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.
Sensitive paper-based analytical device for fast colorimetric detection of nitrite with smartphone Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-19 Xiu-Xiu Zhang, Yi-Zhen Song, Fang Fang, Zhi-Yong Wu
On-site rapid monitoring of nitrite as an assessment indicator of the environment, food, and physiological systems has drawn extensive attention. Here, electrokinetic stacking (ES) was combined with colorimetric reaction on a paper-based device (PAD) to achieve colorless nitrite detection with smartphone. In this paper, nitrite was stacked on the paper fluidic channel as a narrow band by electrokinetic stacking. Then, Griess reagent was introduced to visualize the stacking band. Under optimal conditions, the sensitivity of nitrite was 160-fold increased within 5 min. A linear response in the range of 0.075 to 1.0 μg mL−1 (R2 = 0.99) and a limit of detection (LOD) of 73 ng mL−1 (0.86 μM) were obtained. The LOD was 10 times lower than the reported PAD, and close to that achieved by a desktop spectrophotometer. The applicability was demonstrated by nitrite detection from saliva and water with good selectivity, adding 100 times more concentrated co-ions. High recovery (91.0~108.7%) and reasonable intra-day and inter-day reproducibility (RSD < 9%) were obtained. This work shows that the sensitivity of colorless analyte detection-based colorimetric reaction can be effectively enhanced by integration of ES on a PAD.
Online low-field NMR spectroscopy for process control of an industrial lithiation reaction—automated data analysis Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-04 Simon Kern, Klas Meyer, Svetlana Guhl, Patrick Gräßer, Andrea Paul, Rudibert King, Michael Maiwald
Monitoring specific chemical properties is the key to chemical process control. Today, mainly optical online methods are applied, which require time- and cost-intensive calibration effort. NMR spectroscopy, with its advantage being a direct comparison method without need for calibration, has a high potential for enabling closed-loop process control while exhibiting short set-up times. Compact NMR instruments make NMR spectroscopy accessible in industrial and rough environments for process monitoring and advanced process control strategies. We present a fully automated data analysis approach which is completely based on physically motivated spectral models as first principles information (indirect hard modeling—IHM) and applied it to a given pharmaceutical lithiation reaction in the framework of the European Union’s Horizon 2020 project CONSENS. Online low-field NMR (LF NMR) data was analyzed by IHM with low calibration effort, compared to a multivariate PLS-R (partial least squares regression) approach, and both validated using online high-field NMR (HF NMR) spectroscopy.
Physicochemical study of natural fractionated biocolloid by asymmetric flow field-flow fractionation in tandem with various complementary techniques using biologically synthesized silver nanocomposites Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-03 Viorica Railean-Plugaru, Pawel Pomastowski, Tomasz Kowalkowski, Myroslav Sprynskyy, Boguslaw Buszewski
Asymmetric flow field-flow fractionation coupled with use of ultraviolet–visible, multiangle light scattering (MALLS), and dynamic light scattering (DLS) detectors was used for separation and characterization of biologically synthesized silver composites in two liquid compositions. Moreover, to supplement the DLS/MALLS information, various complementary techniques such as transmission electron spectroscopy, Fourier transform infrared spectroscopy, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used. The hydrodynamic diameter and the radius of gyration of silver composites were slightly larger than the sizes obtained by transmission electron microscopy (TEM). Moreover, the TEM results revealed the presence of silver clusters and even several morphologies, including multitwinned. Additionally, MALDI-TOF MS examination showed that the particles have an uncommon cluster structure. It can be described as being composed of two or more silver clusters. The organic surface of the nanoparticles can modify their dispersion. We demonstrated that the variation of the silver surface coating directly influenced the migration rate of biologically synthesized silver composites. Moreover, this study proves that the fractionation mechanism of silver biocolloids relies not only on the particle size but also on the type and mass of the surface coatings. Because silver nanoparticles typically have size-dependent cytotoxicity, this behavior is particularly relevant for biomedical applications.
Silicone wristbands compared with traditional polycyclic aromatic hydrocarbon exposure assessment methods Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-02 Holly M. Dixon, Richard P. Scott, Darrell Holmes, Lehyla Calero, Laurel D. Kincl, Katrina M. Waters, David E. Camann, Antonia M. Calafat, Julie B. Herbstman, Kim A. Anderson
Currently there is a lack of inexpensive, easy-to-use technology to evaluate human exposure to environmental chemicals, including polycyclic aromatic hydrocarbons (PAHs). This is the first study in which silicone wristbands were deployed alongside two traditional personal PAH exposure assessment methods: active air monitoring with samplers (i.e., polyurethane foam (PUF) and filter) housed in backpacks, and biological sampling with urine. We demonstrate that wristbands worn for 48 h in a non-occupational setting recover semivolatile PAHs, and we compare levels of PAHs in wristbands to PAHs in PUFs-filters and to hydroxy-PAH (OH-PAH) biomarkers in urine. We deployed all samplers simultaneously for 48 h on 22 pregnant women in an established urban birth cohort. Each woman provided one spot urine sample at the end of the 48-h period. Wristbands recovered PAHs with similar detection frequencies to PUFs-filters. Of the 62 PAHs tested for in the 22 wristbands, 51 PAHs were detected in at least one wristband. In this cohort of pregnant women, we found more significant correlations between OH-PAHs and PAHs in wristbands than between OH-PAHs and PAHs in PUFs-filters. Only two comparisons between PAHs in PUFs-filters and OH-PAHs correlated significantly (rs = 0.53 and p = 0.01; rs = 0.44 and p = 0.04), whereas six comparisons between PAHs in wristbands and OH-PAHs correlated significantly (rs = 0.44 to 0.76 and p = 0.04 to <0.0001). These results support the utility of wristbands as a biologically relevant exposure assessment tool which can be easily integrated into environmental health studies.
