Analytical and Bioanalytical Chemistry (v.407, #25)

Improving surface and defect center chemistry of fluorescent nanodiamonds for imaging purposes—a review by Andreas Nagl; Simon Robert Hemelaar; Romana Schirhagl (7521-7536).
Diamonds are widely used for jewelry owing to their superior optical properties accounting for their fascinating beauty. Beyond the sparkle, diamond is highly investigated in materials science for its remarkable properties. Recently, fluorescent defects in diamond, particularly the negatively charged nitrogen-vacancy (NV-) center, have gained much attention: The NV- center emits stable, nonbleaching fluorescence, and thus could be utilized in biolabeling, as a light source, or as a Förster resonance energy transfer donor. Even more remarkable are its spin properties: with the fluorescence intensity of the NV- center reacting to the presence of small magnetic fields, it can be utilized as a sensor for magnetic fields as small as the field of a single electron spin. However, a reproducible defect and surface and defect chemistry are crucial to all applications. In this article we review methods for using nanodiamonds for different imaging purposes. The article covers (1) dispersion of particles, (2) surface cleaning, (3) particle size selection and reduction, (4) defect properties, and (5) functionalization and attachment to nanostructures, e.g., scanning probe microscopy tips. Graphical Abstract We review how diamond surface and defect chemistry can be optimized for different (bio) applications
Keywords: Diamonds; Surface chemistry; Sensors; Magnetometry; Biolabels

Laser-induced breakdown spectroscopy (LIBS) has become an established analytical atomic spectrometry technique and is valued for its very compelling set of advantageous analytical and technical characteristics. It is a rapid, versatile, non-contact technique, which is capable of providing qualitative and quantitative analytical information for practically any sample, in a virtually non-destructive way, without any substantial sample preparation. The instrumentation is simple, robust, compact, and even enables remote analysis. This review attempts to give a critical overview of the diverse progress of the field, focusing on the results of the last five years. The advancement of LIBS instrumentation and data evaluation is discussed in detail and selected results of some prominent applications are also described.
Keywords: Laser-induced breakdown spectroscopy; Laser-induced plasma spectroscopy; Laser-induced plasma; Instrumentation; Data evaluation; Applications

Stable isotope dilution assays (SIDAs) are becoming ever commoner in mycotoxin analysis, and the number of synthesized or commercially available isotopically labelled compounds has greatly increased in the 7 years since our last review dealing with this topic. Thus, this review is conceived as an update for new applications or improvements of SIDAs for compounds discussed earlier, but the main focus is on newly introduced labelled substances and the development of SIDAs for, for example, fusarin C, moniliformin or the enniatins. Mycotoxin research has concentrated on the emerging group of Alternaria toxins in recent years, and a series of SIDAs have been developed, including ones for tenuazonic acid, alternariol, altertoxins and tentoxin that are discussed in detail in this review. Information about synthetic routes, isotopic purity and mass-spectrometric characterization of labelled compounds is given, as well as about the development and validation of SIDAs and their application to foods, feeds or biological samples. As the number of commercially available labelled standards is increasing continuously, a general tendency for the use of analytical methods based on liquid chromatography coupled with mass spectrometry capable of identifying a series of mycotoxins simultaneously (“multimethods”) and using one or more labelled internal standards can be observed. An overview of these applications is given, thus demonstrating that SIDAs are increasingly being used in routine analysis.
Keywords: Alternaria toxins; Fusarium toxins; Liquid chromatography–tandem mass spectrometry (LC-MS/MS); Mycotoxins; Stable isotope dilution assay

is a research scientist in the Chemical Metrology Division, Health Sciences Authority, Singapore. Her main fields of research involve the development of reference measurement methods for biomarkers in human serum, peptide and protein analysis, production of certified reference materials, and purity assessment of small molecules and peptides. is a research scientist in the Chemical Metrology Division, Health Sciences Authority, Singapore. He studied medicinal chemistry at the Department of Chemistry, National University of Singapore and received his PhD degree in 2011. His main fields of research focus on the development of LC–MS–MS-based methods for biomarkers in biological fluids, and the development of quantitative NMR methods for purity assessment of chemical compounds. is a research scientist in the Chemical Metrology Division, Health Sciences Authority, Singapore. She studied chemistry at the Department of Chemistry, National University of Singapore and received her MSc degree in 2012. Her main fields of research involve measurement of biomarkers in biological fluids by isotope-dilution mass spectrometry, and the purity assessment of custom synthesized peptides. is a senior research scientist and a team leader in the Chemical Metrology Division, Health Sciences Authority (HSA), Singapore. His main research interests are development of reference measurement methods for biomarkers in biological fluids, purity assessment of peptides and proteins, and development of certified reference materials. He is also the coordinator of the accuracy-based HSA External Quality Assessment Programme for clinical laboratories. is the Division Director of the Chemical Metrology Division, Health Sciences Authority (HSA), Singapore. The division was established in 2008 to develop the relevant measurement capabilities and spearhead the chemical metrology programme in HSA. The development of reference measurement methods for hemoglobin A1c (HbA1c) is important for quality assurance in diabetes management. The IFCC reference method using purified proteins as calibration standards is the recommended accuracy-based reference method for the standardization of HbA1c measurement. We developed a highly precise and accurate liquid chromatography–isotope-dilution tandem mass spectrometry (LC–IDMS/MS) procedure, which can serve as an alternative accuracy-based method for HbA1c measurement. In this method, enzymatic proteolysis was applied to sample preparation, followed by LC–IDMS/MS measurement of hemoglobin A0 (HbA0) and HbA1c, using two “signature” hexapeptides for calibration. The concentrations of the signature hexapeptide calibration solutions were, in turn, determined using a hydrolysis method with HCl, followed by LC–IDMS/MS measurement using amino acid solutions as calibration standards. These solutions were gravimetrically prepared from pure amino acid certified reference materials (CRMs). The developed LC–IDMS/MS method was used in participation in an IFCC ring trial for reference laboratories (RELA 2013 and 2014) for HbA1c, where our results were compared with those using the IFCC reference method. The deviations were found to be 0.4–1.7 mmol mol−1 [or 0.04–0.16 % in National Glygohemoglobin Standardization Program (NGSP) units], revealing good comparability with the IFCC reference method. The relative expanded uncertainty of the LC–IDMS/MS was in the range of 2.6 % to 2.8 % (1.6 % to 2.2 % after converting to NGSP units). With excellent method precision, good comparability with the IFCC reference method, and a small measurement uncertainty, the developed LC–IDMS/MS method may be used as an alternative accuracy-based reference method for HbA1c measurement. Graphical Abstract The procedure of LC-IDMS/MS method for the measurement of HbA1c
Keywords: Hemoglobin A1c ; diabetes; isotope-dilution mass spectrometry (IDMS); peptide hydrolysis; enzymatic proteolysis; reference measurement method

