Analytical and Bioanalytical Chemistry (v.402, #2)

New Editorial Board members from China by Steffen Pauly (557-558).

Solvent-free microextraction techniques in gas chromatography by Jens Laaks; Maik A. Jochmann; Torsten C. Schmidt (565-571).
Microextraction techniques represent a major part of modern sample preparation in the analysis of organic micropollutants. This article provides a short overview of recent developments in solvent-free microextraction techniques. From the first open-tubular trap techniques in the mid-1980s to recent packed-needle devices, different implementations of in-needle packings for microextraction are discussed with their characteristic benefits, shortcomings and possible sampling modes. Special emphasis is placed on methods providing full automation and solvent exclusion. In this context, in-tube extraction and the needle trap are discussed, with an overview of current research on new sorbent materials, together with the requirements for more efficient method development.
Keywords: Microextraction; Gas chromatography; Solid-phase microextraction; Solid-phase dynamic extraction; Needle trap; In-tube extraction

The herbalome—an attempt to globalize Chinese herbal medicine by Xiuli Zhang; Yanfang Liu; Zhimou Guo; Jiatao Feng; Jun Dong; Qing Fu; Chaoran Wang; Xingya Xue; Yuansheng Xiao; Xinmiao Liang (573-581).
The herbalome is a project with the objective of globalizing Chinese herbal medicine (CHM) by clarification of its composition, structure, and function; by establishing a standard resource library; and by interpreting the synergistic and complementary mechanisms of multi-components on multi-targets. In phase I, it focuses on the development of systematic separation methodology for resolving and analyzing the complex components in CHM and establishment of a comprehensive resource library. This review summarizes recent advances in the herbalome project with regard to innovative separation techniques and demonstration of a resource library. Figure The research strategy of the herbalome
Keywords: Herbalome; TCM; Chinese herbal medicine; Separation technique; Fine multi-components; Sample library

Nanoparticles in aquatic systems by Markus Delay; Fritz H. Frimmel (583-592).
Nanoparticles (NP) are ubiquitous in environmental and technical aquatic systems. Understanding the role and the fate of NP in these systems is an interdisciplinary challenge requiring innovative experimental, theoretical and analytical approaches and critical reflection of classical concepts. This contribution critically reviews the outstanding properties of NP and the resulting consequences for their behaviour in environmental and technical aquatic systems considering natural NP which are mostly geogenic or biogeogenic, and engineered NP. Owing to the severe lack of data on the occurrence of NP in environmental aquatic systems, it is a key task of researchers to further develop analytical methods for the sensitive detection of NP directly in aqueous samples. There is urgent need for standardisation of analytical methods for detection and characterisation of NP, and for toxicity tests to assess possible adverse effects of NP. In this context, NP reference materials have to be defined as a common fundament for research in this field.
Keywords: Nanoparticles; Engineered nanoparticles; Colloids; Analytical coupling techniques; Water quality; Water technology

Amorphous carbon nanoparticles: a versatile label for rapid diagnostic (immuno)assays by Geertruida A. Posthuma-Trumpie; Jan H. Wichers; Marjo Koets; Luciënne B. J. M. Berendsen; Aart van Amerongen (593-600).
Carbon nanoparticles (CNPs) labeled with reporter molecules can serve as signaling labels in rapid diagnostic assays as an alternative to gold, colored latex, silica, quantum dots, or up-converting phosphor nanoparticles. Detailed here is the preparation of biomolecule-labeled CNPs and examples of their use as a versatile label. CNPs can be loaded with a range of biomolecules, such as DNA, antibodies, and proteins (e.g., neutravidin or a fusion protein of neutravidin with an enzyme), and the resulting conjugates can be used to detect analytes of high or low molecular mass. Figure Scheme of a NALFIA-, NAMIA, or NALMIA. Neutravidin adsorbed onto CNPs detects biotin-labelled amplicons; the discriminating tag is recognized by its respective antibody, which is immobilized onto nitrocellulose membranes or pads
Keywords: Carbon nanoparticles; Lateral flow assays; Protein microarray-based assays; Nucleic acid immunoassays (DNA/RNA); Immunochemical assays; Diagnostics

Nanomaterials have emerging importance in laser desorption ionization mass spectrometry (LDI–MS) with the ultimate objective being to overcome some of the most important limitations intrinsically related to the use of conventional organic matrices in matrix-assisted (MA) LDI–MS. This review provides a critical overview of the most recent literature on the use of gold nanomaterials as non-conventional desorption ionization promoters in LDI–MS, with particular emphasis on bioanalytical applications. Old seminal papers will also be discussed to provide a timeline of the most significant achievements in the field. Future prospects and research needs are also briefly discussed.
Keywords: Laser desorption ionization; Mass spectrometry; Gold nanomaterial; SALDI; SELDI; NALDI

This review covers recent developments in mass spectrometry-based applications dealing with functional proteomics with special emphasis on enzymology. The introduction of mass spectrometry into this research field has led to an enormous increase in knowledge in recent years. A major challenge is the identification of “biologically active substances” in complex mixtures. These biologically active substances are, on the one hand, potential regulators of enzymes. Elucidation of function and identity of those regulators may be accomplished by different strategies, which are discussed in this review. The most promising approach thereby seems to be the one-step procedure, because it enables identification of the functionality and identity of biologically active substances in parallel and thus avoids misinterpretation. On the other hand, besides the detection of regulators, the identification of endogenous substrates for known enzymes is an emerging research field, but in this case studies are quite rare. Moreover, the term biologically active substances may also encompass proteins with diverse biological functions. Elucidation of the functionality of those—so far unknown—proteins in complex mixtures is another branch of functional proteomics and those investigations will also be discussed in this review. Figure Mass spectrometric monitoring of enzymatic reactions
Keywords: Protein function; mass spectrometry; inhibitor screening; continuous-flow assays