Multiple reaction monitoring targeted LC-MS analysis of potential cell death marker proteins for increased bioprocess control Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-02 Simone Albrecht, Christian Kaisermayer, David Reinhart, Monica Ambrose, Renate Kunert, Anna Lindeberg, Jonathan Bones
The monitoring of protein biomarkers for the early prediction of cell stress and death is a valuable tool for process characterization and efficient biomanufacturing control. A representative set of six proteins, namely GPDH, PRDX1, LGALS1, CFL1, TAGLN2 and MDH, which were identified in a previous CHO-K1 cell death model using discovery LC-MSE was translated into a targeted liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) platform and verified. The universality of the markers was confirmed in a cell growth model for which three Chinese hamster ovary host cell lines (CHO-K1, CHO-S, CHO-DG44) were grown in batch culture in two different types of basal media. LC-MRM-MS was also applied to spent media (n = 39) from four perfusion biomanufacturing series. Stable isotope-labelled peptide analogues and a stable isotope-labelled monoclonal antibody were used for improved protein quantitation and simultaneous monitoring of the workflow reproducibility. Significant increases in protein concentrations were observed for all viability marker proteins upon increased dead cell numbers and allowed for discrimination of spent media with dead cell densities below and above 1 × 106 dead cells/mL which highlights the potential of the selected viability marker proteins in bioprocess control.
Pipette-tip solid-phase extraction using polypyrrole as efficient adsorbent for extraction of avermectins and milbemycins in milk Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-04-02 Diego Hernando Ângulo Florez, Roseane Andrade Teixeira, Ricky Cássio Santos da Silva, Bruna Carneiro Pires, Flávia Viana Avelar Dutra, Keyller Bastos Borges
In this work, we developed a HPLC method for the multidetermination of avermectins (AVM) (abamectin—ABA 1b and ABA 1a, eprinomectin—EPR, and ivermectin—IVM) and milbemycins (moxidectin—MOX) in milk samples using polypyrrole (PPy) as adsorbent material in pipette-tip solid-phase extraction (PT–PPy–SPE). PPy was characterized by scanning electron microscopy, thermogravimetric analysis, Fourier transform infrared spectroscopy, and X-ray diffraction and the data agreed with the literature. The sample preparation included the clean-up of the milk by protein precipitation (PP) with acetonitrile and extraction of the analytes by PT–PPy–SPE. The chromatographic method was developed in reverse phase and isocratic mode with flow rate at 1.2 mL min−1 and ultraviolet detection at 250 nm. The mobile phase composition was acetonitrile:methanol:water (55:25:20, v/v/v). The studied parameters and the optimized conditions for the sample preparation were washing solvent (300 μL water), volume and type of eluent (500 μL methanol), volume and pH of sample (1 mL and pH 10), amount of adsorbent material (50 mg PPy), and without addition of salt (NaCl). The method was linear over the concentration range from 20 to 3000 ng mL−1 with coefficients of correlation (r) ≥ 0.99 for all analytes and recoveries around 100%. The method developed and validated was used for the analyses of real milk samples from cow treated with Ivomec® (IVM 3.5%), in which were found 21.51 ± 2.94 ng mL−1 of IVM. Finally, the results proved that PT–PPy–SPE coupled to HPLC–UV was economical, simple, and easy-to-perform technique.
Non-targeted analysis of unexpected food contaminants using LC-HRMS Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-29 Marco Kunzelmann, Martin Winter, Magnus Åberg, Karl-Erik Hellenäs, Johan Rosén
A non-target analysis method for unexpected contaminants in food is described. Many current methods referred to as “non-target” are capable of detecting hundreds or even thousands of contaminants. However, they will typically still miss all other possible contaminants. Instead, a metabolomics approach might be used to obtain “true non-target” analysis. In the present work, such a method was optimized for improved detection capability at low concentrations. The method was evaluated using 19 chemically diverse model compounds spiked into milk samples to mimic unknown contamination. Other milk samples were used as reference samples. All samples were analyzed with UHPLC-TOF-MS (ultra-high-performance liquid chromatography time-of-flight mass spectrometry), using reversed-phase chromatography and electrospray ionization in positive mode. Data evaluation was performed by the software TracMass 2. No target lists of specific compounds were used to search for the contaminants. Instead, the software was used to sort out all features only occurring in the spiked sample data, i.e., the workflow resembled a metabolomics approach. Procedures for chemical identification of peaks were outside the scope of the study. Method, study design, and settings in the software were optimized to minimize manual evaluation and faulty or irrelevant hits and to maximize hit rate of the spiked compounds. A practical detection limit was established at 25 μg/kg. At this concentration, most compounds (17 out of 19) were detected as intact precursor ions, as fragments or as adducts. Only 2 irrelevant hits, probably natural compounds, were obtained. Limitations and possible practical use of the approach are discussed.
Fluorescence turn-on detection of target sequence DNA based on silicon nanodot-mediated quenching Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-28 Yanan Zhang, Xinping Ning, Guobin Mao, Xinghu Ji, Zhike He
We have developed a new enzyme-free method for target sequence DNA detection based on the dynamic quenching of fluorescent silicon nanodots (SiNDs) toward Cy5-tagged DNA probe. Fascinatingly, the water-soluble SiNDs can quench the fluorescence of cyanine (Cy5) in Cy5-tagged DNA probe in homogeneous solution, and the fluorescence of Cy5-tagged DNA probe can be restored in the presence of target sequence DNA (the synthetic target miRNA-27a). Based on this phenomenon, a SiND-featured fluorescent sensor has been constructed for “turn-on” detection of the synthetic target miRNA-27a for the first time. This newly developed approach possesses the merits of low cost, simple design, and convenient operation since no enzymatic reaction, toxic reagents, or separation procedures are involved. The established method achieves a detection limit of 0.16 nM, and the relative standard deviation of this method is 9% (1 nM, n = 5). The linear range is 0.5–20 nM, and the recoveries in spiked human fluids are in the range of 90–122%. This protocol provides a new tactic in the development of the nonenzymic miRNA biosensors and opens a promising avenue for early diagnosis of miRNA-associated disease.