Direct profiling of the phospholipid composition of adult Caenorhabditis elegans using whole-body imaging mass spectrometry by Saira Hameed; Koji Ikegami; Eiji Sugiyama; Shoko Matsushita; Yoshishige Kimura; Takahiro Hayasaka; Yuki Sugiura; Noritaka Masaki; Michihiko Waki; Isao Ohta; Md Amir Hossen; Mitsutoshi Setou (7589-7602).
A protocol for the direct analysis of the phospholipid composition in the whole body of adult soil nematode, Caenorhabditis elegans (C. elegans), was developed, which combined freeze-cracking of the exoskeletal cuticle and matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). Biomolecules in the m/z range from 700 to 900 were more effectively detected in the freeze-cracked than from simple frozen adult nematode bodies. Different distribution of biomolecules was observed in a nematode body when the matrix was applied with a sublimation deposition method. The whole-body IMS technique was applied on genetically deficient mutant C. elegans to combine whole-body lipidomics and genetics, by comparing the fatty acid compositions, especially of the phosphatidylcholine (PC) species, between the wild-type and fat-1 mutants, which lack the gene encoding an n-3 fatty acid desaturase. A significant reduction of PC(20:5/20:5) and PC(20:4/20:5) and a marked increase of PC(20:4/20:4), PC(20:3/20:4), and PC(20:3/20:3) were detected in the fat-1 mutants in positive ion mode. In addition, phospholipid compositions other than PCs were analyzed in negative ion mode. A loss of a possible phosphatidylinositol (PI) with 18:0/20:5 and a compensative accumulation of putative PI(18:0/20:4) were detected in the fat-1 mutants. In conclusion, the whole-body MALDI-IMS technique is useful for the profiling of multiple biomolecules in C. elegans in both intra- and inter-individual levels.
Keywords: Caenorhabditis elegans ; Cuticle; Exoskeleton; Freeze-cracking; Matrix-assisted laser desorption/ionization-imaging mass spectrometry; Phosphatidylcholine; Phosphatidylinositol

Correcting mass shifts: A lock mass-free recalibration procedure for mass spectrometry imaging data by Purva Kulkarni; Filip Kaftan; Philipp Kynast; Aleš Svatoš; Sebastian Böcker (7603-7613).
Mass spectrometry imaging (MSI) has become widely popular because of its potential to map the spatial distribution of thousands of compounds in a single measurement directly from tissue surfaces. With every MSI experiment, it is important to maintain high mass accuracy for correct identification of the observed ions. Many times this can be compromised due to different experimental factors, leading to erroneous assignment of peaks. This makes recalibration a crucial preprocessing step. We describe a lock mass-free mass spectra recalibration method, which enables to significantly reduce these mass shift effects. The recalibration method is applied in three steps: First, we decide on an order to process all the spectra. Herein, we describe three different methods for ordering the spectra—minimum spanning tree (MST), topological greedy (TG), and crystal growth (CG). Second, we construct a reference (consensus) spectrum, from the ordered spectra, and third, all spectra are individually corrected against this consensus spectrum. The performance of the recalibration method is demonstrated on three imaging datasets acquired from matrix-assisted laser desorptionionization (MALDI) and laser desorption/ionization (LDI) mass spectrometry imaging of whole-body Drosophila melanogaster fly. The applied recalibration method is shown to strongly reduce the observed mass shifts in the imaging datasets. Among the three ordering methods, CG and MST perform comparatively better than TG and highly decrease the overall standard deviation of the mass error distribution. Lock mass correction of MSI data is practically difficult, as not all spectra contain the selected lock mass peak. Our method eliminates this need.
Keywords: Mass spectrometry imaging; Recalibration; Mass shift correction; Data processing

Rapid determination of phenylethanolamine A in biological samples by enzyme-linked immunosorbent assay and lateral-flow immunoassay by Xiangmei Li; Wenjun Wang; Limiao Wang; Qi Wang; Xingyao Pei; Haiyang Jiang (7615-7624).
Phenylethanolamine A (PA) is a β-adrenergic agonist, which was first used in animal husbandry as a growth promoter in China in 2010. In this study, a monoclonal-antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) and lateral-flow immunoassay (LFA) for the detection of PA in swine urine and pork were developed. The immunogen was prepared by linking PA hapten with carrier protein via a diazotization method. The IC50 value of the optimized icELISA was 0.44 ng mL−1. The limits of detection of the icELISA for PA in swine urine and pork were 0.13 ng mL−1 and 0.39 ng g−1, respectively. The recoveries of PA from spiked swine urine and pork were in the range 82.0–107.4 % and 81.8–113.3 %, respectively, with the coefficients of variation in the range 4.1–16.2 % and 1.2–6.3 %, respectively. The mAbs had negligible cross reactivity with 10 other β-agonists. In contrast, the LFA had a cut-off level of 5 ng mL−1 (g) in swine urine and pork, and the results could be achieved within 5 min. Ten blind samples of swine urine were analyzed simultaneously by icELISA, LFA, and ultra-high-performance liquid chromatography–tandem mass spectrometry, and the results of the three methods agreed well. Therefore, the combination of two immunoassays provides an effective and rapid screening method for detection of PA residues in biological samples. Graphical Abstract Determination of phenylethanolamine A in biological samples by immunological methods
Keywords: β-Adrenergic agonist; Phenylethanolamine A; Indirect competitive ELISA; Lateral-flow immunoassay; Biological sample

