Analytical and Bioanalytical Chemistry (v.393, #6-7)

Tribute to Günter Gauglitz by Frieder W. Scheller (1555-1556).

Direct optical detection in fragment-based screening by Florian Pröll; Peter Fechner; Günther Proll (1557-1562).
Label-free biosensors based on direct optical detection principles are widely used in many different fields of research. Currently the higher level of automation and the increasing throughput of this technology are stimulating the interest of pharmaceutical companies. The information gained with label-free biosensors can be extremely valuable during the drug design process, particularly in combination with complementary techniques, including NMR, mass spectrometry and X-ray crystallography. In this article we focus on the advantages of direct optical biosensors especially in the field of fragment-based drug design, which is a widely used and extremely promising concept. Furthermore, we present optical biosensors as versatile tools for fragment-based screening and the future drug design process.
Keywords: Fragment-based drug design; Biosensor; Label-free screening

Affinity capturing for targeting proteins into micro and nanostructures by Changjiang You; Maniraj Bhagawati; Andreas Brecht; Jacob Piehler (1563-1570).
Protein immobilization into micro and nanoscaled patterns opens exciting possibilities in fundamental and applied research. Developing efficient capturing techniques while preserving the structural and functional integrity of the proteins on surfaces is a key challenge for surface scientists. In this paper, current techniques for site-specific protein immobilization into engineered surface architectures are reviewed. Fundamental principles for functional protein immobilization on solid supports are discussed and popular affinity-based recognition pairs and their application for capturing proteins into nano and microstructures are presented.
Keywords: Protein immobilization; Microstructure; Nanostructure; Patterning; Nitrilotriacetic acid; Biotinylation

Portable Raman explosives detection by David S. Moore; R. Jason Scharff (1571-1578).
Recent advances in portable Raman instruments have dramatically increased their application to emergency response and forensics, as well as homeland defense. This paper reviews the relevant attributes and disadvantages of portable Raman spectroscopy, both essentially and instrumentally, to the task of explosives detection in the field.
Keywords: Raman spectroscopy; Field analysis; Fluorescence interference; Explosives detection

A label-free and time-resolved biosensor based on reflectometric interference spectroscopy (RIfS) has been developed to evaluate the agonistic or antagonistic effects of potential ligands with unknown behavior. The biosensor utilizes the specific interaction between the estrogen receptor α (ERα) and short specific peptides. The unique feature of these peptides allows the investigation of the behavior of ligands and the discrimination between the agonistic and antagonistic effects caused by conformational changes of the receptor. Thus, this developed biosensor allows not only the differentiation between ligands and nonligands of a receptor, but also the potential of these ligands to influence conformational changes in the receptor, leading to activation or inhibition of the receptor-dependent pathways. Owing to the robustness of the direct optical detection principle used, the biosensor is applicable to complex biological matrices, even crude cell extracts. Moreover, the reliability of the biosensor, including regeneration steps when performing subsequent measurements, has been verified.
Keywords: Reflectometric interference spectroscopy (RIfS); Endocrine disruptor (EDC); Estrogen receptor (ER); Biosensor; Small interacting peptides; Label free

The N terminus of peptides generated by AspN is restricted to about 40 dipeptide motifs starting with D or E. These motifs are visible upon collision-induced dissociation (CID) as b2 ions, which are often the most abundant low-mass fragment ions. It was observed that b2 ions are accompanied by a set of sequence-specific neutral losses of CO, H2O, NH3, and some other small units. To test the utility of these profiles as additional parameters for reliable assignment of the b2 ion motif besides its m/z value, the CID spectra of 221 different AspN-generated peptides covering all N-terminal D-X and E-X motifs were recorded. Qualitatively, the b2 ion fragmentation profiles of individual motifs were found to exhibit little dependency on the rest of the peptide sequence. Thus, it is concluded that the set of b2 ion fragmentation profiles recorded in this study can be used as reference set. Knowledge of these profiles provides an increased specificity for b2 ion annotation of AspN-generated peptides compared to the use of only a solitary b2 ion m/z value. Recognition of the b2 ion motif provides a two-amino-acid sequence including its direction; it provides the location of this motif at the N terminus, and it sets a starting point for further extension of the b ion series.
Keywords: Peptide fragmentation; b2 ions; Tandem mass spectrometry; Collision-induced dissociation

