Analytical and Bioanalytical Chemistry (v.410, #20)

European analytical column number 46 by Wolfgang Buchberger; Sibel A. Özkan; Slavica Razic (4765-4766).

is the Charles A. Dana Professor of Chemistry at Bates College in Lewiston, ME. He currently carries out research with the aid of undergraduate students in the area of chiral NMR shift reagents. His research accomplishments were recognized with the 2010 American Chemical Society Award for Research at an Undergraduate Institution. He is active in efforts to reform the undergraduate analytical chemistry curriculum to include inquiry- and project-based experiences. His educational activities were recognized through receipt of the 1999 J.C. Giddings Award for Excellence in Education sponsored by the Analytical Division of the American Chemical Society. More information about his activities can be found at http://www.bates.edu/chemistry-biochemistry/faculty/thomas-wenzel/.

Development and validation of LC/APCI-MS method for the quantification of oat ceramides in skin permeation studies by Efrem N. Tessema; Tsige Gebre-Mariam; Andrej Frolov; Johannes Wohlrab; Reinhard H. H. Neubert (4775-4785).
Ceramides (CERs) are the backbone of the intercellular lipid lamellae of the stratum corneum (SC), the outer layer of the skin. Skin diseases such as atopic dermatitis, psoriasis, and aged skin are characterized by dysfunctional skin barrier and dryness which are associated with reduced levels of CERs. Replenishing the depleted epidermal CERs with exogenous CERs has been shown to have beneficial effects in improving the skin barrier and hydration. The exogenous CERs such as phyto-derived CERs (PhytoCERs) can be delivered deep into the SC using novel topical formulations. This, however, requires investigating the rate and extent of skin permeation of CERs. In this study, an LC/APCI-MS method to detect and quantify PhytoCERs in different layers of the skin has been developed and validated. The method was used to investigate the skin permeation of PhytoCERs using Franz diffusion cells after applying an amphiphilic cream containing PhytoCERs to the surface of ex vivo human skin. As plant-specific CERs are not commercially available, well-characterized CERs isolated from oat (Avena abyssinica) were used as reference standards for the development and validation of the method. The method was linear over the range of 30–1050 ng/mL and sensitive with limit of detection and quantification of 10 and 30 ng/mL, respectively. The method was also selective, accurate, and precise with minimal matrix effect (with mean matrix factor around 100%). Even if more than 85% of oat CERs in the cream remained in the cream after the incubation periods of 30, 100, and 300 min, it was possible to quantify the small quantities of oat CERs distributed across the SC, epidermis, and dermis of the skin indicating the method’s sensitivity. Therefore, the method can be used to investigate the skin permeation of oat CERs from the various pharmaceutical and cosmeceutical products without any interference from the skin constituents such as the epidermal lipids. Graphical abstractᅟ
Keywords: Phyto-derived ceramide; Oat ceramide; Stratum corneum ; Skin; LC/APCI-MS; Skin permeability

In this communication, a gold-coated polydimethylsiloxane (PDMS) chip with cell-sized microwells was prepared through a stamping and spraying process that was applied directly for high-throughput electrochemiluminescence (ECL) analysis of intracellular glucose at single cells. As compared with the previous multiple-step fabrication of photoresist-based microwells on the electrode, the preparation process is simple and offers fresh electrode surface for higher luminescence intensity. More luminescence intensity was recorded from cell-retained microwells than that at the planar region among the microwells that was correlated with the content of intracellular glucose. The successful monitoring of intracellular glucose at single cells using this PDMS chip will provide an alternative strategy for high-throughput single-cell analysis. Graphical abstractᅟ
Keywords: Single cell analysis; PDMS microwells; Electrochemiluminescence imaging; Intracellular glucose; Cellular heterogeneity

Facile determination of sphingolipids under alkali condition using metal-free column by LC-MS/MS by Siddabasave Gowda B. Gowda; Kazutaka Ikeda; Makoto Arita (4793-4803).
Extraction and analysis of sphingolipids from biological samples is a critical step in lipidomics, especially for minor species such as sphingoid bases and sphingosine-1-phosphate. Although several liquid chromatography-mass spectrometry methods enabling the determination of sphingolipid molecular species have been reported, they were limited in analytical sensitivity and reproducibility by causing significant peak tailing, especially by the presence of phosphate groups, and most of the extraction techniques are laborious and do not cover a broad range of sphingolipid metabolites. In this study, we developed a rapid single-phase extraction and highly sensitive analytical method for the detection and quantification of sphingolipids (including phosphates) comprehensively using liquid chromatography-triple quadruple mass spectrometry. After validating the reliability of the method, we analyzed the intestinal tissue sphingolipids of germ-free (GF) and specific pathogen-free (SPF) mice and found significantly higher levels of free sphingoid bases and sphingosine-1-phosphate in the GF condition as compared to the SPF condition. This method enables a rapid extraction and highly sensitive determination of sphingolipids comprehensively at low femtomolar ranges. Graphical abstractDiagrammatic comparision of sphingolipid (phosphates) analysis between conventional and this method.
Keywords: Sphingosine-1-phosphate; Phytosphingosine; Single-phase extraction; Intestine; Liquid chromatography; Mass spectrometry

