Analytical and Bioanalytical Chemistry (v.407, #6)

Combining capillary electrophoresis and next-generation sequencing for aptamer selection by Kathryn R. Riley; Jason Gagliano; Jiajie Xiao; Kara Libby; Shingo Saito; Guo Yu; Roger Cubicciotti; Jed Macosko; Christa L. Colyer; Martin Guthold; Keith Bonin (1527-1532).
Next-generation sequencing (NGS) machines can sequence millions of DNA strands in a single run, such as oligonucleotide (oligo) libraries comprising millions to trillions of discrete oligo sequences. Capillary electrophoresis is an attractive technique to select tight binding oligos or “aptamers” because it requires minimal sample volumes (e.g., 100 nL) and offers a solution-phase selection environment through which enrichment of target-binding oligos can be determined quantitatively. We describe here experiments using capillary transient isotachophoresis (ctITP)-based nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) as a method for selecting aptamers from a randomized library containing a known (29mer) thrombin-binding aptamer. Our capillary electrophoresis (CE)-selected samples were sequenced by the Ion Torrent Personal Genome Machine (PGM) and analyzed for selection efficiency. We show that a single round of CE selection can enrich a randomer synthetic DNA oligo mixture for thrombin-binding activity from 0.4 % aptamer content before selection to >15 % aptamer content.
Keywords: Separations; Instrumentation; Capillary electrophoresis; Electrophoresis; Next-generation sequencing; Bioanalytical methods

Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices by Maria Chiara Giuffrida; Laura Maria Zanoli; Roberta D’Agata; Alessia Finotti; Roberto Gambari; Giuseppe Spoto (1533-1543).
is Post-Doc at Consorzio Interuniversitario Istituto Nazionale Biostrutture e Bioimmagini (I.N.B.B.). Her research activity is focused on analytical chemistry with specific expertise in microfluidics and biosensors. Her current work is focused on developing ultrasensitive techniques for the detection of DNA, RNA, and protein biomarkers by using droplet-based microfluidic devices. is PhD in Nanoscience at Scuola Superiore di Catania, University of Catania. Her research activity has focused on microfluidics, with specific interest in integration of isothermal amplification methods. Recently she moved to ARPA, Ravenna. is PhD in Chemistry and Assistant Professor at the University of Catania. She has substantial expertise in surface-plasmon-resonance imaging and microfluidic-based techniques. She is involved in studies of biointerfaces, surface functionalization, and development of hybridization procedures for the detection of DNA and RNA targets based on peptide-nucleic-acid (PNA) probes. Other research interests focus on preparation and characterization of biofunctionalized gold nanoparticles, and analysis by mass-spectrometry electrospray (ESI) ionization and matrix-assisted laser desorption (MALDI). is a post-doc fellow at the Department of Life Sciences and Biotechnology, University of Ferrara, and contract professor in “Molecular Biology of the Cell” (University of Urbino) and “Biomolecular Drugs” (University of Ferrara). Her research interests are regulation of gene expression, delivery strategies for microRNA therapeutics, studies of DNA methylation, development of transgenic mice, and molecular diagnosis of cystic fibrosis and thalassemia. She is the external collaborator of projects funded by the FFC (the Italian Cystic Fibrosis Foundation). Alessia Finotti has authored over 30 publications. is Full Professor of Biochemistry at the Department of Life Sciences and Biotechnology, University of Ferrara, and Director of the ThalLab (Laboratory for the Development of Gene and Pharmacogenomic Therapy of Thalassemia). His research interests are regulation of gene expression, DNA methylation, study of DNA-binding drugs, peptide nucleic acids (PNAs) and PNA–DNA chimeras, molecular diagnosis of genetic diseases based on SPR, lab-on-a-chip technology, and microRNA therapeutics. He was the coordinator of several AIRC and THELETHON projects. At present he is the coordinator of the FP7 European Project THALAMOSS (THALAssaemia MOdular Stratification System for personalized therapy of beta-thalassemia). Roberto Gambari has authored over 380 publications and is coauthor of over 12 patents; h-INDEX: 40. is Full Professor of Analytical Chemistry at the Chemistry Department, University of Catania, and responsible for the Catania Unit of Consorzio Interuniversitario Istituto Nazionale Biostrutture e Bioimmagini (I.N.B.B.). He has for several years been studying surface and interface processes with specific focus on bioanalytical detection processes, including biosensors and microfluidics. He is a member of INBB Management Committee, coordinator of the Analytical Spectroscopy divisional group of the Italian Chemical Society, and Board of Directors member of the Consorzio Catania Ricerche, a not-for-profit consortium whose main objectives are know-how transfer, technological innovation spreading, and applied research. He is a recipient of the Accademia Gioienia Award for Chemistry (1994) and of The Best Researcher Award, University of Catania (2013). Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L−1–pmol L−1 detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L−1–pmol L−1 microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10−18 mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells.
Keywords: Digital microfluidics; Nucleic-acid amplification; MicroRNA; Isothermal amplification; Circular-strand-displacement polymerization