Rapid detection of Mycobacterium tuberculosis cells by using microtip-based immunoassay by Woon-Hong Yeo; Shieng Liu; Jae-Hyun Chung; Yaling Liu; Kyong-Hoon Lee (1593-1600).
This paper describes a microtip-based approach of concentrating target analytes for a highly sensitive bioassay. As an example, rapid screening of bacterial whole cells is presented to detect Mycobacterium tuberculosis (MTB), a pathogenic bacterium for human tuberculosis (TB). The concentration and detection is performed with three sequential steps of (1) attracting bacterial whole cells in the vicinity of a microtip by alternating current electroosmotic flow; (2) capturing the cells onto the microtip by capillary action; (3) binding fluorophore-labeled polyclonal antibodies to the cells followed by fluorescence measurement (immunofluorescence). Through this mechanism, MTB cells have been detected to the concentration of 8,000 cells/mL within 10 min. This sensitivity is comparable to that of Ziehl–Neelsen smear microscopy, a common culture-free screening method for diagnosis of TB. For comparison, Escherichia coli O157:H7 cells have also been detected to the concentration of 30,000 cells/mL in the same way.
Keywords: Immunoassay; Immunofluorescence; Mycobacterium tuberculosis ; Biosensor; AC electroosmosis; Capillary action

Bilayer lipid membranes from falling droplets by Michele Zagnoni; Mairi E. Sandison; Phedra Marius; Hywel Morgan (1601-1605).
We describe a system that provides a rapid and simple way of forming suspended lipid bilayers within a microfluidic platform from an aqueous droplet. Bilayer lipid membranes are created in a polymeric device by contacting monolayers formed at a two-phase liquid–liquid interface. Microdroplets, containing membrane proteins, are injected onto an electrode positioned above an aperture machined through a conical cavity that is filled with a lipid–alkane solution. The formation of the BLM depends solely on the device geometry and leads to spontaneous formation of lipid bilayers simply by dispensing droplets of buffer. When an aqueous droplet containing transmembrane proteins or proteoliposomes is injected, straightforward electrophysiology measurements are possible. This method is suitable for incorporation into lab-on-a-chip devices and allows for buffer exchange and electrical measurements. Figure Bilayer lipid membranes are formed in a polymeric device by injecting water droplets, containing membrane proteins, directly onto an electrode positioned above an aperture machined into a conical cavity, which is initially filled with a lipid-alkane solution. The water droplet slides down the electrode to the aperture at the bottom of the conical reservoir. The geometry of this system enables the spontaneous formation of a BLM. Ion channel activity is recorded between an electrode in the bottom channel and the electrode in the droplet. The technique is scalable and could be configured as a high throughput multi-site biosensing or drug screening platform.
Keywords: Biosensors; Self-assembled monolayers; Electrophysiology

Hydrolysis of 3,4-methylenedioxymethamphetamine (MDMA) metabolite conjugates in human, squirrel monkey, and rat plasma by Melanie Mueller; Erin A. Kolbrich-Spargo; Frank T. Peters; Marilyn A. Huestis; George A. Ricaurte; Hans H. Maurer (1607-1617).
Characterizing the formation of metabolites of 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”) in different species (rat, squirrel monkey, and human) may provide insight into mechanisms of MDMA neurotoxicity. Two prominent MDMA metabolites, 3,4-dihydroxymethamphetamine (HHMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA), are conjugated with glucuronic or sulfuric acid, but reference standards are not available; therefore, quantification is only possible after conjugate cleavage. Different concentrations of HHMA and HMMA were obtained in human, squirrel monkey, and rat plasma specimens when acid or enzymatic cleavage was performed. Our data document that these differences are due to species-specific influences on conjugate cleavage. Acidic hydrolysis should be used for analyzing free HHMA and HMMA in human or squirrel monkey plasma, while enzymatic hydrolysis with glucuronidase or sulfatase maximizes recovery of free HHMA and HMMA in rat plasma. Optimization of cleavage conditions showed that sulfate conjugates were more readily cleaved by acid hydrolysis and glucuronides by glucuronidase.
Keywords: MDMA; Metabolites; LC-ESI-MS; Hydrolysis; Human; Squirrel monkey; Rat; Plasma

Optical diagnosis of peritoneal metastases by infrared microscopic imaging by Valérie Untereiner; Olivier Piot; Marie-Danielle Diebold; Olivier Bouché; Elodie Scaglia; Michel Manfait (1619-1627).
Fourier transform infrared (FTIR) spectroscopy is nowadays widely accepted as a technique with high potential for diagnosis of cancerous tissues. This study presents an example of the investigation of peritoneal metastases by FTIR microimaging. Peritoneal malignancies are generally secondary localizations of primary visceral cancers such as ovarian, stomach or colon cancers. By analysing simultaneously both formalin-fixed paraffin-embedded and frozen specimens, we examined malignant and non-malignant (i.e. fibrotic and cicatricial) peritoneal lesions. Paraffin-embedded tissues were analysed without any previous dewaxing. Multivariate statistical approaches, based on the classification of infrared data by hierarchical cluster analysis, allowed the discrimination of these various samples. Microimaging also permits the revelation of the heterogeneity of the tissue: it was possible to localize precisely the cancerous areas, and to distinguish, on the basis of their spectral signatures, the peritumoral neighbouring connective tissue close to the carcinomatous areas from the connective tissue distant from the cancerous areas. These spectral differences could be useful as complementary information to study molecular changes associated with the malignancy.
Keywords: Micro Fourier transform infrared analysis; Peritoneal metastases; Optical diagnosis; Multivariate statistical treatment