Colorimetric determination of glutathione in human serum and cell lines by exploiting the peroxidase-like activity of CuS-polydopamine-Au composite by Yanying Wang; Yaqin Liu; Fang Ding; Xiaoyan Zhu; Li Yang; Ping Zou; Hanbing Rao; Qingbiao Zhao; Xianxiang Wang (4805-4813).
In this study, we developed a simple colorimetric approach to detect glutathione (GSH). The proposed approach is based on the ability of CuS-PDA-Au composite material to catalytically oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) to ox-TMB to induce a blue color with an absorption peak centered at 652 nm. However, the introduction of GSH can result in a decrease in oxidized TMB; similarly, it can combine with Au nanoparticles (Au NPs) on the surface of CuS-PDA-Au composite material. Both approaches can result in a fading blue color and a reduction of the absorbance at 652 nm. Based on this above, we proposed a technique to detect GSH quantitatively and qualitatively through UV-Vis spectroscopy and naked eye, respectively. This approach demonstrates a low detection limit of 0.42 μM with a broad detection range of 5 × 10−7–1 × 10−4 M with the assistance of UV-Vis spectroscopy. More importantly, this approach is convenient and rapid. This method was successfully applied to GSH detection in human serum and cell lines. Graphical abstractA colorimetric approach has been developed by exploiting the peroxidase-like activity of CuS-polydopamine-Au composite for sensitive glutathione detection.
Keywords: Glutathione; CuS-PDA-Au composite material; Peroxidase-like activity; Cancer cells

We explored the applicability of different metal oxide nanoparticles (NPs; ZnO, TiO2, Fe2O3, and CeO2) for the optical imaging and mass spectrometric determination of small drug molecules in latent fingerprints (LFPs). Optical imaging was achieved using a dry method—simply dusting the LFPs with a minute amount of NP powder—and still images were captured using a digital microscope and a smartphone camera. Mass spectrometric determination was performed using the NPs as substrates for surface-assisted laser desorption ionization/mass spectrometry (SALDI-MS), which enabled the detection of small drug molecules with high signal intensities. The reproducibility of the results was studied by calculating the % error, SD, and RSD in the results obtained with the various metal oxide NPs. Collectively, the findings showed that using NPs can boost the intensity of the detected signal while minimizing background noise which is an issue predominantly associated with conventional organic matrices of MALDI-MS. Among the four metal oxide NPs, utilization of the Fe2O3 NPs led to the best SALDI performance and the highest detection sensitivity for the analytes of interest. The study was then extended by investigating the influence of time elapsed since the generation of the LFP on the detection of drug molecules in the LFP. The results demonstrated that this method allows the analysis of drug molecules after as long as one week at low and intermediate temperatures (0 and 25 °C). Therefore, the SALDI analysis of small molecules using inorganic NPs, which can be implemented in forensic laboratories for screening and detection purposes, as a powerful alternative to the use of organic matrices. Graphical abstractᅟ
Keywords: Metal oxide NPs; SALDI; Latent fingerprints; Small molecules; Visualization

The scarcity of data about the occurrence of pharmaceuticals in water bodies in Malaysia prompted us to develop a suitable analytical method to address this issue. We therefore developed a method based on solid-phase extraction combined with liquid chromatography–time of flight/mass spectrometry (SPE-LC-TOF/MS) for the analysis of sixteen prescribed and two nonprescribed pharmaceuticals that are potentially present in water samples. The levels of these pharmaceuticals, which were among the top 50 pharmaceuticals consumed in Malaysia during the period 2011–2014, in influent and effluent of five sewage treatment plants (STPs) in Bangi, Malaysia, were then analyzed using the developed method. All of the pharmaceuticals were separated chromatographically using a 5 μm, 2.1 mm × 250 mm C18 column at a flow rate of 0.3 mL/min. Limits of quantification (LOQs) were 0.3–8.2 ng/L, 6.5–89 ng/L, and 11.1–93.8 ng/L in deionized water (DIW), STP effluent, and STP influent, respectively, for most of the pharmaceuticals. Recoveries were 51–108%, 52–118%, and 80–107% from the STP influent, STP effluent, and DIW, respectively, for most of the pharmaceuticals. The matrix effect was also evaluated. The signals from carbamazepine, diclofenac sodium, and mefenamic acid were found to be completely suppressed in the STP influent. The signals from other compounds were found to be influenced by matrix effects more strongly in STP influent (enhancement or suppression of signal ≤180%) than in effluent (≤94%). The signal from prednisolone was greatly enhanced in the STP influent, indicating a matrix effect of −134%. Twelve pharmaceuticals were frequently detected in all five STPs, and caffeine, prazosin, and theophylline presented the highest concentrations among all the pharmaceuticals monitored: up to 7611, 550, and 319 ng/L in the STP influent, respectively. To the best of our knowledge, this is the first time that prazosin has been detected in a water matrix in Malaysia. Graphical abstractᅟ
Keywords: Prescribed and nonprescribed pharmaceuticals; LC-TOF/MS; Solid-phase extraction; Pharmaceutical consumption; Sewage treatment plants

Solid-phase extraction of phospholipids using mesoporous silica nanoparticles: application to human milk samples by Héctor Martínez Pérez-Cejuela; Isabel Ten-Doménech; Jamal El Haskouri; Pedro Amorós; Ernesto F. Simó-Alfonso; José Manuel Herrero-Martínez (4847-4854).
In this study, mesoporous silica materials (MSMs) with bimodal pore systems (namely, UVM-7), MCM-41 silica, and a commercial silica-based material were evaluated as solid-phase extraction (SPE) sorbents for the isolation of phospholipids (PLs) using phosphatidylcholine as a test compound. Morphological characterization (including TEM, surface, and size pore measurements) of these materials was carried out. The mechanism of interaction of the target analyte with the MSMs was also studied. With regard to the SPE process, several experimental parameters that affect the extraction performance (e.g., loading and elution solvent, breakthrough volume, loading capacity, and reusability) were investigated. The recommended protocol was applied to the extraction of PLs in human milk fat samples. The extracted PLs were then determined by hydrophilic interaction liquid chromatography (HILIC) using evaporative light scattering detection (ELSD). This work reports the first application of silica-based mesoporous materials to preconcentrate PLs in these complex matrices. Graphical abstractᅟ
Keywords: Mesoporous silica material; UVM-7; Phospholipids; SPE; HILIC-ELSD

