanie Hambye, Ede Bodoki, Claudio Palmieri, Eugénie Ansseau, Alexandra Belayew, Radu Oprean, Bertrand Blankert
Myotonic dystrophy type 1 (DM1) is an autosomal dominantly inherited degenerative disease with a slow progression. At the present, there is no commercially available treatment, but sustained effort is currently undertaken for the development of a promising lead compound. In the present paper we report the development of a fast, versatile, and cost-effective affinity capillary electrophoresis (ACE) method for the screening and identification of potential drug candidates targeting pathological ARN probes relevant for DM1. The affinity studies were conducted in physiologically relevant conditions using 50 mM HEPES buffer (pH 7.4) in a fused silica capillary dynamically coated with poly(ethylene oxide), by testing a library of potential ligands against (CUG)50 RNA as target probe with a total run time of 4–5 h/ligand. For the most promising ligands, their affinity parameters were assessed and some results formerly reported on the affinity of pentamidine (PTMD) and neomycin against CUG repeats were confirmed. To the best of the authors’ knowledge, the estimated binding stoichiometry for some of the tested compounds (i.e., ~ 121:1 for PTMD against the tested RNA probe) is reported for the first time. Additionally, the potential of a novel pentamidine like compound, namely 1,2-ethane bis-1-amino-4-benzamidine (EBAB) with much lower in vivo toxicity than its parent compound has also been confirmed studying its effect on a live cell model by fluorescence microscopy. Further tests, such as the evaluation of the rescue in the mis-splicing of the involved genes, can be performed to corroborate the potential therapeutic value of EBAB in DM1 treatment.
Qualitative characterization of three combustion-related standard reference materials for polycyclic aromatic sulfur heterocycles and their alkyl-substituted derivatives via normal-phase liquid chromatography and gas chromatography/mass spectrometry Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-07 Walter B. Wilson, Hugh V. Hayes, Andres D. Campiglia, Stephen A. Wise
The research described here provides the most comprehensive qualitative characterization of three combustion-related standard reference materials (SRMs) for polycyclic aromatic sulfur heterocycles (PASHs) and some alkyl-substituted (alkyl-) derivatives to date: SRM 1597a (coal tar), SRM 1991 (coal tar/petroleum extract), and SRM 1975 (diesel particulate extract). An analytical approach based on gas chromatography/mass spectrometry (GC/MS) is presented for the determination of three-, four-, and five-ring PASH isomers and three- and four-ring alkyl-PASHs in the three SRM samples. The benefit of using a normal-phase liquid chromatography (NPLC) fractionation procedure prior to GC/MS analysis was demonstrated for multiple isomeric PASH groups. Using a semi-preparative aminopropyl (NH2) LC column, the three combustion-related samples were fractionated based on the number of aromatic carbon atoms. The NPLC-GC/MS method presented here allowed for the following identification breakdown: SRM 1597a – 35 PASHs and 59 alkyl-PASHs; SRM 1991–31 PASHs and 58 alkyl-PASHs; and SRM 1975–13 PASHs and 25 alkyl-PASHs. These identifications were based on NPLC retention data, the GC retention times of reference standards, and the predominant molecular ion peak in the mass spectrum. Prior to this study, only 11, 1, and 0 PASHs/alkyl-PASHs had been identified in SRM 1597a, SRM 1991, and SRM 1975, respectively.
Multiobjective optimization of liquid chromatography–triple-quadrupole mass spectrometry analysis of underivatized human urinary amino acids through chemometrics Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-07 Manuel David Peris-Díaz, Miguel A. Sentandreu, Enrique Sentandreu
Optimization of instrumental settings of a triple-quadrupole mass analyzer was performed by Box–Behnken design, support vector machines, and a Pareto-optimality approach. This time-saving, stepped chemometric strategy was used to model the signal response of underivatized human urinary amino acids. Drying gas flow, nebulizer pressure, sheath gas flow, and capillary voltage settings were exhaustively studied beyond the parameters conventionally optimized in triple-quadrupole devices (multiple reaction monitoring transitions, fragmentor and collision energy voltages). The results indicate that the best signal response for high-abundance and low-abundance underivatized amino acids was achieved with drying gas flow of 9 L/min, nebulizer pressure of 60 psi, sheath gas flow of 13 L/min, and capillary voltage of 3000 V. Compared with the widely standardized settings tested, chemometric analysis led to signal intensities 74% and 68% higher for high-abundance and low-abundance amino acids, respectively. The flexibility, speed, and efficiency of this method allows its affordable implementation in all mass spectrometry-based research to obtain superior results compared with those obtained with conventionally optimized mass spectrometry instrumental parameters.
Identification of methylated tubulin through analysis of methylated lysine Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-07 Rika Suzuki, Takashi Funatsu, Makoto Tsunoda
Post-translational modifications to tubulin such as acetylation and detyrosination play important roles in microtubule functions. Methylation is an important post-translational modification; however, to date, few methylated tubulins have been identified. In the present study, we developed a method for analyzing methylated lysine with the aim of identifying methylated tubulin. This method involves four steps: (1) acid hydrolysis of tubulin into amino acids, (2) selective extraction of methylated lysine using a monolithic-silica disk-packed spin column, (3) fluorescence derivatization of methylated lysine with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and (4) separation of NBD-methylated lysine on a column consisting of C18, cation and anion ligand, and fluorescence detection. Using the newly developed method, the dimethylation of lysine in tubulin was identified. This new method could be applied to searches for other methylated proteins.
Identification of intentionally and non-intentionally added substances in plastic packaging materials and their migration into food products Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-07 Verónica García Ibarra, Ana Rodríguez Bernaldo de Quirós, Perfecto Paseiro Losada, Raquel Sendón
Plastic materials are widely used in food packaging applications; however, there is increased concern because of the possible release of undesirable components into foodstuffs. Migration of plastic constituents not only has the potential to affect product quality but also constitutes a risk to consumer health. In order to check the safety of food contact materials, analytical methodologies to identify potential migrants are required. In the first part of this work, a GC/MS screening method was developed for the identification of components from plastic packaging materials including intentionally and “non-intentionally added substances” (NIAS) as potential migrants. In the second part of this study, the presence of seven compounds (bis (2-ethylhexyl) phthalate (DEHP), diethyl phthalate (DEP), diisobutyl phthalate (DIBP), dibutyl phthalate (DBP), butylated hydroxytoluene (BHT), acetyl tributyl citrate (ATBC), benzophenone (BP)) previously identified in packaging materials were investigated in food products (corn and potatoes snacks, cookies, and cakes). For this purpose, a suitable extraction method was developed and quantification was performed using GC-MS. The developed method was validated in terms of linearity, recovery, repeatability, and limits of detection and quantification. The spiked recoveries varied between 82.7 and 116.1%, and relative standard deviation (RSD) was in the range of 2.22–15.9%. The plasticizer ATBC was the most detected compound (94% samples), followed by DEP (65%), DEHP (47%), BP (44%), DBP (35%), DIBP (21%), and BHT (12%). Regarding phthalates, DEP and DEHP were the most frequently detected compounds in concentrations up to 1.44 μg g−1. In some samples, only DBP exceeded the European SML of 0.3 mg kg−1 established in Regulation 10/2011.
Uniform polysaccharide composite microspheres with controllable network by microporous membrane emulsification technique Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-07 Hongwu Zhang, Lan Zhao, Yongdong Huang, Kai Zhu, Qibao Wang, Rui Yang, Zhiguo Su, Guanghui Ma
High resolution has been constantly pursued in both preparative and analytical chromatography. Chromatographic media are a key factor during the entire separation process. Tailor-made chromatographic media have gained more attention because of their adjustable structure appropriate for application. Uniform polysaccharide composite microspheres were prepared with a mixture of agarose and dextran solution by membrane emulsification technique for the first time. Their pore structure was deliberately regulated by adjusting both the polysaccharide composition and the molecular weight of dextran. Compared with pure agarose microspheres, polysaccharide composite microspheres had a higher separation resolution and their separation range was controllable. By increasing agarose concentration and decreasing dextran concentration at the same time during the preparation of composite microspheres, the mean pore size increased first and then decreased later, and also the pore size distribution became narrower. By increasing the molecular weight of dextran, the pores became smaller with a narrower pore size distribution. Microspheres with a composition of 10% agarose/2% dextran T40 or 8% agarose/4% dextran T150 showed a higher separation resolution for proteins within range of low molecular weight. Furthermore, the mechanical strength of this composite microsphere was improved by adjusting its composition. Atomic force microscope (AFM) results showed that pores were distributed evenly on both the surface and the inner part of microspheres, beneficial for the passage of biomolecules. These novel uniform polysaccharide composite microspheres have great potential for high-resolution bioseparation.