To address food safety concerns of the public regarding the potential transfer of recombinant DNA (cry1Ab) and protein (Cry1Ab) into the milk of cows fed genetically modified maize (MON810), a highly specific and sensitive quantitative real-time PCR (qPCR) and an ELISA were developed for monitoring suspicious presence of novel DNA and Cry1Ab protein in bovine milk. The developed assays were validated according to the assay validation criteria specified in the European Commission Decision 2002/657/EC. The detection limit and detection capability of the qPCR and ELISA were 100 copies of cry1Ab μL−1 milk and 0.4 ng mL−1 Cry1Ab, respectively. Recovery rates of 84.9% (DNA) and 97% (protein) and low (<15%) imprecision revealed the reliable and accurate estimations. A specific qPCR amplification and use of a specific antibody in ELISA ascertained the high specificity of the assays. Using these assays for 90 milk samples collected from cows fed either transgenic (n = 8) or non-transgenic (n = 7) rations for 6 months, neither cry1Ab nor Cry1Ab protein were detected in any analyzed sample at the assay detection limits. Figure Schematic formats for quantitative real-time PCR and ELISA for the quantification of cry1Ab DNA and Cry1Ab protein
Keywords: Bovine milk; ELISA; MON810; Quantitative real-time PCR; Validation

Microarray of DNA–protein complexes on poly-3-hydroxybutyrate surface for pathogen detection by Tae Jung Park; Seung Min Yoo; Ki Chang Keum; Sang Yup Lee (1639-1647).
A novel strategy was developed for the specific immobilization of DNA probes on poly-3-hydroxybutyrate (PHB) surface by using the substrate-binding domain (SBD) of PHB depolymerase as an active binding motif. To demonstrate whether this method can be used for the detection of clinical pathogens, the pathogen-specific biotin-labeled DNA probes were immobilized via core streptavidin (cSA) fused to the SBD. The pathogen-specific 15-mer oligonucleotide probes were designed for four model pathogens, while the target DNAs were prepared by PCR using universal primers. The complex of pathogen-specific probes and cSA-SBD fusion protein was immobilized on the PHB-coated slide by microspotting. This DNA–protein complex microarray was able to successfully diagnose Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Furthermore, the specific pathogens could be diagnosed in the presence of other microorganisms. Thus, the DNA–protein complex microarray platform technology employing PHB and the SBD reported here can be widely used for the detection of DNA–DNA and DNA–biomolecule interactions without synthetic or chemical modification of biomolecules or solid surface.
Keywords: Protein microarray; DNA chip; Poly-3-hydroxybutyrate; Pathogen detection; PHB depolymerase; Substrate-binding domain

Phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) from urine of patients with breast cancer were qualitatively and quantitatively analyzed by nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (nLC-ESI-MS-MS). Urinary phospholipids (PLs) were extracted from three different categories of patients (non-cancer controls and breast cancer patients before and after surgery) by first lyophilizing only 1 mL of urine sample to enrich PLs. Next, nLC-ESI-MS-MS analysis of intact urinary phospholipids was performed, resulting in structural identification of 21 PCs and 12 PEs, followed by quantitative analysis using a multiple standard addition method. This study demonstrated that nLC-ESI-MS-MS can be powerfully utilized for the study of relative changes in the contents and concentration of urinary PCs and PEs from breast cancer patients: total concentration of PCs and PEs of patient sample increased to (144 ± 9)% and (171 ± 11)%, respectively, compared to control sample but they decreased significantly following surgery.
Keywords: Phospholipids; Quantitative analysis; nLC-ESI-MS-MS; Urine; Breast cancer

