Analytical and Bioanalytical Chemistry (v.395, #8)

The Analytical Sciences Digital Library (ASDL) by Cynthia K. Larive (2425-2429).
is Professor of Analytical Chemistry at the University of California Riverside. She has an active research program involving bioanalytical and environmental analytical applications of NMR spectroscopy (for more information, see http://www.chem.ucr.edu/faculty/larive/larive.html ). Dr. Larive is also active in curricular reform and the promotion of undergraduate research. She is editor-in-chief and principal investigator of the Analytical Sciences Digital Library ( http://www.asdlib.org ).

DGMS 2009: focus on bioanalytical mass spectrometry by Michael Przybylski (2441-2441).
has been Full Professor of Analytical Chemistry and Biopolymer Chemistry at the University of Konstanz since 1988. His research interests focus on peptide and protein structure analysis, biopolymer mass spectrometry, and proteome analysis. Further research areas include molecular recognition and supramolecular association spectrometry, vaccine peptides and epitope analysis, and neurodegenerative peptides and proteins.

Towards a proteome signature for invasive ductal breast carcinoma derived from label-free nanoscale LC-MS protein expression profiling of tumorous and glandular tissue by Claudia Röwer; Johannes P. C. Vissers; Cornelia Koy; Marc Kipping; Michael Hecker; Toralf Reimer; Bernd Gerber; Hans-Jürgen Thiesen; Michael O. Glocker (2443-2456).
As more and more alternative treatments become available for breast carcinoma, there is a need to stratify patients and individual molecular information seems to be suitable for this purpose. In this study, we applied label-free protein quantitation by nanoscale LC-MS and investigated whether this approach could be used for defining a proteome signature for invasive ductal breast carcinoma. Tissue samples from healthy breast and tumor were collected from three patients. Protein identifications were based on LC-MS peptide fragmentation data which were obtained simultaneously to the quantitative information. Hereby, an invasive ductal breast carcinoma proteome signature was generated which contains 60 protein entries. The on-column concentrations for osteoinductive factor, vimentin, GAP-DH, and NDKA are provided as examples. These proteins represent distinctive gene ontology groups of differentially expressed proteins and are discussed as risk markers for primary tumor pathogenesis. The developed methodology has been found well applicable in a clinical environment in which standard operating procedures can be kept; a prerequisite for the definition of molecular parameter sets that shall be capable for stratification of patients.
Keywords: Breast carcinoma; Proteome analysis; Protein expression signature; Biomarker; Therapeutic target; Label-free quantitation; Nanoscale LC-MS; Data-independent alternate scanning (LC-MSE); Western blot; Immunohistochemistry

UV-laser ablation of ionic liquid matrices by Nils Hellwig; Alexander Thrun; Tassilo Muskat; Jürgen Grotemeyer (2457-2463).
Ionic liquid matrices are a new class of matrices used in MALDI mass spectrometry. The ablation process of several ionic liquid matrices was studied by determining the velocity distribution of ablated neutral matrix molecules. This was done by a postionization approach, where the neutrals were ionized in the ablation plume by a second laser pulse. It was found that a second, time-delayed ablation event occurs consisting completely of neutral molecules. To explain this, the reflected-shockwave model is used, which assumes that the shockwave emerging from the laser ablation is reflected at the sample holder surface. When the shockwave arrives at the sample surface it causes a second ablation.
Keywords: Laser ablation; MALDI; Ionic liquids; Mass spectrometry

