Analytical and Bioanalytical Chemistry (v.408, #7)

Mohr’s method challenge by Juris Meija; Anna Maria Michałowska-Kaczmarczyk; Tadeusz Michałowski (1721-1722).

Solution to doubling spectroscopy challenge by Reinhard Meusinger (1723-1724).

Analytical applications of biomimetic recognition elements by María Cruz Moreno-Bondi; Elena Benito-Peña (1725-1726).
received her Ph.D. in Analytical Chemistry at Complutense University (UCM) of Madrid (Spain) in 1990. In 2008 she was promoted to full professor at the Department of Analytical Chemistry of UCM. She has carried out several research stages as a research fellow and as visiting professor at Columbia University (New York, 1988/89 and 2013), the Naval Research Lab (Washington DC, 2015), the Oak Ridge National Lab (Tennessee, 1997), and the Karl-Franzens University (Austria, 1989), working on the characterization of dendrimers as well as on the development of fluorescent based optical fiber sensors, biosensors, and microarrays. She has received the Young Researcher’s Award from the Spanish Society of Analytical Chemistry in 1993 and the Research Award in Analytical Chemistry from the Royal Spanish Society of Chemistry in 2010. Her research interests lie in the development of optical chemical sensors and biosensors and their applications to environmental and food analysis as well as in the development of molecularly imprinted polymers for sensing and separation purposes. She is currently Department Chair of the Analytical Chemistry Department at UCM, President of the Society of Applied Spectroscopy, and co-leader of the Chemical Optosensors and Applied Photochemistry Group (GSOLFA). She serves as a member of the International Advisory Board of the journal Analytical and Bioanalytical Chemistry and in the Permanent Steering Committee of ASCOS (“Advanced Study Courses on Optical Sensors”). received her Ph.D. in Chemistry from Complutense University (Madrid) in 2006. In 2005 she moved to the private sector, working at the Chromatography Lab of Interlab Group (now Labaqua). In 2006, she returned to Complutense University, where she is currently a Postdoctoral Research Associate in the GSOLFA group at the Department of Analytical Chemistry. In 2009 she was awarded a Postdoctoral fellowship from the Spanish Government (MEC-FECYT) to work with Professor David Walt at Tufts University (2009–2011) in the development of multiplexed optical fiber bundle-based biosensor microarrays and InfoBiology. Her research interests are optical sensors and biosensors, microarray biosensing platforms, synthesis of biomimetic elements for sensor and separation purposes, and their application to food, clinical, and environmental analysis.

Molecular imprinting is a very powerful synthetic method for preparation of robust materials with pre-designed molecular selectivity. Molecularly imprinted polymers (MIPs) are attractive substitutes for antibodies in many analytical and bioanalytical applications, e.g. for development of biosensors and for drug assays. In addition to selective molecular binding, new functions are being added to MIPs to make the synthetic materials responsive to different environmental conditions, making it possible to modulate the binding and release of different molecular targets and to simplify affinity separation. Introduction of signal-transduction functionality into MIPs also brings in new, more easily operated chemical sensors for detection and quantification of important analytical targets. Graphical Abstract Multi-functional molecularly imprinted polymers
Keywords: Molecular imprinting; Multi-functionality; Nanoparticle; Composite material; Controlled radical polymerization

Analytical applications of MIPs in diagnostic assays: future perspectives by Thomas S. Bedwell; Michael J. Whitcombe (1735-1751).
Many efforts have been made to produce artificial materials with biomimetic properties for applications in binding assays. Among these efforts, the technique of molecular imprinting has received much attention because of the high selectivity obtainable for molecules of interest, robustness of the produced polymers, simple and short synthesis, and excellent cost efficiency. In this review, progress in the field of molecularly imprinted sorbent assays is discussed—with a focus on work conducted from 2005 to date. Graphical Abstract A growing trend in recent years has been the use of molecularly imprinted polymers as replacements for antibodies in various assay formats, as indicated by a steady increase in publications in the area (see graph)
Keywords: Molecularly imprinted polymers; Molecularly imprinted sorbent assay; Immunoassay; Radioassay; Fluoroimmunoassay; BELISA

