Analytical and Bioanalytical Chemistry (v.407, #7)

Solubility product challenge by Anna Maria Michałowska-Kaczmarczyk; Tadeusz Michałowski (1789-1791).

Solution to the Ariadne’s thread NMR challenge by Reinhard Meusinger (1793-1794).

is Full Professor of Physical Chemistry at Eberhard Karls Universty of Tübingen in Germany. His main research interests center on spectroscopy of single molecules, quantum dots or plasmonic nanoparticles, with very high spatial resolution using advanced optical techniques such as cylindrical vector beams, optical micro-resonators and tip enhanced near-field optical microscopy with a parabolic mirror microscope.

1,1,3,3-Tetramethylguanidine (TMG), methanol and carbon dioxide were investigated as switchable polarity solvents (SPS) in the simultaneous derivatization and extraction of triacylglycerols for the gas chromatographic (GC) characterization of olive oil. Three commercial olive oils were used as test samples. Results of the developed method did not differ statistically from those provided by reference derivatization procedures. The transesterification reaction was carried out under a very mild condition, one step and in situ, and no particular matrix interferences were evidenced. The method represented the first example of the use of a switchable polarity mixture for the preparation of methyl ester derivatives of fatty acids (FAME).
Keywords: Fatty acid methyl esters (FAME); Gas chromatography (GC); Olive oil; Carbon dioxide; Switchable polarity solvents (SPS); 1,1,3,3-Tetramethylguanidine (TMG)

Towards optoelectronic urea biosensors by Marta Pokrzywnicka; Robert Koncki; Łukasz Tymecki (1807-1812).
Integration of immobilized enzymes with light-emitting diodes (LEDs) leads to the development of optoelectronic enzyme-based biosensors. In this work, urease, used as a model enzyme, immobilized in the form of an open-tubular microbioreactor or biosensing membrane that has been integrated with two red LEDs. It forms complete, fiberless, miniaturized, and extremely economic biooptoelectronic devices useful for nonstationary measurements under flow analysis conditions. Both enzyme-based biodevices, operating according to the paired emitter detector diode (PEDD) principle, allow relatively fast, highly sensitive, and well-reproducible urea detection in the millimolar range of concentrations. Potential analytical applications of the developed urea bioPEDDs have been announced. Both presented constructions will be easily adapted for the development of other optoelectronic biosensors exploring various enzyme-based schemes of biodetection.
Keywords: Biosensor; Bioreactor; Urease; Light-emitting diodes; Instrumentation; Flow analysis

Raman spectroscopic investigation of 13CO2 labeling and leaf dark respiration of Fagus sylvatica L. (European beech) by Robert Keiner; Marie-Cécile Gruselle; Beate Michalzik; Jürgen Popp; Torsten Frosch (1813-1817).
An important issue, in times of climate change and more extreme weather events, is the investigation of forest ecosystem reactions to these events. Longer drought periods stress the vitality of trees and promote mass insect outbreaks, which strongly affect ecosystem processes and services. Cavity-enhanced Raman gas spectrometry was applied for online multi-gas analysis of the gas exchange rates of O2 and CO2 and the labeling of Fagus sylvatica L. (European beech) seedlings with 13CO2. The rapid monitoring of all these gases simultaneously allowed for the separation of photosynthetic uptake of CO2 by the beech seedlings and a constant 12CO2 efflux via respiration and thus for a correction of the measured 12CO2 concentrations in course of the labeling experiment. The effects of aphid infestation with the woolly beech aphid (Phyllaphis fagi L.) as well as the effect of a drought period on the respirational gas exchange were investigated. A slightly decreased respirational activity of drought-stressed seedlings in comparison to normally watered seedlings was found already for a low drought intensity. Cavity-enhanced Raman gas monitoring of O2, 12CO2, and 13CO2 was proven to be a powerful new tool for studying the effect of drought stress and aphid infestation on the respirational activity of European beech seedlings as an example of important forest species in Central Europe.
Keywords: Cavity-enhanced Raman spectroscopy; Raman gas sensing; 13CO2 labeling; Environmental sensing; Tree seedlings