A metabonomics approach based on high-resolution magic-angle spinning (HRMAS) 1H NMR spectroscopy was applied to investigate the metabolite composition in intact hepatic tissues and renal cortical tissues from db/db mice of 8 weeks old, an animal model of type 2 diabetes mellitus. Compared to the control group, the hepatic tissues of diabetic mice have elevated levels of triglyceride and bile acid and declined levels of trimethylamine-N-oxide, phosphocholine, glycerophosphocholine, and choline. The biochemical changes are less obvious in renal cortical tissues of diabetic mice. The WET_CPMG pulse sequence was selected for our metabonomics study after the quality and reproducibility of the spectra obtained from the NOEPR, NOEPR_CPMG, and WET_CPMG pulse sequences were analyzed together with principal component analysis. The influence of line-broadening factor of exponential window function for spectral manipulation on class separation was paid attention to for the first time, and an optimal value was obtained under our experimental conditions. These studies show the efficiency of HRMAS 1H NMR spectroscopy for tissue metabonomics study in combination with multivariate statistical analysis, which may help to explore the etiological factor of diabetes mellitus from a new perspective. Comparative 1H MAS NMR spectra of liver and kidney tissues
Keywords: NMR; Metabonomics; Magic-angle spinning (MAS); Principal component analysis (PCA); Type 2 diabetes mellitus (T2DM); Tissues

This paper reports on a rapid and sensitive method for the simultaneous determination of ascorbic acid (H2A), dehydroascorbic acid (DHA), and total vitamin C by electrochemiluminescence (ECL) using a thin-layer electrochemical cell. Significant ECL signals can be generated by the anodic oxidation of Ru(bpy)3 2+ in the presence of H2A or DHA in pH 8.8 phosphate buffer solution. Because of the extremely small dead volume of the thin-layer cell (approximately 1.5 μL), almost all amount of H2A is assumed to be completely oxidized to DHA with a short pre-electrolysis step. As a result, it is possible to determine the reductive vitamin C (H2A) by square wave voltammetry before the pre-electrolysis step, while total vitamin C (sum of H2A and DHA) is able to be determined at a subsequent ECL step. The method was employed for the determination of vitamin C in commercial beverages with the analytical results in good agreement with the certified values. Figure (A) A novel thin-layer electrochemical cell is designed for the determination of ascorbic acid, dehydroascorbic acid (DHA) by Ru(bpy)3 2+ based electrochemiluminescence (ECL) protocol. (B) ECL responses for DHA with different concentration levels
Keywords: Electrochemiluminescence; Tris(2; 2′-bipyridine)ruthenium; Thin-layer electrochemical cell; Ascorbic acid; Dehydroascorbic acid; Vitamin C

Highly sensitive sensor for detection of NADH based on catalytic growth of Au nanoparticles on glassy carbon electrode by Lin Tang; Guangming Zeng; Guoli Shen; Yi Zhang; Yuanping Li; Changzheng Fan; Can Liu; Chenggang Niu (1677-1684).
In this work, an electrochemical dihydronicotinamide adenine dinucleotide (NADH) sensor based on the catalytic growth of Au nanoparticles (Au NPs) on glassy carbon electrode was developed. Catalyzed by Au NPs immobilized on pretreated glassy carbon electrode, the reduction of AuCl4 in the presence of hydroquinone and cetyltrimethyl ammonium chloride led to the formation of enlarged Au NPs on the electrode surface. Spectrophotometry and high-resolution scanning electronic microscope (SEM) analysis of the sensor morphologies before and after biocatalytic reaction revealed a diameter growth of the nanoparticles. The catalytic growth of Au NPs on electrode surface remarkably facilitated the electron transfer and improved the performance of the sensor. Under optimal conditions, NADH could be detected in the range from 1.25 × 10−6 to 3.08 × 10−4 M, and the detection limit was 2.5 × 10−7 M. The advantages of the proposed sensor, such as high precision and sensitivity, fast response, low cost, and good storage stability, made it suitable for on-line detection of NADH in complex biological systems and contaminant degradation processes. Figure Schematic presentation of the bioelectrocatalytic sensing of NADH
Keywords: NADH; Sensor; Catalytic growth; Au nanoparticles; Glassy carbon electrode

In this study, we aimed at optimizing a sensitive and reliable method for a simultaneous determination of 31 pharmaceuticals belonging to predominant therapeutic classes identified in different types of sewage sludge proceeding from conventional and advanced wastewater treatment. Freeze-dried sewage sludge was extracted by pressurized liquid extraction technique using accelerated solvent extractor Dionex 300. In order to minimize interferences with matrix components and to preconcentrate target analytes, solid phase extraction was introduced in the method as a clean-up step. The entire method was validated for linearity, precision, accuracy, and method detection limits (MDLs). The method turned out to be specific, sensitive, and reliable for the analysis of sludge of different composition, type, and retention time in the process. The developed sample preparation protocol and previously published method for LC-MS/MS analysis (Gros et al., Talanta 70:678–690, 2006) were successfully applied to monitor the target pharmaceuticals in different types of sewage sludge, i.e., primary sludge, secondary sludge, treated sludge, and sludge proceeding from pilot-scale membrane bioreactors (MBRs) operating in parallel to the conventional activated sludge treatment. Among the investigated pharmaceuticals, 20 were detected in the sludge proceeding from full-scale installations, whereas the MBR sludge concentrations were below MDLs for several compounds. The highest concentrations were recorded for treated and primary sludge. For example, the mean concentration of ibuprofen in the digested sludge was 299.3 ± 70.9 ng g−1 dw, whereas in the primary sludge, it was enriched up to 741.1 ng g−1 dw. Other pharmaceuticals detected at relatively high concentrations were diclofenac, erythromycin, glibenclamide, ketoprofen, ofloxacin, azithromycin (up to 380.7, 164.2, 190.7, 336.3, 454.7, 299.6 ng g−1 dw in the primary sludge, respectively), gemfibrozil, loratidine, and fluoxetine (up to 189.1, 189.7 and 174.1 ng g−1 dw in the treated sludge, respectively).
Keywords: Sewage sludge; Pharmaceuticals; Multi-residue; Pressurized liquid extraction (PLE)