Characterization of Dickeya and Pectobacterium species by capillary electrophoretic techniques and MALDI-TOF MS by Jiří Šalplachta; Anna Kubesová; Jaroslav Horký; Hana Matoušková; Marie Tesařová; Marie Horká (7625-7635).
Dickeya and Pectobacterium species represent an important group of broad-host-range phytopathogens responsible for blackleg and soft rot diseases on numerous plants including many economically important plants. Although these species are commonly detected using cultural, serological, and molecular methods, these methods are sometimes insufficient to classify the bacteria correctly. On that account, this study was undertaken to investigate the feasibility of three individual analytical techniques, capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), for reliable classification of Dickeya and Pectobacterium species. Forty-three strains, representing different Dickeya and Pectobacterium species, namely Dickeya dianthicola, Dickeya dadantii, Dickeya dieffenbachiae, Dickeya chrysanthemi, Dickeya zeae, Dickeya paradisiaca, Dickeya solani, Pectobacterium carotovorum, and Pectobacterium atrosepticum, were selected for this purpose. Furthermore, the selected bacteria included one strain which could not be classified using traditional microbiological methods. Characterization of the bacteria was based on different pI values (CIEF), migration velocities (CZE), or specific mass fingerprints (MALDI-TOF MS) of intact cells. All the examined strains, including the undetermined bacterium, were characterized and classified correctly into respective species. MALDI-TOF MS provided the most reliable results in this respect.
Keywords: Dickeya ; Pectobacterium ; CZE; CIEF; MALDI

Fast analysis of glibenclamide and its impurities: quality by design framework in capillary electrophoresis method development by Sandra Furlanetto; Serena Orlandini; Benedetta Pasquini; Claudia Caprini; Paola Mura; Sergio Pinzauti (7637-7646).
A fast capillary zone electrophoresis method for the simultaneous analysis of glibenclamide and its impurities (IA and IB) in pharmaceutical dosage forms was fully developed within a quality by design framework. Critical quality attributes were represented by IA peak efficiency, critical resolution between glibenclamide and IB, and analysis time. Experimental design was efficiently used for rapid and systematic method optimization. A 35//16 symmetric screening matrix was chosen for investigation of the five selected critical process parameters throughout the knowledge space, and the results obtained were the basis for the planning of the subsequent response surface study. A Box–Behnken design for three factors allowed the contour plots to be drawn and the design space to be identified by introduction of the concept of probability. The design space corresponded to the multidimensional region where all the critical quality attributes reached the desired values with a degree of probability π ≥ 90%. Under the selected working conditions, the full separation of the analytes was obtained in less than 2 min. A full factorial design simultaneously allowed the design space to be validated and method robustness to be tested. A control strategy was finally implemented by means of a system suitability test. The method was fully validated and was applied to real samples of glibenclamide tablets. Graphical Abstract Identification of sequence variants in IgG Fc mixtures
Keywords: Capillary electrophoresis; Design of experiments; Design space; Glibenclamide; Impurities; Quality by design

Synchrotron radiation nanoscale computed tomography (SR nano-CT) is a powerful analysis tool and can be used to perform chemical identification, mapping, or speciation of carbon and other elements together with X-ray fluorescence and X-ray absorption near edge structure (XANES) imaging. In practical applications, there are often challenges for SR nano-CT due to the misaligned geometry caused by the sample stage axial vibration. It occurs quite frequently because of experimental constraints from the mechanical error of manufacturing and assembly and the thermal expansion during the time-consuming scanning. The axial vibration will lead to the structure overlap among neighboring layers and degrade imaging results by imposing artifacts into the nano-CT images. It becomes worse for samples with complicated axial structure. In this work, we analyze the influence of axial vibration on nano-CT image by partial derivative. Then, an axial vibration calibration method for SR nano-CT is developed and investigated. It is based on the cross correlation of plane integral curves of the sample at different view angles. This work comprises a numerical study of the method and its experimental verification using a dataset measured with the full-field transmission X-ray microscope nano-CT setup at the beamline 4W1A of the Beijing Synchrotron Radiation Facility. The results demonstrate that the presented method can handle the stage axial vibration. It can work for random axial vibration and needs neither calibration phantom nor additional calibration scanning. It will be helpful for the development and application of synchrotron radiation nano-CT systems.
Keywords: Synchrotron radiation nanoscale computed tomography; Axial vibration; Calibration; Cross correlation; Plane integral curve

Seeds of milk thistle, Silybum marianum (L.) Gaertn., are used for treatment and prevention of liver disorders and were identified as a high priority ingredient requiring a validated analytical method. An AOAC International expert panel reviewed existing methods and made recommendations concerning method optimization prior to validation. A series of extraction and separation studies were undertaken on the selected method for determining flavonolignans from milk thistle seeds and finished products to address the review panel recommendations. Once optimized, a single-laboratory validation study was conducted. The method was assessed for repeatability, accuracy, selectivity, LOD, LOQ, analyte stability, and linearity. Flavonolignan content ranged from 1.40 to 52.86 % in raw materials and dry finished products and ranged from 36.16 to 1570.7 μg/mL in liquid tinctures. Repeatability for the individual flavonolignans in raw materials and finished products ranged from 1.03 to 9.88 % RSDr, with HorRat values between 0.21 and 1.55. Calibration curves for all flavonolignan concentrations had correlation coefficients of >99.8 %. The LODs for the flavonolignans ranged from 0.20 to 0.48 μg/mL at 288 nm. Based on the results of this single-laboratory validation, this method is suitable for the quantitation of the six major flavonolignans in milk thistle raw materials and finished products, as well as multicomponent products containing dandelion, schizandra berry, and artichoke extracts. It is recommended that this method be adopted as First Action Official Method status by AOAC International.
Keywords: Silybum marianum (L.) Gaertn; Flavonolignans; Silymarins; Dietary supplements; High-performance liquid chromatography; Validation

Glucaminium ionic liquid-functionalized stationary phase for the separation of nucleosides in hydrophilic interaction chromatography by Qiong Jiang; Mingliang Zhang; Xusheng Wang; Yong Guo; Hongdeng Qiu; Shusheng Zhang (7667-7672).
A glucaminium-based ionic liquid stationary phase was prepared via facile epoxy-amine reaction and subsequent quaternization. Successful immobilization of glucaminium-based ionic liquid onto silica surface was validated by elemental analysis and infrared spectroscopy. The new stationary phase was evaluated for the separation of nucleosides in hydrophilic interaction liquid chromatography (HILIC). Effects of various factors, such as acetonitrile concentration, salt concentration, pH value, as well as column temperature, on the chromatographic behavior toward nucleosides were studied in detail. The results indicated that this new stationary phase can be used for separation of water-soluble polar substances in HILIC mode. The retention of solutes on the stationary phase was influenced by a mixed-mode retention mechanism with a combination of adsorptive and partitioning interactions.
Keywords: Ionic liquid; Glucaminium∙stationary phase; Hydrophilic interaction; High-performance liquid chromatography