Numerous stationary phases have been developed with the aim to provide desired performances during chromatographic analysis of the basic solutes in their protonated form. In this work, the procedure for the characterization of bonded stationary phase performance, when both qualitative and quantitative chromatographic factors were varied in chaotropic chromatography, was proposed. Risperidone and its three impurities were selected as model substances, while acetonitrile content in the mobile phase (20–30%), the pH of the aqueous phase (3.00–5.00), the content of chaotropic agents in the aqueous phase (10–100 mM), type of chaotropic agent (NaClO4, CF3COONa), and stationary phase type (Zorbax Eclipse XDB, Zorbax Extend) were studied as chromatographic factors. The proposed procedure implies the combination of D-optimal experimental design, indirect modeling, and polynomial-modified Gaussian model, while grid point search method was selected for the final choice of the experimental conditions which lead to the best possible stationary phase performance for basic solutes. Good agreement between experimentally obtained chromatogram and simulated chromatogram for chosen experimental conditions (25% acetonitrile, 75 mM of NaClO4, pH 4.00 on Zorbax Eclipse XDB column) confirmed the applicability of the proposed procedure. The additional point was selected for the verification of proposed procedure ability to distinguish changes in solutes’ elution order. Simulated chromatogram for 21.5% acetonitrile, 85 mM of NaClO4, pH 5.00 on Zorbax Eclipse XDB column was in line with experimental data. Furthermore, the values of left and right peak half-widths obtained from indirect modeling were used in order to evaluate performances of differently modified stationary phases applying a half-width plots approach. The results from half-width plot approach as well as from the proposed procedure indicate higher efficiency and better separation performance of the stationary phase extra densely bonded and double end-capped with trimethylsilyl group than the stationary phase with the combination of end-capping and bidentate silane bonding for chromatographic analysis of basic solutes in RP–HPLC systems with chaotropic agents. Graphical abstractᅟ
Keywords: Indirect modeling; Polynomial modified Gaussian model; Chromatogram simulation; Half-width plots approach

Proteins and antibodies in serum, plasma, and whole blood—size characterization using asymmetrical flow field-flow fractionation (AF4) by Mats Leeman; Jaeyeong Choi; Sebastian Hansson; Matilda Ulmius Storm; Lars Nilsson (4867-4873).
The analysis of aggregates of therapeutic proteins is crucial in order to ensure efficacy and patient safety. Typically, the analysis is performed in the finished formulation to ensure that aggregates are not present. An important question is, however, what happens to therapeutic proteins, with regard to oligomerization and aggregation, after they have been administrated (i.e., in the blood). In this paper, the separation of whole blood, plasma, and serum is shown using asymmetric flow field-flow fractionation (AF4) with a minimum of sample pre-treatment. Furthermore, the analysis and size characterization of a fluorescent antibody in blood plasma using AF4 are demonstrated. The results show the suitability and strength of AF4 for blood analysis and open new important routes for the analysis and characterization of therapeutic proteins in the blood.
Keywords: Whole blood; Antibodies; Plasma; Serum; Asymmetric flow field-flow fractionation (AF4); Fluorescence labelling

Highly selective and ratiometric fluorescent nanoprobe for the detection of cysteine and its application in test strips by Fengyang Wang; Yingying Zhu; Jie Xu; Zhiai Xu; Guiying Cheng; Wen Zhang (4875-4884).
Cysteine (Cys) is a bithiol that plays a vital role in many physiological processes. However, it is difficult to discriminate Cys from homocysteine (Hcy) and glutathione (GSH), due to their similar chemical structures and reactivity. Herein, we have developed a polymeric nanoprobe, nanoHFA, for ratiometric, highly selective, and sensitive detection of Cys based on 7-hydroxycoumarin-3-carboxylic acid (HC) and fluorescein isothiocyanate (FITC)-acrylate (FITC-A) group-functionalized lipopolymer DSPE-PEG. The probe nanoHFA showed a strong fluorescence emission peak centered at 450 nm attributed to HC and a weak fluorescence emission peak centered at 520 nm due to the photoinduced electron transfer (PET) process of FITC induced by acrylate group. In the presence of Cys, the fluorescence signal at 520 nm could be lit up and the ratio of F 520nm/F 450nm showed a good linear relationship in the range of 5–60 μM with a low detection limit of 0.37 μM. The probe also displayed excellent water solubility and high selectivity to Cys over other biothiols such as Hcy and GSH. Moreover, we further used probe nanoHFA to detect Cu2+ ions in the range of 100–550 nM with a detection limit of 77 nM. The nanoprobe was successfully applied for the quantitative detection of Cys in fetal bovine serum, and fluorescent strips were developed for facile and visual detection of Cys and Cu2+ ions. Graphical abstractᅟ
Keywords: Nanoprobe; Cysteine; Copper ions; Ratiometric; Test strips

An enzyme-free homogenous electrochemical assay for sensitive detection of the plasmid-mediated colistin resistance gene mcr-1 by Bo Li; Zhixin Chai; Xiaohui Yan; Chunchen Liu; Bo Situ; Ye Zhang; Weilun Pan; Shihua Luo; Jianhua Liu; Lei Zheng (4885-4893).
Antibiotic resistance associated with the mcr-1 gene of Gram-negative bacteria, which confers resistance to drugs of last resort and has the potential to spread via plasmids, is one of the most pressing issues facing global health today. Point-of-care testing for the mcr-1 gene is needed to aid in the identification of colistin resistance in the field and to control its horizontal transmission. Here, we report the successful development of an enzyme-free homogenous electrochemical strategy for sensitive detection of the antibiotic resistance gene mcr-1 using the hybridization chain reaction and mcr-1-specific toehold probe. The long double-stranded DNA polymer produced using this strategy could be detected by assessing the diffusion of methylene blue towards the surface of a screen-printed gold electrode. Under optimized conditions, a linear relationship was observed between the variation of peak current and the natural logarithm of the mcr-1 gene concentration in the range of 1 nM to 1 μM with a detection limit of 0.78 nM (S/N = 3). This enzyme-free, isothermal platform is a rapid, portable, disposable, and sensitive method for detection of plasmid-mediated colistin resistance.
Keywords: Colistin; Electrochemical assay; Hybridization chain reaction; Isothermal amplification; Mcr-1 ; Resistance