Highly sensitive detection of a small molecule by a paired labels recognition system based lateral flow assay Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-28 Leina Dou, Bingxin Zhao, Tong Bu, Wentao Zhang, Qiong Huang, Lingzhi Yan, Lunjie Huang, Yanru Wang, Jianlong Wang, Daohong Zhang
Small molecules are difficult to detect by conventional gold lateral flow assay (GLFA) sensitively because the test system must satisfy the conflict requirements between enough signal intensity and limited antibody (Ab) amount. In this work, a paired labels recognition (PLR)-based biosensor was designed by utilizing the specific binding of Ab and secondary antibody (anti-Ab) to enhance signal intensity and reduce antibody amount applied in small molecule detection. The PLR amplification system is fabricated by self-assembling the common detection probe, Au-labeled Ab (Au-Ab), and the signal booster, Au-labeled anti-Ab (Au-anti-Ab). Benefiting from this, a powerful network structure can be generated to accumulate numerous gold nanoparticles (GNPs) and thus significantly strengthen the signal intensity of detection. Therefore, a lower Ab amount will be applied to offer adequate signal strength, and further, the limit of detection will be obviously downregulated due to the more effective competition reaction. Using furazolidone (FZD) as a model analyte, we achieve a detection limit of as low as 1 ng mL−1, which was at least fivefold improved over that of the traditional GLFA. Furthermore, the practicality of this strategy was certificated in five different food samples.
Comprehensive 2D gas chromatography–time-of-flight mass spectrometry with 2D retention indices for analysis of volatile compounds in frankincense ( Boswellia papyrifera ) Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-27 Ming Jiang, Chadin Kulsing, Philip J. Marriott
Frankincense gum resin secreted from Boswellia papyrifera was analysed by comprehensive 2D gas chromatography hyphenated with accurate mass time-of-flight mass spectrometry (GC×GC−accTOFMS). Direct multiple injection experiments with stepwise isothermal temperature programming were then performed to construct isovolatility curves for reference alkane series in GC×GC. This provides access to calculation of second dimensional retention indices (2I). More than 500 peaks were detected and 220 compounds mainly comprising monoterpenes, sesquiterpenes, diterpenes and oxygenated forms of these compounds were identified according to their 1I, 2I and accurate mass data. The study demonstrates the capability of GC×GC−accTOFMS with retention data on two separate column phases, as an approach for improved component identification. A greater number of identified and/or tentatively identified terpenoids in this traditional Chinese medicine allow for a more comprehensive coverage of the volatile composition of frankincense.
Naked eye detection of infertility based on sperm protamine-induced aggregation of heparin gold nanoparticles Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-26 Raj Vidya, Alex Saji
The development of an easy to use, one-pot, environmentally friendly, non-invasive and label-free colorimetric probe for the determination of semen protamines, the biochemical marker of male fertility, using heparin gold nanoparticles (HAuNPs) is presented. The affinity of HAuNPs for protamines was due to the electrostatic interactions between polycationic protamine and polyanionic heparin. The binding of HAuNPs to protamine was characterized by variation in the plasmon absorption spectra followed by a visibly observable colour change of the solution from red to blue. We observed a red shift in the plasmon peak and the method exhibited linearity in the range of 10–70 ng/mL with a detection limit of 5 ng/mL, which is much lower than that reported for colorimetric sensors of protamine. The colour change and the variation in the absorbance of HAuNPs were highly specific for protamines in the presence of different interfering compounds and the method was successfully applied for determining protamine in real samples of semen and serum. Rather than a quantitative estimation, it seems that the method provides a quick screening between a large array of positive and negative samples and, moreover, it maintains the privacy of the user. The method appears to be simple and would be very useful in third-world countries where high-tech diagnostic aids are inaccessible to the majority of the population.
Consumer-friendly food allergen detection: moving towards smartphone-based immunoassays Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-26 Georgina M. S. Ross, Monique G. E. G. Bremer, Michel W. F. Nielen
In this critical review, we provide a comprehensive overview of immunochemical food allergen assays and detectors in the context of their user-friendliness, through their connection to smartphones. Smartphone-based analysis is centered around citizen science, putting analysis into the hands of the consumer. Food allergies represent a significant worldwide health concern and consumers should be able to analyze their foods, whenever and wherever they are, for allergen presence. Owing to the need for a scientific background, traditional laboratory-based detection methods are generally unsuitable for the consumer. Therefore, it is important to develop simple, safe, and rapid assays that can be linked with smartphones as detectors to improve user accessibility. Smartphones make excellent detection systems because of their cameras, embedded flash functions, portability, connectivity, and affordability. Therefore, this review has summarized traditional laboratory-based methods for food allergen detection such as enzyme-linked-immunosorbent assay, flow cytometry, and surface plasmon resonance, and the potential to modernize these methods by interfacing them with a smartphone readout system, based on the aforementioned smartphone characteristics. This is the first review focusing on smartphone-based food-allergen detection methods designed with the intention of being consumer-friendly.