Molecularly imprinted polymers (MIPs) are potent and established recognition phases in separation and enrichment applications. Because of their robustness, versatility and format adaptability, they also constitute very promising sensing phases, especially when the active sensing element is directly integrated into the MIP. Fluorescent MIPs incorporating fluorescent monomers are perhaps the best developed and most successful approach here. This article reviews the state of the art in this field, discussing the pros and cons of the use of fluorescent dye and probe derivatives as such monomers, the different molecular interaction forces for template complexation, signalling modes and a variety of related approaches that have been realized over the years, including Förster resonance energy transfer processes, covalent imprinting, postmodification attachment of fluorescent units and conjugated polymers as MIPs; other measurement schemes and sensing chemistries that use MIPs and fluorescence interrogation to solve analytical problems (fluorescent competitive assays, fluorescent analytes, etc.) are not covered here. Throughout the article, photophysical processes are discussed to facilitate understanding of the effects that can occur when one is planning for a fluorescence response to happen in a constrained polymer matrix. The article concludes with a concise assessment of the suitability of the different formats for sensor realization.
Keywords: Chemical sensors; Fluorescence; Molecularly imprinted polymers; Dyes; Sol–gel

Plasmonic SERS biosensing nanochips for DNA detection by Hoan T. Ngo; Hsin-Neng Wang; Andrew M. Fales; Tuan Vo-Dinh (1773-1781).
The development of rapid, cost-effective DNA detection methods for molecular diagnostics at the point-of-care (POC) has been receiving increasing interest. This article reviews several DNA detection techniques based on plasmonic-active nanochip platforms developed in our laboratory over the last 5 years, including the molecular sentinel-on-chip (MSC), the multiplex MSC, and the inverse molecular sentinel-on-chip (iMS-on-Chip). DNA probes were used as the recognition elements, and surface-enhanced Raman scattering (SERS) was used as the signal detection method. Sensing mechanisms were based on hybridization of target sequences and DNA probes, resulting in a distance change between SERS reporters and the nanochip’s plasmonic-active surface. As the field intensity of the surface plasmon decays exponentially as a function of distance, the distance change in turn affects SERS signal intensity, thus indicating the presence and capture of the target sequences. Our techniques were single-step DNA detection techniques. Target sequences were detected by simple delivery of sample solutions onto DNA probe-functionalized nanochips and measuring the SERS signal after appropriate incubation times. Target sequence labeling or washing to remove unreacted components was not required, making the techniques simple, easy-to-use, and cost-effective. The usefulness of the nanochip platform-based techniques for medical diagnostics was illustrated by the detection of host genetic biomarkers for respiratory viral infection and of the dengue virus gene.
Keywords: DNA detection; Multiplex DNA detection; DNA biosensor; Surface-enhanced Raman scattering; Molecular sentinel-on-chip

Recent advances in biosensing using magnetic glyconanoparticles by Raluca M. Fratila; María Moros; Jesús M. de la Fuente (1783-1803).
In this critical review we discuss the most recent advances in the field of biosensing applications of magnetic glyconanoparticles. We first give an overview of the main synthetic routes to obtain magnetic-nanoparticle–carbohydrate conjugates and then we highlight their most promising applications for magnetic relaxation switching sensing, cell and pathogen detection, cell targeting and magnetic resonance imaging. We end with a critical perspective of the field, identifying the main challenges to be overcome, but also the areas where the most promising developments are likely to happen in the coming decades.
Keywords: Magnetic glyconanoparticles; Magnetic relaxation switching; Biosensing; Carbohydrates; Nanotechnology