Systematic investigations of endogenous cortisol and cortisone in nails by LC-MS/MS and correlation to hair by Tina M. Binz; Franziska Gaehler; Clarissa D. Voegel; Mathias Hofmann; Markus R. Baumgartner; Thomas Kraemer (4895-4903).
Hair samples are increasingly used for measuring the long-term stress mediator cortisol. However, hair is not always available and nails (finger or toe), as a keratinized matrix, may be an alternative to hair. In order to measure cortisol and cortisone in the nail matrix, an LC-MS/MS method has been developed and validated using 13C3-labeled surrogate analytes. Both analytes were measured in ESI negative mode as formic acid adducts. Different sample preparation techniques were assessed, and single-step extraction in methanol was established for determination of cortisone and cortisol in the nail matrix. The method was successfully validated with limits of detection (LOD) and limits of quantification (LOQ) of 0.5 and 1.0 pg/mg for cortisol and cortisone, respectively. The calibration curve was linear up to a concentration of 500 pg/mg. Recovery was good for both analytes and showed values over 50%. Matrix effects with ion suppression occurred for both substances but could be corrected by the use of internal standard. Accuracy and precision were in the accepted range of ± 20% for both substances. The method was successfully applied to determine cortisol and cortisone concentrations in authentic nail samples. Cortisol and cortisone concentrations varied significantly among different fingernails, being highest in the little fingernails and lowest in the thumbnails. It could be shown that even in only 1 mg nail sample cortisol and cortisone can be reliably quantified. No correlation between hair and nail cortisol and cortisone concentrations could be found. Furthermore, cortisol and cortisone concentrations were significantly higher in hair. Graphical abstract
Keywords: Endogenous hair cortisol and cortisone; LC-MS/MS; Nails; Hair

Serum vitamin D metabolite levels are of interest as biomarkers for vitamin D status, which has influence on numerous body functions and pathologies. The determination of vitamin D metabolite levels by liquid chromatography/mass spectrometry (LC/MS) is challenging due to their low concentrations and relatively low ionization efficiencies. Three ionization sources, dielectric barrier discharge ionization (DBDI), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI), were compared regarding achievable limits of detection and occurring matrix effects. The latter were mainly caused by phospholipids. Therefore, in addition to a conventional solid phase extraction (SPE) stationary phase, a material for selective removal of phospholipids was examined. The selective removal of phospholipids significantly reduced observed matrix effects, especially when ESI was applied. Achievable limits of detection and observed matrix effects were lowest for APCI and with some limitations, also for DBDI. Graphical abstract
Keywords: Vitamin D metabolites; Liquid chromatography/mass spectrometry; High resolution mass spectrometry; Dielectric barrier discharge ionization (DBDI); Matrix effects

In this work, the feasibility of negative-ion atmospheric pressure chemical ionisation (APCI) and atmospheric pressure photoionisation (APPI) for ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) determination of fluorotelomer alcohols (FTOHs), fluorinated octanesulfonamides (FOSAs) and fluorinated octanesulfonamido-ethanols (FOSEs) was evaluated. The study of the effect of mobile phase composition on the atmospheric pressure ionisation of these compounds indicated that methanol/water mixtures provided the best responses in APCI, while acetonitrile/water with a post-column addition of toluene as dopant was the most appropriated mixture in APPI. Under the optimal working conditions, most of the target compounds produced the ion [M–H] as base peak, although in-source collision-induced dissociation fragment ions in APCI and APPI and superoxide adduct ions [M+O2]−• in APPI were also present. These ions proved to be more useful as precursor ions for MS/MS determination than the adduct ions generated in electrospray. Although the UHPLC-APCI-MS/MS method allowed the determination of these semi-volatile compounds at low concentration levels, the analysis by UHPLC-APPI-MS/MS provided the lowest limits of detection and it was applied to the analysis of water samples in combination with solid-phase extraction. Quality parameters demonstrated the good performance of the proposed method, providing low method limits of detection (0.3–6 ng L−1), good precision (RSD % < 5%) and an accurate quantification (relative error % < 14%). Among the river water samples analysed by the developed method, 4:2 FTOH and N-EtFOSA were determined at 30 and 780 ng L−1, respectively.
Keywords: Fluorotelomer alcohols; Fluorinated sulfonamides; Fluorinated sulfonamido-ethanols; Atmospheric pressure chemical ionisation; Atmospheric pressure photoionisation; Liquid chromatography tandem mass spectrometry

