Analytical and Bioanalytical Chemistry (v.406, #28)

is a post-doc in the Laboratory of Bioanalytical Chemistry at the Faculty of Chemistry, Adam Mickiewicz University in Poznan, Poland. She received her Ph.D. in Chemistry and BSc in Biotechnology from Adam Mickiewicz University. Her research is focused on DNAzymes with peroxidase activity and their bioanalytical applications. received her MSc degree from Paris-Sud University after completing the two-year master’s program SERP-Chem. During her master’s project, which she performed in the Laboratory of Bioanalytical Chemistry at the Faculty of Chemistry, Adam Mickiewicz University, she worked with fluorogenic substrate for peroxidase-mimicking DNAzyme. is an adjunct in the Laboratory of Bioanalytical Chemistry at the Faculty of Chemistry, Adam Mickiewicz University. Her research interests include the synthesis of new carbazole ligands, potential inhibitors of telomerase, and the study of ligand interactions with different DNA structures. is a Head of Laboratory of Bioanalytical Chemistry at the Faculty of Chemistry, A. Mickiewicz University in Poznan, Poland. His research interests include application of fluorescence, fluorescence quenching, photoisomerization, and FRET processes for sensing purposes, synthesis of selective ligands for G-quadruplex binding, and development of oligonucleotide-based fluorescent-sensing probes including DNAzymes. Characterization and optimization studies of N-methyl-4-hydrazino-7-nitrobenzofurazan (MNBDH) as a new fluorogenic substrate in the peroxidation reaction catalyzed by DNAzyme are reported. The effects of pH, H2O2 concentration, metal-cation type, and the concentration and type of surfactant on the fluorescence intensity were investigated. The optimized reaction was subsequently used for the development of an assay for DNA detection based on a molecular-beacon probe. The use of a fluorogenic substrate enabled the detection of a single-stranded DNA target with a 1 nmol L−1 detection limit. Graphical Abstract ᅟ
Keywords: Bioanalytical methods; Fluorescence; Nucleic acids; Molecular beacon; DNAzyme

Nile-red and Nile-blue-based near-infrared fluorescent probes for in-cellulo imaging of hydrogen sulfide by Xiao-Dong Liu; Chen Fan; Ru Sun; Yu-Jie Xu; Jian-Feng Ge (7059-7070).
was a master’s course student in Professor Ge’s lab from Soochow University. His research interests include the design and synthesis of fluorescent materials. is studying for a master’s at Soochow University, China. Her research interest is preparation and characterization of nuclei targeting fullerenol nanostructured lipid carriers. is an associate professor, and received her Ph.D. degree in 2008 from Soochow University, China. Her main interests are in spectra analysis and quantum chemical calculations. is an associate professor of Soochow University, China. His principal areas of research are tracer experiments of biomolecules or chemicals with radioisotopes and other fluorescent dyes, special radioprotectors for nuclear accidents, and nanoparticle delivery systems for targeting therapy to human tumors. received his Ph.D. degree in 2003 from Soochow University, China. He is a full professor of applied chemistry and is engaged in research on functional dyes for biological and material purposes. Hydrogen sulfide has recently been identified as a biologically responsive species. The design and synthesis of fluorescence probes, which are constructed with Nile-red or Nile-blue fluorophores and a fluorescence-controllable dinitrophenyl group, for hydrogen sulfide are reported in this paper. The Nile-red–dinitrophenyl-ether-group-based probe (1a) is essentially non-fluorescent because of the inhibition of the photo-induced electron-transfer process; when the dinitrobenzene moiety is removed by nucleophilic substitution with the hydrosulfide anion, probe 1a is converted into hydroxy Nile red, eliciting a H2S-induced fluorescence turn-on signal. Furthermore, probe 1a has high selectivity and sensitivity for the hydrosulfide anion, and its potential for biological applications was confirmed by using it for real-time fluorescence imaging of hydrogen sulfide in live HeLa cells. The Nile-blue–dinitrobenzene-based probe (1b) has gradually diminishing brightness in the red-emission channel with increased hydrogen-sulfide concentration. Thus, this paper reports a comparative study of Nile-red and Nile-blue-based hydrogen-sulfide probes. Graphical Abstract ᅟ
Keywords: Chemo sensor; Optical probes; Near-infrared; Nile red and Nile blue; Hydrogen sulfide

Same-single-cell analysis using the microfluidic biochip to reveal drug accumulation enhancement by an amphiphilic diblock copolymer drug formulation by Avid Khamenehfar; Chung Ping Leon Wan; Paul C. H. Li; Kevin Letchford; Helen M. Burt (7071-7083).
Multidrug resistance (MDR) is one of the major obstacles in drug delivery, and it is usually responsible for unsuccessful cancer treatment. MDR may be overcome by using MDR inhibitors. Among different classes of these inhibitors that block drug efflux mediated by permeability-glycoprotein (P-gp), less toxic amphiphilic diblock copolymers composed of methoxypolyethyleneglycol-block-polycaprolactone (MePEG-b-PCL) have been studied extensively. The purpose of this work is to evaluate how these copolymer molecules can reduce the efflux, thereby enhancing the accumulation of P-gp substrates (e.g., daunorubicin or DNR) in MDR cells. Using conventional methods, it was found that the low-molecular-weight diblock copolymer, MePEG17-b-PCL5 (PCL5), enhanced drug accumulation in MDCKII-MDR1 cells, but the high-molecular-weight version, MePEG114-b-PCL200 (PCL200), did not. However, when PCL200 was mixed with PCL5 (and DNR) in order to encapsulate them to facilitate drug delivery, there was no drug enhancement effect attributable to PCL5, and the reason for this negative result was unclear. Since drug accumulation measured on different cell batches originated from single cells, we employed the same-single-cell analysis in the accumulation mode (SASCA-A) to find out the reason. A microfluidic biochip was used to select single MDR cells, and the accumulation of DNR was fluorescently measured in real time on these cells in the absence and presence of PCL5. The SASCA-A method allowed us to obtain drug accumulation information faster in comparison to conventional assays. The SASCA-A results, and subsequent curve-fitting analysis of the data, have confirmed that when PCL5 was encapsulated in PCL200 nanoparticles as soon as they were synthesized, the ability of PCL5 to enhance DNR accumulation was retained, thus suggesting PCL200 as a promising delivery system for encapsulating P-gp inhibitors, such as PCL5. Graphical Abstract ᅟ
Keywords: Same-single-cell analysis; Multidrug resistance; Microchip; Amphiphilic diblock copolymer