Bioavailability and biotransformation of sulforaphane and erucin metabolites in different biological matrices determined by LC–MS–MS by Stefanie Platz; Ann Liza Piberger; Julia Budnowski; Corinna Herz; Monika Schreiner; Michael Blaut; Andrea Hartwig; Evelyn Lamy; Laura Hanske; Sascha Rohn (1819-1829).
born 1987, is a PhD student at the University of Hamburg, Hamburg School of Food Science, Germany. Her analytical work is focused on the identification and quantification of glucosinolate metabolites in different biological matrices (e.g. urine, plasma) using different mass-spectrometry techniques. The objective of her research is to examine the bioavailability and the kinetics of glucosinolate metabolites. is a postdoctoral researcher at the Karlsruhe Institute of Technology (Department of Food Chemistry and Toxicology) in Karlsruhe, Germany. Her scientific work is focused on the identification and understanding of cellular effects induced by the broccoli-borne isothiocyanate sulforaphane using a variety of molecular and/or cellular biological and analytical techniques. In this context, she is particularly interested in DNA repair processes and the maintenance of genomic stability. has finished her PhD at the German Institute of Human Nutrition Potsdam-Rehbruecke in the Department of Gastrointestinal Microbiology. She investigated the effect of human intestinal bacteria on bioactivation of glucosinolates and has work experience with germfree and gnotobiotic animals. obtained her PhD in biology from the University of Freiburg, Germany in 2008. She is a research associate in the group Molecular Preventive Medicine at the Institute of Environmental Health Sciences, Freiburg University Medical Center, Germany. Her major interest is investigating the cancer-preventive efficacy of plants and natural compounds. obtained her Master of International Agricultural Development and her PhD at the Technische Universität, Berlin. She worked as a Postdoctoral Scientist at the Leibniz-Institute of Agricultural Engineering, Bornim before becoming the head of the Department of Quality at the Leibniz-Institute of Vegetable and Ornamental Crops, Großbeeren/Erfurt and Professor at the Leibniz Universität, Hannover. Her major research interests focus on the study of secondary plant metabolites—particularly glucosinolates—and their functions. is head of the Department of Gastrointestinal Microbiology at the German Institute of Human Nutrition and Professor of Gastrointestinal Microbiology at the Institute of Nutrition Science at the University of Potsdam. Main research topics: function of intestinal microbiota in obesity and inflammatory bowel disease and in the conversion of secondary plant metabolites. is Full Professor and chair for Food Chemistry and Toxicology at the Karlsruhe Institute of Technology, Germany. Her main research topic focuses on the effect of carcinogenic metal compounds, metal-based nanomaterials, essential trace elements, and bioactive food components on genomic stability, with special emphasis on DNA damage induction and effects on DNA repair, gene expression, cell-cycle control, and tumor suppressor functions. Her special focus is on interactions with so-called zinc finger proteins, including their redox regulation. born 1979, obtained her PhD in human biology at the Medical Faculty of the University of Giessen in 2008. She is a Junior Group Leader at the Institute of Environmental Health Sciences, Freiburg University Medical Center, Germany. Her major research interests focus on the anti-cancer effects of plant compounds, especially on signaling of selective cell aging and telomerase regulation. has been working as a postdoctoral research fellow in Professor Blaut’s laboratory for Gastrointestinal Microbiology at the German Institute of Human Nutrition Potsdam-Rehbruecke. She has been working for several years on investigating the effect of gut bacteria on the bioavailability of secondary plant compounds and has been conducting research with germfree and gnotobiotic animals. born 1973, is Full Professor for Food Chemistry at the University of Hamburg, Hamburg School of Food Science, Germany. His group deals with the analysis of secondary plant metabolites, their antioxidant activity, and especially the reactivity and stability of these bioactive compounds. The objective is to identify degradation products that serve as process markers during food and/or feed processing or as biomarkers in nutritional physiology. The food-related isothiocyanate sulforaphane (SFN), a hydrolysis product of the secondary plant metabolite glucoraphanin, has been revealed to have cancer-preventive activity in experimental animals. However, these studies have often provided inconsistent results with regard to bioavailability, bioaccessibility, and outcome. This might be because the endogenous biotransformation of SFN metabolites to the structurally related erucin (ERN) metabolites has often not been taken into account. In this work, a fully validated liquid chromatography tandem mass spectrometry (LC–MS–MS) method was developed for the simultaneous determination of SFN and ERN metabolites in a variety of biological matrices. To reveal the importance of the biotransformation pathway, matrices including plasma, urine, liver, and kidney samples from mice and cell lysates derived from colon-cancer cell lines were included in this study. The LC–MS–MS method provides limits of detection from 1 nmol L−1 to 25 nmol L−1 and a mean recovery of 99 %. The intra and interday imprecision values are in the range 1–10 % and 2–13 %, respectively. Using LC–MS–MS, SFN and ERN metabolites were quantified in different matrices. The assay was successfully used to determine the biotransformation in all biological samples mentioned above. For a comprehensive analysis and evaluation of the potential health effects of SFN, it is necessary to consider all metabolites, including those formed by biotransformation of SFN to ERN and vice versa. Therefore, a sensitive and robust LC–MS–MS method was validated for the simultaneous quantification of mercapturic-acid-pathway metabolites of SFN and ERN. Graphical Abstract Biotransformation of sulforaphane and erucin metabolites in mice and cell culture
Keywords: Mass spectrometry; Sulforaphane; Erucin; Bioactive compounds; Bioavailability; Biotransformation

DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials by Philippe Corbisier; Leonardo Pinheiro; Stéphane Mazoua; Anne-Marie Kortekaas; Pui Yan Jenny Chung; Tsvetelina Gerganova; Gert Roebben; Hendrik Emons; Kerry Emslie (1831-1840).
The value assignment for properties of six certified reference materials (ERM-AD623a–f), each containing a plasmid DNA solution ranging from 1 million to 10 copies per μL, by using digital PCR (dPCR) with the BioMark™ HD System (Fluidigm) has been verified by applying droplet digital PCR (ddPCR) using the QX100 system (Bio-Rad). One of the critical factors in the measurement of copy number concentrations by digital PCR is the partition volume. Therefore, we determined the average droplet volume by optical microscopy, revealing an average droplet volume that is 8 % smaller than the droplet volume used as the defined parameter in the QuantaSoft software version 1.3.2.0 (Bio-Rad) to calculate the copy number concentration. This observation explains why copy number concentrations estimated with ddPCR and using an average droplet volume predefined in the QuantaSoft software were systematically lower than those measured by dPCR, creating a significant bias between the values obtained by these two techniques. The difference was not significant anymore when the measured droplet volume of 0.834 nL was used to estimate copy number concentrations. A new version of QuantaSoft software (version 1.6.6.0320), which has since been released with Bio-Rad’s new QX200 systems and QX100 upgrades, uses a droplet volume of 0.85 nL as a defined parameter to calculate copy number concentration. Graphical Abstract Monolayer of droplets generated by the droplet generator and observed under an optical microscope
Keywords: Digital PCR; Droplet digital PCR; Reference materials; Optical microscopy; Areal equivalent diameter

Quantitative evaluation of bias in PCR amplification and next-generation sequencing derived from metabarcoding samples by Marta Pawluczyk; Julia Weiss; Matthew G. Links; Mikel Egaña Aranguren; Mark D. Wilkinson; Marcos Egea-Cortines (1841-1848).
Unbiased identification of organisms by PCR reactions using universal primers followed by DNA sequencing assumes positive amplification. We used six universal loci spanning 48 plant species and quantified the bias at each step of the identification process from end point PCR to next-generation sequencing. End point amplification was significantly different for single loci and between species. Quantitative PCR revealed that Cq threshold for various loci, even within a single DNA extraction, showed 2,000-fold differences in DNA quantity after amplification. Next-generation sequencing (NGS) experiments in nine species showed significant biases towards species and specific loci using adaptor-specific primers. NGS sequencing bias may be predicted to some extent by the Cq values of qPCR amplification.
Keywords: Metabarcoding; Next-generation sequencing; Ion torrent; Cq value; PCR efficiency

Tag-femtosecond laser-induced breakdown spectroscopy for the sensitive detection of cancer antigen 125 in blood plasma by Yuri Markushin; Poopalasingam Sivakumar; Denise Connolly; Noureddine Melikechi (1849-1855).
Successful treatment of cancers requires detecting early signs of the disease. One promising way to approach this is to develop minimally invasive tests for the sensitive and specific detection of biomarkers in blood. Irrespective of the detection approach one uses, this remains a challenging task because biomarkers are typically present in low concentrations and there are signals that interfere strongly with prevailing compounds of human fluids. In this paper, we show that elemental encoded particle assay coupled with femtosecond laser-induced breakdown spectroscopy for simultaneous multi-elemental analysis can significantly improve biomarker detectability. An estimated near single molecule per particle efficiency of this method leads to sensitive detection of ovarian cancer biomarker CA125 in human blood plasma. This work opens new ways for earlier detection of cancers and for multiplex assay developments in various analytical applications from proteomics, genomics, and neurology fields.
Keywords: Femtosecond laser-induced breakdown spectroscopy; Ovarian cancer biomarker; CA 125; In vitro diagnostics; Element encoded immunoassay; Magnetic separation

