Analytical and Bioanalytical Chemistry (v.409, #26)

The only constant is change by Adam T. Woolley (6053-6053).
is Chair Editor of Analytical and Bioanalytical Chemistry, and University Professor in the Department of Chemistry and Biochemistry at Brigham Young University in Provo, Utah, USA. His current research focuses on 3D printed integrated microfluidics for biomarker measurement, analytical systems for the identification of bacteria and antibiotic resistance genes in sepsis, and biotemplated fabrication of nanoelectronics.

is a PhD student at Aalen University and Helmholtz Zentrum München. He has been working on the analysis of small-molecule drugs, complex proteins, and N-glycans by applying capillary zone electrophoresis–mass spectrometry. His PhD project is related to the development and application of two-dimensional electromigrative separation techniques coupled with mass spectrometry. His latest interest is focused on the characterization of monoclonal antibody charge variants, including detailed interpretation of mass-spectrometric information. is a former PhD student at Aalen University and the University of Tübingen. During his PhD studies he worked on the development and application of two-dimensional electromigrative separation techniques coupled with mass spectrometry, especially for the characterization of capillary isoelectric focused proteins. Since February 2017, he has been a product manager and support engineer for workflow and instrument diagnostic software at Agilent Technologies. In this role, he focuses on new und intuitive workflows for method scouting and development. is a group head in the Analytical Development and Quality Control Department of Pharma Technical Development Europe (Biologics) Analytics at F. Hoffmann-La Roche Ltd, Basel, Switzerland. He works in the field of developing and validating methods for the physicochemical analysis of proteins, mainly antibodies, for release and stability testing with focus on separation techniques such as chromatography and capillary electrophoresis. is a group head in the Analytical Development and Quality Control Department of Pharma Technical Development Europe (Biologics) Analytics at F. Hoffmann-La Roche Ltd, Basel, Switzerland. He works in the field of developing, validating, and characterizing methods for the physicochemical analysis of proteins, mainly antibodies, for release and stability testing with focus on separation techniques such as capillary electrophoresis. is Full Professor in the Faculty of Chemistry at Aalen University. His research interests include sophisticated separation techniques coupled with (high-resolution) mass spectrometry. He is an expert in capillary electrophoresis–mass spectrometry with various applications, especially in the biopharmaceutical field. His latest research focus is on the development of two-dimensional electromigrative separation techniques coupled with mass spectrometry. Capillary zone electrophoresis (CZE) is a powerful tool that is progressively being applied for the separation of monoclonal antibody (mAb) charge variants. Mass spectrometry (MS) is the desired detection method concerning identification of mAb variants. In biopharmaceutical applications, there exist optimized and validated electrolyte systems for mAb variant quantification. However, these electrolytes interfere greatly with the electrospray ionization (ESI) process. Here, a heart-cut CZE–CZE–MS setup with an implemented mechanical four-port valve interface was developed that used a generic ε-aminocaproic acid based background electrolyte in the first dimension and acetic acid in the second dimension. Interference-free, highly precise mass data (deviation less than 1 Da) of charge variants of trastuzumab, acting as model mAb system, were achieved. The mass accuracy obtained (low parts per million range) is discussed regarding both measured and calculated masses. Deamidation was detected for the intact model antibody, and related mass differences were significantly confirmed on the deglycosylated level. The CZE–CZE–MS setup is expected to be applicable to a variety of antibodies and electrolyte systems. Thus, it has the potential to become a compelling tool for MS characterization of antibody variants separated in ESI-interfering electrolytes. Graphical Abstract Two-dimensional capillary zone electrophoresis mass spectrometry for the characterization of intact monoclonal antibody (mAb) charge variants. A generic, but highly electrospray-interfering electrolyte system was used as first dimension for mAb charge variant separation and coupled to a volatile electrolyte system as second dimension via a four-port nanoliter valve. In this way, interference-free and precise mass spectrometric data of separated mAb charge variants, including deamidation products, were obtained
Keywords: Antibody analysis; ε-Aminocaproic acid; Electrospray ionization; Capillary zone electrophoresis–mass spectrometry; Two-dimensional interface; Mechanical valve; Pharmaceutical application