Determination of ganglioside composition and structure in human brain hemangioma by chip-based nanoelectrospray ionization tandem mass spectrometry by Catalin Schiopu; Corina Flangea; Florina Capitan; Alina Serb; Željka Vukelić; Svjetlana Kalanj-Bognar; Eugen Sisu; Michael Przybylski; Alina D. Zamfir (2465-2477).
We report here on a preliminary investigation of ganglioside composition and structure in human hemangioma, a benign tumor in the frontal cortex (HFC) in comparison to normal frontal cortex (NFC) tissue using for the first time advanced mass spectrometric methods based on fully automated chip-nanoelectrospray (nanoESI) high-capacity ion trap (HCT) and collision-induced dissociation (CID). The high ionization efficiency, sensitivity and reproducibility provided by the chip-nanoESI approach allowed for a reliable MS-based ganglioside comparative assay. Unlike NFC, ganglioside mixture extracted from HFC was found dominated by species of short glycan chains exhibiting lower overall sialic acid content. In HFC, only GT1 (d18:1/20:0), and GT3 (d18:1/25:1) polysialylated species were detected. Interestingly, none of these trisialylated forms was detected in NFC, suggesting that such components might selectively be associated with HFC. Unlike the case of previously investigated high malignancy gliosarcoma, in HFC one modified O-Ac-GD2 and one modified O-Ac-GM4 gangliosides were observed. This aspect suggests that these O-acetylated structures could be associated with cerebral tumors having reduced malignancy grade. Fragmentation analysis by CID in MS2 mode using as precursors the ions corresponding to GT1 (d18:1/20:0) and GD1 (d18:1/20:0) provided data corroborating for the first time the presence of the common GT1a and GT1b isomers and the incidence of unusual GT1c and GT1d glycoforms in brain hemangioma tumor. Human brain biomarker discovery by advanced chipbased nanoelectrospray mass spectrometry
Keywords: Gangliosides; Brain hemangioma; Chip-based nanoelectrospray; Tandem mass spectrometry; Brain tumor biomarker

Phosphatidylcholines and -ethanolamines can be easily mistaken in phospholipid mixtures: a negative ion MALDI-TOF MS study with 9-aminoacridine as matrix and egg yolk as selected example by Beate Fuchs; Annabell Bischoff; Rosmarie Süß; Kristin Teuber; Martin Schürenberg; Detlev Suckau; Jürgen Schiller (2479-2487).
Phospholipids (PL) are increasingly analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). As in the case of polar molecules, however, the careful selection of the matrix is crucial for optimum results. 9-Aminoacridine (9-AA) was recently suggested as the matrix of choice to analyze PL mixtures because of (a) the improved sensitivity and (b) the reduction of suppression effects compared to other matrices. However, the distinction of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the negative ion mode is obscured as PC is also detectable as –CH 3 + ion if 9-AA is used as matrix. This may result in the erroneous assignment of PC as a PE species. Using an organic extract from hen egg yolk as example it will be shown that the contribution of PC must be taken into consideration if the negative ion mass spectra are used to evaluate the fatty acyl compositions of PE mixtures. 9-AA can as well be used in hyphenated thin-layer chromatography (TLC)-MALDI-TOF MS where PC and PE are chromatographically well separated for unequivocal assignments. Figure Comparison of negative ion MALDI-TOF mass spectra of isolated 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-sn-phosphatidylethanolamine (POPE) using either DHB (blue) or 9-AA (red) as matrix. The spectra differ significantly as a function of the matrix used. In case of 9-AA, POPC is detectable as negative ion subsequent to the loss of a -CH3 group, which complicates peak assignments when complex mixtures are analyzed
Keywords: MALDI-TOF MS; Phospholipid analysis; Phosphatidylcholine; Phosphatidylethanolamine; 9-Aminoacridine; Hen egg yolk