In-house validation of a rapid and efficient procedure for simultaneous determination of ergot alkaloids and other mycotoxins in wheat and maize Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-24 Natalia Arroyo-Manzanares, Karl De Ruyck, Valdet Uka, Laura Gámiz-Gracia, Ana M. García-Campaña, Sarah De Saeger, José Diana Di Mavungu
A fundamental step in addressing the global problem of mycotoxins is the development of highly sensitive, multi-class extraction and detection methods. This constitutes a field of research that has in recent years enjoyed a steady advance. Such methods, generally based on liquid chromatography coupled to mass spectrometry, are widely reported successfully detecting various mycotoxins in different food and feed samples. In this work, an innovative approach to multi-class mycotoxin control is proposed, offering specific advantages: a broader inclusion of more mycotoxin classes, robust and thorough extraction for all target compounds despite their varied chemical properties, and determination of all analytes from a single injection. The method involved the extraction and quantification of the main mycotoxins produced by Aspergillus, Fusarium, and Penicillium fungi, as well as their reported derivatives, together with 12 other compounds most commonly produced by Claviceps purpurea. The popularly reported QuEChERS technique has been reduced to a simple “salting-out liquid-liquid extraction” (SO-LLE) to obtain the most efficient extraction of the aforementioned mycotoxin classes in a very short time. This is in particular extremely important in ensuring correct determination of individual ergot alkaloids, for which short and robust sample preparation as well as short analytical sequences were key for minimizing the epimerization during analysis. The analyses of wheat and maize samples were performed using ultra-high performance liquid chromatography coupled with tandem mass spectrometry. Matrix-matched calibration curves were established and limits of quantification were below the maximum levels established by the EU regulation. The precision (repeatability and intermediate precision) was lower than 13% in all cases and recoveries ranged between 60 and 98% in maize and between 62 and 103% in wheat, fulfilling the current legislation. The method was applied to study the co-occurrence of these mycotoxins in wheat (n = 13) and maize (n = 15) samples from six European countries. A successful quantification of 23 different mycotoxins, from all major classes, in 85% of wheat and 93% of maize samples was achieved.
Critical assessment of digital PCR for the detection and quantification of genetically modified organisms Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-24 Tigst Demeke, David Dobnik
The number of genetically modified organisms (GMOs) on the market is steadily increasing. Because of regulation of cultivation and trade of GMOs in several countries, there is pressure for their accurate detection and quantification. Today, DNA-based approaches are more popular for this purpose than protein-based methods, and real-time quantitative PCR (qPCR) is still the gold standard in GMO analytics. However, digital PCR (dPCR) offers several advantages over qPCR, making this new technique appealing also for GMO analysis. This critical review focuses on the use of dPCR for the purpose of GMO quantification and addresses parameters which are important for achieving accurate and reliable results, such as the quality and purity of DNA and reaction optimization. Three critical factors are explored and discussed in more depth: correct classification of partitions as positive, correctly determined partition volume, and dilution factor. This review could serve as a guide for all laboratories implementing dPCR. Most of the parameters discussed are applicable to fields other than purely GMO testing.
The influence of pH and divalent/monovalent cations on the internal electron transfer (IET), enzymatic activity, and structure of fructose dehydrogenase Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-22 Paolo Bollella, Yuya Hibino, Kenji Kano, Lo Gorton, Riccarda Antiochia
We report on the influence of pH and monovalent/divalent cations on the catalytic current response, internal electron transfer (IET), and structure of fructose dehydrogenase (FDH) by using amperometry, spectrophotometry, and circular dichroism (CD). Amperometric measurements were performed on graphite electrodes, onto which FDH was adsorbed and the effect on the response current to fructose was investigated when varying the pH and the concentrations of divalent/monovalent cations in the contacting buffer. In the presence of 10 mM CaCl2, a current increase of up to ≈ 240% was observed, probably due to an intra-complexation reaction between Ca2+ and the aspartate/glutamate residues found at the interface between the dehydrogenase domain and the cytochrome domain of FDH. Contrary to CaCl2, addition of MgCl2 did not show any particular influence, whereas addition of monovalent cations (Na+ or K+) led to a slight linear increase in the maximum response current. To complement the amperometric investigations, spectrophotometric assays were carried out under homogeneous conditions in the presence of a 1-electron non-proton-acceptor, cytochrome c, or a 2-electron-proton acceptor, 2,6-dichloroindophenol (DCIP), respectively. In the case of cytochrome c, it was possible to observe a remarkable increase in the absorbance up to 200% when 10 mM CaCl2 was added. However, by further increasing the concentration of CaCl2 up to 50 mM and 100 mM, a decrease in the absorbance with a slight inhibition effect was observed for the highest CaCl2 concentration. Addition of MgCl2 or of the monovalent cations shows, surprisingly, no effect on the electron transfer to the electron acceptor. Contrary to the case of cytochrome c, with DCIP none of the cations tested seem to affect the rate of catalysis. In order to correlate the results obtained by amperometric and spectrophotometric measurements, CD experiments have been performed showing a great structural change of FDH when increasing the concentration CaCl2 up to 50 mM, at which the enzyme molecules start to agglomerate, hindering the substrate access to the active site probably due to a chelation reaction occurring at the enzyme surface with the glutamate/aspartate residues.
Recent trends in water analysis triggering future monitoring of organic micropollutants Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-21 Torsten C. Schmidt
Water analysis has been an important area since the beginning of analytical chemistry. The focus though has shifted substantially: from minerals and the main constituents of water in the time of Carl Remigius Fresenius to a multitude of, in particular, organic compounds at concentrations down to the sub-nanogram per liter level nowadays. This was possible only because of numerous innovations in instrumentation in recent decades, drivers of which are briefly discussed. In addition to the high demands on sensitivity, high throughput by automation and short analysis times are major requirements. In this article, some recent developments in the chemical analysis of organic micropollutants (OMPs) are presented. These include the analysis of priority pollutants in whole water samples, extension of the analytical window, in particular to encompass highly polar compounds, the trend toward more than one separation dimension before mass spectrometric detection, and ways of coping with unknown analytes by suspect and nontarget screening approaches involving high-resolution mass spectrometry. Furthermore, beyond gathering reliable concentration data for many OMPs, the question of the relevance of such data for the aquatic system under scrutiny is becoming ever more important. To that end, effect-based analytics can be used and may become part of future routine monitoring, mostly with a focus on adverse effects of OMPs in specific test systems mimicking environmental impacts. Despite advances in the field of water analysis in recent years, there are still many challenges for further analytical research.
