Analytical and Bioanalytical Chemistry (v.410, #8)

What is novel about certified reference materials? by Stephen A. Wise (2045-2049).
is a Scientist Emeritus at the National Institute of Standards and Technology (NIST) in Gaithersburg, Maryland. During his 40-year career at NIST, he was primarily involved in the development of Standard Reference Materials (SRMs) for trace organic constituents in environmental, clinical, food, and dietary supplement matrices. He is currently a Scientific Consultant in the Office of Dietary Supplements at the National Institutes of Health (NIH-ODS) where he provides support for the Analytical Methods and Reference Materials Program. For his contributions to the development of SRMs, he received the 2006 Harvey W. Wiley Award from AOAC International, the 2014 Reference Material Achievement Award from the Technical Division on Reference Materials of AOAC International, and the 2015 Hillebrand Prize from the Chemical Society of Washington. He is one of the founding editors of Analytical and Bioanalytical Chemistry.

is Assistant Professor of Biochemistry at the University of Utah. His research focuses on using cryo-EM to characterize the atomic-scale mechanisms of macromolecular complexes involved in maintaining protein quality control. The 2017 Nobel Prize in Chemistry was awarded to Jacques Dubochet, Joachim Frank, and Richard Henderson for “developing cryo-electron microscopy (cryo-EM) for the high-resolution structure determination of biomolecules in solution.” This feature article summarizes some of the major achievements leading to the development of cryo-EM and recent technological breakthroughs that have transformed the method into a mainstream tool for structure determination.
Keywords: Cryo-EM; Nobel Prize; Structural biology; TEM

Post-translational modifications in tumor biomarkers: the next challenge for aptamers? by Ana Díaz-Fernández; Rebeca Miranda-Castro; Noemí de-los-Santos-Álvarez; María Jesús Lobo-Castañón (2059-2065).
received her BS in chemistry in 2015 and her MS in analytical and bioanalytical sciences in 2016 from Oviedo University. She is now a PhD student in the Chemical, Biochemical and Structural Analysis and Computational Modeling doctoral program of the University of Oviedo. Her PhD is focused on the selection of aptamers for the development of new aptamer-based biosensors for tumor biomarkers. is a postdoctoral researcher in the Electroanalysis Research Group headed by Prof. M.J. Lobo-Castañón. Her research interests focus on the development of electrochemical sensors for clinical diagnosis and food analysis using molecular recognition elements based on nucleic acids (genosensors and aptasensors). is an Associate Professor at Universidad de Oviedo (Spain). Her field of expertise is Electroanalysis. Her first studies were conducted on electrocatalytic processes and characterization of intermetallic materials for fuel cells (during her postdoc stage at Cornell University). Her current interests are focused on genosensing and aptamer selection (allergens, viruses and cancer biomarkers) to be used in biosensors for diagnostics. is a Professor at University of Oviedo, Department of Physical and Analytical Chemistry, Spain, where she leads the Electroanalysis research group. Her research interests focus on the development of electrochemical sensors for clinical diagnosis and food analysis, using different molecular recognition elements, such as enzymes, DNA, aptamers, and molecularly imprinted polymers. She is the author or co-author of over 100 scientific articles and several book chapters in the field. Advances in proteomics have fueled the search for novel cancer biomarkers with higher selectivity. Differential expression of low abundant proteins has been the usual way of finding those biomarkers. The existence of a selective receptor for each biomarker is compulsory for their use in diagnostic/prognostic assays. Antibodies are the receptors of choice in most cases although aptamers are becoming familiar because of their facile and reproducible synthesis, chemical stability as well as comparable affinity and selectivity. In recent years, it has been reported that the pattern of post-translational modifications, altered under neoplastic disease, is a better predictive biomarker than the total protein level. Among others, abnormal glycosylation is attracting great attention. Lectins and antibodies are being used for identification and detection of the carbohydrate moiety with low level of discrimination among various glycoproteins. Such level of selectivity is critical to bring next-generation biomarkers to the clinic. Aptamers that can be rationally tailored for a certain molecule domain can become the golden receptor to specifically detect aberrant glycosylation at each protein or even at each glycosylation site, providing new diagnostic tools for early detection of cancer. Graphical abstractAptamers may specifically differentiate normal from aberrant glycoproteins
Keywords: Aptamers; Cancer diagnostics; Glycans; Tumor biomarkers

