Analytical and Bioanalytical Chemistry (v.402, #9)

Stable isotope dilution assay for the accurate determination of mycotoxins in maize by UHPLC-MS/MS by Elisabeth Varga; Thomas Glauner; Robert Köppen; Katharina Mayer; Michael Sulyok; Rainer Schuhmacher; Rudolf Krska; Franz Berthiller (2675-2686).
A fast, easy-to-handle and cost-effective analytical method for 11 mycotoxins currently regulated in maize and other cereal-based food products in Europe was developed and validated for maize. The method is based on two extraction steps using different acidified acetonitrile–water mixtures. Separation is achieved using ultrahigh-performance liquid chromatography (UHPLC) by a linear water–methanol gradient. After electrospray ionisation, tandem mass spectrometric detection is performed in dynamic multiple reaction monitoring mode. Since accurate mass spectrometric quantification is hampered by matrix effects, uniformly [13C]-labelled mycotoxins for each of the 11 compounds were added to the sample extracts prior to UHPLC-MS/MS analysis. Method performance parameters were obtained by spiking blank maize samples with mycotoxins before as well as after extraction on six levels in triplicates. The twofold extraction led to total recoveries of the extraction steps between 97% and 111% for all target analytes, including fumonisins. The [13C]-labelled internal standards efficiently compensated all matrix effects in electrospray ionisation, leading to apparent recoveries between 88% and 105% with reasonable additional costs. The relative standard deviations of the whole method were between 4% and 11% for all analytes. The trueness of the method was verified by the measurement of several maize test materials with well-characterized concentrations. In conclusion, the developed method is capable of determining all regulated mycotoxins in maize and presuming similar matrix effects and extraction recovery also in other cereal-based foods.
Keywords: Mycotoxins; Stable isotope dilution assay; Multi-target analysis; Ultrahigh-performance liquid chromatography; Tandem mass spectrometry; Maize

A general approach for the purification and quantitative glycomic analysis of human plasma by Samnang Tep; Marina Hincapie; William S. Hancock (2687-2700).
The development of a general method for the purification and quantitative glycomic analysis of human plasma samples to characterize global glycosylation changes shall be presented. The method involves multiple steps, including the depletion of plasma via multi-affinity chromatography to remove high abundant proteins, the enrichment of the lower abundant glycoproteins via multi-lectin affinity chromatography, the isotopic derivatization of released glycans, and quantitative analysis by MALDI-TOF MS. Isotopic derivatization of glycans is accomplished using the well-established chemistry of reductive amination to derivatize glycans with either a light analog (12C anthranilic acid) or a heavy analog (13C7 anthranilic acid), which allows for the direct comparison of the alternately labeled glycans by MALDI-TOF MS. The method displays a tenfold linear dynamic range for both neutral and sialylated glycans with sub-picomolar sensitivity. Additionally, by using anthranilic acid, a very sensitive fluorophore, as the derivatization reagent, the glycans can be analyzed by chromatography with fluorescence detection. The utility of this methodology is highlighted by the many diseases and disorders that are known to either show or be the result of changes in glycosylation. A method that provides a generic approach for sample preparation and quantitative data will help to further advance the field of glycomics.
Keywords: Isotopic label; Glycomics; MALDI-TOF MS; Human plasma

Using immobilized enzymes to reduce RNase contamination in RNase mapping of transfer RNAs by mass spectrometry by Annika Butterer; Margret Zorc; Colette M. Castleberry; Patrick A. Limbach (2701-2711).
RNase (ribonuclease) mapping by nucleobase-specific endonucleases combined with mass spectrometry (MS) is a powerful analytical method for characterizing ribonucleic acids such as transfer RNAs. Typical free solution enzymatic digestion of RNA samples results in a significant amount of RNase being present in the sample solution analyzed by MS. In some cases, the RNase can lead to contamination of the high performance liquid chromatography and MS instrumentation. Here we investigate and compare several different approaches for reducing or eliminating contaminating RNase from the digested RNA sample before LC-MS analysis. Approaches using immobilized RNases were found to be most effective, with no enzyme carryover into the digested sample detected. Among the various options for immobilized RNases, we show that carbodiimide-based reactions can be used to couple RNases to carboxylic acid-terminated magnetic beads. The immobilized enzymes retain biological activity, are re-usable, and do not interfere with subsequent LC-MS analysis of the expected RNase digestion products. The use of immobilized RNases provides a simple approach for eliminating enzyme contamination in mass spectrometry-based RNase mapping experiments. Figure Immobilizing RNases eliminates contamination during RNase mapping analysis by mass spectrometry
Keywords: tRNA; RNA sequencing; Enzyme immobilization; Enzyme contamination; ncRNA

Standard-based real-time or quantitative polymerase chain reaction quantitation of an unknown sample’s DNA concentration (i.e., [DNA]unk) assumes that the concentration dependence of the standard and unknown reactions (related to reaction efficiency, E) are equivalent. In our work with background food-borne organisms which can interfere with pathogen detection, we have found that it is generally possible to achieve an acceptable E (1 ± 0.05) for standard solutions by optimizing the PCR conditions, template purity, primer sequence, and amplicon lengths. However, this is frequently not true for the solutions containing unknown amounts of target DNA inasmuch as cell extracts are more chemically complex than the standards which have been amplified (230-fold) as well as undergone a purification process. When significant differences in E occur, it is not possible to accurately estimate unknown target DNA concentration from the standard solution’s slope and intercept (from threshold cycle number, or C T , versus Log[DNA] data). What is needed is a standard-mediated intercept which can be specifically coupled with an unknown solution’s PCR concentration dependence. In this work, we develop a simple mathematical procedure to generate a new standard curve with a slope (∂C T /∂Log[Dilution]unk) derived from at least three dilutions of the unknown target DNA solution ([DNA]unk) and an intercept calculated from the unknown’s C T  s, DNA concentrations interpolated from the standard curve (i.e., the traditional estimate of [DNA]unk), and ∂C T /∂Log[Dilution]unk. We were able to achieve this due to our discovery of the predictable way in which the observed and ideal C T versus Log[DNA] slopes and intercepts deviate from one another. This “correction” in the standard-based [DNA]unk determination is typically 20–60% when the difference in the standard and unknown E is >0.1.
Keywords: Real-time PCR or qPCR; 16S rDNA or rRNA gene; DNA quantitation