Interactions between elastin-like peptides and an insulating poly(ortho-aminophenol) membrane investigated by AFM and XPS by Maria Elvira Carbone; Rosanna Ciriello; Pasquale Moscarelli; Federica Boraldi; Giuliana Bianco; Antonio Guerrieri; Brigida Bochicchio; Antonietta Pepe; Daniela Quaglino; Anna Maria Salvi (4925-4941).
This investigation was undertaken to explore the mutual recognition of the pentapeptide (ValGlyGlyValGly) n , a hydrophobic elastin-like peptide (ELP), suspended in deionized water in monomer (n = 1) and trimer (n = 3) forms and the outer surface of a very thin, insulating polymer, poly(ortho-aminophenol) (PoAP), electrochemically grown on a platinum foil by cyclic voltammetry in a neutral medium (phosphate-buffered saline, I = 0.1M) immersed in the suspension. As a prior task, the proved propensity of the ValGlyGlyValGly sequence, at the given minimal length (three or more repeats), to self-assemble into amyloid-like fibrils when solubilized in an aqueous environment was considered within the framework of testing PoAP surfaces for the specific detection of amyloid precursors. From our knowledge of the chemical structure and physical properties of both biomacromolecule families obtained in previous studies, we focused on the efficacy of the binding sites offered to ELP fibrils by PoAP in its as-prepared form or properly modified either by postsynthesis oxidation or by adsorption/entrapping of ELP monomer(s) with or without protecting terminal groups. Consistent with all methods of preparation, the best surfaces, recognizable by the trimer fibrils, are those modified to carry a larger number of carbonyls, particularly by entrapment of ELP monomer(s) during PoAP electrosynthesis using an imprinting-inspired method. The degree of attachment of fibrillar aggregates, detected by atomic force microscopy and X-ray photoelectron spectroscopy, provides unequivocal evidence of the cooperative forces involving PoAP–ELP interactions. The results obtained suggest the prospect of using the proposed Pt/PoAP/ELP systems as biodetectors in Alzheimer disease. Graphical abstractSynthesis steps of Pt/PoAP/ELP electrodes for amyloid detection. AFM = Atomic Force Microscopy, CV = Cyclic Voltammetry, ELPs = Elastin like Peptides, PoAP = Poly ortho-Aminophenol, Pt = Platinum, XPS = X-ray Photoelectron Spectroscopy
Keywords: X-ray photoelectron spectroscopy; Atomic force microscopy; Poly(ortho-aminophenol); Elastin-like peptides; Amyloids; Peptide-imprinted cyclic voltammetry polymerization

A colorimetric assay of DNA methyltransferase activity based on peroxidase mimicking of DNA template Ag/Pt bimetallic nanoclusters by Hanie Ahmadzade Kermani; Morteza Hosseini; Andrea Miti; Mehdi Dadmehr; Giampaolo Zuccheri; Saman Hosseinkhani; Mohammad Reza Ganjali (4943-4952).
DNA methylation catalyzed by DNA methyl transferase (MTase) is a significant epigenetic process for modulating gene expression. Abnormal levels of DNA MTase enzyme have been regarded as a cancer biomarker or a sign of bacterial diseases. We developed a novel colorimetric method to assay M.SssI MTase activity employing peroxidase-like activity of DNA template Ag/Pt NCs without using restriction enzymes. Based on inhibiting the peroxidase reaction that occurred in the TMB-H2O2 system, in the presence of MTase, a highly sensitive and selective colorimetric biosensor was fabricated with a detection limit (LOD) of 0.05 U/mL and a linear range from 0.5 to 10 U/mL. The changes in absorption intensity were monitored to quantify the M.SssI activity. This strategy had a high selectivity over other proteins. Furthermore, it is also demonstrated that this method can be used for the evaluation and screening of inhibitors for DNA MTase.
Keywords: Ag/Pt nanoclusters; DNA methyltransferase; Colorimetric detection; Enzyme mimic

Sensitive electrochemical detection of sodium azide based on the electrocatalytic activity of the pasting liquid of a carbon paste electrode by Kaixuan Li; Moyan Han; Fengxia Wu; Anaclet Nsabimana; Wei Zhang; Jianping Li; Guobao Xu (4953-4957).
Sodium azide (NaN3) is highly toxic and widely used in, for example, automobile airbags and biochemical laboratories. The electrochemical detection of sodium azide on commonly used electrodes is challenging due to sluggish electron transfer, but it has been achieved using a boron-doped diamond thin-film electrode and a highly oriented pyrolytic graphite electrode. Utilizing the electrocatalytic activity of the pasting liquid of a carbon paste electrode, we developed an effective method for the electrochemical detection of sodium azide in which silicone oil was employed as the pasting liquid of the carbon paste electrode. This simple and cheap silicone-oil-based carbon paste electrode exhibited comparable sensitivity to the boron-doped diamond thin-film electrode and highly oriented pyrolytic graphite electrode. The limit of detection for sodium azide at the silicone-oil-based carbon paste electrode was found to be 0.1 μM. Recoveries from diluted human serum samples were between 97.2 and 101.3%. Graphical abstractᅟ
Keywords: Sodium azide; Pasting liquid; Electrocatalysis; Carbon paste electrode; Detection

Insight into the biological effects of acupuncture points by X-ray absorption fine structure by Chenglin Liu; Qinghua Liu; Dongming Zhang; Wei Liu; Xiaohui Yan; Xinyi Zhang; Hiroyuki Oyanagi; Zhiyun Pan; Fengchun Hu; Shiqiang Wei (4959-4965).
Exploration of the biological effects of transition metal ions in acupuncture points is essential to clarify the functional mechanism of acupuncture treatment. Here we show that in the SP6 acupuncture point (Sanyinjiao) the Fe ions are in a high-spin state of approximately t 2g 4.5 e g 1.5 in an Fe–N(O) octahedral crystal field. The Fe K-edge synchrotron radiation X-ray absorption fine structure results reveal that the Fe–N and Fe–O bond lengths in the SP6 acupuncture point are 2.05 and 2.13 Å, respectively, and are 0.05–0.10 Å longer than those in the surrounding tissue. The distorted atomic structure reduces the octahedral symmetry and weakens the crystal field around the Fe ions by approximately 0.3 eV, leading to the high-spin configuration of the Fe ions, which is favorable for strengthening the magnetotransport and oxygen transportation properties in the acupuncture point by the enhanced spin coherence. This finding might provide some insight into the microscopic effect of the atomic and electronic interactions of transition metal ions in the acupuncture point. Graphical Abstractᅟ
Keywords: Acupuncture point; Biological effect; Spin configuration; Synchrotron radiation; X-ray absorption fine structure

