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Analytical and Bioanalytical Chemistry (v.401, #8)

A “little western flower” challenge by Reinhard Meusinger (pp. 2319-2325).

Solution to living cell spectroscopy challenge by Allison Stelling; Gerald Steiner (pp. 2327-2327).

Sebastian Schlücker (Ed.): Surface enhanced Raman spectroscopy: analytical, biophysical and life science applications by Jiří Homola (pp. 2329-2330).

ManMohan Srivastava (Ed.): High-performance thin-layer chromatography (HPTLC), 1st ed by Gertrud Morlock (pp. 2331-2332).

Comprehensive multidimensional separations by James Harynuk; Philip Marriott (pp. 2333-2334).

received his PhD from the University of Waterloo in 2004 where, in the laboratory of Tadeus Górecki, he was the first Canadian student to study the emerging field of comprehensive 2D gas chromatography. Thereafter he continued the study of multidimensional separations with Philip Marriott at the Royal Melbourne Institute of Technology. In 2007 he started his own research group at the University of Alberta which is working on the advancement of multidimensional separations science. His current research interests include studies of the relationships between molecular structure and gas chromatographic retention using a thermodynamic approach which now extends to the development of retention models to aid in determining the structures of analytes analysed by gas chromatography. He is developing chemometric tools for quickly extracting information from chromatographic data and has invented the concept of cluster resolution—a robust metric that permits the automated construction, feature selection, and optimisation for chemometric models. As recently highlighted, these tools have been used to develop chemometric models for the identification of ignitable liquids in simulated fire debris. is Deputy Director of the Centre for Green Chemistry, School of Chemistry, at Monash University, Australia. He had previous appointments at the University of Bristol (UK), National University of Singapore, and at RMIT University, Australia. He has been involved in GC × GC and MDGC technology developments for almost 15 years. He has published 280 papers and book chapters, largely on GC. His key research interests have been in fundamental relationships in and descriptions of GC and GC × GC, chromatography reactor studies, chiral separations, extraction methods, detector technologies, and capillary electrophoresis. He has an interest in applying these technologies to a wide range of applications in order to demonstrate these new tools to a broad base of users of chromatography. Recent work has involved offline method development in MDGC/NMR and GC/NMR, with an aim to improving the absolute structural assignment of resolved molecular species.

Modulation in comprehensive two-dimensional gas chromatography: 20 years of innovation by Matthew Edwards; Ahmed Mostafa; Tadeusz Górecki (pp. 2335-2349).

With almost 20 years having passed since John B. Phillips described the first comprehensive two-dimensional gas chromatography (GC × GC) separation, much has occurred in this ever-expanding field of separation science. GC × GC is currently one of the most effective techniques for the separation and analysis of complex mixtures, offering significantly greater peak capacities than conventional chromatographic methods. The technique is generally based upon separations performed on two chromatographic columns characterized by considerably different selectivities, joined together through a modulating interface. The modulator periodically traps or samples the primary column effluent, usually refocuses it into a narrow chromatographic band and injects the focused fraction into the secondary column. The modulator is often referred to as the ‘heart’ of the instrument, since a GC × GC separation is impossible without its use. This article reviews major innovations in GC × GC modulator development since its first use by Phillips in 1991. Emphasis has been placed on modulator design and function.

Keywords: Comprehensive two-dimensional gas chromatography; Instrumentation; Modulation; Review

Retention indices in comprehensive two-dimensional gas chromatography by Carin von Mühlen; Philip J. Marriott (pp. 2351-2360).

The identification of compounds by using gas chromatography (GC) in samples with significant complexity comprising a range of isomeric species, where characterization is based on peak retention times and mass spectra, generates uncertainty for the analyst. This leads to identification errors. The most reliable way to confirm the identification of each compound is based on authentic standard co-injection, which in several cases is economically prohibitive, and often unachievable in the time available for analysis. Retention index procedures are important tools to minimize misidentification of compounds in conventional chromatography. The introduction of comprehensive two-dimensional GC (GC × GC) for analysis of complex samples was a decisive step to increase the analytical capacity of chromatographic techniques. For many samples, the chromatographic resolution increase leads to quantitative expansion in the number of peaks identified, compared with conventional GC analysis. Notwithstanding this improved resolution, limitations still persist in correct peak identification, which suggests the use of retention indices may assist in supporting component identification in this important technique. In this work, approaches to use of the retention index in GC × GC are discussed, based on an evaluation of the literature in this area. Interpretation of effective chain length data for fatty acid methyl esters in the first and second dimensions is presented.

Keywords: Comprehensive two-dimensional gas chromatography; Linear-temperature-programmed retention index; Retention index; Kovats index; Fatty acid methyl esters; GC × GC retention index

Comprehensive two-dimensional gas chromatography applied to illicit drug analysis by Blagoj Mitrevski; Paul Wynne; Philip J. Marriott (pp. 2361-2371).

Multidimensional gas chromatography (MDGC), and especially its latest incarnation—comprehensive two-dimensional gas chromatography (GC × GC)—have proved advantageous over and above classic one-dimensional gas chromatography (1D GC) in many areas of analysis by offering improved peak capacity, often enhanced sensitivity and, especially in the case of GC × GC, the unique feature of ‘structured’ chromatograms. This article reviews recent advances in MDGC and GC × GC in drug analysis with special focus on ecstasy, heroin and cocaine profiling. Although 1D GC is still the method of choice for drug profiling in most laboratories because of its simplicity and instrument availability, GC × GC is a tempting proposition for this purpose because of its ability to generate a higher net information content. Effluent refocusing due to the modulation (compression) process, combined with the separation on two ‘orthogonal’ columns, results in more components being well resolved and therefore being analytically and statistically useful to the profile. The spread of the components in the two-dimensional plots is strongly dependent on the extent of retention ‘orthogonality’ (i.e. the extent to which the two phases possess different or independent retention mechanisms towards sample constituents) between the two columns. The benefits of ‘information-driven’ drug profiling, where more points of reference are usually required for sample differentiation, are discussed. In addition, several limitations in application of MDGC in drug profiling, including data acquisition rate, column temperature limit, column phase orthogonality and chiral separation, are considered and discussed. Although the review focuses on the articles published in the last decade, a brief chronological preview of the profiling methods used throughout the last three decades is given.

Keywords: Comprehensive two-dimensional gas chromatography; Illicit drugs; Profiling

Chemometrics in comprehensive multidimensional separations by Zhong-Da Zeng; Helmut M. Hugel; Philip J. Marriott (pp. 2373-2386).