Nuclear magnetic resonance (NMR) spectroscopy is one of the most important and powerful instrumental analytical techniques for structural elucidation of unknown small and large (complex) isolated and synthesized compounds in organic and inorganic chemistry. X-ray crystallography, neutron scattering (neutron diffraction), and NMR spectroscopy are the only suitable methods for three-dimensional structure determination at atomic resolution. Moreover, these methods are complementary. However, by means of NMR spectroscopy, reaction dynamics and interaction processes can also be investigated. Unfortunately, this technique is very insensitive in comparison with other spectrometric (e.g., mass spectrometry) and spectroscopic (e.g., infrared spectroscopy) methods. Mainly through the development of stronger magnets and more sensitive solenoidal microcoil flow probes, this drawback has been successfully counteracted. Capillary NMR spectroscopy increases the mass-based sensitivity of the NMR spectroscopic analysis up to 100-fold compared with conventional 5-mm NMR probes, and thus can be coupled online and off-line with other microseparation and detection techniques. It offers not only higher sensitivity, but in many cases provides better quality spectra than traditional methods. Owing to the immense number of compounds (e.g., of natural product extracts and compound libraries) to be examined, single microcoil flow probe NMR spectroscopy will soon be far from being sufficiently effective as a screening method. For this reason, an inevitable trend towards coupled microseparation–multiple microcoil flow probe NMR techniques, which allow simultaneous online and off-line detection of several compounds, will occur. In this review we describe the current status and possible future developments of single and multiple microcoil capillary flow probe NMR spectroscopy and its application as a high-throughput tool for the analysis of a large number of mass-limited samples. The advantages and drawbacks of different coupled microseparation–capillary NMR spectroscopy techniques, such as capillary high-performance liquid chromatography–NMR spectroscopy, capillary electrophoresis–NMR spectroscopy, and capillary gas chromatography–NMR spectroscopy, are discussed and demonstrated by specific applications. Another subject of discussion is the progress in parallel NMR detection techniques. Furthermore, the applicability and mixing capability of tiny reactor systems, termed “microreactors” or “micromixers,” implemented in NMR probes is demonstrated by carbamate- and imine-forming reactions.
Keywords: Microcoil NMR; Capillary techniques; Multiple microprobe; Solenoidal microcoil; Parallel detection; Microseparation; Microreactor

In this article, assays on the analytical determination of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), two important isoprenoid intermediates at biochemically relevant branching points in the mevalonate pathway, are summarized and reviewed. There is considerable recent interest in the measurement of these two isoprenoids because of their direct involvement in several diseases, for example, statins lower cholesterol by inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase but equally affect other metabolite biosyntheses. The isoprenoids FPP and GGPP are key intermediates due to their role as CaaX-specific substrates for posttranslational modification of proteins (protein prenylation). Disease pathologies and therapeutic efficacy of different treatments (e.g., cholesterol-lowering drugs) may lead to a reduction in isoprenoid levels and an accompanying reduction in prenylation of specific proteins. To understand the exact biochemical role of the isoprenoids FPP and GGPP, we need to know their levels. Several recent studies have shown exact levels of FPP and GGP in plasma and relevant tissues and their modulation following treatment. Furthermore, by directly measuring the extent of protein prenylation and identifying target proteins, further insight into the exact biochemical nature of the pathology and regulatory mechanisms will be possible. This short review aims to highlight the relevant literature on the analytical determination of the free isoprenoids FPP and GGPP in biological tissue as well as techniques for directly measuring prenylated proteins. Figure Isoprenoid derivatives for labeling peptides and proteins via prenylation reactions. (1) Dye-containing derivatives; (2) derivatives for orthogonal labeling after prenylation (a); (b) via Staudinger reaction with azide isoprenoids or (c) via Click chemistry ([3+2] cycloaddition) with azide or alkyne isoprenoids (circles, dye and biotinyl group)
Keywords: Farnesyl pyrophosphate (FPP); Geranylgeranyl pyrophosphate (GGPP); Mevalonate pathway; Protein prenylation; Liquid chromatography; Mass spectrometry

Direct molecule-specific glucose detection by Raman spectroscopy based on photonic crystal fiber by Xuan Yang; Alissa Y. Zhang; Damon A. Wheeler; Tiziana C. Bond; Claire Gu; Yat Li (687-691).
This paper reports the first step toward the development of a glucose biosensor based on Raman spectroscopy and a photonic crystal fiber (PCF) probe. Historically, it has been very challenging to detect glucose directly by Raman spectroscopy due to its inherently small Raman scattering cross-section. In this work, we report the first quantitative glucose Raman detection in the physiological concentration range (0–25 mM) with a low laser power (2 mW), a short integration time (30 s), and an extremely small sampling volume (∼50 nL) using the highly sensitive liquid-filled PCF probe. As a proof of concept, we also demonstrate the molecular specificity of this technique in the presence of a competing sugar, such as fructose. High sensitivity, flexibility, reproducibility, low cost, small sampling volume, and in situ remote sensing capability make PCF a very powerful platform for potential glucose detection based on Raman spectroscopy.
Keywords: Glucose detection; Raman spectroscopy; Photonic crystal fiber; Fiber sensor

Development of a multiplex DNA-based traceability tool for crop plant materials by Marleen M. Voorhuijzen; Jeroen P. van Dijk; Theo W. Prins; A. M. Angeline Van Hoef; Ralf Seyfarth; Esther J. Kok (693-701).
The authenticity of food is of increasing importance for producers, retailers and consumers. All groups benefit from the correct labelling of the contents of food products. Producers and retailers want to guarantee the origin of their products and check for adulteration with cheaper or inferior ingredients. Consumers are also more demanding about the origin of their food for various socioeconomic reasons. In contrast to this increasing demand, correct labelling has become much more complex because of global transportation networks of raw materials and processed food products. Within the European integrated research project ‘Tracing the origin of food’ (TRACE), a DNA-based multiplex detection tool was developed—the padlock probe ligation and microarray detection (PPLMD) tool. In this paper, this method is extended to a 15-plex traceability tool with a focus on products of commercial importance such as the emmer wheat Farro della Garfagnana (FdG) and Basmati rice. The specificity of 14 plant-related padlock probes was determined and initially validated in mixtures comprising seven or nine plant species/varieties. One nucleotide difference in target sequence was sufficient for the distinction between the presence or absence of a specific target. At least 5% FdG or Basmati rice was detected in mixtures with cheaper bread wheat or non-fragrant rice, respectively. The results suggested that even lower levels of (un-)intentional adulteration could be detected. PPLMD has been shown to be a useful tool for the detection of fraudulent/intentional admixtures in premium foods and is ready for the monitoring of correct labelling of premium foods worldwide.
Keywords: Authenticity; DNA; Ligation; Detection; Traceability; Multiplex

Initially, a poly (glycidyl methacrylate-co-acrylamide-co-methylenebisacrylamide) monolith was prepared in the 100 μm i.d. capillary, and then was grafted with polyethylenimine (Mw, ∼25,000) for adsorbing Cu2+, followed by chelating trypsin. As a result, efficient digestion for BSA (100 ng/μL) was completed within 50 s via such immobilized enzyme reactor (IMER); yielding 47% sequence coverage by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Compared with the conventional method for preparing the metal-ion chelated IMER, the regeneration of such IMER can be achieved facilely by the respective 30 min desorption and re-adsorption of trypsin, and 51% sequence coverage was obtained for 50 s BSA digestion after regeneration. BSA down to femtomole was also efficiently digested by the prepared regenerable IMER. Meanwhile, after the consecutive digestion of myoglobin and BSA, there was not any mutual interference for both during MALDI-TOF MS identification, indicating the low nonspecific adsorption of such regenerable IMER. To test the applicability of regenerable IMER for complex sample profiling, proteins (150 ng) extracted from Escherichia coli were digested within 80 s by the regenerable IMER and further analyzed by nanoreversed phase liquid chromatography–electrospray ionization–mass spectrometry successfully, showing its practicability for the high throughput analysis of complex samples.
Keywords: Metal-ion chelation; Polyacrylamide monolith; Polyethylenimine; Tryptic digestion; Regenerable immobilized enzyme reactor

