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Analytical and Bioanalytical Chemistry (v.405, #12)

Solution to the copper pots and jam challenge by Hervé This (pp. 3913-3914).

XV International Symposium on Luminescence Spectrometry—ISLS 2012 by Montserrat Pujol Cubells (pp. 3915-3916).

Michael A. Carpenter, Sanjay Mathur and Andrei Kolmakov (Eds.): Metal oxide nanomaterials for chemical sensors by Antonio Doménech-Carbó (pp. 3917-3918).

Metal oxide nanomaterials for chemical sensorsMichael A. Carpenter, Sanjay Mathur and Andrei Kolmakov (eds.)Series: Integrated Analytical SystemsSpringerISBN 978-1-4614-5394-9Hardcover, 548 pages,2013, €181.85

Forensic toxicology by Kazuhito Watanabe; Satoshi Chinaka (pp. 3919-3920).

is a professor at the Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Japan. His research interests include drug metabolism and drugs of abuse. In particular, he is consistently concentrating his research career on biochemical toxicology of marijuana, and has published over 150 original papers on research into cannabinoids. is a senior researcher at the Forensic Science Laboratory, Ishikawa Prefectural Police Headquarters, Japan. He is an expert not only in the analysis of drugs and poisons but also of analysis of industrial materials collected from crime scenes. His research interests are in the application of electrophoresis, chromatography, and mass spectrometry to the analysis of drugs and their metabolites.

Methadone concentrations in blood, plasma, and oral fluid determined by isotope-dilution gas chromatography–mass spectrometry by Ya-Ching Hsu; Bud-Gen Chen; Shu-Ching Yang; Yu-Shan Wang; Shiao-Ping Huang; Mei-Han Huang; Tai-Jui Chen; Hsu-Chun Liu; Dong-Liang Lin; Ray H. Liu; A. Wayne Jones (pp. 3921-3928).

Methadone (MTD) is widely used for detoxification of heroin addicts and also in pain management programs. Information about the distribution of methadone between blood, plasma, and alternative specimens, such as oral fluid (OF), is needed in clinical, forensic, and traffic medicine when analytical results are interpreted. We determined MTD and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in blood, plasma, blood cells, and OF by gas chromatography–mass spectrometry (GC-MS) after adding deuterium-labeled internal standards. The analytical limits of quantitation for MTD and EDDP by this method were 20 and 3 ng/mL, respectively. The amounts of MTD and EDDP were higher in plasma (80.4 % and 76.5 %) compared with blood cells (19.6 % and 23.5 %) and we found that repeated washing of blood cells with phosphate–buffered saline increased the amounts in plasma (93.6 % and 88.6 %). Mean plasma/blood concentration ratios of MTD and EDDP in spiked samples (N = 5) were 1.27 and 1.21, respectively. In clinical samples from patients (N = 46), the concentrations of MTD in plasma and whole blood were highly correlated (r = 0.92, p < 0.001) and mean (median) plasma/blood distribution ratios were 1.43 (1.41). The correlations between MTD in OF and plasma (r = 0.46) and OF and blood (r = 0.52) were also statistically significant (p < 0.001) and the mean OF/plasma and OF/blood distribution ratios were 0.55 and 0.77, respectively. The MTD concentration in OF decreased as salivary pH increased (more basic). These results will prove useful in clinical and forensic medicine when MTD concentrations in alternative specimens are compared and contrasted.

Keywords: Blood; Plasma; Oral fluid; Methadone; EDDP; GC-MS

A fast and inexpensive procedure for the isolation of synthetic cannabinoids from ‘Spice’ products using a flash chromatography system by Bjoern Moosmann; Stefan Kneisel; Ariane Wohlfarth; Volker Brecht; Volker Auwärter (pp. 3929-3935).

In the age of the Internet, the variety of drugs offered online is constantly increasing, and new drugs emerge every month. One group of drugs showing such an enormous increase is that of synthetic cannabinoids. Since their first identification in ‘herbal mixtures’, new structural modifications continue to appear on the market. In order to keep up with this process, toxicological screening methods need to be up to date. This can become extremely difficult if no reference material is available. In this article, a fast and effective way to extract and purify synthetic cannabinoids from ‘herbal mixtures’ is presented. This method opens a new opportunity for a timely reaction by obtaining reference material straight out of the ‘herbal mixtures’ ordered via the Internet. Isolation was carried out on a flash chromatography system with gradient elution on a C18 column using methanol and 0.55 % formic acid as mobile phases. The obtained purity of all compounds exceeded 99 %. In addition to the isolation of single compounds, the method proved to be suitable for the separation of various synthetic cannabinoids in one mixture, including the diastereomers cis- and trans-CP-47,497-C8. This approach for obtaining pure standards of new drugs proved to be effective, inexpensive and much quicker than waiting for the substances to be commercially available as reference material. Figure Flash chromatography method for the isolation of synthetic cannabinoids from ‘herbal mixtures’ to obtain pure reference standards.

Keywords: Flash chromatography; Spice; Synthetic cannabinoids; Reference compounds

Simultaneous analysis of synthetic cannabinoids in the materials seized during drug trafficking using GC-MS by Hyeyoung Choi; Sewoong Heo; Sanggil Choe; Wonkyung Yang; Yuran Park; Eunmi Kim; Heesun Chung; Jaesin Lee (pp. 3937-3944).

A rapid and simple gas chromatography–mass spectrometry (GC-MS) method was developed and validated to identify and quantify synthetic cannabinoids in the materials seized during drug trafficking. Accuracy and reproducibility of the method were improved by using deuterated JWH-018 and JWH-073 as internal standards. Validation results of the GC-MS method showed that it was suitable for simultaneous qualitative and quantitative analyses of synthetic cannabinoids, and we analyzed synthetic cannabinoids in seized materials using the validated GC-MS method. As a result of the analysis, ten species of synthetic cannabinoids were identified in dried leaves (n = 40), bulk powders (n = 6), and tablets (n = 14) seized in Korea during 2009–2012, as a single ingredient or as a mixture with other active co-ingredients. JWH-018 and JWH-073 were the most frequently identified compounds in the seized materials. Synthetic cannabinoids in the dried leaves showed broad concentration ranges, which may cause unexpected toxicity to abusers. The bulk powders were considered as raw materials used to prepare legal highs, and they contained single ingredient of JWH-073, JWH-019, or JWH-250 with the purity over 70 %. In contrast, JWH-018 and JWH-073 contents in the tablets were 7.1–13.8 and 3.0–10.2 mg/g, respectively. Relatively low contents in the tablets suggest that the synthetic cannabinoids may have been added to the tablets as supplements to other active co-ingredients.

Keywords: Synthetic cannabinoid; Identification; Quantitation; Gas chromatography–mass spectrometry

Time-course measurements of caffeine and its metabolites extracted from fingertips after coffee intake: a preliminary study for the detection of drugs from fingerprints by Kenji Kuwayama; Kenji Tsujikawa; Hajime Miyaguchi; Tatsuyuki Kanamori; Yuko T. Iwata; Hiroyuki Inoue (pp. 3945-3952).

The aim of this study was to determine whether an ingested drug and its metabolites could be detected in the subject’s fingerprints. Caffeine (CF) was chosen as the model drug. Three healthy subjects were asked to consume a cup of coffee (ca. 100 mL) containing 80 micro micro mg CF as the total dose, which is the normal amount in one cup of coffee. After washing hands with water to remove external contaminants, each subject pressed the index fingertip to a collecting matrix just before consuming the test cup of coffee, and then again pressed the index fingertip to the collecting matrix after 1, 3, 5, and 7 h. The time curve of the amounts of CF and its metabolites—theobromine (TB), paraxanthine (PX), and theophylline (TP)—in fingerprints and blood was determined using liquid chromatography/tandem mass spectrometry (LC/MS). A filter paper wetted with water (50 μL) was an efficient collecting matrix for extracting the analytes from the fingertip. With optimized sample preparation and LC/MS conditions, the total operating time, from taking the fingerprints to obtaining the analytical result, was approximately 10 min. The lower limits of quantification for CF, TB, PX, and TP were 0.5, 5, 0.5, and 5 ng/fingerprint, respectively. The amount of CF or PX determined in fingerprints obtained over 7 h after coffee intake was significantly greater than the amount determined in fingerprints taken before drinking coffee. Fingerprints were a more efficient source for drug testing than other biological samples, such as blood and sweat, because the procedures for sampling and extracting the drugs were simpler and took less time. The method could be used to prove drug intake in criminal investigations.