Chemometric methods have critical importance for the discovery of the information/knowledge buried or concealed in high-dimensional datasets acquired from comprehensive multidimensional separations (CMDS), and for interpretation of experiments or chemical processes. In this work, employment of new developments in chemometrics making full use of the data to maximize the potential of CMDS to resolve mathematically a variety of practical problems is reviewed whilst providing the authors' point of view. During the past several years, chemometrics has been successfully applied to many areas of concern to CMDS investigation, including experimental parameter optimization, data quality improvement, identification and quantification of target chemical components, pattern recognition technique for clustering and classification, multivariate model establishment to correlate chromatographic properties and molecular descriptors. On the basis of the high-dimensionality characteristics of CMDS, some special aspects such as evaluation of orthogonality and image processing have also been included in this review. It is expected that an overview of the diverse ways in which chemometrics can aid CMDS investigations will prove valuable to interested users in this area through a comprehensive survey of previous research contributions. Chemometrics lends itself well to the powerful separation capability of CMDS.

Keywords: Chemometrics; Comprehensive multidimensional separations; Qualitative and quantitative analysis; Principal component analysis; Quantitative structure–retention relationships; Image processing

Toward a global analysis of metabolites in regulatory mutants of yeast by Elizabeth M. Humston; Kenneth M. Dombek; Benjamin P. Tu; Elton T. Young; Robert E. Synovec (pp. 2387-2402).

The AMP-activated protein kinase in yeast, Snf1, coordinates expression and activity of numerous intracellular signaling and developmental pathways, including those regulating cellular differentiation, response to stress, meiosis, autophagy, and the diauxic transition. Snf1 phosphorylates metabolic enzymes and transcription factors to change cellular physiology and metabolism. Adr1 and Cat8, transcription factors that activate gene expression after the diauxic transition, are regulated by Snf1; Cat8 through direct phosphorylation and Adr1 by dephosphorylation in a Snf1-dependent manner. Adr1 and Cat8 coordinately regulate numerous genes encoding enzymes of gluconeogenesis, the glyoxylate cycle, β-oxidation of fatty acids, and the utilization of alternative fermentable sugars and nonfermentable substrates. To determine the roles of Adr1, Cat8, and Snf1 in metabolism, two-dimensional gas chromatography coupled to time-of-flight mass spectrometry and liquid chromatography coupled to tandem mass spectrometry were used to identify metabolites whose levels change after the diauxic transition in wild-type-, ADR1-, CAT8-, and SNF1-deficient yeast. A discovery-based approach to data analysis utilized chemometric algorithms to identify, quantify, and compare 63 unique metabolites between wild type, adr1∆, cat8∆, adr1∆cat8∆, and snf1∆ strains. The primary metabolites found to differ were those of gluconeogenesis, the glyoxylate and tricarboxylic acid cycles, and amino acid metabolism. In general, good agreement was observed between the levels of metabolites derived from these pathways and the levels of transcripts from the same strains, suggesting that transcriptional control plays a major role in regulating the levels of metabolites after the diauxic transition. Figure Metabolite data for wild type and mutant yeast strains were studied utilizing GC × GC-TOFMS and LC-MS/MS combined with chemometric data analysis. Using PCA as a comparison tool, the mutant strain adr1∆ behaved similar to the wildtype, while snf1∆, cat8∆, and adr1∆cat8∆ behaved similar to each other, when all strains were grown in nonfermentable media

Keywords: GC × GC–TOFMS; Chemometrics; PARAFAC; Snf1 protein complex

A routine accredited method for the analysis of polychlorinated biphenyls, organochlorine pesticides, chlorobenzenes and screening of other halogenated organics in soil, sediment and sludge by GCxGC-μECD by Alina M. Muscalu; Eric J. Reiner; Steven N. Liss; Tony Chen; Gerry Ladwig; David Morse (pp. 2403-2413).

The analysis of persistent organic pollutants is a real challenge due to the large number of compounds with varying chemical and physical properties. Gas chromatography with electron capture detection or mass spectrometry has been the method of choice for the past 50 years. Comprehensive two-dimensional gas chromatography (GCxGC) coupled with micro-electron capture detector (μECD) is a new method that can analyze polychlorinated biphenyls (PCBs), organochlorine pesticides (OCs) and chlorobenzenes (CBz) in a single analytical run with enhanced selectivity and sensitivity over single column methods and can also be used to screen for other halogenated organics in environmental samples. An accredited routine method using commercially available LECO GCxGC-μECD and a column combination DB-1 × Rtx-PCB has been developed to analyse PCBs/OCs/CBz in soils, sediments and sludges. The method provides quantification of Aroclors and Aroclor mixtures to within 15% of target values and sub-nanogrammes per gramme detection limits. Figure Enhanced separation by GCxGC

Keywords: GCxGC; Comprehensive two-dimensional gas chromatography; PCB; Organochlorine pesticides; Chlorobenzenes; μECD; Routine method; POPs

Study of alkyl phosphates in industrial petroleum mixtures by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry by James J. Harynuk; Aleisha D. Rossé; G. Bryce McGarvey (pp. 2415-2422).

Dialkyl phosphate esters used as gellants in some oil well fracturing processes for conventional oil production can result in contamination of the collected crude. Though the exact mechanism is unclear, such compounds form volatile phosphorus that compromises refinery processes. Our initial research involved producing a comprehensive two-dimensional gas chromatographic method (GC × GC) for the detection and quantification of alkyl phosphate esters in petroleum samples, which surpassed the current method employed in sensitivity and speciation capabilities. However, selective detection is required for such analytes in petroleum matrices. This article describes the application of GC × GC with time-of-flight mass spectrometry for selective detection to the analysis of di- and tri-alkyl phosphates in petroleum samples. Features in the electron impact mass spectra of alkyl phosphates are discussed along with the GC × GC retention characteristics of the compounds. Based on these discussions, a preliminary classification and quantification of alkyl phosphate contamination in a suite of industrial samples is then presented.

Keywords: GC × GC; Petroleum; TOFMS; Phosphate ester; Volatile phosphorus; Fracturing fluid

Using GC × GC-FID profiles to estimate the age of weathered gasoline samples by Brianne M. Zorzetti; James J. Harynuk (pp. 2423-2431).