Increasing phosphoproteomic coverage through sequential digestion by complementary proteases by Jason M. Gilmore; Arminja N. Kettenbach; Scott A. Gerber (711-720).
Protein phosphorylation is a reversible post-translational modification known to regulate protein function, subcellular localization, complex formation, and protein degradation. Detailed phosphoproteomic information is critical to kinomic studies of signal transduction and for elucidation of cancer biomarkers, such as in non-small-cell lung adenocarcinoma, where phosphorylation is commonly dysregulated. However, the collection and analysis of phosphorylation data remains a difficult problem. The low concentrations of phosphopeptides in complex biological mixtures as well as challenges inherent in their chemical nature have limited phosphoproteomic characterization and some phosphorylation sites are inaccessible by traditional workflows. We developed a sequential digestion method using complementary proteases, Glu-C and trypsin, to increase phosphoproteomic coverage and supplement traditional approaches. The sequential digestion method is more productive than workflows utilizing only Glu-C and we evaluated the orthogonality of the sequential digestion method relative to replicate trypsin-based analyses. Finally, we demonstrate the ability of the sequential digestion method to access new regions of the phosphoproteome by comparison to existing public phosphoproteomic databases. Our approach increases coverage of the human lung cancer phosphoproteome by accessing both new phosphoproteins and novel phosphorylation site information.
Keywords: Genomics/proteomics; Mass spectrometry/ICP-MS; Bioanalytical methods

Ultra-thin layer MALDI mass spectrometry of membrane proteins in nanodiscs by Michael T. Marty; Aditi Das; Stephen G. Sligar (721-729).
Nanodiscs have become a leading technology to solubilize membrane proteins for biophysical, enzymatic, and structural investigations. Nanodiscs are nanoscale, discoidal lipid bilayers surrounded by an amphipathic membrane scaffold protein (MSP) belt. A variety of analytical tools has been applied to membrane proteins in nanodiscs, including several recent mass spectrometry studies. Mass spectrometry of full-length proteins is an important technique for analyzing protein modifications, for structural studies, and for identification of proteins present in binding assays. However, traditional matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry methods for analyzing full-length membrane proteins solubilized in nanodiscs are limited by strong signal from the MSP belt and weak signal from the membrane protein inside the nanodisc. Herein, we show that an optimized ultra-thin layer MALDI sample preparation technique dramatically enhances the membrane protein signal and nearly completely eliminates the MSP signal. First-shot MALDI and MALDI imaging are used to characterize the spots formed by the ultra-thin layer method. Furthermore, the membrane protein enhancement and MSP suppression are shown to be independent of the type of membrane protein and are applicable to mixtures of membrane proteins in nanodiscs. Figure A model of cytochrome P450 3A4 (CYP 3A4) in a soluble lipid bilayer nanodisc is shown (left). Membrane proteins in nanodiscs demonstrate poor spectra with MALDI mass spectrometry using the conventional dried-drop method (middle). The ultra-thin layer method shows remarkable enhancement of membrane protein signal and nearly complete suppression of the membrane scaffold protein belt (MSP) signal (right).
Keywords: MALDI-TOF mass spectrometry; Membrane proteins; Nanodisc; Rhodopsin; Cytochrome p450 reductase; Cytochrome P450 3A4

A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide by Omid Motabar; Ehud Goldin; William Leister; Ke Liu; Noel Southall; Wenwei Huang; Juan J. Marugan; Ellen Sidransky; Wei Zheng (731-739).
Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening.
Keywords: Glucocerebrosidase; Natural substrate; Gaucher disease; Enzyme substrate; Inhibitor

Direct immobilization of DNA probes on non-modified plastics by UV irradiation and integration in microfluidic devices for rapid bioassay by Yi Sun; Ivan Perch-Nielsen; Martin Dufva; David Sabourin; Dang Duong Bang; Jonas Høgberg; Anders Wolff (741-748).
DNA microarrays have become one of the most powerful tools in the field of genomics and medical diagnosis. Recently, there has been increased interest in combining microfluidics with microarrays since this approach offers advantages in terms of portability, reduced analysis time, low consumption of reagents, and increased system integration. Polymers are widely used for microfluidic systems, but fabrication of microarrays on such materials often requires complicated chemical surface modifications, which hinders the integration of microarrays into microfluidic systems. In this paper, we demonstrate that simple UV irradiation can be used to directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on many different types of plastics without any surface modification. On average, five- and fourfold improvement in immobilization and hybridization efficiency have been achieved compared to surface-modified slides with aminated DNA probes. Moreover, the TC tag only costs 30% of the commonly used amino group modifications. Using this microarray fabrication technique, a portable cyclic olefin copolymer biochip containing eight individually addressable microfluidic channels was developed and used for rapid and parallel identification of Avian Influenza Virus by DNA hybridization. The one-step, cost-effective DNA-linking method on non-modified polymers significantly simplifies microarray fabrication procedures and permits great flexibility to plastic material selection, thus making it convenient to integrate microarrays into plastic microfluidic systems.
Keywords: Microarray; Microfluidic; Non-modified plastic; UV; DNA immobilization

Preparation and value assignment of standard reference material 968e fat-soluble vitamins, carotenoids, and cholesterol in human serum by Jeanice B. Thomas; David L. Duewer; Isaac O. Mugenya; Karen W. Phinney; Lane C. Sander; Katherine E. Sharpless; Lorna T. Sniegoski; Susan S. Tai; Michael J. Welch; James H. Yen (749-762).
Standard Reference Material 968e Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum provides certified values for total retinol, γ- and α-tocopherol, total lutein, total zeaxanthin, total β-cryptoxanthin, total β-carotene, 25-hydroxyvitamin D3, and cholesterol. Reference and information values are also reported for nine additional compounds including total α-cryptoxanthin, trans- and total lycopene, total α-carotene, trans-β-carotene, and coenzyme Q10. The certified values for the fat-soluble vitamins and carotenoids in SRM 968e were based on the agreement of results from the means of two liquid chromatographic methods used at the National Institute of Standards and Technology (NIST) and from the median of results of an interlaboratory comparison exercise among institutions that participate in the NIST Micronutrients Measurement Quality Assurance Program. The assigned values for cholesterol and 25-hydroxyvitamin D3 in the SRM are the means of results obtained using the NIST reference method based upon gas chromatography-isotope dilution mass spectrometry and liquid chromatography-isotope dilution tandem mass spectrometry, respectively. SRM 968e is currently one of two available health-related NIST reference materials with concentration values assigned for selected fat-soluble vitamins, carotenoids, and cholesterol in human serum matrix. This SRM is used extensively by laboratories worldwide primarily to validate methods for determining these analytes in human serum and plasma and for assigning values to in-house control materials. The value assignment of the analytes in this SRM will help support measurement accuracy and traceability for laboratories performing health-related measurements in the clinical and nutritional communities.
Keywords: Standard reference material; Fat-soluble vitamins; Carotenoids; Frozen human serum; Cholesterol; Value assignment