Aptamer-facilitated mass cytometry Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-20 Gleb G. Mironov, Alexandre Bouzekri, Jessica Watson, Olga Loboda, Olga Ornatsky, Maxim V. Berezovski
Mass cytometry is a novel cell-by-cell analysis technique, which uses elemental tags instead of fluorophores. Sample cells undergo rapid ionization in inductively coupled plasma and the ionized elemental tags are then analyzed by means of time-of-flight mass spectrometry. Benefits of the mass cytometry approach are in no need for compensation, the high number of detection channels (up to 100) and low background noise. In this work, we applied a biotinylated aptamer against human PTK7 receptor for characterization of positive (human acute lymphoblastic leukemia) and negative (human Burkitt’s lymphoma) cells by a mass cytometry instrument. Our proof of principal experiments showed that biotinylated aptamers in conjunction with metal-labeled neutravidin can be successfully utilized for mass cytometry experiments at par with commercially available antibodies.
Problems and solutions of polyethylene glycol co-injection method in multiresidue pesticide analysis by gas chromatography-mass spectrometry: evaluation of instability phenomenon in type II pyrethroids and its suppression by novel analyte protectants Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-20 Kazuhiko Akutsu, Yoko Kitagawa, Masato Yoshimitsu, Satoshi Takatori, Naoki Fukui, Masakazu Osakada, Kotaro Uchida, Emiko Azuma, Keiji Kajimura
Polyethylene glycol 300 is commonly used as a base material for “analyte protection” in multiresidue pesticide analysis via gas chromatography-mass spectrometry. However, the disadvantage of the co-injection method using polyethylene glycol 300 is that it causes peak instability in α-cyano pyrethroids (type II pyrethroids) such as fluvalinate. In this study, we confirmed the instability phenomenon in type II pyrethroids and developed novel analyte protectants for acetone/n-hexane mixture solution to suppress the phenomenon. Our findings revealed that among the examined additive compounds, three lipophilic ascorbic acid derivatives, 3-O-ethyl-L-ascorbic acid, 6-O-palmitoyl-L-ascorbic acid, and 6-O-stearoyl-L-ascorbic acid, could effectively stabilize the type II pyrethroids in the presence of polyethylene glycol 300. A mixture of the three ascorbic acid derivatives and polyethylene glycol 300 proved to be an effective analyte protectant for multiresidue pesticide analysis. Further, we designed and evaluated a new combination of analyte protectant compounds without using polyethylene glycol or the troublesome hydrophilic compounds. Consequently, we obtained a set of 10 medium- and long-chain saturated fatty acids as an effective analyte protectant suitable for acetone/n-hexane solution that did not cause peak instability in type II pyrethroids. These analyte protectants will be useful in multiresidue pesticide analysis by gas chromatography-mass spectrometry in terms of ruggedness and reliable quantitativeness.
More and enhanced glyphosate analysis is needed Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-19 Carolin Huhn
Glyphosate is the world’s most heavily applied herbicide. Worldwide, re-approval processes by authorities are ongoing, accompanied by intense public and political discussions on its possible carcinogenic effects. Further aspects involve human dietary exposure, its fate in the environment, and its impact on ecosystems. Many of these aspects are not yet fully understood. In many instances, the analytical strategies developed and applied so far have strong limitations, given the challenging physicochemical characteristics of glyphosate and its metabolites. Analytical chemists are still faced with problems in method development, reachable precision, and detection limits. Thus, not all open research questions can be answered with current strategies. This feature article wishes to address further needs in glyphosate analysis to foster enhancement of analytical strategies.
Determination of lysine content based on an in situ pretreatment and headspace gas chromatographic measurement technique Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-19 Xiao-fang Wan, Bao-lian Liu, Teng Yu, Ning Yan, Xin-Sheng Chai, You-ming Li, Guang-xue Chen
This work reports on a simple method for the determination of lysine content by an in situ sample pretreatment and headspace gas chromatographic measurement (HS-GC) technique, based on carbon dioxide (CO2) formation from the pretreatment reaction (between lysine and ninhydrin solution) in a closed vial. It was observed that complete lysine conversion to CO2 could be achieved within 60 min at 60 °C in a phosphate buffer medium (pH = 4.0), with a minimum molar ratio of ninhydrin/lysine of 16. The results showed that the method had a good precision (RSD < 5.23%) and accuracy (within 6.80%), compared to the results measured by a reference method (ninhydrin spectroscopic method). Due to the feature of in situ sample pretreatment and headspace measurement, the present method becomes very simple and particularly suitable to be used for batch sample analysis in lysine-related research and applications.
Development and validation of a rapid, selective, and sensitive LC–MS/MS method for simultaneous determination of d - and l -amino acids in human serum: application to the study of hepatocellular carcinoma Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-01 Minlu Han, Mengyu Xie, Jun Han, Daoyi Yuan, Tian Yang, Ying Xie
A validated liquid chromatography–tandem mass spectrometry method was developed for the simultaneous determination of d- and l-amino acids in human serum. Under the optimum conditions, except for dl-proline, l-glutamine, and d-lysine, the enantioseparation of the other 19 enantiomeric pairs of proteinogenic amino acids and nonchiral glycine was achieved with a CROWNPAK CR-I(+) chiral column within 13 min. The lower limits of quantitation for l-amino acids (including glycine) and d-amino acids were 5–56.25 μM and 0.625–500 nM, respectively, in human serum. The intraday precision and interday precision for all the analytes were less than 15%, and the accuracy ranged from −12.84% to 12.37% at three quality control levels. The proposed method, exhibiting high rapidity, enantioresolution, and sensitivity, was successfully applied to the quantification of d- and l-amino acid levels in serum from hepatocellular carcinoma patients and healthy individuals. The serum concentrations of l-arginine, l-isoleucine, l-aspartate, l-tryptophan, l-alanine, l-methionine, l-serine, glycine, l-valine, l-leucine, l-phenylalanine, l-threonine, d-isoleucine, d-alanine, d-glutamate, d-glutamine, d-methionine, and d-threonine were significantly reduced in the hepatocellular carcinoma patients compared with the healthy individuals (P < 0.01). d-Glutamate and d-glutamine were identified as the most downregulated serum markers (fold change greater than 1.5), which deserves further attention in hepatocellular carcinoma research.