Real-time quantification of protein expression and translocation at individual cell resolution using imaging-dish-based live cell array by Orit Ravid-Hermesh; Naomi Zurgil; Yana Shafran; Maria Sobolev; Moti Galmidi; Yoav Badihi; Liron Limor Israel; Jean Paul Lellouche; Emmanuel Lellouche; Shulamit Michaeli; Mordechai Deutsch (7085-7101).
Cell populations represent intrinsically heterogeneous systems with a high level of spatiotemporal complexity. Monitoring and understanding cell-to-cell diversity is essential for the research and application of intra- and interpopulation variations. Optical analysis of live cells is challenging since both adherent and nonadherent cells change their spatial location. However, most currently available single-cell techniques do not facilitate treatment and monitoring of the same live cells over time throughout multistep experiments. An imaging-dish-based live cell array (ID-LCA) has been developed and produced for cell handling, culturing, and imaging of numerous live cells. The dish is composed of an array of pico scale cavities—pico wells (PWs) embossed on its glass bottom. Cells are seeded, cultured, treated, and spatiotemporally measured on the ID-LCA, while each cell or small group of cells are locally constrained in the PWs. Finally, predefined cells can be retrieved for further evaluation. Various types of ID-LCAs were used in this proof-of-principle work, to demonstrate on-ID-LCA transfection of fluorescently tagged chimeric proteins, as well as the detection and kinetic analysis of their induced translocation. High variability was evident within cell populations with regard to protein expression levels as well as the extent and dynamics of protein redistribution. The association of these parameters with cell morphology and functional parameters was examined. Both the new methodology and the device facilitate research of the translocation process at individual cell resolution within large populations and thus, can potentially be used in high-throughput fashion. Graphical Abstract ᅟ
Keywords: Fluorescence; Microfabrication; Biochip; Single-cell analysis; Imaging; Kinetics

Nano-LC-MS/MS for the quantitation of prostanoids in immune cells by D. Thomas; J. Suo; T. Ulshöfer; H. Jordan; N. de Bruin; K. Scholich; G. Geisslinger; N. Ferreirós (7103-7116).
Prostanoids, derivatives of arachidonic acid, are involved in inflammation and immune reactions. To understand the role of prostanoids produced by diverse immune cells, a highly sensitive quantitation method for prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), 6-keto prostaglandin F (6-keto PGF), prostaglandin F (PGF), and thromboxane B2 (TXB2) by means of nano-liquid chromatography-tandem mass spectrometry has been developed. It was validated according to the guidelines of the Food and Drug Administration (FDA) in terms of linearity, precision, accuracy, recovery, stability, and lower limit of quantitation (LLOQ). The LLOQ were 25 pg/mL in the injected solution (75 fg on column (o.c.)) for PGE2 and PGD2 and 37.5 pg/mL (112.5 fg on column) for 6-keto PGF, PGF, and TXB2, respectively. It was successfully applied to murine mast cells isolated from paws after zymosan injection and to CD4+ and CD8+ T lymphocytes from blood of sensitized versus non-sensitized mice in context of a delayed type hypersensitivity model. About 5,000 (T cells) to 40,000 (mast cells) cells were sufficient for quantitation. In the mast cells, the production of PGE2 increased at a significantly higher extent than the synthesis of the other prostanoids. The T lymphocytes did not show any difference in prostanoid production, no matter whether they were obtained from sensitized mice or non-sensitized mice.
Keywords: Nano-LC; Tandem mass spectrometry; Prostanoids; Immune cells

Microscale depletion of high abundance proteins in human biofluids using IgY14 immunoaffinity resin: analysis of human plasma and cerebrospinal fluid by Seok-Won Hyung; Paul D. Piehowski; Ronald J. Moore; Daniel J. Orton; Athena A. Schepmoes; Therese R. Clauss; Rosalie K. Chu; Thomas L. Fillmore; Heather Brewer; Tao Liu; Rui Zhao; Richard D. Smith (7117-7125).
Removal of highly abundant proteins in plasma is often carried out using immunoaffinity depletion to extend the dynamic range of measurements to lower abundance species. While commercial depletion columns are available for this purpose, they generally are not applicable to limited sample quantities (<20 μL) due to low yields stemming from losses caused by nonspecific binding to the column matrix and concentration of large eluent volumes. Additionally, the cost of the depletion media can be prohibitive for larger-scale studies. Modern LC-MS instrumentation provides the sensitivity necessary to scale-down depletion methods with minimal sacrifice to proteome coverage, which makes smaller volume depletion columns desirable for maximizing sample recovery when samples are limited, as well as for reducing the expense of large-scale studies. We characterized the performance of a 346 μL column volume microscale depletion system, using four different flow rates to determine the most effective depletion conditions for ∼6-μL injections of human plasma proteins and then evaluated depletion reproducibility at the optimum flow rate condition. Depletion of plasma using a commercial 10-mL depletion column served as the control. Results showed depletion efficiency of the microscale column increased as flow rate decreased, and that our microdepletion was reproducible. In an initial application, a 600-μL sample of human cerebrospinal fluid (CSF) pooled from multiple sclerosis patients was depleted and then analyzed using reversed phase liquid chromatography-mass spectrometry to demonstrate the utility of the system for this important biofluid where sample quantities are more commonly limited.
Keywords: Microscale depletion; IgY-14 immunoaffinity resin; Human plasma; Cerebrospinal fluid; MS

Ultrahigh-performance liquid chromatography has been used for the separation and analysis of unmodified and modified antisense oligonucleotides. For this reason, we tested various columns of low particle sizes in our analysis of unmodified and phosphorothioate oligonucleotides. The influence of both the type and concentration of ion-pair reagent on the retention of the studied biomolecules was tested. The developed methods were used for separation of unmodified oligonucleotides and to determine antisense oligonucleotides in human serum samples. The results proved that octadecyl and phenyl columns are the most selective in the resolution of oligonucleotides which differ in the position of single nucleotides in the sequence. The phenyl column was selected and applied for the analysis of phosphorothioate oligonucleotides in serum samples. The calibration plots showed good linearity within the test concentration ranges. The intra-day CV of the calibration curve slopes was in the range of 1.6 to 4.2 %. The limits of detection (LODs) were in the range of 0.11–0.16 μg mL−1, while the limit of quantification (LOQ) values were between 0.35 and 0.51 μg mL−1. Figure Determination of antisense phosphorothioate oligonucleotides in serum
Keywords: Oligonucleotides; Ultrahigh-performance liquid chromatography; Chromatographic column; Ion-pair chromatography; Separation; Human serum