Screening of salivary volatiles for putative breast cancer discrimination: an exploratory study involving geographically distant populations Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-07 Carina Cavaco, Jorge A. M. Pereira, Khushman Taunk, Ravindra Taware, Srikanth Rapole, Hampapathalu Nagarajaram, José S. Câmara
Saliva is possibly the easiest biofluid to analyse and, despite its simple composition, contains relevant metabolic information. In this work, we explored the potential of the volatile composition of saliva samples as biosignatures for breast cancer (BC) non-invasive diagnosis. To achieve this, 106 saliva samples of BC patients and controls in two distinct geographic regions in Portugal and India were extracted and analysed using optimised headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME/GC-MS, 2 mL acidified saliva containing 10% NaCl, stirred (800 rpm) for 45 min at 38 °C and using the CAR/PDMS SPME fibre) followed by multivariate statistical analysis (MVSA). Over 120 volatiles from distinct chemical classes, with significant variations among the groups, were identified. MVSA retrieved a limited number of volatiles, viz. 3-methyl-pentanoic acid, 4-methyl-pentanoic acid, phenol and p-tert-butyl-phenol (Portuguese samples) and acetic, propanoic, benzoic acids, 1,2-decanediol, 2-decanone, and decanal (Indian samples), statistically relevant for the discrimination of BC patients in the populations analysed. This work defines an experimental layout, HS-SPME/GC-MS followed by MVSA, suitable to characterise volatile fingerprints for saliva as putative biosignatures for BC non-invasive diagnosis. Here, it was applied to BC samples from geographically distant populations and good disease separation was obtained. Further studies using larger cohorts are therefore very pertinent to challenge and strengthen this proof-of-concept study.
A novel fluorescent aptasensor for the highly sensitive and selective detection of cardiac troponin I based on a graphene oxide platform Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-04 Dongkui Liu, Xing Lu, Yiwen Yang, Yunyun Zhai, Jian Zhang, Lei Li
Acute myocardial infarction (AMI) is one of the leading risks to global health. Thus, the rapid, accurate early diagnosis of AMI is highly critical. Human cardiac troponin I (cTnI) has been regarded as a golden biomarker for AMI due to its excellent selectivity. In this work, a novel fluorescent aptasensor based on a graphene oxide (GO) platform was developed for the highly sensitive and selective detection of cTnI. GO binds to the fluorescent anti-cTnI aptamer and quenches its fluorescence. In the presence of cTnI, the fluorescent anti-cTnI aptamer leaves the surface of GO, combines with cTnI because of the powerful affinity of the fluorescent anti-cTnI aptamer and cTnI, and then restores the fluorescence of the fluorescent anti-cTnI aptamer. Fluorescence-enhanced detection is highly sensitive and selective to cTnI. The method exhibited good analytical performance with a reasonable dynamic linearity at the concentration range of 0.10–6.0 ng/mL and a low detection limit of 0.07 ng/mL (S/N = 3). The fluorescent aptasensor also exhibited high selectivity toward cTnI compared with other interference proteins. The proposed method may be a potentially useful tool for cTnI determination in human serum.
Inter-laboratory validation of an inexpensive streamlined method to measure inorganic arsenic in rice grain Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-04 Rufus L. Chaney, Carrie E. Green, Steven J. Lehotay
With the establishment by CODEX of a 200 ng/g limit of inorganic arsenic (iAs) in polished rice grain, more analyses of iAs will be necessary to ensure compliance in regulatory and trade applications, to assess quality control in commercial rice production, and to conduct research involving iAs in rice crops. Although analytical methods using high-performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) have been demonstrated for full speciation of As, this expensive and time-consuming approach is excessive when regulations are based only on iAs. We report a streamlined sample preparation and analysis of iAs in powdered rice based on heated extraction with 0.28 M HNO3 followed by hydride generation (HG) under control of acidity and other simple conditions. Analysis of iAs is then conducted using flow-injection HG and inexpensive ICP-atomic emission spectroscopy (AES) or other detection means. A key innovation compared with previous methods was to increase the acidity of the reagent solution with 4 M HCl (prior to reduction of As5+ to As3+), which minimized interferences from dimethylarsinic acid. An inter-laboratory method validation was conducted among 12 laboratories worldwide in the analysis of six shared blind duplicates and a NIST Standard Reference Material involving different types of rice and iAs levels. Also, four laboratories used the standard HPLC-ICP-MS method to analyze the samples. The results between the methods were not significantly different, and the Horwitz ratio averaged 0.52 for the new method, which meets official method validation criteria. Thus, the simpler, more versatile, and less expensive method may be used by laboratories for several purposes to accurately determine iAs in rice grain.
Analysis of isocyanates in indoor dust Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-04 Kanae Bekki, Shigehisa Uchiyama, Naoki Kunugita
Isocyanates are harmful semi-volatile organic compounds that are emitted from various consumer products like polyurethane foam-based mattresses. Although it is a concern that isocyanates might accumulate in indoor dust, causing infants and toddlers, in particular, to be exposed to them, little information is available on the levels of isocyanates in the indoor environment. In this study, we investigated the suitability of an analytical method for determining the presence and level of isocyanates in the indoor dust. The method we developed displayed acceptable linearity, accuracy, and precision in the analysis of eleven different isocyanates. By using this analytical method, we could detect five isocyanates (ICA, MIC, EIC, PIC, and PHI) and quantify three isocyanates (MIC, EIC, and PHI) in indoor dust collected in different houses. This study is the first to focus on the pollution of indoor dust by isocyanates, and the tested method is suitable for the estimation of the level of isocyanate exposure.
Detection of triacetone triperoxide by thermal decomposition peroxy radical chemical amplification coupled to cavity ring-down spectroscopy Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-04 Youssef M. Taha, Matthew T. Saowapon, Hans D. Osthoff
Triacetone triperoxide (TATP) is frequently used in improvised explosive devices because of its ease of manufacture and tremendous explosive force. In this paper, we describe a new method for detection of TATP, thermal decomposition peroxy radical chemical amplification cavity ring-down spectroscopy (TD-PERCA-CRDS). In this method, air is sampled through a heated inlet to which ~ 1 ppmv nitric oxide (NO) is added. To verify the purity of synthetic standards, the mid-infrared spectrum of TATP vapor was recorded. The thermal decomposition of TATP is shown to produce radicals which oxidize NO to nitrogen dioxide (NO2), whose concentration increase is monitored by optical absorption at 405 nm using a blue diode laser CRDS. The sensitivity could be improved through addition of ~ 1% ethane (C2H6), which fuels catalytic conversion of NO to NO2. The limit of detection of TD-PERCA-CRDS with respect to TATP is 22 pptv (1 s data), approximately six orders of magnitude below TATP’s saturation vapor pressure. Insights into the mechanism of TATP thermal decomposition, TD-PERCA-CRDS interferences, and the suitability of TD-PERCA-CRDS as a peroxy radical explosive detection method at security check points are discussed.
Large-scale identification and visualization of human liver N-glycome enriched from LO2 cells Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-03 Kaijie Xiao, Yuyin Han, Zhixin Tian
Aberrant glycosylation has been commonly observed in various physiological and pathological disorders (including cancers), and quite a few glycoproteins have been approved by the US Food and Drug Administration (FDA) as markers for early diagnosis. Each glycoprotein may have multiple glycoforms, and cancer-related ones can be only some specific glycoforms which have much higher sensitivity and specificity; for example, AFP glycoform AFP-L3 with N-glycan of 01Y(61F)41Y41M(31M41Y41L41S61M41Y41L41S is of bigger diagnostic value for hepatocellular carcinoma than total AFP (i.e., combination of all glycoforms). Mass spectrometry-based glycomics is currently the state-of-the-art instrumental analytical pipeline for high-throughput characterization of various glycoforms, where not only monosaccharide composition but also comprehensive structural information (sequence and linkage) of N-glycans are now reported thanks to our recently developed N-glycan database search engine GlySeeker. With this new capability, here, we report our large-scale characterization of human liver N-glycome with primary structures; 214 unique N-glycans with unique primary structures were identified and visualized with spectrum-level false discovery rate ≤ 1% and number of best hits of 1. The LO2 N-glycans reported here serve as a basic reference for future liver N-glycome study, and further quantitative analysis will enable characterization of differentially expressed N-glycans and discovery of more effective markers for liver and other diseases. Data are available via ProteomeXchange with identifier PXD008158.