Current urinary bladder cancer diagnosis is commonly based on a biopsy obtained during cystoscopy. This invasive method causes discomfort and pain in patients. Recently, taurine and several other compounds such as L-phenylalanine and hippuric acid in urine were found to be indicators of bladder cancer. However, because of a lack of sensitive and accurate analytical techniques, it is impossible to detect these compounds in urine at low levels. In this study, using liquid chromatography–tandem mass spectrometry (LC-MS/MS), a noninvasive method was developed to separate and detect these compounds in urine. 15N2-L-glutamine was used as the internal standard, and creatinine acted as an indicator for urine dilution. A phenyl-hexyl column was used for the separation at an isocratic condition of 0.2% formic acid in water and 0.2% formic acid in methanol. Analytes were detected in multiple-reaction monitoring with positive ionization mode. The limit of detection range is 0.18–6 nM and the limit of quantitation ranges from 0.6 to 17.6 nM. The parameters affecting separation and quantification were also investigated and optimized. Proper clinical validation of these biomarkers can be done using this reliable, fast, and simple method. Furthermore, with simple modifications, this method could be applied to other physiological fluids and other types of diseases.
Keywords: Taurine; Biomarkers; LC-MS/MS; Bladder cancer; Urine

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase and an established biomarker for endothelial function, while symmetric dimethylarginine (SDMA), an emerging biomarker for renal function, has been shown to outperform creatinine-based equations for estimated glomerular filtration rate. In order to study these analytes for clinical research, a fast and simple method for measuring arginine (ARG), SDMA, and ADMA in plasma by liquid chromatography–tandem mass spectrometry (LC-MS/MS) has been developed. Plasma (50 μL) was mixed with 50 μL of internal standard of 13C-arginine and d7-ADMA followed by protein precipitation with methanol containing 1% ammonium acetate (300 μL). After centrifugation, the supernatant (100 μL) was mixed with 300 μL of acetonitrile with 1% formic acid, and the mixture was injected onto a silica column monitored by a mass spectrometer. The analytical cycle time was 5.0 min. The method was linear from 5.7 to 489.7 μM for ARG, 0.06 to 5.15 μM for SDMA, and from 0.34 to 5.65 μM for ADMA, with an accuracy of 99.0–120.0%. Total coefficients of variation for all analytes ranged from 2.7% to 7.7% for three concentration levels. The effects of hemolysis, lipemia, uremia, icterus, specimen tube types, storage at different temperature, and freeze/thaw were thoroughly investigated. Reference ranges were established using 51 well-defined reference subjects (12 men and 39 women, age 19–64 years): 53.1–129.7 μM for ARG, 0.32–0.65 μM for SDMA, and 0.36–0.67 μM for ADMA. In conclusion, the validated LC-MS/MS method described here offers a fast and reliable ARG, SDMA, and ADMA quantitation in plasma with minimum sample preparation.
Keywords: Arginine; Symmetric dimethylarginine; Asymmetric dimethylarginine; LC-MS/MS; Reference range

Combined use of liquid chromatography with mass spectrometry and nuclear magnetic resonance for the identification of degradation compounds in an erythromycin formulation by M. Pendela; S. Béni; E. Haghedooren; L. Van den Bossche; B. Noszál; A. Van Schepdael; J. Hoogmartens; E. Adams (781-790).
A commercial erythromycin formulation containing erythromycin A (EA) as the major compound showed the presence of an unknown degradation compound that was co-eluted with erythromycin E (EE) in the European Pharmacopoeia (Ph. Eur.) liquid chromatographic (LC) method. The amount of the degradation compound increased with respect to time. To separate this unknown (UNK1), investigation was performed with different LC methods coupled to ultraviolet detection (LC-UV). With the present Ph. Eur. method, the degradation compound could not be well separated. However, with the most selective LC-UV method (XTerra method), two more degradation products (UNK2 and UNK3) were found in the formulation which could not be observed using other methods because of their poor separation. By combining the results obtained with LC-UV, LC/MS and LC/NMR, the degradation products were identified as pseudoerythromycin A hemiketal (PsEAHK), erythromycin A enol ether carboxylic acid and erythromycin C enol ether carboxylic acid. PsEAHK is known to be a base-catalysed degradation product of EA, whereas the other two degradation products were newly identified.
Keywords: Erythromycin; Degradation; LC-UV; LC/MS; LC/NMR

Alendronate is an important representative of bisphosphonates, strongly polar compounds that lack chromophores. With rare exceptions, derivatization of the analytes is necessary for bioanalysis. In this study, a rapid liquid chromatography–tandem mass spectrometry method employing pre-column derivatization was developed and validated for the determination of alendronate concentrations in human plasma. The procedure was based on derivatization with trimethylsilyldiazomethane during solid-phase extraction on a weak anion-exchange solid-phase cartridge, which integrated sample purification and derivatization into one step. The alendronate derivative was eluted with methanol. Chromatographic separation was performed on a Capcell PAK-C18 column. The total run time was 6.5 min. The calibration curve was linear in the range 1.00–1,000 ng/mL using d6-alendronate as the internal standard. The lower limit of quantification was 1.00 ng/mL. The intra- and inter-assay precision (in RSD) calculated from quality control samples was less than 15%, and the accuracy was between 98.1% and 100.2%. The validated method was successfully applied to characterize the pharmacokinetic profiles of alendronate following the intravenous infusion of 5 or 10 mg alendronate sodium to healthy volunteers. Figure Derivatization and determination procedures of alendronate in human plasma
Keywords: Alendronate; Derivatization; LC-MS/MS; Trimethylsilyldiazomethane; Pharmacokinetics

An interferometry-based method was developed for detection of a small molecule, argininamide. The quantification of argininamide was demonstrated using aptamers immobilized on silicone oxynitride sensor surface via avidin–biotin binding. The aptamers formed a thin film over avidin layer corresponding to a thickness of 1.2 nm, consistent with a molecular positioning of multipoint attachment to the surface. The binding of argininamide did not cause any significant changes in the thickness of the aptamer film, suggesting that the specific binding did not affect the overall conformation of the aptamer molecules after adaptive rearrangement of aptamer molecules. However, the binding results in clearly detectable changes in mass calculated from multiple parameters determined by mass deposition and structural changes. The limit of detection of the developed sensor was determined to be 5 μM. The sensor can monitor real-time changes in argininamide concentrations with high reliability and sensitivity. The model system demonstrated that a combined measurement considering structural and mass changes through interferometry-based techniques can overcome one of the major problems associated with real-time monitoring of small mass analytes.
Keywords: Aptamers; Biosensors; Dual-polarization interferometry; Amino acid; Small molecules; Optical monitoring

