Analytical and Bioanalytical Chemistry (v.409, #10)

Measurement uncertainty challenge by Juris Meija (2497-2497).

Solution to NMR hide-and-seek challenge by Reinhard Meusinger (2499-2500).

Field flow fractionation (FFF) techniques are used to successfully characterize several nanomaterials by sizing nano-entities and producing information about the aggregation/agglomeration state of nanoparticles. By coupling FFF techniques to specific detectors, researchers can determine particle-size distributions (PSDs), expressed as mass-based or number-based PSDs. This review considers FFF applications in the food, biomedical, and environmental sectors, mostly drawn from the past 4 y. It thus underlines the prominent role of asymmetrical flow FFF within the FFF family. By concisely comparing FFF techniques with other techniques suitable for sizing nano-objects, the advantages and the disadvantages of these instruments become clear. A consideration of select recent publications illustrates the state of the art of some lesser-known FFF techniques and innovative instrumental set-ups.
Keywords: Field flow fractionation; Nanoparticles; Quantum dots; Food additives; Drug delivery systems; Environmental nanoparticles

Determination of true ratios of different N-glycan structures in electrospray ionization mass spectrometry by Clemens Grünwald-Gruber; Andreas Thader; Daniel Maresch; Thomas Dalik; Friedrich Altmann (2519-2530).
is a postdoctoral researcher in the Laboratory for Glycobiology and Analytical Biochemistry at the University of Natural Resources and Life Sciences, Vienna. He is the chief operator of the mass spectrometry equipment of the proteomics core facility of the university’s biotechnology branch. was a doctoral student and a postdoctoral researcher in the working group of F. Altmann at the University of Natural Resources and Life Sciences, Vienna. He specialized in the cloning and expression of glycosyltransferases and their application to generate stable-isotope-labeled N-glycans of various structures. works at the Laboratory for Glycobiology and Analytical Biochemistry at the University of Natural Resources and Life Sciences, Vienna, where his responsibility is to provide mass spectrometric know-how to the participants of a doctoral program in protein biotechnology. is a long-time member of the Laboratory for Glycobiology and Analytical Biochemistry at the University of Natural Resources and Life Sciences, Vienna. His specialty is the preparation of glycopeptides, glycans, and natural enzymes required for the research work of the group. He is responsible for the production of the CCD-blocker that can be used to prevent glycoprotein-based false positives in allergy diagnosis ( http://www.proglycan.com ). is Head of the Laboratory for Glycobiology and Analytical Biochemistry at the University of Natural Resources and Life Sciences, Vienna. His main interests are in advancing the structural analysis of glycoproteins and in the biological impact of protein glycosylation, for example, in the context of allergy diagnosis, where nonhuman glycosylation causes false positive diagnostic results. An ideal method for the analysis of N-glycans would both identify the isomeric structure and deliver a true picture of the relative, if not absolute, amounts of the various structures in one sample. Porous graphitic carbon chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) detection has emerged as a method with a particularly high potential of resolving isomeric oligosaccharides, but little attention has so far been paid to quantitation of the results obtained. In this work, we isolated a range of structures from Man5 to complex type N-glycans with zero to four sialic acids and blended them into an equimolar “glyco tune mix”. When subjected to liquid chromatography–ESI-MS in positive and negative modes, the glyco tune mix clearly demonstrated the futility of quantitation of N-glycans of different overall composition, different number of sialic acids, and strongly differing size without compensation for their very different molar responses. Relative quantitation of human plasma N-glycans was performed with correction factors deduced from this external glyco tune mix. Addition of just one isotope-coded internal standard with enzymatically added 13C-galactose led to absolute quantification in the same experiment. Graphical Abstract Discrepancy between desirable (grey bars) and real (green bars) relative ion abundance of equimolar amounts of glycans in positive mode ESI-MS.
Keywords: N-Glycan; Sialylation; Quantitative glycomics; Mass spectrometry; Electrospray ionization

Although o-quinonemethide (6-methylene-2,4-cyclohexadien-1-one) has been proposed as a key intermediate in char formation during the pyrolysis of guaiacol (2-methoxyphenol), direct evidence of this (e.g., spectroscopic data) has not yet been provided. Using in situ FTIR spectroscopy, the pyrolysis of guaiacol was investigated from 30 °C to 630 °C at 40 °C/min. The IR profiles showed direct evidence of o-quinonemethide production at about 350 °C, which vanished rapidly at around 420 °C in the vapor phase, indicating char formation. In addition, at 400 °C, salicyl aldehyde was observed, which decomposed slowly at about 500 °C. In combination with the known products of guaiacol pyrolysis, these results allowed the major reaction pathways of guaiacol pyrolysis to be discerned. Density functional theory calculations were performed, and the results were found to be in good agreement with the experimentally obtained IR profiles. These findings provide guidance on how to suppress secondary reactions of guaiacol during lignin pyrolysis. Graphical abstract On-line analysis of pyrolysis process of guaiacol using in situ FTIR
Keywords: Guaiacol; o-Quinonemethide; In situ FTIR; Lignin pyrolysis