Rapid determination of designer benzodiazepines, benzodiazepines, and Z-hypnotics in whole blood using parallel artificial liquid membrane extraction and UHPLC-MS/MS by Linda Vårdal; Gladys Wong; Åse Marit Leere Øiestad; Stig Pedersen-Bjergaard; Astrid Gjelstad; Elisabeth Leere Øiestad (4967-4978).
Benzodiazepines (BZD) and Z-hypnotics are frequently analyzed in forensic laboratories, and in 2012, the designer benzodiazepines (DBZD) emerged on the illegal drug scene. DBZD represent a particular challenge demanding new analytical methods. In this work, parallel artificial liquid membrane extraction (PALME) is used for sample preparation of DBZD, BZD, and Z-hypnotics in whole blood prior to UHPLC-MS/MS analysis. PALME of BZD, DBZD, and Z-hypnotics was performed from whole blood samples, and the analytes were extracted across a supported liquid membrane (SLM) and into an acceptor solution of dimethyl sulfoxide and 200 mM formic acid (75:25, v/v). The method was validated according to EMA guidelines. The method was linear throughout the calibration range (R 2 > 0.99). Intra- and inter-day accuracy and precision, as well as matrix effects, were within the guideline limit of ± 15%. LOD and LLOQ ranged from 0.10 to 5.0 ng mL−1 and 3.2 to 160 ng mL−1, respectively. Extraction recoveries were reproducible and above 52%. The method was specific, and the analytes were stable in the PALME extracts for 4 and 10 days at 10 and − 20 °C. No carry-over was observed within the calibration range. PALME and UHPLC-MS/MS for the determination of DBZD, BZD, and Z-hypnotics in whole blood are a green and low-cost alternative that provides high sample throughput (96-well format), extensive sample clean-up, good sensitivity, and high reproducibility. The presented method is also the first method incorporating analysis of DBZD, BZD, and Z-hypnotics in whole blood in one efficient analysis. Graphical abstract
Keywords: Designer benzodiazepines; Benzodiazepines; Z-hypnotics; Parallel artificial liquid membrane extraction; LC-MS/MS; Whole blood samples

The potential of capillary electrophoresis (CE) with ultraviolet (UV)-excited fluorescence detection for sensitive chiral analysis of amino acids (AAs) was investigated. dl-AAs were derivatized with 9-fluorenylmethoxycarbonyl chloride (FMOC)-Cl to allow their fluorescence detection and enhance enantioseparation. Fluorescence detection was achieved employing optical fibers, leading UV excitation light (< 300 nm) from a Xe-Hg lamp to the capillary window, and fluorescence emission to a spectrograph equipped with a charge-coupled device (CCD). Signal averaging over time and emission wavelength intervals was carried out to improve the signal-to-noise ratio of the FMOC-AAs. A background electrolyte (BGE) of 40 mM sodium tetraborate (pH 9.5), containing 15% isopropanol (v/v), 30 mM sodium dodecyl sulfate (SDS), and 30 mM β-cyclodextrin (β-CD), was found optimal for AA chemo- and enantioseparation. Enantioresolutions of 1.0 or higher were achieved for 16 proteinogenic dl-AAs. Limits of detection (LODs) were in the 10–100-nM range (injected concentration) for the d-AA enantiomers, except for FMOC-d-tryptophan (536 nM) which showed intramolecular fluorescence quenching. Linearity (R 2 > 0.997) and repeatability for peak height (relative standard deviations (RSDs) < 7.0%; n = 5) and electrophoretic mobility (RSDs < 0.6%; n = 5) of individual AA enantiomers were established for chiral analysis of dl-AA mixtures. The applicability of the method was investigated by the analysis of cerebrospinal fluid (CSF). Next to l-AAs, endogenous levels of d-glutamine and d-aspartic acid could be measured in CSF revealing enantiomeric ratios of 0.35 and 19.6%, respectively. This indicates the method’s potential for the analysis of low concentrations of d-AAs in presence of abundant l-AAs.
Keywords: Amino acids; Chiral separation; Capillary electrophoresis; FMOC derivatization; Fluorescence detection; Cerebrospinal fluid

A novel assay for histidine and cysteine has been constructed based on modulation of fluorescent copper nanoclusters (CuNCs) by molecular switches. In our previous work, a dumbbell DNA template with a poly-T (thymine) loop has been developed as an excellent template for the formation of strongly fluorescent CuNCs. Herein, for the first time, we established this biosensor for sensing two amino acids by using dumbbell DNA-templated CuNCs as the single probe. Among 20 natural amino acids, only histidine and cysteine can selectively quench fluorescence emission of CuNCs, because of the specific interaction of these compounds with copper ions. Furthermore, by using nickel ions (Ni2+) and N-ethylmaleimide as the masking agents for histidine and cysteine respectively, an integrated logic gate system was designed by coupling with the fluorescent CuNCs and demonstrated selective and sensitive detection of cysteine and histidine. Under optimal conditions, cysteine can be detected in the concentration ranges of 0.01–10.0 μM with the detection limit (DL) of as low as 98 pM, while histidine can be detected in the ranges of 0.05–40.0 μM with DL of 1.6 nM. In addition, histidine and cysteine can be observed with the naked eye under a hand-held UV lamp (DL, 50 nM), which can be easily adapted to automated high-throughput screening. Finally, the strategy has been successfully utilized for biological fluids. The proposed system can be conducted in homogeneous solution, eliminating the need for organic cosolvents, separation processes of nanomaterials, or any chemical modifications. Overall, the assay provides an alternative method for simultaneous detection of cysteine and histidine by taking the advantages of high speed, no label and enzyme requirement, and good sensitivity and specificity, and will satisfy the great demand for determination of amino acids in fields such as food processing, biochemistry, pharmaceuticals, and clinical analysis. Graphical abstract
Keywords: Dumbbell DNA template; Histidine; Cysteine; Logic gate; Fluorescent copper nanoclusters