Dielectrophoretic characterization of antibiotic-treated Mycobacterium tuberculosis complex cells by Shinnosuke Inoue; Hyun-Boo Lee; Annie L. Becker; Kris M. Weigel; Jong-Hoon Kim; Kyong-Hoon Lee; Gerard A. Cangelosi; Jae-Hyun Chung (7673-7680).
Multi-drug resistant tuberculosis (MDR-TB) has become a serious concern for proper treatment of patients. As a phenotypic method, dielectrophoresis can be useful but is yet to be attempted to evaluate Mycobacterium tuberculosis complex cells. This paper investigates the dielectrophoretic behavior of Mycobacterium bovis (Bacillus Calmette-Guérin, BCG) cells that are treated with heat or antibiotics rifampin (RIF) or isoniazid (INH). The experimental parameters are designed on the basis of our sensitivity analysis. The medium conductivity (σ m) and the frequency (f) for a crossover frequency (f xo1) test are decided to detect the change of σ m-f xo1 in conjunction with the drug mechanism. Statistical modeling is conducted to estimate the distributions of viable and nonviable cells from the discrete measurement of f xo1. Finally, the parameters of the electrophysiology of BCG cells, C envelope and σ cyto, are extracted through a sampling algorithm. This is the first evaluation of the dielectrophoresis (DEP) approach as a means to assess the effects of antimicrobial drugs on M. tuberculosis complex cells.
Keywords: Dielectrophoresis; Mycobacterium tuberculosis ; Drug resistance; Sampling algorithm; Sensitivity analysis

A novel surface imprinting polymer based on magnetic carbon nanotubes was prepared using dendritic polyethyleneimine as functional monomer to amplify the number of imprinted cavities. The characteristics of resulting polymers were evaluated by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), and vibrating sample magnetometer (VSM). Results suggest that magnetic nanoparticles are deposited onto the surface of multiwalled carbon nanotubes and the imprinted shell is coated on the surface of magnetic carbon nanotubes with a thickness of approximately 8 nm. Magnetic imprinted polymers are sensitive to magnetic fields and can be easily separated within 3 s using an external magnet. The adsorption results indicate that the obtained imprinted polymers have fast kinetics, an ultrahigh adsorption capacity of 479.9 mg g−1, and satisfactory selectivity towards the template molecule. The prepared materials have excellent stability with no obvious deterioration after six adsorption–regeneration cycles. In addition, a method for determination of gallic acid (GA) in pomegranate rind was developed, using a combination of the prepared polymers used as solid-phase extraction (SPE) sorbents and high-performance liquid chromatography (HPLC) for rapid isolation and determination of GA. The limit of detection of the proposed method is 0.001 μg mL−1, and the intra and inter-day relative standard deviations (RSDs) are lower than 3.8 % and 5.3 %, respectively. The recoveries of GA from pomegranate rind extract are in the range 98.2–103.6 % with RSDs lower than 4.3 %. Graphical Abstract Selective and rapid extraction of gallic acid in pomegranate rind
Keywords: Magnetic carbon nanotubes; Dendritic polyethyleneimine; Surface imprinting; Gallic acid

Fast determination of underivatized gentamicin C components and impurities by LC-MS using a porous graphitic carbon stationary phase by Manuela Rodriquez; Daniel S. Cretoso; Maria Anna Euterpio; Paola Russo; Carlo Crescenzi; Rita P. Aquino (7691-7701).
Gentamicin C antibiotics are important because they are active against many multidrug-resistant Gram-negative bacilli. Unfortunately, their clinical usefulness is limited by their toxicity. Because of the difficulty involved in separating its different components, the US and European pharmacopeias both specify that the composition of gentamicin C should be determined by liquid chromatography with pulsed electrochemical detection. Here, we assess the usefulness of a porous graphitic carbon (PGC) HPLC column for separating the components of gentamicin C, and report chromatographic conditions that enable its direct characterization by PGC chromatography directly coupled to electrospray mass spectrometry. Native major components of gentamicin and impurities in commercial formulations were retained and separated on the PGC column without any need for derivatization, using mobile phases basified with ammonium hydroxide. When coupled with detection by conventional electrospray ion trap mass spectrometry (ESI-IT-MS), several previously reported impurities were detected easily, including the most polar gentamicin impurity, garamine. When operating in full-scan mode, it was possible to identify and quantitate gentamicin-related compounds using injected samples of only a few picograms. Under the described conditions, all analytes were eluted in less than 10 min and the LC-MS analyses exhibited excellent stability and linearity. The method’s effectiveness was evaluated by analyzing commercial gentamicin batches and in-house formulations. When the PGC chromatographic system was coupled to an evaporative light-scattering detector, detection limits of 40–70 ng were achieved for various major gentamicin components. The chromatographic method was applied on a semi-preparative scale to purify the five major components.
Keywords: Gentamicin; HPLC-MS; Porous graphitic carbon (PGC); Evaporative light scattering detector (ELSD); ESI-MS; Gentamicin impurities