A “turn-on” fluorescent sensor for ozone using bovine serum albumin-directed gold nanoclusters (BSA-Au NCs) via energy transfer was developed. The spectral overlap of fluorescent spectrum of BSA-Au NCs with absorption spectrum of indigo carmine (IDS) was utilized. Ozone cleaves C = C bond of IDS and suppresses energy transfer from BSA-Au NCs to IDS. Therefore, this proposed fluorescent sensor is a “turn-on” detection motif. It is the first application of fluorescent nanoclusters in sensitively detecting ozone from 0.2 to 12 μM with the limit of detection of 35 nM (the volume of 500 μL, 1.68 ppb). The proposed fluorescent sensor for ozone is more sensitive and faster (within 2 min) than most methods and is with good selectivity for ozone detection against other reactive oxygen species, reactive nitrogen, or metallic ions. Besides, the proposed method is also utlized in ozone detection in ambient air by monitoring 1 h (60 min) in Qijiang district in Chongqing city. The average of concentration of ozone in ambient air ranges from 44.97 to 52.85 μg/m3. The results are compared with the automatic monitoring data provided by Qijiang Environmental Monitoring Station and the relative deviations range, respectively, from 2.1 to 5.6%, which suggests that it is a promising fluorescent sensor for ozone in ambient air. This study not only develops a new model of energy transfer motif using BSA-Au NCs as donor and IDS as acceptor but also expands the application of BSA-Au NCs in environmental science. Graphical abstract A “turn-on” fluorescent sensor for ozone detection using bovine serum albumin-directed gold nanoclusters (BSA-Au NCs) via energy transfer is developed. It is the first time to utilize spectral overlap of fluorescent spectrum of BSA-Au NCs with absorption spectrum of indigo carmine and to achieve fast, sensitive, and selective ozone detection with a limit of detection of down to 35 nM (the volume of 500 μL, 1.68 ppb).
Keywords: Fluorescent sensor; Ozone; Gold nanoclusters; Energy transfer; Indigo carmine

Use of surface plasmon resonance in the binding study of vitamin D, metabolites and analogues with vitamin D binding protein by Pilar Canoa; Marcos L. Rivadulla; Jonathan Popplewell; René van Oosten; Generosa Gómez; Yagamare Fall (2547-2558).
Vitamin D3 and its metabolites are lipophilic molecules with low aqueous solubility and must be transported bound to plasma carrier proteins, primarily to vitamin D binding protein (DBP). The biological functions of vitamin D3 metabolites are intimately dependent on the protein, hence the importance of determining their affinity for DBP. In this study, we developed a novel procedure for measuring the kinetic and equilibrium constants of human-DBP with vitamin D3 and three metabolites: 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], 25-hydroxyvitamin D3 (25OHD3) and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] by surface plasmon resonance (SPR). At the same time, five different analogues, synthetized in our laboratory, were evaluated in order to compare the affinity values with the metabolites. Real-time SPR measurements showed that 25OHD3 and 24,25(OH)2D3 had higher affinity (0.3 μM) than 1,25(OH)2D3 (5 μM), with the higher affinity of 25OHD3 and 24,25(OH)2D3 due to dissociation constants 1 order of magnitude slower. In the case of the analogues, the affinity values were lower, with 1-hydroxy-25-nitro-vitamin D3 (NO2-446), structurally closer to 1,25(OH)2D3, showing the highest value with a K D of 50 μM. (24R)-1,25-dihydroxyvitamin-24-buthyl-28-norvitamin D2 (Bu-471) and (24R)-1,25-dihydroxyvitamin-24-phenyl-28-norvitamin D2 (Ph-491), structurally similar, had affinities of 180 and 170 μM, respectively. (22R,23E)-1-hydroxy-22-ethenyl-25-methoxy-23-dehydrovitamin D3 (MeO-455) and 1-hydroxy-20(R)-[5(S)-(2,2-dimethyltetrahydropyran-5-yl)]-22,23-dinor vitamin D3 (Oxan-429) had affinities of 310 and 100 μM, respectively. The binding of the metabolites and analogues was reversible allowing the rapid capture of data for replicates. The kinetic and equilibrium data for both the metabolites and the analogues fitted to the Langmuir model describing a 1:1 interaction. Graphical Abstract Label-free, real time binding study between vitamin D binding protein immmobilized on the surface of a SPR sensor chip and the vitamin D, metabolites and analogues passed over it as analytes
Keywords: Vitamin D binding protein; Vitamin D metabolites; Vitamin D analogues; Surface plasmon resonance; Kinetics; Affinity