Using direct infusion mass spectrometry for serum metabolomics in Alzheimer’s disease by R. González-Domínguez; T. García-Barrera; J. L. Gómez-Ariza (7137-7148).
Currently, there is no cure for Alzheimer’s disease and early diagnosis is very difficult, since no biomarkers have been established with the necessary reliability and specificity. For the discovery of new biomarkers, the application of omics is emerging, especially metabolomics based on the use of mass spectrometry. In this work, an analytical approach based on direct infusion electrospray mass spectrometry was applied for the first time to blood serum samples in order to elucidate discriminant metabolites. Complementary methodologies of extraction and mass spectrometry analysis were employed for comprehensive metabolic fingerprinting. Finally, the application of multivariate statistical tools allowed us to discriminate Alzheimer patients and healthy controls, and identify some compounds as potential markers of disease. This approach provided a global vision of disease, given that some important metabolic pathways could be studied, such as membrane destabilization processes, oxidative stress, hypometabolism, or neurotransmission alterations. Most remarkable results are the high levels of phospholipids containing saturated fatty acids, respectively, polyunsaturated ones and the high concentration of whole free fatty acids in Alzheimer’s serum samples. Thus, these results represent an interesting approximation to understand the pathogenesis of disease and the identification of potential biomarkers. Graphical Abstract ᅟ
Keywords: Alzheimer’s disease; Direct infusion mass spectrometry; Metabolomics; Biomarkers

Validation of the Endopep-MS method for qualitative detection of active botulinum neurotoxins in human and chicken serum by Kristian Björnstad; Annica Tevell Åberg; Suzanne R. Kalb; Dongxia Wang; John R. Barr; Ulf Bondesson; Mikael Hedeland (7149-7161).
Botulinum neurotoxins (BoNTs) are highly toxic proteases produced by anaerobic bacteria. Traditionally, a mouse bioassay (MBA) has been used for detection of BoNTs, but for a long time, laboratories have worked with alternative methods for their detection. One of the most promising in vitro methods is a combination of an enzymatic and mass spectrometric assay called Endopep-MS. However, no comprehensive validation of the method has been presented. The main purpose of this work was to perform a validation for the qualitative analysis of BoNT-A, B, C, C/D, D, D/C, and F in serum. The limit of detection (LOD), selectivity, precision, stability in matrix and solution, and correlation with the MBA were evaluated. The LOD was equal to or even better than that of the MBA for BoNT-A, B, D/C, E, and F. Furthermore, Endopep-MS was for the first time successfully used to differentiate between BoNT-C and D and their mosaics C/D and D/C by different combinations of antibodies and target peptides. In addition, sequential antibody capture was presented as a new way to multiplex the method when only a small sample volume is available. In the comparison with the MBA, all the samples analyzed were positive for BoNT-C/D with both methods. These results indicate that the Endopep-MS method is a valid alternative to the MBA as the gold standard for BoNT detection based on its sensitivity, selectivity, and speed and that it does not require experimental animals.
Keywords: Botulinum neurotoxin; Endopep-MS; Method validation; Botulism; MALDI-Q-TOF

Isotachophoresis (ITP) has long been used alone but also as a preconcentration technique for capillary electrophoresis (CE). Unfortunately, up to now, its application is restricted to relatively strong acids and bases as either the degree of (de)protonation is too low or the water dissociation is too high, evoking zone electrophoresis. With the comprehensive ITP analysis of all 20 proteinogenic amino acids as model analytes, we, here, show that non–aqueous ITP using dimethylsulfoxide as a solvent solves this ITP shortcoming. Dimethylsulfoxide changes the pH regime of analytes and electrolytes but, more importantly, strongly reduces the proton mobility by prohibiting hydrogen bonds and thus, the so-called Zundel–Eigen–Zundel electrical conduction mechanism of flipping hydrogen bonds. The effects are demonstrated in an electrolyte system with taurine or H+ as terminator, and imidazole as leader together with strong acids such as oxalic and even trifluoroacetic acid as counterions, both impossible to use in aqueous solution. Mass spectrometric as well as capacitively coupled contactless conductivity detection (C4D) are used to follow the ITP processes. To demonstrate the preconcentration capabilities of ITP in a two-dimensional set-up, we, here, also demonstrate that our non-aqueous ITP method can be combined with capillary electrophoresis–mass spectrometry in a column-coupling system using a hybrid approach of capillaries coupled to a microfluidic interface. For this, C4D was optimized for on-chip detection with the electrodes aligned on top of a thin glass lid of the microfluidic chip.
Keywords: Non-aqueous isotachophoresis; Capacitively coupled contactless conductivity detection; ITP/CE-MS; Weak bases; Amino acids

An organic-inorganic hybrid monolithic column based on 1-vinyl-3-dodecylimidazolium bromide (VC12Im+Br) has been prepared in a single step by combining radical copolymerization with a non-hydrolytic sol-gel (NHSG) process. The NHSG process was significantly shortened to 6 h by using formic acid as catalyst. For comparison, we also prepared polymeric ionic liquid (PIL) monolithic columns by hydrolytic sol-gel and organic polymeric process, respectively. The resulting monolithic columns were characterized by Fourier transform infrared spectra, scanning electron microscopy, and Brunauer-Emmett-Teller. Under the capillary electrochromatography mode, these columns were applied to separate alkylbenzenes, anilines, and proteins, respectively. The results indicated that the NHSG-based hybrid PIL monolithic column exhibited the highest column efficiency among the three types of columns; organic solvent, commonly required by the traditional columns to achieve satisfactory separation efficiency for proteins, was absent in the NHSG-based hybrid PIL monolithic column because of the biocompatibility of the VC12Im+Br, which was beneficial to analysis of protein containing samples. In order to demonstrate its application potential, the developed NHSG-based hybrid PIL monolithic column was also employed to separate egg white sample.
Keywords: Capillary electrochromatography; Hybrid monolithic column; Non-hydrolytic sol-gel; Protein separation

Identification of 34 N-glycan isomers in human serum by capillary electrophoresis coupled with laser-induced fluorescence allows improving glycan biomarker discovery by Christian Schwedler; Matthias Kaup; Stefan Weiz; Maria Hoppe; Elena Iona Braicu; Jalid Sehouli; Berthold Hoppe; Rudolf Tauber; Markus Berger; Véronique Blanchard (7185-7193).
Alterations in glycosylation have been observed in many human diseases and specific changes in glycosylation have been proposed as relevant diagnostic information. Capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) is a robust method to quantify desialylated N-glycans that are labeled with 8-aminopyrene-1,3,6-trisulfonic acid prior to analysis. To date, only a maximum of 12 glycan structures, the most abundant ones, have been identified by CE-LIF to characterize glycome modulations of total serum in the course of the diseases. In most forms of cancer, findings using CE-LIF were limited to the increase of triantennary structures carrying a Lewisx epitope. In this work, we identified 32 linkage and positional glycan isomers in healthy human serum using exoglycosidase digestions as well as standard glycoproteins, for which we report the assignment of novel structures. It was possible to identify and quantify 34 glycan isomers in the serum of primary epithelial ovarian cancer patients (EOC). Reduced levels of diantennary structures and of high-mannose 5 were statistically significant in the EOC samples, and also, elevated branching as well as increased antennary fucosylation were observed. For the first time, we could demonstrate that not only antennary fucosylation was of relevance in tetraantennary structures but also core-fucosylated tetraantennary N-glycans were statistically increased in EOC patients. The results of the current study provide an improved dataset to be used in glycan biomarker discovery. Graphical abstract ᅟ
Keywords: CE-LIF; N-glycan; Isomer; Assignment; Biomarker discovery