The NISTmAb Reference Material 8671 lifecycle management and quality plan by John E. Schiel; Abigail Turner (2067-2078).
Comprehensive analysis of monoclonal antibody therapeutics involves an ever expanding cadre of technologies. Lifecycle-appropriate application of current and emerging techniques requires rigorous testing followed by discussion between industry and regulators in a pre-competitive space, an effort that may be facilitated by a widely available test metric. Biopharmaceutical quality materials, however, are often difficult to access and/or are protected by intellectual property rights. The NISTmAb, humanized IgG1κ Reference Material 8671 (RM 8671), has been established with the intent of filling that void. The NISTmAb embodies the quality and characteristics of a typical biopharmaceutical product, is widely available to the biopharmaceutical community, and is an open innovation tool for development and dissemination of results. The NISTmAb lifecyle management plan described herein provides a hierarchical strategy for maintenance of quality over time through rigorous method qualification detailed in additional submissions in the current publication series. The NISTmAb RM 8671 is a representative monoclonal antibody material and provides a means to continually evaluate current best practices, promote innovative approaches, and inform regulatory paradigms as technology advances. Graphical abstractThe NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing.
Keywords: Reference material; NISTmAb; Monoclonal antibody; Biotherapeutic; Biopharmaceutical; System suitability; Biosimilar

Qualification of NISTmAb charge heterogeneity control assays by Abigail Turner; John E. Schiel (2079-2093).
The NISTmAb is a monoclonal antibody Reference Material from the National Institute of Standards and Technology; it is a class-representative IgG1κ intended serve as a pre-competitive platform for harmonization and technology development in the biopharmaceutical industry. The publication series of which this paper is a part describes NIST’s overall control strategy to ensure NISTmAb quality and availability over its lifecycle. In this paper, the development and qualification of methods for monitoring NISTmAb charge heterogeneity are described. Capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF) assays were optimized and evaluated as candidate assays for NISTmAb quality control. CIEF was found to be suitable as a structural characterization assay yielding information on the apparent pI of the NISTmAb. CZE was found to be better suited for routine monitoring of NISTmAb charge heterogeneity and was qualified for this purpose. This paper is intended to provide relevant details of NIST’s charge heterogeneity control strategy to facilitate implementation of the NISTmAb as a test molecule in the end user’s laboratory. Graphical AbstractRepresentative capillary zone electropherogram of the NIST monoclonal antibody (NISTmAb). The NISTmAb is a publicly available research tool intended to facilitate advancement of biopharmaceutical analytics.
Keywords: Reference Material; NISTmAb; Monoclonal antibody; Biotherapeutic; Biopharmaceutical; System suitability; Biosimilar; Capillary electrophoresis

Development of orthogonal NISTmAb size heterogeneity control methods by Abigail Turner; Katharina Yandrofski; Srivalli Telikepalli; Jason King; Alan Heckert; James Filliben; Dean Ripple; John E. Schiel (2095-2110).
The NISTmAb is a monoclonal antibody Reference Material from the National Institute of Standards and Technology; it is a class-representative IgG1κ intended to serve as a pre-competitive platform for harmonization and technology development in the biopharmaceutical industry. The publication series of which this paper is a part describes NIST’s overall control strategy to ensure NISTmAb quality and availability over its lifecycle. In this paper, the development of a control strategy for monitoring NISTmAb size heterogeneity is described. Optimization and qualification of size heterogeneity measurement spanning a broad size range are described, including capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), size exclusion chromatography (SEC), dynamic light scattering (DLS), and flow imaging analysis. This paper is intended to provide relevant details of NIST’s size heterogeneity control strategy to facilitate implementation of the NISTmAb as a test molecule in the end user’s laboratory. Graphical abstractRepresentative size exclusion chromatogram of the NIST monoclonal antibody (NISTmAb). The NISTmAb is a publicly available research tool intended to facilitate advancement of biopharmaceutical analytics. HMW = high molecular weight (trimer and dimer), LMW = low molecular weight (2 fragment peaks). Peak labeled buffer is void volume of the column from L-histidine background buffer.
Keywords: Reference material; NISTmAb; Monoclonal antibody; Biotherapeutic; Biopharmaceutical; System suitability; Biosimilar; Capillary electrophoresis; Protein particle; Aggregation