Predicting the amount of time that a petroleum mixture has been exposed to weathering effects has applications in areas of environmental and other forensic investigations, such as aiding in determining the cause and intent of a fire. Historically, research on the evaporation rates of hydrocarbon mixtures has focused on forensic oil spill identification and predicting if a fresh sample could be weathered to give an observed composition in an aged sample. Relatively little attention has focused on approaching the problem from the other direction: estimating exposure time based on the observed composition of a weathered sample at a given time and assuming a prior composition. Here, we build upon our previous research into the weathering of model mixtures by extending our work to gasoline. Samples of gasoline with varying octane ratings and from several vendors were weathered under controlled conditions and their composition monitored over time by two-dimensional gas chromatography (GC × GC). A variety of chemometric models were explored, including partial least squares (PLS), nonlinear PLS (PolyPLS) and locally weighted regression (LWR). A hierarchical application of multivariate techniques was able to predict the time for which a sample had been exposed to evaporative weathering. Partial least squares discriminant analysis could predict whether a sample was relatively fresh (<12 h exposure time) or highly weathered (>20 h exposure time). Subsequent regression models for these classes were evaluated for accuracy using the root mean square error of prediction. LWR was the most successful, whereby fresh and highly weathered samples were predicted to within 30 min and 5 h of exposure, respectively.

Keywords: GC × GC; Flow modulation; Weathering; PLS-DA; LWR; Gasoline; Integration template

Identification of organic sulfur compounds in coal bitumen obtained by different extraction techniques using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometric detection by Maria Elisabete Machado; Fernando Cappelli Fontanive; José Vladimir de Oliveira; Elina Bastos Caramão; Cláudia Alcaraz Zini (pp. 2433-2444).

The determination of organic sulfur compounds (OSC) in coal is of great interest. Technically and operationally these compounds are not easily removed and promote corrosion of equipment. Environmentally, the burning of sulfur compounds leads to the emission of SO x gases, which are major contributors to acid rain. Health-wise, it is well known that these compounds have mutagenic and carcinogenic properties. Bitumen can be extracted from coal by different techniques, and use of gas chromatography coupled to mass spectrometric detection enables identification of compounds present in coal extracts. The OSC from three different bitumens were tentatively identified by use of three different extraction techniques: accelerated solvent extraction (ASE), ultrasonic extraction (UE), and supercritical-fluid extraction (SFE). Results obtained from one-dimensional gas chromatography (1D GC) coupled to quadrupole mass spectrometric detection (GC–qMS) and from two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GC × GC–TOFMS) were compared. By use of 2D GC, a greater number of OSC were found in ASE bitumen than in SFE and UE bitumens. No OSC were identified with 1D GC–qMS, although some benzothiophenes and dibenzothiophenes were detected by use of EIM and SIM modes. GC × GC–TOFMS applied to investigation of OSC in bitumens resulted in analytical improvement, as more OSC classes and compounds were identified (thiols, sulfides, thiophenes, naphthothiophenes, benzothiophenes, and benzonaphthothiophenes). The roof-tile effect was observed for OSC and PAH in all bitumens. Several co-elutions among analytes and with matrix interferents were solved by use of GC×GC.

Keywords: Sulfur compounds; Coal bitumen; Comprehensive two-dimensional gas chromatography; Extraction techniques

Characterization of currently marketed heparin products: analysis of molecular weight and heparinase-I digest patterns by Cynthia D. Sommers; Hongping Ye; Richard E. Kolinski; Moheb Nasr; Lucinda F. Buhse; Ali Al-Hakim; David A. Keire (pp. 2445-2454).

We evaluated polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) approaches to determine weight-average molecular weight (M _w) and polydispersity (PD) of heparins. A set of unfractionated heparin sodium (UFH) and low-molecular-weight heparin (LMWH) samples obtained from nine manufacturers which supply the US market were assessed. For SEC-MALLS, we measured values for water content, refractive index increment (dn/dc), and the second virial coefficient (A ₂) for each sample prior to molecular weight assessment. For UFH, a mean ± standard deviation value for M _w of 16,773 ± 797 was observed with a range of 15,620 to 18,363 (n = 20, run in triplicate). For LMWHs by SEC-MALLS, we measured mean M _w values for dalteparin, tinzaparin, and enoxaparin of 6,717 ± 71 (n = 4), 6,670 ± 417 (n = 3), and 3,959 ± 145 (n = 3), respectively. PAGE analysis of the same UFH, dalteparin, tinzaparin, and enoxaparin samples showed values of 16,135 ± 643 (n = 20), 5,845 ± 45 (n = 4), 6,049 ± 95 (n = 3), and 4,772 ± 69 (n = 3), respectively. These orthogonal measurements are the first M _w results obtained with a large heparin sample set on product being marketed after the heparin crisis of 2008 changed the level of scrutiny of this drug class. In this study, we compare our new data set to samples analyzed over 10 years earlier. In addition, we found that the PAGE analysis of heparinase digested UFH and neat LMWH samples yield characteristic patterns that provide a facile approach for identification and assessment of drug quality and uniformity.

Keywords: Heparin sodium; Low-molecular-weight heparin; Polyacrylamide gel electrophoresis; Size exclusion chromatography; Multi-angle laser light scattering

Cancer, pre-cancer and normal oral cells distinguished by dielectrophoresis by H. J. Mulhall; F. H. Labeed; B. Kazmi; D. E. Costea; M. P. Hughes; M. P. Lewis (pp. 2455-2463).

Most oral cancers are oral squamous cell carcinomas (OSCC) that arise from the epithelial lining of the oral mucosa. Given that the oral cavity is easily accessible, the disease lends itself to early detection; however, most oral cancers are diagnosed at a late stage, and approximately half of oral cancer sufferers do not survive beyond five years, post-diagnosis. The low survival rate has been attributed to late detection, but there is no accepted, reliable and convenient method for the detection of oral cancer and oral pre-cancer. Dielectrophoresis (DEP) is a label-free technique which can be used to obtain multi-parametric measurements of cell electrical properties. Parameters such as cytoplasmic conductivity and effective membrane capacitance (C _Eff) can be non-invasively determined by the technique. In this study, a novel lab-on-a-chip device was used to determine the cytoplasmic conductivity and C _Eff of primary normal oral keratinocytes, and pre-cancerous and cancerous oral keratinocyte cell lines. Our results show that the electrical properties of normal, pre-cancerous and cancerous oral keratinocytes are distinct. Furthermore, increasing C _Eff and decreasing cytoplasmic conductivity correlate with disease progression which could prove significant for diagnostic and prognostic applications. DEP has the potential to be used as a non-invasive technique to detect oral cancer and oral pre-cancer. Clinical investigation is needed to establish the reliability and temporal relationship of the correlation between oncologic disease progression and the electrical parameters identified in this study. To use this technique as an OSCC detection tool in a clinical setting, further characterisation and refinement is warranted.