is a PhD student at the Department of Experimental and Clinical Toxicology, Saarland University. Her research interests focus on the in vivo and in vitro metabolism of novel psychotropic substances using GC-MS and LC-(HR)-MSn techniques. finished his PhD in 2014 under the supervision of Professor Hans H. Maurer at the Department of Experimental and Clinical Toxicology, Saarland University. His research interests focus on the in vivo and in vitro metabolism of new legal highs using GC-MS and LC-(HR)-MSn techniques. is the technical and scientific supervisor of the NMR spectrometers of Faculty 8, Saarland University. His scientific interests concern the structural elucidation and biogenesis of natural compounds. He has published more than 70 articles in this field. is full Professor of Pharmacology and Toxicology at the Faculty of Medicine and of Pharmacy, Saarland University, since 1992. He is head of the Department of Experimental and Clinical Toxicology in Homburg, Germany. He has published over 270 original papers and invited reviews on his main two areas of research, analytical toxicology (GC-MS, LC-(HR)-MS) as well as toxicokinetics and metabolism of xenobiotics. He is editorial board member of various international journals and member of executive boards of scientific societies in his field. He received several international scientific awards, among which the title of Doctor Honoris Causa (honorary doctorate) in 2007 by the University of Ghent, Belgium. Lefetamine (N,N-dimethyl-1,2-diphenylethylamine, L-SPA) was marketed as an opioid analgesic in Japan and Italy. After being widely abused, it became a controlled substance. It seems to be a pharmaceutical lead for designer drugs because N-ethyl-1,2-diphenylethylamine (NEDPA) and N-iso-propyl-1,2-diphenylethylamine (NPDPA) were confiscated by the German police. In contrast to these derivatives, metabolism and detectability of lefetamine were not studied yet. Therefore, phase I and II metabolism should be elucidated and correlated to the derivatives. Also the detectability using the authors’ standard urine screening approaches (SUSA) needed to be checked. As lefetamine was commercially unavailable, it had to be synthesized first. For metabolism studies, a high dose of lefetamine was administered to rats and the urine samples worked up in different ways. Separation and analysis were achieved by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS). In accordance with NEPDA and NPDPA, the following metabolic steps could be proposed: N-oxidation, N-dealkylation, mono- and bis-hydroxylation of the benzene ring, and hydroxylation of the phenyl ring only after N-dealkylation. The di-hydroxy metabolites were conjugated by methylation of one hydroxy group, and hydroxy metabolites by glucuronidation or sulfation. All initial metabolites could also be detected in human liver preparations. After a therapeutic lefetamine dose, the bis-nor, bis-nor-hydroxy, nor-hydroxy, nor-di-hydroxy metabolites could be detected using the authors’ GC-MS SUSA and the nor-hydroxy-glucuronide by the LC-MSn SUSA. Thus, an intake of lefetamine should be detectable in human urine assuming similar pharmacokinetics.
Keywords: Drugs of abuse; Lefetamine; Metabolism; GC-MS; LC-HR-MS/MS

Study of the distribution of actinides in human tissues using synchrotron radiation micro X-ray fluorescence spectrometry by Eva Vergucht; Björn De Samber; Andrei Izmer; Bart Vekemans; Karen Appel; Sergei Tolmachev; Laszlo Vincze; Frank Vanhaecke (1559-1566).
This study aims at evaluating the capabilities of synchrotron radiation micro X-ray fluorescence spectrometry (SR micro-XRF) for qualitative and semi-quantitative elemental mapping of the distribution of actinides in human tissues originating from individuals with documented occupational exposure. The investigated lymph node tissues were provided by the United States Transuranium and Uranium Registries (USTUR) and were analyzed following appropriate sample pre-treatment. Semi-quantitative results were obtained via calibration by external standards and demonstrated that the uranium concentration level in the detected actinide hot spots reaches more than 100 μg/g. For the plutonium hot spots, concentration levels up to 31 μg/g were found. As illustrated by this case study on these unique samples, SR micro-XRF has a high potential for this type of elemental bio-imaging owing to its high sensitivity, high spatial resolution, and non-destructive character.ᅟ Graphical Abstract SR micro-XRF study of the distribution of actinitides in human tissues. Left Location of the U-contaminated tissue sample in the human body. Middle U distribution derived from the high resolution SR micro-XRF scan on the tissue sample, indication of five U hot spots. Right Detail of the point measurement spectrum of U hot spot 3, intense U-Lα fluorescence peak located at 13.6 keV.
Keywords: SR micro-XRF; Actinides; Elemental distributions; Human tissues; Bio-imaging

Organically modified silica nanoparticles doped with new acridine-1,2-dioxetane analogues as thermochemiluminescence reagentless labels for ultrasensitive immunoassays by Massimo Di Fusco; Arianna Quintavalla; Marco Lombardo; Massimo Guardigli; Mara Mirasoli; Claudio Trombini; Aldo Roda (1567-1576).
Doped organically modified silica nanoparticles (ORMOSIL NPs) with luminescent molecules represent a potent approach to signal amplification in biomolecule labeling. Herein, we report the synthesis of new ORMOSIL NPs incorporating thermochemiluminescent (TCL) 1,2-dioxetane derivatives to prepare TCL labels for ultrasensitive immunoassay, displaying a detectability comparable to those offered by other conventional luminescence-based systems. Amino-functionalized ORMOSIL NPs were synthesized for inclusion of acridine-containing 1,2-dioxetane derivatives with a fluorescence energy acceptor. The doped ORMOSIL NPs were further functionalized with biotin for binding to streptavidin-labeled species to be used as universal detection reagents for immunoassays. A quantitative non-competitive immunoassay for streptavidin has been developed by immobilizing anti-streptavidin antibody to capture streptavidin, then the antibody-bound streptavidin was detected by the biotinylated TCL ORMOSIL NPs. The analytical performance was similar to that obtained by chemiluminescent (CL) detection using horseradish peroxidase (HRP) as label, being the limits of detection 2.5–3.8 and 0.8 ng mL−1 for TCL and CL detection, respectively. In addition, since the TCL emission is simply initiated by thermolysis of the label, chemical reagents were not required, thus allowing reagentless detection with a simplification of the analytical protocols. A compact mini dark box device based on the use of a cooled charge-coupled device (CCD) and a miniaturized heater has been developed and used to quantify the light emission after heat decomposition of the label at a temperature of 90–120 °C. These characteristics make TCL-doped ORMOSIL NPs ideal universal nanoprobes for ultrasensitive bioassays such as immuno- and DNA-based assay. Graphical Abstract Schematic representation of the silica nanoparticles decorated with biotin and containing 1,2-dioxetane derivatives and the fluorescent energy acceptor BPEA (left); thermochemiluminescence images obtained for a model streptavidin immunoassay (right)
Keywords: ORMOSIL nanoparticles; Thermochemiluminescence; 1,2-Dioxetanes; Biotin conjugation; Bioassays; Imaging