The analysis of intact glycopeptides is a challenge because of the structural variety of the complex conjugates. In this work, we used separation involving hydrophilic interaction liquid chromatography using a superficially porous particle HALO® penta-HILIC column with tandem mass spectrometric detection for the analysis of N-glycopeptides of hemopexin. We tested the effect of the mobile phase composition on retention and separation of the glycopeptides. The results indicated that the retention of the glycopeptides was the combination of partitioning and adsorption processes. Under the optimized conditions, our HILIC method showed the ability to efficiently separate the glycoforms of the same peptide backbone including separation of the isobaric glycoforms. We achieved efficient separation of core and outer arm linked fucose of bi-antennary and tri-antennary glycoforms of the SWPAVGNCSSALR peptide and bi-antennary glycoform of the ALPQPQNVTSLLGCTH peptide, respectively. Moreover, we demonstrated the separation of antennary position of sialic acid linked via α2-6 linkage of the monosialylated glycopeptides. Glycopeptide isomers are often differentially associated with various biological processes. Therefore, chromatographic separation of the species without the need for an extensive sample preparation appears attractive for their identification, characterization, and reliable quantification.
Keywords: Glycoproteomics; Glycopeptides; Hemopexin; Hydrophilic interaction liquid chromatography; LC-MS/MS

Agricultural intensification, and in particular the use of pesticides, leads over the years to a loss of biodiversity and a decline of ecosystem services in cultivated zones and agricultural landscapes. Among the animal communities involved in the functioning of agro-ecosystems, earthworms are ubiquitous and recognized as indicators of land uses and cultural practices. However, little data is available on the levels of pesticides in such organisms in natura, which would allow estimating their actual exposure and the potentially resulting impacts. Thus, the objective of this study was to develop a sensitive analytical methodology to detect and quantify 27 currently used pesticides in earthworms (Allolobophora chlorotica). A modified QuEChERS extraction was implemented on individual earthworms. This step was followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The whole analytical method was validated on spiked earthworm blank samples, with regard to linearity (from 1 to 100 method limit of quantification, r 2 > 0.95), intra-day precision (relative standard deviation (RSD) < 15%), inter-day precision (RSD < 20%), recoveries (mainly in the range 70–110%), and limits of detection and of quantification (inferior to 5 ng/g for most of the pesticides). The developed method was successfully applied to determine the concentrations of pesticides in nine individuals collected in natura. Up to five of the selected pesticides have been detected in one individual. Graphical abstract
Keywords: Pesticide residues; Earthworm; LC-MS/MS; Ecotoxicology; Trace analysis; Bioaccumulation

Strain-level typing and identification of bacteria – a novel approach for SERS active plasmonic nanostructures by Evelin Witkowska; Dorota Korsak; Aneta Kowalska; Anna Janeczek; Agnieszka Kamińska (5019-5031).
One of the potential applications of surface-enhanced Raman spectroscopy (SERS) is the detection of biological compounds and microorganisms. Here we demonstrate that SERS coupled with principal component analysis (PCA) serves as a perfect method for determining the taxonomic affiliation of bacteria at the strain level. We demonstrate for the first time that it is possible to distinguish different genoserogroups within a single species, Listeria monocytogenes, which is one of the most virulent foodborne pathogens and in some cases contact with which may be fatal. We also postulate that it is possible to detect additional proteins in the L. monocytogenes cell envelope, which provide resistance to benzalkonium chloride and cadmium. A better understanding of this infectious agent could help in selecting the appropriate pharmaceutical product for enhanced treatment. Graphical abstractᅟ
Keywords: Listeria monocytogenes ; SERS; Bacteria detection; Bacteria identification; Strain level discrimination; bcrABC ; BcrB; BcrC; CadA1; CadA2; PCA

The lipid fluidity of various lipid nanoemulsions (LNEs) without and with flutamide (FT) and containing one of two neutral lipids, one of four phosphatidylcholines as a surfactant, and sodium palmitate as a cosurfactant was investigated by the combination of 1H nuclear magnetic resonance (NMR) spectroscopy and principal component analysis (PCA). In the 1H NMR spectra, the peaks from the methylene groups of the neutral lipids and surfactants for all LNE preparations showed downfield shifts with increasing temperature from 20 to 60 °C. PCA was applied to the 1H NMR spectral data obtained for the LNEs. The PCA resulted in a model in which the first two principal components (PCs) extracted 88% of the total spectral variation; the first PC (PC-1) axis and second PC (PC-2) axis accounted for 73 and 15%, respectively, of the total spectral variation. The Score-1 values for PC-1 plotted against temperature revealed the existence of two clusters, which were defined by the neutral lipid of the LNE preparations. Meanwhile, the Score-2 values decreased with rising temperature and reflected the increase in lipid fluidity of each LNE preparation, consistent with fluorescence anisotropy measurements. In addition, the changes of Score-2 values with temperature for LNE preparations with FT were smaller than those for LNE preparations without FT. This indicates that FT encapsulated in LNE particles markedly suppressed the increase in lipid fluidity of LNE particles with rising temperature. Thus, PCA of 1H NMR spectra will become a powerful tool to analyze the lipid fluidity of lipid nanoparticles. Graphical abstractᅟ
Keywords: 1H NMR spectroscopy; Principal component analysis; Lipid fluidity; Lipid nanoemulsion; Flutamide; Fluorescence anisotropy