Keywords: OSCC; Oral cancer; Dielectrophoresis; DEP; Diagnostics; Detection

Generation of a miniaturized free-flow electrophoresis chip based on a multi-lamination technique—isoelectric focusing of proteins and a single-stranded DNA fragment by Britta Walowski; Wilhelm Hüttner; Hainer Wackerbarth (pp. 2465-2471).

Free-flow electrophoresis techniques have been applied for separations in various areas of chemistry and biochemistry. Here we focus on the generation of a free-flow electrophoresis chip and direct monitoring of the separation of different molecules in the separation bed of the miniaturized chip. We demonstrate a fast and efficient way to generate a low-cost micro-free-flow electrophoresis (μFFE) chip with a filling capacity of 9.5 μL based on a multi-lamination technique. Separating webs realized by two transfer-adhesive tapes avoid the problem of gas bubbles entering the separation area. The chip is characterized by isoelectric focusing markers (IEF markers). The functionality of the chip is demonstrated by free-flow isoelectric focusing (FFIEF) of the proteins BSA (bovine serum albumin) and avidin and a single-stranded DNA (ssDNA) fragment in the pH range 3 to 10. The separation voltage ranges between 167 V cm⁻¹ and 422 V cm⁻¹, depending on the application.

Keywords: Isoelectric focusing; Free-flow electrophoresis; Miniaturized free-flow electrophoresis chip; Fluorescence detection

Cation exchange HPLC analysis of desmosines in elastin hydrolysates by Joanna Perła-Kaján; Agnieszka Gryszczyńska; Sebastian Mielcarek; Hieronim Jakubowski (pp. 2473-2479).

Desmosine crosslinks are responsible for the elastic properties of connective tissues in lungs and cardiovascular system and are often compromised in disease states. We developed a new, fast, and simple cation exchange HPLC assay for the analysis of desmosine and isodesmosine in animal elastin. The method was validated by determining linearity, accuracy, precision, and desmosines stability and was applied to measure levels of desmosines in porcine and murine organs. The detection and quantification limits were 2 and 4 pmol, respectively. The run-time was 8 min. Our cation exchange column does not separate desmosine and isodesmosine, but their level can be quantified from absorbance at different wavelengths. Using this assay, we found that desmosines levels were significantly lower in elastin isolated from various organs of immunodeficient severe combined immunodeficiency mice compared with wild-type animals. We also found that desmosines levels were lower in lung elastin isolated from hyperhomocysteinemic Pcft ^−/− mice deficient in intestinal folate transport compared with wild-type Pcft ^+/+ animals.

Keywords: Cation exchange HPLC; Desmosine; Isodesmosine; Elastin; Homocysteine; Pcft mouse; SCID mouse

Development of an LC-MS/MS method for the analysis of serotonin and related compounds in urine and the identification of a potential biomarker for attention deficit hyperactivity/hyperkinetic disorder by Merisa Moriarty; Aoife Lee; Brendan O’Connell; Ann Kelleher; Helen Keeley; Ambrose Furey (pp. 2481-2493).

Serotonin is a major neurotransmitter and affects various functions both in the brain and in the rest of the body. It has been demonstrated that altered serotinergic function is implicated in various psychiatric disorders including depression and schizophrenia. Serotonin has also been implicated along with dopamine in attention deficit–hyperkinetic disorder (AD-HKD). This study provides a versatile validated method for the analysis of serotonin, hydroxyindole acetic acid and dopamine in urine using LC-MS/MS. This method was then used to quantify these analytes in a test group of 17 children diagnosed with severe AD-HKD. This group was compared to a matched control group to investigate the possibility that one of these compounds may be a potential biomarker for this condition. The developed method provided good linear calibration curves for the multiplex assay of analytes in urine (0.05–3.27 nmol/L; R ² ≥ 0.9977). Acceptable inter-day repeatability was achieved for all analytes with RSD values (n = 9) ranging from 1.1% to 9.3% over a concentration range of 0.11–3.27 μmol/L in urine. Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with LODs of 8.8–18.2 nmol/L and the LOQs of 29.4–55.7 nmol/L for analytes in urine. Recoveries were in the ranges of 98–104%, 100–106% and 91–107% for serotonin, 5-HIAA and dopamine, respectively. An appropriate sample clean-up procedure for urine was developed to ensure efficient recovery and reproducibility on analysis. Evaluation of matrix effects was also carried out and the influence of ion suppression on analytical results reported. Confirmatory analysis was carried out on a linear trap quadrupole-Orbitrap mass spectrometer to obtain high mass accuracy data of the target analytes in the clinical samples.

Keywords: Attention deficit–hyperactivity/hyperkinetic disorder; AD-HKD; LC-MS/MS; Serotonin; Dopamine; Hydroxyindole acetic acid; Urine

High-throughput mass finger printing and Lewis blood group assignment of human milk oligosaccharides by Dennis Blank; Sabine Gebhardt; Kai Maass; Günter Lochnit; Viktoria Dotz; Jennifer Blank; Rudolf Geyer; Clemens Kunz (pp. 2495-2510).

The structural diversity of human milk oligosaccharides (HMOs) strongly depends on the Lewis (Le) blood group status of the donor which allows a classification of these glycans into three different groups. Starting from 50 μL of human milk, a new high-throughput, standardized, and widely automated mass spectrometric approach has been established which can be used for correlation of HMO structures with the respective Lewis blood groups on the basis of mass profiles of the entire mixture of glycans together with selected fragment ion spectra. For this purpose, the relative abundance of diagnostically relevant compositional species, such as Hex₂Fuc₂ and Hex₃HexNAc₁Fuc₂, as well as the relative intensities of characteristic fragment ions obtained thereof are of key importance. For each Lewis blood group, i.e., Le(a − b+), Le(a + b−), and Le(a − b−), specific mass profile and fragment ion patterns could be thus verified. The described statistically proven classification of the derived glycan patterns may be a valuable tool for analysis and comparison of large sets of milk samples in metabolic studies. Furthermore, the outlined protocol may be used for rapid screening in clinical studies and quality control of milk samples donated to milk banks. Figure MALDI-TOF-MS HMO profiles of Le(a − b+) colostrum samples obtained in the positive-ion mode

Keywords: Automated sample processing; Discriminant analysis; Human milk oligosaccharides; Lewis blood group; Mass spectrometry; Oligosaccharide screening

Potential application of Raman spectroscopy for determining burial duration of skeletal remains by Gregory McLaughlin; Igor K. Lednev (pp. 2511-2518).