is a scientist in the Chemical Metrology Division, Health Sciences Authority, Singapore. She obtained her MSc in chemistry from the National University of Singapore in 2014. Her main research interests are development of reference measurement methods for additives and contaminants in food and beverages, as well as purity assessment of organic compounds using the mass balance approach. is a scientist in the Chemical Metrology Division, Health Sciences Authority, Singapore. She obtained her PhD in chemistry from the University of Wisconsin–Madison in 2009. Her main research interests are development measurement methods for contaminates and additives in food and beverages, as well as purity assessment of organic compounds using quantitative nuclear magnetic resonance spectroscopy. is the Laboratory Director and the Team Leader of the Proficiency Testing & Certified Reference Materials Unit in the Chemical Metrology Division, Health Sciences Authority, Singapore. She received her PhD in chemistry from the National University of Singapore in 2007. Her main interests are the development of reference methods for contaminants and additives in food and beverages, as well as purity assessment of organic compounds using mass balance and quantitative nuclear magnetic resonance spectroscopic methods. is a senior scientist in the Chemical Metrology Division, Health Sciences Authority, Singapore. She obtained her MSc in chemistry from the National University of Singapore in 2008. Her main interests are the development of certified reference materials and statistical methods for use in chemical testing, CRM production and proficiency testing. is the Division Director of the Chemical Metrology Division, Health Sciences Authority, Singapore. He has overseen the development of the measurement capabilities for the chemical metrology programme since 2008. A gas chromatography–isotope dilution mass spectrometry (GC–IDMS) technique was developed for the quantification of two heavy polyaromatic hydrocarbons (PAHs), benz[a]anthracene and benzo[a]pyrene, in yerba maté tea (maté). The optimisation of two extraction methods, namely liquid–liquid extraction and accelerated solvent extraction, was carried out. Both optimised methods were validated using a certified reference material of fine dust and the results were within the expanded uncertainties at 95% confidence level. Recoveries of 99.2–106.7% with RSD of measurements of 1.1–2.3% were achieved for benz[a]anthracene. Recoveries of 95.7–101.9% with RSD of measurements of 0.4–1.4% were achieved for benzo[a]pyrene. The validated methods were applied for the extraction of benz[a]anthracene and benzo[a]pyrene in maté powder from NIST. A metrological approach was undertaken to ensure the traceability of measurement results. The uncertainties associated with the results were rigorously evaluated and also reported herein. Graphical abstract Quantification of benz[a]anthracene and benzo[a]pyrene using IDMS
Keywords: Isotope dilution mass spectrometry; Polycyclic aromatic hydrocarbon; Traceability; Yerba maté tea; Measurement uncertainty

The concentration of L-cysteine (Cys) and glutathione (GSH) is closely related to the critical risk of various diseases. In our study, a new rapid method for the determination of Cys and GSH in water and urine samples has been developed using a fluorescent probe technique, which was based on crystal violet (CV)-functionalized CdTe quantum dots (QDs). The original QDs emitted fluorescence light, which was turned off upon adding CV. This conjugation of CV and QDs could be attributed to electrostatic interaction between COO of mercaptopropionic acid (MPA) on the surface of QDs and N+ of CV in aqueous solution. In addition, Förster resonance energy transfer (FRET) also occurred between CdTe QDs and CV. After adding Cys or GSH to the solution, Cys or GSH exhibited a stronger binding preference toward Cd2+ than Cd2+-MPA, which disturbed the interaction between MPA and QDs. Thus, most MPA was able to be separated from the surface of QDs because of the participation of Cys or GSH. Then, the fluorescence intensity of the CdTe QDs was enhanced. Good linear relationships were obtained in the range of 0.02–40 μg mL−1 and 0.02–50 μg mL−1, and the detection limits were calculated as 10.5 ng mL−1 and 8.2 ng mL−1, for Cys and GSH, respectively. In addition, the concentrations of biological thiols in water and urine samples were determined by the standard addition method using Cys as the standard; the quantitative recoveries were in the range of 97.3–105.8%, and relative standard deviations (RSDs) ranged from 2.5 to 3.7%. The method had several unique properties, such as simplicity, lower cost, high sensitivity, and environmental acceptability. Graphical abstract Crystal violet-functionalized CdTe quantum dots for detecting L-cysteine and glutathione with switch-on fluorescent strategy
Keywords: L-Cysteine; Glutathione; Quantum dot; Fluorescent; Switch-on