Determination of sulfation pattern in brain glycosaminoglycans by chip-based electrospray ionization ion trap mass spectrometry by Corina Flangea; Catalin Schiopu; Eugen Sisu; Alina Serb; Michael Przybylski; Daniela G. Seidler; Alina D. Zamfir (2489-2498).
Chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans display variability of sulfation in their constituent disaccharide repeats during chain elongation. Since a large proportion of the extracellular matrix of the central nervous system (CNS) is composed of proteoglycans, CS/DS disaccharide degree and profile of sulfation play important roles in the functional diversity of neurons, brain development, and some of its pathological states. To investigate the sulfation pattern of CS/DS structures expressed in CNS, we introduced here a novel method based on an advanced system encompassing fully automated chip nanoelectrospray ionization (nanoESI) in the negative ion mode and high capacity ion trap multistage mass spectrometry (MS2–MS3) by collision-induced dissociation (CID). This method, introduced here for the first time in glycomics of brain glycosaminoglycans, was particularly applied to structural investigation of disaccharides obtained by β-elimination and digestion with chondroitin B and AC I lyase of hybrid CS/DS chains from wild-type mouse brain. Screening in the chip-MS mode of DS disaccharide fraction resulting after depolymerization with chondroitin B lyase revealed molecular ions assigned to monosulfated disaccharide species having a composition of 4,5-Δ-[IdoA-GalNAc]. By optimized CID MS2–MS3, fragment ions supporting the localization of sulfate ester group at C4 within GalNAc were produced. Chip ESI MS profiling of CS disaccharide fraction obtained by depolymerization of the same CS/DS chain using chondroitin AC I lyase indicated the occurrence of mono- and bisulfated 4,5-Δ-[GlcA-GalNAc]. The site of oversulfation was determined by MS2–MS3, which provided sequence patterns consistent with a rare GlcA-3-sulfate–GalNAc-6-sulfate structural motif. Figure Mouse brain GlcA-3-sulfate-GalNAc-6-sulfate structural motif
Keywords: Chondroitin/dermatan sulfate; Mouse brain; Fully automated chip-based nanoelectrospray; Ion trap mass spectrometry

Determination of basic drugs of abuse in human serum by online extraction and LC–MS/MS by Nerea Ferreirós Bouzas; Sebastian Dresen; Barbara Munz; Wolfgang Weinmann (2499-2507).
A new quantitation method for the determination of drugs of abuse (opiates, amphetamine and derivatives, cocaine, methadone and metabolites) in serum by using online extraction coupled to liquid chromatography (LC)–mass spectrometry (MS)/MS has been developed. The online extraction is carried out using two extraction columns simultaneously and one analytical column. One extraction column is loaded, while the other one is eluted by a gradient. The elution gradient also separates the analytes in the analytical column. For the sample preparation, serum is spiked with a mixture of deuterated analogues of the drugs. After protein precipitation with methanol/zinc sulphate, centrifugation, evaporation and reconstitution, the sample is injected into the LC system. The quantitation is based on the analysis of two multiple reaction monitoring transitions per drug. The recovery of the protein precipitation step is over 80% for all analytes. Intra- and interday precision, as relative standard deviation, is lower than 6%, and in the case of accuracy, RE is lower than 15%. Only the most polar analytes showed matrix effects. The limits of quantitation for the analysed compounds vary between 0.5 and 2.8 ng/mL. The developed method was used to quantify basic drugs in samples “from driving under the influence of drugs” cases. The results were compared with those obtained by using solid-phase extraction–GC–MS.
Keywords: Online extraction; Drugs of abuse; Tandem mass spectrometry; Quantitation; Validation