O-glycosylation-site characterization of individual glycoproteins is a major challenge because of the heterogeneity of O-glycan core structures. In proteomic studies, O-glycosylation-site analysis is even more difficult because of the complexity of the sample. In this work, we designed a rapid and convenient workflow for characterizing the O-glycosylation sites of individual proteins and the human-plasma proteome. A mixture of exoglycosidases was used to partially remove O-glycan chains and leave an N-acetylgalacosamine (GalNAc) residue attached to the Ser or Thr residues. The O-glycosylated peptides could then be identified by using liquid chromatography–tandem mass spectrometry (LC–MS–MS) to detect the 203 Da mass increase. Jacalin was used to selectively isolate O-GalNAc glycopeptides before LC–MS–MS analysis, which is optional for individual proteins and necessary for complex human-plasma proteins. Bovine fetuin and human chorionic gonadotropin (hCG) were used to test the analytical workflow. The workflow indicated superior sensitivity by not only covering most previously known O-glycosylation sites but also discovering several novel sites. Using only one drop of blood, a total of 49 O-GalNAc-linked glycopeptides from 36 distinctive glycoproteins in human plasma were identified unambiguously. The approach described herein is simple, sensitive, and global for site analysis of core 1 through core 4 O-glycosylated proteins.
Keywords: O-glycosylation site; hCG; Human-plasma proteins; Mass spectrometry; Exoglycosidases; Jacalin

High-resolution mass spectrometry provides novel insights into products of human metabolism of organophosphate and brominated flame retardants by Mohamed Abou-Elwafa Abdallah; Jinkang Zhang; Gopal Pawar; Mark R. Viant; J. Kevin Chipman; Kyle D’Silva; Maciej Bromirski; Stuart Harrad (1871-1883).
The high resolution, accurate mass, and fast scanning features of the OrbitrapTM mass spectrometer, combined with the separation power of ultrahigh-performance liquid chromatography were applied for the first time to study the metabolic profiles of several organic flame retardants (FRs) present in indoor dust. To mimic real-life exposure, in vitro cultured HepG2 human hepatocyte cell lines were exposed simultaneously to various FRs in an indoor dust extract for 24 h. Target parent FRs, hexabromocyclododecanes (α-, β-, and γ-HBCDs), tris-2-chloroethyl phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCIPP), and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), were separated in a single run for the first time using alternating positive and negative heated ESI source. Further metabolite separation and identification was achieved using full scan (70,000 full width at half maximum (FWHM)), accurate mass (up to 1 ppm) spectrometry. Structural confirmation was performed via all ion fragmentation (AIF) spectra using the optional higher collisional dissociation (HCD) cell and MS/MS analysis. First insights into human metabolism of HBCDs revealed several hydroxylated and debrominated phase I metabolites, in addition to conjugated phase II glucuronides. Furthermore, various hydroxylated, oxidized, and conjugated metabolites of chlorinated phosphorous FRs were identified, leading to the suggestion of α-oxidation as a significant metabolic pathway for these compounds. Graphical abstract Orbitrap™-MS provides novel insights into human metabolism of flame retardants
Keywords: High-resolution mass spectrometry; Orbitrap; Metabolism; Indoor dust; HBCD; Organophosphate flame retardants