Impurity determination for hepcidin by liquid chromatography-high resolution and ion mobility mass spectrometry for the value assignment of candidate primary calibrators by Pauline Bros; Ralf D. Josephs; Norbert Stoppacher; Guillaume Cazals; Sylvain Lehmann; Christophe Hirtz; Robert I. Wielgosz; Vincent Delatour (2559-2567).
In metrology institutes, the state-of-the-art for purity analysis of peptides/proteins mainly addresses short and unfolded peptides. Important developments are anticipated for the characterization of nonlinear peptides or proteins. Hepcidin 1-25 is an interesting model system because this small protein contains four disulfide bridges with a particular connectivity that is difficult to reproduce and could induce a bias in quantification. Hepcidin 1-25 is involved in iron-related disorders and anemia, in an inflammatory context, and its clinical relevance in neurodegenerative disorders is under investigation. It is also an emerging biomarker. Recent inter-laboratory studies showed a need for standardization of hepcidin assay and the need to produce certified reference materials. This paper discusses two hepcidin standards from different synthesis pathways that have been characterized by high-resolution mass spectrometry and ion mobility mass spectrometry.
Keywords: Hepcidin; Purity assessment; High-resolution mass spectrometry; Ion mobility, conformers; Disulfide bridges

Magnetical hollow fiber bar collection of extract in homogenous ionic liquid microextraction of triazine herbicides in water samples by Kun Wang; Jia Jiang; Mingqin Kang; Dan Li; Shuang Zang; Sizhu Tian; Hanqi Zhang; Aimin Yu; Ziwei Zhang (2569-2579).
The homogeneous ionic liquid microextraction combined with magnetical hollow fiber bar collection was developed for extracting triazine herbicides from water samples. These analytes were separated and determined by high performance liquid chromatography. The triazines were quickly extracted into ionic liquid microdroplets dispersed in solution, and then these microdroplets were completely collected with magnetical hollow fiber bars; the pores of which were impregnated with hydrophobic ionic liquid, which makes the phase separation simplified with no need of centrifugation. Some experimental parameters, such as the type of ionic liquid, ultrasonic immersion time of hollow fiber, pH of sample solution, volume of hydrophilic ionic liquid, amount of ion-pairing agent NH4PF6, NaCl concentration, number of magnetical hollow fiber bar, stirring rate, and collection time were investigated and optimized. When the present method was applied to the analysis of real water samples, the precision and recoveries of six triazine herbicides vary from 0.1 to 9.2% and 73.4 to 118.5%, respectively. The detection limits for terbumeton, ametryn, prometryn, terbutryn, trietazine, and dimethametryn were 0.48, 0.15, 0.15, 0.14, 0.35, and 0.16 μg L−1, respectively.
Keywords: Homogeneous ionic liquid microextraction; Ionic liquid; Magnetical hollow fiber bar collection; Triazine herbicides; Environmental water samples

Compound-specific stable isotope analysis (CSIA) is among the most promising tools for studying the fate of organic pollutants in the environment. However, the feasibility of multidimensional CSIA was limited by the availability of a robust method for precise isotope analysis of heteroatom-bearing organic compounds. We developed a method for δ 13C and δ 2H analysis of eight organophosphorus compounds (OPs) with different chemical properties. In particular, we aimed to compare high-temperature conversion (HTC) and chromium-based HTC (Cr/HTC) units to explore the limitations of hydrogen isotope analysis of heteroatom-bearing compounds. Analysis of the amount dependency of the isotope values (linearity analysis) of OPs indicated that the formation of HCl was a significant isotope fractionation process leading to inaccurate δ 2H analysis in HTC. In the case of nonchlorinated OPs, by-product formation of HCN, H2S, or PH3 in HTC was observed but did not affect the dynamic range of reproducible isotope values above the limit of detection. No hydrogen-containing by-products were found in the Cr/HTC process by use of ion trap mass spectrometry analysis. The accuracy of gas chromatography – isotope ratio mass spectrometry was validated in comparison with elemental analyzer – isotope ratio mass spectrometry. Dual-isotope fractionation yielded Λ values of 0 ± 0 at pH 7, 7 ± 1 at pH 9, and 30 ± 6 at pH 12, indicating the potential of 2D CSIA to characterize the hydrolysis mechanisms of OPs. This is the first report on the combination of δ 2H and δ 13C isotope analysis of OPs, and this is the first study providing a systematic evaluation of HTC and Cr/HTC for hydrogen isotope analysis using OPs as target compounds. Graphical Abstract Comparison of δ2H measurement of non-chlorinated and chlorinated OPs via GC-Cr/HTC-IRMS and GC-HTC-IRMS system
Keywords: Organophosphorus compounds; Compound-specific stable isotope analysis; Hydrogen isotope; Pesticides; Chromium-based high-temperature conversion