Molecular identification of common Salmonella serovars using multiplex DNA sensor-based suspension array Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-02-19 Muhsin Aydin, Jacqueline Carter-Conger, Ning Gao, David F. Gilmore, Steven C. Ricke, Soohyoun Ahn
Salmonella is one of major foodborne pathogens and the leading cause of foodborne illness-related hospitalizations and deaths. It is critical to develop a sensitive and rapid detection assay that can identify Salmonella to ensure food safety. In this study, a DNA sensor-based suspension array system of high multiplexing ability was developed to identify eight Salmonella serovars commonly associated with foodborne outbreaks to the serotype level. Each DNA sensor was prepared by activating pre-encoded microspheres with oligonucleotide probes that are targeting virulence genes and serovar-specific regions. The mixture of 12 different types of DNA sensors were loaded into a 96-well microplate and used as a 12-plex DNA sensor array platform. DNA isolated from Salmonella was amplified by multiplex polymerase chain reaction (mPCR), and the presence of Salmonella was determined by reading fluorescent signals from hybridization between probes on DNA sensors and fluorescently labeled target DNA using the Bio-Plex® system. The developed multiplex array was able to detect synthetic DNA at the concentration as low as 100 fM and various Salmonella serovars as low as 100 CFU/mL within 1 h post-PCR. Sensitivity of this assay was further improved to 1 CFU/mL with 6-h enrichment. The array system also correctly and specifically identified serotype of tested Salmonella strains without any cross-reactivity with other common foodborne pathogens. Our results indicate the developed DNA sensor suspension array can be a rapid and reliable high-throughput method for simultaneous detection and molecular identification of common Salmonella serotypes.
Cellular dielectrophoresis coupled with single-cell analysis Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-02-23 Min Li, Robbyn K. Anand
In this review, recent advances that leverage dielectrophoretic approaches to accomplish single-cell analysis (both “on-chip” and ”off-chip”) are discussed with special emphasis on eukaryotic cells. Dielectrophoresis as an electric-field-induced force utilized for cell manipulation can confer selectivity without labeling. Recent technical improvements have increased the volumetric throughput of the separation of cells from complex mixtures, introduced new strategies for massively parallel single-cell confinement for subsequent on-chip analysis, made possible selective transport of individual cells off-chip, and integrated preconcentration and prefocusing steps to enhance dielectrophoretic performance. Collectively, these studies potentiate all-in-one platforms capable of taking as their input complex mixtures of cells and accomplishing single-cell analysis. Assays requiring small reaction volumes (e.g., enzymatic assays, fluorescent in situ hybridization, and immunostaining) have been demonstrated. Still greater opportunities to unravel cell-to-cell variations and for point-of-care applications can be realized by making possible on-chip gene amplification, live-cell assays, and either dielectrophoretic manipulation in native media or on-chip exchange of media. We therefore conclude with a discussion of emerging capabilities in these areas.
Fabrication and application of noble metal nanoclusters as optical sensors for toxic metal ions Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-02-01 Gaozhi Ou, Jing Zhao, Peng Chen, Changjin Xiong, Fan Dong, Biao Li, Xiaojun Feng
Atomically precise noble metal nanoclusters with ultrasmall physical sizes in the subnanometer range have emerged as a new class of probing fluorophores and have attracted considerable research interest because of their intrinsic physical, chemical, optical, biological, and electrical properties, such as stability, biocompatibility, and molecule-like photoluminescence. In comparison with traditional fluorophores such as organic dyes and quantum dots, noble metal nanoclusters have significant advantages, including low toxicity toward the environment and biological tissues, high stability when exposed to irradiation, and small size, that make them more suitable for biological sensing or biological labeling applications. Several reviews have summarized the fabrication of noble metal nanoclusters, including gold, silver, copper, and bimetallic nanoclusters. However, these reviews focused either on various facile preparation methods or multidisciplinary application areas. Here, we focus on the application of noble metal nanoclusters as optical sensing materials for toxic metal ions, including new synthetic approaches and discussion of the detection mechanism. We briefly summarize the development of metal cation monitoring technology that uses ultrasmall nanoclusters as the sensing probes. We also provide a fresh opinion on research expectations in the field of inorganic nanoscience and nanotechnology Finally, perspectives for future research hot topics are discussed.
Detection of mycoplasma in contaminated mammalian cell culture using FTIR microspectroscopy Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-17 Katia Wehbe, Marzia Vezzalini, Gianfelice Cinque
Mycoplasma contamination represents a significant problem to the culture of mammalian cells used for research as it can cause disastrous effects on eukaryotic cells by altering cellular parameters leading to unreliable experimental results. Mycoplasma cells are very small bacteria therefore they cannot be detected by visual inspection using a visible light microscope and, thus, can remain unnoticed in the cell cultures for long periods. The detection techniques used nowadays to reveal mycoplasma contamination are time consuming and expensive with each having significant drawbacks. The ideal detection should be simple to perform with minimal preparation time, rapid, inexpensive, and sensitive. To our knowledge, for the first time, we employed Fourier transform infrared (FTIR) microspectroscopy to investigate whether we can differentiate between control cells and the same cells which have been infected with mycoplasmas during the culturing process. Chemometric methods such as HCA and PCA were used for the data analysis in order to detect spectral differences between control and intentionally infected cells, and spectral markers were revealed even at low contamination level. The preliminary results showed that FTIR has the potential to be used in the future as a reliable complementary detection technique for mycoplasma-infected cells.
