Analytical and Bioanalytical Chemistry (v.406, #7)

The absinthe challenge by Lucia D’Ulivo (1815-1816).

Solution to angels’ share challenge by Reinhard Meusinger (1817-1818).

is Professor of Physical Chemistry at the University of Oviedo (Principado de Asturias, Spain). He carried out postdoctoral research on atomic structure at the University of Alberta (Canada) with Prof. Serafín Fraga and collaborated with Prof. Enrico Clementi at IBM-Kingston (New York) in the development of amino acid–amino acid ab initio pair potentials. His present interests include chemical kinetics studies using statistical theories.

Discovery and validation of urinary exposure markers for different plant foods by untargeted metabolomics by Maj-Britt Schmidt Andersen; Mette Kristensen; Claudine Manach; Estelle Pujos-Guillot; Sanne Kellebjerg Poulsen; Thomas Meinert Larsen; Arne Astrup; Lars Dragsted (1829-1844).
is about to finalize her PhD studies at Department of Nutrition, Exercise and Sports at the University of Copenhagen. Her work is focused on the discovery of new exposure and compliance markers in urine for intake of foods and dietary patterns by application of untargeted LC-MS based metabolomics. While metabolomics is increasingly used to investigate the food metabolome and identify new markers of food exposure, limited attention has been given to the validation of such markers. The main objectives of the present study were to (1) discover potential food exposure markers (PEMs) for a range of plant foods in a study setting with a mixed dietary background and (2) validate PEMs found in a previous meal study. Three-day weighed dietary records and 24-h urine samples were collected three times during a 6-month parallel intervention study from 107 subjects randomized to two distinct dietary patterns. An untargeted UPLC-qTOF-MS metabolomics analysis was performed on the urine samples, and all features detected underwent strict data analyses, including an iterative paired t test and sensitivity and specificity analyses for foods. A total of 22 unique PEMs were identified that covered 7 out of 40 investigated food groups (strawberry, cabbages, beetroot, walnut, citrus, green beans and chocolate). The PEMs reflected foods with a distinct composition rather than foods eaten more frequently or in larger amounts. We found that 23 % of the PEMs found in a previous meal study were also valid in the present intervention study. The study demonstrates that it is possible to discover and validate PEMs for several foods and food classes in an intervention study with a mixed dietary background, despite the large variability in such a dataset. Final validation of PEMs for intake of foods should be performed by quantitative analysis. Figure Examples of two urinary exposure markers for cabbage (left) and beetroot (right) found in the study from an untargeted LC‐MS metabolomics analysis of urine samples and self‐reported food intake data
Keywords: Untargeted metabolomics; UPLC-MS; Food exposure markers; Urine; Dietary intervention study; Humans

is a Postdoc in the Department of Experimental and Clinical Toxicology, Saarland University, Homburg (Germany). His research is focused on the toxicokinetics of drugs of abuse, for example metabolism and interaction studies, and analytical toxicology and biotransformation of drugs in unusual matrices, for example wastewater. In 2008, he received the TIAFT young scientist award in La Martinique is a PhD student in the Department of Experimental and Clinical Toxicology, Saarland University, Homburg (Germany). As part of her degree she is currently working in her practical year at the University Hospital, Homburg Saar is a PhD student in the Department of Experimental and Clinical Toxicology, Saarland University, Homburg (Germany). Her research interests focus on use of GC–MS and LC–(HR)MS n to study the in-vivo and in-vitro metabolism of new designer drugs. In 2013, she received the IATDMCT young scientist award in Salt Lake City is full Professor of Pharmacology and Toxicology in the Faculty of Medicine and the Faculty of Pharmacy, Saarland University (Homburg, Germany) and head of the Department of Experimental and Clinical Toxicology. His main areas of research are analytical toxicology (GC–MS, LC–(HR)MS), toxicokinetics, and the metabolism of xenobiotics. He is an editorial board member of several international journals and is a member of the executive boards of a variety of scientific societies in his field. He has received several international awards for his outstanding scientific work, and in 2007 was awarded the title Doctor Honoris Causa (honorary doctorate) by the University of Ghent, Belgium Dimethocaine (DMC, larocaine), a synthetic derivative of cocaine, is a widely distributed “legal high” consumed as a “new psychoactive substance” (NPS) without any safety testing, for example studies of metabolism. Therefore, the purpose of this work was to study its in-vivo and in-vitro metabolism by use of liquid chromatography–(high resolution) mass spectrometry (LC–HRMS n ). DMC was administered to male Wistar rats (20 mg kg−1) and their urine was extracted either by solid-phase extraction after enzymatic cleavage of conjugates or by use of protein precipitation (PP). The metabolites were separated and identified by LC–HRMS n . The main phase I reactions were ester hydrolysis, deethylation, hydroxylation of the aromatic system, and a combination of these. The main phase II reaction was N-acetylation of the p-aminobenzoic acid part of the unchanged parent compound and of several phase I metabolites. The metabolites identified were then used for identification of DMC in rat urine after application of a common user’s dose. By use of GC–MS and LC–MS n standard urine-screening approaches (SUSAs), DMC and its metabolites could be detected in the urine samples.
Keywords: Designer drug; DMC; Cocaine; Metabolism; Urine screening; GC–MS; LC–MS n ; LC–HRMS n