Due to the complexity and variation of the chemical constituents in authentic black cohosh (Actaea racemosa) and its potential adulterant species, an accurate and feasible method for black cohosh authentication is not easy. A high-resolution accurate mass (HRAM) LC-MS fingerprinting method combined with chemometric approach was employed to discover new marker compounds. Seven hydroxycinnamic acid amide (HCAA) glycosides are proposed as potential marker compounds for differentiation of black cohosh from related species, including two Asian species (A. foetida, A. dahurica) and two American species (A. pachypoda, A. podocarpa). These markers were putatively identified by comparing their mass spectral fragmentation behavior with those of their authentic aglycone compounds and phytochemistry reports. Two isomers of feruloyl methyldopamine 4-O-hexoside ([M + H]+ 506) and one feruloyl tyramine 4-O-hexoside ([M + H]+ 476) contributed significantly to the separation of Asian species in principle component analysis (PCA) score plot. The efficacy of the models built on four reasonable combinations of these markers in differentiating black cohosh and its adulterants were evaluated and validated by partial least-square discriminant analysis (PLS-DA). Two models based on these reduced dataset achieved 100% accuracy based on the current sample collection, including the model that used only three feruloyl dopamine-O-hexoside isomers ([M + H]+ 492) and one feruloyl dopamine-O-dihexoside ([M + H-hexosyl]+ at m/z 492). Graphical abstract Hydroxycinnamic acid amide glycosides are proposed as potential marker compounds for authentication of black cohosh
Keywords: Hydroxycinnamic acid amide glycoside; Hydroxycinnamic acid ester; UHPLC-HRMS; Nitrogen-containing; PCA; PLS-DA

Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA by Jernej Pavšič; Alison Devonshire; Andrej Blejec; Carole A. Foy; Fran Van Heuverswyn; Gerwyn M. Jones; Heinz Schimmel; Jana Žel; Jim F. Huggett; Nicholas Redshaw; Maria Karczmarczyk; Erkan Mozioğlu; Sema Akyürek; Müslüm Akgöz; Mojca Milavec (2601-2614).
Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework.
Keywords: Digital PCR; DNA quantification; Inter-laboratory assessment; Human cytomegalovirus; Virus reference materials

Functionalized ZnO nanowires for microcantilever biosensors with enhanced binding capability by Stefano Stassi; Alessandro Chiadò; Valentina Cauda; Gianluca Palmara; Giancarlo Canavese; Marco Laurenti; Carlo Ricciardi (2615-2625).
An efficient way to increase the binding capability of microcantilever biosensors is here demonstrated by growing zinc oxide nanowires (ZnO NWs) on their active surface. A comprehensive evaluation of the chemical compatibility of ZnO NWs brought to the definition of an innovative functionalization method able to guarantee the proper immobilization of biomolecules on the nanostructured surface. A noteworthy higher amount of grafted molecules was evidenced with colorimetric assays on ZnO NWs-coated devices, in comparison with functionalized and activated silicon flat samples. ZnO NWs grown on silicon microcantilever arrays and activated with the proposed immobilization strategy enhanced the sensor binding capability (and thus the dynamic range) of nearly 1 order of magnitude, with respect to the commonly employed flat functionalized silicon devices. Graphical Abstract An efficient way to increase the binding capability of microcantilever biosensors is represented by growing zinc oxide nanowires (ZnO NWs) on their active surface. ZnO NWs grown on silicon microcantilever arrays and activated with an innovative immobilization strategy enhanced the sensor binding capability of nearly 1 order of magnitude, with respect to the commonly employed flat functionalized silicon devices.
Keywords: Zinc oxide; Nanowires; Biosensors; Microcantilever; Functionalization; Protein immobilization

Estrogen measurements are important in the assessment of female reproductive function and have expanding roles in other fields. A simple, accurate, highly sensitive and specific isotope-dilution liquid chromatography–tandem mass spectrometry method was developed and evaluated to simultaneously measure three endogenous estrogens in serum: estrone (E1), 17β-estradiol (E2), and estriol (E3). Chromatographic separation was achieved on a C18 column before electrospray ionization triple-quadrupole mass spectrometry in multiple reaction monitoring mode. The sample preparation in this assay requires no derivatization and extraction by liquid–liquid extraction. After optimization of the extraction conditions, the final extraction efficiency of E1, E2, and E3 was 83.8%, 78.9%, and 77.3% respectively. The metabolites and structural analogs that have the same molecular masses as the estrogens were separated under the optimized liquid chromatography conditions. Method validation showed satisfactory linearity over the concentration range of 20–10000 pg mL-1 for all three estrogens (r 2 > 0.997). The limits of quantification were 5, 10, and 10 pg mL-1 for E1, E2, and E3 respectively, and their recoveries ranged from 94.7% to 103.5%. The accuracy of the proposed method was further evaluated with use of certified reference materials BCR-576, BCR-577, and BCR-578 for E2 and 2014 International Federation of Clinical Chemistry and Laboratory Medicine External Quality Assessment Scheme for Reference Laboratories in Laboratory Medicine samples for E3, whose certified values were determined by reference methods. Great agreement was observed between the measured values and the certified values. Satisfactory precision (coefficients of variation less than 7.44%) was also obtained for the three estrogens. Moreover, the proposed method was successfully applied to measure the three estrogens in serum samples of pregnant women in the second trimester and to assess the accuracy of chemiluminescent immunoassays in clinical laboratories by determination of E2 and unconjugated E3 in serum samples. Graphical Abstract Schematic representation of the simultaneous quantitation of three major endogenous estrogens in human serum by ID-LC-MS/MS
Keywords: Endogenous estrogens; Mass spectrometry; Liquid–liquid extraction; Serum samples; Clinical laboratory