Peripheral endocannabinoid microdialysis: in vitro characterization and proof-of-concept in human subjects by Alexander A. Zoerner; Christin Rakers; Stefan Engeli; Sandor Batkai; Marcus May; Jens Jordan; Dimitrios Tsikas (2727-2735).
In vivo endocannabinoid (EC) microdialysis has only seldom been performed, mostly in rodent brain tissue. Low solubility in aqueous media, adsorption to surfaces, and instability with co-present human serum albumin (HSA) are the major obstacles in EC microdialysis. The addition of hydroxypropyl-ß-cyclodextrine (HPCD) to the perfusion fluid has been previously described to facilitate lipid microdialysis, but the general biophysical properties of HPCD, especially with respect to peripheral EC microdialysis, have not been described before. We report on the characterization of EC microdialysis using an in vitro system using Ringer’s solution with 10% HPCD as the perfusion fluid and with fatty acid-free HSA as the matrix fluid. The endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2AG) were measured using LC-MS/MS. AEA was stable in the perfusion and matrix fluids, whereas 2AG was only stable in the perfusion fluid. In the matrix fluid, 2AG underwent rapid isomerization to 1-arachidonoyl glycerol. A relative recovery of 3.5% for AEA was found with 10% HPCD in the perfusion fluid and a flow rate of 1 μL/min. For 2AG, a similar relative recovery of 3.5% was estimated. Since 2AG was found unstable in the matrix fluid, a reliable calculation of the relative recovery rates was not possible. Delivery and recovery experiments revealed unequal inward and outward EC transport across the microdialysis membrane. Contrary to usual microdialysis findings, we observed increasing recovery rates for AEA with increasing flow rates. Long equilibration times of several hours were necessary to obtain constant relative recovery rates. In a proof-of-concept study in humans, we collected AEA from subcutaneous abdominal adipose tissue employing the described methodology. Our study suggests that the microdialysis technique is not suitable for the exact quantification of tissue EC concentrations, but it allows for their rough estimation. Figure Microdialysis of peripheral endocannabinoids, especially anandamide (AEA).
Keywords: Microdialysis; Endocannabinoids; Anandamide; 2-Arachidonoyl glycerol; LC-MS/MS; Hydroxypropyl-ß-cyclodextrine

Chromatography, mass spectrometry, and molecular modeling studies on ammodytoxins by Marija Brgles; Branimir Bertoša; Wolfgang Winkler; Tihana Kurtović; Günter Allmaier; Martina Marchetti-Deschmann; Beata Halassy (2737-2748).
The ammodytoxins (Atxs) are neurotoxic phospholipases which occur in Vipera ammodytes ammodytes (Vaa) snake venom. There are three Atx isoforms, A, B, and C, which differ in only five amino acid positions at the C-terminus but differ substantially in their toxicity. The objective of this study was to establish an analytical method for unambiguous identification of all three isoforms and to use the method to assess a procedure for purification of the most toxic phospholipase, AtxA, from the venom. Isolation procedure for AtxA consisted of isolation of Atx-cross-reactive material (proteins recognized by anti-Atx antibodies), by use of an affinity column, then cation exchange on CIM (Convective Interaction Media) disks. The purification procedure was monitored by means of reversed-phase chromatography (RPC) and mass spectrometry (MS). Although previous cation exchange of the pure isoforms enabled separate elution of AtxA from B and C, separation of AtxA from Atxs mixture was not accomplished. RPC was not able to separate the Atx isoforms, whereas an MS based approach proved to be more powerful. Peptides resulting from tryptic digestion of Atxs which enable differentiation between the three isoforms were successfully detected and their sequences were confirmed by post-source decay (PSD) fragmentation. Separation of Atx isoforms by ion-exchange chromatography is most presumably prevented by Atxs heterodimer formation. The tendency of Atxs to form homodimers and heterodimers of similar stability was confirmed by molecular modeling.
Keywords: Ammodytoxin (Atx); MALDI mass spectrometry; Molecular modeling; Chromatography; Convective Interaction Media (CIM)

The analytical methodology for the in vivo study of selenium metabolism using two enriched selenium isotopes has been modified, allowing for the internal correction of spectral interferences and mass bias both for total selenium and speciation analysis. The method is based on the combination of an already described dual-isotope procedure with a new data treatment strategy based on multiple linear regression. A metabolic enriched isotope (77Se) is given orally to the test subject and a second isotope (74Se) is employed for quantification. In our approach, all possible polyatomic interferences occurring in the measurement of the isotope composition of selenium by collision cell quadrupole ICP-MS are taken into account and their relative contribution calculated by multiple linear regression after minimisation of the residuals. As a result, all spectral interferences and mass bias are corrected internally allowing the fast and independent quantification of natural abundance selenium (natSe) and enriched 77Se. In this sense, the calculation of the tracer/tracee ratio in each sample is straightforward. The method has been applied to study the time-related tissue incorporation of 77Se in male Wistar rats while maintaining the natSe steady-state conditions. Additionally, metabolically relevant information such as selenoprotein synthesis and selenium elimination in urine could be studied using the proposed methodology. In this case, serum proteins were separated by affinity chromatography while reverse phase was employed for urine metabolites. In both cases, 74Se was used as a post-column isotope dilution spike. The application of multiple linear regression to the whole chromatogram allowed us to calculate the contribution of bromine hydride, selenium hydride, argon polyatomics and mass bias on the observed selenium isotope patterns. By minimising the square sum of residuals for the whole chromatogram, internal correction of spectral interferences and mass bias could be accomplished. As a result, the tracer/tracee ratio could be calculated for each selenium-containing species and a time relationship for synthesis and degradation established. Both selenite and selenized yeast labelled with 77Se were employed for comparative purposes.
Keywords: Mass spectrometry; ICP-MS; Speciation; Biological samples