Fluorescent protein-based FRET sensor for intracellular monitoring of redox status in bacteria at single cell level by Bobin George Abraham; Ville Santala; Nikolai V. Tkachenko; Matti Karp (7195-7204).
Monitoring of intracellular redox status in a bacterial cell provides vital information about the physiological status of the cell, which can be exploited in several applications such as metabolic engineering and computational modeling. Fluorescent protein-based genetically encoded sensors can be used to monitor intracellular oxidation/reduction status. This study reports the development of a redox sensor for intracellular measurements using fluorescent protein pairs and the phenomenon of Förster resonance energy transfer (FRET). For the development of the sensor, fluorescent proteins Citrine and Cerulean were genetically modified to carry reactive cysteine residues on the protein surface close to the chromophore and a constructed FRET pair was fused using a biotinylation domain as a linker. In oxidized state, the FRET pairs are in close proximity by labile disulfide bond formation resulting in higher FRET efficiency. In reducing environment, the FRET is diminished due to the increased distance between FRET pairs providing large dynamic measurement range to the sensor. Intracellular studies in Escherichia coli mutants revealed the capability of the sensor in detecting real-time redox variations at single cell level. The results were validated by intensity based and time resolved measurements. The functional immobilization of the fluorescent protein-based FRET sensor at solid surfaces for in vitro applications was also demonstrated. Graphical Abstract Schematic representation of FRET-based redox sensor
Keywords: Biosensors; FRET; Fluorescent proteins; Redox; Site-directed mutagenesis

Enzyme-linked immunosorbent assays are routinely used in laboratories around the world and ensure highly specific protein detection. Often, more than one analyte needs to be determined in a single sample and numerous protein arrays for multianalyte detection of a single sample have been developed to address this problem. They have the potential to analyze several dozen or even more analytes in an assay volume of usually around 100 μL. However, due to the presence of numerous different antibodies, these multianalyte sandwich immunoassays suffer from undesired cross-reactivities between the antibodies which lead to a loss of assay specificities. Here, we present an assay principle which allows, e.g., a detection of an analyte in a sample volume of only 1 μL in a normal 96-microtiter well plate, so that up to 100 analytes can be determined from a 100 μL sample volume, but in separate wells. This eliminates antibody cross-reactivities. The assay is based on the biotinylated time-resolved fluorophore EuLH used as a PEG11-dye conjugate in combination with ExtrAvidin® to ensure high signal-to-background ratios. The model protein epidermal growth factor (EGF) was detected with the established sandwich immunoassay and showed assay parameters comparable to commercially available ones. Furthermore, the assay principle enables a spatial resolution of the assay signal. Here, we demonstrated the application of the new detection system for universal imaging-based analysis of individual spots in one single 96-microtiter well by applying it to multisample and also multianalyte detections. In the case of the multisample analysis approach, a considerable reduction of the required sample volume to only 1 μL in a single 96 microtiter well could be achieved.
Keywords: Immunoassay; Solid-phase detection; Imaging; Time-resolved fluorescence; Bioanalytical methods; Bioassays

A core-shell surface magnetic molecularly imprinted polymers with fluorescence for λ-cyhalothrin selective recognition by Lin Gao; Jixiang Wang; Xiuying Li; Yongsheng Yan; Chunxiang Li; Jianming Pan (7213-7220).
In this study, we report here a general protocol for making core-shell magnetic Fe3O4/SiO2-MPS/MIPs (MPS = 3-(methacryloxyl) propyl trimethoxysilane, MIPs = molecularly imprinted polymers, Fe3O4/SiO2-MPS as core, MIPs as shell) via a surface molecular imprinting technique for optical detection of trace λ-cyhalothrin. The fluorescent molecularly imprinted polymer shell was first prepared by copolymerization of acrylamide with a small quantity of allyl fluorescein in the presence of λ-cyhalothrin to form recognition sites without doping. The magnetic Fe3O4/SiO2-MPS/MIPs exhibited paramagnetism, high fluorescence intensity, and highly selective recognition. Using fluorescence quenching as a detecting tool, Fe3O4/SiO2-MPS/MIPs were successfully applied to selectively and sensitively detect λ-cyhalothrin, and a linear relationship could be obtained covering a wide concentration range of 0–50 nM with a correlation coefficient of 0.9962 described by the Stern-Volmer equation. The experimental results of practical detection revealed that magnetic Fe3O4/SiO2-MPS/MIPs as an attractive recognition element was satisfactory for determination of trace λ-cyhalothrin in honey samples. This study, therefore, demonstrated the potential of MIPs for detection of λ-cyhalothrin in food.
Keywords: Polymers; Optical sensors; Fluorescence/luminescence

Thermal preparation of lysozyme-imprinted microspheres by using ionic liquid as a stabilizer by Li-Wei Qian; Xiao-Ling Hu; Ping Guan; Bo Gao; Dan Wang; Chao-Li Wang; Ji Li; Chun-Bao Du; Wen-Qi Song (7221-7231).
Thermal preparation of lysozyme-imprinted microspheres was firstly investigated by using biocompatible ionic liquid (IL) as a thermal stabilizer. The imprinted microspheres made with IL could obtain the good recognition ability to template protein, whereas the imprinted polymer synthesized in the absence of it had a similar adsorption capacity to the non-imprinted one. Furthermore, the preparation conditions of imprinted polymers (MIPs) including the content of IL, temperature of polymerization, and types of functional monomers and crosslinkers were systematically analyzed via circular dichroism spectrum and activity assay. The results illustrated that using hydroxyethyl acrylate as the functional monomer, ethylene glycol dimethacrylate as the crosslinker, 5 % IL as the stabilizer, and 75 °C as the reaction temperature could retain the structure of template protein as much as possible. The obtained MIPs showed excellent recognition ability to the template protein with the separation factor and selectivity factor value of 4.30 and 2.21, respectively. Consequently, it is an effective way to accurately imprint and separate template protein by cooperatively using circular dichroism spectroscopy and activity assay during the preparation of protein MIPs. The method of utilizing IL to stabilizing protein at high temperature would offer a good opportunity for various technologies to improve the development of macromolecules imprinting. Graphical Abstract ᅟ
Keywords: Protein imprinting; Ionic liquids; Lysozyme; Thermal stability of protein