Keywords: Fingerprint; Drug; Sweat; Caffeine; LC/MS

Determination of seven selected antipsychotic drugs in human plasma using microextraction in packed sorbent and gas chromatography–tandem mass spectrometry by B. M. da Fonseca; I. E. D. Moreno; M. Barroso; S. Costa; J. A. Queiroz; E. Gallardo (pp. 3953-3963).

A method using microextraction by packed sorbent (MEPS) and gas chromatography–tandem mass spectrometry (GC-MS/MS) is described for the determination of seven antipsychotic drugs in human plasma. The studied compounds were chlorpromazine (CPZ), haloperidol (HAL), cyamemazine, quetiapine, clozapine, olanzapine (OLZ), and levomepromazine; promazine, protriptyline, and deuterated CPZ were used as internal standards. The validation parameters included selectivity, linearity and limits of detection and quantitation, intra- and interday precision and trueness, recovery, and stability and were studied according to internationally accepted guidelines. The method was found to be linear between the lower limit of quantitation and 1000 ng/mL, except for OLZ and HAL (200 ng/mL), with determination coefficients higher than 0.99 for all analytes, and extraction efficiencies ranged from 62 to 92 %. Intra- and interday precision ranged from 0.24 to 10.67 %, while trueness was within a ±15 % interval from the nominal concentration for all analytes at all studied levels. MEPS has shown to be a rapid procedure for the determination of the selected antipsychotic drugs in human plasma, allowing reducing the handling time and the costs of analysis. Furthermore, GC-MS/MS has demonstrated to be a powerful tool for the simultaneous quantitation of the studied compounds, enabling obtaining adequate selectivity and sensitivity using a sample volume of as low as 0.25 mL.

Keywords: MEPS; GC-MS/MS; Antipsychotic drugs; Plasma

A novel homogeneous immunoassay for anthrax detection based on the AlphaLISA method: detection of B. anthracis spores and protective antigen (PA) in complex samples by Adva Mechaly; Noam Cohen; Shay Weiss; Eran Zahavy (pp. 3965-3972).

Amplified Luminescent Proximity Homogeneous Assay (AlphaLISA) technology is an energy-transfer-based assay, utilizing singlet oxygen as an energy donor to a fluorescent acceptor. The long singlet oxygen migration distance allows the energy transfer mechanism to go up to ∼200 nm, facilitating flexible and sensitive homogeneous immunoassays. While soluble protein detection using AlphaLISA was previously described, the detection of particles such as bacteria and viruses was not reported. In this work, we show for the first time the implementation of the AlphaLISA technology for the detection of a particulate antigen, i.e., Bacillus anthracis spores. Here, we show that an efficient particle immunoassay requires a high acceptor-to-donor ratio (>4:1). The results suggested that the high acceptor/donor ratio is required to avoid donor aggregation (“islands”) on the spore surface, hence facilitating donor/acceptor interaction. The developed assay enabled the detection of 10⁶ spores/mL spiked in PBS. We also demonstrate the development of a highly sensitive AlphaLISA assay for the detection of the main toxin component of anthrax, protective antigen (PA). The assay enabled the detection of 10 and 100 pg/mL PA in buffer and spiked naïve rabbit sera, respectively, and was successfully implemented in sera of anthrax-infected rabbits. To summarize, this study demonstrates that AlphaLISA enables detection of anthrax spores and toxin, utilizing short homogeneous assays. Moreover, it is shown for the first time that this technology facilitates the detection of particulate entities and might be suitable for the detection of other bacteria or viruses.

Keywords: Anthrax; PA; Spores; Immunoassay; AlphaLISA; FRET; Antibodies; Immuno-diagnostic

Design and application of GB virus C (GBV-C) peptide microarrays for diagnosis of GBV-C/HIV-1 co-infection by Leticia Fernández; M. José Bleda; M. José Gómara; Isabel Haro (pp. 3973-3982).

The main objectives of the design of GB virus C (GBV-C) peptide microarrays are the miniaturisation of antigen–antibody interaction assays, the simultaneous analysis of several peptide sequences and the reduction in the volume of serum required from patients since this always represents a limiting factor in studies to develop new systems for diagnosing human diseases. We herein report the design of a microarray immunoassay based on synthetic peptides derived from the GBV-C E2 protein to evaluate their diagnostic value in detecting anti-E2 antibodies in HIV-1 patients. To this end, peptide microarrays were initially prepared to identify the most relevant epitopes in the GBV-C E2 protein. Thus, 124 peptides composed of 18 amino acids covering the whole E2-protein sequence, with 15 residue overlaps, were spotted in triplicate onto γ-aminopropyl silane-functionalised adsorbent binding slides. The procedure to select the E2 protein epitopes was carried out using serum samples from HIV-1-infected patients. The samples had previously been tested for the presence or absence of GBV-C anti-E2 antibodies by means of the Abbott test. Thus, 11 specific epitopes in the GBV-C E2 protein were identified. Subsequently, peptide antigen microarrays were constructed using the E2 epitopes identified to detect GBV-C anti-E2 antibodies in the serum of HIV-1-infected patients with no known GBV-C co-infection. The 11 peptides selected identified anti-E2 GBV-C antibodies among HIV-1-infected patients, and a reactivity of 47 % was established. The potential antigenic peptides selected could be considered a useful tool for designing a new diagnostic system based on peptide microarrays to determine anti-GBV-C E2 antibodies in the serum of HIV-1-infected patients.

Keywords: GBV-C/HIV-1 co-infection; Diagnosis; GBV-C peptides; Microarrays

The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging by Sandrine Poëa-Guyon; Hélène Pasquier; Fabienne Mérola; Nicolas Morel; Marie Erard (pp. 3983-3987).

pH is an important parameter that affects many functions of live cells, from protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive fluorescent proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations can be performed either by ratiometric or fluorescence lifetime detection. However, most available genetically encoded pH sensors are based on green and yellow fluorescent proteins and are not compatible with multicolor approaches. Taking advantage of the strong pH sensitivity of enhanced cyan fluorescent protein (ECFP), we demonstrate here its suitability as a sensitive pH sensor using fluorescence lifetime imaging. The intracellular ECFP lifetime undergoes large changes (32 %) in the pH 5 to pH 7 range, which allows accurate pH measurements to better than 0.2 pH units. By fusion of ECFP with the granular chromogranin A, we successfully measured the pH in secretory granules of PC12 cells, and we performed a kinetic analysis of intragranular pH variations in living cells exposed to ammonium chloride.

Keywords: Enhanced cyan fluorescent protein or ECFP; pH sensor; Fluorescent protein; FLIM; PC12 cells; Granular pH

Limitations in detection of ¹⁵N incorporation by mass spectrometry in protein-based stable isotope probing (protein-SIP) by Martin Taubert; Martin von Bergen; Jana Seifert (pp. 3989-3996).

The method of protein-based stable isotope probing (protein-SIP) has previously been shown to allow the modeling of carbon fluxes in microbial communities, thus tackling one of the key questions in microbial ecology. The method allows the analysis of stable isotope distribution in peptides, revealing metabolic activities of the species present in an ecosystem. Besides carbon, an application of protein-SIP with nitrogen is of interest for resolving the nitrogen fluxes in microbial communities. Thus, the sensitivity and reliability of a protein-SIP approach employing ¹⁵N was analyzed. For this, cultivations of Pseudomonas fluorescens ATCC 17483 with different ratios of ¹⁴N/¹⁵N were performed, from 10 % down to 0.1 % ¹⁵N. After incubation leading to complete labeling of biomass, proteins were extracted and separated by one-dimensional gel electrophoresis, followed by tryptic digest and UPLC Orbitrap MS/MS analysis. ¹⁵N relative isotope abundance (RIA) was calculated based on isotopic patterns from identified peptides in mass spectra. Proteomics data have been deposited to ProteomeXchange with identifier PXD000127. The distribution of ¹⁵N RIA values among peptides was analyzed in samples with different ¹⁵N amount, and potential causes for variations within individual samples of either technical or biological origin were investigated. Using a number of 50 peptides, significant differences (p ≤ 0.05) in ¹⁵N incorporation were found between samples of different ¹⁵N RIA down to 0.1 %. The study demonstrates that protein-SIP using ¹⁵N is sufficiently sensitive for quantitative investigation of microbial activity in nitrogen cycling processes.

Keywords: Stable isotope probing; Protein-SIP; Mass spectrometry; Relative isotope abundance; ¹⁵N incorporation

Probing site-specific ¹³C/¹⁵N-isotope enrichment of spider silk with liquid-state NMR spectroscopy by Xiangyan Shi; Jeffery L. Yarger; Gregory P. Holland (pp. 3997-4008).