Glycoproteins secreted or expressed on the cell surface at specific pathophysiological stages are well-recognized disease biomarkers and therapeutic targets. While mapping of specific glycan structures can be performed at the level of released glycans, site-specific glycosylation and identification of specific protein carriers can only be determined by analysis of glycopeptides. A key enabling step in mass spectrometry (MS)-based glycoproteomics is the ability to selectively or non-selectively enrich for the glycopeptides from a total pool of a digested proteome for MS analysis since the highly heterogeneous glycopeptides are usually present at low abundance and ionize poorly compared with non-glycosylated peptides. Among the most common approaches for non-destructive and non-glycan-selective glycopeptide enrichment are strategies based on various forms of hydrophilic interaction liquid chromatography (HILIC). We present here a variation of this method using amine-derivatized Fe3O4 nanoparticles, in concert with in situ peptide N-glycosidase F digestion for direct matrix-assisted laser desorption/ionization–mass spectrometry analysis of N-glycosylation sites and the released glycans. Conditions were also optimized for efficient elution of the enriched glycopeptides from the nanoparticles for on-line nanoflow liquid chromatography–MS/MS analysis. Successful applications to single glycoproteins as well as total proteomic mixtures derived from biological fluids established the unrivaled practical versatility of this method, with enrichment efficiency comparable to other HILIC-based methods.
Keywords: Glycoproteomics; N-glycosylation sites; Glycopeptide capture; Nanoparticles; Mass spectrometry

Multi-residue analysis of free and conjugated hormones and endocrine disruptors in rat testis by QuEChERS-based extraction and LC-MS/MS by Charlène Pouech; Mikaël Tournier; Nadia Quignot; Agneta Kiss; Laure Wiest; Florent Lafay; Marie-Magdeleine Flament-Waton; Emmanuel Lemazurier; Cécile Cren-Olivé (2777-2788).
Endocrine disrupting compounds (EDCs) are suspected to be responsible for many disorders of the human reproductive system. To establish a causality relationship between exposure to endocrine disruptors and disease, experiments on animals must be performed with improved or new analytical tools. Therefore, a simple, rapid, and effective multi-residue method was developed for the determination of four steroid hormones (i.e., testosterone, androstenedione, estrone, and estradiol), glucuronide and sulfate conjugates of estrone and estradiol and four endocrine disruptors in rat testis (i.e., bisphenol A, atrazine, and active metabolites of methoxychlor and vinclozolin). The sample preparation procedure was based on the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) approach. An analytical method was then developed to quantify these compounds at ultra-trace levels by liquid chromatography coupled to tandem mass spectrometry. The QuEChERS extraction was optimized with regard to the acetonitrile/water ratio used in the extraction step, the choice of the cleanup method and the acetonitrile/hexane ratio used in the cleanup step. The optimized extraction method exhibited recoveries between 89% and 108% for all tested compounds except the conjugates (31% to 58%). The detection limits of all compounds were below 20 ng g−1 of wet weight of testis. The method was subsequently applied to determine the levels of hormones and EDCs in seven rat testis samples. Figure
Keywords: LC-MS/MS; Multi-residue; QuEChERS; Endocrine disruptors; Hormones; Testis

Qualitative detection of desmopressin in plasma by liquid chromatography–tandem mass spectrometry by Simone Esposito; Koen Deventer; Guy T’Sjoen; Anna Vantilborgh; Frans T. Delbeke; An-Sofie Goessaert; Karel Everaert; Peter Van Eenoo (2789-2796).
This work describes a liquid chromatography–electrospray tandem mass spectrometry method for detection of desmopressin in human plasma in the low femtomolar range. Desmopressin is a synthetic analogue of the antidiuretic hormone arginine vasopressin and it might be used by athletes as a masking agent in the framework of blood passport controls. Therefore, it was recently added by the World Anti-Doping Agency to the list of prohibited substances in sport as a masking agent. Mass spectrometry characterization of desmopressin was performed with a high-resolution Orbitrap-based mass spectrometer. Detection of the peptide in the biological matrix was achieved using a triple-quadrupole instrument with an electrospray ionization interface after protein precipitation, weak cation solid-phase extraction and high performance liquid chromatography separation with an octadecyl reverse-phase column. Identification of desmopressin was performed using three product ions, m/z 328.0, m/z 120.0, and m/z 214.0, from the parent ion, m/z 535.5. The extraction efficiency of the method at the limit of detection was estimated as 40% (n = 10), the ion suppression as 5% (n = 10), and the limit of detection was 50 pg/ml (signal-to-noise ratio greater than 3). The selectivity of the method was verified against several endogenous and synthetic desmopressin-related peptides. The performance and the applicability of the method were tested by analysis of clinical samples after administration of desmopressin via intravenous, oral, and intranasal routes. Only after intravenous administration could desmopressin be successfully detected.
Keywords: Desmopressin; Liquid chromatography–mass spectrometry; Doping; Plasma

Dynamic monitoring of glucagon secretion from living cells on a microfluidic chip by Jonathan G. Shackman; Kendra R. Reid; Colleen E. Dugan; Robert T. Kennedy (2797-2803).
A rapid microfluidic based capillary electrophoresis immunoassay (CEIA) was developed for on-line monitoring of glucagon secretion from pancreatic islets of Langerhans. In the device, a cell chamber containing living islets was perfused with buffers containing either high or low glucose concentration. Perfusate was continuously sampled by electroosmosis through a separate channel on the chip. The perfusate was mixed on-line with fluorescein isothiocyanate-labeled glucagon (FITC-glucagon) and monoclonal anti-glucagon antibody. To minimize sample dilution, the on-chip mixing ratio of sampled perfusate to reagents was maximized by allowing reagents to only be added by diffusion. Every 6 s, the reaction mixture was injected onto a 1.5-cm separation channel where free FITC-glucagon and the FITC-glucagon–antibody complex were separated under an electric field of 700 V cm−1. The immunoassay had a detection limit of 1 nM. Groups of islets were quantitatively monitored for changes in glucagon secretion as the glucose concentration was decreased from 15 to 1 mM in the perfusate revealing a pulse of glucagon secretion during a step change. The highly automated system should be enable studies of the regulation of glucagon and its potential role in diabetes and obesity. The method also further demonstrates the potential of rapid CEIA on microfluidic systems for monitoring cellular function.
Keywords: Electrophoresis; Immunoassay; Cells on chips; Microfluidics; Glucagon