We developed and validated a simple and fast UFLC-MS/MS method for the accurate determination of 3-nitrotyrosine (3-NT) in human urine as a noninvasive biomarker for oxidative stress. The method, involving tailored 96-well μElution solid-phase extraction (SPE) combined with UFLC-MS/MS, allows 3-NT to be determined in biological samples without the need for hydrolysis, derivatization, evaporation, and two-dimensional LC for the first time. Using ammonium acetate (pH 9, 25 mM) as an elution buffer was found to improve SPE selectivity. Fast chromatographic elution of 3-NT with a total run time of 7 min was achieved on a PFPP column (150 mm × 2.1 mm, 3 μm). This fine-tuned integrated method delivered significantly improved throughput, specificity, and sensitivity while reducing the matrix effect, solvent usage, and waste disposal. Using this simple and rapid method, two plates of urine samples (n = 192) can be processed within 24 h. The lower limit of quantification for 3-NT is 10 pg/mL, which represents a notable sensitivity enhancement over reported methods. Less than 6.0 % variations for intraday and interday assay precisions and 97.7–106.3 % for accuracies in terms of recovery were obtained. The applicability and reliability of the method were demonstrated by determining the reference range in human urine for 82 healthy people. Considering the noninvasive and inexpensive nature of urine sampling, this novel method could be used to re-evaluate the role of 3-NT as an oxidative stress biomarker in pre-clinical and clinical studies.
Keywords: 3-Nitrotyrosine; LC-MS/MS; 96-well μElution; Solid-phase extraction; Oxidative stress biomarker; Urine

Human excretory products of selenium are natural constituents of marine fish muscle by Nina Kroepfl; Kenneth B. Jensen; Kevin A. Francesconi; Doris Kuehnelt (7713-7719).
A selenosugar (selenosugar 1, methyl-2-acetamido-2-deoxy-1-seleno-β-D-galactopyranoside) was identified in aqueous extracts of muscle tissue of three marine fish species, mackerel (Scomber scombrus), sardine (Sardina pilchardus), and tuna (Thunnus albacares), by high-performance liquid chromatography coupled to elemental and high-resolution molecular mass spectrometry. Selenoneine (2-selenyl-Nα, Nα, Nα-trimethyl-L-histidine), a known selenium compound in fish, was the major form of selenium in the aqueous extracts, and the methylated derivative of selenoneine, namely Se-methylselenoneine, was also identified as a minor natural constituent in the fish. Selenosugar 1, a major urinary excretion product of selenium often found in organs and body fluids related to selenium excretion, has so far not been reported in muscle tissue. Se-methylselenoneine has been proposed as the main urinary metabolite from selenoneine. This first report of selenosugar 1 and Se-methylselenoneine as natural constituents of fish muscle tissue opens up a new perspective on the role of these compounds in selenium metabolism and is relevant to selenium supplementation studies.
Keywords: Selenosugar; Se-methylselenoneine; HPLC-mass spectrometry; Fish muscle

Intact cell mass spectrometry as a rapid and specific tool for the differentiation of toxic effects in cell-based ecotoxicological test systems by Sascha Liane Kober; Henriette Meyer-Alert; Desirée Grienitz; Henner Hollert; Marcus Frohme (7721-7731).
In the last few decades, MALDI-TOF MS has become a useful technique not only in proteomics, but also as a fast and specific tool for whole cell analysis through intact cell mass spectrometry (IC-MS). The present study evaluated IC-MS as a novel tool for the detection of distinct patterns that can be observed after exposure to a certain toxin or concentration by utilizing the eukaryotic fish cell line RTL-W1. Two different viability assays were performed to define the range for IC-MS investigations, each of which employing copper sulfate, acridine, and β-naphthoflavone (BNF) as model compounds for several classes of environmental toxins. The IC-MS of RTL-W1 cells revealed not only specific spectral patterns for the various toxins, but also that the concentration used had an effect on RTL-W1 profiles. After the exposure with copper sulfate and acridine, the spectra of RTL-W1 showed a significant increase of certain peaks in the higher mass range (m/z >7000), which is probably attributed to the apoptosis of RTL-W1. On the contrary, exposure to BNF showed a distinct change of ion abundances only in the lower mass range (m/z <7000). Furthermore, a set of mass peaks could be identified as a specific biomarker for a single toxin treatment, so IC-MS demonstrates a new method for the distinction of toxic effects in fish cells. Due to fast sample preparation and high throughput, IC-MS offers great potential for ecotoxicological studies to investigate cellular effects of different substances and complex environmental samples. Graphical Abstract Use of intact cell MALDI-TOF mass spectrometry (IC-MS) to detect and differentiate toxic effects of environmental toxins in rainbow trout liver cell line RTL-W1
Keywords: RTL-W1; Intact cell mass spectrometry; MALDI; Acridine; β-Naphthoflavone; Copper sulfate

Ginkgo biloba is one of the most widely sold herbal supplements and medicines in the world. Its popularity stems from having a positive effect on memory and the circulatory system in clinical studies. As ginkgo popularity increased, non-proprietary extracts were introduced claiming to have a similar phytochemical profile as the clinically tested extracts. The standardized commercial extracts of G. biloba leaf used in ginkgo supplements contain not less than 6 % sesquiterpene lactones and 24 % flavonol glycosides. While sesquiterpene lactones are unique constituents of ginkgo leaf, the flavonol glycosides are found in many other botanical extracts. Being a high value botanical, low quality ginkgo extracts may be subjected to adulteration with flavonoids to meet the requirement of 24 % flavonol glycosides. Chemical analysis by ultra high performance liquid chromatography-mass spectrometry revealed that adulteration of ginkgo leaf extracts in many of these products is common, the naturally flavonol glycoside-rich extract being spiked with pure flavonoids or extracts made from another flavonoid-rich material, such as the fruit/flower of Japanese sophora (Styphnolobium japonicum), which also contains the isoflavone genistein. Recently, genistein has been proposed as an analytical marker for the detection of adulteration of ginkgo extracts with S. japonicum. This study confirms that botanically authenticated G. biloba leaf and extracts made therefrom do not contain genistein, and the presence of which even in trace amounts is suggestive of adulteration. In addition to the mass spectrometric approach, a high performance thin layer chromatography method was developed as a fast and economic method for chemical fingerprint analysis of ginkgo samples.
Keywords: Ginkgo biloba L.; HPTLC; UHPLC-DAD-QToF-MS; Dietary supplements; Styphnolobium japonicum (L.) Schott