Selective trapping of single mammalian breast cancer cells by insulator-based dielectrophoresis by Sanchari Bhattacharya; Tzu-Chiao Chao; Nethmi Ariyasinghe; Yvette Ruiz; Douglas Lake; Robert Ros; Alexandra Ros (1855-1865).
The trapping or immobilization of individual cells at specific locations in microfluidic platforms is essential for single cell studies, especially those requiring cell stimulation and downstream analysis of cellular content. Selectivity for individual cell types is required when mixtures of cells are analyzed in heterogeneous and complex matrices, such as the selection of metastatic cells within blood samples. Here, we demonstrate a microfluidic device based on direct current (DC) insulator-based dielectrophoresis (iDEP) for selective trapping of single MCF-7 breast cancer cells from mixtures with both mammalian peripheral blood mononuclear cells (PBMC) as well MDA-MB-231 as a second breast cancer cell type. The microfluidic device has a teardrop iDEP design optimized for the selective capture of single cells based on their differential DEP behavior under DC conditions. Numerical simulations adapted to experimental device geometries and buffer conditions predicted the trapping condition in which the dielectrophoretic force overcomes electrokinetic forces for MCF-7 cells, whereas PBMCs were not trapped. Experimentally, selective trapping of viable MCF-7 cells in mixtures with PBMCs was demonstrated in good agreement with simulations. A similar approach was also executed to demonstrate the selective trapping of MCF-7 cells in a mixture with MDA-MB-231 cells, indicating the selectivity of the device for weakly invasive and highly invasive breast cancer cells. The DEP studies were complemented with cell viability tests indicating acceptable cell viability over the course of an iDEP trapping experiment. Figure ᅟ
Keywords: Dielectrophoresis; Selective trapping; Cancer cell; Insulator-based; Numerical simulation

A fluorescence-based high throughput assay for the determination of small molecule−human serum albumin protein binding by Megan M. McCallum; Alan J. Pawlak; William R. Shadrick; Anton Simeonov; Ajit Jadhav; Adam Yasgar; David J. Maloney; Leggy A. Arnold (1867-1875).
Herein, we describe the development of a fluorescence-based high throughput assay to determine the small molecule binding towards human serum albumin (HSA). This innovative competition assay is based on the use of a novel fluorescent small molecule Red Mega 500 with unique spectroscopic and binding properties. The commercially available probe displays a large fluorescence intensity difference between the protein-bound and protein-unbound state. The competition of small molecules for HSA binding in the presence of probe resulted in low fluorescence intensities. The assay was evaluated with the library of pharmacological active compounds (LOPAC) small molecule library of 1,280 compounds identifying known high protein binders. The small molecule competition of HSA−Red Mega 500 binding was saturable at higher compound concentrations and exhibited IC50 values between 3 and 24 μM. The compound affinity toward HSA was confirmed by isothermal titration calorimetry indicating that the new protein binding assay is a valid high throughput assay to determine plasma protein binding. Figure A high throughput fluorescence-based serum protein binding assay
Keywords: Drug–protein binding; High throughput screening; Red Mega 500; Human serum albumin