Lysergic acid diethylamide (LSD) is a widely used recreational drug. The aim of the present study is to develop a quantitative turboflow LC-MS/MS method that can be used for rapid quantification of LSD and its main metabolite 2-oxo-3-hydroxy LSD (O-H-LSD) in serum and urine in emergency toxicological cases without time-consuming extraction steps. The method was developed on an ion-trap LC-MS/MS instrument coupled to a turbulent-flow extraction system. The validation data showed no significant matrix effects and no ion suppression has been observed in serum and urine. Mean intraday accuracy and precision for LSD were 101 and 6.84 %, in urine samples and 97.40 and 5.89 % in serum, respectively. For O-H-LSD, the respective values were 97.50 and 4.99 % in urine and 107 and 4.70 % in serum. Mean interday accuracy and precision for LSD were 100 and 8.26 % in urine and 101 and 6.56 % in serum, respectively. For O-H-LSD, the respective values were 101 and 8.11 % in urine and 99.8 and 8.35 % in serum, respectively. The lower limit of quantification for LSD was determined to be 0.1 ng/ml. LSD concentrations in serum were expected to be up to 8 ng/ml. 2-Oxo-3-hydroxy LSD concentrations in urine up to 250 ng/ml. The new method was accurate and precise in the range of expected serum and urine concentrations in patients with a suspected LSD intoxication. Until now, the method has been applied in five cases with suspected LSD intoxication where the intake of the drug has been verified four times with LSD concentrations in serum in the range of 1.80–14.70 ng/ml and once with a LSD concentration of 1.25 ng/ml in urine. In serum of two patients, the O-H-LSD concentration was determined to be 0.99 and 0.45 ng/ml. In the urine of a third patient, the O-H-LSD concentration was 9.70 ng/ml.
Keywords: LSD; O-H-LSD; LC-MS; Lysergic acid diethylamide; 2-Oxo-3-hydroxy LSD; Blood; Urine

Dried blood spot analysis of creatinine with LC-MS/MS in addition to immunosuppressants analysis by Remco A. Koster; Ben Greijdanus; Jan-Willem C. Alffenaar; Daan J. Touw (1585-1594).
In order to monitor creatinine levels or to adjust the dosage of renally excreted or nephrotoxic drugs, the analysis of creatinine in dried blood spots (DBS) could be a useful addition to DBS analysis. We developed a LC-MS/MS method for the analysis of creatinine in the same DBS extract that was used for the analysis of tacrolimus, sirolimus, everolimus, and cyclosporine A in transplant patients with the use of Whatman FTA DMPK-C cards. The method was validated using three different strategies: a seven-point calibration curve using the intercept of the calibration to correct for the natural presence of creatinine in reference samples, a one-point calibration curve at an extremely high concentration in order to diminish the contribution of the natural presence of creatinine, and the use of creatinine-[2H3] with an eight-point calibration curve. The validated range for creatinine was 120 to 480 μmol/L (seven-point calibration curve), 116 to 7000 μmol/L (1-point calibration curve), and 1.00 to 400.0 μmol/L for creatinine-[2H3] (eight-point calibration curve). The precision and accuracy results for all three validations showed a maximum CV of 14.0 % and a maximum bias of −5.9 %. Creatinine in DBS was found stable at ambient temperature and 32 °C for 1 week and at −20 °C for 29 weeks. Good correlations were observed between patient DBS samples and routine enzymatic plasma analysis and showed the capability of the DBS method to be used as an alternative for creatinine plasma measurement. Graphical Abstract From blood spot to chromatogram
Keywords: Dried blood spot; Creatinine; Tacrolimus; Sirolimus; Everolimus; Cyclosporin A

Quantitative evaluation of peptide-extraction methods by HPLC–triple-quad MS–MS by Yan Du; Dapeng Wu; Qian Wu; Yafeng Guan (1595-1605).
In this study, the efficiency of five peptide-extraction methods—acetonitrile (ACN) precipitation, ultrafiltration, C18 solid-phase extraction (SPE), dispersed SPE with mesoporous carbon CMK-3, and mesoporous silica MCM-41—was quantitatively investigated. With 28 tryptic peptides as target analytes, these methods were evaluated on the basis of recovery and reproducibility by using high-performance liquid chromatography–triple-quad tandem mass spectrometry in selected-reaction-monitoring mode. Because of the distinct extraction mechanisms of the methods, their preferences for extracting peptides of different properties were revealed to be quite different, usually depending on the pI values or hydrophobicity of peptides. When target peptides were spiked in bovine serum albumin (BSA) solution, the extraction efficiency of all the methods except ACN precipitation changed significantly. The binding of BSA with target peptides and nonspecific adsorption on adsorbents were believed to be the ways through which BSA affected the extraction behavior. When spiked in plasma, the performance of all five methods deteriorated substantially, with the number of peptides having recoveries exceeding 70 % being 15 for ACN precipitation, and none for the other methods. Finally, the methods were evaluated in terms of the number of identified peptides for extraction of endogenous plasma peptides. Only ultrafiltration and CMK-3 dispersed SPE performed differently from the quantitative results with target peptides, and the wider distribution of the properties of endogenous peptides was believed to be the main reason. Graphical Abstract Distribution of the recoveries of target peptides after treatment by the five peptide-extraction methods
Keywords: Method evaluation; Peptide extraction; Plasma; Triple-quad MS–MS

