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Analytical and Bioanalytical Chemistry (v.403, #1)

Markus Sauer, Johan Hofkens, and Jörg Enderlein: Handbook of fluorescence spectroscopy and imaging: from ensemble to single molecules by Massimo Guardigli (pp. 1-2).

Kevin Goodner and Russel Rouseff (Eds.): Practical analysis of flavor and fragrance materials by R. G. Berger (pp. 3-4).

Small – smaller – μTAS by Conni Hanke; Petra S. Dittrich (pp. 5-6).

Biomolecule immobilization techniques for bioactive paper fabrication by Fanzhi Kong; Yim Fun Hu (pp. 7-13).

Research into paper-based sensors or functional materials that can perform analytical functions with active recognition capabilities is rapidly expanding, and significant research effort has been made into the design and fabrication of bioactive paper at the biosensor level to detect potential health hazards. A key step in the fabrication of bioactive paper is the design of the experimental and operational procedures for the immobilization of biomolecules such as antibodies, enzymes, phages, cells, proteins, synthetic polymers and DNA aptamers on a suitably prepared paper membrane. The immobilization methods are concisely categorized into physical absorption, bioactive ink entrapment, bioaffinity attachment and covalent chemical bonding immobilization. Each method has individual immobilization characteristics. Although every biomolecule–paper combination has to be optimized before use, the bioactive ink entrapment method is the most commonly used approach owing to its general applicability and biocompatibility. Currently, there are four common applications of bioactive paper: (1) paper-based bioassay or paper-based analytical devices for sample conditioning; (2) counterfeiting and countertempering in the packaging and construction industries; (3) pathogen detection for food and water quality monitoring; and (4) deactivation of pathogenic bacteria using antimicrobial paper. This article reviews and compares the different biomolecule immobilization techniques and discusses current trends. Current, emerging and future applications of bioactive paper are also discussed. Online Abstract Figure Trend Paper: Biomolecule immobilization techniques for bioactive paper fabrication

Keywords: Bioactive paper; Biomolecule immobilization; Paper-based analytical devices; Antimicrobial paper; Pathogen detection; Biosensor

Exploring the feasibility of bioaerosol analysis as a novel fingerprinting technique by Josemar A. Castillo; Sarah J. R. Staton; Thomas J. Taylor; Pierre Herckes; Mark A. Hayes (pp. 15-26).

The purpose of this review is to investigate the feasibility of bioaerosol fingerprinting based on current understanding of cellular debris (with emphasis on human-emitted particulates) in aerosols and arguments regarding sampling, sensitivity, separations, and detection schemes. Target aerosol particles include cellular material and proteins emitted by humans, animals, and plants and can be regarded as information-rich packets that carry biochemical information specific to the living organisms present where the sample is collected. In this work we discuss sampling and analysis techniques that can be integrated with molecular (e.g. protein)-detection procedures to properly assess the aerosolized cellular material of interest. Developing a detailed understanding of bioaerosol molecular profiles in different environments suggests exciting possibilities of bioaerosol analysis with applications ranging from military defense to medical diagnosis and wildlife identification Figure Feasibility of identifying humans via bioaerosols is examined.

Keywords: Bioaerosols; Skin debris; Fingerprinting; Identification

Surface-enhanced Raman spectroscopy (SERS): progress and trends by Dana Cialla; Anne März; René Böhme; Frank Theil; Karina Weber; Michael Schmitt; Jürgen Popp (pp. 27-54).

Surface-enhanced Raman spectroscopy (SERS) combines molecular fingerprint specificity with potential single-molecule sensitivity. Therefore, the SERS technique is an attractive tool for sensing molecules in trace amounts within the field of chemical and biochemical analytics. Since SERS is an ongoing topic, which can be illustrated by the increased annual number of publications within the last few years, this review reflects the progress and trends in SERS research in approximately the last three years. The main reason why the SERS technique has not been established as a routine analytic technique, despite its high specificity and sensitivity, is due to the low reproducibility of the SERS signal. Thus, this review is dominated by the discussion of the various concepts for generating powerful, reproducible, SERS-active surfaces. Furthermore, the limit of sensitivity in SERS is introduced in the context of single-molecule spectroscopy and the calculation of the ‘real’ enhancement factor. In order to shed more light onto the underlying molecular processes of SERS, the theoretical description of SERS spectra is also a growing research field and will be summarized here. In addition, the recording of SERS spectra is affected by a number of parameters, such as laser power, integration time, and analyte concentration. To benefit from synergies, SERS is combined with other methods, such as scanning probe microscopy and microfluidics, which illustrates the broad applications of this powerful technique. Figure Various SERS substrates visualized using scanning electron microscopy

Keywords: Surface-enhanced Raman spectroscopy (SERS); Plasmonics; Plasmonic array; Microfluidics; Tip-enhanced Raman spectroscopy (TERS); Single-molecule detection; SERS enhancement factor; Theoretical description of SERS spectra; Parameters for SERS detection

Analytical methods for tracing plant hormones by Fuyou Du; Guihua Ruan; Huwei Liu (pp. 55-74).