Simultaneous determination of organophosphorus pesticides and phthalates in baby food samples by ultrasound–vortex-assisted liquid–liquid microextraction and GC–IT/MS Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-17 Ivan Notardonato, Elisabetta Salimei, Mario Vincenzo Russo, Pasquale Avino
Baby foods are either a soft, liquid paste or an easily chewed food since babies lack developed muscles and teeth to chew effectively. Babies typically move to consuming baby food once nursing or formula is not sufficient for the child’s appetite. Some commercial baby foods have been criticized for their contents. This article focuses on the simultaneous determination of organophosphorus pesticides and phthalates by means of a method based on ultrasound–vortex-assisted liquid–liquid microextraction coupled with gas chromatography–ion trap mass spectrometry (GC–IT/MS). The protocol developed allowed the determination of six phthalates [dimethyl phthalate, diethyl phthalate, dibutyl phthalate, isobutyl cyclohexyl phthalate, benzyl butyl phthalate, bis(2-ethylhexyl) phthalate] and 19 organophosphorus pesticides. Freeze-dried product samples (0.1-0.2 g) were dissolved in 10 mL of warm distilled water along with 5 μL of an internal standard (anthracene at 10 mg mL-1 in acetone): the choice of extraction solvent was studied, with the most suitable being n-heptane, which is used for phthalate determination in similar matrices. The solution, held for 5 min in a vortex mixer and for 6 min in a 100-W ultrasonic bath to favor solvent dispersion and consequently analyte extraction, was centrifuged at 4000 rpm for 30 min. Then 1 μL was injected into the GC–IT/MS system (SE-54 capillary column; length 30 m, inner diameter 250 μm, film thickness 0.25 μm). All analytical parameters investigated are discussed in depth. The method was applied to real commercial freeze-dried samples: significant contaminant concentrations were not found.
MALDI imaging facilitates new topical drug development process by determining quantitative skin distribution profiles Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-15 David Bonnel, Raphaël Legouffe, André H. Eriksson, Rasmus W. Mortensen, Fabien Pamelard, Jonathan Stauber, Kim T. Nielsen
Generation of skin distribution profiles and reliable determination of drug molecule concentration in the target region are crucial during the development process of topical products for treatment of skin diseases like psoriasis and atopic dermatitis. Imaging techniques like mass spectrometric imaging (MSI) offer sufficient spatial resolution to generate meaningful distribution profiles of a drug molecule across a skin section. In this study, we use matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to generate quantitative skin distribution profiles based on tissue extinction coefficient (TEC) determinations of four different molecules in cross sections of human skin explants after topical administration. The four drug molecules: roflumilast, tofacitinib, ruxolitinib, and LEO 29102 have different physicochemical properties. In addition, tofacitinib was administrated in two different formulations. The study reveals that with MALDI-MSI, we were able to observe differences in penetration profiles for both the four drug molecules and the two formulations and thereby demonstrate its applicability as a screening tool when developing a topical drug product. Furthermore, the study reveals that the sensitivity of the MALDI-MSI techniques appears to be inversely correlated to the drug molecules’ ability to bind to the surrounding tissues, which can be estimated by their Log D values.
A guide to large-scale RNA sample preparation Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-15 Lorenzo Baronti, Hampus Karlsson, Maja Marušič, Katja Petzold
RNA is becoming more important as an increasing number of functions, both regulatory and enzymatic, are being discovered on a daily basis. As the RNA boom has just begun, most techniques are still in development and changes occur frequently. To understand RNA functions, revealing the structure of RNA is of utmost importance, which requires sample preparation. We review the latest methods to produce and purify a variation of RNA molecules for different purposes with the main focus on structural biology and biophysics. We present a guide aimed at identifying the most suitable method for your RNA and your biological question and highlighting the advantages of different methods.
Detection of target DNA with a novel Cas9/sgRNAs-associated reverse PCR (CARP) technique Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-15 Beibei Zhang, Qiao Wang, Xinhui Xu, Qiang Xia, Feifei Long, Weiwei Li, Yingchun Shui, Xinyi Xia, Jinke Wang
This study develops a new method for detecting target DNA based on Cas9 nuclease, which was named as CARP, representing CRISPR- or Cas9/sgRNAs-associated reverse PCR. This technique detects target DNA in three steps: (1) cleaving the detected DNA sample with Cas9 in complex with a pair of sgRNAs specific to target DNA; (2) ligating the cleaved DNA with DNA ligase; (3) amplifying target DNA with PCR. In the ligation step, the Cas9-cut target DNA was ligated into intramolecular circular or intermolecular concatenated linear DNA. In the PCR step, the ligated DNA was amplified with a pair of reverse primers. The technique was verified by detecting HPV16 and HPV18 L1 genes in nine different human papillomavirus (HPV) subtypes. The technique also detected the L1 and E6–E7 genes of two high-risk HPVs, HPV16 and HPV18, in the genomic DNA of two HPV-positive cervical carcinoma cells (HeLa and SiHa), in which no L1 and E6–E7 genes were detected in the HPV-negative cervical carcinoma cell, C-33a. By performing these proof-of-concept experiments, this study provides a new CRISPR-based DNA detection and typing method. Especially, the CARP method developed by this study is ready for the clinical HPV detection, which was supported by the final clinical sample detection.
Insert-based microfluidics for 3D cell culture with analysis Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-14 Chengpeng Chen, Alexandra D. Townsend, Elizabeth A. Hayter, Hannah M. Birk, Scott A. Sell, R. Scott Martin
We present an insert-based approach to fabricate scalable and multiplexable microfluidic devices for 3D cell culture and integration with downstream detection modules. Laser-cut inserts with a layer of electrospun fibers are used as a scaffold for 3D cell culture, with the inserts being easily assembled in a 3D-printed fluidic device for flow-based studies. With this approach, the number and types of cells (on the inserts) in one fluidic device can be customized. Moreover, after an investigation (i.e., stimulation) under flowing conditions, the cell-laden inserts can be removed easily for subsequent studies including imaging and cell lysis. In this paper, we first discuss the fabrication of the device and characterization of the fibrous inserts. Two device designs containing two (channel width = 260 μm) and four (channel width = 180 μm) inserts, respectively, were used for different experiments in this study. Cell adhesion on the inserts with flowing media through the device was tested by culturing endothelial cells. Macrophages were cultured and stimulated under different conditions, the results of which indicate that the fibrous scaffolds under flow conditions result in dramatic effects on the amount and kinetics of TNF-α production (after LPS stimulation). Finally, we show that the cell module can be integrated with a downstream absorbance detection scheme. Overall, this technology represents a new and versatile way to culture cells in a more in vivo fashion for in vitro studies with online detection modules.