Microfluidic chip based nano liquid chromatography coupled to tandem mass spectrometry for the determination of abused drugs and metabolites in human hair by Kevin Y. Zhu; K. Wing Leung; Annie K. L. Ting; Zack C. F. Wong; Winki Y. Y. Ng; Roy C. Y. Choi; Tina T. X. Dong; Tiejie Wang; David T. W. Lau; Karl W. K. Tsim (2805-2815).
A microfluidic chip based nano-HPLC coupled to tandem mass spectrometry (nano-HPLC-Chip-MS/MS) has been developed for simultaneous measurement of abused drugs and metabolites: cocaine, benzoylecgonine, cocaethylene, norcocaine, morphine, codeine, 6-acetylmorphine, phencyclidine, amphetamine, methamphetamine, MDMA, MDA, MDEA, and methadone in the hair of drug abusers. The microfluidic chip was fabricated by laminating polyimide films and it integrated an enrichment column, an analytical column and a nanospray tip. Drugs were extracted from hairs by sonication, and the chromatographic separation was achieved in 15 min. The drug identification and quantification criteria were fulfilled by the triple quardropule tandem mass spectrometry. The linear regression analysis was calibrated by deuterated internal standards with all of the R 2 at least over 0.993. The limit of detection (LOD) and the limit of quantification (LOQ) were from 0.1 to 0.75 and 0.2 to 1.25 pg/mg, respectively. The validation parameters including selectivity, accuracy, precision, stability, and matrix effect were also evaluated here. In conclusion, the developed sample preparation method coupled with the nano-HPLC-Chip-MS/MS method was able to reveal the presence of drugs in hairs from the drug abusers, with the enhanced sensitivity, compared with the conventional HPLC-MS/MS.
Keywords: Microfluidic chip; LC-MS; Drug abuse; Hair analysis; Method development

A label-free electrochemical biosensor for detecting DNA hybridisation was developed by monitoring the change in the voltammetric activity of ferrocenecarboxylic acid at the biosensor–solution interface. The biosensor was constructed by initially immobilising on a glassy carbon electrode an anchoring layer consisting of chitosan, carboxyl group functionalised carbon nanofibres and glutaraldehye. Chitosan acted as an adhering agent and carbon nanofibres were strategically used to provide a large surface area with binding points for DNA immobilisation, while glutaraldehye was a linker for DNA probes on the electrode surface. Based on a two-factorial design, cyclic voltammetry of [Fe(CN)6]3−/4− was performed to optimise the composition of the anchoring layer. Next, a 17-base pair DNA probe was attached to the anchoring layer, followed by its complementary target. Zr(IV) ion, known to exhibit affinity for oxygen-containing electroactive markers, for example, ferrocenecarboxylic acid, was then coordinated in the DNA duplex. In this way, ferrocenecarboxylic acid was attracted towards the biosensor for oxidation. A change in the voltammetric oxidation current of ferrocenecarboxylic acid pre- and post-hybridisation was used to provide an indication of hybridisation. A linear dynamic range between 0.5 and 40 nM and a detection limit of 88 pM of DNA target were then achieved. In addition, the biosensor exhibited good selectivity, repeatability and stability for the determination of DNA sequences.
Keywords: Electrochemical DNA sensor; Label-free detection; Zr(IV)-coordinated DNA duplex; Carbon nanofibre; Chitosan

Surface plasmon resonance detection of silver ions and cysteine using DNA intercalator-based amplification by Chia-Chen Chang; Shenhsiung Lin; Shih-Chung Wei; Yu Chu-Su; Chii-Wann Lin (2827-2835).
We report the development of a surface plasmon resonance sensor based on the silver ion (Ag+)-induced conformational change of a cytosine-rich, single-stranded DNA for the detection of Ag+ and cysteine (Cys) in aqueous solutions. In the free state, single-stranded oligonucleotides fold into double-helical structures through the addition of Ag+ to cytosine–cytosine (C–C) mismatches. However, in the presence of Cys, which competitively binds to Ag+, the formation of the C–Ag+–C assembly is inhibited, resulting in free-state, single-stranded oligonucleotides. To enhance sensitivity, the DNA intercalator, daunorubicin, was employed to achieve signal enhancement. The detection limit for Ag+ was 10 nM with a measurement range of 50–2,000 nM, and the detection limit for Cys was 50 nM with a measurement range of 50–2,000 nM. This simple assay was also used to individually determine the spiked Ag+ concentration in water samples and Cys concentrations in biological fluid samples.
Keywords: Silver ion; Cysteine; Surface plasmon resonance; DNA intercalator; Cytosine–cytosine mismatch

Sr isotope measurements in beef—analytical challenge and first results by S. Rummel; C. H. Dekant; S. Hölzl; S. D. Kelly; M. Baxter; N. Marigheto; C. R. Quetel; R. Larcher; G. Nicolini; H. Fröschl; H. Ueckermann; J. Hoogewerff (2837-2848).
The strontium isotope ratio (87Sr/86Sr) in beef, derived from 206 European cattle, has been measured. These cattle were located in 12 different European regions within France, Germany, Greece, Ireland, Italy, Spain and the UK. As animal protein is known to be a difficult material on which to conduct Sr isotope analysis, several investigations were undertaken to develop and improve the sample preparation procedure. For example, Sr isotope analysis was performed directly on freeze-dried meat and defatted dry mass from the same samples. It was found that enormous differences—sometimes exceeding the measurement uncertainty—could occur between the fractions and also within one sample even if treated in the same manner. These variations cannot be definitely allocated to one cause but are most likely due to inhomogeneities caused by physiological and biochemical processes in the animals as post mortem contamination during analytical processing could be excluded. For further Sr isotope measurements in meat, careful data handling is recommended, and for the authentic beef samples within this project, it was decided to use only freeze-dried material. It can be demonstrated, however, that Sr isotope measurements in beef proteins are a valuable tool for authentication of geographic origin. Although partly overlapping, some of the European sampling sites could be discriminated even by only using 87Sr/86Sr. Figure Box plot diagram displaying 87Sr/86Sr in authentic beef samples ordered by Trace sampling sites
Keywords: Sr/86Sr; TIMS; Isotopes; Beef; Authenticity control; Geographic origin