Plant hormones play important roles in regulating numerous aspects of plant growth, development, and response to stress. In the past decade, more analytical methods for the accurate identification and quantitative determination of trace plant hormones have been developed to better our understanding of the molecular mechanisms of plant hormones. As sample preparation is often the bottleneck in analysis of plant hormones in biological samples, this review firstly discusses sample preparation techniques after a brief introduction to the classes, roles, and methods used in the analysis of plant hormones. The analytical methods, especially chromatographic techniques and immuno-based methods, are reviewed in detail, and their corresponding advantages, limitations, applications, and prospects are also discussed. This review mainly covers reports published from 2000 to the present on methods for the analysis of plant hormones.

Keywords: Plant hormones; Sample preparation; Chromatographic techniques; Mass spectrometry; Immuno-based analysis; Trace analysis

Analytical methods for tracing plant hormones by Fuyou Du; Guihua Ruan; Huwei Liu (pp. 55-74).

Keywords: Plant hormones; Sample preparation; Chromatographic techniques; Mass spectrometry; Immuno-based analysis; Trace analysis

Biosensors for the analysis of microbiological and chemical contaminants in food by T. F. McGrath; C. T. Elliott; T. L. Fodey (pp. 75-92).

Increases in food production and the ever-present threat of food contamination from microbiological and chemical sources have led the food industry and regulators to pursue rapid, inexpensive methods of analysis to safeguard the health and safety of the consumer. Although sophisticated techniques such as chromatography and spectrometry provide more accurate and conclusive results, screening tests allow a much higher throughput of samples at a lower cost and with less operator training, so larger numbers of samples can be analysed. Biosensors combine a biological recognition element (enzyme, antibody, receptor) with a transducer to produce a measurable signal proportional to the extent of interaction between the recognition element and the analyte. The different uses of the biosensing instrumentation available today are extremely varied, with food analysis as an emerging and growing application. The advantages offered by biosensors over other screening methods such as radioimmunoassay, enzyme-linked immunosorbent assay, fluorescence immunoassay and luminescence immunoassay, with respect to food analysis, include automation, improved reproducibility, speed of analysis and real-time analysis. This article will provide a brief footing in history before reviewing the latest developments in biosensor applications for analysis of food contaminants (January 2007 to December 2010), focusing on the detection of pathogens, toxins, pesticides and veterinary drug residues by biosensors, with emphasis on articles showing data in food matrices. The main areas of development common to these groups of contaminants include multiplexing, the ability to simultaneously analyse a sample for more than one contaminant and portability. Biosensors currently have an important role in food safety; further advances in the technology, reagents and sample handling will surely reinforce this position. Figure ASSET Technology Centre

Keywords: Biosensor; Food analysis; Pathogen; Toxin; Pesticide; Veterinary drug residues

Analytical methods for determination of new fluoroquinolones in biological matrices and pharmaceutical formulations by liquid chromatography: a review by Joana Sousa; Gilberto Alves; Ana Fortuna; Amílcar Falcão (pp. 93-129).

Fluoroquinolones are one of the most promising and intensively studied drugs of contemporary anti-infective chemotherapy. New fluoroquinolone antibacterials with improved pharmacokinetic properties and a broad spectrum of activity have been developed, opening new windows of opportunity for clinical use. To our knowledge, no comprehensive and critical review of the analytical methods for the determination of these agents, which correspond to the third- and fourth-generation quinolones, has yet been published. This work summarizes for the first time most of the liquid chromatographic methods reported in the literature for the separation and quantification of the new fluoroquinolones in biological matrices and pharmaceutical formulations. A systematic and detailed survey of physicochemical properties, sample preparation procedures, and chromatographic and detection conditions is presented herein. In the course of this review several liquid chromatographic methods are discussed: reversed-phase high-performance liquid chromatography (RP-HPLC), ion-exchange high-performance liquid chromatography (IEX-HPLC), hydrophilic interaction liquid chromatography (HILIC), high-performance thin-layer chromatography (HPTLC) and other chiral chromatographic methods. Their advantages, applicability and limitations are also examined. Figure Liquid chromatographic methods for determination of new fluoroquinolones in biological matrices and pharmaceutical formulations.

Keywords: Fluoroquinolones; Analytical methods; Bioanalysis; Pharmaceuticals; Liquid chromatography

Destruction of Raman biosignatures by ionising radiation and the implications for life detection on Mars by Lewis R. Dartnell; Kristian Page; Susana E. Jorge-Villar; Gary Wright; Tasnim Munshi; Ian J. Scowen; John M. Ward; Howell G. M. Edwards (pp. 131-144).

Raman spectroscopy has proven to be a very effective approach for the detection of microorganisms colonising hostile environments on Earth. The ExoMars rover, due for launch in 2018, will carry a Raman laser spectrometer to analyse samples of the martian subsurface collected by the probe’s 2-m drill in a search for similar biosignatures. The martian surface is unprotected from the flux of cosmic rays, an ionising radiation field that will degrade organic molecules and so diminish and distort the detectable Raman signature of potential martian microbial life. This study employs Raman spectroscopy to analyse samples of two model organisms, the cyanobacterium Synechocystis sp. PCC 6803 and the extremely radiation resistant polyextremophile Deinococcus radiodurans, that have been exposed to increasing doses of ionising radiation. The three most prominent peaks in the Raman spectra are from cellular carotenoids: deinoxanthin in D. radiodurans and β-carotene in Synechocystis. The degradative effect of ionising radiation is clearly seen, with significant diminishment of carotenoid spectral peak heights after 15 kGy and complete erasure of Raman biosignatures by 150 kGy of ionising radiation. The Raman signal of carotenoid in D. radiodurans diminishes more rapidly than that of Synechocystis, believed to be due to deinoxanthin acting as a superior scavenger of radiolytically produced reactive oxygen species, and so being destroyed more quickly than the less efficient antioxidant β-carotene. This study highlights the necessity for further experimental work on the manner and rate of degradation of Raman biosignatures by ionising radiation, as this is of prime importance for the successful detection of microbial life in the martian near subsurface.