Raman spectroscopy was used to study trends in chemical composition of bones in a burial environment. A turkey bone was sectioned and buried for short intervals between 12 and 62 days. Buried sections were analyzed using Raman microspectroscopy with 785 nm excitation. The results indicate that chemical changes in bone due to soil bacteria are time-dependent. Spectroscopic trends within buried bone segments were correlated to burial duration. A preliminary model was constructed using peak integration of Raman bands. Data collected within buried bone segments fit very well in this model. The model constructed is sensitive to changes in bone composition in a scale of days. This study illustrates the great potential of Raman spectroscopy as a non-destructive method for estimating the burial duration of bone for forensic purposes. Figure

Keywords: Raman spectroscopy; Forensic anthropology; Forensic science; Collagen; Cortical bone; Time since death

Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry by Carol Davis; Natasha Gordon; Sinéad Murphy; Ishwar Singh; Kevin Kavanagh; Stephen Carberry; Sean Doyle (pp. 2519-2529).

Gliotoxin is produced by non-ribosomal peptide synthesis and secreted from certain fungi, including Aspergillus fumigatus. It is an epipolythiodioxopiperazine that contains an intact disulphide bridge and is the focus of intense research as a consequence of its negative immunomodulatory properties. Gliotoxin detection is generally enabled by reversed-phase–high-performance liquid chromatography (RP-HPLC), with absorbance detection (220–280 nm), or liquid chromatography-mass spectrometry, yet detection is not readily achievable by matrix-assisted laser desorption ionisation–time-of-flight mass spectrometry (MALDI-ToF MS). We have developed a single-pot derivatisation strategy which uses sodium borohydride-mediated reduction of gliotoxin followed by immediate alkylation of exposed thiols by 5′-iodoacetamidofluorescein to yield a stable product, diacetamidofluorescein-gliotoxin (GT-(AF)₂), of molecular mass 1103.931 Da ((M + H)+). This product is readily detectable by RP-HPLC and exhibits a 6.8-fold increase in molar absorptivity compared with gliotoxin, which results in a higher sensitivity of detection (40 ng; 125 pmoL). GT-(AF)₂ also fluoresces (excitation/emission, 492:518 nm). Unlike free gliotoxin, the product (>800 fmol) is detectable by MALDI-ToF MS. Sporidesmin A can also be detected by RP-HPLC and MALDI-ToF MS (>530 fmol) using this strategy. We also demonstrate that the strategy facilitates detection of gliotoxin (mean ± SD = 3.55 ± 0.07 μg 100 μL⁻¹; n = 2) produced by A. fumigatus, without the requirement for organic extraction of culture supernatants and associated solvent removal. GT-(AF)₂ is also detectable (150 ng; 460 pmol) by thin-layer chromatography. Figure The gliotoxin derivative, diacetamidofluorescein-gliotoxin (GT-(AF)2), generated by single-pot reduction and alkylation, is completely stable during MALDI-ToF MS analysis with m/z 1103.93.

Keywords: Aspergillus fumigatus ; Epipolythiodioxopiperazine; Gliotoxin; NRPS; Redox; Sporidesmin A.

Biliary excretion of essential trace elements in rats under oxidative stress caused by selenium deficiency by Kosuke Yamasaki; Yasunobu Sakuma; Junya Sasaki; Ken-ichiro Matsumoto; Kazunori Anzai; Keisuke Matsuoka; Chikako Honda; Masamichi Tsukada; Kazutoyo Endo; Shuichi Enomoto (pp. 2531-2538).

The excretion of essential trace elements, namely, Se, Sr, As, Mn, Co, V, Fe, and Zn into the bile of Se-deficient (SeD) Wistar male rats was studied using the multitracer (MT) technique, and instrumental neutron activation analysis (INAA). Normal and Se-control (SeC) rat groups were used as reference groups to compare the effects of Se levels on the behaviors of the essential trace elements. The excretion (% dose) of Se, Sr, As, Mn, Co, and V increased with Se levels in the liver. The biliary excretion of Mn and As dramatically enhanced for SeC rats compared with SeD rats, while that of V accelerated a little for SeC rats. The radioactivity levels of ⁵⁹Fe and ⁶⁵Zn in the MT tracer solution were insufficient to measure their excretion into bile. The role of glutathione and bilirubin for biliary excretion of the metals was discussed in relation to Se levels in rat liver. Figure Cumulative excretion (% dose) of V, Mn, Co, As, Sr, and Se into bile of SeD, normal, and SeC rats 120 min after intravenous administration of multitracer. Values are means ± SD for the three rats in each respective group

Keywords: Essential trace element; Oxidative stress; Glutathione peroxidase; Glutathione; Bile; Biliary excretion; INAA; Multitracer

Quantitative analysis of eletriptan in human plasma by HPLC-MS/MS and its application to pharmacokinetic study by Venkata Suresh Ponnuru; B. R. Challa; Ramarao Nadendla (pp. 2539-2548).

Authors developed a simple, sensitive, selective, rapid, rugged, and reproducible liquid chromatography–tandem mass spectrometry method for the quantification of eletriptan (EP) in human plasma using naratriptan (NP) as an internal standard (IS). Chromatographic separation was performed on Ascentis Express C18, 50 × 4.6 mm, 2.7 μm column. Mobile phase was composed of 0.1% formic acid: methanol (40:60 v/v), with 0.5 mL/min flow rate. Drug and IS were extracted by liquid–liquid extraction. EP and NP were detected with proton adducts at m/z 383.2→84.3 and 336.2→97.8 in multiple reaction monitoring (MRM) positive mode, respectively. The method was validated with the correlation coefficients of (r ²) ≥ 0.9963 over a linear concentration range of 0.5–250.0 ng/mL. This method demonstrated intra- and inter-day precision within 1.4–9.2% and 4.4–5.5% and accuracy within 96.8–103% and 98.5–99.8% for EP. This method is successfully applied in the bioequivalence study of 24 human volunteers.