Application of bacteriophages in sensor development by Riikka Peltomaa; Irene López-Perolio; Elena Benito-Peña; Rodrigo Barderas; María Cruz Moreno-Bondi (1805-1828).
Bacteriophage-based bioassays are a promising alternative to traditional antibody-based immunoassays. Bacteriophages, shortened to phages, can be easily conjugated or genetically engineered. Phages are robust, ubiquitous in nature, and harmless to humans. Notably, phages do not usually require inoculation and killing of animals; and thus, the production of phages is simple and economical. In recent years, phage-based biosensors have been developed featuring excellent robustness, sensitivity, and selectivity in combination with the ease of integration into transduction devices. This review provides a critical overview of phage-based bioassays and biosensors developed in the last few years using different interrogation methods such as colorimetric, enzymatic, fluorescence, surface plasmon resonance, quartz crystal microbalance, magnetoelastic, Raman, or electrochemical techniques.
Keywords: Bacteriophage; Phage display technology; Biosensing; Biosensor; Biorecognition element

Affinity interactions of human immunoglobulin G with short peptides: role of ligand spacer on binding, kinetics, and mass transfer by Fei Shen; Orlando J. Rojas; Jan Genzer; Patrick V. Gurgel; Ruben G. Carbonell (1829-1841).
The interaction affinity between human IgG and a short peptide ligand (hexameric HWRGWV) was investigated by following the shifts in frequency and energy dissipation in a quartz crystal microbalance (QCM). HWRGWV was immobilized by means of poly(ethylene glycol) tethered on QCM sensors coated with silicon oxide, which enhanced the accessibility of the peptide to hIgG and also passivated the surface. Ellipsometry and ToF-SIMS were employed for surface characterization. The peptide ligand density was optimized to 0.88 chains nm−2, which enabled the interaction of each hIgG molecule with at least one ligand. The maximum binding capacity was found to be 4.6 mg m−2, corresponding to a monolayer of hIgG, similar to the values for chromatographic resins. Dissociation constants were lower than those obtained from resins, possibly due to overestimation of bound mass by QCM. Equilibrium thermodynamic and kinetic parameters were determined, shedding light on interfacial effects important for detection and bioseparation. Graphical Abstract The interaction affinity between human IgG and a short peptide ligand was investigated by using quartz crystal microgravimetry, ellipsometry and ToF-SIMS. Equilibrium thermodynamic and kinetics parameters were determined, shedding light on interfacial effects important for detection and bioseparation.
Keywords: Peptide ligands; Immunoglobulin G; Peptide HWRGWV; Spacer arms; Quartz crystal microbalance

Molecularly imprinted polymer beads for clean-up and preconcentration of β-lactamase-resistant penicillins in milk by Javier L. Urraca; Raquel Chamorro-Mendiluce; Guillermo Orellana; Maria C. Moreno-Bondi (1843-1854).
This work describes the development and application of class-selective molecularly imprinted polymers (MIPs) for the analysis of beta-lactamase-resistant penicillins, namely cloxacillin (CLOXA), oxacillin (OXA), and dicloxacillin (DICLOXA), in milk samples. Our method is based on molecularly imprinted solid-phase extraction (MISPE) coupled to high-performance liquid chromatography (HPLC) with diode-array detection (DAD). 2-Biphenylylpenicillin (2BPEN), a surrogate with a close resemblance to beta-lactamase-resistant penicillins in terms of size, shape, hydrophobicity, and functionality, was synthesized and used as the template for the polymer synthesis. A MIP library was prepared and screened to select the optimum functional monomer, N-(2-aminoethyl)methacrylamide, and cross-linker, trimethylolpropane trimethacrylate, that provided the best recognition for the target antibiotics. For the MISPE application, the MIPs were prepared in the form of microspheres, using porous silica beads (40–75 μm) as sacrificial scaffolds. The developed MISPE method enables efficient extraction from aqueous samples and analysis of the antimicrobials, when followed by a selective washing with 2 mL acetonitrile–water (20:80 v/v) and elution with 1 mL 0.05 mol L−1 tetrabutylammonium in methanol. The analytical method was validated according to EU guideline 2002/657/EC. The limits of quantification (S/N = 10) were in the 5.3–6.3 μg kg−1 range, well below the maximum residue limits (MRLs) currently established. Inter-day mean recoveries were in the range 99–102 % with RSDs below 9 %, improving on the performance of previously reported MISPE methods for the analysis of CLOXA, OXA, or DICLOXA in milk samples.
Keywords: Penicillin; Molecularly imprinted polymers; Surrogate template; MISPE; Milk; Method validation