Keywords: Raman spectroscopy; Cosmic rays; Biosignature; Mars; Astrobiology; Microbe

Quantification of ligand packing density on gold nanoparticles using ICP-OES by Sherrie Elzey; De-Hao Tsai; Savelas A. Rabb; Lee L. Yu; Michael R. Winchester; Vincent A. Hackley (pp. 145-149).

In this study, a prototypical thiolated organic ligand, 3-mercaptopropionic acid (MPA), was conjugated on gold nanoparticles (AuNPs), and packing density was measured on an ensemble-averaged basis using inductively coupled plasma optical emission spectrometry. The effects of sample preparation, including centrifugation and digestion, as well as AuNP size and concentration, on recovery were investigated. For AuNPs with diameters of 5, 10, 30, 60, and 100 nm, calculated packing density is independent of size, averaging 7.8 nm⁻² and ranging from 6.7 to 9.0 nm⁻², and is comparable to reported values for MPA and similar short-chain ligands on AuNPs. These preliminary data provide fundamental information on the advantages and limitations of ICP-based analyses of conjugated AuNP systems. Moreover, they provide necessary information for the development of more broadly applicable methods for quantifying nanoparticle–ligand conjugates of critical importance to nanomedicine applications. Figure Inductively coupled plasma optical emission spectrometry was used to determine the packing density of S-containing ligands on gold nanoparticles based on the ratio of the measured S and Au mass fractions per square nanometer of surface area.

Keywords: Functional ligand; Gold nanoparticle; ICP-OES; Packing density; Quantitative analysis

Effect of a high surface-to-volume ratio on fluorescence-based assays by Radoslaw Kwapiszewski; Karina Ziolkowska; Kamil Zukowski; Michal Chudy; Artur Dybko; Zbigniew Brzozka (pp. 151-155).

In the work discussed in this paper, the effect of a high surface-to-volume ratio of a microfluidic detection cell on fluorescence quenching was studied. It was found that modification of the geometry of a microchannel can provide a wider linear range. This is a phenomenon which should be taken into consideration when microfluidic systems with fluorescence detection are developed. The dependence of the linear range for fluorescein on the surface-to-volume ratio was determined. Both fluorescence inner-filter effects and concentration self-quenching were taken into consideration. It was found that inner-filter effects have little effect on the extent of the linear range on the microscale. Figure Dependence of the linear range on surface-to-volume ratio in microfluidic detection.

Keywords: Lab-on-a-chip; Microfluidics; Optical detection; Fluorescence; Fluorescein; Surface-to-volume ratio

Multiplex time-reducing quantitative polymerase chain reaction assay for determination of telomere length in blood and tissue DNA by Jingjing Jiao; Jing X. Kang; Rui Tan; Jingdong Wang; Yu Zhang (pp. 157-166).

In this paper we describe a multiplex time-reducing quantitative polymerase chain reaction (qPCR) method for determination of telomere length. This multiplex qPCR assay enables two pairs of primers to simultaneously amplify telomere and single copy gene (albumin) templates, thus reducing analysis time and labor compared with the previously established singleplex assay. The chemical composition of the master mix and primers for the telomere and albumin were systematically optimized. The thermal cycling program was designed to ensure complete separation of the melting processes of the telomere and albumin. Semi-log standard curves of DNA concentration versus cycle threshold (C _t) were established, with a linear relationship over an 81-fold DNA concentration range. The well-performed intra-assay (RSD range 2.4–4.7%) and inter-assay (RSD range: 3.1–5.0%) reproducibility were demonstrated to ensure measurement stability. Using wild-type, Lewis lung carcinoma and H22 liver carcinoma C57BL/6 mouse models, significantly different telomere lengths among different DNA samples were not observed in wild-type mice. However, the relative telomere lengths of the tumor DNA in the two strains of tumor-bearing mice were significantly shorter than the lengths in the surrounding non-tumor DNA of tumor-bearing mice and the tissue DNA of wild-type mice. These results suggest that the shortening of telomere lengths may be regarded as an important indicator for cancer control and prevention. Quantification of telomere lengths was further confirmed by the traditional Southern blotting method. This method could be successfully used to reduce the time needed for rapid, precise measurement of telomere lengths in biological samples. Figure Schematic diagram of the general procedures of the multiplex time-reducing qPCR method for determination of telomere length

Keywords: Multiplex qPCR; Time-reducing; Telomere length; Genomic DNA; Cancer

Accurate and reliable quantification of total microalgal fuel potential as fatty acid methyl esters by in situ transesterification by Lieve M. L. Laurens; Matthew Quinn; Stefanie Van Wychen; David W. Templeton; Edward J. Wolfrum (pp. 167-178).