Combined QCM-D/GE as a tool to characterize stimuli-responsive swelling of and protein adsorption on polymer brushes grafted onto 3D-nanostructures by Meike Koenig; Tadas Kasputis; Daniel Schmidt; Keith B. Rodenhausen; Klaus-Jochen Eichhorn; Angela K. Pannier; Mathias Schubert; Manfred Stamm; Petra Uhlmann (7233-7242).
A combined setup of quartz crystal microbalance and generalized ellipsometry can be used to comprehensively investigate complex functional coatings comprising stimuli-responsive polymer brushes and 3D nanostructures in a dynamic, noninvasive in situ measurement. While the quartz crystal microbalance detects the overall change in areal mass, for instance, during a swelling or adsorption process, the generalized ellipsometry data can be evaluated in terms of a layered model to distinguish between processes occurring within the intercolumnar space or on top of the anisotropic nanocolumns. Silicon films with anisotropic nanocolumnar morphology were prepared by the glancing angle deposition technique and further functionalized by grafting of poly-(acrylic acid) or poly-(N- isopropylacrylamide) chains. Investigations of the thermoresponsive swelling of the poly-(N-isopropylacrylamide) brush on the Si nanocolumns proved the successful preparation of a stimuli-responsive coating. Furthermore, the potential of these novel coatings in the field of biotechnology was explored by investigation of the adsorption of the model protein bovine serum albumin. Adsorption, retention, and desorption triggered by a change in the pH value is observed using poly-(acrylic acid) functionalized nanostructures, although generalized ellipsometry data revealed that this process occurs only on top of the nanostructures. Poly-(N-isopropylacrylamide) is found to render the nanostructures non-fouling properties.
Keywords: Thin films; Biomaterials; Interface/surface analysis; Nanostructures; Polymer brushes; Protein adsorption

Structural changes of ultrasonicated bovine serum albumin revealed by hydrogen–deuterium exchange and mass spectrometry by Qiuting Zhang; Zongcai Tu; Hui Wang; Xiaoqin Huang; Xiaomei Sha; Hui Xiao (7243-7251).
The structural changes of bovine serum albumin (BSA) under high-intensity ultrasonication were investigated by fluorescence spectroscopy and mass spectrometry. Evidence for the ultrasonication-induced conformational changes of BSA was provided by the intensity changes and maximum-wavelength shift in fluorescence spectrometry. Matrix-assisted laser desorption–ionization time-of-flight mass spectroscopy (MALDI-TOF MS) revealed the increased intensity of the peak at the charge state +5 and a newly emerged peak at charge state +6, indicating that the protein became unfolded after ultrasonication. Prevalent unfolding of BSA after ultrasonication was revealed by hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS). Increased intensity and duration of ultrasonication further promoted the unfolding of the protein. The unfolding induced by ultrasonication goes through an intermediate state similar to that induced by a low concentration of denaturant.
Keywords: BSA structure; Ultrasound; H–D exchange; Mass spectrometry

Electrochemistry combined with (liquid chromatography) high resolution mass spectrometry was used to simulate the general reductive metabolism of three biologically important nitro aromatic molecules: 3-trifluoromethyl-4-nitrophenol (TFM), niclosamide, and nilutamide. TFM is a pesticide used in the Laurential Great Lakes while niclosamide and nilutamide are used in cancer therapy. At first, a flow-through electrochemical cell was directly connected to a high resolution mass spectrometer to evaluate the ability of electrochemistry to produce the main reduction metabolites of nitro aromatic, nitroso, hydroxylamine, and amine functional groups. Electrochemical experiments were then carried out at a constant potential of −2.5 V before analysis of the reduction products by LC-HRMS, which confirmed the presence of the nitroso, hydroxylamine, and amine species as well as dimers. Dimer identification illustrates the reactivity of the nitroso species with amine and hydroxylamine species. To investigate xenobiotic metabolism, the reactivity of nitroso species to biomolecules was also examined. Binding of the nitroso metabolite to glutathione was demonstrated by the observation of adducts by LC-ESI+-HRMS and the characteristics of their MSMS fragmentation. In conclusion, electrochemistry produces the main reductive metabolites of nitro aromatics and supports the observation of nitroso reactivity through dimer or glutathione adduct formation. Graphical Abstract Electrochemical simulation of nitro aromatics metabolism using hyphenation of electrochemistry and massspectrometry
Keywords: Electrochemistry; Nitro aromatic metabolism; Nitroso; Reactive metabolite; Glutathione binding; Lampricide

In this work, a novel ionic liquid (IL) chemically bonded sol–gel coating was prepared for stir bar sorptive extraction (SBSE) of nonsteroidal anti-inflammatory drugs (NSAIDs) followed by high-performance liquid chromatography-ultraviolet detection (HPLC-UV). By using γ-(methacryloxypropyl)trimethoxysilane (KH-570) as a bridging agent, 1-allylimidazolium tetrafluoroborate ([AIM][BF4]) was chemically bonded onto the bare stir bar, and the prepared IL-bonded sol–gel stir bar coating showed higher extraction efficiency and better adsorption/desorption kinetics for target NSAIDs over other polydimethylsiloxane (PDMS)-based or monolithic stir bar coatings. The mechanical strength and durability (chemical/thermal stability) of the prepared IL-bonded sol–gel coating were excellent. The influencing factors of SBSE, such as sample pH, salt effect, stirring rate, extraction time, desorption solvent, and desorption time, were optimized, and the analytical performance of the developed SBSE-HPLC-UV method was evaluated under the optimized conditions. The limits of detection (LODs) of the proposed method for three NSAIDs were in the range of 0.23–0.31 μg L−1, and the enrichment factors (EFs) were in the range of 51.6–56.3 (theoretical enrichment factor was 100). The reproducibility was also investigated at concentrations of 5, 20, and 100 μg L−1, and the relative standard deviations (RSDs) were found to be less than 9.5, 7.5, and 7.6 %, respectively. The proposed method was successfully applied for the determination of NSAIDs in environmental water, urine, and milk samples. Graphical Abstract ᅟ
Keywords: Stir bar sorptive extraction; Ionic liquid bonded sol–gel coating; High-performance liquid chromatography; Nonsteroidal anti-inflammatory drugs; Environmental waters; Milk

Metabolomic results on human blood plasma largely depend on the sample preparation protocols employed for protein precipitation and metabolite extraction. Five different extraction methods were examined, which can be grouped into two categories, liquid-liquid extraction and protein precipitation methods, including long-standing protocols such as the Folch extraction and Bligh-Dyer extraction in comparison to modern methods such as the Matyash protocol and two global metabolite extraction methods. Extracts were subjected to analysis of blood plasma lipids and primary metabolites by using chip-based direct infusion nanoelectrospray tandem mass spectrometry and gas chromatography coupled to time-of-flight mass spectrometry, respectively. Optimal extraction schemes were evaluated based on the number of identified metabolites, extraction efficiency, compound diversity, reproducibility, and convenience for high-throughput sample preparations. Results showed that Folch and Matyash methods were equally valid and robust for lipidomic assessments while primary metabolites were better assessed by the protein precipitation methods with organic solvent mixtures. Graphical Abstract Schematic workflow of five extraction methods and subsequent mass spectrometry analysis using GC-TOF MS and nanoelectrospray direct-infusion ion trap MS/MSᅟ
Keywords: Extraction method; Lipid profiling; Primary metabolite profiling; Human blood plasma; GC-MS; Nanoelectrospray direct-infusion MS