Smart coumarin-tagged imprinted polymers for the rapid detection of tamoxifen by Judith V. Ray; Fosca Mirata; Celine Pérollier; Michel Arotcarena; Sami Bayoudh; Marina Resmini (1855-1861).
A signalling molecularly imprinted polymer was synthesised for easy detection of tamoxifen and its metabolites. 6-Vinylcoumarin-4-carboxylic acid (VCC) was synthesised from 4-bromophenol to give a fluorescent monomer, designed to switch off upon binding of tamoxifen. Clomiphene, a chlorinated analogue, was used as the template for the imprinting, and its ability to quench the coumarin fluorescence when used in a 1:1 ratio was demonstrated. Tamoxifen and 4-hydroxytamoxifen were also shown to quench coumarin fluorescence. Imprinted and non-imprinted polymers were synthesised using VCC, methacrylic acid as a backbone monomer and ethylene glycol dimethacrylate as cross-linker, and were ground and sieved to particle sizes ranging between 45 and 25 μm. Rebinding experiments demonstrate that the imprinted polymer shows very strong affinity for both clomiphene and tamoxifen, while the non-imprinted polymer shows negligible rebinding. The fluorescence of the imprinted polymer is quenched by clomiphene, tamoxifen and 4-hydroxytamoxifen. The switch off in fluorescence of the imprinted polymer under these conditions could also be detected under a UV lamp with the naked eye, making this matrix suitable for applications when coupled with a sample preparation system.
Keywords: Molecularly imprinted polymer; Tamoxifen; 6-Vinylcoumarin-4-carboxylic acid; Clomiphene

A simple and rapid method for detection of potassium ion (K+) based on a guanine chemiluminescence (CL) system is presented. In this system, one guanine-rich DNA molecule is used as the recognition element. K+ can cause the guanine-rich DNA to form a G-quadruplex conformation, resulting in remarkable quenching of the guanine CL intensity of guanine-rich DNA. The CL intensity of this CL system decreased with increasing K+ concentration, revealing a linear relationship in K+ concentration range from 3 × 10−5 to 1 × 10−3 M. A complete detection process can be accomplished in about 5 min. Other common cations (such as Na+, NH4 +, Mg2+, Ca2+, Zn2+, and Pb2+) did not notably interfere with K+ detection. The mechanism of this strategy is also discussed. The sensing strategy is low cost and simple without the requirement of complex labeling of probe DNA. The scheme is applicable to the detection of other guanine-rich aptamer-binding chemicals or biomolecules. Graphical Abstract A simple and homogenous CL assay of K+ with K+-induced G-quadruplex and guanine CL
Keywords: Guanine; Chemiluminescence; G-quadruplex; Potassium ion

Analysis of saccharides in beverages by HPLC with direct UV detection by Thomas Schmid; Benedikt Baumann; Markus Himmelsbach; Christian W. Klampfl; Wolfgang Buchberger (1871-1878).
The present study demonstrates the suitability of direct UV detection for saccharide analysis in HPLC. Under highly alkaline conditions, the non-UV absorbing saccharides are converted by a photo-initiated chemical reaction in the detection cell into malonenolate, which can be detected at 266 nm. A straightforward method for such direct UV detection of saccharides after their separation by anion-exchange chromatography was developed and successfully applied to several beverage samples. Investigation and optimization of the influencing factors using design of experiment resulted in a baseline separation of glucose, fructose, and sucrose within 6 min and LOD values below 0.2 mg L−1. In addition, a fast, simple and cost-effective flow injection method was developed to estimate the total saccharide concentration. The results of this method applied to beverage samples are in good agreement with the chromatographic method as well as to the saccharide concentration stated by the manufacturer. Finally, a comparison of different commercially available UV detectors and detector cells revealed that sensitive detection requires the use of recently introduced flow cells with extended path length. Graphical Abstract Online direct UV detection of saccharides in HPLC, which is possible due to a photo-initiated conversion of the saccharides into malonenolate
Keywords: Anion-exchange chromatography; Direct UV detection; HPLC; Saccharide