In the context of algal biofuels, lipids, or better aliphatic chains of the fatty acids, are perhaps the most important constituents of algal biomass. Accurate quantification of lipids and their respective fuel yield is crucial for comparison of algal strains and growth conditions and for process monitoring. As an alternative to traditional solvent-based lipid extraction procedures, we have developed a robust whole-biomass in situ transesterification procedure for quantification of algal lipids (as fatty acid methyl esters, FAMEs) that (a) can be carried out on a small scale (using 4–7 mg of biomass), (b) is applicable to a range of different species, (c) consists of a single-step reaction, (d) is robust over a range of different temperature and time combinations, and (e) tolerant to at least 50% water in the biomass. Unlike gravimetric lipid quantification, which can over- or underestimate the lipid content, whole biomass transesterification reflects the true potential fuel yield of algal biomass. We report here on the comparison of the yield of FAMEs by using different catalysts and catalyst combinations, with the acid catalyst HCl providing a consistently high level of conversion of fatty acids with a precision of 1.9% relative standard deviation. We investigate the influence of reaction time, temperature, and biomass water content on the measured FAME content and profile for 4 different samples of algae (replete and deplete Chlorella vulgaris, replete Phaeodactylum tricornutum, and replete Nannochloropsis sp.). We conclude by demonstrating a full mass balance closure of all fatty acids around a traditional lipid extraction process. Figure Accurate quantification of different types of lipids in algal biomass using a comprehensive study of reaction parameters.

Keywords: Fatty acids; Fuels; Catalysts; GC; Bioanalytical methods; Algae

Molecular beacon probes for the detection of cisplatin-induced DNA damage by Zahra J. Shire; Glen R. Loppnow (pp. 179-184).

Cisplatin (cis-diamminedichloroplatinum(II)) causes crosslinking of DNA at AG and GG sites in cellular DNA, inhibiting replication, and making it a useful anti-cancer drug. Several techniques have been used previously to detect nucleic acid damage but most of these tools are labour-intensive, time-consuming, and/or expensive. Here, we describe a sensitive, robust, and quantitative tool for detecting cisplatin-induced DNA damage by using fluorescent molecular beacon probes (MB). Our results show a decrease of fluorescence in the presence of cisplatin-induced DNA damage, confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The decrease in fluorescence upon damage scales with the number of AG and GG sites, indicating the ability of MB to quantitatively detect DNA damage by cisplatin. Figure The MB fluorescence drop for various DNA sequences. The cisplatin lesion sites are shown in red. The greater the number of lesions, the greater the damage response as measured by fluorescence loss.

Keywords: Cisplatin; DNA damage; Fluorescence; Mass spectrometry; Molecular beacon; Nucleic acids

Design, characterization, and evaluation of peptide arrays allowing the direct monitoring of MMP activities by Mohamed-Anis Alouini; El-Farouck Moustoifa; Sandra Albenque Rubio; Aghleb Bartegi; Thomas Berthelot; Gérard Déléris (pp. 185-194).

Characterization of matrix metalloprotease (MMP) activities is of increasing interest for cancer prognosis or treatment follow-up. Indeed, MMP-1, -2 and -9 are widely involved in the growth of many tumors and progression steps such as angiogenesis, invasion, and metastasis. Fluorogenic peptide MMP substrates were previously synthesized with the aim of detecting MMP activities. One of their drawbacks is their limited solubility in biological media. Grafting them onto a solid support represented a novel way to yield efficient analysis devices whilst at the same time decreasing the quantities of peptides used. Novel peptide arrays were designed in order to detect MMP activities in biological fluids. Silicon plates were used as the solid support for the design of these novel tools. These were functionalized by organic self-assembled monolayers (SAMs) on which fluorogenic peptides were covalently coupled. SAM and peptide grafting on silicon plates were confirmed by epifluorescence, ellipsometry, and FT-IR analysis. Enzymatic assays were monitored by fluorescence spectrometry and showed that immobilized linear peptides were recognized and cleaved by MMPs.

Keywords: Matrix metalloprotease; Silanized support; Activity measurement; Fluorescence; Molecular imaging; Cancer

Highly sensitive colorimetric enzyme-linked oligonucleotide assay based on cyclodextrin-modified polymeric surfaces by Mayreli Ortiz; Mabel Torréns; Alex Fragoso; Ciara K. O’Sullivan (pp. 195-202).

In this paper, we describe the development of an enzyme-linked oligonucleotide assay for the detection of a human leukocyte antigen allele associated with celiac disease based on cyclodextrin-modified polymeric surfaces. The surface of maleimide-pre-coated plates was modified with a layer of thiolated cyclodextrin polymer and used for the supramolecular capture of adamantane or ferrocene-modified carboxymethylcellulose polymers bearing DNA probes. The assay was optimised in terms of incubation time, temperature, and surface chemistry and applied to the highly sensitive and selective detection of HLA sequences with a limit of detection of 0.7 nM. A real sample analysed using this platform showed an excellent correlation with maleimide-activated plates using thiolated DNA probes.