Facile one-pot synthesis of multifunctional polyphosphazene nanoparticles as multifunctional platform for tumor imaging Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-03 Zongfang Wang, Min Hu, Shugang Hu, Jia Han, Zhenzhen Wang, Yanke Chen, Cheng Huang, Lei Fu, Zhenxi Zhang
By integrating imaging and drug-delivery in a single system, fluorescent nano-multifunctional imaging platforms can offer simultaneous diagnosis and therapy to diseases like cancer. However, the synthesis of such system involves a tedious, time-consuming, and multi-step process. Herein we report a facile method based on simple ultrasonication to synthesize highly cross-linked, monodispersed fluorescent polyphosphazene nanoparticles from hexachlorocyclotriphosphazene (HCCP) and dichlorofluorescein (FD). Various functional groups (folic acid, PEG-NH2, and methylene blue) can be “fastened” in situ onto the poly(cyclotriphosphazene-co-dichlorofluorescein) (PCTPDF) nanoparticles to expand its application as nano-multifunctional platform. All the nanoparticles were characterized spectrophotometrically, and morphology was established by the images obtained from scanning electron microscope (SEM). The synthesized multifunctional nanoparticles exhibited low toxicity and penetrated through the cytomembranes of human colon cancer (HCT 116) cells. When applied to in vivo tumor imaging using biologically engineered mouse model, methylene blue functionalized (PCTPDF@MB) nanoparticles exhibited excellent photodynamic activity and imaging ability. Thus, PCTPDF nanoplatform based on multi-functional fluorescent nanoparticles might offer an efficient solution to new age theranostics. Apart from diagnostics application, PCTPDF, as a nanoplatform, could also be utilized to achieve more comprehensive application in modern analytic chemistry.
Using a portable Raman spectrometer to detect carotenoids of halophilic prokaryotes in synthetic inclusions in NaCl, KCl, and sulfates Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-03 Jan Jehlička, Adam Culka, Lilly Mana, Aharon Oren
Cell suspensions of the haloarchaea Halorubrum sodomense and Halobacterium salinarum and the extremely halophilic bacterium Salinibacter ruber (Bacteroidetes) in saturated solutions of chlorides and sulfates (NaCl, KCl, MgSO4·7H2O, K2SO4, and (NH4)Al(SO4)2·12H2O) were left to evaporate to produce micrometric inclusions in laboratory-grown crystals. Raman spectra of these pinkish inclusions were obtained using a handheld Raman spectrometer with green excitation (532 nm). This portable instrument does not include any microscopic tool. Acceptable Raman spectra of carotenoids were obtained in the range of 200–4000 cm−1. This detection achievement was related to the mode of illumination and collection of scattered light as well as due to resonance Raman enhancement of carotenoid signals under green excitation. The position of diagnostic Raman carotenoid bands corresponds well to those specific carotenoids produced by a given halophile. To our best knowledge, this is the first study of carotenoids included in the laboratory in crystalline chlorides and sulfates, using a miniature portable Raman spectrometer.
Validated reverse transcription droplet digital PCR serves as a higher order method for absolute quantification of Potato virus Y strains Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-03 Nataša Mehle, David Dobnik, Maja Ravnikar, Maruša Pompe Novak
RNA viruses have a great potential for high genetic variability and rapid evolution that is generated by mutation and recombination under selection pressure. This is also the case of Potato virus Y (PVY), which comprises a high diversity of different recombinant and non-recombinant strains. Consequently, it is hard to develop reverse transcription real-time quantitative PCR (RT-qPCR) with the same amplification efficiencies for all PVY strains which would enable their equilibrate quantification; this is specially needed in mixed infections and other studies of pathogenesis. To achieve this, we initially transferred the PVY universal RT-qPCR assay to a reverse transcription droplet digital PCR (RT-ddPCR) format. RT-ddPCR is an absolute quantification method, where a calibration curve is not needed, and it is less prone to inhibitors. The RT-ddPCR developed and validated in this study achieved a dynamic range of quantification over five orders of magnitude, and in terms of its sensitivity, it was comparable to, or even better than, RT-qPCR. RT-ddPCR showed lower measurement variability. We have shown that RT-ddPCR can be used as a reference tool for the evaluation of different RT-qPCR assays. In addition, it can be used for quantification of RNA based on in-house reference materials that can then be used as calibrators in diagnostic laboratories.
A fast and accurate method for the identification of peroxidase inhibitors from Radix Salvia Miltiorrhizae by on-flow biochemical assay coupled with LC/Q-TOF-MS: comparison with ultrafiltration-based affinity selection Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-03 Si-Qi Wu, Hui-Peng Song, Bin Li, Run-Zhou Liu, Hua Yang, Ling He, Ping Li
Development of fast and accurate methods to discover lead compounds for drug candidates is highly important. In this study, a reliable and effective post-column on-flow biochemical assay (POBA) was established to screen potent peroxidase inhibitors from complex chemical mixtures (e.g., natural product extracts). Multiple factors such as flow rate, organic phase, detection wavelength, and reaction coil were carefully investigated. To better understand the features of POBA, another emerging technology of ultrafiltration LC-MS was used for comparison. The result showed that POBA had advantages in saving time, avoiding false positives, and improving the accuracy. To illustrate the practicality of the method, Radix Salvia Miltiorrhizae, a traditional herb for cardiovascular disease treatment, was applied as the research objective. Finally, six compounds including tanshinol, protocatechuic aldehyde, salvianolic acid D, rosmarinic acid, lithospermic acid, and salvianolic acid B were determined as novel peroxidase inhibitors. Their bioactivities were validated by microplate-based assay, molecular docking, and pharmacophore modeling. This study demonstrates a great potential of POBA in the efficient and accurate discovery of drug candidates.
Inter-laboratory study for the certification of trace elements in seawater certified reference materials NASS-7 and CASS-6 Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-05-02 Lu Yang, Kenny Nadeau, Juris Meija, Patricia Grinberg, Enea Pagliano, Francisco Ardini, Marco Grotti, Christian Schlosser, Peter Streu, Eric P. Achterberg, Yoshiki Sohrin, Tomoharu Minami, Linjie Zheng, Jingfeng Wu, Gedun Chen, Michael J. Ellwood, Clara Turetta, Ana Aguilar-Islas, Robert Rember, Géraldine Sarthou, Manon Tonnard, Hélène Planquette, Tomáš Matoušek, Steven Crum, Zoltán Mester
Certification of trace metals in seawater certified reference materials (CRMs) NASS-7 and CASS-6 is described. At the National Research Council Canada (NRC), column separation was performed to remove the seawater matrix prior to the determination of Cd, Cr, Cu, Fe, Pb, Mn, Mo, Ni, U, V, and Zn, whereas As was directly measured in 10-fold diluted seawater samples, and B was directly measured in 200-fold diluted seawater samples. High-resolution inductively coupled plasma mass spectrometry (HR-ICPMS) was used for elemental analyses, with double isotope dilution for the accurate determination of B, Cd, Cr, Cu, Fe, Pb, Mo, Ni, U, and Zn in seawater NASS-7 and CASS-6, and standard addition calibration for As, Co, Mn, and V. In addition, all analytes were measured using standard addition calibration with triple quadrupole (QQQ)-ICPMS to provide a second set of data at NRC. Expert laboratories worldwide were invited to contribute data to the certification of trace metals in NASS-7 and CASS-6. Various analytical methods were employed by participants including column separation, co-precipitation, and simple dilution coupled to ICPMS detection or flow injection analysis coupled to chemiluminescence detection, with use of double isotope dilution calibration, matrix matching external calibration, and standard addition calibration. Results presented in this study show that majority of laboratories have demonstrated their measurement capabilities for the accurate determination of trace metals in seawater. As a result of this comparison, certified/reference values and associated uncertainties were assigned for 14 elements in seawater CRMs NASS-7 and CASS-6, suitable for the validation of methods used for seawater analysis.