Blood cells, particularly erythrocytes, present a significant compartment for distribution of drugs and endogenous compounds and have been suggested to be factored in pharmacokinetic and pharmacodynamic evaluations. We previously detected the binding of polyphenols to red blood cells and found indications for a facilitated uptake of the bioactive procyanidin metabolite δ-(3,4-dihydroxy-phenyl)-γ-valerolactone (M1) into human erythrocytes. The purpose of the present investigation was to develop an effective, sensitive and robust liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify low concentrations of polyphenols in human blood cells. Various sample preparation methods including classic sample clean-up techniques and variations of the QuEChERS (quick, easy, cheap, effective, rugged and safe) approach were compared regarding compound recovery, matrix effects and overall process efficiency. The QuEChERS technique which involves a liquid-liquid extraction and clean-up by dispersive solid-phase extraction yielded best results. The method was fully validated for the six analytes: (+)-catechin, ferulic acid, M1, taxifolin, caffeic acid and δ-3-methoxy-4-hydroxy-phenyl- γ-valerolactone (M2) in human blood cells with an optimised QuEChERS sample preparation and prior enzymatic hydrolysis of analyte conjugates. The lower limits of quantification for the analytes ranged from 0.12 ng/mL for M1, M2 and taxifolin to 48.40 ng/mL for caffeic acid. The application of the method to a blood cell sample of a volunteer ingesting 100 mg/day of the standardised pine bark extract Pycnogenol® over the course of 3 weeks revealed measurable steady-state concentrations of catechin, M1, taxifolin, ferulic acid and M2. To our knowledge, this is the first report of using the QuEChERS approach for detection and quantification of plant-derived compounds in human blood cells. The method can be applied in pharmacokinetic studies to determine the distribution of polyphenols and their metabolites in human whole blood, blood cells or erythrocytes. This might contribute in gaining deeper insights into the in vivo distribution of polyphenols and their metabolites. Graphical abstract Preparation of human blood cell samples using the QuEChERS (quick, easy, cheap, effective, rugged and safe) approach. A Cells before centrifugation immediately after addition of the extraction solvent and salts. B Cells after centrifugation. A decolorized supernatant is obtained, which can be subsequently purified by dispersive solid-phase extraction (d-SPE).
Keywords: QuEChERS; Pine bark; Human; Procyanidins; Metabolism

Cross-platform metabolic profiling: application to the aquatic model organism Lymnaea stagnalis by Sara Tufi; Marja H. Lamoree; Jacob De Boer; Pim E. G. Leonards (1901-1912).
The freshwater pond snail Lymnaea stagnalis is used in several studies on molecular and behavioral neurobiology and ecotoxicology showing its successful application as a model organism. In the present study, a cross-platform metabolomic approach has been evaluated to characterize the organ molecular phenotypes of L. stagnalis central nervous system (CNS), digestive gland (DG), and albumen gland (AG). Two types of tissue disruption methods were evaluated of which beads beating was the preferred method. To obtain a broad picture of the hydrophilic and lipophilic metabolome, two complementary analytical platforms have been employed: liquid chromatography (LC) and gas chromatography (GC) coupled to high-resolution mass spectrometry. Furthermore, to increase the power to separate small polar metabolites, hydrophilic interaction liquid chromatography (HILIC) was applied. The analytical platform performances have been evaluated based on the metabolome coverage, number of molecular features, reproducibility, and multivariate data analysis (MVDA) clustering. This multiplatform approach is a starting point for future global metabolic profiling applications on L. stagnalis.
Keywords: Metabolomics; Lymnaea stagnalis ; Cross-platform; LC-MS; GC-MS; HILIC

Application of magnetic iron oxide nanoparticles for the analysis of PCBs in water and soil leachates by gas chromatography–tandem mass spectrometry by Rosa Ana Pérez; Beatriz Albero; José Luis Tadeo; Encarnación Molero; Consuelo Sánchez-Brunete (1913-1924).
Two magnetic solid-phase extraction methods (mSPE) were developed and compared for the extraction and preconcentration of polychlorinated biphenyls (PCBs) from water and soil leachates. Analyses were carried out by gas chromatography coupled to triple quadrupole mass spectrometry. The mSPE extraction parameters were optimised using Fe3O4 nanoparticles coated with palmitate or oleate. Differences were found between the developed mSPE methods depending on the magnetic nanoparticle coating. The extraction efficiency of both sorbents was studied by spiking soil leachates at three concentration levels (from 0.6 to 0.18 ng ml−1 and from 0.4 to 0.04 ng ml−1 using palmitate or oleate coated nanoparticles, respectively) and recoveries from 86 to 109 % were obtained. The developed method provided a preconcentration factor of 250. The detection limits were about 29 times lower with the oleate-coated nanoparticles. Although both mSPE procedures could be used for the extraction of PCBs from water and soil leachates, oleate-coated nanoparticles gave the best extractive conditions and lower quantifications limits. Finally, the mSPE using oleate-coated nanoparticles was applied to the analysis of PCBs in river waters and in soil leachates obtained from soil with different physico-chemical characteristics. The levels of PCBs present in the leachates depended on the soil sample. The present work demonstrates the applicability of both mSPE methods to the determination of PCBs in water and soil leachates, which is of interest for mobility and bioavailability studies of these compounds in soil. Graphical Abstract Determination of PCBs by mSPE and GC–MS/MS
Keywords: Magnetic solid-phase extraction; Magnetic nanoparticles; Gas chromatography–tandem mass spectrometry; Polychlorinated biphenyls; Water; Soil leachate