Development of an LC-MS/MS peptide mapping protocol for the NISTmAb by Trina Mouchahoir; John E. Schiel (2111-2126).
Peptide mapping is a component of the analytical toolbox used within the biopharmaceutical industry to aid in the identity confirmation of a protein therapeutic and to monitor degradative events such as oxidation or deamidation. These methods offer the advantage of providing site-specific information regarding post-translational and chemical modifications that may arise during production, processing or storage. A number of such variations may also be induced by the sample preparation methods themselves which may confound the ability to accurately evaluate the true modification levels. One important focus when developing a peptide mapping method should therefore be the use of sample preparation conditions that will minimize the degree of artificial modifications induced. Unfortunately, the conditions that are amenable to effective reduction, alkylation and digestion are often the same conditions that promote unwanted modifications. Here we describe the optimization of a tryptic digestion protocol used for peptide mapping of the NISTmAb IgG1κ which addresses the challenge of balancing maximum digestion efficiency with minimum artificial modifications. The parameters on which we focused include buffer concentration, digestion time and temperature, as well as the source and type of trypsin (recombinant vs. pancreatic; bovine vs porcine) used. Using the optimized protocol we generated a peptide map of the NISTmAb which allowed us to confirm its identity at the level of primary structure. Graphical abstractPeptide map of the NISTmAb RM 8671 monoclonal antibody. Tryptic digestion was performed using an optimized protocol and followed by LC-UV-MS analysis. The trace represents the total ion chromatogram. Each peak was mapped to peptides identified using mass spectrometry data.
Keywords: Peptide mapping; NISTmAb; RM 8671; Tryptic digestion; Mass spectrometry; Optimization

The NISTmAb Reference Material 8671 value assignment, homogeneity, and stability by John E. Schiel; Abby Turner; Trina Mouchahoir; Katharina Yandrofski; Srivalli Telikepalli; Jason King; Paul DeRose; Dean Ripple; Karen Phinney (2127-2139).
The NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing. It must therefore embody the quality and characteristics of a typical biopharmaceutical product and be available long-term in a stable format with consistent product quality attributes. A stratified sampling and analysis plan using a series of qualified analytical and biophysical methods is described that assures RM 8671 meets these criteria. Results for the first three lots of RM 8671 highlight the consistency of material attributes with respect to size, charge, and identity. RM 8671 was verified to be homogeneous both within and between vialing lots, demonstrating the robustness of the lifecycle management plan. It was analyzed in concert with the in-house primary sample 8670 (PS 8670) to provide a historical link to this seminal material. RM 8671 was verified to be fit for its intended purpose as a technology innovation tool, external system suitability control, and cross-industry harmonization platform. Graphical abstractThe NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing.
Keywords: Reference Material; NISTmAb; Monoclonal antibody; Biotherapeutic; Biopharmaceutical; System suitability; Biosimilar

Characterization of the NISTmAb Reference Material using small-angle scattering and molecular simulation by Maria Monica Castellanos; Steven C. Howell; D. Travis Gallagher; Joseph E. Curtis (2141-2159).
Both conformational and colloidal stability of therapeutic proteins must be closely monitored and thoroughly characterized to assess the long-term viability of drug products. We characterized the IgG1 NISTmAb reference material in its histidine formulation buffer and report our findings on the higher order structure and interactions of NISTmAb under a range of conditions. In this paper we present the analysis of experimental small-angle scattering data with atomistic molecular simulations to characterize the monodisperse dilute solution of NISTmAb. In part II we describe the characterization of the NISTmAb at high protein concentration (Castellanos et al. 2018). The NISTmAb was found to be a flexible protein with a radius of gyration of 49.0 ± 1.2 Å in histidine formulation buffer using a variety of neutron and X-ray scattering measurements. Scattering data were then modeled using molecular simulation. After building and validating a starting NISTmAb structure from the Fc and Fab crystallographic coordinates, molecular dynamics and torsion-angle Monte Carlo simulations were performed to explore the configuration space sampled in the NISTmAb and obtain ensembles of structures with atomistic detail that are consistent with the experimental data. Our results indicate that the small-angle scattering profiles of the NISTmAb can be modeled using ensembles of flexible structures that explore a wide configuration space. The NISTmAb is flexible in solution with no single preferred orientation of Fc and Fab domains, but with some regions of configuration space that are more consistent with measured scattering profiles. Analysis of inter-domain atomistic contacts indicated that all ensembles contained configurations where residues between domains are ≤ 4 Å, although few contacts were observed for variable and C H 3 regions. Graphical AbstractHeavy atom self contact maps of the NISTmAb indicate a highly-flexible structure.
Keywords: Small-angle scattering; NISTmAb reference material; Antibody structure; Protein conformation; Antibody flexibility; Higher order structure