Molecular beacon-based fluorescence biosensor for the detection of gene fragment and PCR amplification products related to chronic myelogenous leukemia by Ailin Liu; Zhouliang Sun; Kun Wang; Xuhai Chen; Xiongwei Xu; Yong Wu; Xinhua Lin; Yuanzhong Chen; Min Du (805-812).
A novel fluorescence method has been established for the determination of gene fragment and PCR amplification products related to chronic myelogenous leukemia (CML). A molecular beacon (MB) which comprises a DNA loop section, a pair of fluorophore (tetramethoxyl rhodamine, TAMRA), and a quencher (4-(2-methyl-on-amino-azobenzene) benzoate, DABCYL) was designed. The loop sequence of MB was designed according to the DNA sequence relating to CML (type b3a2) which contained a single-stranded oligonucleotide. Before hybridization, the fluorescence from the TAMRA had been quenched by the DABCYL. After hybridization with the complementary DNA, the quencher will become far away from the TAMRA, and the fluorescence intensity detected will increase. Changes in the fluorescence intensity have a linear relationship with the concentration of complementary DNA (C) in the range of 4.0 × 10−9–3.2 × 10−8 mol/L, with a correlation coefficient of 0.9973; the detection limit was 6.0 × 10−10 mol/L (S/N = 3). The developed method has high selectivity, which can be used to discriminate single-base mismatch sequence. The method has been applied to detect the short-stranded CML DNA fragment (278 bp) with high sensitivity. This approach is a promising method for the detection of CML in real samples for medical diagnostics.
Keywords: Chronic myelogenous leukemia; Molecular beacon; Fluorescence spectrophotometry

A CMOS image sensor to recognize the cardiovascular disease markers troponin I and C-reactive protein by Jasmine Pramila Devadhasan; Mohana Marimuthu; Sanghyo Kim; Min-Gon Kim (813-821).
A complementary metal oxide semiconductor (CMOS) image sensor was utilized to detect the interaction of cardiovascular disease markers, troponin I and C-reactive protein. Each marker with its respective antibodies was adsorbed to an indium nanoparticle (InNP)-coated glass substrate. Dielectric layers of antigens and antibodies bound onto and interacted on conducting InNPs. Normal room light passed through these protein-layer-bound substrates and hit the CMOS image sensor surface, and the number of photons was detected and converted into digital form. We tested this approach for real-time monitoring of cardiac disease markers based on photon count, demonstrating its low cost and its capacity to detect antigens with high sensitivity at picogram per milliliter concentration.
Keywords: Complementary metal oxide semiconductor sensor; Indium nanoparticle; Troponin I protein; C-reactive protein

Data processing for image-based chemical sensors: unsupervised region of interest selection and background noise compensation by Francesca Dini; Eugenio Martinelli; Roberto Paolesse; Daniel Filippini; Detlev Schild; Ingemar Lundström; Corrado DI Natale (823-832).
Natural olfaction suggests that numerous replicas of small sensors can achieve large sensitivity. This concept of sensor redundancy can be exploited by use of optical chemical sensors whose use of image sensors enables the simultaneous measurement of several spatially distributed indicators. Digital image sensors split the framed scene into hundreds of thousands of pixels each corresponding to a portion of the sensing layer. The signal from each pixel can be regarded as an independent sensor, which leads to a highly redundant sensor array. Such redundancy can eventually be exploited to increase the signal-to-noise ratio. In this paper we report an algorithm for reduction of the noise of pixel signals. For this purpose, the algorithm processes the output of groups of pixels whose signals share the same time behavior, as is the case for signals related to the same indicator. To define these groups of pixels, unsupervised clustering, based on classification of the indicator colors, is proposed here. This approach to signal processing is tested in experiments on the chemical sensitivity of replicas of eight indicators spotted on to a plastic substrate. Results show that the groups of pixels can be defined independently of the geometrical arrangement of the sensing spots, and substantial improvement of the signal-to-noise ratio is obtained, enabling the detection of volatile compounds at any location on the distributed sensing layer.
Keywords: Optical sensors; Chemical sensors; Chemometrics/Statistics

Salvia divinorum is a hallucinogenic herb that is internationally regulated. In this study, salvinorin A, the active compound in S. divinorum, was extracted from S. divinorum plant leaves using a 5-min extraction with dichloromethane. Four additional Salvia species (Salvia officinalis, Salvia guaranitica, Salvia splendens, and Salvia nemorosa) were extracted using this procedure, and all extracts were analyzed by gas chromatography–mass spectrometry. Differentiation of S. divinorum from other Salvia species was successful based on visual assessment of the resulting chromatograms. To provide a more objective comparison, the total ion chromatograms (TICs) were subjected to principal components analysis (PCA). Prior to PCA, the TICs were subjected to a series of data pretreatment procedures to minimize non-chemical sources of variance in the data set. Successful discrimination of S. divinorum from the other four Salvia species was possible based on visual assessment of the PCA scores plot. To provide a numerical assessment of the discrimination, a series of statistical procedures such as Euclidean distance measurement, hierarchical cluster analysis, Student’s t tests, Wilcoxon rank-sum tests, and Pearson product moment correlation were also applied to the PCA scores. The statistical procedures were then compared to determine the advantages and disadvantages for forensic applications.
Keywords: Controlled substance identification; Salvia divinorum ; Salvinorin A; Salvia species; Multivariate statistical procedures; Principal components analysis

Salvia divinorum is a plant material that is of forensic interest due to the hallucinogenic nature of the active ingredient, salvinorin A. In this study, S. divinorum was extracted and spiked onto four different plant materials (S. divinorum, Salvia officinalis, Cannabis sativa, and Nicotiana tabacum) to simulate an adulterated sample that might be encountered in a forensic laboratory. The adulterated samples were extracted and analyzed by gas chromatography–mass spectrometry, and the resulting total ion chromatograms were subjected to a series of pretreatment procedures that were used to minimize non-chemical sources of variance in the data set. The data were then analyzed using principal components analysis (PCA) to investigate association of the adulterated extracts to unadulterated S. divinorum. While association was possible based on visual assessment of the PCA scores plot, additional procedures including Euclidean distance measurement, hierarchical cluster analysis, Student’s t tests, Wilcoxon rank-sum tests, and Pearson product moment correlation were also applied to the PCA scores to provide a statistical evaluation of the association observed. The advantages and limitations of each statistical procedure in a forensic context were compared and are presented herein.
Keywords: Controlled substance identification; Salvia divinorum ; Salvinorin A; Adulterated samples; Principal components analysis; Statistical procedures