Dopamine-mediated immunoassay for bacteria detection by Yi Wan; Guiyou Zhu (6091-6096).
Traditional enzyme-linked immunosorbent assay (ELISA) with sufficient sensing specificity is a useful analytical approach for the detection of toxicologically important substances in in vivo systems or complicated biological systems. Increasing worldwide demand for analyses of bacteria by signal amplification and increasing concern regarding their safe development and use require a simple, stable, and sensitive detection assay for target evaluation and clinical diagnosis. A sensitive and selective immunoassay for detection of bacteria is constructed that combines horseradish peroxidase (HRP)-catalyzed signal amplification with the strong linker of the polydopamine–biotin complex on the surface of solid substances or biomolecules. The incorporation of HRP labeling and amplification increases the detection sensitivity by about one to two orders of magnitude compared with conventional ELISA systems. A linear relationship between the response and the logarithm of the bacterial concentration was observed in the range from 1.5 × 102 to 1.5 × 107 colony-forming units per milliliter. This work demonstrates a new signal-amplification-based dopamine-mediated process for the development of a sensitive method. This dopamine-mediated immunoassay may be broadly applied in clinical diagnoses and for the monitoring of water environmental pollution. The approach proposed is distinct with simple protocols and easy processes, which allow it to be applied in a broad area.
Keywords: Dopamine; Immunoassay; Signal amplification; Bacterial detection

ToF-SIMS and principal component analysis of lipids and amino acids from inflamed and dysplastic human colonic mucosa by Marco Urbini; Valentina Petito; Francesco de Notaristefani; Franco Scaldaferri; Antonio Gasbarrini; Luca Tortora (6097-6111).
Here, time of flight secondary ion mass spectrometry (ToF-SIMS) and multivariate analysis were combined to study the role of ulcerative colitis (UC), a type of inflammatory bowel disease (IBD), in the colon cancer progression. ToF-SIMS was used to obtain mass spectra and chemical maps from the mucosal surface of human normal (NC), inflamed (IC), and dysplastic (DC) colon tissues. Chemical mapping with a lateral resolution of ≈ 1 μm allowed to evaluate zonation of fatty acids and amino acids as well as the morphological condition of the intestinal glands. High mass resolution ToF-SIMS spectra showed chemical differences in lipid and amino acid composition as a function of pathological state. In positive ion mode, mono- (MAG), di- (DAG), and triacylglycerol (TAG) signals were detected in NC tissues, while in IC and DC tissues, the only cholesterol was present as lipid class representative. Signals from fatty acids, collected in negative ion mode, were subjected to principal component analysis (PCA). PCA showed a strict correlation between IC and DC samples, due to an increase of stearic, arachidonic, and linoleic acid. In the same way, differences in the amino acid composition were highlighted through multivariate analysis. PCA revealed that glutamic acid, leucine/isoleucine, and valine fragments are related to IC tissues. On the other hand, tyrosine, methionine, and tryptophan peaks contributed highly to the separation of DC tissues. Finally, a classification of NC, IC, and DC patients was also achieved through hierarchical cluster analysis of amino acid fragments. In this case, human colonic inflammation showed a stronger relationship with normal than dysplastic condition. Graphical Abstract ᅟ
Keywords: ToF-SIMS; PCA; Colon cancer; IBD; Ulcerative colitis

Magnetic graphene nanoparticles coated with a new deep eutectic solvent (Fe3O4@GO-DES) were developed for efficient preconcentration of methadone. The extracted methadone was then analyzed by gas chromatography–flame ionization detection (GC–FID) or gas chromatography–mass spectrometry (GC–MS). Fe3O4@GO-DES were characterized by Fourier transform IR and X-ray diffraction techniques. Ultrasound was used to enhance the dispersion of the sorbent, with a high extraction recovery. Some parameters affecting the extraction recovery, such as pH, type of deep eutectic solvent, sample volume, amount of sorbent, extraction time, and type of eluent, were investigated. Under optimum conditions, the method developed was linear in the concentration range from 3 to 45,000 μg L-1 for GC–FID and from 0.1 to 500 μg L-1 for GC–MS, with a detection limit of 0.8 μg L-1 for GC–FID and 0.03 μg L-1 for GC–MS. The relative standard deviations (n = 6) as the intraday and interday precisions of the methadone spike at a concentration of 100 μg L-1 were 5.8% and 8.4% respectively for GC–FID. The preconcentration factor was 250. Relative recoveries from spiked plasma, urine, and water samples ranged from 95.1% to 101.5%.
Keywords: Deep eutectic solvent; Magnetic graphene nanoparticles; Ultrasonic; Methadone; Plasma and urine