Keywords: ELONA; Celiac disease; DNA detection; Cyclodextrin

Serum metabolomics reveals the deregulation of fatty acids metabolism in hepatocellular carcinoma and chronic liver diseases by Lina Zhou; Quancai Wang; Peiyuan Yin; Wenbin Xing; Zeming Wu; Shili Chen; Xin Lu; Yong Zhang; Xiaohui Lin; Guowang Xu (pp. 203-213).

Patients with chronic liver diseases (CLD) including chronic hepatitis B and hepatic cirrhosis (CIR) are the major high-risk population of hepatocellular carcinoma (HCC). The differential diagnosis between CLD and HCC is a challenge. This work aims to study the related metabolic deregulations in HCC and CLD to promote the discovery of the differential metabolites for distinguishing the different liver diseases. Serum metabolic profiling analysis from patients with CLD and HCC was performed using a liquid chromatography–mass spectrometry system. The acquired large amount of metabolic information was processed with the random forest–recursive feature elimination method to discover important metabolic changes. It was found that long-chain acylcarnitines accumulated, whereas free carnitine, medium and short-chain acylcarnitines decreased with the severity of the non-malignant liver diseases, accompanied with corresponding alterations of enzyme activities. However, the general changing extent was smaller in HCC than in CIR, possibly due to the special energy-consumption mechanism of tumor cells. These observations may help to understand the mechanism of HCC occurrence and progression on the metabolic level and provide information for the identification of early and differential metabolic markers for HCC.

Keywords: Hepatocellular carcinoma; Metabolomic profiling; Random forest–recursive feature elimination; Acylcarnitine; Fatty acid oxidation

De novo subtype and strain identification of botulinum neurotoxin type B through toxin proteomics by Suzanne R. Kalb; Jakub Baudys; Jon C. Rees; Theresa J. Smith; Leonard A. Smith; Charles H. Helma; Karen Hill; Skadi Kull; Sebastian Kirchner; Martin B. Dorner; Brigitte G. Dorner; James L. Pirkle; John R. Barr (pp. 215-226).

Botulinum neurotoxins (BoNTs) cause the disease botulism, which can be lethal if untreated. There are seven known serotypes of BoNT, A–G, defined by their response to antisera. Many serotypes are distinguished into differing subtypes based on amino acid sequence, and many subtypes are further differentiated into toxin variants. Previous work in our laboratory described the use of a proteomics approach to distinguish subtype BoNT/A1 from BoNT/A2 where BoNT identities were confirmed after searching data against a database containing protein sequences of all known BoNT/A subtypes. We now describe here a similar approach to differentiate subtypes BoNT/B1, /B2, /B3, /B4, and /B5. Additionally, to identify new subtypes or hitherto unpublished amino acid substitutions, we created an amino acid substitution database covering every possible amino acid change. We used this database to differentiate multiple toxin variants within subtypes of BoNT/B1 and B2. More importantly, with our amino acid substitution database, we were able to identify a novel BoNT/B subtype, designated here as BoNT/B7. These techniques allow for subtype and strain level identification of both known and unknown BoNT/B rapidly with no DNA required. Figure Identification of an existing or new BoNT/B can be accomplished through MS/MS analysis of digestion fragments of the protein.

Keywords: Botulinum neurotoxin; Botulism; Mass spectrometry; Proteomics

Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase IIa by peptide chip and proteome analysis: a pilot study with colorectal carcinoma patient samples by Michael Linnebacher; Peter Lorenz; Cornelia Koy; Annika Jahnke; Nadine Born; Felix Steinbeck; Johannes Wollbold; Tobias Latzkow; Hans-Jürgen Thiesen; Michael O. Glocker (pp. 227-238).

Patient-specific sequential epitopes were identified by peptide chip analysis using 15mer peptides immobilized on glass slides that covered the topoisomerase IIa protein with a frameshift of five amino acids. Binding specificities of serum antibodies against sequential epitopes were confirmed as being mono-specific by peptide chip re-analysis of epitope-affinity-purified antibody pools. These results demonstrate that serum samples from colon carcinoma patients contain antibodies against sequential epitopes from the topoisomerase IIa antigen. Interactions of patients’ antibodies with sequential epitopes displayed by peptides on glass surfaces may thus mirror disease-specific immune situations. Consequently, these data suggest epitope–antibody reactivities on peptide chips as potential diagnostic readouts of individual immune response characteristics, especially because monospecific antibodies can be interrogated. Subsequently, the clonality of the antibodies present in the mono-specific antibody pools was characterized by 2D gel electrophoresis. This analysis suggested that the affinity-purified antibodies were oligoclonal. Similarly to large-scale screening approaches for specific antigen–antibody interactions in order to improve disease diagnostic, we suggest that “protein-wide” screening for specific epitope–paratope interactions may help to develop novel assays for monitoring of personalized therapies, since individual properties of antigen–antibody interactions remain distinguishable. Figure

Keywords: Topoisomerase IIa; Peptide chip analysis; Epitope mapping; Epitope–antibody reactivities; Antibody profiling; Mass spectrometry

Ion-exchange-membrane-based enzyme micro-reactor coupled online with liquid chromatography–mass spectrometry for protein analysis by Zhigui Zhou; Youyou Yang; Jialing Zhang; Zhengxiang Zhang; Yu Bai; Yiping Liao; Huwei Liu (pp. 239-246).