Solid-state nuclear magnetic resonance (NMR) has been extensively used to elucidate spider silk protein structure and dynamics. In many of these studies, site-specific isotope enrichment is critical for designing particular NMR methods for silk structure determination. The commonly used isotope analysis techniques, isotope-ratio mass spectroscopy and liquid/gas chromatography-mass spectroscopy, are typically not capable of providing the site-specific isotope information for many systems because an appropriate sample derivatization method is not available. In contrast, NMR does not require any sample derivatization or separation prior to analysis. In this article, conventional liquid-state ¹H NMR was implemented to evaluate incorporation of ¹³C/¹⁵N-labeled amino acids in hydrolyzed spider dragline silk. To determine site-specific ¹³C and ¹⁵N isotope enrichments, an analysis method was developed to fit the ¹H–¹³C and ¹H–¹⁵N J-splitting (J _CH and J _NH) ¹H NMR peak patterns of hydrolyzed silk fiber. This is demonstrated for Nephila clavipes spiders, where [U–¹³C₃,¹⁵N]-Ala and [1-¹³C,¹⁵N]-Gly were dissolved in their water supplies. Overall, contents for Ala and Gly isotopomers are extracted for these silk samples. The current methodology can be applied to many fields where site-specific tracking of isotopes is of interest.

Keywords: Spider dragline silk; NMR; Isotope labeling; Site-specific isotope enrichment

Nanodisc-solubilized membrane protein library reflects the membrane proteome by Michael T. Marty; Kyle C. Wilcox; William L. Klein; Stephen G. Sligar (pp. 4009-4016).

The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library. Figure A Nanodisc-solubilized membrane protein library is formed by extracting a population of membrane proteins into detergent and then incorporating these proteins into a heterogeneous Nanodisc library, which models the membrane proteome

Keywords: Nanodisc; Membrane proteins; Proteomics; Solubilized membrane protein library

Superior performance of liposomes over enzymatic amplification in a high-throughput assay for myoglobin in human serum by Katie A. Edwards; Katherine J. Meyers; Barbara Leonard; Antje J. Baeumner (pp. 4017-4026).

Myoglobin is one of several cardiac markers which become elevated in the blood following an acute myocardial infarction and can aid in the diagnosis of a heart attack. Here, a sandwich immunoassay for myoglobin was developed, including a thorough optimization of fluorescent dye-encapsulating liposomes versus enzymatic amplification (alkaline phosphatase and horseradish peroxidase) at each step. The optimized microtiter plate-based assay was capable of detecting as low as 11.3 pg/mL myoglobin and was successfully applied for the quantification of myoglobin in human serum. In comparison to enzymatic approaches, the liposomes demonstrated lower limits of detection, significantly reduced limits of quantification, improved signal discrimination through substantial signal enhancement, and reduced assay time. Liposomes were stable and functional at ambient temperatures for over 400 days. Finally, ease of use was greater due to lack of reliance on additional reagents, non-time-based signal enhancement, and excellent photostability. Optimal conditions identified for enzymatic approaches can also be used for liposome amplification, which makes substitution of these liposomes into existing assays straightforward. Thus, the extensive studies carried out here suggest that liposomes may be incorporated into formats currently utilizing enzymatic enhanced fluorescence with a potential for increased performance on various levels. Sandwich immunoassay for the cardiac marker myoglobin. Excellent performance of fluorescent dye-encapsulating liposomes for signal enhancement versus enzymes using commercially available fluorescent substrates was demonstrated.

Keywords: Sandwich immunoassay; Liposomes; Myoglobin; Enzymes; Fluorescence

Monoclonal antibodies with group specificity toward sulfonamides: selection of hapten and antibody selectivity by Zhanhui Wang; Ross C. Beier; Yajie Sheng; Suxia Zhang; Wenxiao Jiang; Zhaopeng Wang; Jin Wang; Jianzhong Shen (pp. 4027-4037).

Immunoassays based on the current available antibodies for large multi-sulfonamide screening programs have suffered from high selectivity for individual sulfonamides and a wide range of selectivities for different sulfonamides. In this study, five synthesized haptens, HS, BS, CS, SA10, and TS and two sulfonamides, SG and SMX were used as haptens, which may or may not contain a ring structure at the N1 position of the sulfonamides, were selected to evaluate the effectiveness for producing group-specific monoclonal antibodies (MAbs). Mice immunized with three different two-ring haptens were used for hybridoma production, which resulted in three unique MAbs recognizing 10, 13, and 15 sulfonamides showing 50 % inhibition (IC₅₀) at concentrations below 100 ng mL^–1. MAb 4D11 derived from one novel immunizing hapten could recognize 12 sulfonamides with IC₅₀ values ranging from 1.2 to 12.4 ng mL^–1, almost within 1 order of magnitude. These produced MAbs show lower IC₅₀ values in addition to significantly improved group specificity compared with previously generated MAbs. This study clearly indicates that the careful selection of the immunizing hapten has an important effect on the specificity of the generated antibodies. Figure Generation of broad-specific monoclonal antibodies against sulfonamides.

Keywords: Sulfonamides; Group specificity; Monoclonal antibody; Hapten

Localisation and quantification of benzalkonium chloride in eye tissue by TOF-SIMS imaging and liquid chromatography mass spectrometry by Nicolas Desbenoit; Isabelle Schmitz-Afonso; Christophe Baudouin; Olivier Laprévote; David Touboul; Françoise Brignole-Baudouin; Alain Brunelle (pp. 4039-4049).

Benzalkonium (BAK) chloride is the most commonly used preservative in eye drops. It is generally composed of benzyldimethyldodecylammonium C₁₂ and benzyldimethyltetradecylammonium C₁₄ and is supposed to increase penetration of active compounds. However, numerous studies have reported its toxic effect to ocular surface especially in long-term treatments like against glaucoma, a sight-threatening disease. Albino rabbits were treated with a hyperosmolar solution and a high concentration of BAK solution for 1 month. Enucleated eyes were cryo-sectioned and analysed by mass spectrometry. Mass spectrometry imaging using time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been used to characterize the spatial distribution and to determine the relative quantity of BAK at the surface of rabbit eye sections. Liquid chromatography coupled with mass spectrometry (LC-MS) using a hybrid linear ion trap-Orbitrap® mass spectrometer was used to obtain relative quantification of BAK at the sample surface. TOF-SIMS images of BAK ions indicated a distribution at the ocular surface and in deeper structures. Didecyldimethylammonium (DDMAC), which is used in hospitals as a substitute for BAK, was also detected and showed an accumulation around the eyes. After extraction with acetonitrile and chromatographic separation using a Gemini C18 column and an original elution gradient, the relative quantities of BAK and DDMAC present in the whole eye section surface were determined. This LC-MS method was validated in terms of limits of quantification, linearity, repeatability and reproducibility and its feasibility was evaluated in surgically obtained human samples. Specimens of iris, lens capsule or trabecular meshwork were found with significant levels of BAK and DDMAC, thus confirming the penetration of BAK in deep ocular structures, with potential deleterious effects induced by this cytotoxic compound. The analytical method developed here could therefore be of primary interest in the field of pharmaco-toxicology in order to localise, identify and quantify drugs or xenobiotic compounds present at biological sample surfaces. Figure Mass spectrometry image (TOF-SIMS) of rabbit eye conjunctiva treated with benzalkonium chloride

Keywords: LC-MS; Mass spectrometry imaging; TOF-SIMS; Glaucoma; Benzalkonium chloride; DDMAC

Signal-amplification detection of small molecules by use of Mg²⁺- dependent DNAzyme by Zhijun Guo; Jiahai Wang; Erkang Wang (pp. 4051-4057).

Because small molecules can be beneficial or toxic in biology and the environment, specific and sensitive detection of small molecules is one of the most important objectives of the scientific community. In this study, new signal amplification assays for detection of small molecules based on Mg²⁺-dependent DNAzyme were developed. A cleavable DNA substrate containing a ribonucleotide, the ends of which were labeled with black hole quencher (BHQ) and 6-carboxyfluorescein (FAM), was used for fluorescence detection. When the small molecule of interest is added to the assay solution, the Mg²⁺-dependent DNAzyme is activated, facilitating hybridization between the Mg²⁺-dependent DNAzyme and the DNA substrate. Binding of the substrate to the DNAzyme structure results in hydrolytic cleavage of the substrate in the presence of Mg²⁺ ions. The fluorescence signal was amplified by continuous cleavage of the enzyme substrate. Ochratoxin A (OTA) and adenosine triphosphate (ATP) were used as model analytes in these experiments. This method can detect OTA specifically with a detection limit as low as 140 pmol L⁻¹ and detect ATP specifically with a detection limit as low as 13 nmol L⁻¹. Moreover, this method is potentially extendable to detection of other small molecules which are able to dissociate the aptamer from the DNAzyme, leading to activation of the DNAzyme.