Towards quantification of toxicity of lithium ion battery electrolytes - development and validation of a liquid-liquid extraction GC-MS method for the determination of organic carbonates in cell culture materials by Jenny Strehlau; Till Weber; Constantin Lürenbaum; Julia Bornhorst; Hans-Joachim Galla; Tanja Schwerdtle; Martin Winter; Sascha Nowak (6123-6131).
A novel method based on liquid-liquid extraction with subsequent gas chromatography separation and mass spectrometric detection (GC-MS) for the quantification of organic carbonates in cell culture materials is presented. Method parameters including the choice of extraction solvent, of extraction method and of extraction time were optimised and the method was validated. The setup allowed for determination within a linear range of more than two orders of magnitude. The limits of detection (LODs) were between 0.0002 and 0.002 mmol/L and the repeatability precisions were in the range of 1.5–12.9%. It could be shown that no matrix effects were present and recovery rates between 98 and 104% were achieved. The methodology was applied to cell culture models incubated with commercial lithium ion battery (LIB) electrolytes to gain more insight into the potential toxic effects of these compounds. The stability of the organic carbonates in cell culture medium after incubation was studied. In a porcine model of the blood-cerebrospinal fluid (CSF) barrier, it could be shown that a transfer of organic carbonates into the brain facing compartment took place. Graphical abstract Schematic setup for the investigation of toxicity of lithium ion battery electrolytes
Keywords: Liquid-liquid extraction; GC-MS; Lithium ion battery (LIB); Organic carbonates; Cell culture materials

Development of neutron imaging quantitative data treatment to assess conservation products in cultural heritage by Marco Realini; Chiara Colombo; Claudia Conti; Francesco Grazzi; Enrico Perelli Cippo; Jan Hovind (6133-6139).
Distribution, penetration depth and amount of new mineralogical phases formed after the interaction between an inorganic treatment and a matrix are key factors for the evaluation of the conservation treatment behaviour. Nowadays, the conventional analytical methodologies, such as vibrational spectroscopies, scanning electron microscopy and X-ray diffraction, provide only qualitative and spot information. Here, we report, for the first time, the proof of concept of a methodology based on neutron imaging able to achieve quantitative data useful to assess the formation of calcium oxalate in a porous carbonatic stone treated with ammonium oxalate. Starting from the neutron attenuation coefficient of Noto stone-treated specimens, the concentrations of newly formed calcium oxalate and the diffusion coefficient have been calculated for both sound and decayed substrates. These outcomes have been also used for a comparative study between different treatment modalities. Graphical abstract Horizontal slice at 300 mm depth and CaOx molar density profile by NEUTRA output
Keywords: Neutron imaging; Ammonium oxalate; Cultural heritage; Treatment; Noto stone

A new LC-MS/MS bioanalytical method for perindopril and perindoprilat in human plasma and milk by Ei Mon Phyo Lwin; Cobus Gerber; Yunmei Song; Catherine Leggett; Usha Ritchie; Sean Turner; Sanjay Garg (6141-6148).
A first of its kind, simple, rapid, and sensitive liquid chromatography mass spectrometry (LC-MS/MS) method was developed and validated for quantification of perindopril and perindoprilat in both human plasma and breast milk. The analytes and internal standards (phenazone and acetyl salicylic acid) were extracted from biological matrices by protein precipitation. A Phenomenex® C-18 column was used to provide an appropriate chromatographic separation of the analytes, followed by detection with tandem mass spectrometry. Gradient chromatographic and mass spectrometric detection conditions with mobile phases (A: 5% methanol + 0.1% formic acid in water v/v, and B: 95% methanol + 0.1% formic acid in water v/v) were developed to achieve a LOQ of 0.5 ng/mL in both human plasma and milk. The method was suitable of evaluating clinical samples. The mass transition was followed as m/z 369.10/172.00 for perindopril, m/z 339.00/168.10 for perindoprilat, m/z 188.90/55.95 for phenazone, and m/z 179.04/137.02 for acetyl salicylic acid. The developed method was optimized and validated with a linear range of 0.1–200 ng/mL (r 2 = better than 0.99 for both perindopril and perindoprilat). The precision and accuracy values were within 15% CV. The overall recovery of the analytes was 80–110%. The method has good specificity and repeatability. Stability studies were conducted in both human plasma and bovine milk for up to 3 months, at the storage conditions of 25, 4, and −80 °C.
Keywords: Perindopril; Perindoprilat; LC-MS/MS; Human plasma; Human breast milk; Lactation