Rapid on-site TLC–SERS detection of four antidiabetes drugs used as adulterants in botanical dietary supplements by Qingxia Zhu; Yongbing Cao; Yingying Cao; Yifeng Chai; Feng Lu (1877-1884).
A novel facile method has been established for rapid on-site detection of antidiabetes chemicals used to adulterate botanical dietary supplements (BDS) for diabetes. Analytes and components of pharmaceutical matrices were separated by thin-layer chromatography (TLC) then surface-enhanced Raman spectroscopy (SERS) was used for qualitative identification of trace substances on the HPTLC plate. Optimization and standardization of the experimental conditions, for example the method used for preparation of silver colloids, the mobile phase, and the concentration of colloidal silver, resulted in a very robust and highly sensitive method which enabled successful detection when the amount of adulteration was as low as 0.001 % (w/w). The method was also highly selective, enabling successful identification of some chemicals in extremely complex herbal matrices. The established TLC–SERS method was used for analysis of real BDS used to treat diabetes, and the results obtained were verified by liquid chromatography–triple quadrupole mass spectrometry (LC–MS–MS). The study showed that TLC–SERS could be used for effective separation and detection of four chemicals used to adulterate BDS, and would have good prospects for on-site qualitative screening of BDS for adulterants. Figure Experimental procedure of TLC-SERS method
Keywords: Thin-layer chromatography; Surface-enhanced Raman spectroscopy; Adulterant; Botanical dietary supplement

Using a silver-enhanced microarray sandwich structure to improve SERS sensitivity for protein detection by Xuefang Gu; Yuerong Yan; Guoqing Jiang; Jason Adkins; Jian Shi; Guomin Jiang; Shu Tian (1885-1894).
A simple and sensitive method, based on surface-enhanced Raman scattering (SERS), for immunoassay and label-free protein detection is reported. A series of bowl-shaped silver cavity arrays were fabricated by electrodeposition using a self-assembled polystyrene spheres template. The reflection spectra of these cavity arrays were recorded as a function of film thickness, and then correlated with SERS enhancement using sodium thiophenolate as the probe molecule. The results reveal that SERS enhancement can be maximized when the frequency of both the incident laser and the Raman scattering approach the frequency of the localized surface plasmon resonance. The optimized array was then used as the bottom layer of a silver nanoparticle–protein–bowl-shaped silver cavity array sandwich. The second layer of silver was introduced by the interactions between the proteins in the middle layer of the sandwich architecture and silver nanoparticles. Human IgG bound to the surface of this microcavity array can retain its recognition function. With the Raman reporter molecules labeled on the antibody, a detection limit down to 0.1 ng mL−1 for human IgG is easily achieved. Furthermore, the SERS spectra of label-free proteins (catalase, cytochrome C, avidin and lysozyme) from the assembled sandwich have excellent reproducibility and high quality. The results reveal that the proposed approach has potential for use in qualitative and quantitative detection of biomolecules. Schematic diagram of sandwich structure for labelled and label-free protein detection.
Keywords: SERS; Bowl-shaped silver cavity; Silver enhancement; Immunoassay; Label-free protein detection

Ligation-triggered fluorescent silver nanoclusters system for the detection of nicotinamide adenine dinucleotide by Zhijuan Cao; Pei Wang; Xue Qiu; Choiwan Lau; Jianzhong Lu (1895-1902).
Herein, we demonstrate a novel silver nanocluster-based fluorescent system for the detection of nicotinamide adenine dinucleotide (NAD+), an important biological small molecule involved in a wide range of biological processes. A single-stranded dumbbell DNA probe was designed and used for the assay, which contained a nick in the stem, a poly-cytosine nucleotide loop close to 5′ end as the template for the formation of highly fluorescent silver nanoclusters (Ag NCs) and another loop close to 3′ end. Only in the presence of NAD+, the probe was linked at 5′ and 3′ ends by Escherichia coli DNA ligase, which blocked the DNA polymerase-based extension reaction, ensuring the formation of fluorescent Ag NCs. This technique provided a logarithmic linear relationship in the range of 1 pM–500 nM with a detection limit of as low as 1 pM NAD+, and exhibited high selectivity against its analogues, and was then successfully used for the detection of NAD+ level in four kinds of cell homogenates. In addition, this new approach was conducted in an isothermal and homogeneous condition without the need of any thermal cycling, washing, and separation steps, making it very simple. Overall, this label-free protocol offers a promising alternative for the detection of NAD+, taking advantage of specificity, sensitivity, cost-efficiency, and simplicity. Figure Ligation triggered fluorescent silver nanoclusters system for nicotinamide adenine dinucleotide sensing
Keywords: Nicotinamide adenine dinucleotide; Poly-cytosine nucleotide loop; Silver nanoclusters; Ligation reaction; Fluorescent biosensor