Bioaccessibility of total arsenic and arsenic species in seafood as determined by a continuous online leaching method by Axelle Leufroy; Laurent Noël; Diane Beauchemin; Thierry Guérin (2849-2859).
A continuous leaching method coupled online with inductively coupled plasma mass spectrometry (ICP-MS) detection was used to assess the maximum bioaccessibility of arsenic (As) in seafood samples. The method simulates continuous-flow digestion by successively pumping artificial saliva, gastric and intestinal juices through a mini-column of powdered sample directly connected to the nebuliser of an ICP-MS instrument. The method allows the real-time measurement of As being released by a given reagent. Because the analyte is continuously removed from the system, in contrast to batch methods, the dissolution equilibrium is driven to the right, hence quickly providing information about the worst-case scenario. Following consecutive leaching by the digestive reagents, the leachates were subject to speciation analysis by ion-exchange chromatography with ICP-MS detection to determine the arsenic species released. Finally, the remaining residue from the mini-column was fully digested to verify mass balance. The method was used to determine the bioaccessibility of total As and As species in four certified reference materials and in several real seafood samples. The mass balance was verified in each case. Generally speaking, the non-toxic form was easily released whereas the inorganic forms were poorly bioaccessible.
Keywords: Bioaccessibility; Arsenic; Speciation; IEC/ICP-MS; Seafood

Displacement immunoassay for the detection of ochratoxin A using ochratoxin B modified glass beads by Vasilica Lates; Cheng Yang; Ionel Catalin Popescu; Jean-Louis Marty (2861-2870).
We report here the development of a new assay for the detection of ochratoxin A (OTA) based on the use of its dechlorinated analogue, ochratoxin B (OTB), in a displacement immunoassay. OTB was immobilised on controlled-pore glass beads followed by the binding of anti-OTA antibody, with anti-IgG antibody peroxidase conjugate used as a label. In this manner, an original bio-sensing material was obtained. Upon incubation of this material with OTA, the toxin competes with OTB for the binding sites of the anti-OTA antibodies and releases the antibody-tagged peroxidase complex into the solution. Compared to classic competitive immunoassays, this newly developed displacement immunoassay presents a similar detection limit and assay time. Moreover, the detection, based on the activity of the horseradish peroxidase, is performed for the first time in situ using wine samples. Figure Steps in the construction of the OTB based bio-sensing glass material (1–5) and the displacement reaction (step 6) upon incubation with ochratoxin A
Keywords: Ochratoxin B; Displacement immunoassay; Controlled-pore glass; Ochratoxin A

Novel approaches to analysis of 3-chloropropane-1,2-diol esters in vegetable oils by Eliska Moravcova; Lukas Vaclavik; Ondrej Lacina; Vojtech Hrbek; Katerina Riddellova; Jana Hajslova (2871-2883).
A sensitive and accurate method utilizing ultra-high performance liquid chromatography (U-HPLC) coupled to high resolution mass spectrometry based on orbitrap technology (orbitrapMS) for the analysis of nine 3-chloropropane-1,2-diol (3-MCPD) diesters in vegetable oils was developed. To remove the interfering triacylglycerols that induce strong matrix effects, a clean-up step on silica gel column was used. The quantitative analysis was performed with the use of deuterium-labeled internal standards. The lowest calibration levels estimated for the respective analytes ranged from 2 to 5 μg kg−1. Good recovery values (89–120%) and repeatability (RSD 5–9%) was obtained at spiking levels of 2 and 10 mg kg−1. As an alternative, a novel ambient desorption ionization technique, direct analysis in real time (DART), hyphenated with orbitrapMS, was employed for no separation, high-throughput, semi-quantitative screening of 3-MCPD diesters in samples obtained by chromatographic fractionation. Additionally, the levels of 3-MCPD diesters measured in real-life vegetable oil samples (palm oil, sunflower oil, rapeseed oil) using both methods are reported. Relatively good agreement of the data generated by U-HPLC-orbitrapMS and DART-orbitrapMS were observed. With regard to a low ionization yield achieved for 3-MCPD monoesters, the methods presented in this paper were not yet applicable for the analysis of these contaminants at the naturally occurring levels.
Keywords: 3-MCPD esters; Vegetable oils; U-HPLC-MS; DART-MS

Biodegradation studies of N 4-acetylsulfapyridine and N 4-acetylsulfamethazine in environmental water by applying mass spectrometry techniques by María Jesús García-Galán; Tobias Frömel; Jutta Müller; Manuela Peschka; Thomas Knepper; Silvia Díaz-Cruz; Damiá Barceló (2885-2896).
This work evaluates the biodegradation of N 4-acetylsulfapyridine (AcSPY) and N 4-acetylsulfamethazine (AcSMZ), metabolites of two of the most commonly used sulfonamides (SAs) in human and veterinary medicine, respectively. Aerobic transformation in effluent wastewater was simulated using aerated fixed-bed bioreactors. No visible changes in concentration were observed in the AcSMZ reactor after 90 days, whereas AcSPY was fully degraded after 32 days of experiment. It was also demonstrated that AcSPY transformed back to its parent compound sulfapyridine (SPY). The environmental presence of these two metabolites in wastewater effluent had been previously investigated and confirmed, together with three more SA acetylated metabolites and their corresponding parent compounds, in 18 different wastewater treatment plants in Hesse (Germany). Sulfamethoxazole (SMX) and SPY were the two SAs detected most frequently (90% and 89% of the samples, respectively) and in the highest concentrations (682 ng L−1 for SMX and 532 ng L−1 for SPY). To conclude, hazard quotients were calculated whenever toxicity data were available. None of the SAs studied posed an environmental risk.
Keywords: Sulfonamides; Metabolites; Biodegradation; HPLC-MS/MS analysis; Wastewater