Catecholamines are a class of biogenic amines that play an important role as neurotransmitters and hormones. We developed and validated a rapid, specific and sensitive LC-MS/MS method for quantitative determination of catecholamines in human urine. Linearity, specificity, sensitivity, precision, accuracy, matrix effect, carryover, analyte stability, method comparison and reference range were evaluated. The catecholamine measurements were not affected by 35 structurally-related drugs and metabolites. The outstanding specificity was achieved by use of a specific diphenylborate-based solid phase extraction and subsequent selective LC-MS/MS analysis. Excellent sensitivity, accuracy and precision (average intra-assay variations <2.9 % and inter-assay variations <4.6 %) were obtained. The method was successfully applied in the study of day-to-day biological within- and between-subject variations of 25 healthy people under free-living conditions over three consecutive days. We observed that catecholamine excretions for second morning sampling had least day-to-day within-subject variation and excellent reproducibility. This work is one of the rare studies on these topics and represents the first utilization of advanced LC-MS/MS technology. Additionally, we found significant correlations between spot and conventional 24 h collections of human urine (n = 22, r > 0.853, p < 0.0001). These findings suggest that determining the catecholamine concentrations in the second morning urine sample presents accurate, convenient and reliable measurement of catecholamine excretions. In addition, consistent and significant diurnal variations for norepinephrine and epinephrine excretions were observed during the three-day period, while dopamine did not exhibit a diurnal rhythm. The LC-MS/MS method presented here is rapid, sensitive and specific, which could be an advantage in clinical laboratories. Graphical Abstract Diurnal variation of urinary catecholamines for 25 healthy people in three consecutive days
Keywords: LC-MS/MS; Catecholamine; Biological variation; Diurnal rhythm; Method validation; Urine

Analysis of 21 progestagens in various matrices by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) with diverse sample pretreatment by Shuang-Shuang Liu; Guang-Guo Ying; Shan Liu; Hua-Jie Lai; Zhi-Feng Chen; Chang-Gui Pan; Jian-Liang Zhao; Jun Chen (7299-7311).
In this study, a highly sensitive and robust method using an ultra-high-performance liquid chromatography-tandem mass spectrometry combined with solid-phase extraction and ultrasonic extraction for pretreatment and silica gel purification steps has been developed for determination of 21 natural and synthetic progestagens in river surface water and sediments, and influents, effluents, and sludge from municipal wastewater treatment plants, and flush water and feces from swine farms. For the various matrices considered, the optimized method showed satisfactory performance with recoveries of 70–129 % (except AD, 5α-DHP, DPT, HPC), the limits of quantification below 2.30 ng/L for liquid samples and 2.59 ng/g for solid samples (except AD), and good linearity and reproducibility. This developed method was successfully applied in the analysis of progestagens in environmental samples from Liuxi Reservoir, Xintang municipal wastewater treatment plant, and Shunfeng swine farm in South China. Six analytes were detected at trace levels in surface water, effluent, and sediment samples. Seven analytes (0.7 (HPA)–35.1 ng/L (DGT)) were found in the influent samples and three analytes (5.6 (DGT)–11.8 ng/g (5α-DHP)) in the dewatered sludge samples. Moreover, 13 analytes were detected in swine farm, with high concentrations ranging from 23.8 ng/L (ET) to 5,024 ng/L (P) in flush water, and from 20.0 ng/g (MPA) to 1952 ng/g (P) in feces.
Keywords: Progestagens; Environment matrices; Extraction; Purification; UHPLC-MS/MS

Quantification of total fatty acids in microalgae: comparison of extraction and transesterification methods by Lillie R. Cavonius; Nils-Gunnar Carlsson; Ingrid Undeland (7313-7322).
Determination of microalgaes’ fatty acid content is often done with chloroform and methanol according to the Bligh and Dyer extraction, though faster methods exist. A number of comparisons between the Bligh and Dyer and faster methods have resulted in contradicting data, possibly due to differences in algae used and the different versions of the Bligh and Dyer method applied. Here, various forms of direct-transesterification (D-TE) and two-step transesterification (2-TE), including three versions developed in our lab, are compared with the original Bligh and Dyer (Can J Biochem Physiol 37: 911–917, 1959) extraction and two modifications thereof (Lee et al. J AOAC Int 79:487–492, 1996, and our own acidified version) on microalgae with different cell walls: Isochrysis galbana, Nannochloropsis oculata, and Phaeodactylum tricornutum. In total, fatty acid extracts from 11 methods were separated and quantified by gas chromatography with mass spectrometry. Results show that, for N. oculata and P. tricornutum, methods based on chloroform–methanol underestimated the fatty acid content compared with the 2-TE and D-TE methods, which gave similar results. Moreover, D-TE methods are faster than chloroform–methanol methods and use chemicals that are less toxic. Of the D-TE methods, the ones using hydrochloric acid and sulfuric acid recovered the most fatty acids, while boron trifluoride recovered slightly less. The main qualitative difference between the fatty acids recovered was that the chloroform–methanol methods recovered less saturated fatty acids in P. tricornutum.
Keywords: Microalgae; Fatty acids; Bligh and Dyer extraction; Direct transesterification

Suspect and nontarget screening approaches to identify organic contaminant records in lake sediments by Aurea C. Chiaia-Hernandez; Emma L. Schymanski; Praveen Kumar; Heinz P. Singer; Juliane Hollender (7323-7335).
Sediment cores provide a valuable record of historical contamination, but so far, new analytical techniques such as high-resolution mass spectrometry (HRMS) have not yet been applied to extend target screening to the detection of unknown contaminants for this complex matrix. Here, a combination of target, suspect, and nontarget screening using liquid chromatography (LC)-HRMS/MS was performed on extracts from sediment cores obtained from Lake Greifensee and Lake Lugano located in the north and south of Switzerland, respectively. A suspect list was compiled from consumption data and refined using the expected method coverage and a combination of automated and manual filters on the resulting measured data. Nontarget identification efforts were focused on masses with Cl and Br isotope information available that exhibited mass defects outside the sample matrix, to reduce the effect of analytical interferences. In silico methods combining the software MOLGEN-MS/MS and MetFrag were used for direct elucidation, with additional consideration of retention time/partitioning information and the number of references for a given substance. The combination of all available information resulted in the successful identification of three suspect (chlorophene, flufenamic acid, lufenuron) and two nontarget compounds (hexachlorophene, flucofuron), confirmed with reference standards, as well as the tentative identification of two chlorophene congeners (dichlorophene, bromochlorophene) that exhibited similar time trends through the sediment cores. This study demonstrates that complementary application of target, suspect, and nontarget screening can deliver valuable information despite the matrix complexity and provide records of historical contamination in two Swiss lakes with previously unreported compounds.
Keywords: High-resolution mass spectrometry; Suspect screening; Nontarget screening; Sediment matrix; Organic contaminants; Mass accuracy; Chlorophenes