A simple, inexpensive yet highly selective colorimetric indicator-displacement assay array for the simultaneous detection and identification of three important biothiols at micromolar concentrations under physiological conditions and in real samples has been developed in this work. With use of an array composed of metal indicators and metal ions, clear differentiation among cysteine, homocysteine and glutathione was achieved. On the basis of the colour change of the array, quantification of each analyte was accomplished easily, and different biothiols were identified readily using standard chemometric approaches (hierarchical clustering analysis). Moreover, the colorimetric sensor array was not responsive to changes with 19 other natural amino acids, and it showed excellent reproducibility. Importantly, the sensor array developed was successfully applied to the determination and identification of the three biothiols in a real biological sample. Figure A simple, inexpensive yet highly selective colorimetric indicator-displacement assay array for the simultaneous detection and identification of three important biothiols was developed in this work
Keywords: Colorimetric sensor array; Biothiols; Indicator-displacement assay; Hierarchical clustering analysis

On-line species-unspecific isotope dilution analysis in the picomolar range reveals the time- and species-depending mercury uptake in human astrocytes by Christoph A. Wehe; Imke Pieper; Michael Holtkamp; Georgina M. Thyssen; Michael Sperling; Tanja Schwerdtle; Uwe Karst (1909-1916).
In order to reveal the time-depending mercury species uptake by human astrocytes, a novel approach for total mercury analysis is presented, which uses an accelerated sample introduction system combined on-line with an inductively coupled plasma mass spectrometer equipped with a collision/reaction cell. Human astrocyte samples were incubated with inorganic mercury (HgCl2), methylmercury chloride (MeHgCl), and thimerosal. After 1-h incubation with Hg2+, cellular concentrations of 3 μM were obtained, whereas for organic species, concentrations of 14–18 μM could be found. After 24 h, a cellular accumulation factor of 0.3 was observed for the cells incubated with Hg2+, whereas the organic species both showed values of about 5. Due to the obtained steady-state signals, reliable results with relative standard deviations of well below 5 % and limits of detection in the concentration range of 1 ng L−1 were obtained using external calibration and species-unspecific isotope dilution analysis approaches. The results were further validated using atomic fluorescence spectrometry. Figure ᅟ
Keywords: Mercury; Thimerosal; Astrocytes; ICP-MS; Isotope dilution analysis; Automation

A lipidomic platform establishment for structural identification of skin ceramides with non-hydroxyacyl chains by Jung-Hoon Shin; Jong Cheol Shon; Kyohoon Lee; Sunki Kim; Chang Seo Park; Eung Ho Choi; Choong Hwan Lee; Hye Suk Lee; Kwang-Hyeon Liu (1917-1932).
The stratum corneum (SC) is the outermost layer of skin that functions as a barrier and protects against environmental influences and transepidermal water loss. Its unique morphology consists of keratin-enriched corneocytes embedded in a distinctive mixture of lipids containing mainly ceramides, free fatty acids, and cholesterol. Ceramides are sphingolipids consisting of sphingoid bases, which are linked to fatty acids by an amide bond. Typical sphingoid bases in the skin are composed of dihydrosphingosine (dS), sphingosine (S), phytosphingosine (P), and 6-hydroxysphingosine (H), and the fatty acid acyl chains are composed of non-hydroxy fatty acid (N), α-hydroxy fatty acid (A), ω-hydroxy fatty acid (O), and esterified ω-hydroxy fatty acid (E). The 16 ceramide classes include several combinations of sphingoid bases and fatty acid acyl chains. Among them, N-type ceramides are the most abundant in the SC. Mass spectrometry (MS)/MS analysis of N-type ceramides using chip-based direct infusion nanoelectrospray-ion trap mass spectrometry generated the characteristic fragmentation pattern of both acyl and sphingoid units, which could be applied to structural identification of ceramides. Based on the MS/MS fragmentation patterns of N-type ceramides, comprehensive fragmentation schemes were proposed. In addition, mass fragmentation patterns, which are specific to the sphingoid backbone of N-type ceramides, were found in higher m/z regions of tandem mass spectra. These characteristic and general fragmentation patterns were used to identify N-type ceramides in human SC. Based on established MS/MS fragmentation patterns of N-type ceramides, 52 ceramides (including different classes of NS, NdS, NP, and NH) were identified in human SC. The MS/MS fragmentation patterns of N-type ceramides were characterized by interpreting their product ion scan mass spectra. This information may be used to identify N-type ceramides in the SC of human, rat, and mouse skin.
Keywords: Ceramide; Mass spectrometry; Skin lipid; Stratum corneum