Membrane-assisted solvent extraction coupled to large volume injection–gas chromatography–mass spectrometry for the determination of a variety of endocrine disrupting compounds in environmental water samples by Arantza Iparraguirre; Patricia Navarro; Ailette Prieto; Rosario Rodil; Maitane Olivares; Luis-Ángel Fernández; Olatz Zuloaga (2897-2907).
Membrane-assisted solvent extraction coupled to large volume injection in a programmable temperature vaporisation injector using gas chromatography–mass spectrometry analysis was optimised for the simultaneous determination of a variety of endocrine disrupting compounds in environmental water samples (estuarine, river and wastewater). Among the analytes studied, certain hormones, alkylphenols and bisphenol A were included. The nature of membranes, extraction solvent, extraction temperature, solvent volume, extraction time, ionic strength and methanol addition were evaluated during the optimisation of the extraction. Matrix effects during the extraction step were studied in different environmental water samples: estuarine water, river water and wastewater (influent and effluent). Strong matrix effects were observed for most of the compounds in influent and effluent samples. Different approaches were studied in order to correct or minimise matrix effects, which included the use of deuterated analogues, matrix-matched calibration, standard addition calibration, dilution of the sample and clean-up of the extract using solid-phase extraction (SPE). The use of deuterated analogues corrected satisfactorily matrix effect for estuarine and effluent samples for most of the compounds. However, in the case of influent samples, standard addition calibration and dilution of the sample were the best approaches. The SPE clean-up provided similar recoveries to those obtained after correction with the corresponding deuterated analogue but better chromatographic signal was obtained in the case of effluent samples. Method detection limits in the 5–54 ng L−1 range and precision, calculated as relative standard deviation, in the 2–25% range were obtained.
Keywords: Endocrine disrupting compounds; Environmental water samples; Membrane-assisted solvent extraction; Gas chromatography–mass spectrometry; Large volume injection

Three sequential extraction procedures were evaluated for the study of fractionation of arsenic in environmental solid samples. The procedures considered were as follows: i) the standardized and widely recognised BCR procedure, conceived for the study of the partitioning of heavy metals; ii) the procedure developed by Manful, who adapted a phosphorus scheme for arsenic fractionation; and iii) a novel sequential extraction scheme especially devised for arsenic. The efficiency and suitability of these methods and the corresponding extraction steps for partitioning arsenic obtained from the most important solid forms were tested by application of the methods to real sediment samples heavily polluted by mining activity. Results showed the BCR scheme was inappropriate for arsenic fractionation. The procedure could, nevertheless, be a first approach for the assessment of arsenic partitioning, because its first extraction step can be regarded as adequate for the estimation of the most easily mobilizable arsenic. Although the Manful scheme results in a more differentiated arsenic pattern, some drawbacks arise from the lack of selectivity of some of the reagents used, for example overlapping of specific target phases, overestimation of adsorbed arsenate because of inadequate coprecipitation processes, and the inability to discriminate among amorphous and crystalline oxyhydroxides which are mainly responsible for arsenic retention. The novel procedure achieves the most suitable arsenic fractionation, because the main phases retaining arsenic are selectively targeted according to mobilization potential. In addition, the simplicity of its extraction steps would enable automation in a continuous flow scheme.
Keywords: Arsenic; Fractionation; Comparison; Sequential extraction scheme; Sediment

A reversed-phase capillary ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) method for comprehensive top-down/bottom-up lipid profiling by Xiaoli Gao; Qibin Zhang; Da Meng; Giorgis Isaac; Rui Zhao; Thomas L. Fillmore; Rosey K. Chu; Jianying Zhou; Keqi Tang; Zeping Hu; Ronald J. Moore; Richard D. Smith; Michael G. Katze; Thomas O. Metz (2923-2933).
Lipidomics is a critical part of metabolomics and aims to study all the lipids within a living system. We present here the development and evaluation of a sensitive capillary UPLC-MS method for comprehensive top-down/bottom-up lipid profiling. Three different stationary phases were evaluated in terms of peak capacity, linearity, reproducibility, and limit of quantification (LOQ) using a mixture of lipid standards representative of the lipidome. The relative standard deviations of the retention times and peak abundances of the lipid standards were 0.29% and 7.7%, respectively, when using the optimized method. The linearity was acceptable at >0.99 over 3 orders of magnitude, and the LOQs were sub-fmol. To demonstrate the performance of the method in the analysis of complex samples, we analyzed lipids extracted from a human cell line, rat plasma, and a model human skin tissue, identifying 446, 444, and 370 unique lipids, respectively. Overall, the method provided either higher coverage of the lipidome, greater measurement sensitivity, or both, when compared to other approaches of global, untargeted lipid profiling based on chromatography coupled with MS.
Keywords: Ultra-performance liquid chromatography (UPLC); Tandem mass spectrometry (MS/MS); Electrospray ionization (ESI); Top-down/bottom-up lipid profiling

Enantioseparations of racemic nonsteroidal anti-inflammatory drugs (naproxen, ibuprofen, ketoprofen, flurbiprofen, suprofen, indoprofen, cicloprofen, and carprofen) were performed by nano-liquid chromatography, employing achiral capillary columns and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD) or hydroxylpropyl-β-cyclodextrin (HP-β-CD) as a chiral mobile phase additive (CMPA). Working under the same experimental conditions (in terms of mobile phase and linear velocity), the performance of a RP-C18 monolithic column was compared with that of a RP-C18 packed column of the same dimensions (100 μm i.d. × 10 cm). Utilizing a mobile phase composed of 30% ACN (v/v) buffered with 50 mM sodium acetate at pH 3, and containing 30 mM TM-β-CD, the monolithic column provided faster analysis but lower resolution than the packed column. This behavior was ascribed to the high permeability of the monolithic column, as well as to its minor selectivity. HP-β-CD was chosen as an alternative to TM-β-CD. Employing the monolithic column, the effects of different parameters such as HP-β-CD concentration, mobile phase composition, and pH on the retention factor and the chiral resolution of the analytes were studied. For the most of the analytes, enantioresolution (which ranged from R s = 1.80 for naproxen to R s = 0.86 for flurbiprofen) was obtained with a mobile phase consisting of sodium acetate buffer (25 mM, pH 3), 10% MeOH, and 15 mM HP-β-CD. When the same experimental conditions were used with the packed column, no compound eluted within 1 h. Upon increasing the percentage of organic modifier to favor analyte elution, only suprofen eluted within 30 min, with an R s value of 1.14 (20% MeOH). Replacing MeOH with ACN resulted in a loss of enantioresolution, except for naproxen (R s = 0.89). Figure Cyclodextrin used as a chiral mobile phase additive in chromatography
Keywords: Enantioseparations; Nano-liquid chromatography; Monolithic column; Chiral mobile phase additive; Cyclodextrins; NSAIDs