Keywords: Signal amplification; Small molecule; Ochratoxin A; ATP; DNAzyme

Combination of pentafluorophenylhydrazine derivatization and isotope dilution LC-MS/MS techniques for the quantification of apurinic/apyrimidinic sites in cellular DNA by Jie Li; Elvis M. K. Leung; Martin M. F. Choi; Wan Chan (pp. 4059-4066).

Apurinic/apyrimidinic (AP) sites are common DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and base-excision repair mechanisms of the modified bases. Due to the strong association of AP site formation with physically/chemically induced DNA damage, quantifying AP sites provides important information for risk assessment of exposure to genotoxins and oxidative stress. However, rigorous quantification of AP sites in DNA has been hampered by technical problems relating to the sensitivity and selectivity of existing analytical methods. We have developed a new isotope dilution liquid chromatography–coupled tandem mass spectrometry (LC-MS/MS) method for the rigorous quantification of AP sites in genomic DNA. The method entails enzymatic digestion of AP site-containing DNA by endo- and exonucleases, derivatization with pentafluorophenylhydrazine (PFPH), addition of an isotopically labeled PFPH derivative as internal standard, and quantification by LC-MS/MS. The combination of PFPH derivatization with LC-MS/MS analysis on a triple quadrupole mass spectrometer allows for sensitive and selective quantification of AP sites in DNA at a detection limit of 6.5 fmol, corresponding to 4 AP sites/10⁹ nt in 5 μg of DNA, which is at least ten times more sensitive than existing analytical methods. The protocol was validated by AP site-containing oligonucleotides and applied in quantifying methyl methanesulfonate-induced formation of AP sites in cellular DNA. Fig Chemistry of apurinic/apyrimidinic site formation

Keywords: Apurinic/apyrimidinic sites; LC-MS/MS; Isotope dilution; Pentafluorophenylhydrazine; Derivatization

Oral fluid and plasma 3,4-methylenedioxymethamphetamine (MDMA) and metabolite correlation after controlled oral MDMA administration by Nathalie A. Desrosiers; Allan J. Barnes; Rebecca L. Hartman; Karl B. Scheidweiler; Erin A. Kolbrich-Spargo; David A. Gorelick; Robert S. Goodwin; Marilyn A. Huestis (pp. 4067-4076).

Oral fluid (OF) offers a noninvasive sample collection for drug testing. However, 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) in OF has not been adequately characterized in comparison to plasma. We administered oral low-dose (1.0 mg/kg) and high-dose (1.6 mg/kg) MDMA to 26 participants and collected simultaneous OF and plasma specimens for up to 143 h after dosing. We compared OF/plasma (OF/P) ratios, time of initial detection (t _first), maximal concentrations (C _max), time of peak concentrations (t _max), time of last detection (t _last), clearance, and 3,4-methylenedioxyamphetamine (MDA)-to-MDMA ratios over time. For OF MDMA and MDA, C _max was higher, t _last was later, and clearance was slower compared to plasma. For OF MDA only, t _first was later compared to plasma. Median (range) OF/P ratios were 5.6 (0.1–52.3) for MDMA and 3.7 (0.7–24.3) for MDA. OF and plasma concentrations were weakly but significantly correlated (MDMA: R ² = 0.438, MDA: R ² = 0.197, p < 0.0001). Median OF/P ratios were significantly higher following high dose administration: MDMA low = 5.2 (0.1–40.4), high = 6.0 (0.4–52.3, p < 0.05); MDA low = 3.3 (0.7–17.1), high = 4.1 (0.9–24.3, p < 0.001). There was a large inter-subject variation in OF/P ratios. The MDA/MDMA ratios in plasma were higher than those in OF (p < 0.001), and the MDA/MDMA ratios significantly increased over time in OF and plasma. The MDMA and MDA concentrations were higher in OF than in plasma. OF and plasma concentrations were correlated, but large inter-subject variability precludes the estimation of plasma concentrations from OF. Figure Oral fluid and plasma 3,4-methylenedioxymethamphetamine (MDMA) concentrations in all simultaneously collected paired-positive specimens collected −0.25 to 143 h after 1.0 and 1.6 mg/kg oral MDMA administration to 26 adult participants

Keywords: Toxicology; 3,4-Methylenedioxymethamphetamine; MDMA; Oral fluid

Metabolism of levamisole and kinetics of levamisole and aminorex in urine by means of LC-QTOF-HRMS and LC-QqQ-MS by Cornelius Hess; Natalie Ritke; Sebastian Broecker; Burkhard Madea; Frank Musshoff (pp. 4077-4088).

The antihelminthic drug Levamisole can enhance cocaine effects by conversion into the amphetamine-like drug aminorex. We describe an LC-MS method for the determination of levamisole and its metabolite aminorex in human urine. Selectivity is given, calibration curves were linear within the calibration range 2.5–250 ng/mL; limits of the method were LoD 0.51 ng/mL, LoQ 1.02 ng/mL for levamisole and LoD 0.65 ng/mL, LoQ 0.76 ng/mL for aminorex. Precision data was in accordance with the guidelines (intraday precision for aminorex ranged between 5.75 and 11.0 % for levamisole between 8.36 and 10.9 %; interday precision for levamisole 10.9–16.9 % and for aminorex 7.64–12.7 %; accuracy data for levamisole −1.96 to –14.3 % and for aminorex−11.9 to–18.5 %). The validated method was successfully applied to study the urinary excretion of levamisole after the administration of 100 mg of levamisole orally. Levamisole and aminorex could be detected in post-administration urine samples. Levamisole could be detected up to 39 h after ingestion, while aminorex was detectable up to 54 h. Maximum aminorex concentrations were 45 ng/mL urine. Further metabolites of levamisole after oral ingestion by means of liquid chromatography hybrid quadrupole time-of-flight high-resolution mass spectrometry (LC-QTOF-HRMS) were identified. Only 0.5 % of the ingested drug was quantified as unchanged levamisole in urine. Besides aminorex, five isomers of aminorex and 4 hydroxy-metabolites of aminorex or its isomers were found. Furthermore, levamisole is also hydroxylated and eliminated free or conjugated with sulfate or glucuronide into urine.

Keywords: Levamisole; Aminorex; LC-MS; LC-QTOF-HRMS; Metabolism

A quantitative and comprehensive method to analyze human milk oligosaccharide structures in the urine and feces of infants by Maria Lorna A. De Leoz; Shuai Wu; John S. Strum; Milady R. Niñonuevo; Stephanie C. Gaerlan; Majid Mirmiran; J. Bruce German; David A. Mills; Carlito B. Lebrilla; Mark A. Underwood (pp. 4089-4105).

Human milk oligosaccharides (HMOs), though non-nutritive to the infant, shape the intestinal microbiota and protect against pathogens during early growth and development. Infant formulas with added galacto-oligosaccharides have been developed to mimic the beneficial effects of HMOs. Premature infants have an immature immune system and a leaky gut and are thus highly susceptible to opportunistic infections. A method employing nanoflow liquid chromatography time-of-flight mass spectrometry (MS) is presented to simultaneously identify and quantify HMOs in the feces and urine of infants, of which 75 HMOs have previously been fully structurally elucidated. Matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance MS was employed for high-resolution and rapid compositional profiling. To demonstrate this novel method, samples from mother–infant dyads as well as samples from infants receiving infant formula fortified with dietary galacto-oligosaccharides or probiotic bifidobacteria were analyzed. Ingested oligosaccharides are demonstrated in high abundance in the infant feces and urine. While the method was developed to examine specimens from preterm infants, it is of general utility and can be used to monitor oligosaccharide consumption and utilization in term infants, children, and adults. This method may therefore provide diagnostic and therapeutic opportunities. Figure Quantification of human milk oligosacchairdes in the milk, feces, and urine of a mother-infant dyad by MALDI FT-ICR (spectra) and nano-LC MS (pie charts)

Keywords: Mass spectrometry; Human milk oligosaccharides; Galacto-oligosaccharides; Preterm infants; Breast milk; Prebiotic; Infant formula

Liquid chromatography/time-of-flight mass spectrometry analysis of postmortem blood samples for targeted toxicological screening by Markus Roman; Lena Ström; Helena Tell; Martin Josefsson (pp. 4107-4125).