Characterization of the NISTmAb Reference Material using small-angle scattering and molecular simulation by Maria Monica Castellanos; Kevin Mattison; Susan Krueger; Joseph E. Curtis (2161-2171).
Protein-protein interactions in monoclonal antibody solutions are important for the stability of a therapeutic drug and directly influence viscosity in concentrated protein solutions. This study describes the use of small-angle scattering to estimate protein-protein interactions at high concentrations of the IgG1 NISTmAb reference material and validate colloidal models for interacting molecules. In particular, we studied the colloidal stability of the NISTmAb at high protein concentrations and analyzed protein-protein interactions upon adding sodium chloride and its effect on viscosity. Isotropic colloidal models for interacting molecules were combined with an ensemble of atomistic structures from molecular simulation to account for the flexibility of the NISTmAb in solution. In histidine formulation buffer, net repulsive electrostatic interactions are important for the colloidal stability of the NISTmAb at high concentrations. Addition of sodium chloride increased the viscosity of the NISTmAb and decreased the colloidal stability due to charge screening of the repulsive interactions. The interactions at high concentrations (up to ~ 250 mg/mL) were consistent with those from light scattering at low concentrations (below ~ 20 mg/mL). However, in the presence of sodium chloride, the screening of charges was less pronounced with increasing protein concentration and the interactions approached those of the repulsive hard-sphere models. Additionally, we studied the NISTmAb under frozen conditions using in situ neutron scattering to analyze the crowded state as proteins are excluded from the water-rich phase. In the frozen samples, where protein concentration can reach hundreds of mg/mL in the protein-rich phase, sodium chloride did not affect the molecular spacing and crowding of the NISTmAb. Graphical AbstractNet repulsive interactions in concentrated NISTmAb solutions assessed by small-angle neutronscattering.
Keywords: Small-angle scattering; NISTmAb reference material; Protein-protein interactions; Concentrated protein solutions; Frozen protein solutions

Nanocomposite materials are potentially revolutionizing many technologies, including sensors. In this paper, we described the application of “PANI/MWCNTs/Starch” modified carbon paste electrode (PCS-CPE) as a simple and highly sensitive cholesterol sensor. This novel nano-composite material has integrated nano-morphology, where polyaniline could interact effectively with the additives; pi-pi stacking “MWCNTs,” and covalently bonded with starch. Specific binding sites (sugar chains), better electro-catalytic properties and fast electron transfer facilitated the oxidation of cholesterol. Fourier transform infrared spectra confirmed the interaction of cholesterol with the composite material. The sensing response of PCS was measured by cyclic voltammetry and chronoamperometry (0.1 M PBS-5 used as supporting electrolyte). As the amount of cholesterol increased in the test solution, cyclic voltammograms showed a rise of peak current (cathodic and anodic). Under the normal experimental conditions, the developed sensor exhibited wide linear dynamic range (0.032 to 5 mM) (upper limit is due to lack of solubility of cholesterol), high sensitivity (800 μAmM−1 cm−2), low detection limit (0.01 mM) and shorter response time (within 4–6 s). Analytical specificity, selectivity, and sensitivity during cholesterol estimation were compared with the response of some other analytes (ascorbic acid, glucose, l-dopa, urea and lactic acid). This novel sensor was successfully applied to estimate cholesterol in cow milk (used as a model real sample). The sensing platform is highly sensitive and shows a linear response towards cholesterol without using any additional redox mediator or enzyme, thus this material is extremely promising for the realization of a low-cost integrated cholesterol sensor device. Graphical abstractCyclic voltammetric response of cholesterol of composite modified carbon paste capillary electrode
Keywords: Cholesterol sensor; Polyaniline; MWCNTs; Starch; Composite material; Real sample analysis