The present study developed an high-performance liquid chromatography (HPLC) method for the simultaneous determination of urinary metabolites of endogenous cortisol, 6α-hydroxycortisol (6α-OHF) and 6β-hydroxycortisol (6β-OHF), in human urine, using 6α-hydroxycorticosterone as internal standard. 6α-OHF and 6β-OHF were extracted from urine with ethyl acetate by using a Sep-Pak C18 plus cartridge. Separation of the stereoisomers was achieved on a reversed-phase hybrid column by a gradient elution of (A) 0.05 M KH2PO4–0.01 M CH3COOH (pH 3.77) and (B) 0.05 M KH2PO4–0.01 M CH3COOH/acetonitrile (2:3, v/v). 6α-OHF and 6β-OHF were well separated on an XTerra MS C18 5 μm column using two types of stepwise gradient elution program (programs 2 and 3). Resolutions of 6α-OHF and 6β-OHF were Rs = 4.41 for program 2 and Rs = 4.60 for program 3. The analysis was performed within 23~26 min, monitored by UV absorbance at 239 nm. The lower limits of detection of 6α-OHF and 6β-OHF were 0.80 ng per injection (s/n = ca. 8), and the lower limits of quantification were 5.02 ng/ml for 6α-OHF and 41.08 ng/ml for 6β-OHF, respectively. The within-day reproducibilities in the amounts of 6α-OHF and 6β-OHF determined were in good agreement with the actual amounts added, the relative errors being −5.37% and −3.73% (gradient 2) and −5.69% and −3.96% (gradient 3) for both 6α-OHF and 6β-OHF, respectively. The inter-assay precisions (RSDs) for 6α-OHF and 6β-OHF were less than 1.99% (gradient 2) and 2.61% (gradient 3), respectively. The present HPLC method was applied to the measurement of 6α-OHF and 6β-OHF in urine to evaluate the time courses of 6α-hydroxylation and 6β-hydroxylation clearances of cortisol during 40 days for phenotyping CYP3A in a healthy subject.
Keywords: 6α-hydroxycortisol; 6β-Hydroxycortisol; HPLC-UV; Cortisol 6β-hydroxylation clearance

Four disulfide-reducing agents, dithiothreitol (DTT), 2,3-dimercaptopropanesulfonate (DMPS), and the newly tested 2-mercaptoethanesulfonate (MESNA) and Tris(hydroxypropyl)phosphine (THP), were investigated in detail for release of sulfur amino acids in human plasma. After protein precipitation with trichloroacetic acid (TCA), the plasma supernatant was treated with methyl, ethyl, or propyl chloroformate via the well-proven derivatization–extraction technique and the products were subjected to gas chromatographic–mass spectrometric (GC–MS) analysis. All the tested agents proved to be rapid and effective reducing agents for the assay of plasma thiols. When compared with DTT, the novel reducing agents DMPS, MESNA, and THP provided much cleaner extracts and improved analytical performance. Quantification of homocysteine, cysteine, and methionine was performed using their deuterated analogues, whereas other analytes were quantified by means of 4-chlorophenylalanine. Precise and reliable assay of all examined analytes was achieved, irrespective of the chloroformate reagent used. Average relative standard deviations at each analyte level were ≤6%, quantification limits were 0.1–0.2 μmol L−1, recoveries were 94–121%, and linearity was over three orders of magnitude (r 2 equal to 0.997–0.998). Validation performed with the THP agent and propyl chloroformate derivatization demonstrated the robustness and reliability of this simple sample-preparation methodology.
Keywords: Plasma amino acids; Disulfide-reducing agents; Trichloroacetic acid deproteinization; Chloroformate-treated supernatant; Validated GC–MS assay

Traditional activity-guided fractionation of natural products is a time-consuming, labor intensive, and expensive strategy, which cannot compete with high-throughput and rapid screening of natural products. Therefore, more efficient approaches are necessary for searching active compounds from natural products. Three main methods based on high-performance liquid chromatography (HPLC) analysis combined with 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assay, DPPH spiking HPLC analysis, on-line post-column HPLC-DPPH analysis, and HPLC-based DPPH activity profiling, were then developed for the rapid screening of antioxidants from complex mixtures. In the present study, a comparative study of these three methods has been conducted to identify antioxidants from an ethyl acetate fraction of Pueraria lobata flowers. The parameters in HPLC analysis and DPPH assay were optimized. The results indicated that all three methods could achieve similar information with regard to antioxidants, without the need for preparative isolation techniques. However, there were differences in instrumental set-up, sensitivity, and efficiency. DPPH spiking HPLC analysis seemed to be more sensitive and effective with simpler instrumental set-up and easier operation, which could also detect the total antioxidant capacity of color complexes. Eighteen antioxidants were tentatively screened and identified from P. lobata flowers by DPPH spiking HPLC-MS/MS. Among them, ten compounds including one new compound were first isolated from P. lobata flowers, and the DPPH radical scavenging activity of the new compound was reported for the first time.
Keywords: HPLC; DPPH; Antioxidant; Rapid screening; Pueraria lobata flower

FTIR imaging investigation in MIR and in an enlarged MIR–NIR spectral range by Tommaso Poli; Oscar Chiantore; Annamaria Giovagnoli; Anna Piccirillo (2977-2984).
The study of polished cross sections is a well-assessed and practical method to investigate the stratigraphy of paintings and multilayer polychromies on works of art, in general. Analyses on cross sections allow us to characterize, at once, all the layers in the stratigraphy, giving information about the artists technique, the number of layers and their composition and sometimes about the conservation history of the artefact. In this paper, the application of an imaging detector focal plane array (FPA) coupled to an infrared (IR) microscope has been studied, focusing on the characteristics and potential of the different working methodologies (attenuated total reflectance (ATR) and total reflection). FPA detector coupled with ATR crystal can “localize” IR information coming from a 30 × 30μm sample area, in a 64 × 64 dot matrix detector. In particular, an innovative analysis methodology has been tested for the total reflectance measurements in order to obtain maximum information with single measurements. Micro-infrared total reflection measurements have been carried out in an extended IR range (from 1,000 to 5,266 cm−1) exploiting the broad spectral response of mercury cadmium telluride detector in order to include overtones and combination bands from near-infrared spectral range without any modification of the standard mid-infrared micro-FT instrumentation. The potentialities of this new approach have been successfully transferred in the imaging/mapping investigations with a minimal tuning of the apparatus. Results obtained on a polished cross section coming from a modern painting and on a micro-sample of a wood polychromy from an undated historic polyptic are shown for demonstration.
Keywords: IR spectroscopy/Raman spectroscopy; FTIR imaging; NIR; Archeometry/fine arts; MIR total reflectance