A liquid chromatography/time-of-flight mass spectrometry (LC-TOF-MS) method for targeted toxicological screening in human postmortem blood samples from forensic autopsy cases has been developed, validated and compared with a previously used method using gas chromatography with nitrogen–phosphorus detection (GC-NPD). Separation was achieved within 12 min by high-resolution gradient chromatography. Ions were generated in positive and negative electrospray ionization mode and were detected in 2-GHz single mass spectrometry mode, m/z range 50–1,000. Before injection, 0.25 g blood was prepared by protein precipitation with 500 μL of a mixture of acetonitrile and ethanol containing deuterated internal standards. An in-house database comprising 240 drugs and metabolites was built by analysing solutions from certified standards or other documented reference material available. Identification was based on scoring of retention time, accurate mass measurement and isotopic pattern. Validation was performed on spiked blood samples and authentic postmortem blood samples. The thresholds defined as minimum required performance levels were for most compounds in the range from 0.01 to 0.10 μg/g. Typically, a mass error of less than 2 ppm and a precision of area measurements of less than 5 % coefficient of variation were achieved. Positive identification was confirmed at concentrations up to 500 μg/g. Most compounds were determined in positive ionization mode, but for a limited number of compounds (fewer than 4 %) negative ionization was needed and a few early-eluted compounds could not be identified owing to substantial influence of interferences from the matrix and were thus not included in the screening. A robust and valid toxicological screening by LC-TOF-MS for postmortem blood samples, covering 50 % more compounds, and with higher precision and sensitivity than the previously used screening by GC-NPD was achieved.

Keywords: Postmortem; Blood; Liquid chromatography; Mass spectrometry; Time of flight; Liquid chromatography/time-of-flight mass spectrometry

Analysis of polyfluoroalkyl substances and bisphenol A in dried blood spots by liquid chromatography tandem mass spectrometry by Wanli Ma; Kurunthachalam Kannan; Qian Wu; Erin M. Bell; Charlotte M. Druschel; Michele Caggana; Kenneth M. Aldous (pp. 4127-4138).

Dried blood spots (DBS), collected as part of the newborn screening program (NSP) in the USA, is a valuable resource for studies on environmental chemical exposures and associated health outcomes in newborns. Nevertheless, determination of concentrations of environmental chemicals in DBS requires assays with great sensitivity, as the typical volume of blood available on a DBS with 16-mm diameter disc is approximately 50 μL. In this study, we developed a liquid–liquid extraction and high-performance liquid chromatography/tandem mass spectrometry method for the detection of perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), and bisphenol A (BPA) in DBS. The method was validated for accuracy, precision, and sensitivity, by spiking of target chemicals at different levels on Whatman 903 filter cards, which is used in the collection of DBS by the NSP. Contamination arising from collection, storage, and handling of DBS is an important issue to be considered in the analysis of trace levels of environmental chemicals in DBS. For the evaluation of the magnitude of background contamination, field blanks were prepared from unspotted portions of DBS filter cards collected by the NSP. The method was applied for the measurement of PFOS, PFOA, and BPA in 192 DBS specimens provided by NSP of New York State. PFOS and PFOA were detected in 100 % of the specimens analyzed. The concentrations of PFOS and PFOA measured in DBS were similar to those reported earlier in the whole blood samples of newborns. BPA was also found in 86 % of the specimens at concentrations ranging from 0.2 to 36 ng/mL (excluding two outliers). Further studies are needed to evaluate the sources of BPA exposures and health outcomes in newborns.

Keywords: Dried blood spot; Newborn screening; PFOS; PFOA; BPA; HPLC-MS/MS

GC-MS analysis of ethanol and other volatile compounds in micro-volume blood samples—quantifying neonatal exposure by Rebecca L. Cordell; Hitesh Pandya; Marie Hubbard; Mark A. Turner; Paul S. Monks (pp. 4139-4147).

A static headspace gas chromatography coupled mass spectrometry (GC-MS) method was developed and fully validated for the quantitative measurement of acetaldehyde, acetone, methanol, ethanol and acetic acid in the headspace of micro-volumes of blood using n-propanol as an internal standard. The linearity of the method was established over the range 0.2–100 mg/L (R ² > 0.99) and the limits of detection were 0.1–0.2 mg/L and lower limits quantification 0.5–1 mg/L. Precision and accuracies fell within acceptable limits (20 % for LLOQ and 15 %) for both intra- and inter-day analyses for all compounds except acetaldehyde which had inter-day variability of ≤25 %. The method was applied to analyse blood samples from neonatal patients receiving courses of ethanol excipient containing medications. Baseline levels of acetaldehyde, acetone, methanol and ethanol could be measured in patients before dosing commenced and an increase in levels of some volatiles were observed in several neonates after receiving ethanol-containing medications.

Keywords: GC-MS; Ethanol; Micro-volume; Excipient; Neonate

Determination of tenuazonic acid in human urine by means of a stable isotope dilution assay by Stefan Asam; Katharina Habler; Michael Rychlik (pp. 4149-4158).

The content of tenuazonic acid in human urine was determined by a stable isotope dilution assay (SIDA) that was recently developed for the analysis of food commodities and extensively re-validated for urine matrix in this study. Linearity of the response curve was proven between molar ratios n(labeled standard)/n(analyte) of 0.02–100. The limits of detection and determination were 0.2 and 0.6 μg/L, respectively. The mean recovery of the stable isotope dilution assay was 102 ± 3 % in the range between 1.0 and 100 μg/L. Interassay precision was 6.7 % (relative standard deviation of three triplicate analyses of a human urine sample during 3 weeks). The method was applied to two studies dealing with urinary excretion of tenuazonic acid: In the first study, tenuazonic acid was quantified in the 24-h urine of six volunteers from Germany (three female, three male) in a concentration range of 1.3–17.3 μg/L or 2.3–10.3 ng/mg⁻¹ creatinine, respectively. In the second study, two volunteers (one female, one male) ingested 30 μg tenuazonic acid by consumption of naturally contaminated whole meal sorghum infant cereals and tomato juice, respectively. The urinary excretion of the ingested tenuazonic acid was 54–81 % after 6 h, depending on matrix and volunteer. After 24 h, 87–93 % of the ingested amount of tenuazonic acid was excreted, but the fate of the remaining about 10 % is open. Thus, it is not possible to exclude potential health hazards for the consumer, completely. Figure Urinary excretion of tenuazonic acid (TA) by two volunteers (A and B) after ingestion of sorghum infant cereals [1] and tomato juice [2]

Keywords: Tenuazonic acid; Mycotoxin; Stable isotope dilution assay; SIDA; Human urine

Characterization of liquid chromatography-tandem mass spectrometry method for the determination of acrylamide in complex environmental samples by Patrick D. DeArmond; Amanda L. DiGoregorio (pp. 4159-4166).

This work describes the characterization of a solid-phase extraction (SPE) and liquid-chromatography-tandem mass spectrometry-based method for the analysis of acrylamide (AA) in complex environmental waters. The method involved the SPE of AA using activated carbon, and the AA was detected with tandem mass spectrometry after separating on an ion exclusion high-performance liquid chromatography column. The method incorporated two labeled AA standards for quantification using isotope dilution and to assess absolute extraction recovery. The method was evaluated for inter- and intra-day precision and accuracy. The method was both accurate (i.e., <30 % error) and precise (i.e., <20 % relative standard deviation), with absolute extraction recoveries averaging 37 %. The mass spectrometry provided excellent sensitivity, with instrumental limits of detection and quantitation values of 23 and 75 pg, respectively. The method detection limit was determined to be 0.021 μg/L. The analysis of AA was successfully performed in real-world samples that contained total dissolved solids concentrations ranging from 23,600 to 297,000 mg/L and AA concentrations ranging from 0.082 to 1.0 μg/L. Figure Product ion spectra of, from top to bottom, acrylamide, acrylamide-1-¹³C, and acrylamide-2,3,3-d₃. The predominant peak in each spectrum was used for quantitation

Keywords: Acrylamide; Liquid chromatography-tandem mass spectrometry; Ion exclusion; Activated carbon; Solid-phase extraction

High sensitivity liquid chromatography tandem mass spectrometric methods for the analysis of dioctyl sulfosuccinate in different stages of an oil spill response monitoring effort by Cesar E. Ramirez; Sudha Rani Batchu; Piero R. Gardinali (pp. 4167-4175).