Application of solvent floatation to separation and determination of triazine herbicides in honey by high-performance liquid chromatography by Kun Wang; Jia Jiang; Xinping Lv; Shuang Zang; Sizhu Tian; Hanqi Zhang; Aimin Yu; Ziwei Zhang; Yong Yu (2183-2192).
Based on the foaming property of the honey, a rapid, simple, and effective method solvent floatation (SF) was developed and firstly applied to the extraction and separation of triazine herbicides in honey. The analytes were determined by high-performance liquid chromatography. Some parameters affecting the extraction efficiencies, such as the type and volume of extraction solvent, type of salt, amount of (NH4)2SO4, pH value of sample solution, gas flow rate, and floatation time, were investigated and optimized. The limits of detection for analytes are in the range of 0.16–0.56 μg kg−1. The recoveries and relative standard deviations for determining triazines in five real honey samples are in the range of 78.2–112.9 and 0.2–9.2%, respectively.
Keywords: Solvent floatation; Honey; Triazine herbicides; High-performance liquid chromatography

A comprehensive evaluation of tyrosol and hydroxytyrosol derivatives in extra virgin olive oil by microwave-assisted hydrolysis and HPLC-MS/MS by Lucia Bartella; Fabio Mazzotti; Anna Napoli; Giovanni Sindona; Leonardo Di Donna (2193-2201).
A rapid and reliable method to assay the total amount of tyrosol and hydroxytyrosol derivatives in extra virgin olive oil has been developed. The methodology intends to establish the nutritional quality of this edible oil addressing recent international health claim legislations (the European Commission Regulation No. 432/2012) and changing the classification of extra virgin olive oil to the status of nutraceutical. The method is based on the use of high-performance liquid chromatography coupled with tandem mass spectrometry and labeled internal standards preceded by a fast hydrolysis reaction step performed through the aid of microwaves under acid conditions. The overall process is particularly time saving, much shorter than any methodology previously reported. The developed approach represents a mix of rapidity and accuracy whose values have been found near 100% on different fortified vegetable oils, while the RSD% values, calculated from repeatability and reproducibility experiments, are in all cases under 7%. Graphical abstractSchematic of the methodology applied to the determination of tyrosol and hydroxytyrosol ester conjugates
Keywords: Olive oil; Isotope dilution mass spectrometry; Microwave hydrolysis; 432/2012 EU regulation; Hydroxytyrosol; Phenolic compound

Alkyne is unique, specific and biocompatible in the Raman-silent region of the cell, but there still remains a challenge to achieve ultrasensitive detection in living systems due to its weak Raman scattering. Herein, a terminal alkyne ((E)-2-[4-(ethynylbenzylidene)amino]ethane-1-thiol (EBAE)) with surface-enhanced Raman scattering is synthesized. The EBAE molecule possesses S- and C-termini, which can be directly bonded to gold nanoparticles and dopamine/silver by forming the Au-S chemical bond and the carbon-metal bond, respectively. The distance between Raman reporter and AuNPs/AgNPs can be reduced, contributing to forming hot-spot-based SERS substrate. The alkyne functionalized nanoparticles are based on Au core and encapsulating polydopamine shell, defined as Au-core and dopamine/Ag-shell (ACDS). The bimetallic ACDS induce strong SERS signals for molecular imaging that arise from the strong electromagnetic field. Furthermore, the EBAE provides a distinct peak in the cellular Raman-silent region with nearly zero background interference. The EBAE Raman signals could be tremendously enhanced when the Raman reporter is located at the middle of the Au-core and dopamine/Ag-shell. Therefore, this work could have huge potential benefits for the highly sensitive detection of intercellular information delivery by connecting the recognition molecules in biomedical diagnostics. Graphical abstractTerminal-alkyne-functionalized Au-core and silver/dopamine-shell nanotags for live-cell surface-enhanced Raman scattering imaging
Keywords: (E)-2-[4-(ethynylbenzylidene)amino]ethane-1-thiol (EBAE); Surface-enhanced Raman scattering imaging (SERS); Live-cell imaging; Au-core and dopamine/silver-shell (ACDS)