The isotopic double-spike method allows for the determination of stable isotope ratios by multi-collector inductively coupled plasma-mass spectrometry (MC-ICP-MS) with accuracy and precision in the range of ∼0.02 ‰ amu−1, but its adoption has been hindered by the perceived difficulties in double-spike calibration and implementation. To facilitate the implementation of the double-spike approach, an explanation of the calibration and validation of a 97Mo-100Mo double-spike protocol is given in more detail than has been presented elsewhere. The long-term external standard reproducibility is 0.05 ‰ on δ98/95Mo measurements of standards. δ98/95Mo values for seawater and U.S. Geological Survey (USGS) reference materials SDO-1 and BCR-2 measured in this study are 2.13 ± 0.04 ‰ (2 SD, n = 3), 0.79 ± 0.05 ‰ (2 SD, n = 11), and −0.04 ± 0.10 ‰ (2 SD, n = 3) relative to the NIST-SRM-3134. The double-spike method corrects for laboratory and instrumental fractionation which are not accounted for using other mass bias correction methods. Spike/sample molar ratios between 0.4 and 0.8 provide accurate isotope measurements; outside of this range, isotope measurements are inaccurate but corrections are possible when standards and samples are spiked at a similar ratio.
Keywords: Molybdenum; Double-spike; MC-ICP-MS; Stable isotopes; Reference material

Integrated comparative metabolite profiling via MS and NMR techniques for Senna drug quality control analysis by Mohamed A. Farag; Andrea Porzel; Engy A. Mahrous; Mo’men M. El-Massry; Ludger A. Wessjohann (1937-1949).
Senna alexandrina Mill (Cassia acutifolia and Cassia angustifolia) are used for the laxative medicine Senna. Leaves and pods from two geographically different sources were distinguished from each other via proton nuclear magnetic resonance (1H-NMR) and ultra performance liquid chromatography-mass spectrometry (UPLC-MS) analysis. Under optimized conditions, we were able to simultaneously quantify and identify 107 metabolites including 21 anthraquinones, 24 bianthrones (including sennosides), 5 acetophenones, 25 C/O-flavonoid conjugates, 5 xanthones, 3 naphthalenes, 2 further phenolics, and 9 fatty acids. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) were used to define both similarities and differences among samples. For sample classification based on tissue type (leaf and pod), both UPLC-MS and NMR were found to be more effective in separation than on geographical origin. Results reveal that the amounts of the major classes of bioactives in Senna, i.e., flavonoids and sennosides, varied significantly among organs. Leaves contained more flavonoids and ω-3 fatty acids but fewer benzophenone derivatives than pods. In contrast, pods were more enriched in bianthrones (sennosides). PCA analysis was found to be equally effective in predicting the origin of the commercial Senna preparation using NMR and/or UPLC-MS datasets. Furthermore, a selection of six so far uninvestigated Senna species were analyzed by UPLC-MS. Results revealed that the Senna alata leaf in terms of secondary metabolite composition is the most closely related species to S. alexandrina Mill, showing the highest levels of the anthraquinone “rhein” and flavonoid conjugates. To the best of our knowledge, this study provides the first approach utilizing both UPLC-MS and NMR to reveal secondary metabolite compositional differences among Senna species.
Keywords: Senna alexandrina ; S. bicapsularis ; S. corymbosa ; S. didymobotrya ; S. alata ; S. sophera ; NMR; UPLC-MS; Sennosides; Principal component analysis; Laxative