Aggressive dereplication using UHPLC–DAD–QTOF: screening extracts for up to 3000 fungal secondary metabolites by Andreas Klitgaard; Anita Iversen; Mikael R. Andersen; Thomas O. Larsen; Jens Christian Frisvad; Kristian Fog Nielsen (1933-1943).
In natural-product drug discovery, finding new compounds is the main task, and thus fast dereplication of known compounds is essential. This is usually performed by manual liquid chromatography-ultraviolet (LC-UV) or visible light-mass spectroscopy (Vis-MS) interpretation of detected peaks, often assisted by automated identification of previously identified compounds. We used a 15 min high-performance liquid chromatography–diode array detection (UHPLC–DAD)–high-resolution MS method (electrospray ionization (ESI)+ or ESI), followed by 10–60 s of automated data analysis for up to 3000 relevant elemental compositions. By overlaying automatically generated extracted-ion chromatograms from detected compounds on the base peak chromatogram, all major potentially novel peaks could be visualized. Peaks corresponding to compounds available as reference standards, previously identified compounds, and major contaminants from solvents, media, filters etc. were labeled to differentiate these from compounds only identified by elemental composition. This enabled fast manual evaluation of both known peaks and potential novel-compound peaks, by manual verification of: the adduct pattern, UV–Vis, retention time compared with log D, co-identified biosynthetic related compounds, and elution order. System performance, including adduct patterns, in-source fragmentation, and ion-cooler bias, was investigated on reference standards, and the overall method was used on extracts of Aspergillus carbonarius and Penicillium melanoconidium, revealing new nitrogen-containing biomarkers for both species.
Keywords: Metabolomics; Mycotoxin; NRPS; LC–MS; UPLC; Polyketide; Nonribosomal peptide

Presence of fatty acid ethyl esters (FAEE), ethyl glucuronide (EtG), and ethyl sulfate (EtS) in meconium, the first neonatal feces, identifies maternal alcohol consumption during pregnancy. Current meconium alcohol marker assays require separate analyses for FAEE and EtG/EtS. We describe development and validation of the first quantitative liquid chromatography tandem mass spectrometry assay for 9 FAEEs, EtG, and EtS in 100 mg meconium. For the first time, these alcohol markers are analyzed in the same meconium aliquot, enabling comparison of the efficiency of gestational ethanol exposure detection. 100 mg meconium was homogenized in methanol and centrifuged. The supernatant was divided, and applied to two different solid phase extraction columns for optimized analyte recovery. Limits of quantification for ethyl laurate, myristate, linolenate, palmitoleate, arachidonate, linoleate, palmitate, oleate, and stearate ranged from 25–50 ng/g, with calibration curves to 2,500–5,000 ng/g. EtG and EtS linear dynamic ranges were 5–1,000 and 2.5–500 ng/g, respectively. Mean bias and between-day imprecision were <15 %. Extraction efficiencies were 51.2–96.5 %. Matrix effects ranged from −84.7 to 16.0 %, but were compensated for by matched deuterated internal standards when available. All analytes were stable (within ±20 % change from baseline) in 3 authentic positive specimens, analyzed in triplicate, after 3 freeze/thaw cycles (−20 °C). Authentic EtG and EtS also were stable after 12 h at room temperature and 72 h at 4 °C; some FAEE showed instability under these conditions, although there was large inter-subject variability. This novel method accurately detects multiple alcohol meconium markers and enables comparison of markers for maternal alcohol consumption. Figure ᅟ
Keywords: Meconium; Ethyl glucuronide; Fatty acid ethyl esters; Alcohol; Liquid chromatography-tandem mass spectrometry