Electron microscopy of Staphylococcus epidermidis fibril and biofilm formation using image-enhancing ionic liquid by Chisato Takahashi; Golap Kalita; Noriko Ogawa; Keiichi Moriguchi; Masaki Tanemura; Yoshiaki Kawashima; Hiromitsu Yamamoto (1607-1613).
We established an optimized biofilm observation method using a hydrophilic ionic liquid (IL), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]). In the present study, a biofilm was formed by Staphylococcus epidermidis. Using field emission (FE) scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the colonization of assemblages formed by microbial cells was observed as a function of the cultivation time. FE-TEM analysis revealed that the fibril comprises three types of protein. In addition, the ultrastructure of each protein monomer was visualized. It was expected that the curly-structured protein plays an important role in extension during fibril formation. Compared to the conventional sample preparation method for electron microscopy, a fine structure was easily obtained by the present method using IL. This observation technique can provide valuable information to characterize the ultrastructure of the fibril and biofilm that has not been revealed till date. Furthermore, these findings of the molecular architecture of the fibril and the colonization behavior of microbial cells during biofilm formation are useful for the development of antibacterial drugs and microbial utilization.
Keywords: Fibril; Gram-positive bacteria; Staphylococcus epidermidis ; Ionic liquid; Electron microscopy

A combination of thin-film microextaction based on an aptamer immobilized on modified Whatman cellulose paper followed by electrospray ionization ion mobility spectrometry has been developed for the analysis of codeine in urine samples. The immobilization is based on the covalent linking of an amino-modified anticodeine aptamer to aldehyde groups of the oxidized cellulose paper. The covalent bonds were examined by infrared spectroscopy and elemental analysis. The effect of the extraction parameters, including the elution conditions (solvent type and volume), extraction time, and extraction temperature, on the extraction efficiency were investigated. Under the optimized conditions, the linear dynamic range was found to be 10-300 ng/mL with a detection limit of 3.4 ng/mL for codeine in urine. The relative standard deviation was 6.8 % for three replicate measurements of codeine at 100 ng/mL in urine. Furthermore, the samples were analyzed with a standard method for the analysis of codeine using high-performance liquid chromatography with ultraviolet detection. The comparison of the results validates the accuracy of the proposed method as an alternative method for the analysis of codeine in urine samples. Graphical Abstract A combination of TFME based on aptamer immobilized on modified Whatman cellulose paper with ESIIMS has been developed. This method was used for the determination of codeine in urine samples
Keywords: Aptamer; Cellulose paper; Codeine; Ion mobility spectrometry

Determination of ω-6 and ω-3 PUFA metabolites in human urine samples using UPLC/MS/MS by Ai Sasaki; Hayato Fukuda; Narumi Shiida; Nobuaki Tanaka; Ayako Furugen; Jiro Ogura; Satoshi Shuto; Nariyasu Mano; Hiroaki Yamaguchi (1625-1639).
The ω-6 and ω-3 polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are the precursors of various bioactive lipid mediators including prostaglandins, thromboxanes, leukotrienes, hydroxyeicosatetraenoic acid, isoprostanes, lipoxins, and resolvins (Rvs). These lipid mediators play important roles in various physiological and pathological processes. The quantitative determination of PUFA metabolites seems necessary for disease research and for developing biomarkers. However, there is a paucity of analytical methods for the quantification of ω-6 and ω-3 PUFA metabolites—the specialized pro-resolving mediators (SPMs) present in the human urine. We developed a method for the quantification of ω-6 and ω-3 PUFA metabolites present in human urine using ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS). The developed method shows good linearity, with a correlation coefficient >0.99 for all of the analytes. The validation results indicate that our method is adequately reliable, accurate, and precise. The method was successfully used to examine urine samples obtained from 43 healthy volunteers. We could identify 20 PUFA metabolites, and this is the first report of the quantitative determination of RvD1, 17(R)-RvD1, 11-dehydro thromboxane B3, RvE2, and 5(S)-HETE in human urine. The urinary 8-iso PGF and PGE2 levels were significantly higher in the men smokers than in the men nonsmokers (p < 0.05). In this study, we developed an accurate, precise, and novel analytical method for estimating the ω-6 and ω-3 PUFA metabolites, and this is the first report that the SPMs derived from EPA and DHA are present in human urine.
Keywords: Eicosanoids; Polyunsaturated fatty acids; Specialized pro-resolving mediators; UPLC/MS/MS; Urine