Comparative characterization of rat hippocampal plasma membrane and mitochondrial membrane proteomes based on a sequential digestion-centered combinative strategy Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-19 Jianying Shen, Jian Zhou, Yong Lin, Zhen Liu, Ping Chen, Xianchun Wang
Plasma membrane (PM) and mitochondrial membrane (MM) proteins of rat hippocampal neurons were identified and comparatively characterized on the basis of a sequential digestion-centered combinative strategy for sample treatment. A total of 478 membrane proteins were identified, of which 240 had PM localization, 170 had MM localization, and 33 had both of the two subcellular localizations. Compared with the PM proteome, the MM proteome not only was smaller, more basic, and more hydrophobic, but also had a narrower protein molecular mass distribution range and a higher proportion of transmembrane proteins. By functional enrichment analysis, 287 molecular function terms for the PM proteome and 173 for the MM proteome were obtained. The MM proteome had a lower percentage of binding function terms and a higher percentage of catalysis function terms than the PM proteome, suggesting that mitochondrial proteins were more inclined to affect the physiological and biochemical processes by binding various molecules and as enzymes. Biological process enrichment showed that the genes of the PM and MM proteomes were mapped to 1104 and 460 biological processes, respectively. The biological processes with the most mapped genes of the PM proteome included those involved in vesicle recycling, transmitter release, neuronal development, protein and ion transport, etc., whereas those involved in electron transport, ATP synthesis, mitochondrial transport, mitochondrial apoptosis, etc., were the most gene-mapped biological processes for the MM proteome. The present work has deepened our understanding of the structure and function of hippocampal neurons and provided reference methods for research in the related field.
A rapid solution-based method for determining the affinity of heroin hapten-induced antibodies to heroin, its metabolites, and other opioids Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-19 Oscar B. Torres, Alexander J. Duval, Agnieszka Sulima, Joshua F. G. Antoline, Arthur E. Jacobson, Kenner C. Rice, Carl R. Alving, Gary R. Matyas
We describe for the first time a method that utilizes microscale thermophoresis (MST) technology to determine polyclonal antibody affinities to small molecules. Using a novel type of heterologous MST, we have accurately measured a solution-based binding affinity of serum antibodies to heroin which was previously impossible with other currently available methods. Moreover, this mismatch approach (i.e., using a cross-reactive hapten tracer) has never been reported in the literature. When compared with equilibrium dialysis combined with ultra-performance liquid chromatography/tandem mass spectrometry (ED-UPLC/MS/MS), this novel MST method yields similar binding affinity values for polyclonal antibodies to the major heroin metabolites 6-AM and morphine. Additionally, we herein report the method of synthesis of this novel cross-reactive hapten, MorHap-acetamide—a useful analog for the study of heroin hapten–antibody interactions. Using heterologous MST, we were able to determine the affinities, down to nanomolar accuracies, of polyclonal antibodies to various abused opioids. While optimizing this method, we further discovered that heroin is protected from serum esterase degradation by the presence of these antibodies in a concentration-dependent manner. Lastly, using affinity data for a number of structurally different opioids, we were able to dissect the moieties that are crucial to antibody binding. The novel MST method that is presented herein can be extended to the analysis of any ligand that is prone to degradation and can be applied not only to the development of vaccines to substances of abuse but also to the analysis of small molecule/protein interactions in the presence of serum.
Magnetic ionic liquid-based dispersive liquid-liquid microextraction technique for preconcentration and ultra-trace determination of Cd in honey Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-19 Emiliano F. Fiorentini, Leticia B. Escudero, Rodolfo G. Wuilloud
A simple, highly efficient, batch, and centrifuge-less dispersive liquid-liquid microextraction method based on a magnetic ionic liquid (MIL-DLLME) and electrothermal atomic absorption spectrometry (ETAAS) detection was developed for ultra-trace Cd determination in honey. Initially, Cd(II) was chelated with ammonium diethyldithiophosphate (DDTP) at pH 0.5 followed by its extraction with the MIL trihexyl(tetradecyl)phosphonium tetrachloroferrate(III) ([P6,6,6,14]FeCl4) and acetonitrile as dispersant. The MIL phase containing the analyte was separated from the aqueous phase using only a magnet. A back-extraction procedure was applied to recover Cd from the MIL phase using diluted HNO3 and this solution was directly injected into the graphite furnace of ETAAS instrument. An extraction efficiency of 93% and a sensitivity enhancement factor of 112 were obtained under optimal experimental conditions. The detection limit (LOD) was 0.4 ng L−1 Cd, while the relative standard deviation (RSD) was 3.8% (at 2 μg L−1 Cd and n = 10), calculated from the peak height of absorbance signals. This work reports the first application of the MIL [P6,6,6,14]FeCl4 along with the DLLME technique for the successful determination of Cd at trace levels in different honey samples.
Rapid and sensitive determination of nine bisphenol analogues, three amphenicol antibiotics, and six phthalate metabolites in human urine samples using UHPLC-MS/MS Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-19 Yuan Yao, Yijun Shao, Ming Zhan, Xiaoli Zou, Weidong Qu, Ying Zhou
Bisphenol analogues, amphenicol antibiotics, and phthalate have widely aroused public concerns due to their adverse effects on human health. In this study, a rapid and sensitive method for determination of nine bisphenol analogues, three amphenicol antibiotics, and six phthalate metabolites in the urine based on ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry was developed and validated. The sample pretreatment condition on the base of mixed-mode anion-exchange (Oasis MAX) SPE was optimized to separate bisphenol analogues and amphenicol antibiotics from phthalate metabolites: the former were detected with a mobile phase of 0.1% ammonium water solution/methanol containing 0.1% ammonium water solution in negative mode, whereas the latter were determined with a mobile phase of 0.1% acetic acid solution/acetonitrile containing 0.1% acetic acid in negative mode. The limits of detection were less than 0.26 ng/mL for bisphenol analogues, 0.12 ng/mL for amphenicol antibiotics, and 0.14 ng/mL for phathalate metabolites. The recoveries of all target analytes in three fortification levels ranged from 72.02 to 117.64% with the relative standard deviations of no larger than 14.51%. The matrix effect was adjusted by isotopically labeled internal standards. This proposed method was successfully applied to analyze 40 actual urines and 13 out of 18 studied compounds were detected.
Improved LC-MS/MS method for the quantification of hepcidin-25 in clinical samples Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-18 Ioana M. Abbas, Holger Hoffmann, María Montes-Bayón, Michael G. Weller
Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the “sticky” character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing a LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend a LC-MS/MS-based quantification method with a dynamic range of 0.5–40 μg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials.
Biochemical profiling of rat embryonic stem cells grown on electrospun polyester fibers using synchrotron infrared microspectroscopy Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-18 Ernesto Doncel-Pérez, Gary Ellis, Christophe Sandt, Peter S. Shuttleworth, Agatha Bastida, Julia Revuelta, Eduardo García-Junceda, Alfonso Fernández-Mayoralas, Leoncio Garrido
Therapeutic options for spinal cord injuries are severely limited; current treatments only offer symptomatic relief and rehabilitation focused on educating the individual on how to adapt to their new situation to make best possible use of their remaining function. Thus, new approaches are needed, and interest in the development of effective strategies to promote the repair of neural tracts in the central nervous system inspired us to prepare functional and highly anisotropic polymer scaffolds. In this work, an initial assessment of the behavior of rat neural progenitor cells (NPCs) seeded on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) fiber scaffolds using synchrotron-based infrared microspectroscopy (SIRMS) is described. Combined with a modified touch imprint cytology sample preparation method, this application of SIRMS enabled the biochemical profiles of NPCs on the coated polymer fibers to be determined. The results showed that changes in the lipid and amide I–II spectral regions are modulated by the type and coating of the substrate used and the culture time. SIRMS studies can provide valuable insight into the early-stage response of NPCs to the morphology and surface chemistry of a biomaterial, and could therefore be a useful tool in the preparation and optimization of cellular scaffolds.