A proficiency test for the analysis of pesticide residues in brown rice was carried out to support upgrading in analytical skills of participant laboratories. Brown rice containing three target pesticides (etofenprox, fenitrothion, and isoprothiolane) was used as the test samples. The test samples were distributed to the 57 participants and analyzed by appropriate analytical methods chosen by each participant. It was shown that there was no significant difference among the reported values obtained by different types of analytical method. The analytical results obtained by National Metrology Institute of Japan (NMIJ) were 3 % to 10 % greater than those obtained by participants. The results reported by the participant were evaluated by using two types of z-scores, that is, one was the score based on the consensus values calculated from the analytical results of participants, and the other one was the score based on the reference values obtained by NMIJ with high reliability. Acceptable z-scores based on the consensus values and NMIJ reference values were achieved by 87 % to 89 % and 79 % to 94 % of the participants, respectively. Graphical Abstract Distribution of z and zNMIJ-scores for isoprothiolane
Keywords: Proficiency test; Quality assurance/quality control; Pesticide residues; Brown rice

Survey of pyrrolizidine alkaloids in teas and herbal teas on the Swiss market using HPLC-MS/MS by Caroline Mathon; Patrick Edder; Stefan Bieri; Philippe Christen (7345-7354).
Pyrrolizidine alkaloids (PAs) are a large class of natural compounds amongst which the esterified 1,2-unsaturated necine base is toxic for humans and livestock. In the present study, a method was developed and validated for the screening and quantification of nine PAs and one PA N-oxide in teas (Camellia sinensis (L.) O. Kuntze) and herbal teas (camomile, fennel, linden, mint, rooibos, verbena). Samples were analysed by HPLC on a RP-column, packed with sub-2 μm core-shell particles, and quantified using tandem mass spectrometry operating in the positive electrospray ionisation mode. These PAs and some of their isomers were detected in a majority of the analysed beverages (50/70 samples). In 24 samples, PA concentrations were above the limit of quantification and the sum of the nine targeted PAs was between 0.021 and 0.954 μg per cup of tea. Thus, in some cases, total concentrations exceed the maximum daily intake recommended by the German Federal Institute for Risk Assessment and the UK’s Committee On Toxicity (i.e. 0.007 μg kg−1 bw). Graphical Abstract ᅟ
Keywords: Pyrrolizidine alkaloids; Hepatotoxicity; Herbal teas; HPLC-MS/MS; Food safety; Quality control

The distribution of free and bound forms of B-group vitamins (B1, B2, B3, B5, and B6) was quantified in quinoa seeds using LC-MS-TOF combined with a stable isotope dilution assay. The effectiveness of liberating thiamine, riboflavin, nicotinic acid, pantothenic acid, pyridoxal, and pyridoxine from the food matrix and cofactors was evaluated for a variety of extraction conditions, including the addition of enzymes. Phosphatase and protease inhibitors, as well as ultrafiltration, were evaluated for their ability to suppress vitamer liberation via enzymes endogenous to quinoa. Cold extraction, together with a mixture of phosphatase and protease inhibitors, is identified as the most efficient treatment to prevent the conversion of cofactors into simple vitamers. Overnight incubation at 37 °C both with and without additional hydrolytic enzyme preparations containing phosphatase and β-glucosidase activity was almost equally effective in releasing the bound forms of the vitamers. This indicates that the endogenous enzymes within quinoa seeds have high activity. β-Glucosidase should be used when the total pyridoxine content is to be determined, and thermal treatment followed by enzymatic treatment with phosphatase activity is recommended to liberate the bound forms of pyridoxal prior to quantification. Graphical Abstract Free and bound forms of B-group vitamins in quinoa
Keywords: B-group vitamins; Isotope dilution mass spectrometry; Liquid chromatography; MS-TOF; Quinoa

Integrated plasma and urine metabolomics coupled with HPLC/QTOF-MS and chemometric analysis on potential biomarkers in liver injury and hepatoprotective effects of Er-Zhi-Wan by Weifeng Yao; Haiwei Gu; Jiangjiang Zhu; Gregory Barding; Haibo Cheng; Beihua Bao; Li Zhang; Anwei Ding; Wei Li (7367-7378).
Metabolomics techniques are the comprehensive assessment of endogenous metabolites in a biological system and may provide additional insight into the molecular mechanisms. Er-Zhi-Wan (EZW) is a traditional Chinese medicine formula, which contains Fructus Ligustri Lucidi (FLL) and Herba Ecliptae (HE). EZW is widely used to prevent and treat various liver injuries through the nourishment of the liver. However, the precise molecular mechanism of hepatoprotective effects has not been comprehensively explored. Here, an integrated metabolomics strategy was designed to assess the effects and possible mechanisms of EZW against carbon tetrachloride-induced liver injury, a commonly used model of both acute and chronic liver intoxication. High-performance chromatography/quadrupole time-of-flight mass spectrometry (HPLC/QTOF-MS) combined with chemometric approaches including principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used to discover differentiating metabolites in metabolomics data of rat plasma and urine. Results indicate six differentiating metabolites, tryptophan, sphinganine, tetrahydrocorticosterone, pipecolic acid, l-2-amino-3-oxobutanoic acid and phosphoribosyl pyrophosphate, in the positive mode. Functional pathway analysis revealed that the alterations in these metabolites were associated with tryptophan metabolism, sphingolipid metabolism, steroid hormone biosynthesis, lysine degradation, glycine, serine and threonine metabolism, and pentose phosphate pathway. Of note, EZW has a potential pharmacological effect, which might be through regulating multiple perturbed pathways to the normal state. Our findings also showed that the robust integrated metabolomics techniques are promising for identifying more biomarkers and pathways and helping to clarify the function mechanisms of traditional Chinese medicine. Graphical Abstract Overview of the integrated metabolomics strategy
Keywords: Integrated metabolomics; Er-Zhi-Wan (EZW); Hepatoprotective; HPLC/QTOF-MS; Chemometrics