Veterinary drug residues in animal-derived foods must be monitored to ensure food safety, verify proper veterinary practices, enforce legal limits in domestic and imported foods, and for other purposes. A common goal in drug residue analysis in foods is to achieve acceptable monitoring results for as many analytes as possible, with higher priority given to the drugs of most concern, in an efficient and robust manner. The U.S. Department of Agriculture has implemented a multiclass, multi-residue method based on sample preparation using dispersive solid phase extraction (d-SPE) for cleanup and ultrahigh-performance liquid chromatography–tandem quadrupole mass spectrometry (UHPLC-QQQ) for analysis of >120 drugs at regulatory levels of concern in animal tissues. Recently, a new cleanup product called “enhanced matrix removal for lipids” (EMR-L) was commercially introduced that used a unique chemical mechanism to remove lipids from extracts. Furthermore, high-resolution quadrupole–time-of-flight (Q/TOF) for (U)HPLC detection often yields higher selectivity than targeted QQQ analyzers while allowing retroactive processing of samples for other contaminants. In this study, the use of both d-SPE and EMR-L sample preparation and UHPLC-QQQ and UHPLC-Q/TOF analysis methods for shared spiked samples of bovine muscle, kidney, and liver was compared. The results showed that the EMR-L method provided cleaner extracts overall and improved results for several anthelmintics and tranquilizers compared to the d-SPE method, but the EMR-L method gave lower recoveries for certain β-lactam antibiotics. QQQ vs. Q/TOF detection showed similar mixed performance advantages depending on analytes and matrix interferences, with an advantage to Q/TOF for greater possible analytical scope and non-targeted data collection. Either combination of approaches may be used to meet monitoring purposes, with an edge in efficiency to d-SPE, but greater instrument robustness and less matrix effects when analyzing EMR-L extracts. Graphical abstract Comparison of cleanup methods in the analysis of veterinary drug residues in bovine tissues
Keywords: Veterinary drug residue analysis; Sample preparation; Liquid chromatography–tandem mass spectrometry; Quadrupole–high-resolution time-of-flight mass spectrometry; Bovine tissues

Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification by Wei Fu; Pengyu Zhu; Shuang Wei; Du Zhixin; Chenguang Wang; Xiyang Wu; Feiwu Li; Shuifang Zhu (2655-2664).
Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for the final evaluation. After the second evaluation, the final amplification curves and melting curves have been achieved
Keywords: ME-qPCR; GMO; Multiplex; High-throughput detection; Highly sensitive

A fast, simple, economical, and environmentally friendly magnetic solid-phase extraction (MSPE) procedure has been developed to preconcentrate 2,4,6-trinitrotoluene (TNT) from water samples prior to determination by liquid chromatography-UV-Vis employing graphene oxide/Fe3O4 nanocomposite as sorbent. The nanocomposite synthesis was investigated, and the MSPE was optimized by a multivariate approach. The optimum MSPE conditions were 40 mg of nanocomposite, 10 min of vortex extraction, 1 mL of acetonitrile as eluent, and 6 min of desorption in an ultrasonic bath. Under the optimized experimental conditions, the method was evaluated to obtain a preconcentration factor of 153. The linearity of the method was studied from 1 to 100 μg L−1 (N = 5), obtaining a correlation coefficient of 0.994. The relative standard deviation and limit of detection were found to be 12% (n = 6, 10 μg L−1) and 0.3 μg L−1, respectively. The applicability of the method was investigated, analyzing three types of water samples (i.e., reservoir and drinking water and effluent wastewater) and recovery values ranged between 87 and 120% (50 μg L−1 spiking level), showing that the matrix had a negligible effect upon extraction. Finally, the semiquantitative Eco-Scale metrics confirmed the greenness of the developed method.
Keywords: Graphene oxide/Fe3O4 nanocomposite; Liquid chromatography-UV-Vis; Magnetic solid-phase extraction; 2,4,6-Trinitrotoluene; Water samples