Comparison of commercial exosome isolation kits for circulating exosomal microRNA profiling Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-18 Meng Ding, Cheng Wang, Xiaolan Lu, Cuiping Zhang, Zhen Zhou, Xi Chen, Chen-Yu Zhang, Ke Zen, Chunni Zhang
Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. Here, we analyzed the advantages and weaknesses of four commonly used commercial kits for exosomal miRNA profiling and their application to the sample of serum and/or plasma, respectively. NanoSight and Western blotting were conducted to evaluate the efficiency and purity of the isolated exosomes. In our conditions, the size distribution of the isolated particles was appropriate (40–150 nm), and ExoQuick™ Exosome Precipitation Solution (EXQ) generated a relatively high yield of exosomes. Nevertheless, albumin impurity was ubiquitous for all the four kits, and Total Exosome Isolation for serum or plasma (TEI) yielded a relatively pure isolation. We further performed Illumina sequencing combined with RT-qPCR to determine the ability of these kits for miRNA profiling. There was significant correlation of the exosomal miRNA profile and specific miRNAs between kits, but with differences depending on methods. exoRNeasy Serum/Plasma Midi Kit (EXR) and EXQ performed better in the specific exosomal miRNAs recovery. Intraassay CVs for specific miRNA measurement were 0.88–3.82, 1.19–3.77, 0–2.70, and 1.23–9.11% for EXR, TEI, EXQ, and RIBO™ Exosome Isolation Reagent (REI), respectively. In each kit, serum yielded a higher abundance of exosomes and exosomal miRNAs than plasma, yet with more albumin impurity. In conclusion, our data provide some valuable guidance for the methodology of disease biomarker identification of circulation exosomal miRNAs.
A novel fluorescent biosensor for adrenaline detection and tyrosinase inhibitor screening Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-17 Ziping Liu, Shasha Liu
In this work, a novel simple fluorescent biosensor for the highly sensitive and selective detection of adrenaline was established. Firstly, water-soluble CuInS2 quantum dots (QDs) capped by L-Cys were synthesized via a hydrothermal synthesis method. Then, the positively charged adrenaline was assembled on the surface of CuInS2 QDs due to the electrostatic interactions and hydrogen bonding, which led to the formation of adrenaline-CuInS2 QD (Adr-CuInS2 QD) electrostatic complexes. Tyrosinase (TYR) can catalyze adrenaline to generate H2O2, and additionally oxidize the adrenaline to adrenaline quinone. Both the H2O2 and the adrenaline quinone can quench the fluorescence of the CuInS2 QDs through the electron transfer (ET) process. Thus, the determination of adrenaline could be facilely achieved by taking advantage of the fluorescence “turn off” feature of CuInS2 QDs. Under the optimum conditions, the fluorescence quenching ratio If/If0 (If and If0 were the fluorescence intensity of Adr-CuInS2 QDs in the presence and absence of TYR, respectively) was proportional to the logarithm of adrenaline concentration in the range of 1 × 10−8–1 × 10−4 mol L−1 with the detection limit of 3.6 nmol L−1. The feasibility of the proposed biosensor in real sample assay was also studied and satisfactory results were obtained. Significantly, the proposed fluorescent biosensor can also be utilized to screen TYR inhibitors.
Magnetic bead/capture DNA/glucose-loaded nanoliposomes for amplifying the glucometer signal in the rapid screening of hepatitis C virus RNA Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-17 Haijian Tu, Kun Lin, Yongzhi Lun, Liuming Yu
A digital detection strategy based on a portable personal glucometer (PGM) was developed for the simple, rapid, and sensitive detection of hepatitis C virus (HCV) RNA, involving the release of glucose-loaded nanoliposomes due to coupling-site-specific cleavage by the endonuclease BamHI. The glucose-loaded nanoliposomes were synthesized using a reversed-phase evaporation method and provided an amplified signal at the PGM in the presence of HCV RNA. Initially, a 21-mer oligonucleotide complementary to HCV RNA was covalently conjugated to a magnetic bead through the amino group at the 5′ end of the oligonucleotide, and then bound to a glucose-loaded liposome by typical carbodiimide coupling at its 3′ end. In the presence of the target HCV RNA, the target hybridized with the oligonucleotide to form double-stranded DNA. The symmetrical duplex sequence 5′-GGATCC-3′ between guanines was then catalytically cleaved by BamHI, which detached the glucose-loaded liposome from the magnetic bead. Following magnetic separation of the bead, the detached glucose-loaded liposome was lysed using Triton X-100 to release the glucose molecules within it, which were then detected as an amplified signal at the digital PGM. Under optimal conditions, the PGM signal increased with increasing HCV RNA, and displayed a strongly linear dependence on the level of HCV RNA for concentrations ranging from 10 pM to 1.0 μM. The detection limit (LOD) of the system was 1.9 pM. Good reproducibility and favorable specificity were achieved in the analysis of the target HCV RNA. Human serum samples containing HCV RNA were analyzed using this strategy, and the developed sensing platform was observed to yield satisfactory results based on a comparison with the corresponding results from a Cobas® Amplicor HCV Test Analyzer.
FractionOptimizer: a method for optimal peptide fractionation in bottom-up proteomics Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-17 Elizaveta M. Solovyeva, Anna A. Lobas, Arthur T. Kopylov, Irina Y. Ilina, Lev I. Levitsky, Sergei A. Moshkovskii, Mikhail V. Gorshkov
Recent advances in mass spectrometry and separation technologies created the opportunities for deep proteome characterization using shotgun proteomics approaches. The “real world” sample complexity and high concentration range limit the sensitivity of this characterization. The common strategy for increasing the sensitivity is sample fractionation prior to analysis either at the protein or the peptide level. Typically, fractionation at the peptide level is performed using linear gradient high-performance liquid chromatography followed by uniform fraction collection. However, this way of peptide fractionation results in significantly suboptimal operation of the mass spectrometer due to the non-uniform distribution of peptides between the fractions. In this work, we propose an approach based on peptide retention time prediction allowing optimization of chromatographic conditions and fraction collection procedures. An open-source software implementing the approach called FractionOptimizer was developed and is available at http://hg.theorchromo.ru/FractionOptimizer. The performance of the developed tool was demonstrated for human embryonic kidney (HEK293) cell line lysate. In these experiments, we improved the uniformity of the peptides distribution between fractions. Moreover, in addition to 13,492 peptides, we found 6787 new peptides not identified in the experiments without fractionation and up to 800 new proteins (or 25%).
Geochemical wolframite fingerprinting – the likelihood ratio approach for laser ablation ICP-MS data Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-17 Agnieszka Martyna, Hans-Eike Gäbler, Andreas Bahr, Grzegorz Zadora
Wolframite has been specified as a ‘conflict mineral’ by a U.S. Government Act, which obliges companies that use these minerals to report their origin. Minerals originating from conflict regions in the Democratic Republic of the Congo shall be excluded from the market as their illegal mining, trading, and taxation are supposed to fuel ongoing violent conflicts. The German Federal Institute for Geosciences and Natural Resources (BGR) developed a geochemical fingerprinting method for wolframite based on laser ablation inductively coupled plasma-mass spectrometry. Concentrations of 46 elements in about 5300 wolframite grains from 64 mines were determined. The issue of verifying the declared origins of the wolframite samples may be framed as a forensic problem by considering two contrasting hypotheses: the examined sample and a sample collected from the declared mine originate from the same mine (H1), and the two samples come from different mines (H2). The solution is found using the likelihood ratio (LR) theory. On account of the multidimensionality, the lack of normal distribution of data within each sample, and the huge within-sample dispersion in relation to the dispersion between samples, the classic LR models had to be modified. Robust principal component analysis and linear discriminant analysis were used to characterize samples. The similarity of two samples was expressed by Kolmogorov-Smirnov distances, which were interpreted in view of H1 and H2 hypotheses within the LR framework. The performance of the models, controlled by the levels of incorrect responses and the empirical cross entropy, demonstrated that the proposed LR models are successful in verifying the authenticity of the wolframite samples.