After the incident on the Deepwater Horizon platform, around 1.8 million gallons of dispersants were used in the field as part of the response cleanup efforts. This study describes an online solid phase extraction–liquid chromatography (LC)–tandem mass spectrometry (MS/MS) method and a direct-injection LC-MS/MS method for the analysis of dioctyl sulfosuccinate (DOSS; a component in Corexit® EC9500A) in seawater at trace levels, with method detection limits (MDLs) of 7.0 and 440 ng/L and run times of 7 and 17 min, respectively. Stability and preservation studies demonstrated that samples at 4.7 μg/L could be preserved for up to 150 days without loss of analyte when stored with 33 % acetonitrile in glass containers. Data acquisition was performed by heated electrospray ionization (ESI) MS/MS operating in negative mode. Methods were validated in terms of percent recovery in fortified blank and matrix samples and to evaluate carryover. A simple modification of the direct-injection method allowed quantitation of 2-butoxyethanol, a dispersant component specific to the Corexit® EC9527A formulation. This method was used to simultaneously quantify DOSS and 2-butoxyethanol in two Corexit® formulations and extracts from an MC-252 source oil standard. MDLs in crude oil were 0.723 and 4.46 mg/kg, respectively, with recoveries of (92 ± 9)% for DOSS and (104 ± 8)% for 2-butoxyethanol. Detection of both indicators was achieved in a single chromatographic run by ESI-MS/MS operating sequentially in positive and negative mode. Corexit® EC9500A and Corexit® EC9527A were found to contain (21 ± 2)% and (22 ± 5)% w/w DOSS and 0 and (37 ± 2)% w/w 2-butoxyethanol, respectively.

Keywords: Corexit components; Dioctyl sulfosuccinate; 2-Butoxyethanol; Deepwater Horizon; Seawater; Crude oil

Targeted multidimensional gas chromatography using a heart-cutting device and cryogenic focusing for the determination of benzophenone derivatives in foodstuffs by Aurélie Bugey; Yves Janin; Patrick Edder; Stefan Bieri (pp. 4177-4185).

Photoinitiators are used to promote the polymerization process during the curing of varnishes or inks on cartonboard packaging. Depending on storage conditions and shelf life, these substances are able to migrate through the packaging layer into the foodstuff. This type of contamination phenomenon is therefore becoming a critical issue in terms of food safety. In order to tackle this problem, a fast and selective method was developed for the determination of benzophenone and three methylbenzophenone isomers in cereal-based foodstuffs and their cardboard packaging. Food samples or packages were efficiently extracted by pressurized liquid extraction using acetonitrile, and the extracts were directly injected onto the analytical system. The analysis was performed by multidimensional gas chromatography–mass spectrometry using a heart-cutting approach to reduce the background noise from complex matrices. The strategy employed two distinct cuts each containing its proper deuterated internal standard leading to accurate quantification. By integrating a cryofocusing effect, an enhancement in signal/noise ratio was achieved by a factor >10, which markedly decreased the sensitivity threshold. Moreover, baseline separation of the critical isomers allowed their unequivocal determination. The method was fully validated on cereal-based foodstuffs based upon an analysis of variance, and excellent performances were obtained at the decision limit making this method well suited for official food controls.

Keywords: Multidimensional gas chromatography; Cryotrapping; Food contact material; Packaging; Photoinitiators

Assessment of silicone as support to investigate the transformation routes of organic chemicals under environmental conditions and UV exposure. Application to selected fungicides by T. Rodríguez-Cabo; I. Rodríguez; M. Ramil; R. Cela (pp. 4187-4198).

The suitability of bulk silicone as support to follow the degradation of chemical compounds under environmental conditions and UV radiation is illustrated selecting three fungicides (fenhexamid, FEN; triadimenol, TRI and difenoconazole, DIF) as model compounds. These precursor species were first absorbed in silicone supports (10 mm length × 2 mm i.d. and 0.5 mm thickness) and then kept outdoors for several days (up to 2 months) or exposed to UV radiation (254 nm), from a low pressure mercury lamp, in the laboratory. Degradation of precursor fungicides and by-products formation was followed by liquid chromatography (LC) quadrupole time-of-flight (QTOF) mass spectrometry (MS), after desorption of silicone supports using 0.5 mL of acetonitrile. Half-lives (t _1/2) measured under UV exposure varied from 5 to 100 min. As regards environmental conditions, the most stable fungicide was DIF, degraded by just 15 % after 2 months; whereas, t _1/2 values of 30 and 83 h were calculated for FEN during summer and autumn, respectively. Supports contained by-products arising from precursor species through de-chlorination, cleavage, hydroxylation, intra-molecular cyclation and oligomerization reactions. Most of them have been previously identified in soil surface, vegetable leaves and water after application of fungicides in agriculture fields. The low cost of silicone tubes (ca. 0.4 Euros), added to their excellent chemical stability and capability to retain precursor species and their by-products, make them ideal supports to follow the transformation routes of organic compounds under environmental and simulated conditions, even for relatively stable species with t _1/2 in the range of weeks or months. Figure Scheme of degradation studies using fungicides impregnated silicone supports

Keywords: Silicone supports; Fungicides; Degradation; Liquid chromatography; Time-of flight-mass spectrometry

Profiling of secondary metabolites in tissues from Rheum palmatum L. using laser microdissection and liquid chromatography mass spectrometry by Zhitao Liang; Tungting Sham; Guangyi Yang; Ling Yi; Hubiao Chen; Zhongzhen Zhao (pp. 4199-4212).

Evaluating the quality of herbal medicines by morphological features is a convenient, quick, and practical method compared with other methods that mostly depend on modern instruments. Here, laser microdissection and ultra-performance liquid chromatography are combined with mass spectrometry to map the distribution of secondary metabolites in cells or tissues of a herb itself for correlating its bioactive components and morphological features. The root and rhizome of Rheum palmatum L. were taken as research target, which is the Chinese medicine, Radix et Rhizoma Rhei. According to fluorescent microscopic characteristics, 12 herbal cells or tissues of Radix et Rhizoma Rhei were separated by laser microdissection. Thirty-eight compounds were identified or tentatively characterized in the microdissected tissues. (+)-Catechin, 1-O-galloyl-2-O-cinnamoyl-β-d-glucose, and emodin were found to be the major components in most of the tissues. The brown ergastic substances found in rays of normal and anomalous vascular bundles as well as the parenchymatous cells of rhizome pith and the parenchymatous cells of root xylem contained higher than average amounts of these three components and more kinds of secondary metabolites. Overall, results suggest that Radix et Rhizoma Rhei of larger size and with conspicuous “brocaded patterns” and star spots are of higher quality as they tend to have greater contents of bioactive components. The study provides quantitative and specific criteria by which the quality of Radix et Rhizoma Rhei can be judged. This research also established a new, reliable, and practical method for direct profiling and imaging of secondary metabolites in any herbal tissue. Figure Linking macroscopic features with bioactive components by tissue-specific chemical profiling

Keywords: Secondary metabolites; Herbal tissues; Rheum palmatum L.; Laser microdissection; Liquid chromatography-mass spectrometry

Combination of preparative HPLC and HSCCC methods to separate phosphodiesterase inhibitors from Eucommia ulmoides bark guided by ultrafiltration-based ligand screening by Shu-Yun Shi; Mi-Jun Peng; Yu-Ping Zhang; Sheng Peng (pp. 4213-4223).

Phosphodiesterase (PDE) inhibitors are widely used because of their various pharmacological properties, and natural products are considered the most productive source of PDE inhibitors. In this work, a new ultrafiltration–high-performance liquid chromatography (HPLC)–diode-array detection–mass spectrometry based ligand screening was developed for the first screening of PDE inhibitors from Eucommia ulmoides bark, and then the target bioactive compounds were prepared by combination of stepwise preparative HPLC and high-speed countercurrent chromatography (HSCCC) methods. Experiments were conducted to optimize the parameters in ultrafiltration, stepwise preparative HPLC, and HSCCC to allow rapid and effective screening and isolation of active compounds from complex mixtures. Seven lignans with purity over 97 % were isolated and identified by their UV, electrospray ionization mass spectrometry, and NMR data as (+)-pinoresinol-4,4′-di-O-β-D-glucopyranoside (1), (+)-pinoresinol-4-O-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranoside (2), (+)-medioresinol-4,4′-di-O-β-D-glucopyranoside (3), (+)-syringaresinol-4,4′-di-O- β-D-glucopyranoside (4), (−)-olivil-4′-O-β-D-glucopyranoside (5), (−)-olivil-4-O-β-D- glucopyranoside (6), and (+)-pinoresinol-4-O-β-D-glucopyranoside (7). Compound 2 was first isolated from the genus Eucommia. Lignan diglucopyranosides (compounds 1–4) shower a greater inhibitory effect than lignan monoglucopyranosides (compounds 5–7). The method developed could be widely applied for high-throughput screening and preparative isolation of PDE inhibitors from natural products.