Online solid-phase extraction was combined with nano-liquid chromatography coupled to high-resolution mass spectrometry (HRMS) for the analysis of micropollutants in environmental samples from small volumes. The method was validated in surface water, Microcystis aeruginosa cell lysate, and spent Microcystis growth medium. For 41 analytes, quantification limits of 0.1–28 ng/L (surface water) and 0.1–32 ng/L (growth medium) were obtained from only 88 μL of sample. In cell lysate, quantification limits ranged from 0.1–143 ng/L or 0.33–476 ng/g dry weight from a sample of 88 μL, or 26 μg dry weight, respectively. The method matches the sensitivity of established online and offline solid-phase extraction–liquid chromatography–mass spectrometry methods but requires only a fraction of the sample used by those techniques, and is among the first applications of nano-LC-MS for environmental analysis. The method was applied to the determination of bioconcentration in Microcystis aeruginosa in a laboratory experiment, and the benefit of coupling to HRMS was demonstrated in a transformation product screening.
Keywords: Nano-LC; Environmental analytics; High-resolution MS; Miniaturization; Online SPE; Biological samples

Orbitrap high resolution mass spectrometry (HRMS) with electrospray ionization in both positive and negative polarity was conducted on Suwannee River fulvic acid (SRFA), Pony Lake fulvic acid (PLFA) standards, and dissolved organic matter (DOM) released by freshwater phytoplankton (Scenedesmus obliquus, Euglena mutabilis, and Euglena gracilis). Three-dimensional van Krevelen diagrams expressing various oxygenation states of sulfur molecules and abundance plots of sulfur-containing species were constructed. Orbitrap HRMS analysis of SRFA found a high density of peaks in the lignin region (77 %) and low density of protein material (6.53 %), whereas for PLFA, 25 % of the total peaks were lignin related compared to 56 % of peaks in protein regions, comparable with other HRMS studies. Phytoplankton-derived DOM of S. obliquus, E. mutabilis, and E. gracilis was dominated by protein molecules at respective percentages of 36, 46, and 49 %, and is consistent with previous experiments examining phytoplankton-derived DOM composition. The normalized percentage of SO-containing compounds was determined among the three phytoplankton to be 56 % for Scenedesmus, 54 % for E. mutabilis, and 47 % for E. gracilis, suggesting variation between sulfur content in phytoplankton-derived DOM and differences in metal binding capacities. These results suggest the level of resolution by Orbitrap mass spectrometry is sufficient for preliminary characterization of phytoplankton DOM at an affordable cost relative to other HRMS techniques. Graphical Abstract Euglena mutabilis-derived DOM examined using Orbitrap mass spectrometry (a) with the percentage representing various elemental compositions where blue regions are CHO, orange CHNO, grey CHOS and yellow CHNOS (b). 2D (c) and 3D (d) van Krevelen diagrams display abundant groups with similar elemental ratio properties and various oxygen states of sulfur molecules produced where SH=red, SO=green, SO2=black (d). Double bond equivalence and carbon number provide structural characteristics of sulfur compounds (e), where contour intensities range from 5-30% relative intensity
Keywords: Dissolved organic matter; High resolution mass spectrometry; Phytoplankton; Sulfur; Van Krevelen