Keywords: Liquid chromatography; Mass spectrometry; Eletriptan; Human plasma; Bioequivalence

Fast DNA and protein microarray tests for the diagnosis of hepatitis C virus infection on a single platform by Stuart W. J. Ember; Holger Schulze; Alan J. Ross; Julie Luby; Mizanur Khondoker; Gerard Giraud; Jonathan G. Terry; Ilenia Ciani; Chaker Tlili; Jason Crain; Anthony J. Walton; Andrew R. Mount; Peter Ghazal; Till T. Bachmann; Colin J. Campbell (pp. 2549-2559).

Hepatitis C virus (HCV) is a major cause of chronic liver disease and liver cancer, and remains a large health care burden to the world. In this study we developed a DNA microarray test to detect HCV RNA and a protein microarray to detect human anti-HCV antibodies on a single platform. A main focus of this study was to evaluate possibilities to reduce the assay time, as a short time-to-result (TTR) is a prerequisite for a point-of-care test. Significantly reducing hybridisation and washing times did not impair the assay performance. This was confirmed first using artificial targets and subsequently using clinical samples from an HCV seroconversion panel derived from a HCV-infected patient. We were able to reduce the time required for the detection of human anti-HCV antibodies to only 14 min, achieving nanomolar sensitivity. The protein microarray exhibited an analytical sensitivity comparable to that of commercial systems. Similar results were obtained with the DNA microarray using a universal probe which covered all different HCV genotypes. It was possible to reduce the assay time after PCR from 150 min to 16 min without any loss of sensitivity. Taken together, these results constitute a significant step forward in the design of rapid, microarray-based diagnostics for human infectious disease, and show that the protein microarray is currently the most favourable candidate to fill this role.

Keywords: DNA microarray; Protein microarray; HCV; Time-to-result; Point-of-care; Seroconversion panel

A rapid fluorescence polarization immunoassay for the determination of T-2 and HT-2 toxins in wheat by Vincenzo Lippolis; Michelangelo Pascale; Stefania Valenzano; Valeria Pluchinotta; Sabine Baumgartner; Rudolf Krska; Angelo Visconti (pp. 2561-2571).

A rapid fluorescence polarization (FP) immunoassay has been developed for the simultaneous determination of T-2 and HT-2 toxins in naturally contaminated wheat samples. Syntheses of four fluorescein-labelled T-2 or HT-2 toxin tracers were carried out and their binding response with seven monoclonal antibodies was evaluated. The most sensitive antibody-tracer combination was obtained by using an HT-2-specific antibody and a fluorescein-HT-2 tracer. The developed competitive FP immunoassay in solution showed high cross-reactivity for T-2 toxin (CR% = 100%) while a very low CR% for neosolaniol (0.12%) and no cross-reactivity with other mycotoxins frequently occurring in wheat. A rapid extraction procedure using 90% methanol was applied to wheat samples prior to FP immunoassay. The average recovery from spiked wheat samples (50 to 200 μg kg⁻¹) was 96% with relative standard deviation generally lower than 8%. A limit of detection of 8 μg kg⁻¹ for the combined toxins was determined. Comparative analyses of 45 naturally contaminated and spiked wheat samples by both the FP immunoassay and high-performance liquid chromatography/immunoaffinity clean-up showed a good correlation (r = 0.964). These results, combined with the rapidity (10 min) and simplicity of the assay, show that this method is suitable for high throughput screening as well as for quantitative determination of T-2 and HT-2 toxins in wheat.

Keywords: T-2 toxin; HT-2 toxin; Fluorescence polarization; Immunoassay; Wheat

Ca²⁺-triggered coelenterazine-binding protein from Renilla as an enzyme-dependent label for binding assay by V. V. Krasitskaya; S. I. Korneeva; A. N. Kudryavtsev; S. V. Markova; G. A. Stepanyuk; L. A. Frank (pp. 2573-2579).

The recombinant Ca²⁺-triggered coelenterazine-binding protein (CBP) from Renilla muelleri was investigated as a biospecifically labeled molecule for in vitro assay applications. The protein was shown to be stable in solutions in the frozen state, as well as stable under heating and to chemical modifications. Conjugates with biotin, oligonucleotide, and proteins were obtained and applied as biospecific molecules in a solid-phase microassay. CBP detection was performed with intact (no modifications were made) Renilla luciferase in the presence of calcium, and the detection limit was found to be 75 amol. Model experiments indicate that this approach shows much promise, especially with regard to the development of multianalytical systems. Figure 3D structures of obelin D12C (a) and CBP (b) (N ends), displaying the introduced SH group. c Plots of bioluminescence of obelin versus the amount of obelin (open triangles) and RmLuc versus the amount of CBP (open circles). d Simultaneous bioluminescence immunoassay of luteinizing hormone (LH; obelin signals, filled triangles) and follicle-stimulating hormone (FSH; RmLuc signals, filled circles) in standard sera

Keywords: Ca²⁺-triggered coelenterazine-binding protein (CBP); Renilla muelleri luciferase; Bioluminescent solid-phase microassay

Development of a liposome-based immunochromatographic strip assay for the detection of Salmonella by Shruti Shukla; Hyerim Leem; Myunghee Kim (pp. 2581-2590).

Salmonella species are ubiquitous human pathogens which pose a dangerous threat to the elderly and children worldwide. In this study, to develop a more efficient assay procedure for the rapid detection of Salmonella Typhimurium, an immunochromatographic strip assay was developed using immunoliposome (anti-Salmonella IgG-tagged) encapsulated with sulforhodamine B (SRB). The detection sensitivity of the developed immunochromatographic assay was compared with a commercial immunochromatographic test strip using colloidal gold nanoparticles. The liposomes were prepared through a reverse-phase evaporation method by using a lipid and phospholipid mixture and SRB, a fluorescence dye, which was encapsulated in the phospholipid bilayer. Furthermore, the outer surface of the SRB-encapsulated liposome was conjugated with antibody (affinity-purified polyclonal goat anti-Salmonella IgG) to form an immunoliposome (size, 223 nm), used as the analytical reagent in the developed immunoassay. For this strip assay, a plastic-backed nitrocellulose strip was immobilized with two antibody zones. The lower zone of the strip referred to Salmonella antigen capture zone (test line), while the other zone served as a positive control (control line). The lower zone was coated with affinity-purified polyclonal goat anti-Salmonella IgG, while the upper zone was coated with rabbit anti-goat IgG. During capillary migration of the wicking solution (diluted liposome and Salmonella culture, each 50 μl), Salmonella was captured with surface-bound immunoliposomes at the antigen capture zone, while the unbound liposomes migrated upward and bound to another zone. The color density of the antigen capture zone was directly proportional to the amount of S. Typhimurium in the test sample. As a result, the detection limit of the immunochromatographic strip assay developed in this study against S. Typhimurium was found to be 10² CFU/ml, which was significantly higher than the detection limit (10⁷ CFU/ml) of the commercial immunochromatographic test strip assay. Figa The concept of immunochromatographic strip assay

Keywords: Immunochromatographic strip assay; Salmonella typhimurium; Immunoliposome; Anti-Salmonella IgG

Electrochemical sensor for blood deoxyribonucleases: design and application to the diagnosis of autoimmune thyroiditis by Irina I. Shakhmaeva; Diana V. Saifullina; Liliya I. Sattarova; Timur I. Abdullin (pp. 2591-2597).