Search of non-ionic surfactants suitable for micellar liquid chromatography by Ester Peris-García; Jorge Rodríguez-Martínez; Juan J. Baeza-Baeza; María Celia García-Alvarez-Coque; María José Ruiz-Angel (5043-5057).
Most reports in reversed-phase liquid chromatography (RPLC) with micellar mobile phases make use of the anionic sodium dodecyl sulfate. This surfactant masks efficiently the silanol groups that are the origin of the poor efficiencies and tailing peaks observed for basic compounds in conventional RPLC. However, it has the handicap of yielding excessive retention, which forces the addition of an organic solvent to reduce the retention times to practical values. Other surfactants, such as the non-ionic polyoxyethylene(23)lauryl ether (Brij-35), are rarely used. Brij-35 allows the separation of a large range of analytes in adequate retention times, without the need of adding an organic solvent to the mobile phase. However, this non-ionic surfactant shows irreversible adsorption on chromatographic columns and peak shape is poorer. Therefore, the search of non-ionic surfactants with similar properties to Brij-35, but showing reversible adsorption and better peak shape, can be of great interest. In this work, the adequacy of several non-ionic surfactants as modifiers in RPLC has been explored, being polyoxyethylene(10)tridecyl ether particularly attractive. The separation of different types of compounds was checked: sulfonamides (acidic), β-adrenoceptor antagonists and tricyclic antidepressants (basic with diverse polarity), and flavonoids (with and without hydroxyl groups on the aromatic rings). The chromatographic behaviors were examined in terms of retention and peak shape. The results were compared with those obtained with Brij-35.
Keywords: Micellar liquid chromatography; Non-ionic surfactants; Polyoxyethylene(10)tridecyl ether; Polyoxyethylene(10)lauryl ether; Polyoxyethylene(23)lauryl ether; Chromatographic behavior

Identification and accurate quantification of structurally related peptide impurities in synthetic human C-peptide by liquid chromatography–high resolution mass spectrometry by Ming Li; Ralf D. Josephs; Adeline Daireaux; Tiphaine Choteau; Steven Westwood; Robert I. Wielgosz; Hongmei Li (5059-5070).
Peptides are an increasingly important group of biomarkers and pharmaceuticals. The accurate purity characterization of peptide calibrators is critical for the development of reference measurement systems for laboratory medicine and quality control of pharmaceuticals. The peptides used for these purposes are increasingly produced through peptide synthesis. Various approaches (for example mass balance, amino acid analysis, qNMR, and nitrogen determination) can be applied to accurately value assign the purity of peptide calibrators. However, all purity assessment approaches require a correction for structurally related peptide impurities in order to avoid biases. Liquid chromatography coupled to high resolution mass spectrometry (LC-hrMS) has become the key technique for the identification and accurate quantification of structurally related peptide impurities in intact peptide calibrator materials. In this study, LC-hrMS-based methods were developed and validated in-house for the identification and quantification of structurally related peptide impurities in a synthetic human C-peptide (hCP) material, which served as a study material for an international comparison looking at the competencies of laboratories to perform peptide purity mass fraction assignments. More than 65 impurities were identified, confirmed, and accurately quantified by using LC-hrMS. The total mass fraction of all structurally related peptide impurities in the hCP study material was estimated to be 83.3 mg/g with an associated expanded uncertainty of 3.0 mg/g (k = 2). The calibration hierarchy concept used for the quantification of individual impurities is described in detail. Graphical abstractᅟ
Keywords: Human C-peptide; Synthetic peptide; Impurity; Liquid chromatography; High resolution mass spectrometry

Fully automated sample preparation procedure to measure drugs of abuse in plasma by liquid chromatography tandem mass spectrometry by Tiphaine Robin; Alan Barnes; Sylvain Dulaurent; Neil Loftus; Sigrid Baumgarten; Stéphane Moreau; Pierre Marquet; Souleiman El Balkhi; Franck Saint-Marcoux (5071-5083).
For the analysis of drugs and pharmaceutical compounds in biological matrices, extraction procedures are typically used for LC-MS/MS analysis often requiring manual steps in sample preparation. In this study, we report a fully automated extraction method carried out by a programable liquid handler directly coupled to an LC-MS/MS system for the determination of 42 components (illicit drugs and/or metabolites) (plus 20 deuterated internal standards). The acquisition was performed in positive ionization mode with up to 15 MRM transitions per compound, each with optimized collision energy (MRM spectrum mode) to enable qualitative library searching in addition to quantitation. After placing the sample tube into the system, no further intervention was necessary: automated preparation used 50 μL of blood or plasma with 3 μL of extracted sample injected for analysis. The method was validated according to the requirements of ISO 15189. The limit of detection and quantification was 1–5 ng/mL depending on the compound. Stability experiments found that historic calibration curve data files could accurately quantify for up to 1 month with less than 20% uncertainty. Comparison to a QuEChERS method was made using patient samples providing a regression correlation R 2 = 0.98 between the two methods. This approach was successfully designed to support parallel sample preparation and analysis therefore significantly increasing sample throughput and reduced cycle times. Graphical abstractᅟ
Keywords: Liquid chromatography; Mass spectrometry; Automated sample preparation; Drugs of abuse

Electrochemical nonenzymatic sensor for cholesterol determination in food by Ksenia Derina; Elena Korotkova; Yekaterina Taishibekova; Lyazat Salkeeva; Bohumil Kratochvil; Jiri Barek (5085-5092).
The treatment of some inborn metabolism errors requires cholesterol substitution therapy. Cholesterol plays a vital role in the human body. Therefore, the majority of cholesterol determination techniques are targeted to blood and blood serum. Nevertheless, cholesterol determination in food is important as well. In this paper, cholesterol determination using differential pulse voltammetry (DPV) in dairy products (e.g., milk, clotted cream, yogurt, butter, etc.) is reported with a novel nonenzymatic sensor based on diphosphonic acid of 1,4-diacetylglycoluril (DPADGU) as an electrode surface modifier. Stable anodic response was obtained from cholesterol on the modified carbon-based electrode. The sensor has high stability, sensitivity (20 μA mol L−1 cm−2), and a wide linear range from 1 up to 200 μM. The LOD and LOQ values are 1.5 and 5.1 μM, respectively. The developed methods were successfully applied to the above mentioned dairy products. Graphical abstractᅟ
Keywords: Cholesterol; Voltammetry; Sensor; Smith-Lemli-Opitz syndrome; Dairy products