Sugar alcohols, such as sorbitol, are commonly used as a replacement for sucrose in the food industry, applied as starting material for vitamin C synthesis, and involved as one of the causative factors in diabetic complications. Therefore, their detection and quantification in aqueous solution are necessary. The reversible covalent interactions between boronic acids and diols are the basis of efficient methods for the detection of saccharides. Herein, we report a new internal charge transfer (ICT) fluorene-based fluorescent boronic acid sensor (1) 2-[(9,9-dimethyl-9H-fluoren-2-yl-amino)methyl] phenyl boronic acid that shows significant fluorescence changes upon addition of saccharides. The boronic acid has high affinity (K a = 1107.9 M−1) and selectivity for sorbitol at pH = 8.31. It showed a linear response toward sorbitol in the concentration range from 1.0 × 10−5 to 6.0 × 10−4 mol L−1 with the detection limit of 7.04 × 10−6 mol L−1. Sensor 1 was used to detect sorbitol in real samples with good recovery. Graphical Abstract A new ICT fluorene-based fluorescent boronic acid sensor (1) that shows high affinity and selectivity for d-sorbitol (K a = 1107.9 M−1) is reported.
Keywords: Boronic acid; Fluorene; Fluorescent sensor; Sugar alcohol

High-throughput miniaturized microfluidic microscopy with radially parallelized channel geometry by Veerendra Kalyan Jagannadh; Bindu Prabhath Bhat; Lourdes Albina Nirupa Julius; Sai Siva Gorthi (1909-1916).
In this article, we present a novel approach to throughput enhancement in miniaturized microfluidic microscopy systems. Using the presented approach, we demonstrate an inexpensive yet high-throughput analytical instrument. Using the high-throughput analytical instrument, we have been able to achieve about 125,880 cells per minute (more than one hundred and twenty five thousand cells per minute), even while employing cost-effective low frame rate cameras (120 fps). The throughput achieved here is a notable progression in the field of diagnostics as it enables rapid quantitative testing and analysis. We demonstrate the applicability of the instrument to point-of-care diagnostics, by performing blood cell counting. We report a comparative analysis between the counts (in cells per μl) obtained from our instrument, with that of a commercially available hematology analyzer.
Keywords: High throughput screening; Clinical / biomedical analysis; Microfluidic biosensors

Comprehensive comparison of the chemical and structural characterization of landfill leachate and leonardite humic fractions by Abdelghani Tahiri; Aurore Richel; Jacqueline Destain; Philippe Druart; Philippe Thonart; Marc Ongena (1917-1928).
Humic substances (HS) are complex and heterogeneous mixtures of organic compounds that occur everywhere in the environment. They represent most of the dissolved organic matter in soils, sediments (fossil), water, and landfills. The exact structure of HS macromolecules has not yet been determined because of their complexity and heterogeneity. Various descriptions of HS are used depending on specific environments of origin and research interests. In order to improve the understanding of the structure of HS extracted from landfill leachate (LHS) and commercial HS from leonardite (HHS), this study sought to compare the composition and characterization of the structure of LHS and HHS using elemental composition, chromatographic (high-performance liquid chromatography (HPLC)), and spectroscopic techniques (UV–vis, FTIR, NMR, and MALDI-TOF). The results showed that LHS molecules have a lower molecular weight and less aromatic structure than HHS molecules. The characteristics of functional groups of both LHS and HHS, however, were basically similar, but there was some differences in absorbance intensity. There were also less aliphatic and acidic functional groups and more aromatic and polyphenolic compounds in the humic acid (HA) fraction than in the fulvic acid (FA) and other molecules (OM) fractions of both origins. The differences between LHS and HHS might be due to the time course of humification. Combining the results obtained from these analytical techniques cold improve our understanding of the structure of HS of different origins and thus enhance their potential use. Graphical Abstract Chemical and structural and characterization of landfill leachate and leonardite humic fractions
Keywords: Humic substances; Landfill leachates; Leonardite; HPLC; UV–vis; FTIR; NMR; MALDI-TOF