The detection and confirmation of cannabinoids in oral fluid are important in forensic toxicology. Currently, the presence of Δ9-tetrahydrocannabinol (THC) is used for the detection of cannabis in oral fluid. A low concentration of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) is found in oral fluid, which suggested a convenient and low-sensitivity confirmation assay can be used in a routine forensic laboratory. In this study, a highly sensitive isotope dilution liquid chromatography–tandem mass spectrometry method following dansylation was successfully developed for simultaneous determination of THC and THC-COOH in oral fluid. The dansylated derivatives dramatically demonstrated and enhanced the sensitivity of THC and THC-COOH. To avoid signal influenced by the matrix, a 5-min liquid chromatography gradient program was evaluated and optimized, which reduced the sample diffusion and caused sharp peaks (less than 12 s) and thus helped to achieve detection at a low level. The sensitivity, accuracy, and precision were also evaluated, and high quantitative accuracy and precision were obtained. The limit of quantitation of this approach was 25 pg/mL for THC and 10 pg/mL for THC-COOH in oral fluid. Finally, the method was successfully applied to eight suspected cannabis users. Among them, in six oral fluid samples THC-COOH was determined at a concentration from 13.1 to 47.2 pg/mL.
Keywords: Forensics; Toxicology, Clinical; Biomedical analysis, Mass spectrometry; Inductively coupled plasma mass spectrometry

Determination of elemental fingerprint profile of 40 commercial beer samples was performed using inductively coupled plasma–mass spectrometry combined with principal component analysis and receiver operating characteristic (ROC) analysis. Fourteen trace elements, 51V, 52Cr, 59Co, 60Ni, 75As, 82Se, 95Mo, 111Cd, 115In, 121Sb, 133Cs, 208Pb, 209Bi, and 238U, were monitored. All 40 beer samples are distinguishable by using the proposed method. ROC analysis showed that individual beer samples can be correctly identified via the magnitude of its correlation coefficient with respect to the other beers with low false positive rate. The obtained results suggested that the elemental fingerprint technique is feasible for sample differentiation and comparison.
Keywords: Elemental fingerprint; Inductively coupled plasma-mass spectrometry (ICP-MS); Beer; Principal component analysis (PCA); Receiver operating characteristic (ROC)

Iron topochemistry and surface reactivity of amphibole asbestos: relations with in vitro toxicity by Alessandro Pacella; Giovanni B. Andreozzi; Jeanine Fournier; Lorenzo Stievano; Federica Giantomassi; Guendalina Lucarini; Maria Rita Rippo; Armanda Pugnaloni (871-881).
Chemical reactivity of asbestos tremolite from Italy and USA localities and Union Internationale Contre le Cancer (UICC) crocidolite was studied in relation to Fe content, oxidation state, and structural coordination. Direct correlation between amount of Fe2+ at the exposed M(1) and M(2) sites of the amphibole structure and fiber chemical reactivity was established. The in vitro toxicity of the same samples was investigated on human alveolar A549 cell line. Relationship between crystal-chemical features and cell toxicity is not straightforward. UICC crocidolite has Fe content and chemical reactivity largely higher than that of tremolite samples, but all show comparable in vitro toxic potential. Results obtained evidenced that Fe topochemistry is not a primary factor for induced cell toxicity, though it accounts for asbestos chemical reactivity (and possibly genotoxicity).
Keywords: Asbestos amphiboles; Fe topochemistry; Surface reactivity; Lipid peroxidation; In vitro toxicity

A new methodology for precise cadmium isotope analyses of seawater by Zichen Xue; Mark Rehkämper; Maria Schönbächler; Peter J. Statham; Barry J. Coles (883-893).
Previous studies have revealed considerable Cd isotope fractionations in seawater, which can be used to study the marine cycling of this micronutrient element. The low Cd concentrations that are commonly encountered in nutrient-depleted surface seawater, however, pose a particular challenge for precise Cd stable isotope analyses. In this study, we have developed a new procedure for Cd isotope analyses of seawater, which is suitable for samples as large as 20 L and Cd concentrations as low as 1 pmol/L. The procedure involves the use of a 111Cd–113Cd double spike, co-precipitation of Cd from seawater using Al(OH)3, and subsequent Cd purification by column chromatography. To save time, seawater samples with higher Cd contents can be processed without co-precipitation. The Cd isotope analyses are carried out by multiple collector inductively coupled plasma mass spectrometry (MC-ICP-MS). The performance of this technique was verified by analyzing multiple aliquots of a large seawater sample that was collected from the English Channel, the SAFe D1 seawater reference material, and several samples from the GEOTRACES Atlantic intercalibration exercise. The overall Cd yield of the procedure is consistently better than 85% and the methodology can routinely provide ε 114/110Cd data with a precision of about ±0.5 ε (2sd, standard deviation) when at least 20–30 ng of natural Cd is available for analysis. However, even seawater samples with Cd contents of only 1–3 ng can be analyzed with a reproducibility of about ±3 to ±5 ε. A number of experiments were furthermore conducted to verify that the isotopic results are accurate to within the quoted uncertainty.
Keywords: Isotopes; Stable isotopes; Mass fractionation; Geochemistry; Marine geochemistry; Chemical oceanography

On the basis of flow injection analysis technology, a simple, accurate, and sensitive method has been developed for the determination of volatile phenols in environmental water samples by using CdTe/ZnSe nanocrystals as a fluorescent probe. The influences of coexisting metal ions and volatile phenol substitutes were also investigated. The method developed for analysis of volatile phenols displayed very good linearity in the range from 1.0 × 10−8 to 4.0 × 10−7 g L−1, with a correlation coefficient greater than 0.995 and a detection limit down to 2.7 × 10−9 g L−1 (signal-to-noise ratio 3). The proposed method was successfully applied to determine the content of volatile phenols in environmental water samples, and the quantitative recoveries were 93.4–106.1%. A possible reaction mechanism for the quenching of fluorescence is discussed using UV–vis absorption spectra, fluorescence spectra, and time-resolved luminescence spectra of volatile phenols obtained by titrating a CdTe/ZnSe nanocrystal aqueous solution and zeta potential data.
Keywords: Volatile phenols; CdTe/ZnSe; Fluorescent probe; Flow injection analysis