Determination of breath acetone in 149 Type 2 diabetic patients using a ringdown breath-acetone analyzer by Meixiu Sun; Zhuying Chen; Zhiyong Gong; Xiaomeng Zhao; Chenyu Jiang; Yuan Yuan; Zhennang Wang; Yingxin Li; Chuji Wang (1641-1650).
Over 90 % of diabetic patients have Type 2 diabetes. Although an elevated mean breath acetone concentration has been found to exist in Type 1 diabetes (T1D), information on breath acetone in Type 2 diabetes (T2D) has yet to be obtained. In this study, we first used gas chromatography–mass spectrometry (GC–MS) to validate a ringdown breath-acetone analyzer based on the cavity-ringdown-spectroscopy technique, through comparing breath acetone concentrations in the range 0.5–2.5 ppm measured using both methods. The linear fitting of R = 0.99 suggests that the acetone concentrations obtained using both methods are consistent with a largest standard deviation of ±0.4 ppm in the lowest concentration of the range. Next, 620 breath samples from 149 T2D patients and 42 healthy subjects were collected and tested using the breath analyzer. Four breath samples were taken from each subject under each of four different conditions: fasting, 2 h post-breakfast, 2 h post-lunch, and 2 h post-dinner. Simultaneous blood glucose levels were also measured using a standard diabetic-management blood-glucose meter. For the 149 T2D subjects, their exhaled breath acetone concentrations ranged from 0.1 to 19.8 ppm; four different ranges of breath acetone concentration, 0.1–19.8, 0.1–7.1, 0.1–6.3, and 0.1–9.5 ppm, were obtained for the subjects under the four different conditions, respectively. For the 42 healthy subjects, their breath acetone concentration ranged from 0.1 to 2.6 ppm; four different ranges of breath acetone concentration, 0.3–2.6, 0.1–2.6, 0.1–1.7, and 0.3–1.6 ppm, were obtained for the four different conditions. The mean breath acetone concentration of the 149 T2D subjects was determined to be 1.5 ± 1.5 ppm, which was 1.5 times that of 1.0 ± 0.6 ppm for the 42 healthy subjects. No correlation was found between the breath acetone concentration and the blood glucose level of the T2D subjects and the healthy volunteers. This study using a relatively large number of subjects provides new data regarding breath acetone in diabetes (T1D and T2D) and suggests that an elevated mean breath acetone concentration also exists in T2D.
Keywords: Breath acetone; Type 2 diabetic patients; Cavity-ringdown breath analyzer; GC–MS validation; Elevated mean acetone concentration

Medical devices (MDs) for infusion and enteral and parenteral nutrition are essentially made of plasticized polyvinyl chloride (PVC). The first step in assessing patient exposure to these plasticizers, as well as ensuring that the MDs are free from di(2-ethylhexyl) phthalate (DEHP), consists of identifying and quantifying the plasticizers present and, consequently, determining which ones are likely to migrate into the patient’s body. We compared three different extraction methods using 0.1 g of plasticized PVC: Soxhlet extraction in diethyl ether and ethyl acetate, polymer dissolution, and room temperature extraction in different solvents. It was found that simple room temperature chloroform extraction under optimized conditions (30 min, 50 mL) gave the best separation of plasticizers from the PVC matrix, with extraction yields ranging from 92 to 100 % for all plasticizers. This result was confirmed by supplemented Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) and gravimetric analyses. The technique was used on eight marketed medical devices and showed that they contained different amounts of plasticizers, ranging from 25 to 36 % of the PVC weight. These yields, associated with the individual physicochemical properties of each plasticizer, highlight the need for further migration studies.
Keywords: Biomaterials; GC; Extraction (SFE | SPE | SPME); IR spectroscopy; Polymers

Fructooligosaccharides (FOS) are popular components of functional foods produced by the enzymatic transfer of fructose units to sucrose. Improving β-fructofuranosidase traits by protein engineering is restricted by the absence of a rapid, direct screening method for the fructooligosaccharide products produced by enzyme variants. The use of standard high-performance liquid chromatography (HPLC) methods involves time-consuming sample preparation and chromatographic and data analysis steps. To overcome these limitations, this work presents a rapid method for screening β-fructofuranosidase variant libraries using Fourier transform mid-infrared attenuated total reflectance (FT-MIR ATR) spectroscopy and calibration using partial least squares (PLS) regression. The method offers notable improvements in terms of sample analysis times and cost, with the added benefit of the absence of toxic eluents. Wavenumber interval selection methods were tested to develop optimised PLS regression models that were successfully applied to quantify of glucose, fructose, sucrose, 1-kestose and nystose, the substrates and products of β-fructofuranosidase activity. To the best of our knowledge, this is the first report on the use of infrared spectroscopy and PLS calibration for the quantification of 1-kestose and nystose. Independent test set-validated results indicated that optimal wavenumber selection by interval PLS (iPLS) served to provide the best models for all sugars, bar glucose. Application of this screening method will facilitate the engineering of β-fructofuranosidases and other glycosyltransferase enzymes by random mutagenesis strategies, as it provides, for the first time, a rapid, direct assay for transferase products that may be adapted to a high-throughput set-up. Graphical Abstract ᅟ
Keywords: β-fructofuranosidase; FT-MIR ATR spectroscopy; Interval PLS (iPLS); Glycosyltransferases; Fructooligosaccharides; Protein engineering