Three different stationary phases were investigated for the analysis of the 15 + 1 EU-priority polycyclic aromatic hydrocarbons (PAHs) by gas chromatography-mass spectrometry. In addition to the most commonly used 5% phenyl methylpolysiloxane, a mid-polar phase (50% phenyl methylpolysiloxane) and a recently commercialised mid-polar to polar phase (Optima® δ-6), were evaluated. Challenging groups of PAHs in terms of separation, such as the pair dibenz[a,h]anthracene-indeno[1,2,3,-cd]pyrene and the two groups benzo[b]fluoranthene-benzo[k]fluoranthene-benzo[j]fluoranthene and cyclopenta[cd]pyrene-benz[a]anthracene-chrysene, were satisfactorily separated by using the mid-polar phase. Moreover, discrimination in terms of peak height for the heaviest PAHs (caused from the strong interaction of these compounds with the stationary phase) was reduced without compromising the resolution of the other target analytes when applying the mid-polar phase in a tailor-made column geometry (20 m × 0.18 mm internal diameter and 0.14 μm film thickness) in combination with optimised chromatographic conditions. A significant enhancement of the analytical sensitivity for dibenzopyrenes is demonstrated with an almost threefold increase of the signal-to-noise (S/N) ratio for dibenzo[a,h]pyrene, the last eluting PAH. The ability of the selected column to separate potentially interfering PAHs from the target analytes in both solvent solutions and food extracts is demonstrated.
Keywords: Polycyclic aromatic hydrocarbons (PAHs); 15 + 1 EU-priority PAHs; GC-MS; Stationary phase; Peak height discrimination; Resolution

Ultra performance liquid chromatography tandem mass spectrometry performance evaluation for analysis of antibiotics in natural waters by Fatima Tamtam; Fabien Mercier; Joëlle Eurin; Marc Chevreuil; Barbara Le Bot (1709-1718).
An ultra performance liquid chromatography electrospray tandem mass spectrometry (UPLC/MS/MS) method was developed and validated for the determination of 17 antibiotics in natural waters in one single extraction and chromatographic procedure. Gradient separation conditions were optimised for 17 compounds belonging to five different antibiotic groups: quinolones (oxolinic acid, nalidixic acid, pipemidic acid, flumequine), fluoroquinolones (enoxacin, ciprofloxacin, norfloxacin, ofloxacin, enrofloxacin, sarafloxacin, danofloxacin, difloxacin, lomefloxacin), sulphonamides (sulphamethoxazole, sulphamethazine), nitro-imidazole (ornidazole) and diaminopyrimidine (trimethoprim). The separation of all compounds, obtained using a 1.7 μm particle size column (100 mm × 2.1 mm), was achieved within 10 min time. Water samples were adjusted to pH 7 and extracted using Oasis hydrophilic–lipophilic balance (HLB) solid phase extraction cartridges. After elution with methanol and concentration, extracts were injected in a C18 column (Acquity UPLC BEH C18) and detected by tandem mass spectrometry. Average recovery from 100 ng L−1 fortified samples was higher than 70% for most of the compounds, with relative standard deviations below 20%. Performances of the method (recoveries, detection limit, quantification limit and relative standard deviation) and matrix effects were studied, and results obtained showed that method was suitable for routine analysis of antibiotics in surface water. Samples analysis from Seine River (France) confirmed the interest of antibiotic contamination evaluation in that area. Fig. a UPLC/MS/MS extracted ion chromatograms of a standard solution containing 17 analytes
Keywords: UPLC; Antibiotics; River water; Quinolones; Sulphonamides; Matrix effects; Solid phase extraction