In this paper, fluorescent Ag nanoclusters (Ag NCs) templated by hyperbranched polyethyleneimine (PEI) are utilized as a versatile probe through the photoinduced electron transfer (PET) between PEI-Ag NCs and G-quadruplex-hemin complexes. In the presence of hemin and target molecule, the specific conjugation with its aptamer induces the conformational change of the DNA sequence, releasing the G-quadruplex sequence part. Once the G-quadruplex-hemin complexes are introduced, electron transfer from the PEI-Ag NCs to G-quadruplex-hemin complexes occurs, resulting in fluorescence quenching. Through changing the sensing DNA sequence, this novel PET system enables the specific detection of target DNA and adenosine triphosphate (ATP) with the wide linear range of 1–200 nM and 5–500 nM, respectively, and the corresponding limit of detection as low as 0.3 nM for target DNA and 1.5 nM for ATP. In addition, the proposed method is successfully applied to the determination of ATP in human serum samples with satisfactory recoveries, and a logic gate is fabricated using target molecules and hemin as inputs and the fluorescence signal of PEI-Ag NCs as an output.
Keywords: Ag nanoclusters; G-Quadruplex-hemin complexes; Adenosine triphosphate; Target DNA; Logic gate

Sample pretreatment and SERS-based detection of ceftriaxone in urine by Natalia E. Markina; Irina Yu. Goryacheva; Alexey V. Markin (2221-2227).
The aim of the work is the development of the procedure for ceftriaxone (antibiotic drug of cephalosporin class) detection in urine using surface-enhanced Raman spectroscopy (SERS). Hydroxylamine stabilized silver nanoparticles were used as SERS-active material. Additional urine pretreatment steps were developed in order to eliminate the influence of creatinine on the ceftriaxone SERS signal. These steps include adjusting of the sample pH to alkaline value (pH 13) and purification of the sample using silica gel column chromatography. Alkali pH increases SERS signal of ceftriaxone, while silica gel separates the analyte from creatinine—the main admixture in urine which provides inappropriate SERS signal background. Additionally, it was found that total protein content up to 0.2 mg/mL (upper level for urine of healthy person) and pH deviation of initial urine do not influence on SERS signal of ceftriaxone. The proposed detection procedure enables fast (~ 10 min) determination of ceftriaxone in artificially spiked urine samples within 5 to 500 μg/mL range of concentrations which matches the range of the drug concentrations in urine after injection of therapeutically required dosages. Limits of detection (3σ) and quantification (10σ) were found to be 0.4 and 2.0 μg/mL, correspondingly. Graphical abstractApplication of urine pretreatment enables the purification of target analyte from intrinsic urine components and improves SERS-based detection of ceftriaxone (antibiotic drug)
Keywords: Surface-enhanced Raman spectroscopy; Cephalosporin antibiotic drug; Silver nanoparticles; Therapeutic drug monitoring; Silica gel

Voltammetric and electrogeneration approaches for the assessment of the oxidative drug metabolism by Laura Rodríguez-Cid; Sonia Sentellas; Javier Saurina (2229-2239).
In this paper, electrochemical (EC) methods have been proposed to evaluate the oxidative behavior of drugs as rapid, simple, and cheap strategies to predict some metabolic features. Various commercial drugs belonging to different therapeutic families have been assayed to deal with a wide variety of biotransformations and to cover different metabolism extents. First, differential pulse voltammetry has been applied to evaluate the oxidative behavior of drugs. Voltammetric assays have demonstrated to be highly efficient to predict the metabolism extent from the current intensity data. The second objective of this work has been the comparison of metabolite profiles from both EC and in vitro methods based on liver microsome assays. The resulting samples have been analyzed by reversed-phase liquid chromatography mode using a core-shell column and UV detection. Chromatographic methods have been established for each particular drug and its metabolites using 0.1% (v/v) formic acid aqueous solution and methanol (MeOH) as the components of the mobile phase. Drug oxidation products from both EC- and microsome-based methodologies have been compared in terms of variety and percentage from the corresponding chromatographic profiles. In general, most of the metabolites occurring in vitro have also been reproduced in the EC runs. Besides, it has been found that compositional profiles from EC experiments are dependent on experimental variables such as pH and potential. In general, acid (pH 2) and basic (pH 10) conditions and too high potentials can contribute to the generation of oxidation artifacts which differ from metabolites while milder potentials and neutral pH values may reproduce more accurately the microsome patterns. The proposed methodology is suitable for a first study of the oxidative behavior of molecules that can be related to relevant metabolic properties. The obtained information could be of great interest to prioritize or discard compounds, as a first screening, on the research of drug candidates.
Keywords: Drug metabolism; Electrochemical assay; Microsome incubation; Liquid chromatography; Experimental design