Vortex-assisted emulsification semimicroextraction is proposed as a one-step solution-extraction procedure for sample preparation in cosmetic products. The procedure allows rapid preparation based on dispersion of the sample in a mixture of 1 mL of n-hexane and 0.5 mL of ethanol, followed by the addition of 0.5 mL of water and centrifugation to obtain two separated phases. This procedure provides good sample clean-up with minimum dilution and is very useful for the determination of ingredients with restricted concentrations, such as bronopol. The procedure was applied to the determination of bronopol by liquid chromatography with UV detection. The best chromatographic separation was obtained by using a C18 column set at 40 °C and performing a stepwise elution with a mixture of ethanol/aqueous 1 % acetic acid solution as mobile phase pumped at 0.5 mL min−1. The detection wavelength was set at 250 nm and the total run time required was 12 min. The method was successfully applied to 18 commercial cosmetic samples including creams, shampoos, and bath gels. Good recoveries and repeatability were obtained, with a limit of detection of 0.9 μg mL−1, which makes the method suitable for the analytical control of cosmetic products. Moreover, it could be considered environmentally friendly, because water, ethanol, and only a low volume of n-hexane are used as solvents. Graphical Abstract Vortex-assisted emulsification semimicroextraction (VAEsME) for the preparation of cosmetic samples
Keywords: Cosmetic products; Bronopol; Vortex-assisted emulsification; Semimicroextraction; Liquid chromatography

Quantification of homocysteine and cysteine by derivatization with pyridoxal 5'-phosphate and hydrophilic interaction liquid chromatography by Rafał Głowacki; Justyna Stachniuk; Kamila Borowczyk; Hieronim Jakubowski (1935-1941).
A simple and rapid assay using pyridoxal 5'-phosphate (PLP) as a derivatizing reagent was developed for the simultaneous determination of homocysteine (Hcy) and cysteine (Cys) in human plasma. Derivatization with PLP affords UV-absorbing tetrahydrothiazine and thiazolidine derivatives of Hcy and Cys, respectively. Separation of these derivatives was achieved in 5 min using a hydrophilic interaction liquid chromatography, followed by UV detection at 330 nm. Linearity in detector response was observed over the range of 0.25–20 μM for Hcy and 10–300 μM for Cys. The limit of quantification (LOQ) values for Hcy and Cys were 0.25 and 2.5 μM, respectively. The method was successfully applied to plasma samples donated by apparently healthy volunteers. Graphical Abstract A flow chart diagram of analytical procedure for total plasma Hcy and Cys determination
Keywords: Plasma; Cysteine; Homocysteine; Derivatization; Pyridoxal 5'-phosphate; Hydrophilic interaction liquid chromatography

Τwo-panel molecular testing for genetic predisposition for thrombosis using multi-allele visual biosensors by Nikolaos Fountoglou; Margarita Petropoulou; Alexandra Iliadi; Theodore K. Christopoulos; Penelope C. Ioannou (1943-1952).
Thrombosis is considered as the most typical example of multigenic/multifactorial disorder. The three most common genetic risk factors for thrombotic disorders are the G1691A mutation in factor V gene (FV Leiden), the G20210Α mutation in prothrombin gene (FII), and the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. An additional panel of biomarkers predisposing for thrombotic events includes the H1299R variant in factor V gene (HR2), A1298C variant in MTHFR gene, the V34L mutation in fibrinogen stabilizing factor XIII (FXIII) gene as well as the 4G/5G polymorphism in plasminogen activator inhibitor type-1 (PAI-1) gene. In this context, we report a novel, rapid and low-cost two-panel diagnostic platform for the simultaneous visual genotyping of the seven mutations (14 alleles). The proposed method comprises the following: (a) a multiplex PCR using genomic DNA isolated from peripheral blood, (b) a multiplex genotyping reaction based on allele-specific primer extension, and (c) visual detection of the genotyping reaction products by means of a multi-allele dipstick-type DNA biosensor, using gold nanoparticles as reporters. The method was applied to 40, previously characterized, and 15 blind clinical samples and the results were 100 % accurate. The proposed assay is simple to perform, requires no specialized and costly equipment, and eliminates multiple pipetting, incubation, and washing steps. Graphical Abstract Genotyping platform for visual detection of point mutations using PEXT reaction and multi-allele DNA biosensor
Keywords: Thrombophilia; Molecular testing; Primer extension reaction; Multi-allele DNA biosensor