Vibrational spectroscopic analysis of peripheral blood plasma of patients with Alzheimer’s disease by Pedro Carmona; Marina Molina; Eduardo López-Tobar; Adolfo Toledano (7747-7756).
Using Raman and infrared spectroscopy, we monitored spectral changes occurring in the blood plasma of patients with Alzheimer’s disease (AD) in relation to healthy controls. The protein secondary structure as reflected by amide I band involves β-sheet enrichment, which may be attributable to Aβ peptide formation and to increasing proportion of the globulins that are β-sheet rich. Likewise, the behavior of the infrared 1200–1000–cm−1 region and the Raman 980–910- and 450–400-cm−1 regions can be explained in terms of the said plasma composition change. Further, the 744-cm−1 Raman band from healthy control plasma shows frequency upshifting in the course of AD, which may be generated by the platelets collected in blood plasma. Linear discrimination analysis and receiver operating characteristic (ROC) analysis have been used to distinguish between patients with AD and age-matched healthy controls with a diagnostic accuracy of about 94 %. Graphical Abstract ROC assessment of sample classifications by Raman spectroscopy (dashed line) and linear combination of infrared and Raman spectroscopies (solid line)
Keywords: Raman spectroscopy; Infrared spectroscopy; Blood plasma; Alzheimer’s disease

Monoterpene separation by coupling proton transfer reaction time-of-flight mass spectrometry with fastGC by Dušan Materić; Matteo Lanza; Philipp Sulzer; Jens Herbig; Dan Bruhn; Claire Turner; Nigel Mason; Vincent Gauci (7757-7763).
Proton transfer reaction mass spectrometry (PTR-MS) is a well-established technique for real-time analysis of volatile organic compounds (VOCs). Although it is extremely sensitive (with sensitivities of up to 4500 cps/ppbv, limits of detection <1 pptv and the response times of approximately 100 ms), the selectivity of PTR-MS is still somewhat limited, as isomers cannot be separated. Recently, selectivity-enhancing measures, such as manipulation of drift tube parameters (reduced electric field strength) and using primary ions other than H3O+, such as NO+ and O2 +, have been introduced. However, monoterpenes, which belong to the most important plant VOCs, still cannot be distinguished so more traditional technologies, such as gas chromatography mass spectrometry (GC-MS), have to be utilised. GC-MS is very time consuming (up to 1 h) and cannot be used for real-time analysis. Here, we introduce a sensitive, near-to-real-time method for plant monoterpene research—PTR-MS coupled with fastGC. We successfully separated and identified six of the most abundant monoterpenes in plant studies (α- and β-pinenes, limonene, 3-carene, camphene and myrcene) in less than 80 s, using both standards and conifer branch enclosures (Norway spruce, Scots pine and black pine). Five monoterpenes usually present in Norway spruce samples with a high abundance were separated even when the compound concentrations were diluted to 20 ppbv. Thus, fastGC-PTR-ToF-MS was shown to be an adequate one-instrument solution for plant monoterpene research.
Keywords: PTR-MS; FastGC; Monoterpenes; VOC; Plant VOCs; Pinene

Measurement of serum aldosterone in picomolar level by LC-MS/MS using charge-tagged technique by Bonnie Mei-Wah Fong; Tak-Shing Siu; Sidney Tam (7765-7774).
Aldosterone is a mineralocorticoid steroid hormone, the measurement of which in the clinical laboratory is principally performed for the investigation of primary hyperaldosteronism. Primary hyperaldosteronism is a specifically treatable and potentially curable form of hypertension, which typically presents as drug-resistant hypertension and, in up to 37 % of cases, hypokalemia. Accurate measurement of aldosterone concentration is essential for correct diagnosis. The serum concentrations of aldosterone are in the picomolar range and therefore sensitive aldosterone assays are required. With the advancement in instrumentation of LC-MS/MS, the picomolar range of aldosterone can be easily measured by the newer models, but for those with a less sensitive instrument, special technique for sample preparation to enhance assay sensitivity is required. This work described the use of charge-tagging for the picomolar measurement of serum aldosterone in a less sensitive LC-MS/MS instrument. The assay was linear up to 3000 pmol/L with lower limit of quantitation at 80 pmol/L. The mean relative recovery was 96.5 % with a range of 89.3–101.6 % for aqueous calibrators and the mean relative recovery was 94.8 % with a range of 87.5–101.4 % for serum calibrators. Intra-assay CVs range from 8.2 % to 11.3 %, and inter-assay CVs ranged from 8.5 % to 13.5 % at concentration range from 229 to 1720 pmol/L. The LC-MS/MS method compared well (y = 1.04x + 8.97) with the in-use radioimmunoassay method. There was no significant difference found (p = 0.7135) between results determined by LC-MS/MS and radioimmunoassay method.
Keywords: Serum aldosterone; LC-MS/MS; Picomolar level; Charge-tagged

The biocompatibility of carbon hydroxyapatite/β-glucan composite for bone tissue engineering studied with Raman and FTIR spectroscopic imaging by Anna Sroka-Bartnicka; James A. Kimber; Leszek Borkowski; Marta Pawlowska; Izabela Polkowska; Grzegorz Kalisz; Anna Belcarz; Krzysztof Jozwiak; Grazyna Ginalska; Sergei G. Kazarian (7775-7785).
The spectroscopic approaches of FTIR imaging and Raman mapping were applied to the characterisation of a new carbon hydroxyapatite/β-glucan composite developed for bone tissue engineering. The composite is an artificial bone material with an apatite-forming ability for the bone repair process. Rabbit bone samples were tested with an implanted bioactive material for a period of several months. Using spectroscopic and chemometric methods, we were able to determine the presence of amides and phosphates and the distribution of lipid-rich domains in the bone tissue, providing an assessment of the composite’s bioactivity. Samples were also imaged in transmission using an infrared microscope combined with a focal plane array detector. CaF2 lenses were also used on the infrared microscope to improve spectral quality by reducing scattering artefacts, improving chemometric analysis. The presence of collagen and lipids at the bone/composite interface confirmed biocompatibility and demonstrate the suitability of FTIR microscopic imaging with lenses in studying these samples. It confirmed that the composite is a very good background for collagen growth and increases collagen maturity with the time of the bone growth process. The results indicate the bioactive and biocompatible properties of this composite and demonstrate how Raman and FTIR spectroscopic imaging have been used as an effective tool for tissue characterisation.
Keywords: IR spectroscopy; Raman spectroscopy; Biomaterials; Bone tissue engineering; Hydroxyapatite composite