Electrochemical sensor for determination of tulathromycin built with molecularly imprinted polymer film by Jiadi Sun; Jian Ji; Yifan Wang; Ying Zhao; Yinzhi Zhang; Xiulan Sun (1951-1959).
A novel tulathromycin (TLTMC) electrochemical sensor based on molecularly imprinted polymer (MIP) membranes was constructed. p-Aminothiophenol (p-ATP) and TLTMC were assembled on the surface of gold nanoparticles (AuNPs) modified on the gold electrode (GE) by the formation of Au-S bonds and hydrogen-bonding interactions. Besides, polymer membranes were formed by electropolymerization in a polymer solution containing p-ATP, tetrachloroaurate(III) acid (HAuCl4), tetrabutylammonium perchlorate (TBAP), and a template molecule TLTMC. A novel molecular imprinted sensor (MIS) in this experiment was achieved after the removal of TLTMC. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were used to illustrate the process of electropolymerization and its optimal conditions. The electrode with MIP obtained the linear of response range, which was between 3.0 × 10–12 mol L–1 and 7.0 × 10–9 mol L–1, and the limit of detection was 1.0 × 10–12 mol L–1. All the obtained results indicate that the MIS tends to be an effective electrochemical technique for the determination of TLTMC in real-time and in a complicated matrix. Graphical Abstract Schematic illustration of the facrication of the tulathromycin (TLTMC) electrochemical sensors
Keywords: Tulathromycin; Electrochemical sensors; Molecular imprinted polymer; Gold electrode

Direct determination of major components in human diets and baby foods by Mari Merce Cascant; Salvador Garrigues; Miguel de la Guardia (1961-1972).
A fast method has been developed for the determination of fat, proteins, carbohydrates, and energy value in baby food, infant fast food, and canteen menus, using near-infrared (NIR) and mid-infrared (MIR) spectroscopy measurements and multivariate calibration methods based on partial least square regression. Reference standard methods were employed to build and validate the infrared methods for direct determination of nutrients. Coefficients of determination obtained between predicted values and reference ones for total fat, proteins, carbohydrates, and energy value were 96.7, 98.1, 98.9, and 96.5 for NIR and 91.0, 93.0, 92.0, and 84.1 for MIR, respectively, with relative root mean square error of prediction (RRMSEP) below or equal to 9 % for NIR and 16 % for MIR. Results obtained indicate that both NIR and MIR techniques have good predictive capabilities, with the NIR method being the most accurate and simple.
Keywords: Near-infrared spectroscopy; Mid-infrared spectroscopy; Total fat; Proteins; Carbohydrates; Energy value; Human diet; Baby food; Infant fast food; Canteen menus; Partial least square (PLS)

A sensitive chemiluminescent immunoassay for point-of-care testing of repaglinide in natural dietary supplements and serum by Lei Zheng; Jing Wang; Yufen Wang; Zhaorui Song; Yaqing Dong; Yongmei Yin; Sergei A. Eremin; Meng Meng; Rimo Xi (1973-1980).
For point-of-care testing of the illegal fortification of repaglinide (Rep) in natural dietary supplements, a competitive chemiluminescent immunoassay (CLIA) was established, using a horseradish peroxidase (HRP)–luminol–H2O2 system for signal amplification. Polyclonal antibodies for Rep were produced via immunization technique. Following optimization of the enzyme reaction time and concentrations of antibody and coating antigen, the method showed a limit of quantification (LOQ) of 1.0 ng/mL in PBS and limit of detection (LOD) of 8.3 ng/mL in serum and 6.0 ng/mL in blank tablets. When applied in natural dietary supplements, the method provided results consistent with those from HPLC, suggesting that the proposed method could be used for rapid screening of Rep in natural dietary supplements and detecting Rep in serum after administration. Graphical Abstract A sensitive chemiluminescent immunoassay for rapid analysis of repaglinide in natural dietarysupplements and serum
Keywords: Repaglinide; Antibody; Chemiluminescent immunoassay; Natural dietary ingredients

A generic method for the quantification of type II collagen in protein-based dietary supplements is described. This quantitative analysis was conducted using liquid chromatography–electrospray ionization–time-of-flight mass spectrometry (LC–ESI–TOF MS). Compared to classical methods with the use of isotope-labeled standards, our method includes, for the first time, the quantification of hydroxyproline using histidine as an internal standard. Separation of the analytes was performed on a Phenomenex Synergi 4 μm Fusion-RP 80 Ǻ column (150 × 2.0 mm, 4.0 μm) with a mobile phase made of 10 mM ammonium formate in water (A) and 10 mM ammonium formate in methanol (B). The assay was fully validated according to FDA guidelines, and the method exhibited sufficient specificity, accuracy, and precision. Intra- and inter-batch accuracy, determined as a deviation between nominal and measured values, ranged from −4.8 to 9.1% and from 0.9 to 6.4 %, respectively. All analytes (hydroxyproline and histidine) at three concentration levels showed extraction recoveries from 89 to 98 %. The method was successfully applied to protein-based dietary supplements of the pharmaceutical industry.
Keywords: Collagen; Quantitative analysis; Method validation; Protein; LC–ESI–TOF MS; Pharmaceutical applications