Modification of polydopamine-coated Fe 3 O 4 nanoparticles with multi-walled carbon nanotubes for magnetic-μ-dispersive solid-phase extraction of antiepileptic drugs in biological matrices Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-16 Ruiqi Zhang, Siming Wang, Ye Yang, Yulan Deng, Di Li, Ping Su, Yi Yang
In this study, multi-walled carbon nanotubes were coated on the surface of magnetic nanoparticles modified by polydopamine. The synthesized composite was characterized and applied to magnetic-μ-dispersive solid-phase extraction of oxcarbazepine (OXC), phenytoin (PHT), and carbamazepine (CBZ) from human plasma, urine, and cerebrospinal fluid samples prior to analysis by a high-performance liquid chromatography-photodiode array detector. The extraction parameters were investigated and the optimum condition was obtained when the variables were set to the following: sorbent type, Fe3O4@polyDA–MWCNTs (length < 2 μm); sample pH, 6; amount of sorbent, 15 mg; sorption time, 1.5 min at room temperature; type and volume of the eluent, 2.5 mL methanol; and salt content, none added. Under the optimized conditions, the calibration curves are linear in the concentration range 2–2000 ng/mL, the limits of detection are in the range 0.4–3.1 ng/mL, and the relative standard deviations and relative recoveries of plasma (spiked at 200 ng/mL) and CSF (spiked at 50 ng/mL) are in the ranges 1.4–8.2% and 92.8–96.5%, respectively. The applicability of the method was successfully confirmed by extraction and determination of OXC, PHT, and CBZ in biological matrices.
Degradation product characterization of therapeutic oligonucleotides using liquid chromatography mass spectrometry Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-14 N. M. Elzahar, N. Magdy, Amira M. El-Kosasy, Michael G. Bartlett
Synthetic antisense phosphorothioate oligonucleotides (PS) have undergone rapid development as novel therapeutic agents. The increasing significance of this class of drugs requires significant investment in the development of quality control methods. The determination of the many degradation pathways of such complex molecules presents a significant challenge. However, an understanding of the potential impurities that may arise is necessary to continue to advance these powerful new therapeutics. In this study, four different antisense oligonucleotides representing several generations of oligonucleotide therapeutic agents were evaluated under various stress conditions (pH, thermal, and oxidative stress) using ion-pairing reversed-phase liquid chromatography tandem mass spectrometry (IP-RPLC-MS/MS) to provide in-depth characterization and identification of the degradation products. The oligonucleotide samples were stressed under different pH values at 45 and 90 °C. The main degradation products were observed to be losses of nucleotide moieties from the 3′- and 5′-terminus, depurination, formation of terminal phosphorothioates, and production of ribose, ribophosphorothioates (Rp), and phosphoribophosphorothioates (pRp). Moreover, the effects of different concentrations of hydrogen peroxide were studied resulting in primarily extensive desulfurization and subsequent oxidation of the phosphorothioate linkage to produce the corresponding phosphodiester. The reaction kinetics for the degradation of the oligonucleotides under the different stress conditions were studied and were found to follow pseudo-first-order kinetics. Differences in rates exist even for oligonucleotides of similar length but consisting of different sequences.
A novel sandwich enzyme-linked immunosorbent assay with covalently bound monoclonal antibody and gold probe for sensitive and rapid detection of bovine β-lactoglobulin Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-14 Shengfa He, Xin Li, Yong Wu, Shandong Wu, Zhihua Wu, Anshu Yang, Ping Tong, Juanli Yuan, Jinyan Gao, Hongbing Chen
Bovine milk is a recognized allergenic food source with β-lactoglobulin (BLG) as its major allergen. Reliable detection of BLG epitopes can, therefore, be a useful marker for the presence of milk in processed food products, and for potential allergenicity. At the present, enzyme-linked immunosorbent assays (ELISA) for the detection of BLG are time-consuming and generally not specific to BLG IgE epitopes. In this study, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-activated anti-BLG IgE epitope monoclonal antibody (mAb 1G9) was covalently bound onto the KOH-treated microtiter plate surface. Using this mAb-bound plate in sandwich combination with biotinylated anti-BLG polyclonal antibody-labeled gold nanoparticles, a linear dynamic range between 31.25 and 64 × 103 ng mL−1 with a limit of detection for BLG of 0.49 ng mL−1 was obtained, which is 32 times wider and 16 times more sensitive than conventional sandwich ELISA (sELISA). Total recovery of BLG in spiked food samples was found, without matrix effects. Also in partially hydrolyzed infant formulas, the allergenic BLG residues were detected quantitatively. Compared with conventional and commercial BLG detection sELISAs, our sELISA is reliable, highly BLG epitope-specific, user-friendly, and time-saving and allows accurate detection of potentially allergenic residues in different types of processed foods. This improved sELISA protocol can be easily extended to detect other well-identified and characterized food allergens.
Biomimetic trapping cocktail to screen reactive metabolites: use of an amino acid and DNA motif mixture as light/heavy isotope pairs differing in mass shift Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-14 Shuto Hosaka, Takuto Honda, Seon Hwa Lee, Tomoyuki Oe
Candidate drugs that can be metabolically transformed into reactive electrophilic products, such as epoxides, quinones, and nitroso compounds, are of special concern because subsequent covalent binding to bio-macromolecules can cause adverse drug reactions, such as allergic reactions, hepatotoxicity, and genotoxicity. Several strategies have been reported for screening reactive metabolites, such as a covalent binding assay with radioisotope-labeled drugs and a trapping method followed by LC–MS/MS analyses. Of these, a trapping method using glutathione is the most common, especially at the early stage of drug development. However, the cysteine of glutathione is not the only nucleophilic site in vivo; lysine, histidine, arginine, and DNA bases are also nucleophilic. Indeed, the glutathione trapping method tends to overlook several types of reactive metabolites, such as aldehydes, acylglucuronides, and nitroso compounds. Here, we introduce an alternate way for screening reactive metabolites as follows: A mixture of the light and heavy isotopes of simplified amino acid motifs and a DNA motif is used as a biomimetic trapping cocktail. This mixture consists of [2H0]/[2H3]-1-methylguanidine (arginine motif, Δ 3 Da), [2H0]/[2H4]-2-mercaptoethanol (cysteine motif, Δ 4 Da), [2H0]/[2H5]-4-methylimidazole (histidine motif, Δ 5 Da), [2H0]/[2H9]-n-butylamine (lysine motif, Δ 9 Da), and [13C0,15N0]/[13C1,15N2]-2′-deoxyguanosine (DNA motif, Δ 3 Da). Mass tag triggered data-dependent acquisition is used to find the characteristic doublet peaks, followed by specific identification of the light isotope peak using MS/MS. Forty-two model drugs were examined using an in vitro microsome experiment to validate the strategy.
The requirements for low-temperature plasma ionization support miniaturization of the ion source Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-13 Andreas Kiontke, Frank Holzer, Detlev Belder, Claudia Birkemeyer
Ambient ionization mass spectrometry (AI-MS), the ionization of samples under ambient conditions, enables fast and simple analysis of samples without or with little sample preparation. Due to their simple construction and low resource consumption, plasma-based ionization methods in particular are considered ideal for use in mobile analytical devices. However, systematic investigations that have attempted to identify the optimal configuration of a plasma source to achieve the sensitive detection of target molecules are still rare. We therefore used a low-temperature plasma ionization (LTPI) source based on dielectric barrier discharge with helium employed as the process gas to identify the factors that most strongly influence the signal intensity in the mass spectrometry of species formed by plasma ionization. In this study, we investigated several construction-related parameters of the plasma source and found that a low wall thickness of the dielectric, a small outlet spacing, and a short distance between the plasma source and the MS inlet are needed to achieve optimal signal intensity with a process-gas flow rate of as little as 10 mL/min. In conclusion, this type of ion source is especially well suited for downscaling, which is usually required in mobile devices. Our results provide valuable insights into the LTPI mechanism; they reveal the potential to further improve its implementation and standardization for mobile mass spectrometry as well as our understanding of the requirements and selectivity of this technique.
Simultaneous determination of amantadine and rimantadine in feed by liquid chromatography-Qtrap mass spectrometry with information-dependent acquisition Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-13 Qi Jia, Dan Li, Xinlu Wang, Shuming Yang, Yongzhong Qian, Jing Qiu
A sensitive method for simultaneous determination of amantadine and rimantadine in feed was developed using an ultra-high-performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry (UHPLC-Qtrap-MS) in the multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) mode, and employing the mixed cation exchange (MCX) solid-phase extraction column as sample cleanup and amantadine-d15 and rimantadine-d4 as internal standards, respectively. Compared to traditional MRM mode, for the targeted drugs in feed simultaneously both the secondary mass spectra and MRM information can be obtained using UHPLC-Qtrap-MS with MRM-IDA-EPI mode, and thus more accurate qualitative confirmation results achieved even at lower concentration of 0.2 μg/L in acceptable purity fit values. After optimization of sample preparation, good linearities (R > 0.9994) were obtained over the concentration range from 1 to 200 μg/L for amantadine and rimantadine. The precision was validated by intra-day and inter-day, and the relative standard deviations were all within 9.61%. Mean recoveries ranged from 76.1 to 112% at spiked concentrations of 0.5–100 μg/kg in three types of feed samples, including formula feed and complex concentrated feed for pigs and premix feed for chicken. The limits of detection (LODs) and quantification (LOQs) were 0.2 and 0.5 μg/kg for both drugs, respectively. The application in real feed samples further proved the accuracy and reliability of the developed method. This method provides an important tool to detect illegal uses of amantadine and rimantadine in feed.