Structural characterization of ß-amyloid oligomer-aggregates by ion mobility mass spectrometry and electron spin resonance spectroscopy by Marius Ionuţ Iuraşcu; Claudia Cozma; Nick Tomczyk; John Rontree; Michael Desor; Malte Drescher; Michael Przybylski (2509-2519).
Formation and accumulation of fibrillar plaques and aggregates of ß-amyloid peptide (Aß) in brain have been recognized as characteristics of Alzheimer’s disease (AD). Oligomeric aggregates of Aß are considered critical intermediates leading to progressive neurodegeneration; however, molecular details of the oligomerization and aggregation pathway and the structures of Aß-oligomers are hitherto unclear. Using an in vitro fibril formation procedure of Aß(1–40), ß-amyloid aggregates were prepared and insoluble aggregates separated from soluble products by centrifugation. In this study, ion mobility mass spectrometry (IM-MS) was applied in combination with electron paramagnetic resonance spectroscopy (EPR) to the identification of the components of Aß-oligomers, and to their structural and topographical characterization. The formation of Aß-oligomers and aggregates was monitored by gel electrophoresis, and Aß-oligomer bands were identified by in-gel tryptic digestion and matrix-assisted laser desorption ionization–mass spectrometry (MALDI-MS) to consist predominantly of Aß(1–40) peptide. First, ion mobility-MS studies of soluble Aß-aggregates prepared by incubation for 5 days were performed on a quadrupole time-of-flight mass spectrometer and revealed (1) the presence of at least two different conformational states, and (2), the formation of Met-35 oxidized products. For estimation of the size of Aß-aggregates using EPR spectroscopy, a modified Aß(1–40) peptide containing an additional N-terminal cysteine residue was prepared, and a 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy radical spin label derivative (IPSL) was coupled by S-alkylation. The EPR spectra of the spin-labeled Cys-Aß(1–40) oligomers were matched with spectra simulations using a multi-component simulation strategy, resulting in complete agreement with the gel electrophoresis results.
Keywords: Alzheimer’s disease; ß-Amyloid; Aß-fibrils; Oligomerization; Ion mobility mass spectrometry; EPR spectroscopy

ESI-MS/MS library of 1,253 compounds for application in forensic and clinical toxicology by Sebastian Dresen; Merja Gergov; Lucia Politi; Claudia Halter; Wolfgang Weinmann (2521-2526).
An electrospray ionization tandem mass spectrometry (ESI-MS/MS) library which contains over 5,600 spectra of 1,253 compounds relevant in clinical and forensic toxicology has been developed using a hybrid tandem mass spectrometer with a linear ion trap. Pure compound solutions—in some cases solutions made of tablets—were prepared and 1 to 2,000 ng of each compound were injected into the system using standard reversed-phase analytical columns with gradient elution. To obtain maximum mass spectral information enhanced product ion spectra were acquired with positive and/or negative ionization at low, medium, and high collision energies and additionally applying collision energy spread. In this mode, all product ions generated by the different collision energies are trapped in the linear ion trap prior to their detection. The applicability of the library for other types of hybrid tandem mass spectrometers with a linear ion trap of the same manufacturer as well as a standard triple-quadrupole tandem mass spectrometer has been investigated with a selection of compounds. The spectra of the developed library can be used to create methods for target analysis, either screening methods or quantitative procedures by generating transitions for multiple reaction monitoring. For those procedures, suitable transitions and convenient collision energies are selected from the library. It also has been utilized to identify compounds with a multi target screening approach for clinical and forensic toxicology with a standardized and automated system. The novel aspects compared to our former library produced with a standard triple-quadrupole mass spectrometer are the enlargement of the ESI-MS/MS library and the additional acquisition of spectra with collision energy spread.
Keywords: Mass spectral library; Forensics/toxicology; Drug monitoring/drug screening; Clinical/biomedical analysis

Alignment of capillary electrophoresis–mass spectrometry datasets using accurate mass information by Ekaterina Nevedomskaya; Rico Derks; André M. Deelder; Oleg A. Mayboroda; Magnus Palmblad (2527-2533).
Capillary electrophoresis–mass spectrometry (CE–MS) is a powerful technique for the analysis of small soluble compounds in biological fluids. A major drawback of CE is the poor migration time reproducibility, which makes it difficult to combine data from different experiments and correctly assign compounds. A number of alignment algorithms have been developed but not all of them can cope with large and irregular time shifts between CE–MS runs. Here we present a genetic algorithm designed for alignment of CE–MS data using accurate mass information. The utility of the algorithm was demonstrated on real data, and the results were compared with one of the existing packages. The new algorithm showed a significant reduction of elution time variation in the aligned datasets. The importance of mass accuracy for the performance of the algorithm was also demonstrated by comparing alignments of datasets from a standard time-of-flight (TOF) instrument with those from the new ultrahigh resolution TOF maXis (Bruker Daltonics).
Keywords: Capillary electrophoresis; Electrophoresis; Mass spectrometry; Software; Bioanalytical methods; Biological samples