Combining different analytical approaches to identify odor formation mechanisms in polyethylene and polypropylene by Helene Hopfer; Nina Haar; Wolfgang Stockreiter; Christian Sauer; Erich Leitner (903-919).
In a previous study, we identified carbonyls as highly odor-active compounds in both unprocessed and processed polypropylene (PP) with higher intensities after processing, indicating a temperature-driven forming mechanism. In the presented work, we studied whether (a) these carbonyls are the major odor drivers to the overall odor of polyolefins, (b) their formation is taking place already at moderate temperatures well below the typical processing temperatures, (c) conventional antioxidants in polyolefins can prevent or reduce their formation, and (d) whether reducing the amount of oxygen present can decrease the overall odor. One polyethylene (PE) and one PP were selected, and both stabilized and unstabilized polymer powder samples were exposed to conditions differing in oxygen concentration and aging time. The changes in the volatile fraction as well as the formation of odor-active compounds were monitored using a multidisciplinary approach by combining analytical methods based on gas chromatography (GC), multivariate data analysis, and sensory methods (GC–olfactometry and a sensory panel). Both investigated materials (PE and PP) showed similar degradation products (aldehydes, ketones, carboxylic acids, alcohols, and lactones) which increased dramatically with increasing aging time and the lack of stabilization. Oxidation products, mainly carbonyl compounds, were responsible for the odor of the investigated materials. The main odor drivers were unsaturated ketones and aldehydes with a chain length between six and nine C-atoms. Interestingly, similar odor patterns were found for both stabilized and unstabilized samples, indicating that similar formation processes take place independent of the stabilization. Figure 1 Combining analytical and sensory odor analysis methods
Keywords: Odor; Polyethylene (PE); Polypropylene (PP); Gas chromatography–olfactometry (GC-O); Gas chromatography–mass spectrometry (GC-MS); Headspace solid-phase microextraction (HS-SPME)

The formation of volatile products during the reaction of As(iii), As(v), MeAsO(OH)2, and Me2AsO(OH) with aqueous NaBH4 has been investigated, and the formation of arsanes, diarsanes, and triarsanes has been detected. The presence of triarsanes is reported here for the first time. Diarsanes and triarsanes are likely formed in condensation cascade reactions, whereas trimethylarsane arises via the transfer of a methyl group. The formation of volatile by-products is considerably reduced by increasing the acidity of the medium and the concentration of NaBH4 or by the addition of thiols, such as cysteine. A reaction scheme is proposed which reconciles the evidence reported herein and elsewhere in the literature that is valid for both analytical (trace analysis) and non-analytical reaction conditions. Figure Condensation cascade and methyl transfer reactions taking place during the hydride generation of As-compounds under non-analytical conditions. They are originated by the interaction of As reaction intermdiates, among them, and with the other As species containing As-H and As-OH bonds.
Keywords: Arsenic; Monomethyarsonic acid; Dimethylarsinic acid; Tetrahydridoborate; Diarsanes; Triarsanes

Determination of nicotine in mushrooms by various GC/MS- and LC/MS-based methods by A. Lozano; M. A. Martínez-Uroz; M. J. Gómez-Ramos; M. M. Gómez-Ramos; M. Mezcua; A. R. Fernández-Alba (935-943).
Due to the basic properties of nicotine, it is not easily integrated into commonly used multiresidue methods. The present work investigates the application of two commonly employed multiresidue methods—the QuEChERS method and the ethyl acetate method—for determining nicotine in mushrooms. Both methods are employed in a modified form and an unmodified form: the former to address the special properties of nicotine and the latter, combined with the use of isotopically labelled nicotine, to compensate for poor recoveries. The QuEChERS-based methods were followed by liquid chromatography–time-of-flight mass spectrometry and those based on ethyl acetate extraction were followed by gas chromatography–triple quadrupole-mass spectrometry. All methods were validated according to European guidelines (document no. SANCO/10684/2009). Recovery studies performed on mushroom spiked at 10 and 100 μg kg−1 yielded average recoveries in the range 80–110% with relative standard deviation (RSD) values below 9%. The linearity of the response over two orders of magnitude was demonstrated (r 2 > 0.995) for all of the determination techniques employed. The limits of detection and quantification obtained were in the 0.7 and 10 μg kg−1 range, depending on the technique, and thus below the maximum residue level established for this toxic alkaloid by current EU legislation. Good repeatability and reproducibility were obtained in terms of the RSD of the analytical methods (0.4–13.2%). The modified QuEChERS method was tested in a proficiency test on nicotine in dried mushrooms obtaining good results. The methods were successfully applied to 20 real samples.
Keywords: Nicotine in mushrooms; Liquid chromatography; Gas chromatography; QuEChERS; Ethyl acetate method; Validation

The electrochemical detection of sugars is favored by the electrocatalytic effects of copper and nickel, which allow their detection with high sensitivity and without the need for derivatization. Also, novel nanotechnological tools known as metallic nanowires (NWs) may offer new possibilities for sensing, since their large active surfaces allow greatly enhanced sensitivity. In this work, nickel (NiNWs) and nickel-copper (Ni-CuNWs 50:50 w/w) nanowires were synthesized by electroplating using an alumina template, and characterized by scanning electronic microscopy (SEM), transmission electronic microscopy (TEM), and energy-dispersive X-ray spectrometry (EDS). Using an alumina template of 200 nm, and under the controlled deposition of 45 C of charge, both of these NWs were obtained with lengths of about 7 μm and widths of about 300 μm. EDS analysis confirmed that the percentage of Ni in the NiNWs was 97%, and the percentages of Ni and Cu in the Ni-CuNWs were 50.6% and 49.4%, respectively. Both of the electrosynthesized NW exhibited enhanced electrocatalytic properties (and therefore improved detection sensitivity) toward the target sugars. Analytical performance was then evaluated by analyzing ten target honeys, utilizing a simplified calibration strategy that employed a glucose:fructose mixture as the standard. Repeatability was excellent when calibration (RSDs ≤ 5%) and sample analysis (RSDs ≤ 6%) protocols were performed. The total sugar content obtained by the electrochemical approach was compared with that obtained using a HPLC-RI (refractive index) method. The novel method gave low systematic errors, revealing that it afforded very accurate results. Also, a high throughput was achieved, as 60 samples were processed in about 25 min (yielding a rate of 2 samples min−1) on a board containing just one electrode, without any loss of performance. While both electrosynthesized NWs exhibited good analytical performance, the NiNWs were judged to be the most suitable for use in a sensor because of their easy preparation, stability, and magnetic properties. Nanowire-based disposable screen-printed detectors that are strategically connected to miniaturized instrumentation represent a novel approach that could be used in standard labs.
Keywords: Electroanalytical methods; Foods; Beverages; Nanoparticles; Nanotechnology