We designed an electrochemical sensor based on a carbon nanotube modified electrode (ME) to analyze DNA-cleaving activity. The cleavage of high molecular weight DNA resulted in an increase in the oxidation current from DNA guanine nucleotides due to a change in DNA adsorptive behavior on the surface of the ME. DNA digestion with DNAse I was accompanied by a linear increase in the DNA signal in proportion to the enzyme activity. We then proposed an assay based on the sensor for the direct assessment of the total deoxyribonuclease activity of blood serum as well as the separate detection of DNAse I and DNA abzymes. The assay was applied to analyze deoxyribonucleases in sera from 21 healthy donors and 17 patients with autoimmune thyroiditis. Our results show that the response of the sensor to DNA cleavage by blood deoxyribonucleases is a promising diagnostic criterion for autoimmune thyroiditis. This sensor can be implemented in a disposable screen-printed electrode format for application in clinical laboratories.

Keywords: Biomarkers; Electrochemical assays; DNAse I; DNA abzymes; Autoimmune thyroiditis; Myocardial ischemia

Development of an amperometric sulfite biosensor based on a gold nanoparticles/chitosan/multiwalled carbon nanotubes/polyaniline-modified gold electrode by Rachna Rawal; Sheetal Chawla; Tulika Dahiya; Chandra Shekhar Pundir (pp. 2599-2608).

A sulfite oxidase (SOx) purified from leaves of Syzygium cumini (Jamun) was immobilized covalently onto a gold nanoparticles (AuNPs)/chitosan (CHIT)/carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite that was electrodeposited onto the surface of a gold (Au) electrode. A novel and highly sensitive sulfite biosensor was developed that used this enzyme electrode (SOx/AuNPs/CHIT/cMWCNT/PANI/Au) as the working electrode, Ag/AgCl as the standard electrode, and Pt wire as the auxiliary electrode. The modified electrode was characterized by Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS) before and after the immobilization of the SOx. The sensor produced its optimum response within 3 s when operated at 50 mVs⁻¹ in 0.1 M phosphate buffer, pH 7.0, and at 35 °C. The linear range and detection limit of the sensor were 0.75–400 μM and 0.5 μM (S/N = 3), respectively. The biosensor was employed to determine sulfite levels in fruit juices and alcoholic beverages. The enzyme electrode was used 300 times over a period of three months when stored at 4 °C. Figure A novel and highly sensitive sulfite biosensor was developed based on covalent immobilization of Syzygium cumini leaf sulfite oxidase (SOX) onto gold nanoparticles (AuNP’s)/chitosan (CHIT)/carboxylated multiwalled carbon nanotubes (cMWCNT)/polyaniline (PANI) layer electrodeposited on the surface of gold (Au) electrode

Keywords: Sulfite; Sulfite oxidase; Sulfite biosensor; CHIT; AuNPs; cMWCNT/PANI

Triacylglycerol profiling of microalgae strains for biofuel feedstock by liquid chromatography–high-resolution mass spectrometry by Karen M. MacDougall; Jesse McNichol; Patrick J. McGinn; Stephen J. B. O’Leary; Jeremy E. Melanson (pp. 2609-2616).

Biofuels from photosynthetic microalgae are quickly gaining interest as a viable carbon-neutral energy source. Typically, characterization of algal feedstock involves breaking down triacylglycerols (TAG) and other intact lipids, followed by derivatization of the fatty acids to fatty acid methyl esters prior to analysis by gas chromatography (GC). However, knowledge of the intact lipid profile could offer significant advantages for discovery stage biofuel research such as the selection of an algal strain or the optimization of growth and extraction conditions. Herein, lipid extracts from microalgae were directly analyzed by ultra-high pressure liquid chromatography–mass spectrometry (UHPLC-MS) using a benchtop Orbitrap mass spectrometer. Phospholipids, glycolipids, and TAGs were analyzed in the same chromatographic run, using a combination of accurate mass and diagnostic fragment ions for identification. Using this approach, greater than 100 unique TAGs were identified over the six algal strains studied and TAG profiles were obtained to assess their potential for biofuel applications. Under the growth conditions employed, Botryococcus braunii and Scenedesmus obliquus yielded the most comprehensive TAG profile with a high abundance of TAGs containing oleic acid. Figure Optical microscope image of Botryococcus braunii and high resolution mass spectrum of triacylglycerol 28:2/18:1/18:1 (inset)

Keywords: Microalgae; Biofuels; Triacylglyerols; Liquid chromatography; Mass spectrometry

Development and validation of a rapid method for microcystins in fish and comparing LC-MS/MS results with ELISA by Lucía Geis-Asteggiante; Steven J. Lehotay; Laurie L. Fortis; George Paoli; Chandi Wijey; Horacio Heinzen (pp. 2617-2630).