Characterization, stoichiometry, and stability of salivary protein–tannin complexes by ESI-MS and ESI-MS/MS by Francis Canon; Franck Paté; Emmanuelle Meudec; Thérèse Marlin; Véronique Cheynier; Alexandre Giuliani; Pascale Sarni-Manchado (2535-2545).
Numerous protein–polyphenol interactions occur in biological and food domains particularly involving proline-rich proteins, which are representative of the intrinsically unstructured protein group (IUP). Noncovalent protein–ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), which also gives access to ligand binding stoichiometry. Surprisingly, the study of interactions between polyphenolic molecules and proteins is still an area where ESI-MS has poorly benefited, whereas it has been extensively applied to the detection of noncovalent complexes. Electrospray ionization mass spectrometry has been applied to the detection and the characterization of the complexes formed between tannins and a human salivary proline-rich protein (PRP), namely IB5. The study of the complex stability was achieved by low-energy collision-induced dissociation (CID) measurements, which are commonly implemented using triple quadrupole, hybrid quadrupole time-of-flight, or ion trap instruments. Complexes composed of IB5 bound to a model polyphenol EgCG have been detected by ESI-MS and further analyzed by MS/MS. Mild ESI interface conditions allowed us to observe intact noncovalent PRP–tannin complexes with stoichiometries ranging from 1:1 to 1:5. Thus, ESI-MS shows its efficiency for (1) the study of PRP–tannin interactions, (2) the determination of stoichiometry, and (3) the study of complex stability. We were able to establish unambiguously both their stoichiometries and their overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Our results prove that IB5·EgCG complexes are maintained intact in the gas phase. Figure Schematic illustrating the interaction between IB5 and EgCG, leading to IB5·EgCG complexes that remain intact in the gas phase during ESI-MS analysis. This system provides a model of biological interest with regards to astringency
Keywords: Polyphenol; Interaction; Proline-rich protein; Saliva; Astringency; IUP

Determination of ketamine and amphetamines in hair by LC/MS/MS by María Jesús Tabernero; Maria Linda Felli; Ana María Bermejo; Marcello Chiarotti (2547-2557).
A liquid chromatography-tandem mass spectrometry method was developed for the determination of ketamine (with its metabolite norketamine) and some amphetamines (amphetamine, methamphetamine, methylenedioxyamphetamine, and 3,4-methylenedioxymethamphetamine). This method was developed to determine these compounds in hair and is able to simultaneously quantify all of them in human hair. Hair samples (20 mg) were washed and pulverized, and an extraction with formic acid (0.01%) and ultrasonication for 4 h was used. Deuterated analogs of the analytes were used as internal standards for quantification. Linearity from 0.5 to 25 ng/mg was obtained for both ketamine (and norketamine) and amphetamines with correlation coefficients exceeding 0.99. The limit of detection and the limit of quantification obtained were 0.1 and 0.5 ng/mg, respectively, for ketamine and amphetamines. A total of 25 hair samples from known drug abusers (relating to designer drug consumption or consumption of amphetamines) were examined by this validated method. The results show that the proposed method is suitable for testing these drugs in a single sample of hair. In addition, it is simpler and faster than analysis by conventional methods such as gas chromatography-mass spectrometry, which usually require a more laborious extraction procedure and, in most of cases, an additional derivatization process.
Keywords: Ketamine; Amphetamines; Hair; Liquid chromatography-tandem mass spectrometry