Investigation into persistent organic pollutants (POPs) in sea turtles is an important area of conservation research due to the harmful effects of these chemicals. However, the analysis of POPs in the green sea turtle (Chelonia mydas) has been limited by methods with relatively high limits of detection and high costs associated with multiple sample injections into complex arrangements of analytical equipment. The present study aimed to develop a method that could detect a large number of POPs in the blood, eggs and tissue of C. mydas at trace concentrations. A gas chromatography with tandem mass spectrometry (GC-MS/MS) method was developed that could report 125 POP compounds to a limit of detection of <35 pg g−1 using a single sample injection. The recoveries of internal standards ranged from 30% to 96%, and the standard reference materials were reported to within 70% of the certified values. The coefficient of variation of ten replicates of pooled egg sample was <20% for all compounds, indicating low within-run variation. This GC-MS/MS method is an improvement of previous methods for analysing POPs in C. mydas in that more compounds can be reported at lower concentrations and the accuracy and precision of the method are sound. This is particularly important for C. mydas as they occupy a low trophic level and have lower concentrations of POPs. This method is also simple to set up, and there are minimal differences in sample preparation for the different tissue types.
Keywords: Chelonia mydas ; Persistent organic pollutants; GC-MS/MS; Chlorinated and brominated compounds

In this stir bar sorptive extraction (SBSE) method, 16 pesticides were extracted from surface water samples by sorption onto 1 mm polydimethylsiloxane layer coated on a 10-mm-length stir bar magnet. After liquid desorption of the analytes with 1 ml of methanol, the detection was performed on a liquid chromatography-tandem mass spectrometry with a triple quadrupole (QqQ) analyzer using selected reaction monitoring mode via electrospray ionization. Parameters affecting SBSE operation, including sample volume, salt addition, extraction time, stirring rate, and desorption conditions, have been evaluated. The optimized SBSE method required two 50 ml aliquots of surface water samples, one aliquot was added of 30% NaCl and stirred at 900 rpm during 1 h for testing five pesticides with log K o/w < 3, and the other aliquot was directly extracted following the same procedure for the rest of the pesticides with log K o/w > 3. The method was validated in spiked surface water samples at limits of quantifications (LOQs) and ten times the LOQs showing recoveries <62%, and the LOQs reached were from 0.03 μg l−1 for diazinon to 3 μg l−1 for simazine. The proposed methodology was applied to the determination of these compounds in samples from Albufera Lake and surrounding channels, showing that SBSE is a powerful tool for routine control analysis of pesticide residues in surface water.
Keywords: Surface water analysis; Stir-bar sorptive extraction; Environmental analysis; Liquid chromatography; Tandem mass spectrometry; Triple quadrupole

Determination of benzimidazole fungicides in water samples by on-line MISPE–HPLC by O. Zamora; E. E. Paniagua; C. Cacho; L. E. Vera-Avila; C. Perez-Conde (1745-1753).
An analytical methodology based on an on-line sample enrichment of water samples by means of an imprinted polymer, and the separation of benzimidazole compounds within a C18 column by ion-pair reversed-phase liquid chromatography, has been developed. The molecularly imprinted polymer has been synthesized by precipitation polymerization using thiabendazole as template molecule, methacrylic acid as functional monomer, and divinylbenzene as cross-linker. Initial experiments carried out by solid-phase extraction on cartridges demonstrated a clear imprint effect for thiabendazole, as well as the ability of the imprinted polymer to selectively rebind several benzimidazole compounds. The developed methodology has been applied to the quantification of thiabendazole, carbendazim, and benomyl in river, tap, and well water samples within a single analytical run at concentration levels below the legislated maximum concentration levels. In this sense, detection limits of 2.3–5.7 ng·L−1 have been obtained for the analysis of benzimidazole fungicides in different water matrices. Recoveries obtained for the determination of benzimidazole fungicides in spiked samples ranged from 87% to 95%, with RSD below 5% in all cases.
Keywords: Benzimidazole fungicides; Molecularly imprinted polymers; On-line SPE; Ion-pair HPLC; Water samples

A rapid and sensitive method has been established for the determination of four carbamate pesticides (carbofuran, carbaryl, pirimicarb, and diethofencarb) in water samples by using dispersive liquid–liquid microextraction coupled with high-performance liquid chromatography–diode array detection. Parameters that affect the extraction efficiency, such as the kind and volume of the extraction and disperser solvent, extraction time, and salt addition, were investigated and optimized. Under the optimum conditions, the enrichment factors were in the range between 101 and 145. The linearity of the method was obtained in the range of 5–500 ng mL−1 with the correlation coefficients (r) ranging from 0.9978 to 0.9997. The method detection limits were 0.4–1.0 ng mL−1. The relative standard deviations varied from 4.7% to 6.5% (n = 5). The relative recoveries of the four carbamates from water samples at spiking levels of 5.0 and 20.0 ng mL−1 were 84.0–92.0% and 86.5–94.0%, respectively. The proposed method has been successfully applied to the analysis of target carbamate residues in river, rain, well, and tap water samples with satisfactory results. Figure 1 The typical chromatogram of tap water sample spiked with carbamate pesticides at each concentration of 10 ng mL−1. Peak identification: (1) carbofuran, (2) carbaryl, (3) pirimicarb, and (4) diethofencarb
Keywords: Dispersive liquid–liquid microextraction; Carbamate pesticides; High-performance liquid chromatography; Water samples