Development and in-house validation of an allergen-specific ELISA for quantification of Bet v 4 in diagnostic and therapeutic birch allergen products by Oliver Dehus; Julia Zimmer; Sascha Döring; Frank Führer; Kay-Martin Hanschmann; Thomas Holzhauser; Florian Neske; Daniel Strecker; Jan-Hendrik Trösemeier; Stefan Vieths; Susanne Kaul (1673-1683).
Birch (Betula) pollen is a major cause of allergy in northern and central Europe. The allergenic potency of products for diagnosis and therapy of birch pollen allergy is adjusted nearly exclusively to the major birch pollen allergen Bet v 1. Although every fifth patient is additionally sensitized to Bet v 4, both content and variability of this minor allergen in birch allergen products remain unclear due to a lack of simple and cost-effective quantitative methods. This study aimed to develop and in-house validate the first Bet v 4-specific sandwich enzyme-linked immunosorbent assay (ELISA). Based on a murine monoclonal antibody in combination with a polyclonal rabbit antiserum, the ELISA proved to be highly sensitive, with a lower limit of quantification of 30 pg/ml Bet v 4. After confirmation of satisfactory accuracy, reproducibility, and robustness, the ELISA was utilized to quantify Bet v 4 in 30 authorized birch allergen products. The allergen was detected in all samples tested, ranging from 0.2 to 4.4 μg/ml. No significant correlation of Bet v 4 was found with the respective amount of Bet v 1. In contrast to Bet v 1, also no correlation of Bet v 4 with total protein content or total allergenic activity could be observed. Thus, it seems presently unfeasible to base birch allergen product standardization additionally on Bet v 4. In light of these results, the continuous monitoring of Bet v 4 in birch allergen products with the presented ELISA will provide a basis for the understanding of the clinical relevance of minor allergens.
Keywords: Bet v 1; Bet v 4; ELISA; Minor birch allergen; Allergen immunotherapy; Standardization

Neuropeptides are structurally the most diverse group of messenger molecules of the nervous system. Regarding neuropeptide identification, distribution, function, and evolution, insects are among the best studied invertebrates. Indeed, more than 100 neuropeptides are known from single species. Most of these peptides can easily be identified by direct tissue or cell profiling using MALDI-TOF MS. In these experiments, protein hormones with extensive post-translational modifications such as inter- and intramolecular disulfides are usually missed. It is evident that an exclusion of these bioactive molecules hinders the utilization of direct profiling methods in comprehensive peptidomic analyses. In the current study, we focus on the detection and structural elucidation of homo- and heterodimeric adipokinetic hormone precursor-related peptides (APRPs) of cockroaches. The physiological relevance of these molecules with highly conserved sequences in insects is still uncertain. Sequence similarities with vertebrate growth hormone-releasing factors have been reported, but remarkably, few data regarding APRP processing exist and these data are restricted to locusts. Here, we elucidated sequences of carbamidomethylated APRP monomers of different cockroaches by means of MALDI-TOF MS2, and we were able to identify a surprisingly large number of APRP sequences, resulting either from intraspecific amino acid substitutions within the APRP sequences or C-terminal truncated APRPs.
Keywords: Adipokinetic hormone; APRP; Blattodea; Cockroach; Insect neuropeptides; MALDI-TOF MS

Identification of natural indigo in historical textiles by GC–MS by Laura Degani; Chiara Riedo; Oscar Chiantore (1695-1704).
The possibility of successfully applying a common GC–MS procedure for identification in one step of all types of dyes from plants of unknown origin and from historical objects is particularly attractive due to the high separation efficiency of the capillary columns, the MS detection sensitivity and the reproducibility of results. In this work, GC–MS analysis, previously and successfully used for the characterization of anthraquinones, flavonoids and tannins from plant extracts and historical samples, has been tested on indigoid dyestuffs. An analytical procedure based on the silylating agent N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) with 1 % trimethylchlorosilane (TMCS) was applied to pure molecules of indigotin and indirubin and to plant extracts of Indigofera tinctoria L. and Isatis tinctoria L. Preliminary tests have been done to establish the chromatographic conditions and the derivatization amounts most suitable for the simultaneous detection of indigoid molecules and of the other natural compounds, such as fatty acids, carboxylic acids and sugars, contained within the plant extracts. In order to assess the capacity and the sensitivity of the analytical procedure in typical archaeometric applications, wool samples dyed in the laboratory with indigo were analysed by mimicking the sample amounts typically available with historical objects. The electron ionization (EI) spectra of the main silylated derivatives of indigoid molecules obtained in this way constitute the necessary data set for the characterization of natural extracts and historical works of art. Subsequently, the procedure has been applied to historical samples for the detection of indigo and of other dyestuffs eventually contained in samples. Additional information, useful for restoration and preservation of works of art, could be also obtained on the nature of stains and smudges present on the sampled textile material. The GC–MS method turns out to be an efficient and fast analytical tool also for the identification of natural indigo in plants and textile artefacts, providing results complementary to those from high-performance liquid chromatography (HPLC).
Keywords: Natural indigo; GC–MS; Historical textiles; Archaeometry fine arts