Microcystins (MCs) are the most common cyanotoxins found worldwide in freshwater, brackish, and marine environments. The rapid and accurate analysis of MCs and nodularin (Nod-R) in fish tissue is important for determining occurrence, following trends, and monitoring exposure for risk assessment and other purposes. The aim of this study was to develop a streamlined and reliable sample preparation method for eight MCs (MC-RR, MC-YR, MC-LR, MC-WR, MC-LA, MC-LY, MC-LW, and MC-LF) and Nod-R in fish, and conduct a validation of the new method using liquid chromatography–tandem mass spectrometry (LC-MS/MS) for analysis and compare the results with a commercial enzyme-linked immunosorbent assay (ELISA) kit. Different sample preparation methods were compared, and a simple extraction protocol with acidified acetonitrile/water (3:1) followed by hexane partitioning cleanup was found to be most effective. Thorough validation of the final method was conducted, and 90–115% recoveries were achieved for all analytes except for MC-RR, which gave 130% average recovery (isotopically labeled internal standards were unavailable to correct for possible biases). The use of electrospray ionization in the negative mode gave few interferences and minimal matrix effects in the LC-MS/MS analysis overall. Precision was typically 10–20% RSD among multiple days in experiments, detection limits were <10 ng/g in the fish tissue (catfish, basa, and swai filets), and no false-positives or false-negatives occurred in blind analyses of many spiked samples. The ELISA was unable to distinguish between MCs but was found to correctly assess the presence or absence of MCs and Nod-R in the blind-fortified fish tissues. The capability of these approaches to measure covalently bound MCs was not assessed.

Keywords: Microcystins; LC-MS/MS; ELISA; Fish tissue; Validation

His₆-OPH enzyme-based bio-hybrid material for organophosphate detection by Nina Frančič; Aljoša Košak; Ilya Lyagin; Elena N. Efremenko; Aleksandra Lobnik (pp. 2631-2638).

In this work, we report on the development of a bio-sensing film for the detection of organophosphorous compounds using sol–gel technology. A novel sol–gel immobilization method employing tetraethoxysilane/3-glycidoxypropyltrimethoxysilane/water hybrid material was developed and used to immobilize the hexahistidine-tagged organophosphorous hydrolase enzyme (His₆-OPH). Bio-sensing layers with encapsulated His₆-OPH of various structures (water/silane, precursor ratios) have been prepared. The optimal (P = 5:1, R = 188) bio-sensing layers retained 90% of the initial enzyme activity. Furthermore, the bio-sensing layer prepared by this method was able to maintain its activity at or above 80% of its initial activity for 2 weeks. The bio-hybrid film also showed excellent reusability and improved activity at neutral pH in comparison to the same enzyme in solution.

Keywords: Enzyme immobilization; His₆-OPH; Sol–gel; Organophosphate detection; Bio-sensing layer

Determination of ultra-trace levels of priority PBDEs in water samples by isotope dilution GC(ECNI)MS using ⁸¹Br-labelled standards by Adriana González-Gago; Sicco H. Brandsma; Pim E. G. Leonards; Jacob de Boer; Juan Manuel Marchante-Gayón; J. Ignacio Garcia Alonso (pp. 2639-2649).

A gas chromatography electron capture negative ionization mass spectrometry (GC(ECNI)MS) procedure for the determination of priority polybrominated diphenyl ethers (PBDEs; congeners 28, 47, 99, 100, 153 and 154) in water samples at regulatory EU levels has been developed. The method is based on the use of ⁸¹Br-labelled PBDEs for isotope dilution analysis and the measurement of ⁷⁹Br/⁸¹Br isotope ratios in gas chromatography peaks with the electron capture negative ionization technique. The suitability of this ion source for the precise and accurate measurement of bromine isotope ratios has been demonstrated. The general ECNI-IDMS procedure was evaluated by the analysis of NIST SRM 1947 (Lake Michigan fish tissue) with satisfactory results. For the analysis of water samples, 500 mL of the samples were spiked with the labelled PBDEs and extracted with 10 mL isooctane for 30 min. The extract was evaporated down to ca. 100 μL and injected in the GC(ECNI)MS. Detection limits ranged from 0.014 ⁻¹ to 0.089 pg mL⁻¹ depending on the congener. Recoveries from real water samples, spiked at a level of 0.5 pg mL⁻¹, ranged from 77% to 102%. Fig. 1 The proposed method is based on the use of ⁸¹Br-labelled PBDE standards for isotope dilution analysis and the measurement of ⁷⁹Br/₈₁Br isotope ratios in gas chromatography peaks with the electron capture negative ionization source

Keywords: Isotope dilution mass spectrometry; Polybrominated diphenyl ethers; ⁸¹Br-labelled standards; Electron capture negative ionization

Multistep liquid-phase lithography for fast prototyping of microfluidic free-flow-electrophoresis chips by Stefan Jezierski; Leonid Gitlin; Stefan Nagl; Detlev Belder (pp. 2651-2656).

We present a fast and versatile method to produce functional micro free-flow electrophoresis chips. Microfluidic structures were generated between two glass slides applying multistep liquid-phase lithography, omitting troublesome bonding steps or cost-intensive master structures. Utilizing a novel spacer-less approach with the photodefinable polymer polyethyleneglycol dimethacrylate (PEG-DA), microfluidic devices with hydrophilic channels of only 25 μm in height were generated. The microfluidic chips feature ion-permeable segregation walls between the electrode channels and the separation bed and hydrophilic surfaces. The performance of the chip is demonstrated by free-flow electrophoretic separation of fluorescent xanthene dyes and fluorescently labeled amino acids.

Keywords: Capillary electrophoresis/electrophoresis; Microfluidics; Microfabrication; Free-flow electrophoresis; Lab-on-a-chip

Hot embossing and thermal bonding of poly(methyl methacrylate) microfluidic chips using positive temperature coefficient ceramic heater by Xia Wang; Luyan Zhang; Gang Chen (pp. 2657-2665).

As a self-regulating heating device, positive temperature coefficient ceramic heater was employed for hot embossing and thermal bonding of poly(methyl methacrylate) microfluidic chip because it supplied constant-temperature heating without electrical control circuits. To emboss a channel plate, a piece of poly(methyl methacrylate) plate was sandwiched between a template and a microscopic glass slide on a positive temperature coefficient ceramic heater. All the assembled components were pressed between two elastic press heads of a spring-driven press while a voltage was applied to the heater for 10 min. Subsequently, the embossed poly(methyl methacrylate) plate bearing negative relief of channel networks was bonded with a piece of poly(methyl methacrylate) cover sheet to obtain a complete microchip using a positive temperature coefficient ceramic heater and a spring-driven press. High quality microfluidic chips fabricated by using the novel embossing/bonding device were successfully applied in the electrophoretic separation of three cations. Positive temperature coefficient ceramic heater indicates great promise for the low-cost production of poly(methyl methacrylate) microchips and should find wide applications in the fabrication of other thermoplastic polymer microfluidic devices.

Keywords: Positive temperature coefficient ceramic; Embossing; Poly(methyl methacrylate); Microchip electrophoresis

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Analytical and Bioanalytical Chemistry (v.401, #8)