Selective solid-phase extraction of ethynylestradiol from river water by molecularly imprinted polymer microcolumns by J. C. Bravo; R. M. Garcinuño; P. Fernández; J. S. Durand (1763-1768).
A selective on-column molecularly imprinted solid-phase extraction (MISPE) for ethynylestradiol (EE2) from water samples was developed. Following a non-covalent molecular imprinting approach, two molecularly imprinted polymers (MIPs) have been synthesised using methacrylic acid (MAA) as functional monomer, ethyleneglycol dimethacrylate (EGDMA) as crosslinker and EE2 as template, in two different polymerisation solvents (acetonitrile or toluene). The optimisation of the on-column MISPE conditions for the selective rebinding of the EE2 resulted in a flow rate of 2.3 mL min−1 and volumes of 1 mL toluene and 1.5 mL methanol–acetic acid (9:1) for washing and elution steps, respectively. The selectivity of the imprinted polymer towards several related estrogens such as estriol (E3), estrone (E1) and estradiol (E2) was evaluated. The recovery of EE2 from a 50-mL spiked river-water sample was 75% when the optimised on-column MISPE was applied.
Keywords: Ethynylestradiol; Estrogens; MIPs; SPE; Waters

A study of complex protein mixtures obtained from biological samples by MS demands proper purification and separation technique. The method of divergent flow isoelectric focusing (DF IEF) promises improvement of sample preparation in proteomic studies. DF IEF was carried out in a separation channel with increasing width. The channel was cut out from a polyester nonwoven web. DC voltage (800 V) was brought to two pairs of electrodes situated on the channel sides. Amphoteric compounds, including proteins, drift through the channel carried by flow (18–25 ml/h) in streamlines given by their isoelectric points. The pH gradient (3–10) and its stability during analysis have been monitored with colored low-molecular mass pI markers. Separated fractions were collected in ten microvials and further analyzed by MS. The suggested method was used for separation and purification of crude protein extract from barley grain, malt, and beer. Collected fractions of separated proteins were characterized by MALDI-MS. Desalting during IEF enhanced significantly the quality of mass spectra. It also simplified monitoring of post-translational modifications and protein changes occurring during malting and brewing. Results have shown the real potential of the suggested DF IEF device lay-out as an efficient preparative tool for separation and purification of complex protein mixtures for further analyses.
Keywords: Isoelectric focusing; MALDI-MS; Protein; Glycation; Post-translational modifications

HPLC-fluorescence detection method for determination of key intermediates of the lincomycin biosynthesis in fermentation broth by Zdeněk Kameník; Jan Kopecký; Markéta Marečková; Dana Ulanová; Jitka Novotná; Stanislav Pospíšil; Jana Olšovská (1779-1787).
The biosynthetic pathway of the clinically important antibiotic lincomycin is not known in details. The precise knowledge of the lincomycin biosynthesis is a prerequisite for generation of improved derivatives by means of combinatorial genetics. Methods allowing determination of the key intermediates are very important tools of the pathway investigation. Two new high-performance liquid chromatography methods with fluorescence detection for determination of lincomycin precursors in fermentation broth of Streptomyces lincolnensis and its lincomycin nonproducing mutants were developed. The first one enables simultaneous analysis of methylthiolincosamide (MTL) and N-demethyllincomycin (NDL), whereas the second one is suitable for 4-propyl-l-proline (PPL) assay. Both methods are based on the pre-column derivatization: MTL and NDL with 4-chloro-7-nitrobenzofurazan; PPL with o-phthaldialdehyde. The methods were validated with lower limit of quantification values of 2.50, 3.75, and 3.75 μg ml−1 for MTL, NDL, and PPL, respectively. The inter- and intra-day accuracies and precisions were all within 12%. Stability of oxidized and derivatized analytes was investigated.
Keywords: Lincomycin precursors; o-Phthaldialdehyde; 4-Chloro-7-nitrobenzofurazan; HPLC; Fluorescence detection