This study investigates the feasibility of using surface-enhanced Raman scattering (SERS) for the quantification of absolute levels of the boar-taint compounds skatole and androstenone in porcine fat. By investigation of different types of nanoparticles, pH and aggregating agents, an optimized environment that promotes SERS of the analytes was developed and tested with different multivariate spectral pre-processing techniques, and this was combined with variable selection on a series of analytical standards. The resulting method exhibited prediction errors (root mean square error of cross validation, RMSECV) of 2.4 × 10−6 M skatole and 1.2 × 10−7 M androstenone, with a limit of detection corresponding to approximately 2.1 × 10−11 M for skatole and approximately 1.8 × 10−10 for androstenone. The method was subsequently tested on porcine fat extract, leading to prediction errors (RMSECV) of 0.17 μg/g for skatole and 1.5 μg/g for androstenone. It is clear that this optimized SERS method, when combined with multivariate analysis, shows great potential for optimization into an on-line application, which will be the first of its kind, and opens up possibilities for simultaneous detection of other meat-quality metabolites or pathogen markers. Graphical abstract Artistic rendering of a laser-illuminated gold colloid sphere with skatole and androstenone adsorbed on the surface
Keywords: Boar taint; Porcine fat; Androstenone; Skatole; Surface-enhanced Raman scattering (SERS); Multivariate calibration

A novel direct spray-from-tissue ionization method for mass spectrometric analysis of human brain tumors by Alexey Kononikhin; Evgeny Zhvansky; Vsevolod Shurkhay; Igor Popov; Denis Bormotov; Yury Kostyukevich; Sofiia Karchugina; Maria Indeykina; Anna Bugrova; Natalia Starodubtseva; Alexander Potapov; Eugene Nikolaev (7797-7805).
Real-time feedback about dissected tissue during the neurosurgical procedure is strongly requested. A novel direct ionization mass spectrometric method for identifying pathological differences in tissues is proposed. The method is based on simultaneous extraction of tissue lipids and electrospray ionization which allows mass spectrometric data to be obtained directly from soft tissues. The advantage of this method is the stable flow of solvent, which leads to stable time-dependent spectra. The tissues included necrotized tissues and tumor tissues in different combinations. Capability for direct analysis of samples of dissected tissues during the neurosurgical procedure is demonstrated. Data validation is conducted by compound identification using precise masses from the MS profile, MS/MS, and isotopic distribution structure analysis. The method can be upgraded and applied for real-time identification of tissues during surgery. This paper describes the technique and its application perspective. For these purposes, other methods were compared with the investigated one and the results were shown to be reproducible. Differences in lipid profiles were observed even in tissues from one patient where distinctions between different samples could be poor. The paper presents a proof of concept for the method to be applied in neurosurgery particularly and in tissue analysis generically. The paper also contains preliminary results proving the possibility of observing differences in mass spectra of different tumors.
Keywords: Ambient mass spectrometry; Brain; Tumor; Diagnostics; Electrospray; FT ICR

A biosensor based on electroactive dipyrromethene-Cu(II) layer deposited onto gold electrodes for the detection of antibodies against avian influenza virus type H5N1 in hen sera by Urszula Jarocka; Róża Sawicka; Anna Stachyra; Anna Góra-Sochacka; Agnieszka Sirko; Włodzimierz Zagórski-Ostoja; Violetta Sączyńska; Anna Porębska; Wim Dehaen; Jerzy Radecki; Hanna Radecka (7807-7814).
This paper describes the development of a biosensor for the detection of anti-hemagglutinin antibodies against the influenza virus hemagglutinin. The steps of biosensor fabrications are as follows: (i) creation of a mixed layer containing the thiol derivative of dipyrromethene and 4-mercapto-1-butanol, (ii) complexation of Cu(II) ions, (iii) oriented immobilization of the recombinant histidine-tagged hemagglutinin, and (iv) filling free spaces with bovine serum albumin. The interactions between recombinants hemagglutinin from the highly pathogenic avian influenza virus type H5N1 and anti-hemagglutinin H5 monoclonal antibodies were explored with Osteryoung square-wave voltammetry. The biosensor displayed a good detection limit of 2.4 pg/mL, quantification limit of 7.2 pg/mL, and dynamic range from 4.0 to 100.0 pg/mL in buffer. In addition, this analytical device was applied for the detection of antibodies in hen sera from individuals vaccinated and non-vaccinated against the avian influenza virus type H5N1. The limit of detection for the assay was the dilution of sera 1: 7 × 106, which is about 200 times better than the enzyme-linked immunosorbent assay. Graphical Abstract Scheme of immunosensor based on dipyrromethene-Cu(II)-Histidine-tagged hemagglutinin deposited on gold electrode
Keywords: Dipyrromethene-Cu(II) layer; Histidine-tagged antigen immobilization; Detection of antibodies in sera; Avian influenza virus

Development of an aptasensor based on a fluorescent particles-modified aptamer for ochratoxin A detection by Akhtar Hayat; Rupesh K. Mishra; Gaelle Catanante; Jean Louis Marty (7815-7822).
The presented work reports a generic fluorescent aptasensing design employing carboxy-modified fluorescent particles as a signal-generating probe and magnetic particles as a solid separation support. Carboxy-modified fluorescent particles were used to modify the aptamer and act as a signal-generating probe. Magnetic beads were used as an immobilization surface to perform the function of a solid separation support. As a proof of concept, the assay was used to detect ochratoxin A (OTA). Fluorescent detection based on the displacement and competition format was performed, and the obtained results were compared. The competition-based assays were characterized with improved analytical characteristics as compared to those based on the displacement principle. The competitive fluorescent assays showed a high sensitivity where the detection limit and IC50 were 0.005 and 7.2 nM respectively. The aptasensing platform was finally demonstrated for the detection of OTA in a beer sample. However, this is a generic approach that can be very easily extended to other matrixes to determine OTA. Additionally, the proposed concept of fluorescent particles as a signal-generating probe in combination with magnetic particles can also be integrated to other fluorescent-based affinity assays.
Keywords: Fluorescence detection; Displacement/competition assays; Magnetic particles; Modified aptamer; Ochratoxin A