Phenolic resin based activated carbon fibers (ACFs) were applied for the first time as a reversed-dispersive solid-phase extraction (r-DSPE) sorbent. A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was applied to determine 26 pesticides (organophosphates, organochlorines, synthetic pyrethroids, and herbicides) in different complex matrices, including cauliflower, cucumber, banana, apple, wheat, and black gram. Different physicochemical characterization techniques were used to investigate the engineering and structural properties of the r-DSPE sorbent. All the chromatographic analyses were performed with a gas chromatograph equipped with an electron capture detector. The recoveries of all 26 pesticides were acceptable (70–120%), with relative standard deviations of less than 15%. The limit of detection and the limit of quantification were 1.13–5.48 ng/g and 3.42–16.60 ng/g, respectively. In the original QuEChERS method, primary secondary amine is extensively used as the r-DSPE sorbent in the cleanup process, but it is eightfold more expensive than the ACFs used in this study. Therefore, the modified QuEChERS method using ACFs during the cleanup process is more efficient, cheaper, and more robust to determine pesticides from different types of matrices, including vegetables, grains, and fruits, and ACFs could be used as a cost-effective alternative to primary secondary amine. Graphical AbstractSample clean-up using PSA and ACF as r-DSPE sorbent in QuEChERS method
Keywords: QuEChERS; Reversed-dispersive solid-phase extraction; Activated carbon fibers; Primary secondary amine; Pesticides; Sample preparation

Paper-based immune-affinity arrays for detection of multiple mycotoxins in cereals by Li Li; Hongpu Chen; Xiaolan Lv; Min Wang; Xizhi Jiang; Yifei Jiang; Heye Wang; Yongfu Zhao; Liru Xia (2253-2262).
Mycotoxins produced by different species of fungi may coexist in cereals and feedstuffs, and could be highly toxic for humans and animals. For quantification of multiple mycotoxins in cereals, we developed a paper-based mycotoxin immune-affinity array. First, paper-based microzone arrays were fabricated by photolithography. Then, monoclonal mycotoxin antibodies were added in a copolymerization reaction with a cross-linker to form an immune-affinity monolith on the paper-based microzone array. With use of a competitive immune-response format, paper-based mycotoxin immune-affinity arrays were successfully applied to detect mycotoxins in samples. The detection limits for deoxynivalenol, zearalenone, T-2 toxin, and HT-2 toxin were 62.7, 10.8, 0.36, and 0.23 μg·kg-1, respectively, which meet relevant requirements for these compounds in food. The recovery rates were 81–86% for deoxynivalenol, 89–117% for zearalenone, 79–86% for T-2 toxin, and 78–83% for HT-2 toxin, and showed the paper-based immune-affinity arrays had good reproducibility. In summary, the paper-based mycotoxin immune-affinity array provides a sensitive, rapid, accurate, stable, and convenient platform for detection of multiple mycotoxins in agro-foods. Graphical abstractPaper-based immune-affinity monolithic array. DON deoxynivalenol, HT-2 HT-2 toxin, T-2 T-2 toxin, PEGDA polyethylene glycol diacrylate, ZEN zearalenone
Keywords: Paper-based array; Immune-affinity array; Mycotoxins; UV-initiated copolymerization; Chemiluminescent detection