Screening of additives and other chemicals in polyurethanes by direct analysis in real time mass spectrometry (DART-MS) by Ákos Kuki; Lajos Nagy; Tibor Nagy; Miklós Zsuga; Sándor Kéki (6149-6162).
Direct analysis in real time mass spectrometry (DART-MS) was used to characterize commercial polyurethane (PUR) samples without sample pretreatment. More than 50 substances, such as catalysts, stabilizers, antioxidants, flame retardants, plasticizers, chain extenders, chain terminators, polyols, solvents, degradation products and contaminants, a few of them presumably toxic, were detected and identified in 18 PUR items. The identification of 16 compounds was further confirmed by DART MS/MS experiments. Catalysts were the largest class of compounds detected in the PURs by DART-MS. In each of the 18 PUR samples, at least one catalyst residue was identified. In addition, DART-MS was able to detect the migration of hazardous chemicals from the PURs to other objects. The collision-induced dissociation (CID) properties of two PUR catalysts, such as the protonated bis[2-(dimethylamino)ethyl] ether (DMAEE) and the protonated 2,2-dimorpholinodiethylether (DMDEE), as well as those of two PUR antioxidants (Antioxidant 1135 and Antioxidant 1076), were explored.
Keywords: Polymers; Catalysts; Mass spectrometry

Investigation of an optimal cell lysis method for the study of the zinc metalloproteome of Histoplasma capsulatum by Anna M. Donnell; Stephanie Lewis; Sami Abraham; Kavitha Subramanian; Julio Landero Figueroa; George S. Deepe Jr; Anne P. Vonderheide (6163-6172).
This work sought to assess optimal extraction conditions in the study of the metalloproteome of the dimorphic fungus Histoplasma capsulatum. One of the body’s responses to H. capsulatum infection is sequestration of zinc within host macrophage (MØ), as reported by Vignesh et al. (Immunity 39:697–710, 2013) and Vignesh et al. (PLOS Pathog 9:E1003815, 2013). Thus, metalloproteins containing zinc were of greatest interest as it plays a critical role in survival of the fungus. One challenge in metalloproteomics is the preservation of the native structure of proteins to retain non-covalently bound metals. Many of the conventional cell lysis, separation, and identification techniques in proteomics are carried out under conditions that could lead to protein denaturation. Various cell lysis techniques were investigated in an effort to both maintain the metalloproteins during lysis and subsequent analysis while, at the same time, serving to be strong enough to break the cell wall, allowing access to cytosolic metalloproteins. The addition of 1% Triton x-100, a non-ionic detergent, to the lysis buffer was also studied. Seven lysis methods were considered and these included: Glass Homogenizer (H), Bead Beater (BB), Sonication Probe (SP), Vortex with 1% Triton x-100 (V, T), Vortex with no Triton x-100 (V, NT), Sonication Bath, Vortex, and 1% Triton x-100 (SB, V, T) and Sonication Bath, Vortex, and no Triton x-100 (SB, V, NT). A Qubit® Assay was used to compare total protein concentration and inductively coupled plasma–mass spectrometry (ICP-MS) was utilized for total metal analysis of cell lysates. Size exclusion chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS) was used for separation of the metalloproteins in the cell lysate and the concentration of Zn over a wide molecular weight range was examined. Additional factors such as potential contamination sources were also considered. A cell lysis method involving vortexing H. capsulatum yeast cells with 500 μm glass beads in a 1% Triton x-100 lysis buffer (V, T) was found to be most advantageous to extract intact zinc metalloproteins as demonstrated by the highest Zn to protein ratio, 1.030 ng Zn/μg protein, and Zn distribution among high, mid, and low molecular weights suggesting the least amount of protein denaturation. Graphical abstract In this work, several cell lysis techniques and two lysis buffers were investigated to evaluate the preservation of the zinc metalloproteome of H. capsulatum while maintaining compatibility with the analytical techniques employed.
Keywords: Cell lysis; Metalloproteomics; Histoplasma capsulatum ; ICP-MS; Zinc