Ternary mixed-mode silica sorbent of solid-phase extraction for determination of basic, neutral and acidic drugs in human serum Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-13 Shupei Jin, Yinghua Qiao, Jun Xing
In this study, a ternary mixed-mode silica sorbent (TMSS) with octamethylene, carboxyl, and amino groups was prepared via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction and a subsequent reduction of azide to primary amine. While used in solid-phase extraction (SPE), the retention behavior of TMSS towards a total of nine kinds of basic, neutral, and acidic drugs was investigated in detail. The results revealed that hydrophobic, ion-exchange interaction, and electrostatic repulsion between TMSS and the analytes were closely related to the retention behavior of TMSS. Besides, the log Kow value of the analyte was also a factor influencing the retention behavior of analytes on TMSS. The nine analytes could be retained by TMSS simultaneously and then, were eluted into two fractions according to the acid-base property of the analytes for further determinations. The acidic and neutral analytes were in one fraction, and the basic ones in the other fraction. When used to treat the human serum spiked with the nine drugs, TMSS offered higher recoveries than BakerBond CBA and comparable recoveries to Oasis WCX. It should be noted TMSS had better purifying capability for human serum than Oasis WCX. Under the optimized SPE conditions, a method of SPE hyphenated to high-performance liquid chromatography-ultraviolet detection (HPLC-UV) for determination of the basic, neutral, and acidic drugs spiked in human serum was established. For the nine drugs, the linear ranges were all between 5.0 and 1000 μg L−1 with correlation coefficients (R2) above 0.9990, and the limits of detection (LODs) were in the range of 0.8–2.3 μg L−1. The intra-day and inter-day relative standard deviations (RSDs) were less than 5.3 and 8.8%, respectively.
Synthesis and application of magnetic molecularly imprinted polymers in sample preparation Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-12 Shuyao Huang, Jianqiao Xu, Jiating Zheng, Fang Zhu, Lijun Xie, Gangfeng Ouyang
Magnetic molecularly imprinted polymers (MMIPs) have superior advantages in sample pretreatment because of their high selectivity for target analytes and the fast and easy isolation from samples. To meet the demand of both good magnetic property and good extraction performance, MMIPs with various structures, from traditional core–shell structures to novel composite structures with a larger specific surface area and more accessible binding sites, are fabricated by different preparation technologies. Moreover, as the molecularly imprinted polymer (MIP) layers determine the affinity, selectivity, and saturated adsorption amount of MMIPs, the development and innovation of the MIP layer are attracting attention and are reviewed here. Many studies that used MMIPs as sorbents in dispersive solid-phase extraction of complex samples, including environmental, food, and biofluid samples, are summarized.
Multielement analysis of Zanthoxylum bungeanum Maxim. essential oil using ICP-MS/MS Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-12 Liang Fu, Hualin Xie, Shuyun Shi
The concentrations of trace elements (Cr, Ni, As, Cd, Hg, and Pb) in Zanthoxylum bungeanum Maxim. essential oil (ZBMEO) were determined by inductively coupled plasma tandem mass spectrometry. The ZBMEO sample was directly analyzed after simple dilution with n-hexane. Aiming for a relatively high vapor pressure of n-hexane and its resultant loading on plasma, we used a narrow injector torch and optimized plasma radio frequency power and carrier gas flow to ensure stable operation of the plasma. An optional gas flow of 20% O2 in Ar was added to the carrier gas to prevent the incomplete combustion of highly concentrated organic carbon in plasma and the deposition of carbon on the sampling and skimmer cone orifices. In tandem mass spectrometry mode, O2 was added to the collision/reaction cell to eliminate the interferences. The limits of detection for Cr, Ni, As, Cd, Hg, and Pb were 2.26, 1.64, 2.02, 1.35, 1.76, and 0.97 ng L-1, respectively. After determination of 23 ZBMEO samples from different regions in China, we found that the average concentration ranges of trace elements in the 23 ZBMEO samples were 0.72–6.02 ng g-1, 0.09–2.87 ng g-1, 0.21–5.84 ng g-1, 0.16–2.15 ng g-1, 0.13–0.92 ng g-1, and 0.17–0.73 ng g-1 for Cr, Ni, As, Cd, Hg, and Pb, respectively. The trace elements in ZBMEO differed significantly when different extraction technologies were used. The study revealed that the contents of the toxic elements As, Cd, Hg, and Pb were extremely low, and hence they are unlikely to pose a health risk following ZBMEO ingestion.
A simple and ultrasensitive fluorescence assay for single-nucleotide polymorphism Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-12 Qian Ma, Zhiqiang Gao
In this report, a simple, label-free and highly efficient nucleic acid amplification technique is developed for ultrasensitive detection of single-nucleotide polymorphism (SNP). Briefly, a designed padlock probe is first circularized by a DNA ligase when it perfectly complements to a mutant gene. Then, the mutant gene functions as a primer to initiate branched rolling circle amplification reaction (BRCA), generating a large number of branched DNA strands and a lot of pyrophosphate molecules which is equivalent to the number of nucleotides consumed. With the addition of a terpyridine–Zn(II) complex, pyrophosphate molecules can be sensitively detected owing to the formation of a fluorescent terpyridine–Zn(II)–pyrophosphate complex. The fluorescence intensity is directly associated with the content of the mutant gene in a sample solution. On the other hand, the circulation of the padlock probe is prohibited when it hybridizes with the wild-type gene. In this assay, the accumulative nature of the BRCA process produces a detection limit of 0.1 pM and an excellent selectivity factor of 1000 toward SNP. As little as 0.1% mutant in the wild-type gene can be successfully detected. The simple procedure, high sensitivity, and high selectivity of this assay offer a potentially viable alternative for routine SNP analysis.
Proteomic approaches beyond expression profiling and PTM analysis Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-10 Jiaqi Fu, Mei Wu, Xiaoyun Liu
Essentially, all cellular functions are executed by proteins. Different physiological and pathological conditions dynamically control various properties of proteins, including expression levels, post-translational modifications (PTMs), protein–protein interactions, enzymatic activity, etc. Thus far, the vast majority of proteomic efforts have been focused on quantitative profiling of protein abundance/expression and their PTMs. In this article, we review some recent exciting progress in the development of proteomic approaches to examine protein functions from perspectives other than expression levels and PTMs. Specifically, we discuss advancements in proximity-based labeling, analysis of protein termini and newly synthesized proteins, and activity-based protein profiling.
Simultaneous determination of sulfur compounds from the sulfur pathway in rat plasma by liquid chromatography tandem mass spectrometry: application to the study of the effect of Shao Fu Zhu Yu decoction Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-10 Yue Zhang, An Kang, Haishan Deng, Le Shi, Shulan Su, Li Yu, Tong Xie, Jinjun Shan, Hongmei Wen, Yumei Chi, Shuying Han, Ruilin Su, Yilin Song, Xi Chen, Armaan Basheer Shaikh
A sensitive, accurate, and time-saving approach was developed for the simultaneous quantification of eight sulfur compounds in the sulfur pathway, which could reflect the status of an organism, including oxidative stress, signal transduction, enzyme reaction, and so on. In order to overcome the instability of highly reactive sulfhydryl compounds, N-ethylmaleimide derivatization was adopted to effectively protect sulfhydryl-containing samples. Using isotope-labeled glutathione (GSH-13C2, 15N), the validated method was demonstrated to offer satisfactory linearity, accuracy, and precision. Separation was done by UHPLC, using a BEH amide column. Accordingly, 0.1% formic acid acetonitrile was selected as the precipitant. A tandem mass spectrometer was coupled to the chromatographic system and afforded a detection limit of 0.2 ng/mL. Good linearity was maintained over a wide concentration range (r2 > 0.994), and the accuracy was in the range of 86.6–114% for all the studied compounds. The precision, expressed in RSD%, ranged from 1.1% to 9.4% as intraday variability and less than 13% as interday precision for all of the analytes. The approach was applied to study the potential therapeutic mechanism of a well-known traditional Chinese medicine, Shao Fu Zhu Yu decoction. The results suggested that Shao Fu Zhu Yu decoction might protect against oxidative damage by increasing the concentrations of sulfhydryl compounds.