Ultra-high-performance liquid chromatography-tandem mass spectrometry for the analysis of free and conjugated natural estrogens in cow milk without deconjugation by Anna Laura Capriotti; Chiara Cavaliere; Patrizia Foglia; Roberto Samperi; Serena Stampachiacchiere; Salvatore Ventura; Aldo Laganà (1705-1719).
A sensitive liquid chromatography/electrospray ionization-tandem mass spectrometry method for the determination of free and conjugated estrogens in cow milk is described. Milk samples were diluted with water and then extracted and cleaned up by solid-phase extraction using graphitized carbon black as adsorbent material, without any enzymatic cleavage, derivatization, and/or protein precipitation step. Two fractions were collected (free and conjugated estrogens) and analyzed separately. Mass spectrometry analysis was performed in negative ion mode using selected reaction monitoring acquisition mode. For all compounds, the coefficients of determination (R 2) ranged between 0.9892 and 0.9997. Analytical recoveries were in the range of 86–109 % for free estrogens and 85–118 % for conjugates, with relative standard deviations below 10 %, and the method detection limit ranged between 3 and 80 ng L−1. Finally, the developed method was used to determine the presence of free and conjugated estrogens in six retail milk samples (five cow milk samples and one goat milk sample) and one goat milk sample provided by a local shepherd. Estrone was found to be the major free estrogen present in commercial milk samples, and estrone 3-sulfate showed the highest concentration among the target conjugated estrogens. Estriol was also observed in some analyzed milk samples, but the concentrations were always below the limit of quantification. Graphical abstract Free and conjugated estrogens were extracted from milk samples and cleaned-up by solid-phase extraction using graphitized carbon black as adsorbent material, without any enzymatic cleavage, derivatization and/or protein precipitation step. Two distinct fractions were collected and analyzed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry.
Keywords: Estrogens; Milk; Graphitized carbon black; Solid-phase extraction (SPE); Liquid chromatography-mass spectrometry (LC-MS)

A novel dual-function mixed-mode stationary phase based on poly(glycidyl methacrylate-co-ethylene dimethacrylate) microspheres was synthesized by thiol–ene click chemistry and characterized by infrared spectroscopy and elemental analysis. The new system displays both hydrophobic interaction chromatography (HIC) character in a high salt concentration mobile phase, and weak cation exchange (WCX) chromatography character in a low salt concentration mobile phase. It can be used to separate proteins in both ion-exchange chromatography (IEC) mode and HIC mode. The resolution and selectivity of the stationary phase were evaluated in both HIC mode and IEC mode using protein standards. In comparison with the conventional WCX and HIC columns, the results were satisfactory and acceptable. Protein mass and bioactivity recoveries of more than 96 % can be achieved in both HIC mode and IEC mode using this column. The results indicate that the novel dual-function mixed-mode column in many cases can replace the use of two individual WCX and HIC columns. In addition, the effects on protein separation of different ligand structures in the dual-function stationary phase and the pH of the mobile phase used were also investigated in detail. The results show that electrostatic interaction of the ligand with proteins must match the hydrophobicity of the ligand, which is an important factor to prepare the dual-function stationary phase. On the basis of this dual-function mixed-mode chromatography column, a new two-dimensional liquid chromatography technology with a single column system was also developed in this study, and was used to renature and purify recombinant human interferon-γ from inclusion bodies. The mass recovery, purity, and specific bioactivity obtained for the purified recombinant human interferon-γ were 87.2 %, 92.4 %, and 2.8 × 107 IU/mg, respectively, in IEC mode, and 83.4 %, 95.2 %, and 4.3 × 107 IU/mg, respectively, in HIC mode. The results indicate that the dual-function mixed-mode stationary phase prepared in this study may aid in the development of new two-dimensional liquid chromatography technology with a single column for intensive proteomic studies, and it may also find applications in recombinant protein drug production since it can save column costs and simplify the biotechnological processes.
Keywords: Mixed-mode chromatography; Click chemistry; Two-dimensional liquid chromatography; Ion-exchange chromatography; Hydrophobic interaction chromatography; Polymer microsphere

Analyses of four endocrine disrupting chemicals (EDCs) in water and milk samples were undertaken by using magnetic molecularly imprinted polymers (MMIPs). These were prepared via the surface molecular imprinting technique using super paramagnetic core-shell nanoparticle as support. Diethylstilbestrol (DES), which is a typical EDC, was employed as the template molecule. The obtained MMIPs were characterized using transmission electron microscope, Fourier transform infrared, X-ray diffraction, and vibrating sample magnetometer. Accordingly, the adsorption capacity and selectivity of prepared MMIPs were investigated. The binding isotherms were obtained for DES and fitted by the Freundlich isotherm model. A corresponding analytical method to determine four EDCs was developed. The recoveries of the spiked samples in pond water and pure milk range from 67.8 to 93.2 % and from 65.3 to 92.5 %, respectively. Coupled with high-performance liquid chromatography analysis, the prepared MMIPs were successfully applied to the analysis of EDCs in water and milk samples. Graphical Abstract The construction and feature of magnetic molecularly imprinted polymers
Keywords: Molecularly imprinted polymers; Super paramagnetic; Endocrine disrupting chemicals; Water and milk

A simple, fast, and universal suspension polymerization method was used to synthesize the molecularly imprinted microspheres (MIMs) for the topical anesthetic benzocaine (BZC). The desired diameter (10–20 μm) and uniform morphology of the MIMs were obtained easily by changing one or more of the synthesis conditions, including type and amount of surfactant, stirring rate, and ratio of organic to water phase. The MIMs obtained were used as a molecular-imprinting solid-phase-extraction (MISPE) material for extraction of BZC in human serum and fish tissues. The MISPE results revealed that the BZC in these biosamples could be enriched effectively after the MISPE operation. The recoveries of BZC on MIMs cartridges were higher than 90 % (n = 3). Finally, an MISPE-HPLC method with UV detection was developed for highly selective extraction and fast detection of trace BZC in human serum and fish tissues. The developed method could also be used for the enrichment and detection of BZC in other complex biosamples.
Keywords: Molecularly imprinted microspheres; Benzocaine; Aqueous-suspension polymerization; Solid-phase extraction; Human serum; Fish tissues