Enantioseparation of nonproteinogenic amino acids by Margit Winkler; Norbert Klempier (1789-1796).
The enantioseparation of structurally related N-protected β-/γ-amino acids, β-/γ-amino amides, and β-/γ-amino nitriles by using six different commercially available chiral stationary phases (CSPs) is reported. The synthetic key step to introduce stereochemical asymmetry into all compounds is an enzymatic kinetic resolution of the racemic nitriles to the respective amino amides and/or amino acids, depending on the class of enzyme (nitrile hydratase or nitrilase) applied. The separation efficiencies of all CSPs with regard to functional groups as well as structural variations of the amino acid derivatives depicted in Fig. 1 are discussed. Figure Enantioseparation of (±)-3-N-Ts-Aminocyclopentanecarboxylic acid
Keywords: Enantioseparation; HPLC; Biotransformation; β-Amino acids; γ-Amino acids; Macrocyclic glycopeptide-based CSPs; Chiral crown ether; Polysaccharide-based CSPs; Protein-based CSPs

Liquid chromatography under critical conditions (LCCC) allows the separation of block copolymers from the corresponding homopolymers as well as the separation of homopolymers according to their functionality. At the transition of exclusion and adsorption mode, the polymer chain becomes “chromatographically invisible,” and thus a separation according to other structural units can be achieved. In the case of block copolymers this situation can be utilized to detect and determine unwanted homopolymers. At critical conditions for the repeat unit, the other block may be eluted in the exclusion or the adsorption regime. In the first case, the block copolymer is eluted before the homopolymer, and its molar mass can be determined as in size-exclusion chromatography. In the second case, it is eluted later than the homopolymer, and the separation of the individual oligomers can be achieved. Advantages and limitations of different approaches in LCCC are discussed. Figure Application of LCCC in the characterization of polypropylene glycol and its block copolymers.
Keywords: Liquid chromatography; Critical conditions; Block copolymers; Functional polymers

A method for the simultaneous analysis of the enantiomers of mefloquine (MQ) and its main metabolite carboxymefloquine (CMQ) in plasma is described for the first time. The assay involves two-step liquid-phase microextraction (LPME) and enantioselective high-performance liquid chromatography. In the first LPME step, the enantiomers of MQ were extracted from an alkalinized sample through a thin layer of di-n-hexyl ether immobilized in the pores of the hollow fiber and into 0.01 M perchloric acid as acceptor solution. In the second LPME step, the same sample was acidified to enable the extraction of CMQ using the same organic solvent and 0.05 M sodium hydroxide as acceptor phase. The analytes were resolved on a Chirobiotic T column in the polar-organic mode of elution and detected at 285 nm. The recovery rates from 1 mL of plasma were in the range 35–38%. The method presented limits of quantification of 50 ng/mL for all analytes and was linear up to 1,500 and 3,000 ng/mL for the enantiomers of MQ and CMQ, respectively. The plasmatic concentrations of (+)-(RS)-MQ were higher than those of (−)-(SR)-MQ after oral administration of the racemic drug to rats. Extraction device for LPME employing hollow fiber membranes used in the developed method
Keywords: Mefloquine enantiomers; Carboxymefloquine; Liquid-phase microextraction; Liquid chromatography; Plasma samples

Elution behavior of poly(lactide-co-succinimide) copolymers studied by SEC-MALS by Maja Gričar; Majda Žigon; Ema Žagar (1815-1823).
We synthesized poly(lactide-co-succinimide) (PLS) copolymers with the ratio of lactide to succinimide units of 3:1 and 6.5:1 and studied their elution behavior by size exclusion chromatography with an on-line light-scattering detection. Since the copolymers contain a certain amount of carboxyl groups, they behave as ionomers in N,N-dimethylacetamide (DMAc) and show a typical polyelectrolyte (PE) effect. The PE effect was eliminated by the addition of simple electrolyte like LiBr, H3PO4, or both in DMAc. The efficiency of the additive decreases in the order: LiBr > LiBr + H3PO4 > H3PO4. The ionic strength of the 0.1 M LiBr/DMAc was high enough for the onset of hydrophobic interactions of PLS lactic acid segments intermolecularly as well as with the column packing material. The drawback of the LiBr + H3PO4/DMAc solvent system is a rather high intensity of the system peaks, which are imposed on the right side of the copolymer signal. System peaks strongly influence the determination of number and to a lesser extent the weight average molar masses of PLS copolymers. An addition of only H3PO4 in high enough concentration to DMAc (0.05 and 0.1 M) successfully eliminated the PE effect of the 6.5:1 PLS copolymer. On the contrary, the PE effect of the 3:1 PLS copolymer having higher charge density compared to 6.5:1 PLS copolymer cannot be entirely canceled out in any of the H3PO4/DMAc solutions examined.
Keywords: Poly(lactide-co-succinimide); Amphiphilic copolymers; Size exclusion chromatography; Light scattering; Elution behavior