Characterization of bioactive compounds of Annona cherimola L. leaves using a combined approach based on HPLC-ESI-TOF-MS and NMR Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-09 Elixabet Díaz-de-Cerio, Luis Manuel Aguilera-Saez, Ana María Gómez-Caravaca, Vito Verardo, Alberto Fernández-Gutiérrez, Ignacio Fernández, David Arráez-Román
Annona cherimola Mill. (cherimoya) has widely been used as food crop. The leaves of this tree possess several health benefits, which are, in general, attributed mainly to its bioactive composition. However, literature concerning a comprehensive characterization based on a combined approach, which consists of nuclear magnetic resonance (NMR) and high-performance liquid chromatography coupled with time-of-flight mass spectrometry (HPLC-TOF-MS), from these leaves is scarce. Thus, the aim of this work was to study the polar profile of full extracts of cherimoya leaves by using these tools. Thus, a total of 77 compounds have been characterized, 12 of which were identified by both techniques. Briefly, 23 compounds were classified as amino acids, organic acids, carbohydrates, cholines, phenolic acid derivatives, and flavonoids by NMR, while 66 metabolites were divided into sugars, amino acids, phenolic acids and derivatives, flavonoids, phenylpropanoids, and other polar compounds by HPLC-TOF-MS. It is worth mentioning that different solvent mixtures were tested and the total phenolic content in the extracts quantified (TPC via HPLC-TOF-MS). The tendency observed was EtOH/water 80/20 (v/v) (17.0 ± 0.2 mg TPC/g leaf dry weight (d.w.)) ≥ acetone/water 70/30 (v/v) (16.1 ± 0.7 mg TPC/g leaf d.w.) > EtOH/water 70/30 (v/v) (14.0 ± 0.3 mg TPC/g leaf d.w.) > acetone/water 80/20 (v/v) (13.5 ± 0.4 mg TPC/g leaf d.w.). Importantly, flavonoids derivatives were between 63 and 76% of the TPC in those extracts. Major compounds were sucrose, glucose (α and β), and proline, and chlorogenic acid and rutin for NMR and HPLC-TOF-MS, respectively.
Evaluating droplet digital PCR for the quantification of human genomic DNA: converting copies per nanoliter to nanograms nuclear DNA per microliter Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-19 David L. Duewer, Margaret C. Kline, Erica L. Romsos, Blaza Toman
The highly multiplexed polymerase chain reaction (PCR) assays used for forensic human identification perform best when used with an accurately determined quantity of input DNA. To help ensure the reliable performance of these assays, we are developing a certified reference material (CRM) for calibrating human genomic DNA working standards. To enable sharing information over time and place, CRMs must provide accurate and stable values that are metrologically traceable to a common reference. We have shown that droplet digital PCR (ddPCR) limiting dilution end-point measurements of the concentration of DNA copies per volume of sample can be traceably linked to the International System of Units (SI). Unlike values assigned using conventional relationships between ultraviolet absorbance and DNA mass concentration, entity-based ddPCR measurements are expected to be stable over time. However, the forensic community expects DNA quantity to be stated in terms of mass concentration rather than entity concentration. The transformation can be accomplished given SI-traceable values and uncertainties for the number of nucleotide bases per human haploid genome equivalent (HHGE) and the average molar mass of a nucleotide monomer in the DNA polymer. This report presents the considerations required to establish the metrological traceability of ddPCR-based mass concentration estimates of human nuclear DNA.
Fabrication of an immunosensor for early and ultrasensitive determination of human tissue plasminogen activator (tPA) in myocardial infraction and breast cancer patients Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-07 Haidar Saify Nabiabad, Khosro Piri, Fatemeh Kafrashi, Abbas Afkhami, Tayyebeh Madrakian
Sensitive detection of biomarkers will mean accurate and early diagnosis of diseases. A tissue plasminogen activator (tPA) has a crucial role in many cardiovascular diseases and it is related to many processes such as angiogenesis in cancer cells. Therefore, sensitive determination of tPA is important in diagnosis and clinical research. tPA monoclonal antibody was covalently attached onto single-wall carbon nanotubes (SWCNTs) using diimide-activated imidation coupling. Functionalized SWCNTs were immobilized onto a glassy carbon electrode and the modification process was investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), SEM, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). Cyclic voltammograms (CVs) in a scan rate of 100 mVs−1 was studied and comparisons were made between the modified glassy carbon electrodes (immobilized with antibodies) as a working electrode before and after the formation of tPA-antibody complex. Results of the SDS-PAGE demonstrated that the antibody was covalently and site directly attached to the SWCNTs. The fabricated biosensor provided a good linear response range from 0.1 to 1.0 ng mL−1 with a low detection limit of 0.026 ng mL−1. The immunosensor showed selectivity, reproducibility, good sensitivity, and acceptable stability. Satisfactory results were observed for early and sensitive determination of tPA in human serum samples. For the first time, such specific biosensor is currently being fabricated for tPA in our laboratories and successfully could determine tPA in myocardial infraction and breast cancer patients.
Identification and quantification of glue-like off-odors in elastic therapeutic tapes Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-06 Philipp Denk, Andrea Buettner
Elastic therapeutic tapes are an important tool in the field of physical therapy and medicine. These tapes contain types of adhesive. However, sensory evaluations revealed the release of pronounced and irritating odors of the tapes. Negative odors were, amongst others, reported in elastic therapeutic tapes containing acrylic adhesives. In this study, the odor of four different tape samples was evaluated applying a descriptive analysis approach carried out by a trained sensory panel. Afterwards, the volatile compounds were recovered from the samples by solvent extraction and isolated by solvent-assisted flavor evaporation (SAFE). The obtained distillates were subsequently analyzed by gas chromatography-olfactometry (GC-O) and two-dimensional GC-O coupled with mass spectrometry (2D-GC-MS/O). To determine the most potent odorants in the distillates, odor extract dilution analyses (OEDA) were carried out. Thirty-one odorants were successfully identified using this approach, which were all described for the first time as odorants in tapes. Amongst the set of volatiles, unsaturated and saturated aldehydes were present, eliciting fatty, soapy, and citrus-like odor impressions, as well as a range of glue-like, moldy, and fruity smelling odor-active volatiles, such as 2-ethyl-1-hexanol, butyl benzoate, and 3-phenyltoluene. Based on their relative intensities, the concentrations of the glue-like smelling substances were determined: 2-ethyl-1-hexanol, present in all samples, was determined with concentrations ranging from 10 to 200 mg/kg in the investigated tapes.
Precise, accurate and user-independent blood collection system for dried blood spot sample preparation Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-05 Ricardo Neto, Andrew Gooley, Michael C. Breadmore, Emily F. Hilder, Florian Lapierre
An accurate and precise 3 μL blood collection and dispensing system is presented for the preparation of dried blood spot (DBS) samples. Using end-to-end glass capillaries in conjugation with pre-punched DBS pads, a blood micro collection system was developed to eliminate the haematocrit dispersion, widely associated with DBS technology, while providing better levels of accuracy and precision during sample preparation. This methodology is compared to traditional micro-volume blood collection systems, such as a pipette and a digitally controlled analytical syringe. Results showed that % of recovery for the capillary methodology was closer to 100% across the three haematocrit (HCT) levels tested and when prepared by two users (98 to 100% for capillaries, 78 to 104% for pipette and 93 to 97% for digital syringe) attesting a higher accuracy. Additionally, by taking advantage of the capillary action mechanism to collect and dispense autonomously the desired volume of blood onto the DBS pad, coefficients of variation between two individuals were significantly lower than with standard methodologies (capillaries—0.05 to 0.77%, pipette—12.71 to 18.53% and digital syringe—0.72 to 1.77%). This alternate aspiration and dispensing methodology could be used by different users without compromising accuracy or precision when handling low volumes of blood during the pre-analytical steps.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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