Layer-by-layer coating of bacteria with noble metal nanoparticles for surface-enhanced Raman scattering by Mehmet Kahraman; Alsu I. Zamaleeva; Rawil F. Fakhrullin; Mustafa Culha (2559-2567).
A simple layer-by-layer method to coat the bacterial cells with gold and silver nanoparticles (AuNPs and AgNPs) for the acquisition of surface-enhanced Raman scattering (SERS) spectra is reported. First, the bacteria cell wall is coated with poly (allylamine hydrochloride) (PAH), a positively charged polymer, and then with citrate reduced Au or AgNPs. In order to increase the stability of the coating, another layer of PAH is prepared on the surface. The SEM and AFM images indicate that the nanoparticles are in the form of both isolated and aggregated nanoparticles on the bacterial wall. The coating of bacterial cells with AgNPs or AuNPs not only serves for their preparation for SERS measurement but also helps to visualize the coated of bacterial cells under the ordinary white-light microscope objective due to efficient light-scattering properties of Au and AgNPs. A comparative study single versus aggregates of bacterial cells is also demonstrated for possible single bacterial detection with SERS. The two bacteria that differ in shape and cell wall biochemical structure, Escherichia coli and Staphylococcus cohnii, Gram-negative and -positive, respectively, are used as models. The preliminary results reveal that the approach could be used for single bacterial cell identification.
Keywords: Layer-by-layer; SERS; Single bacterium; Gold and silver nanoparticles

Studies of interaction of tumor suppressor p53 with apo-MT using surface plasmon resonance by Ning Xia; Lin Liu; Xinyao Yi; Jianxiu Wang (2569-2575).
The interaction of tumor suppressor p53 with apo-metallothionein (apo-MT) has been carried out using a flow injection-surface plasmon resonance (FI-SPR) instrument. MT was first tethered onto the carboxymethylated dextran film. Via incorporation of glycine–HCl (pH 2) to remove the sequestrated metal ions inherent in MT molecules, a more extended and open structure of apo-MT was formed. Substantial SPR angle shift corresponding to the interaction of wild-type p53 with apo-MT was observed. The interaction was originated from the binding between the free sulfhydryl groups of apo-MT and Zn2+ of p53 with the binding constant of 1.4 × 108 M−1. The specific binding of p53 to consensus double-stranded DNA was hindered after metal chelation from p53 by apo-MT. Furthermore, inhibition of the interaction between p53 and apo-MT imposed by p53/DNA complex was observed. The fluorescence measurements also revealed the binding of p53 to apo-MT, being consistent with the SPR results. Thus, SPR could potentially serve as an attractive technique for monitoring p53 conformational change and transcriptional activity regulated by the MT/apo-MT couple.
Keywords: Surface plasmon resonance; Metallothionein; p53; Interaction

Improved rapid assay of plasma uric acid by short-end injection capillary zone electrophoresis by Salvatore Sotgia; Ciriaco Carru; Bastianina Scanu; Elisabetta Pisanu; Manuela Sanna; Gerard Aime Pinna; Leonardo Gaspa; Luca Deiana; Angelo Zinellu (2577-2582).
A rapid and simple short-end injection capillary zone electrophoresis method was developed for the quantification of plasma uric acid. The separation was performed in an uncoated fused-silica capillary (50 μm ID, 60 cm total length, 10.2 cm effective length) by using as a background electrolyte a 75 mmol/L glycylglycine solution titrated with NaOH 5 mol/L to pH 9.0, a voltage of 28 kV, a cartridge temperature of 15 °C, and direct UV detection at 292 nm. Under optimized conditions, uric acid was determinate in little more than 1 min (1.076 minutes). In order to verify the accuracy of the analysis, urate levels were measured in 543 apparently healthy volunteers by the new assay and our previous method, and the obtained data were compared by Passing–Bablock regression, Bland–Altman test, and a new regression-based approach, which showed a good agreement between two methods.
Keywords: Capillary zone electrophoresis; Short-end injection; Uric acid; Method comparison studies

Benzene is classified as a Group I carcinogen by the International Agency for Research on Cancer (IARC). The risk assessment for benzene can be performed by monitoring environmental and occupational air, as well as biological monitoring through biomarkers. The present work developed and validated methods for benzene analysis by GC/MS using SPME as the sampling technique for ambient air and breath. The results of the analysis of air in parks and avenues demonstrated a significant difference, with average values of 4.05 and 18.26 μg m−3, respectively, for benzene. Sampling of air in the occupational environment furnished an average of 3.41 and 39.81 μg m−3. Moreover, the correlations between ambient air and expired air showed a significant tendency to linearity (R 2 = 0.850 and R 2 = 0.879). The results obtained for two groups of employees (31.91 and 72.62 μg m−3) presented the same trend as that from the analysis of environmental air.
Keywords: Benzene; Environmental analysis; Breath analysis; SPME; GC/MS

Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine by Ming-Zhou Zhang; Min-Zi Wang; Zong-Lun Chen; Jie-Hong Fang; Mei-Ming Fang; Jun Liu; Xiao-Ping Yu (2591-2599).
A multianalyte lateral-flow immunochromatographic technique using colloidal gold-labeled polyclonal antibodies was developed for the rapid simultaneous detection of clenbuterol and ractopamine. The assay procedure could be accomplished within 5 min, and the results of this qualitative one-step assay were evaluated visually according to whether test lines appeared or not. When applied to the swine urines, the detection limit and the half maximal inhibitory concentration (IC50) of the test strip under an optical density scanner were calculated to be 0.1 ± 0.01 ng mL−1 and 0.1 ± 0.01 ng mL−1, 0.56 ± 0.08 ng mL−1, and 0.71 ± 0.06 ng mL−1, respectively, the cut-off levels with the naked eye of 1 ng mL−1 and 1 ng mL−1 for clenbuterol and ractopamine were observed. Parallel analysis of swine urine samples with clenbuterol and ractopamine showed comparable results obtained from the multianalyte lateral-flow test strip and GC-MS. Therefore, the described multianalyte lateral-flow test strip can be used as a reliable, rapid, and cost-effective on-site screening technique for the simultaneous determination of clenbuterol and ractopamine residues in swine urine. Figure The colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine.
Keywords: Clenbuterol; Ractopamine; Lateral-flow immunochromatography; Colloidal gold; Multianalyte

Matrix solid-phase dispersion and solid-phase microextraction applied to study the distribution of fenbutatin oxide in grapes and white wine by R. Montes; P. Canosa; J. Pablo Lamas; A. Piñeiro; I. Orriols; R. Cela; I. Rodríguez (2601-2610).
The fate of the acaricide fenbutatin oxide (FBTO) during the elaboration of white wine is evaluated. Matrix solid-phase dispersion (MSPD) and solid-phase microextraction (SPME) were used as sample preparation techniques applied to the semi-solid and the liquid matrices involved in this research, respectively. Selective determination of FBTO was achieved by gas chromatography with atomic emission detection (GC–AED). GC coupled to mass spectrometry was also used to establish the identity of FBTO by-products detected in must and wine samples. MSPD extractions were accomplished using C18 as dispersant and co-sorbent. Sugars and other polar interferences were first removed with water and water/acetone mixtures, then FBTO was recovered with 8 mL of acetone. When used in combination with GC–AED, the MSPD method provided limits of quantification (LOQs) in the low nanogram per gram range, recoveries around 90% and relative standard deviations below 13% for extractions performed in different days. Performance of SPME for must and wine was mainly controlled by the extraction temperature, time and fibre coating. Under final conditions, FBTO was extracted in the headspace mode for 45 min at 100 °C, using a 100 μm poly(dimethylsiloxane)-coated fibre. The achieved LOQs remained around or below 0.1 ng mL−1, depending on the type of sample, and the inter-day precision ranged from 10% to 13%. FBTO residues in grapes stayed mostly on the skin of the fruit. Although FBTO was not removed during must and white wine elaboration, it remained associated with suspended particles existing in must and lees, settled after must fermentation, with a negligible risk of being transferred to commercialised wine. On the other hand, two by-products of FBTO (bis and mono (2-methyl-2-phenylpropyl) tin) were identified, for first time, in must and final white wines obtained from FBTO treated grapes. Found values for the first species ranged from 0.03 to 0.9 ng mL−1.
Keywords: Fenbutatin oxide; Grapes; Wine; By-products; Sample preparation; Organotin species