Rapid screening and detection of XOD inhibitors from S. tamariscina by ultrafiltration LC-PDA–ESI-MS combined with HPCCC by Jing Wang; Shu Liu; Bing Ma; Lina Chen; Fengrui Song; Zhiqiang Liu; Chun-ming Liu (7379-7387).
Xanthine oxidase (XOD) catalyzes the metabolism of hypoxanthine and xanthine to uric acid, the overproduction of which could cause hyperuricemia, a risk factor for gout. Inhibition of XOD is a major treatment for gout, and biflavonoids have been found to act as XOD-inhibitory compounds. In this study, ultrafiltration liquid chromatography with photodiode-array detection coupled to electrospray-ionization tandem mass spectrometry (UF-LC-PDA–ESI-MS) was used to screen and identify XOD inhibitors from S. tamariscina. High-performance counter-current chromatography (HPCCC) was used to separate and isolate the active constituents of these XOD inhibitors. Furthermore, ultrahigh-performance liquid chromatography (UPLC) and triple-quadrupole mass spectrometry (TQ-MS) was used to determine the XOD-inhibitory activity of the obtained XOD inhibitors, and enzyme kinetics was performed with Lineweaver–Burk (LB) plots using xanthine as the substrate. As a result, two compounds in S. tamariscina were screened as XOD inhibitors: 65.31 mg amentoflavone and 0.76 mg robustaflavone were isolated from approximately 2.5 g S. tamariscina by use of HPCCC. The purities of the two compounds obtained were over 98 % and 95 %, respectively, as determined by high-performance liquid chromatography (HPLC). Lineweaver–Burk plot analysis indicated that amentoflavone and robustaflavone were non-competitive inhibitors of XOD, and the IC 50 values of amentoflavone and robustaflavone for XOD inhibition were 16.26 μg mL−1 (30.22 μmol L−1) and 11.98 μg mL−1 (22.27 μmol L−1), respectively. The IC 50 value of allopurinol, used as the standard, was 7.49 μg mL−1 (46.23 μmol L−1). The results reveal that the method for systematic screening, identification, and isolation of bioactive components in S. tamariscina and for detecting their inhibitory activity using ultrafiltration LC–ESI-MS, HPCCC, and UPLC–TQ-MS is feasible and efficient, and could be expected to extend to screening and separation of other enzyme inhibitors. Graphical Abstract ᅟ
Keywords: Selaginella tamariscina ; Ultrafiltration LC-PDA–ESI-MS; Biflavonoids XOD inhibitor; HPCCC; UPLC–TQ-MS

Ceritinib is a highly selective inhibitor of an important cancer target, anaplastic lymphoma kinase (ALK). Because it is an investigational compound, there is a need to develop a robust and reliable analytical method for its quantitative determination in human plasma. Here, we report the validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the rapid quantification of ceritinib in human plasma. The method consists of protein precipitation with acetonitrile, and salting-out assisted liquid-liquid extraction (SALLE) using a saturated solution of sodium chloride prior to analysis by LC-MS/MS with electrospray ionization (ESI) technique in positive mode. Samples were eluted at 0.800 mL min−1 on Ascentis Express® C18 column (50 mm × 2.1 mm, 2.7 μm) with a mobile phase made of 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B). The method run time was 3.6 min and the low limit of quantification (LLOQ) was estimated at 1.00 ng mL−1 when using 0.100 mL of human plasma. The assay was fully validated and the method exhibited sufficient specificity, accuracy, precision, and sensitivity. In addition, recovery data and matrix factor (MF) in normal and in hemolyzed plasmas were assessed, while incurred samples stability (ISS) for ceritinib was demonstrated for at least 21 months at a storage temperature of −65 °C or below. The method was successfully applied to the measurement of ceritinib in clinical samples and the data obtained on incurred samples reanalysis (ISR) showed that our method was reliable and suitable to support the analysis of samples from the clinical studies.
Keywords: Non-small cell lung cancer; Anaplastic lymphoma kinase; Validation; LC-MS/MS; Pharmacokinetic

The evaluation of a fully automated quantitative proton nuclear magnetic resonance spectroscopy (qNMR) processing program, including the determination of its processing uncertainty, and the calculations of the combined uncertainty of the qNMR result, is presented with details on the use of a trimmed purity average. Quantitative NMR spectra (1359) were collected over a 4-month period on various concentrations of pseudoephedrine HCl dissolved in D2O (0.0610 to 93.60 mg/mL) containing maleic acid (the internal standard) to yield signal-to-noise ratios ranging from 3 to 72,000 for analyte integral regions. The resulting 5436 purities exhibited a normal distribution about the best estimate of the true value. The median absolute deviation (MAD) statistical method was used to obtain a model of uncertainty relative to the signal-to-noise of the analyte’s integral peaks. The model was then tested using different concentrations of known purity chloroquine diphosphate. qNMR results of numerous illicit heroin HCl samples were compared to those obtained by capillary electrophoresis. Graphical Abstract ᅟ
Keywords: Uncertainty; Quantitation; Nuclear magnetic resonance; qNMR; Purity determination

Evaluation of a commercial enzymatic test kit regarding the quantitative analysis of different free fatty acids by Mandy Eibisch; Yulia Popkova; Rosmarie Süß; Jürgen Schiller; Dirk Dannenberger (7401-7405).
The quantitative determination of the total free fatty acids (FFAs) is an important analytical task because FFAs exhibit important physiological effects and are also relevant in many other fields, for instance, in food research. Our aim was to investigate whether a commercially available enzymatic test kit developed for the determination of FFAs in human serum is also suitable to determine different physiological and nonphysiological FFAs and to which extent the impact on the sensitivities (i.e., the accuracy by which a given FFA can be determined) differ. It will be shown that the chain length as well as the double bond content has a significant impact on the sensitivity by which a given FFA can be determined. For instance, palmitic acid (16:0) is determined with an approximately 20 times higher sensitivity in comparison to docosahexaenoic acid (22:6n-3). All data were obtained by measuring the concentrations of the FFAs by gas chromatography, and selected FFAs were also determined in a complex matrix of human serum. It is concluded that this kit is not useful if major alterations of the FFA composition of a complex mixture are expected because the individual FFAs are not detected with the same sensitivities: the concentrations of polyunsaturated FFA determined by this kit are wrong. Figure The used enzymatic kit detects different free fatty acids with significantly different sensitivities: the number of carbon atoms and the number of double bonds massively contribute to these differences
Keywords: Free fatty acids; Chain length; Double bond content; Enzymatic test; Gas chromatography

Erratum to: Increased phosphatidylcholine (16:0/16:0) in the folliculus lymphaticus of Warthin tumor by Qian He; Yoshinori Takizawa; Takahiro Hayasaka; Noritaka Masaki; Yukiko Kusama; Jiping Su; Hiroyuki Mineta; Mitsutoshi Setou (7409-7410).

Erratum to: Large-scale analysis of posttranslational modifications in the hippocampus of patients with Alzheimer’s disease using pI shift and label-free quantification without enrichment by Taewook Kang; Jae Ho Kim; Ingie Hong; Nam Hyun Park; Helmut Heinsen; Joo-Yong Lee; Rivka Ravid; Isidro Ferrer; Jong Shin Yoo; Kyung-Hoon Kwon; Young Mok Park (7411-7411).