Comparison of global metabolite extraction strategies for soybeans using UHPLC-HRMS by Iqbal Mahmud; Sandi Sternberg; Michael Williams; Timothy J. Garrett (6173-6180).
Metabolism, downstream effectors of genomics, transcriptomics, and proteomics, can determine the potential of phenotype of an organism including plants. Profiling the global scenario of metabolism requires optimization of different solvent extraction methods. Here, we report an approach comparing three different metabolite extraction strategies, including ammonium acetate/methanol (AAM), water/methanol (WM), and sodium phosphate/methanol (PM) in soybean plant using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). Interestingly, both AAM and WM methods were found to cover a wider range of metabolites and provide better detection of molecular features than the PM method. Various clustering analyses based on multivariate statistical tools revealed that both AAM and WM methods showed tight and overlapping extraction strategy compared with the PM method. Using MatLab-based Mahalanobis distance (D M) calculation, statistically significant score plot separation was observed between AAM and PM, as well as WM and PM. However, no significant separation was observed between AAM and WM, which is expected from the overlap of principal component scores for these two methods. Using differential metabolite expression analysis, we identified that a large number of metabolites were extracted at a significantly higher level using AAM vs. PM. These comparative extraction methods suggest that AAM can effectively be applied for an LC/MS-based plant metabolomics profile study. Graphical abstract Step-by-step outline of three different metabolite extraction methods and data analysis
Keywords: Metabolite profiling; Extraction comparison; Liquid chromatography/mass spectrometry; Multivariate statistics; Metabolomics

An analytical method was developed and validated for the determination of ten pesticides in sewage sludge coming from an agro-food industry. The method was based on the application of Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) extraction for solid sewage sludge and SPE extraction for sludge aqueous phase, followed by liquid chromatography (LC) coupled to hybrid quadrupole/linear ion trap mass spectrometry (QqLIT-MS). The QuEChERS method was reported 14 years ago and nowadays is mainly applied to the analysis of pesticides in food. More recent applications have been reported in other matrices as sewage sludge, but the complexity of the matrix makes necessary the optimization of the cleanup step to improve the efficiency of the analysis. With this aim, several dispersive solid-phase extraction cleanup sorbents were tested, choosing C18 + PSA as a d-SPE sorbent. The proposed method was satisfactorily validated for most compounds investigated, showing recoveries higher than 80% in most cases, with the only exception of prochloraz (71%) at low concentration level. Limits of quantification were lower than 40 ng l−1 in the aqueous phase and below 40 ng g−1 in the solid phase for the majority of the analytes. The method was applied to solid sludge and the sludge aqueous phase coming from an agro-food industry which processes fruits and vegetables. Graphical abstract Application of LC/MS/MS advanced analytical techniques for determination of pesticides contained in sewage sludge
Keywords: Agro-food industry; Industrial wastewater; LC-MS; Pesticides; Sludge; QuEChERS

Phthalocyanine-BODIPY dye: synthesis, characterization, and utilization for pattern recognition of CYFRA 21-1 in whole blood samples by Raluca-Ioana Stefan-van Staden; Ionela Raluca Comnea-Stancu; Hülya Yanık; Meltem Göksel; Anghel Alexandru; Mahmut Durmuş (6195-6203).
Phthalocyanine-BODIPY dye (BODIPY = boron dipyrromethene) was synthesized, fully characterized, and used for molecular recognition of CYFRA 21-1, a lung cancer biomarker, from whole blood samples. Thin films of three magnesium oxides ((MgO) n , where n = 8, 9, or 10)) were deposited on a paper substrate, and they were immersed in a solution of phthalocyanine-BODIPY dye (1.17 × 10−3 mol/L) for the design of stochastic sensors. Limits of determination of picograms per milliliter magnitude order were recorded for the proposed stochastic sensors. CYFRA 21-1 was reliably identified and determined with recoveries higher than 95% and RSD lower than 1% in whole blood samples.
Keywords: CYFRA 21-1; Stochastic sensors; Lung cancer; Phthalocyanine; BODIPY