Advanced analytical strategies for measuring free bioactive milk sugars: from composition and concentrations to human metabolic response Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-13 Aifric O’Sullivan, Jaime Salcedo, Josep Rubert
Our daily food intake provides the nutrients to maintain health. However, in addition to the nutritional values, food can promote health and be beneficial in preventing diseases. Human milk is a unique food source that contains essential nutrients in the right balance and other bioactive factors that make it the ideal food for all healthy term infants. Human milk oligosaccharides (HMOs) play an important role in health, at several levels: acting as prebiotics promoting the growth of beneficial bacterial strains, preventing the growth of pathogenic bacteria in the intestine, and modulating the immune response against bacterial infections. However, despite their biological relevance and the advances made in the analytical field, very few studies have been carried out to better understand HMOs bioactivity mechanisms or to examine human metabolic response to dietary supplementation. This review describes the state-of-the-art of glycomics strategies, recent analytical methods, and future trends for the identification and discovery of bioactive sugars, the known mechanisms of action, and discusses findings of some recent human intervention trials.
Variations of l - and d -amino acid levels in the brain of wild-type and mutant mice lacking d -amino acid oxidase activity Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-12 Siqi Du, Yadi Wang, Choyce A. Weatherly, Kylie Holden, Daniel W. Armstrong
d-amino acids are now recognized to be widely present in organisms and play essential roles in biological processes. Some d-amino acids are metabolized by d-amino acid oxidase (DAO), while d-Asp and d-Glu are metabolized by d-aspartate oxidase (DDO). In this study, levels of 22 amino acids and the enantiomeric compositions of the 19 chiral proteogenic entities have been determined in the whole brain of wild-type ddY mice (ddY/DAO+/+), mutant mice lacking DAO activity (ddY/DAO−/−), and the heterozygous mice (ddY/DAO+/−) using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). No significant differences were observed for l-amino acid levels among the three strains except for l-Trp which was markedly elevated in the DAO+/− and DAO−/− mice. The question arises as to whether this is an unknown effect of DAO inactivity. The three highest levels of l-amino acids were l-Glu, l-Asp, and l-Gln in all the three strains. The lowest l-amino acid level was l-Cys in ddY/DAO+/− and ddY/DAO−/− mice, while l-Trp showed the lowest level in ddY/DAO+/+mice. The highest concentration of d-amino acid was found to be d-Ser, which also had the highest % d value (~ 25%). d-Glu had the lowest % d value (~ 0.01%) in all the three strains. Significant differences of d-Leu, d-Ala, d-Ser, d-Arg, and d-Ile were observed in ddY/DAO+/− and ddY/DAO−/− mice compared to ddY/DAO+/+ mice. This work provides the most complete baseline analysis of l- and d-amino acids in the brains of ddY/DAO+/+, ddY/DAO+/−, and ddY/DAO−/− mice yet reported. It also provides the most effective and efficient analytical approach for measuring these analytes in biological samples. This study provides fundamental information on the role of DAO in the brain and may be relevant for future development involving novel drugs for DAO regulation.
Combination of in situ metathesis reaction with a novel “magnetic effervescent tablet-assisted ionic liquid dispersive microextraction” for the determination of endogenous steroids in human fluids Anal. Bioanal. Chem. (IF 3.431) Pub Date : 2018-03-12 Jia Wu, Zilin Xu, Yixuan Pan, Yi Shi, Xiujie Bao, Jun Li, Yu Tong, Han Tang, Shuyan Ma, Xuedong Wang, Jianxin Lyu
Herein, a novel magnetic effervescence tablet-assisted microextraction coupled to in situ metathesis reaction of ionic liquid (IS-META-ILDM) is presented for the determination of four endogenous steroids in human urine, pregnant women’s blood, and fetal umbilical cord blood. The magnetic effervescent tablets, which were composed of Fe3O4 nanoparticles, sodium carbonate (alkaline source), and tartaric acid (acidic source), were used to disperse the extractant and for convenient magnetic separation. After the effervescent reaction, in situ reaction between NH4PF6 and [C6MIM]BF4 was adopted to change hydrophilic ionic liquid to hydrophobic liquid, which could be separated from the aqueous phase. The newly developed method has three obvious advantages: (1) combination of effervescent dispersion and magnetic nanoparticles’ retrieval is cost-effective and the dispersion and collection of the extractant can be completed almost simultaneously; (2) as compared to temperature-controlled ionic liquid dispersive microextraction and cold-induced solidified microextraction, this method avoids a heating and cooling process which significantly reduces the extraction time and energy cost; and (3) the combination of adsorption by magnetic nanoparticles with extraction by in situ metathesis reaction easily produces high recoveries for target analytes. The optimized composition of effervescent tablet and experimental parameters are as follows: 0.64 g mixture of sodium carbonate and tartaric acid, 7 mg of Fe3O4 (20 nm) as magnetic sorbents, 40 μL of [C6MIM]BF4 as the extraction solvent, 0.15 g NH4PF6, and 300 μL of elution solvent. Under the optimized conditions, the newly developed method provided high extraction recoveries (90.0–118.5%) and low LODs (0.14–0.17 μg L−1) in urine and blood samples. In total, this IS-META-ILDM method provided high extraction efficiency, fast and convenient separation, and underutilization of any organic solvent, and thus it has great potential for the determination of trace endogenous steroids in complex human fluids.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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