Development of sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in plasma by Laura Zehnacker; Marie-Claire Nevers; Véronique Sinou; Dominique Parzy; Christophe Créminon; Daniel Parzy; Stéphane Azoulay (7823-7830).
Despite significant progress in prevention and therapy, malaria is still one of the world’s leading major diseases due to its high morbidity and mortality. Recommended treatments by the World Health Organization include the use of artemisinin and artemisinin derivative-based combination therapies. To allow efficient patient monitoring during antimalarial therapy without the use of expensive apparatus, we developed a sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in biological fluids. To produce specific antibodies against dihydroartemisinin (DHA), a synthetic DHA derivative was coupled to bovine serum albumin as the immunogen. In parallel, a new, rapid, and efficient procedure to covalently link glycoprotein to all amine-containing molecules has been established and the enzyme tracer was prepared by chemically coupling the DHA derivative in combination with SBP rather than the more commonly used HRP. It allowed us to develop, after optimization of the luminescent reagent, a sensitive and stable luminescent EIA, with a LLOQ of 90 pg mL−1. This assay compares favorably with the most efficient HPLC methods previously reported with a LLOQ close to 1 ng mL−1 and shows good precision and efficiency since recovery from human plasma spiked with DHA ranged between 91 and 103 %, with coefficients of variation of <13 %. To date, no immunoassay for DHA has been applied to plasma analysis and this EIA should be very useful in all clinical laboratories for rapid and cost-effective analysis. Graphical Abstract DHA chemical structure and chemiluminescent immunoassay standard curve
Keywords: Malaria; Dihydroartemisinin; Chemiluminescent enzyme immunoassay; Therapeutic drug monitoring; Soybean peroxidase

N,N-Diallyltryptamine (DALT) and 5-methoxy-DALT (5-MeO-DALT) are synthetic tryptamine derivatives commonly referred to as so-called new psychoactive substances (NPS). They have psychoactive effects that may be similar to those of other tryptamine derivatives. The objectives of this work were to study the metabolic fate and detectability, in urine, of DALT and 5-MeO-DALT. For metabolism studies, rat urine obtained after high-dose administration was prepared by precipitation and analyzed by liquid chromatography–high-resolution mass spectrometry (LC–HR–MS–MS). On the basis of the metabolites identified, several aromatic and aliphatic hydroxylations, N-dealkylation, N-oxidation, and combinations thereof are proposed as the main metabolic pathways for both compounds. O-Demethylation of 5-MeO-DALT was also observed, in addition to extensive glucuronidation or sulfation of both compounds after phase I transformation. The cytochrome P450 (CYP) isoenzymes predominantly involved in DALT metabolism were CYP2C19, CYP2D6, and CYP3A4; those mainly involved in 5-MeO-DALT metabolism were CYP1A2, CYP2C19, CYP2D6, and CYP3A4. For detectability studies, rat urine was screened by GC–MS, LC–MS n , and LC–HR–MS–MS after administration of low doses. LC–MS n and LC–HR–MS–MS were deemed suitable for monitoring consumption of both compounds. The most abundant targets were a ring hydroxy metabolite of DALT, the N,O-bis-dealkyl metabolite of 5-MeO-DALT, and their glucuronides. GC–MS enabled screening of DALT by use of its main metabolites only.
Keywords: Designer drugs; DALT; 5-MeO-DALT; Metabolism; SUSA; LC–HR–MS–MS

Fluorescence polarization immunoassay using IgY antibodies for detection of valnemulin in swine tissue by Huiyan Zhang; Tiejun Mi; Oleg Yu Khan; Yajie Sheng; Sergei A. Eremin; Ross C. Beier; Suxia Zhang; Jianzhong Shen; Zhanhui Wang (7843-7848).
Immunoglobulin Y (IgY) is derived from egg yolk and has been identified as a cheap and high-yield immunoreagent. The application of IgY in immunoassays for the detection of chemical contaminants in food samples has rarely been reported. In this work, we describe a rapid and sensitive fluorescence polarization immunoassay (FPIA) for valnemulin (VAL) using IgY which was produced using a previously prepared immunogen. Three fluorophore-labeled VAL tracers were synthesized and the sensitivity of the best tracer (VAL–DTAF) in the optimized FPIA with antibody IgY100 demonstrated an IC50 value of 12 ng mL−1 in buffer. After evaluation of several extraction procedures, acidified acetonitrile was selected to extract VAL from swine tissue. The recoveries of VAL in spiked swine tissue at three levels (50, 100, and 200 μg kg−1) were higher than 79 % with coefficients of variation (CVs) lower than 12 %. The limit of detection (LOD) of the FPIA in swine tissue was 26 μg kg−1 and was lower than the maximum residue limit (MRL) of VAL set by the European Union. The study showed that IgY could be a good substitute for IgG when developing a high-throughput assay for chemical residues. Graphical Abstract FPIA based on IgY for rapid detection of Valnemulin in swine tissue
Keywords: Fluorescence polarization immunoassay; IgY; Chemical residues; Swine tissue; Valnemulin

Simultaneous determination of ten anticoagulant rodenticides in tissues by column-switching UHPLC-ESI-MS/MS by Petr Maršálek; Helena Modrá; Veronika Doubková; Vladimír Večerek (7849-7854).
This paper describes the development of a method for the simultaneous determination of ten anticoagulant rodenticides (coumafuryl, warfarin, pindone, coumatetralyl, coumachlor, difenacoum, bromadiolone, brodifacoum, chlorophacinone and flocoumafen) in the liver and kidney based on column-switching liquid chromatography coupled with heated electrospray ionization tandem mass spectrometry. The simple sample preparation includes extraction with methanol. A C18 trapping column was used for online solid-phase extraction before analytical separation with the mobile phase comprising a mixture of 0.1 % formic acid in water, methanol and acetonitrile. Chromatographic separation was achieved using a Thermo Hypersil ultra high-performance liquid chromatography (UHPLC) C18 column with the mobile phase consisting of 5 mM ammonium formate buffer (pH = 9) and methanol. The column-switching procedure ensured no matrix effects during electrospray ionization (ESI). Extraction recoveries ranged between 91 and 100 % for liver and between 89 and 97 % for kidney. The method showed good linearity up to 750 ng g−1. The limit of detection ranged between 0.001 and 0.022 ng g−1 for liver and between 0.001 and 0.028 ng g−1 for kidney. The developed method was successfully used in several animal poisoning cases.
Keywords: Online SPE; Trapping; Mass spectrometry; Liquid chromatography