Keywords: Phosphodiesterase inhibitors; Eucommia ulmoides ; Lignan; Ultrafiltration; Preparative high-performance liquid chromatography; High-speed countercurrent chromatography

Characterization of radiation-induced luminescence properties and free radicals for the identification of different gamma-irradiated teas by Joong-Ho Kwon; Jae-Jun Ahn; Kashif Akram; Ik-Jae Son; Sang-O Lee (pp. 4225-4234).

Two kinds (20 each) of gamma-irradiated (0, 5, and 10 kGy) tea samples, blended powders and packed in sachets (tea bags), were investigated using photostimulated luminescence (PSL), thermoluminescence (TL), and electron spin resonance spectroscopy (ESR) to identify their irradiation status. PSL-based rapid screening was possible for all the control samples except for one packed and two powdered samples. The irradiated samples presented a good dose-dependent PSL count except two powdered samples with very low PSL sensitivity. TL analysis provided the most reliable results, in which all the irradiated samples were identified using a well-defined high-intensity TL glow curve in a temperature range of 150–250 °C. The TL results were also confirmed by determining the TL ratio (TL₁/TL₂), which was <0.1 in all the non-irradiated samples and >0.1 in the irradiated ones. ESR spectroscopy was effective for only 3 packed and 6 powdered samples showing the radiation-induced cellulosic and sugar radical signals, respectively. Figure TL-based detection of irradiated teas

Keywords: Tea; Irradiation; Photostimulated luminescence analysis; Thermoluminescence analysis; Electron spin resonance spectroscopy

Ultrasound-assisted liquid-phase microextraction based on a nanostructured supramolecular solvent by Morteza Moradi; Yadollah Yamini; Moslem Tayyebi; Hamid Asiabi (pp. 4235-4243).

Novel ultrasonically enhanced supramolecular solvent microextraction (USESSM) then high-performance liquid chromatography with ultraviolet detection have been used for extraction and determination of phthalates in water and cosmetics. Coacervates consisting of decanoic acid-based nano-structured aggregates, specifically reverse micelles, have been used the first time as solvents for ultrasound-assisted emulsification microextraction (USAEME). Sonication accelerated mass transfer of the target analytes into the nano-structured solvent from the aqueous sample, thus reducing extraction time. Several conditions affecting extraction efficiency, for example the concentrations of major components of the supramolecular solvent (tetrahydrofuran and decanoic acid), sample solution pH, salt addition, and ultrasonication time, were investigated and optimized. Under the optimum conditions, preconcentration of the analytes ranged from 176 to 412-fold and the linear range was 0.5–100 μg L⁻¹, with correlation coefficients (R ²) ≥ 0.9984. The detection sensitivity of the method was excellent, with limits of detection (LOD, S/N = 3) in the range 0.10–0.70 μg L⁻¹ and precision in the range 4.1–11.7 % (RSD, n = 5). This method was successfully used for analysis of phthalates in water and cosmetics, with good recovery of spiked phthalates (91.0–108.5 %).

Keywords: Ultrasonically enhanced supramolecular solvent microextraction; Nano-structured solvent; Phthalates; Water samples; Cosmetics

Water-compatible ‘aspartame’-imprinted polymer grafted on silica surface for selective recognition in aqueous solution by Meenakshi Singh; Abhishek Kumar; Nazia Tarannum (pp. 4245-4252).

Molecularly imprinted polymers selective for aspartame have been prepared using N-[2-ammonium-ethyl-piperazinium) maleimidopropane sulfonate copolymer bearing zwitterionic centres along the backbone via a surface-confined grafting procedure. Aspartame, a dipeptide, is commonly used as an artificial sweetener. Polymerisation on the surface was propagated by means of Michael addition reaction on amino-grafted silica surface. Electrostatic interactions along with complementary H-bonding and other hydrophobic interactions inducing additional synergetic effect between the template (aspartame) and the imprinted surface led to the formation of imprinted sites. The MIP was able to selectively and specifically take up aspartame from aqueous solution and certain pharmaceutical samples quantitatively. Hence, a facile, specific and selective technique using surface-grafted specific molecular contours developed for specific and selective uptake of aspartame in the presence of various interferrants, in different kinds of matrices is presented.

Keywords: Aspartame; Dipeptide; Molecularly imprinted polymer; Sulfobetaine polymer

Preparation of guanidinium terminus-molecularly imprinted polymers for selective recognition and solid-phase extraction (SPE) of [arginine]-microcystins by Elbert A. Mbukwa; Titus A. M. Msagati; Bhekie B. Mamba (pp. 4253-4267).

About 70 % of microcystin (MC) congeners reported in literature consist of l-arginine amino acid (R) with its guanidinium terminal extending out of the cyclic moiety of these MCs. Molecularly imprinted polymer (MIP) bearing guanidinium terminus cavities was successfully synthesised using l-arginine as a template. Non-imprinted polymer (NIP; without template) was also synthesised for control purposes. The surface area, total pore volume and average pore diameter of MIP and NIP were 267.13 m²/g, 0.63 cm³/g and 88.39 Å; 249.39 m²/g; 0.54 cm³/g and 87.14 Å, respectively. The polymers were investigated for selective recognition and extraction of [arginine]-MCs in water using solid-phase extraction/liquid chromatography-electrospray ionisation–mass spectrometry (SPE/LC-ESI-MS) method. Representative model standard solutions (0.5–10.0 μg/L) of MC-LR and MC-LY were spiked in distilled water, recovered by SPE and quantified by LC-ESI-MS. In this study, Oasis Waters™ HLB cartridges served as positive control SPE sorbents. The MIP recognised MC-LR with high recoveries (70.8–91.4 %; r 2 = 0.9962) comparable to HLB cartridges (71.0–91.85 %; r 2 = 0.9993), whereas the NIP did not recognise or retain MC-LR. Also, neither MIP nor NIP recognised or retained MC-LY. Extracts of environmental toxic Microcystis aeruginosa were subjected to SPE procedure employing MIP, NIP and HLB cartridges. Microcystin-LR, -YR, -RR, -WR, -(H₄)YR and (D-Asp³, Dha⁷)MC-RR were extracted by MIP and HLB cartridges only as confirmed by LC-ESI-MS. This study demonstrated that the prepared MIP have potential applications for the removal in water and LC-ESI-MS identifications of MCs consisting the guanidinium moiety, i.e.[arginine]-MCs, and in particular targeting commonly encountered toxic congeners, MC-LR, -YR and -RR. Online Abstract Figure 1 Synthesis of guanidinium-terminus-based molecularly imprinted polymers and their applications for selective recognition, binding and solid-phase extraction of MC-LR from aqueous media

Keywords: Guanidinium terminus; MIP; SPE; Selective recognition; [arginine]-Microcystins

Synthesis and evaluation of molecularly imprinted polymeric microspheres for highly selective extraction of an anti-AIDS drug emtricitabine by Jia-ping Lai; Li Xie; Hui Sun; Fang Chen (pp. 4269-4275).

The molecularly imprinted polymeric microspheres (MIPMs, 3∼5 μm), used as high-performance liquid chromatography (HPLC) and solid-phase extraction (SPE) packing materials for anti-AIDS drug emtricitabine (FTC), were synthesized by precipitation polymerization. The effects of ratio of chloroform to acetonitrile on the morphology and diameter of MIPMs were investigated. The prepared MIPMs were characterized by HPLC. The imprinting factor (2.26) suggests that the resultant MIPMs exhibit good recognition and affinity to FTC. In addition, the MIPMs were used in SPE as packing material for separation and enrichment of FTC. The recovery of FTC on MIPMs cartridge was 97.6 % in standard solution. Finally, the MIPMs cartridge was applied to extract the FTC in human serum samples. Impurities in sample have been mostly removed, and the average recovery of 92.5 % was obtained with a detection limit of 0.005 μg/mL and a linear range of 0.02∼4.0 μg/mL. The method established can be used to monitor the FTC in human serum sample with good accuracy and selectivity.