An electrochemiluminescence resonance energy transfer (ECL-RET) system that detects prostate-specific antigen (PSA) was created. Near-infrared (NIR)-emitting NaYF4:Yb,Tm/Mn upconverting nanoparticles (UCNPs) are used as donors, and gold nanorods (GNRs) are used as acceptors. The ECL was enhanced nearly threefold by Mn2+ doping, with an emission peak appearing at an NIR wavelength of 808 nm. Anti-PSA 1 (Ab1) was bound to the surfaces of UCNPs after being modified with poly(acrylic acid) (PAA). As for acceptors, cetyltrimethylammonium bromide (CTAB)-capped GNRs were treated with 11-mercaptoundecanoic acid (MUDA) and then conjugated with Anti-PSA 2 (Ab2). When PSA was added, donors and acceptors were brought in close proximity through specific interactions of antibodies and antigens, resulting in high quenching efficiency levels. Under optimal conditions, the linear range of detection was 3.75–938 pg/mL for PSA (R = 0.999), with a detection limit as low as 3.16 pg/mL. This method can be applied to detect PSA in human serums with satisfactory results. Graphical abstract An electrochemiluminescence resonance energy transfer system was developed for determination of prostate-specific antigen using near-infrared-emitting NaYF4:Yb,Tm/Mn upconverting nanoparticles as donors and gold nanorods as acceptors.
Keywords: NaYF4:Yb,Tm/Mn nanoparticles; Gold nanorods; ECL-RET; Near-infrared emitting; PSA; Detection

An efficient liquid chromatography-high resolution mass spectrometry approach for the optimization of the metabolic stability of therapeutic peptides by Simone Esposito; Riccardo Mele; Raffaele Ingenito; Elisabetta Bianchi; Fabio Bonelli; Edith Monteagudo; Laura Orsatti (2685-2696).
In drug discovery, there is increasing interest in peptides as therapeutic agents due to several appealing characteristics that are typical of this class of compounds, including high target affinity, excellent selectivity, and low toxicity. However, peptides usually present also some challenging ADME (absorption, distribution, metabolism, and excretion) issues such as limited metabolic stability, poor oral bioavailability, and short half-lives. In this context, early preclinical in vitro studies such as plasma metabolic stability assays are crucial to improve developability of a peptidic drug. In order to speed up the optimization of peptide metabolic stability, a strategy was developed for the integrated semi-quantitative determination of metabolic stability of peptides and qualitative identification/structural elucidation of their metabolites in preclinical plasma metabolic stability studies using liquid chromatography-high-resolution Orbitrap™ mass spectrometry (LC-HRMS). Sample preparation was based on protein precipitation: experimental conditions were optimized after evaluating and comparing different organic solvents in order to obtain an adequate extraction of the parent peptides and their metabolites and to minimize matrix effect. Peptides and their metabolites were analyzed by reverse-phase liquid chromatography: a template gradient (total run time, 6 min) was created to allow retention and good peak shape for peptides of different polarity and isoelectric points. Three LC columns were selected to be systematically evaluated for each series of peptides. Targeted and untargeted HRMS data were simultaneously acquired in positive full scan + data-dependent MS/MS acquisition mode, and then processed to calculate plasma half-life and to identify the major cleavage sites, this latter by using the software Biopharma Finder™. Finally, as an example of the application of this workflow, a study that shows the plasma stability improvement of a series of antimicrobial peptides is described. This approach was developed for the evaluation of in vitro plasma metabolic stability studies of peptides, but it could also be applied to other in vitro metabolic stability models (e.g., whole blood, hepatocytes). Graphical Abstract Left: trend plot for omiganan and major metabolites. Right: stability plot for five antimicrobial peptidesafter incubation with mouse plasma
Keywords: Peptide metabolite ID; DMPK; LC-HRMS; Qual/Quan; Peptide ADME; Metabolic stability

Use of chemometrics to optimize a glucose assay on a paper microfluidic platform by Ani Avoundjian; Mehdi Jalali-Heravi; Frank A. Gomez (2697-2703).
We describe the use of a chemometrics-based computational platform to optimize a glucose assay on a microfluidic paper-based analytical device (μPAD). Glucose is colorimetrically detected in the presence of glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI). Using a Y-shaped paper microfluidic chip, the concentration of glucose, volume of reagents, and the length and width of the microfluidic channel were examined. The responses of the microfluidic chips were analyzed at the halfway point of the channel length. Variables affecting the response were screened by using a 24 factorial design, and among them, volume and concentration of the glucose were optimized by applying a rotatable central composite design (CCD). The optimum and experimental responses are 151.58 and 149.80, respectively, with an absolute error of 1.2%.
Keywords: Paper microfluidics; Chemometrics; Glucose oxidase; Point-of-care diagnostic device