Determination of triazine herbicides in fresh vegetables by dynamic microwave-assisted extraction coupled with homogeneous ionic liquid microextraction high performance liquid chromatography by Lijie Wu; Mingzhu Hu; Zhanchao Li; Ying Song; Cui Yu; Yupu Zhang; Hanqi Zhang; Aimin Yu; Qiang Ma; Ziming Wang (1753-1762).
A novel extraction method, dynamic microwave-assisted extraction coupled with homogeneous ionic liquid microextraction, was developed for the determination of triazine herbicides, including desmetryn, terbumeton, propazine, terbuthylazine, dimethametryn, and dipropetryn in fresh vegetable samples by high performance liquid chromatography (HPLC). In the developed method, 120 μL of 1-butyl-3-methylimidazolium tetrafluoroborate ([C4MIM][BF4]) was added to 10 mL of aqueous solution containing 0.3 g of NaCl to obtained the extraction solvent. Six triazines could be extracted completely within 4 min by the present method. Then, [NH4][PF6] was added into the extract to form a water-insoluble ionic liquid [C4MIM][PF6] via a simple metathesis reaction, and the analytes were enriched into the ionic liquid phase. After centrifugation and dilution with acetonitrile, the resulting solution was analyzed directly by HPLC. The effects of some experimental parameters, including type and volume of ionic liquid, volume of extraction solvent, amount of ion-pairing agent [NH4][PF6], salt concentration, microwave power, and flow rate of extraction solvent on the extraction efficiency were investigated and optimized. Under the optimum experimental conditions, the linearity for determining the analytes was in the range of 2.50–250.00 μg kg−1, with the correlation coefficients ranging from 0.9989 to 0.9999. When the present method was applied to the analysis of vegetable samples, satisfactory recoveries were obtained in the range of 76.8 %–106.9 %, and relative standard deviations were lower than 9.8 %. Graphical Abstract Extraction procedure of triazines from vegetable sample
Keywords: Dynamic microwave-assisted extraction; Homogeneous ionic liquid microextraction; In-situ metathesis reaction; High-performance liquid chromatography; Triazine herbicides

A novel effervescent-salt-assisted dispersive micro-solid-phase extraction using mesoporous hybrid materials was developed for the extraction of minute traces of constituents in complicated plant preparations. In this study, a special tablet containing carbon dioxide sources (sodium dihydrogenphosphate and sodium carbonate) and the sorbent (mesoporous hybrid materials) was prepared. The effects of different parameters influencing the extraction efficiency such as the concentration of salts, the type and concentration of mesoporous material, pH of diluent, and desorption solvents were investigated and optimized. Results show that the proposed method using green solvents (water) as extraction solutions required low consumption of sample amount and obtained high enrichment efficiency. Moreover, under optimized conditions, the tested tanshinones exhibited good linearity (r 2 > 0.994) in the concentration range of 0.5 to 80 ng mL−1. The limits of detection values were lower than 0.0803 pg using UV–visible detection. The developed method was successfully applied for the analysis of trace tanshinones in compound Danshen dripping pill and Danqi tablet samples. Graphical Abstract Schematic of effervescent salts assisted dispersive micro-solid-phase extraction using mesoporoushybrid materials
Keywords: Danshen preparations; Dispersive micro-solid-phase extraction; Effervescent salts; Mesoporous hybrid materials; Ultra-performance liquid chromatography

The case of triethylammonium cation loss during purification of certain nucleotide analogues: a cautionary note by Krystian Kolodziej; Joanna Romanowska; Jacek Stawinski; Adam Kraszewski; Michal Sobkowski (1775-1780).
Nucleotides, their analogues, and other phosphate esters and phosphoramidates often contain the triethylammonium cation as a counterion. We found that this may be lost during chromatographic purification or concentration of solutions, yielding products in acidic forms or containing sub-stoichiometric amounts of the counterion. This in turn may be detrimental, e.g., due to possible decomposition of a compound or inaccurate sample preparation. Correlations between the structure of studied compounds and their susceptibility for cation loss were analyzed. Modifications in preparative techniques were developed to obtain the studied compounds with stoichiometric anion to cation ratios.ᅟ Graphical Abstract Triethylammonium salts of phosphate esters and phosphoramidates may lose the cationic component during chromatography or evaporation of solvent
Keywords: Cation loss; Anion to cation ratio; Triethylammonium cation; Phosphates; Nucleotide analogues; Purification techniques; Solvent evaporation

The endocannabinoid system has been considered as a target for pharmacological intervention. Accordingly, inhibition of fatty acid amide hydrolase (FAAH), a degrading enzyme of the endocannabinoids N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG) as well as of the endocannabinoid-like substances N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA), can cause augmented endogenous cannabinoid tone. Using liquid chromatography coupled with positive electrospray ionisation mass spectrometry, we herein describe a method to simultaneously quantify levels of AEA, OEA, PEA and 2-AG in cultured cells. The procedure was developed according to the FDA guidelines for bioanalytical methods validation. The limits of quantification (LOQs) were 0.05 pmol for AEA, 0.09 pmol for OEA, 0.10 pmol for PEA and 0.80 pmol for 2-AG when molecular ion monitoring was used. In H460 human lung carcinoma cells, basal levels of all four analytes ranged between 2 and 17 pmol mg−1 protein with PEA showing the lowest and OEA the highest concentrations. Endocannabinoid levels observed in mesenchymal stem cells were of the same order of magnitude when compared to those in H460 human lung carcinoma cells.
Keywords: LC–MS/MS; Endocannabinoids; Endocannabinoid-like substances; H460 Lung carcinoma cells; Mesenchymal stem cells