Determination of 89 drugs and other micropollutants in unfiltered wastewater and freshwater by LC-MS/MS: an alternative sample preparation approach by Alexandros G. Asimakopoulos; Pranav Kannan; Sean Higgins; Kurunthachalam Kannan (6205-6225).
A liquid chromatography-triple quadrupole-tandem mass spectrometry (LC-qQq-MS/MS) method was developed for simultaneous determination of 89 legal neuropsychiatric pharmaceuticals and illicit drugs (both parent compounds and metabolites) and other micropollutants in unfiltered wastewater and freshwater. The target chemicals fall under the classes of amphetamine-type stimulants, cocaine compounds, opiates and opioids, benzodiazepines, lysergic compounds, antipsychotics, anesthetics, antiepileptics, antidepressants, sympathomimetics, cannabinoids, blood thinners, antihistamines, β-blockers, caffeine derivatives, nicotine derivatives, z-drugs, new designer drugs, and Alzheimer medications. The sample preparation procedure was designed for unfiltered wastewater and freshwater without the need to separate the particulate matter (if any) from the aqueous phase prior to extraction. Samples were pre-concentrated by rotary evaporation in the presence of a solvent. Method precision (absolute values; N = 6 replicate analyses at the fortification level of 50 ng, k = 6 days) for 87 out of 89 target analytes ranged from 2.8 to 34% (RSD %). The limits of detection ranged from 0.11 to 202 ng L−1, and the matrix effects ranged from +16 to −84%. A total of 10 samples, 8 wastewater, 1 drinking water, and 1 lake water, were collected from New York State, USA, and were analyzed for the target compounds to demonstrate the applicability of the developed method. This is the first study to report the analysis of multiple classes of pharmaceuticals, illicit drugs, and other micropollutants in unfiltered wastewater. Graphical abstract Analysis of 89 micropollutants in unfiltered wastewater by LC-MS/MS
Keywords: Drugs; Narcotics; Micropollutants; Wastewater; Freshwater; Illicit drugs

Gold nanoparticle-based localized surface plasmon immunosensor for staphylococcal enterotoxin A (SEA) detection by Maroua Ben Haddada; David Hu; Michèle Salmain; Lu Zhang; Chen Peng; Yi Wang; Bo Liedberg; Souhir Boujday (6227-6234).
We describe the engineering of stable gold nanoparticle (AuNP) bioconjugates for the detection of staphylococcal enterotoxin A (SEA) using localized surface plasmon resonance (LSPR). Two types of AuNP bioconjugates were prepared by covalently attaching anti-SEA antibody (Ab) or SEA to AuNPs. This was achieved by reacting Traut’s reagent with lysine residues of both proteins to generate thiol groups that bind to gold atoms on the AuNP surface. These bioconjugates were characterized in-depth by absorption spectroscopy, cryo-transmission electron microscopy, dynamic light scattering, and zeta potential measurements. Their stability over time was assessed after 1 year storage in the refrigerator at 4 °C. Two formats of homogeneous binding assays were set up on the basis of monitoring of LSPR peak shifts resulting from the immunological reaction between the (i) immobilized antibody and free SEA, the direct assay, or (ii) immobilized SEA and free antibody, the competitive assay. In both formats, a correlation between the LSPR band shift and SEA concentration could be established. Though the competitive format did not meet the expected analytical performance, the direct format, the implementation of which was very simple, afforded a specific and sensitive response within a broad dynamic range—nanogram per milliliter to microgram per milliliter. The limit of detection (LOD) of SEA was estimated to equal 5 ng/mL, which was substantially lower than the LOD obtained using a quartz crystal microbalance. Moreover, the analytical performance of AuNP-Ab bioconjugate was preserved after 1 year of storage at 4 °C. Finally, the LSPR biosensor was successfully applied to the detection of SEA in milk samples. The homogeneous nanoplasmonic immunosensor described herein provides an attractive alternative for stable and reliable detection of SEA in the nanogram per milliliter range and offers a promising avenue for rapid, easy to implement, and sensitive biotoxin detection. Sensitive LSPR Biosensing of SEA in buffer and milk using stable AuNP-Antibody bioconjugates Graphical abstract
Keywords: Immunosensor; Localized surface plasmon resonance; Staphylococcal enterotoxin A; Gold nanoparticles