In this article, we developed a membrane-based enzyme micro-reactor by directly using commercial polystyrene–divinylbenzene cation–exchange membrane as the support for trypsin immobilization via electrostatic and hydrophobic interactions and successfully applied it for protein digestion. The construction of the reactor can be simply achieved by continuously pumping trypsin solution through the reactor for only 2 min, which was much faster than the other enzyme immobilization methods. In addition, the membrane reactor could be rapidly regenerated within 35 min, resulting in a “new” reactor for the digestion of every protein sample, completely eliminating the cross-interference of different protein samples. The amount and the activity of immobilized trypsin were measured, and the repeatability of the reactor was tested, with an RSD of 3.2% for the sequence coverage of cytochrome c in ten digestion replicates. An integrated platform for protein analysis, including online protein digestion and peptide separation and detection, was established by coupling the membrane enzyme reactor with liquid chromatography–quadrupole time-of-flight mass spectrometry. The performance of the platform was evaluated using cytochrome c, myoglobin, and bovine serum albumin, showing that even in the short digestion time of several seconds the obtained sequence coverages was comparable to or higher than that with in-solution digestion. The system was also successfully used for the analysis of proteins from yeast cell lysate. Figure Schemes of the designed ion-exchange-membrane-based enzyme micro-reactor (a) and the online coupling system of the enzyme micro-reactor with LC-QTOF MS (b)

Keywords: Ion exchange membrane; Enzyme micro-reactor; Protein analysis; Liquid chromatography; Mass spectrometry; LC-MS

Determination of standard sample purity using the high-precision ¹H-NMR process by Torsten Schoenberger (pp. 247-254).

This paper discusses the technique for high-precision quantification using ¹H-NMR to determine the purity of analytical standard samples. The procedure described is based on the use of internal reference samples in an ¹H NMR experiment in our laboratories. The sample preparation and all relevant NMR parameters were optimized for minimum uncertainty. The validation of accuracy and precision was performed by comparing different certified reference materials. It was shown that the high-precision measurement is applicable even for relatively small sample amounts down to 2.5 mg. The relative combined uncertainty of measurement was found to be 0.15%. Two different approaches for uncertainty calculation were compared; a complete uncertainty budget was calculated. High-precision ¹H-qNMR for determination of standard sample purity

Keywords: qNMR; Standard; Purity determination; High-precision quantification; Uncertainty of measurement; Impurity detection

Combined time- and space-resolved Raman spectrometer for the non-invasive depth profiling of chemical hazards by Biju Cletus; William Olds; Emad L. Izake; Shankaran Sundarajoo; Peter M. Fredericks; Esa Jaatinen (pp. 255-263).

A time-resolved inverse spatially offset Raman spectrometer was constructed for depth profiling of Raman-active substances under both the lab and the field environments. The system operating principles and performance are discussed along with its advantages relative to traditional continuous wave spatially offset Raman spectrometer. The developed spectrometer uses a combination of space- and time-resolved detection in order to obtain high-quality Raman spectra from substances hidden behind coloured opaque surface layers, such as plastic and garments, with a single measurement. The time-gated spatially offset Raman spectrometer was successfully used to detect concealed explosives and drug precursors under incandescent and fluorescent background light as well as under daylight. The average screening time was 50 s per measurement. The excitation energy requirements were relatively low (20 mW) which makes the probe safe for screening hazardous substances. The unit has been designed with nanosecond laser excitation and gated detection, making it of lower cost and complexity than previous picosecond-based systems, to provide a functional platform for in-line or in-field sensing of chemical substances.

Keywords: Time-resolved SORS; Noninvasive detection; Depth profiling; In-field screening; National security

Multiplex flow-through immunoassay formats for screening of mycotoxins in a variety of food matrices by E. Njumbe Ediage; J. Diana Di Mavungu; I. Y. Goryacheva; C. Van Peteghem; S. De Saeger (pp. 265-278).

Two multi-analyte flow-through immunoassay formats for rapid detection of mycotoxins in a variety of food matrices (peanut cake, maize, and cassava flour) were developed and evaluated. The selected food matrices are typical staple foods and export products for most low-income communities around the world. The assay formats included gel-based and membrane-based flow-through assays and were based on the principle of indirect enzyme-linked immunosorbent assay. Using the same immunoreagents, the performance characteristics of both assays were compared. To the best of our knowledge, this is the first report on such a comparison. The gel-based format was developed to screen for ochratoxin A, fumonisin B₁, deoxynivalenol, and zearalenone detection at cut-off values of 3, 1,250, 1,000, and 200 μg kg⁻¹, respectively, while the membrane-based format can be used to screen ochratoxin A, aflatoxin B_1, deoxynivalenol, and zearalenone at the following cut-offs: 3, 5, 700, and 175 μg kg⁻¹, respectively. The applicability of these assay formats was demonstrated by evaluating the performance characteristics of both tests through performing multiple experiments on different days. Both assays were further evaluated by analyzing naturally contaminated samples in the laboratory and also in the field under tropical conditions (Cameroon, West Africa). The false-negative rate with both formats was less than 5%, which is in good agreement with Commission Decision 2002/657/EC regarding the performance of analytical methods intended for screening purposes.