Natural abundance deuterium 2D NMR spectroscopy in weakly ordering, polypeptide chiral liquid crystals is a powerful technique that enables determination of enantiotopic isotopic ratios (2H/1H) i at the methylene groups of long-chain fatty acids. This technique has been used to study the bioconversion of linoleic acid to vernoleic acid with the objective of establishing the in-vivo site-specific fractionation of 2H associated with this process. The fractionation pattern was investigated in Euphorbia lagascae and Vernonia galamensis, plants that use different enzyme systems to perform the Δ12-epoxidation: a cytochrome P450 monooxygenase in the former and a di-iron dioxygenase in the latter. The specific interest in this study was to ascertain whether different (2H/1H) i isotopic ratios in substrate and product might reflect distinct features of the nature of the reaction centre. However, both the linoleate (substrate) samples and both vernoleate (product) samples isolated from the seed oils of the two plants had remarkably similar 2H isotope profiles, with selection against 2H in the positions around the Δ12-epoxidation site. This is interpreted as indicating that, despite differences in the form in which the activated Fe is presented and in the architecture of the active site, the (2H/1H) i isotopic pattern is determined by features common to the reaction. It is suggested that the effects acting as the overall determinants of the final (2H/1H) i distribution in the product are the encumbrance of the active site pocket and constraints to conformational readjustment during the linoleate to vernoleate transformation. Figure Changes in the (2H/1H)i ratios in converting methyl linoleate to methyl vernoleate in either V. galamensis or E. lagascae indicate isotopic fractionations independent of the type of enzyme reaction centre carrying out the epoxidation
Keywords: Epoxide formation; Isotope ratio profile determination; Liquid-crystalline solvents; Natural abundance deuterium 2D NMR spectroscopy; Vernoleic acid; Linoleic acid

We report herein a new class of metal ion chemosensors and give the first example of a metal-dependent peptidase chemosensor for metal ions. The chemosensor contains the basic specific Ni(II)-dependent peptide bond hydrolysis sequence (Gly-Ala-Ser-Arg-His-Trp-Lys-Phe-Lys). The substrate was labeled with a fluorophore at the N-terminal and a quencher at the C-terminal Lys side chain. Initially, the MOCAc ((7-methoxycoumarin-4-yl)acetyl-) emission was quenched by the nearby quencher. In the presence of Ni(II), the substrate was irreversibly cleaved at the cleavage site, leading to a 20-fold increase in fluorescence intensity. The chemosensor combines the high selectivity of a peptidase (at least greater than tenfold for Ni(II) over other metal ions) with the high sensitivity of fluorescence detection limit of 50 nM and can be applied for the quantitative detection of Ni(II) over a concentration range of three orders of magnitude. Given this degree of selectivity and sensitivity, our molecular engineering design may prove useful in the future development of other peptidase-based probes for different metal ions in toxicological and environmental monitoring. Figure The chemosensor peptidase was coordinated and cleaved by Ni(II), leading to its increased fluorescence intensity
Keywords: Peptidase; Chemosensors; Ni(II); Detection

Liquid chromatography–tandem mass spectrometry has become the preferred technology to measure unconjugated metanephrine and normetanephrine in plasma because of its high sensitivity and specificity over immunoassay and gas chromatography–mass spectrometry. In our earlier study, plasma metanephrines were extracted with offline ion-pairing solid-phase extraction and quantified by liquid chromatography–tandem mass spectrometry with porous graphitic carbon column based chromatography. In this study, we aim to automate the sample preparation with turbulent flow online extraction technology and maintain or improve the analytical performance previously achieved from the offline approach. The online extraction was done with a mixed-mode cation exchange turbulent flow chromatography column assisted with ion-pairing reagent and porous graphitic column was used for chromatographic separation. The total online extraction and analytical LC runtime was 12 min. This method was linear from 6.3 to 455.4 pg/mL for metanephrine; 12.6 to 954.5 pg/mL for normetanephrine with an accuracy of 80.6% to 93.5% and 80.9% to 101.7%, respectively. The lower limit of quantitation was 6.3 pg/mL for metanephrine and 12.6 pg/mL for normetanephrine. Inter-assay and intra-assay precision for metanephrine and normetanephrine at low and high concentration levels ranged from 2.0% to 10.5%. In conclusion, we have developed a fast and sensitive automated online turbulent flow extraction method for the quantitative analysis of plasma metanephrines. Ion-pairing reagent was necessary for the success of this method. Fig Schematic diagrams of turbulent flow and reverse phase LC System for Pmets analysis.
Keywords: Metanephrines; Turbulent flow chromatography; Ion pair; Porous graphitic carbon

Polar and non-polar organic binder characterization in Pompeian wall paintings: comparison to a simulated painting mimicking an “a secco” technique by Gaetano Corso; Monica Gelzo; Carmen Sanges; Angela Chambery; Antimo Di Maro; Valeria Severino; Antonio Dello Russo; Ciro Piccioli; Paolo Arcari (3011-3016).
The use of Fourier transform infrared spectromicroscopy and mass spectrometry (MS) allowed us to characterize the composition of polar and non-polar binders present in sporadic wall paint fragments taken from Pompeii’s archaeological excavation. The analyses of the polar and non-polar binder components extracted from paint powder layer showed the presence of amino acids, sugars, and fatty acids but the absence of proteinaceous material. These results are consistent with a water tempera painting mixture composed of pigments, flours, gums, and oils and are in agreement with those obtained from a simulated wall paint sample made for mimicking an ancient “a secco” technique. Notably, for the first time, we report the capability to discriminate by tandem MS the presence of free amino acids in the paint layer.
Keywords: Pompeii’s wall painting; Cultural heritage; GC-MS; LC-ESI/MS/MS; FT-IR spectromicroscopy

Erratum to: HS-SPME-GC×GC-qMS volatile metabolite profiling of Chrysolina herbacea frass and Mentha spp. leaves by Chiara Cordero; Simon Atsbaha Zebelo; Giorgio Gnavi; Alessandra Griglione; Carlo Bicchi; Massimo E. Maffei; Patrizia Rubiolo (3017-3018).