The concentration of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) in whole blood is used as a parameter for assessing the consumption behavior of cannabis consumers. The blood level of THCCOOH-glucuronide might provide additional information about the frequency of cannabis use. To verify this assumption, a column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid and direct quantification of free and glucuronidated THCCOOH in human whole blood was newly developed. The method comprised protein precipitation, followed by injection of the processed sample onto a trapping column and subsequent gradient elution to an analytical column for separation and detection. The total LC run time was 4.5 min. Detection of the analytes was accomplished by electrospray ionization in positive ion mode and selected reaction monitoring using a triple-stage quadrupole mass spectrometer. The method was fully validated by evaluating the following parameters: linearity, lower limit of quantification, accuracy and imprecision, selectivity, extraction efficiency, matrix effect, carry-over, dilution integrity, analyte stability, and re-injection reproducibility. All acceptance criteria were analyzed and the predefined criteria met. Linearity ranged from 5.0 to 500 μg/L for both analytes. The method was successfully applied to whole blood samples from a large collective of cannabis consumers, demonstrating its applicability in the forensic field.
Keywords: THCCOOH; THCCOOH-glucuronide; Column-switching chromatography; LC-MS/MS; Whole blood; DUI

Plasma proteome coverage is increased by unique peptide recovery from sodium deoxycholate precipitate by Aida Serra; Hongbin Zhu; Xavier Gallart-Palau; Jung Eun Park; Hee Haw Ho; James P. Tam; Siu Kwan Sze (1963-1973).
The ionic detergent sodium deoxycholate (SDC) is compatible with in-solution tryptic digestion and LC-MS/MS-based shotgun proteomics by virtue of being easy to separate from the peptide products via precipitation in acidic buffers. However, it remains unclear whether unique human peptides co-precipitate with SDC during acid treatment of complex biological samples. In this study, we demonstrate for the first time that a large quantity of unique peptides in human blood plasma can be co-precipitated with SDC using an optimized sample preparation method prior to shotgun proteomic analysis. We show that the plasma peptides co-precipitated with SDC can be successfully recovered using a sequential re-solubilization and precipitation procedure, and that this approach is particularly efficient at the extraction of long peptides. Recovery of peptides from the SDC pellet dramatically increased overall proteome coverage (>60 %), thereby improving the identification of low-abundance proteins and enhancing the identification of protein components of membrane-bound organelles. In addition, when we analyzed the physiochemical properties of the co-precipitated peptides, we observed that SDC-based sample preparation improved the identification of mildly hydrophilic/hydrophobic proteins that would otherwise be lost upon discarding the pellet. These data demonstrate that the optimized SDC protocol is superior to sodium dodecyl sulfate (SDS)/urea treatment for identifying plasma biomarkers by shotgun proteomics.
Keywords: Plasma proteome; Mass spectrometry; Shotgun proteomics; Sodium deoxycholate; Trypsin digestion

A high-performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (HPLC-FT-ICR MS) method was developed to study the in vivo metabolism of salidroside for the first time. Plasma, urine, bile, and feces samples were collected from male rats after a single intragastric gavage of salidroside at a dose of 50 mg/kg. Besides the parent drug, a total of seven metabolites (three phase I and four phase II metabolites) were detected and tentatively identified by comparing their mass spectrometry profiles with those of salidroside. Results indicated that metabolic pathways of salidroside in male rats included hydroxylation, dehydrogenation, glucuronidation, and sulfate conjugation. Among them, glucuronidation and sulfate conjugation were the major metabolic reactions. And most important, the detection of the sulfation metabolite of p-tyrosol provides a clue for whether the deglycosylation of salidroside occurs in vivo after intragastric gavage. In summary, results obtained in this study may contribute to the better understanding of the safety and mechanism of action of salidroside.
Keywords: Salidroside; HPLC-FT-ICR MS; Metabolism