Keywords: Immunoassay; Membrane-based; Gel-based; Flow-through; Mycotoxins

Development of a non-aqueous capillary electrophoresis method with UV–visible and fluorescence detection for phenolics compounds in olive oil by M. P. Godoy-Caballero; M. I. Acedo-Valenzuela; I. Durán-Merás; T. Galeano-Díaz (pp. 279-290).

Response surface methodology has been applied to the optimization of a simple and rapid non-aqueous capillary electrophoresis method for the separation and determination of several phenolic compounds belonging to the different families present in olive oil. A Box–Behnken design was employed and a total of 27 experiments were performed using olive oil samples spiked with the phenols and injected directly in the capillary after dilution 1:1 with 1-propanol. Finally, the background electrolyte (BGE) was constituted of 25 mM boric acid and 18 mM KOH in a mixture of 74:26 (v/v) 1-propanol/methanol. The hydrophobicity of the BGE allows its miscibility with the olive oil and, as a consequence, the possibility of characterizing and determining these kinds of compounds in this sample without any pretreatment. A hydrodynamic injection (6 s, −30 mbar) was applied and the separation was carried out using 35 °C and +20 kV of separation temperature and voltage, respectively. A capillary with two detection windows for serial online UV and fluorescence detection was satisfactorily employed. The validation of the method was carried out by setting the calibration curves, and the figures of merit were finally obtained. A lineal relationship between the corrected peak area and concentration and limits of detection in the order of micrograms per milliliter were found.

Keywords: Non-aqueous capillary electrophoresis; Phenolic compounds; Olive oil; Response surface methodology

Determination of phosphatidylethanolamine molecular species in various food matrices by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS²) by Li Zhou; Minjie Zhao; Saïd Ennahar; Françoise Bindler; Eric Marchioni (pp. 291-300).

A liquid chromatographic–electrospray ionization–tandem mass spectrometric (LC–ESI–MS²) method has been developed for determination of the molecular species of phosphatidylethanolamine (PE) in four food matrices (soy, egg yolk, ox liver, and krill oil). The extraction and purification method consisted of a pressurized liquid extraction procedure for total lipid (TL) extraction, purification of phospholipids (PLs) by adsorption on a silica gel column, and separation of PL classes by semi-preparative normal-phase HPLC. Separation and identification of PE molecular species were performed by reversed-phase HPLC coupled with electrospray ionization tandem mass spectrometry (ESI–MS²). Methanol containing 5 mmol L⁻¹ ammonium formate was used as the mobile phase. A variety of PE molecular species were detected in the four food matrices. (C16:0–C18:2)PE, (C18:2–C18:2)PE, and (C16:0–C18:1)PE were the major PE molecular species in soy. Egg yolk PE contained (C16:0–C18:1)PE, (C18:0–C18:1)PE, (C18:0–C18:2)PE, and (C16:0–C18:2)PE as the major molecular species. Ox liver PE was rich in the species (C18:0–C18:1)PE, (C18:0–C20:4)PE, and (C18:0–C18:2)PE. Finally, krill oil which was particularly rich in (C16:0(alkyl)–C22:6(acyl))plasmanylethanolamine (PakE), (C16:0–C22:6)PE, and (C16:0–C20:5)PE, seemed to be an interesting potential source for supplementation of food with eicosapentaenoic acid and docosahexaenoic acid.

Keywords: PLE; Purification; Phosphatidylethanolamine; LC–ESI–MS² ; Molecular species

Simultaneous quantitative analysis of letrozole, its carbinol metabolite, and carbinol glucuronide in human plasma by LC-MS/MS by Jana C. Precht; Boian Ganchev; Georg Heinkele; Hiltrud Brauch; Matthias Schwab; Thomas E. Mürdter (pp. 301-308).

Letrozole is an efficient endocrine treatment of postmenopausal breast cancer, however, not all patients benefit from this treatment, and moreover, severe side-effects like arthralgia frequently lead to discontinuation. To better understand inter-individual variability in drug response and side-effects, plasma analysis of steady-state concentrations of letrozole and its major metabolites is crucial. We developed a rapid, sensitive, and specific method for the simultaneous quantification of letrozole and its metabolites 4,4′-(hydroxymethylene)dibenzonitrile (carbinol) and bis(4-cyanophenyl)methyl hexopyranosiduronic acid (carbinol-gluc) by UHPLC-ESI-MS/MS using in-house synthesized, stable isotope-labeled internal standards. Following solid-phase extraction in BondElut C18 96-well plates, the analytes were separated on a ZORBAX Eclipse XDB-C18 column (1.8 μm, 4.6 × 50 mm) with a gradient of acetonitrile in 0.1% acetic acid in water and detected on a triple quadrupole mass spectrometer with electrospray ionization in the multiple reaction monitoring mode. Lower limits of quantification were 20, 0.2, and 2 nM for letrozole, carbinol, and carbinol-gluc, respectively. The assay has been validated according to FDA guidance and applied to the analysis of 20 plasma samples of postmenopausal breast cancer patients treated with 2.5 mg of letrozole per day. Mean plasma levels (±SD) were 366 ± 173, 0.38 ± 0.09, and 34 ± 12 nM for letrozole, carbinol, and carbinol-gluc, respectively. Our rapid and sensitive mass spectrometry based method enables future pharmacokinetic investigations of letrozole outcome. Figure LC-MS/MS analysis of letrozole and its metabolites in human plasma