4-Methyl-amphetamine (1-(4-methylphenyl)propane-2-amine; 4-MA) and its isomers 2-methyl-amphetamine (2-MA) and 3-methyl-amphetamine (3-MA) belong to the group of amphetamine-type stimulants and of new psychoactive substances. Several studies showed similar potencies in releasing noradrenalin and dopamine, but higher potencies in releasing serotonin than amphetamine. In March 2013, the EU Council decided on an EU-wide control based on the European Monitoring Centre for Drugs and Drug Addiction risk assessment report documenting that 4-MA was sold as amphetamine on the illicit market and detected in several fatal cases. Therefore, 4-MA and its isomers should be covered by drug testing in clinical and forensic toxicology. The aims of the presented work were to study the metabolism and detectability of each isomer in urine samples. For metabolism studies, rat urine samples were isolated by solid-phase extraction without and after enzymatic cleavage of conjugates. The phase I metabolites were separated and identified after acetylation by gas chromatography–mass spectrometry (GC-MS) and/or liquid chromatography–high resolution-linear ion trap mass spectrometry (LC-HR-MS n ) and the phase II metabolites by LC-HR-MS n . From the identified phase I and II metabolites, the following main metabolic pathways were deduced: aromatic hydroxylation, hydroxylation of the phenylmethyl group followed by oxidation to the corresponding carboxylic acid, hydroxylation of the side chain, and glucuronidation and/or sulfation of the hydroxy and carboxy groups. CYP2D6 was involved in the aromatic hydroxylation. Finally, the intake of a commonly used dose of the MAs could be confirmed in rat urine using the authors’ GC-MS and the LC-MS n standard urine screening approaches. Differentiation of the isomers to confirm the intake of a specific isomer was possible with an additional workup in rat urine.
Keywords: Designer drugs; Methyl-amphetamine; Metabolism; GC-MS; LC-MS n ; LC-HR-MS n

To find potential lead compounds for antigout drug discovery, an automated online, restricted-access material coupled with column-switching liquid chromatography with a diode-array detection (RAM–LC–DAD) system was developed for screening of xanthine oxidase (XO) inhibitors and their affinity rankings in complex mixtures. The system was first evaluated by analyzing a mixture of six compounds with known inhibition of XO. Nonspecific binding to the denatured XO was investigated and used as the control for screening. Subsequently, the newly developed system was applied to screening of a natural product, Oroxylum indicum extract, and four compounds which could specifically interact with XO were found and identified as oroxin B, oroxin A, baicalin, and baicalein. The results were verified by a competitive binding test using the known competitive inhibitor allopurinol and were further validated by an inhibition assay in vitro. The online RAM–LC–DAD system developed was shown to be a simple and effective strategy for the rapid screening of bioactive compounds from a complex mixture. Figure Scheme of RAM-LC-DAD assay for affinity screening of xanthine oxidase inhibitors
Keywords: Xanthine oxidase inhibitors; Restricted-access material; Column switching; Liquid chromatography

Selective iteratively reweighted quantile regression for baseline correction by Xinbo Liu; Zhimin Zhang; Pedro F. M. Sousa; Chen Chen; Meilan Ouyang; Yangchao Wei; Yizeng Liang; Yong Chen; Chaoping Zhang (1985-1998).
Extraction of qualitative and quantitative information from large numbers of analytical signals is difficult with drifted baselines, particularly in multivariate analysis. Baseline drift obscures and “fuzzies” signals, and even deteriorates analytical results. In order to obtain accurate and clear results, some effective methods should be proposed and implemented to perform baseline correction before conducting further data analysis. However, most of the classic methods require user intervention or are prone to variability, especially with low signal-to-noise signals. In this study, a novel baseline correction algorithm based on quantile regression and iteratively reweighting strategy is proposed. This does not require user intervention and prior information, such as peak detection. The iteratively reweighting strategy iteratively changes weights of residuals between fitted baseline and original signals. After a series of tests and comparisons with several other popular methods, using various kinds of analytical signals, the proposed method is found to be fast, flexible, robust, and easy to use both in simulated and real datasets. Figure ᅟ
Keywords: Baseline correction; Quantile regression; Iteratively reweighted; Robustness