Vitamin D measurements in biological fluids by mass spectrometry are challenging at very low concentration levels. As a result, chemical derivatization is often employed to enhance the ionization properties of low abundant vitamin D compounds. Cookson-type reagents such as 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) or similar derivatives work well but require careful, water-free experimental conditions, as traces of water inactivate the reagent and inhibit or stop the derivatization reactions, thus making quantitative measurements in aqueous samples impossible. We describe a novel electrospray liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for determining 25-hydroxyvitamin D3 (25(OH)D3) directly in aqueous cellular systems using a new derivatization reagent, the ionic liquid 12-(maleimidyl)dodecyl-tri-n-butylphosphonium bromide (MDBP). The proof-of-concept for the MDBP assay was demonstrated by measuring the levels of 25(OH)D3 in four different human cell types, namely T cells, helper T cells, B cells, and macrophages. In addition to the ability to determine the levels of 25(OH)D3 directly in aqueous samples, the cellular integrity was maintained in our application. We show the time-dependent uptake of 25(OH)D3 into the investigated cells to demonstrate the applicability of the new label. Furthermore, the MDBP derivatization technique may be equally useful in imaging mass spectrometry, where it could be used for response enhancements of spatially localized vitamin D metabolites on wet tissue surfaces, without destroying the integrity of the tissue surface. Graphical Abstract MDBP labelling of 25-hydroxyvitamin D in the extracellular space
Keywords: Vitamin D; LC-MS/MS; Chemical derivatization; Cellular uptake

Solid-phase PCR for rapid multiplex detection of Salmonella spp. at the subspecies level, with amplification efficiency comparable to conventional PCR by Wai Hoe Chin; Yi Sun; Jonas Høgberg; Tran Quang Hung; Anders Wolff; Dang Duong Bang (2715-2726).
Solid-phase PCR (SP-PCR) has attracted considerable interest in different research fields since it allows parallel DNA amplification on the surface of a solid substrate. However, the applications of SP-PCR have been hampered by the low efficiency of the solid-phase amplification. In order to increase the yield of the solid-phase amplification, we studied various parameters including the length, the density, as well as the annealing position of the solid support primer. A dramatic increase in the signal-to-noise (S/N) ratio was observed when increasing the length of solid support primers from 45 to 80 bp. The density of the primer on the surface was found to be important for the S/N ratio of the SP-PCR, and the optimal S/N was obtained with a density of 1.49 × 1011 molecules/mm2. In addition, the use of solid support primers with a short overhang at the 5′ end would help improve the S/N ratio of the SP-PCR. With optimized conditions, SP-PCR can achieve amplification efficiency comparable to conventional PCR, with a limit of detection of 1.5 copies/μl (37.5 copies/reaction). These improvements will pave the way for wider applications of SP-PCR in various fields such as clinical diagnosis, high-throughput DNA sequencing, and single-nucleotide polymorphism analysis. Graphical abstract Schematic representation of solid-phase PCR.
Keywords: Solid-phase PCR; High efficiency; Rapid diagnostics; Multiplex detection

Accurate quantitation of circulating cell-free mitochondrial DNA in plasma by droplet digital PCR by Wei Ye; Xiaojun Tang; Chu Liu; Chaowei Wen; Wei Li; Jianxin Lyu (2727-2735).
To establish a method for accurate quantitation of circulating cell-free mitochondrial DNA (ccf-mtDNA) in plasma by droplet digital PCR (ddPCR), we designed a ddPCR method to determine the copy number of ccf-mtDNA by amplifying mitochondrial ND1 (MT-ND1). To evaluate the sensitivity and specificity of the method, a recombinant pMD18-T plasmid containing MT-ND1 sequences and mtDNA-deleted (ρ0) HeLa cells were used, respectively. Subsequently, different plasma samples were prepared for ddPCR to evaluate the feasibility of detecting plasma ccf-mtDNA. In the results, the ddPCR method showed high sensitivity and specificity. When the DNA was extracted from plasma prior to ddPCR, the ccf-mtDNA copy number was higher than that measured without extraction. This difference was not due to a PCR inhibitor, such as EDTA-Na2, an anti-coagulant in plasma, because standard EDTA-Na2 concentration (5 mM) did not significantly inhibit ddPCR reactions. The difference might be attributable to plasma exosomal mtDNA, which was 4.21 ± 0.38 copies/μL of plasma, accounting for ∼19% of plasma ccf-mtDNA. Therefore, ddPCR can quickly and reliably detect ccf-mtDNA from plasma with a prior DNA extraction step, providing for a more accurate detection of ccf-mtDNA. The direct use of plasma as a template in ddPCR is suitable for the detection of exogenous cell-free nucleic acids within plasma, but not of nucleic acids that have a vesicle-associated form, such as exosomal mtDNA. Graphical Abstract Designs of the present work. *: Module 1, #: Module 2, &: Module 3
Keywords: Circulating cell-free DNA; Mitochondrial DNA; Droplet digital PCR