Purge and trap with monolithic sorbent for gas chromatographic analysis of pesticides in honey by Orapin Chienthavorn; Kanittha Dararuang; Anupop Sasook; Narumol Ramnut (955-964).
Polydivinylbenzene (P-DVB) and silica monolithic materials were synthesised in capillaries and then used to adsorb nine organonitrogen pesticides extracted from honey. After adsorption, each monolith-capillary was connected to a gas chromatograph (GC) with nitrogen-phosphorous detector (NPD). The silica monolith gave better peak shape and intensity than the P-DVB monolith. Formulation of silica monolith with tetramethoxysilane (TMOS) as a sole initiator provided better separation and higher peak intensity, compared to those with methyltrimethoxysilane (MTMS) and (3:1) TMOS/MTMS initiators. The optimum internal diameter and length of silica monolith-capillary were 0.25 mm and 5 cm, respectively. The purge and trap conditions were optimised by purging with nitrogen and heating the honey sample solution at 100 °C for 60 min. Recovery of the method was in a range of 84.95–99.71% and the detection limits of the pesticides determined by using GC-NPD and GC-mass spectrometric detector (MSD) ranged between 0.36–1.75 and 0.13–0.25 ng g−1, respectively.
Keywords: Purge and trap; Monolithic sorbent; Pesticide; Honey

A novel, simple, and rapid reversed-phase liquid chromatography–tandem mass spectrometric methodology was developed for the analysis of natamycin in wine samples. Natamycin was protonated to form singly charged ions in an electrospray positive ion mode. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of three fragment ion transitions (666.3 → 648.2, 666.3 → 503.3, and 666.3 → 485.2) to provide a high degree of sensitivity and specificity. Chromatographic separation was performed on a rapid resolution column using a mobile phase consisting of an acetonitrile/water mixture with a total run time of 5.0 min. After only filtration as pretreatment, the sample was injected into the chromatographic system. The proposed method was validated in terms of selectivity, trueness, precision, decision limit (CCα), and detection capability (CCβ) according to 2002/657/EC Commission decision. The values for trueness, reported as bias (%), agreed with those established by the aforementioned document. Repeatability (intraday variability) values were 12.37% at a concentration of 1.0 μg L−1 and 8.99–4.19% at concentrations between 2.5 and 10 μg L−1. The overall within-laboratory (interday variability) reproducibility was 15.47% at a concentration of 1.0 μg L−1, which was significantly lower than the indicative value reported in the EU decision. The results indicated that the proposed approach is a sensitive, fast, reproducible, and robust methodology suitable for the analysis of natamycin levels in wine samples.
Keywords: Natamycin; Wines; LC-MS/MS; 2002/657/EC European decision

On the stability of the bioactive flavonoids quercetin and luteolin under oxygen-free conditions by Šárka Ramešová; Romana Sokolová; Ilaria Degano; Jana Bulíčková; Ján Žabka; Miroslav Gál (975-982).
The natural flavonoid compounds quercetin (3,3′,4′,5,7-pentahydroxyflavone) and luteolin (3′,4′,5,7-tetrahydroxyflavone) are important bioactive compounds with antioxidative, anti-allergic, and anti-inflammatory properties. However, both are unstable when exposed to atmospheric oxygen, which causes degradation and complicates their analytical determinations. The oxidative change of these flavonoids was observed and followed by UV–visible spectrophotometry, both in aqueous and ethanolic solutions. The distribution of the degradation products in aqueous media was monitored by LC–MS and LC–DAD analysis. The amounts of oxidative reaction products increase with the exposure time. The oxidative degradation reduces the pharmacological efficiency of these antioxidants and renders analytical determination inaccurate. The oxidative changes in flavonoid test solutions can explain the inconsistent dissociation constants reported in the literature. Dissociation constants of quercetin and luteolin were determined both by alkalimetric titration and by UV–visible spectrophotometry under deaerated conditions. The values pK 1 = 5.87 ± 0.14 and pK 2 = 8.48 ± 0.09 for quercetin, and pK 1 = 5.99 ± 0.32 and pK 2 = 8.40 ± 0.42 for luteolin were found. Figure The change of absorption spectra of quercetin during the exposure to the air oxygen
Keywords: Flavonoids; Antioxidants; Stability; Oxidation; Dissociation constant

Raman-spectroscopy-based noninvasive microanalysis of native lignin structure by Pradeep N. Perera; Martin Schmidt; Vincent L. Chiang; P. James Schuck; Paul D. Adams (983-987).
A new robust, noninvasive, Raman microspectroscopic method is introduced to analyze the structure of native lignin. Lignin spectra of poplar, Arabidopsis, and Miscanthus were recovered and structural differences were unambiguously detected. Compositional analysis of 4-coumarate-CoA ligase suppressed transgenic poplar showed that the syringyl-to-guaiacyl ratio decreased by 35% upon the mutation. A cell-specific compositional analysis of basal stems of Arabidopsis showed similar distributions of S and G monolignols in xylary fiber cells and interfascicular cells. Figure Estimated Raman spectra of lignin of Arabidopsis (blue), poplar (red), and Miscanthus (black) with spectral entropy minimization. Raman peaks of Guaiacial (G), Syringyl (S) and Hydroxyphenyl (H) units are indicated.
Keywords: Lignin; Entropy minimization; Chemometrics; Cell wall; Monolignol; Raman

A new strategy for the development of monoclonal antibodies for the determination of human procalcitonin in serum samples by Elisabeth Kremmer; Kathleen Meyer; Friedrich A. Grässer; Andrew Flatley; Miwako Kösters; Peter B. Luppa; Petra M. Krämer (989-995).
Procalcitonin (PCT)—a diagnostic serum parameter for bacterial infection and sepsis—is of great interest in the field of biosensors for point-of-care testing. Its detection needs specific biological recognition elements, such as antibodies. Herein, we describe the development and characterization of rat monoclonal antibodies (mAbs) for PCT, and their application in enzyme-linked immunosorbent assays (ELISAs) for the determination of PCT in patient serum samples. From about 50 mAbs, two mAbs, CALCA 2F3 and CALCA 4A6, were selected as a pair with high affinity for PCT in sandwich immunoassays. Both mAbs could be used either as capture or as detection mAb. They were Protein G-purified and biotinylated when used as detection mAb. The setup of two sandwich ELISAs with standards of human recombinant (hr) PCT, using either CALCA 2F3 (assay A) or CALCA 4A6 (assay B) as capture mAbs and the biotinylated mAbs CALCA 4A6 or CALCA 2F3, respectively, as detection mAbs, led to highly specific determinations of PCT without cross-reactivity to calcitonin and katacalcin. Test midpoints (IC50) of both assays were determined for hrPCT standards in 4% (w/v) human serum albumin and found with 2.5 (assay A) and 2.7 μg L−1 (assay B). With both sandwich ELISAs a collection of eight patient serum samples have been determined in comparison to the determination by the Elecsys BRAHMS PCT assay. Good correlations between our prototype ELISAs and the BRAHMS assay could be demonstrated (R 2: assay A, 0.996 and assay B, 0.990). The use of these newly developed anti-PCT mAbs should find broad applications in immunosensors for point-of-care diagnostics of sepsis and systemic inflammation processes.
Keywords: Human procalcitonin; Katacalcin; Calcitonin; Monoclonal antibody; Sandwich ELISA; Serum samples