Keywords: Letrozole; Aromatase inhibitor; Plasma; Metabolites; Glucuronide; Tandem mass spectrometry

LC-MS/MS identification of the principal in vitro and in vivo phase I metabolites of the novel thiosemicarbazone anti-cancer drug, Bp4eT by Ján Stariat; Vít Šesták; Kateřina Vávrová; Milan Nobilis; Zuzana Kollárová; Jiří Klimeš; Danuta S. Kalinowski; Des R. Richardson; Petra Kovaříková (pp. 309-321).

The iron chelator, 2-benzoylpyridine-4-ethyl-3-thiosemicarbazone (Bp4eT), was identified as a lead compound of the 2-benzoylpyridine thiosemicarbazone series, which were designed as potential anti-cancer agents. This ligand has been shown to possess potent anti-proliferative activity with a highly selective mechanism of action. However, further progress in the development of this compound requires data regarding its metabolism in mammals. The aim of this study was to identify the main in vitro and in vivo phase I metabolites of Bp4eT using liquid chromatography tandem mass spectrometry (LC-MS/MS). Two metabolites were detected after incubation of this drug with rat and human liver microsomal fractions. Based on LC-MSⁿ analysis, the metabolites were demonstrated to be 2-benzoylpyridine-4-ethyl-3-semicarbazone and N ³-ethyl-N ¹-[phenyl(pyridin-2-yl)methylene]formamidrazone, with both resulting from the oxidation of the thiocarbonyl group. The identity of these metabolites was further shown by LC-MS/MS analysis of these latter compounds which were prepared by oxidation of Bp4eT with hydrogen peroxide and their structures confirmed by nuclear magnetic resonance and infrared spectra. Both the semicarbazone and the amidrazone metabolites were detected in plasma, urine, and feces after i.v. administration of Bp4eT to rats. In addition, another metabolite that could correspond to hydroxylated amidrazone was found in vivo. Thus, oxidative pathways play a major role in the phase I metabolism of this promising anti-tumor agent. The outcomes of this study will be further utilized for: (1) the development and validation of the analytical method for the quantification of Bp4eT and its metabolites in biological materials; (2) to design pharmacokinetic experiments; and to (3) evaluate the potential contribution of the individual metabolites to the pharmacodynamics/toxico-dynamics of this novel anti-proliferative agent. Figure a LC-MS chromatogram of the analysis of the sample from in vivo experiment, b proposed metabolic pathway of Bp4eT and c MS/MS fragmentation of the parent compound and metabolites M1-M3

Keywords: 2-Benzoylpyridine-4-ethyl-3-thiosemicarbazone; Bp4eT; Anti-cancer; Anti-tumor chemotherapeutic; LC-MS

Development of a UPLC-ESI-MS/MS method for the determination of larotaxel in beagle dog plasma: application to the pharmacokinetic study by Zhenzhen Liu; Bo Zhang; Zhihong Liu; Song Li; Guofei Li; Lulu Geng; Xu Zhao; Kaishun Bi; Xing Tang; Xiaohui Chen (pp. 323-330).

A UPLC-ESI-MS/MS method has been developed and validated for the determination of larotaxel in beagle dog plasma. After addition of the internal standard, plasma samples were extracted by liquid–liquid extraction with methyl tert-butyl ether and separated on a 50 × 2.1 mm ACQUITY 1.7 μm C₁₈ column (Waters, USA), with acetonitrile and 5 mM ammonium acetate as mobile phase, within a runtime of 3.0 min. The analytes were detected without interference in Multiple Reaction Monitoring mode with positive electrospray ionization. The linear range was 2.5–5,000 ng/mL. The intra-day and inter-day precisions (relative standard deviation, RSD, %) were within 9.3% and 10.2%, respectively, and the accuracy (relative error, RE, %) was less than 11.5%. The validated method was successfully applied to a pharmacokinetic study of larotaxel in beagle dogs after intravenous administration of larotaxel-loaded lipid microsphere with different doses of 0.4, 0.8, and 1.6 mg/kg. The area under the concentration–time curve and the peak concentration of larotaxel seemed to increase with increasing dose proportionally, suggesting linear pharmacokinetics. Figure The concentration–time curves of larotaxel in beagle dog plasma after intravenous administration of LTX-LM at different doses of 0.4, 0.8, and 1.6 mg/kg

Keywords: UPLC-ESI-MS/MS; Pharmacokinetics; Larotaxel-loaded lipid microsphere; Intravenous administration

Erratum to: Microcantilevers and organic transistors: two promising classes of label-free biosensing devices which can be integrated in electronic circuits by Serafina Cotrone; Damiana Cafagna; Stefania Cometa; Elvira De Giglio; Maria Magliulo; Luisa Torsi; Luigia Sabbatini (pp. 331-331).

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Analytical and Bioanalytical Chemistry (v.403, #1)