A rapid analytical method of human whole blood viscosity with low, medium, and high shear rates [WBV(L), WBV(M), and WBV(H), respectively] was developed using visible and near-infrared (Vis-NIR) spectroscopy combined with a moving-window partial least squares (MW-PLS) method. Two groups of peripheral blood samples were collected for modeling and validation. Separate analytical models were established for male and female groups to avoid interference in different gender groups and improve the homogeneity and prediction accuracy. Modeling was performed for multiple divisions of calibration and prediction sets to avoid over-fitting and achieve parameter stability. The joint analysis models for three indicators were selected through comprehensive evaluation of MW-PLS. The selected joint analysis models were 812–1278 nm for males and 670–1146 nm for females. The root-mean-square errors (SEP) and the correlation coefficients of prediction (RP) for all validation samples were 0.54 mPa•s and 0.91 for WBV(L), 0.25 mPa•s and 0.92 for WBV(M), and 0.22 mPa•s and 0.90 for WBV(H). Results indicated high prediction accuracy, with prediction values similar to the clinically measured values. Overall, the findings confirmed the feasibility of whole blood viscosity quantification based on Vis-NIR spectroscopy with MW-PLS. The proposed rapid and simple technique is a promising tool for surveillance, control, and treatment of cardio-cerebrovascular diseases in large populations. Graphical Abstract The caption/legend of the online abstract figure: The selected wavebands and the prediction effects for the three indicators of whole blood viscosity
Keywords: Whole blood viscosity; Shear rate; Visible and near-infrared spectroscopy; Moving-window partial least squares; Joint-analysis model

For a realistic evaluation of the potential hazard emanating from airborne particulate matter (APM), the determination of the total inhaled metal amounts associated with APM is insufficient in risk assessment. Additional information about metal fractions that can be mobilized by the human body is necessary, because only those soluble (also called bioaccessible) fractions can be absorbed by the human body, and thus potentially cause adverse health effects. In the present study, a dynamic flow-through approach as a front end to inductively coupled plasma optical emission spectrometry (ICP-OES) exploiting advanced flow analysis is employed for on-line handling of multiple APM samples and determination of bioaccessible trace metals under worst case extraction scenarios. The method is based on on-line continuous extraction of filter samples with synthetic gastric fluid followed by on-line ICP-OES measurement of the dissolved fraction of trace metals. The assembly permits an automated successive measurement of three sample replicates in less than 19 min. The on-line extraction procedure offers increased sample throughput and reduced risk of sample contamination and overcomes metal re-adsorption processes compared to the traditional batch-wise counterparts. Furthermore, it provides deeper information on the kinetics of the leaching process. The developed procedure was applied to the determination of bioaccessible metal fractions (Al, Ba, Cu, Fe and Mn as model analytes) in PM10 samples from Palma de Mallorca (Spain) and Vienna (Austria). Graphical Abstract On-line gastric bioaccessibility of elements in airborne particulate matter
Keywords: Airborne particulate matter; Bioaccessibility; Gastric fluid; Flow analysis; Metal; Inductively coupled plasma spectrometry

Asymmetrical flow field-flow fractionation of white wine chromophoric colloidal matter by Christian Coelho; Jérémie Parot; Michael Gonsior; Maria Nikolantonaki; Philippe Schmitt-Kopplin; Edith Parlanti; Régis D. Gougeon (2757-2766).
Two analytical separation methods—size-exclusion chromatography and asymmetrical flow field-flow fractionation—were implemented to evaluate the integrity of the colloidal composition of Chardonnay white wine and the impact of pressing and fermentations on the final macromolecular composition. Wine chromophoric colloidal matter, representing UV–visible-absorbing wine macromolecules, was evaluated by optical and structural measurements combined with the description of elution profiles obtained by both separative techniques. The objective of this study was to apply these two types of fractionation on a typical Chardonnay white wine produced in Burgundy and to evaluate how each of them impacted the determination of the macromolecular chromophoric content of wine. UV–visible and fluorescence measurements of collected fractions were successfully applied. An additional proteomic study revealed that grape and microorganism proteins largely impacted the composition of chromophoric colloidal matter of Chardonnay wines. Asymmetrical flow field-flow fractionation appeared to be more reliable and less invasive with respect to the native chemical environment of chromophoric wine macromolecules, and hence is recommended as a tool to fractionate chromophoric colloidal matter in white wines. Graphical Abstract An innovative macromolecular separation method based on Asymmetrical Flow Field-Flow Fractionation was developed to better control colloidal dynamics across Chardonnay white winemaking.
Keywords: Asymmetrical flow field-flow fractionation; White wine; Chromophoric colloidal matter; Wine proteins