Keywords: Emtricitabine; Molecularly imprinted polymeric microspheres (MIPMs); Anti-AIDS drugs; Solid-phase extraction (SPE); Serum

A high-throughput microfluidic biochip to quantify bacterial adhesion to single host cells by real-time PCR assay by Rui Zhang; Hai-Qing Gong; Xu Dong Zeng; Chun Chau Sze (pp. 4277-4282).

A high-throughput microfluidic poly-(dimethylsiloxane) biochip was developed to quantify bacterial adhesion to single host cells by real-time PCR assay. The biochip is simply structured with a two-dimensional array of 900 micro-wells, one inlet, and one outlet micro-channels. Isolation of single infected host cells into the individual micro-wells of the biochip was achieved by one-step vacuum-driven microfluidics. The adhered bacterial cells were then quantified by direct on-chip real-time PCR assay with single-bacterium-detection sensitivity. The performance of this microfluidic platform was demonstrated through profiling of the association of a common bacterial pathogen, Pseudomonas aeruginosa, to single host human lung epithelial A549 cells, revealing an adherence distribution that has not been previously reported. This microfluidic platform offers a simple and effective tool for biologists to analyze pathogen–host interaction at the single-cell level without the necessities of fluorescence labeling. The chip can similarly be used for other PCR-based applications requiring single-cell analysis.

Keywords: Microfluidic; Biochip; High-throughput PCR; Bacterial adhesion; Single cell

Determination of the molecular weight of poly(ethylene glycol) in biological samples by reversed-phase LC–MS with in-source fragmentation by Bethanne M. Warrack; Brian P. Redding; Guodong Chen; Mark S. Bolgar (pp. 4283-4287).

PEGylation has been widely used to improve the biopharmaceutical properties of therapeutic proteins and peptides. Previous studies have used multiple analytical techniques to determine the fate of both the therapeutic molecule and unconjugated poly(ethylene glycol) (PEG) after drug administration. A straightforward strategy utilizing liquid chromatography–mass spectrometry (LC–MS) to characterize high-molecular weight PEG in biologic matrices without a need for complex sample preparation is presented. The method is capable of determining whether high-MW PEG is cleaved in vivo to lower-molecular weight PEG species. Reversed-phase chromatographic separation is used to take advantage of the retention principles of polymeric materials whereby elution order correlates with PEG molecular weight. In-source collision-induced dissociation (CID) combined with selected reaction monitoring (SRM) or selected ion monitoring (SIM) mass spectrometry (MS) is then used to monitor characteristic PEG fragment ions in biological samples. MS provides high sensitivity and specificity for PEG and the observed retention times in reversed-phase LC enable estimation of molecular weight. This method was successfully used to characterize PEG molecular weight in mouse serum samples. No change in molecular weight was observed for 48 h after dosing. Figure Correlation between log PEG MW and retention time observed by reversed-phase LC-MS with in-source fragmentation

Keywords: Poly(ethylene glycol); PEG; Mass spectrometry; LC–MS; In-source collision-induced dissociation; Selected reaction monitoring

Improvement of mass spectrometry analysis of glycoproteins by MALDI-MS using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid by Makoto Watanabe; Kazuya Terasawa; Kaoru Kaneshiro; Hiromasa Uchimura; Rie Yamamoto; Yuko Fukuyama; Kazuharu Shimizu; Taka-Aki Sato; Koichi Tanaka (pp. 4289-4293).

Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.

Keywords: 3-AQ/CHCA; Liquid matrix; Glycopeptide; MALDI; HER2

Simultaneous determination of opiates, methadone, amphetamines, cocaine, and metabolites in human placenta and umbilical cord by LC-MS/MS by Ana de Castro; Ariana Díaz; Beatriz Piñeiro; Elena Lendoiro; Angelines Cruz; Manuel López-Rivadulla; Marta Concheiro (pp. 4295-4305).

LC-MS/MS methods for the quantification of morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, 6-acetylmorphine, cocaine, benzoylecgonine, ecgonine methyl ester, hydroxybenzoylecgonine, cocaethylene, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), methadone, and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in human placenta and umbilical cord were developed and validated. Specimens (1 ± 0.02 g) were homogenized with the Ultra-Turrax T8 disperser and centrifuged, and the supernatant was submitted to solid-phase extraction with Oasis MCX cartridges. Chromatographic separation was performed using an Atlantis T3 analytical column (100 × 2.1 mm, 3 μm) and a gradient of 0.1 % formic acid and acetonitrile. Selectivity was verified in 10 different blank specimens. The method was linear from 1–5 to 100–500 ng/g, depending on the analyte. Limits of detection and quantification ranged from 0.5 to 2.5 ng/g and 1 to 5 ng/g, respectively. Method imprecision was ≤15.3 %, except for MDMA at low quality control (18.1 %); accuracy, 87.1 to 114 %; extraction efficiency, 16.3 to 154.0 % (%CV = 1.8-39.4 %); matrix effect, −75.7 to 449.9 % (%CV = 3.5–50 %); and process efficiency, 8.7 to 316.0 %. The method was applied to authentic placenta and umbilical cord specimens from drug-user pregnant women.

Keywords: Placenta; Umbilical cord; Cocaine; Methadone; Opioids; Amphetamines

Water-based slow injection ultrasound-assisted emulsification microextraction for the determination of deoxynivalenol and de-epoxy-deoxynivalenol in maize and pork samples by Songqing Li; Yanshen Li; Ying Wang; Wenfeng Zhou; Haixiang Gao; Suxia Zhang (pp. 4307-4311).

A novel water-based slow injection ultrasound-assisted emulsification microextraction (SI-USAEME) method followed by ultra-high performance liquid chromatography-tandem mass spectrometry analysis was developed for the rapid pretreatment and determination of deoxynivalenol (DON) and its metabolite, de-epoxy-deoxynivalenol (DOM-1), in maize and pork samples. After optimization, the method recoveries for DON and DOM-1 ranged from 73 to 85 % with intraday and interday variations less than 9.4 and 9.2 %, respectively. The limits of detection for DON were 4.2 μg kg⁻¹ in maize and 6.2 and 5.9 μg kg⁻¹ for DON and DOM-1, respectively, in pork. In addition, an immunoaffinity column (IAC) was prepared. A study comparing the IAC cleanup method, the solid-phase extraction (SPE) cleanup method, and the proposed SI-USAEME method was presented. The water-based SI-USAEME method could become a simple, low-cost alternative to the conventional IAC and SPE method. The method was successfully applied to the analysis of commercial maize and pork products.

Keywords: Water-based slow injection ultrasound-assisted emulsification microextraction; Ultra-high performance liquid chromatography-tandem mass spectrometry; Deoxynivalenol; De-epoxy-deoxynivalenol; Maize; Pork

Rapid and sensitive analysis of phthalate metabolites, bisphenol A, and endogenous steroid hormones in human urine by mixed-mode solid-phase extraction, dansylation, and ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry by He-xing Wang; Bin Wang; Ying Zhou; Qing-wu Jiang (pp. 4313-4319).

Steroid hormone levels in human urine are convenient and sensitive indicators for the impact of phthalates and/or bisphenol A (BPA) exposure on the human steroid hormone endocrine system. In this study, a rapid and sensitive method for determination of 14 phthalate metabolites, BPA, and ten endogenous steroid hormones in urine was developed and validated on the basis of ultra-performance liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry. The optimized mixed-mode solid phase-extraction separated the weakly acidic or neutral BPA and steroid hormones from acidic phthalate metabolites in urine: the former were determined in positive ion mode with a methanol/water mobile phase containing 10 mM ammonium formate; the latter were determined in negative ion mode with a acetonitrile/water mobile phase containing 0.1 % acetic acid, which significantly alleviated matrix effects for the analysis of BPA and steroid hormones. Dansylation of estrogens and BPA realized simultaneous and sensitive analysis of the endogenous steroid hormones and BPA in a single chromatographic run. The limits of detection were less than 0.84 ng/mL for phthalate metabolites and less than 0.22 ng/mL for endogenous steroid hormones and BPA. This proposed method had satisfactory precision and accuracy, and was successfully applied to the analyses of human urine samples. This method could be valuable when investigating the associations among endocrine-disrupting chemicals, endogenous steroid hormones, and relevant adverse outcomes in epidemiological studies. Figure Analytical flowchart of phthalate metabolites, bisphenol A, and endogenous steroid hormones in human urine

Keywords: Phthalate metabolites; Bisphenol A; Endogenous steroid hormones; Mixed-mode solid-phase extraction; Liquid chromatography–tandem mass spectrometry; Urine

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Analytical and Bioanalytical Chemistry (v.405, #12)

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Analytical and Bioanalytical Chemistry (v.405, #12)