A simple and highly sensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) method combined with solid-phase extraction cleanup was established for the comprehensive determination of 16 Environmental Protection Agency (EPA) polycyclic aromatic hydrocarbons (PAHs) in various kinds of Chinese herbal medicines (CHMs). A solid-phase extraction (SPE) purification strategy, including three parallel procedures, was developed depending on sample type, and satisfactory purification performances were achieved for all selected CHMs. The limits of detection ranged from 0.12 to 1.08 μg kg–1 for the analyzed PAHs. The average recoveries were in the range of 65.9 % to 100.8 %, except for naphthalene (43.8 %–75.9 %), and the relative standard deviations were ≤12.8 %. The proposed method was successfully applied to the analysis of PAHs in 24 CHMs including five roots, three stems, four flowers, two fruits, four seeds, three leaves, and three barks. In the samples analyzed, all 16 PAHs are present. Their sum ranges from 21.1 to 2236.3 μg kg–1. The entire procedure was shown to be effective and conveniently fast, and may serve as an alternative screening protocol for the determination of PAHs in CHMs. Graphical Abstract Representative CHMs of seven types and the purification performances compared before and after cleanup
Keywords: Polycyclic aromatic hydrocarbon; Chinese herbal medicine; Solid-phase extraction; Gas chromatography-tandem mass spectrometry

Matrix augmentation is regularly employed in extended multivariate curve resolution-alternating least-squares (MCR-ALS), as applied to analytical calibration based on second- and third-order data. However, this highly useful concept has almost no correspondence in parallel factor analysis (PARAFAC) of third-order data. In the present work, we propose a strategy to process third-order chromatographic data with matrix fluorescence detection, based on an Augmented PARAFAC model. The latter involves decomposition of a three-way data array augmented along the elution time mode with data for the calibration samples and for each of the test samples. A set of excitation–emission fluorescence matrices, measured at different chromatographic elution times for drinking water samples, containing three fluoroquinolones and uncalibrated interferences, were evaluated using this approach. Augmented PARAFAC exploits the second-order advantage, even in the presence of significant changes in chromatographic profiles from run to run. The obtained relative errors of prediction were ca. 10 % for ofloxacin, ciprofloxacin, and danofloxacin, with a significant enhancement in analytical figures of merit in comparison with previous reports. The results are compared with those furnished by MCR-ALS. Graphical Abstract A new modeling strategy for third-order data
Keywords: Liquid chromatography; Third-order data; Fluoroquinolones; Water samples; Augmented parallel factor analysis

An automatic phenolic compounds analyzer is presented. The system performs online magnetic-stirring-assisted dispersive liquid–liquid microextraction before multisyringe chromatography (MSC) using a monolithic Chromolith Flash RP-18e column. The extraction behavior of the following phenolic pollutants: phenol, 2-nitrophenol, 4-nitrophenol, 2-chlorophenol, 2,4-diclorophenol, and 2,4,6-trichlorophenol, has been studied. A critical comparison of extractants (tributyl phosphate, acetonitrile, hexane, and 1-chlorobutane) and disperser solvents (acetone, acetonitrile, ethanol, methanol, 1-propanol, and 2-propanol) was made. Tributyl phosphate and acetonitrile were chosen as the extractant and the disperser solvent, respectively, since these showed the best performance. Phenols were online back-extracted into NaOH and neutralized before multi-isocratic chromatographic separation. The proposed analyzer can be applied for wide linear working ranges, i.e., between 40 and 20,000 μg L-1. The precision of the developed system has been proved, with maximum values for the intraday and interday precision of 4.4 % and 5.2 %, respectively, expressed as relative standard deviation, and high preconcentration factors (9.3–10.5) for most of the compounds studied. The method developed was successfully applied to natural water samples.
Keywords: Magnetic-stirring-assisted dispersive liquid–liquid microextraction; Multisyringe chromatography; Phenolic compounds; Environmental samples; Automation