A new metabolomics-based strategy for identification of endogenous markers of urine adulteration attempts exemplified for potassium nitrite by Andrea E. Steuer; Kim Arnold; Tom D. Schneider; Michael Poetzsch; Thomas Kraemer (6235-6244).
Urine adulteration to circumvent positive drug testing represents a problem for toxicological laboratories. While creatinine is a suitable marker for dilution, detection of chemicals is often performed by dipstick tests associated with high rates of false positives. Several methods would be necessary to check for all possible adulterants. Untargeted mass spectrometry (MS) methods used in metabolomics should theoretically allow detecting concentration changes of any endogenous urinary metabolite or presence of new biomarkers produced by chemical adulteration. As a proof of concept study, urine samples from 10 volunteers were treated with KNO2 and analyzed by high-resolution MS. For statistical data evaluation, XCMSplus and MetaboAnalyst were used. Compound identification was performed by database searches using an in-house database, Chemspider, METLIN, HMDB, and NIST. Principle component analysis revealed clear separation between treated and untreated urine samples. In detail, 307 features showed significant concentration changes with fold changes greater than 2 (79 decreased; 228 increased). Mainly amino acids (e.g., histidine, methylhistidine, di- and trimethyllysine) and purines (uric acid) were detected in lower amounts. 5-HO-isourate was found to be formed as a new compound from uric acid and, e.g., imidazole lactate concentrations increased due to the breakdown of histidine. This metabolomics-based strategy allowed for a broad identification range of markers of urinary adulteration. More studies will be needed to investigate routine applicability of identified potential markers exploring urinary conditions of their formation and stability. Selected markers might then be integrated into routine MS screening procedures allowing for detection of adulteration within routine MS analysis. Graphical Abstract ᅟ
Keywords: Urine adulteration; Biomarker identification; Metabolomics methods; High-resolution mass spectrometry

Formaldehyde passive sampler using an optical chemical sensor: how to limit the humidity interference by Jane Vignau-Laulhere; Pierre Mocho; Hervé Plaisance; Katarzyna Raulin-Woznica; Thu-Hoa Tran-Thi; Valérie Desauziers (6245-6252).
The behaviour of a new formaldehyde diffusive sampler using an optical chemical sensor with respect to high humidity conditions is examined in controlled atmospheres. Five prototypes of the radial diffusion sampler having the same chemical sensor and different designs were tested. In addition, a set of experiments were performed on the chemical sensor to characterise its efficiency of trapping water vapour in the absence and in the presence of the reactants, Fluoral-P and formaldehyde. Differences in humidity interference between the five diffusive sampler prototypes were studied and discussed. From all the results obtained, it was shown that the prototype LDE1.4 combining a small diffusion slot, a reduced internal volume and a sensor shifted upwards from the diffusion slot provided formaldehyde measurements least affected by humidity up to 80% RH at 20 °C. This new type of diffusive sampler with on-site direct reading is intended to ultimately replace conventional passive samplers with DNPH requiring offset laboratory analyses.
Keywords: Radial diffusive sampler; Gas sensor; Fluoral-P; Exposure chamber; Formaldehyde

Micro- versus nano-sized molecularly imprinted polymers in MALDI-TOF mass spectrometry analysis of peptides by Lucia Cenci; Maddalena Bertolla; Andrea Anesi; Emmanuele Ambrosi; Graziano Guella; Alessandra Maria Bossi (6253-6261).
The integration of molecularly imprinted polymers (MIPs) with MALDI-TOF mass spectrometry (MS) combines MIP selectivity with MS sensitivity. Whether the size of the MIP material—micro versus nano—has an effect on the MS analysis was the object of the study. MIPs, targeting respectively the epitope peptide NR11 of cardiac troponin I and the peptide CK13 of human serum transferrin, were synthesized and characterized. The size-related performance of the MIP materials hyphenated with MALDI-TOF-MS analysis was studied by the incubation of the target peptide with the respective micro- or nano-MIP, followed by rinsing to remove non-specific deposition of the MIP to the MALDI target plate, co-crystallization with the organic matrix, and mass analysis. The quality of the MS analysis was assessed comparing the S/N of the mass peaks of the MIP-bound peptide to that of the same quantity of free peptide. Sweet spots and lower S/N (~ 1 order of magnitude) were observed for micro-MIP materials, while in the case of nano-MIP-bound peptide, the S/N was comparable to that of the free peptide, indicating higher compatibility of the nano-MIPs to MALDI-TOF-MS. The nano-MIP/MALDI-TOF-MS permitted the selective determination of the target peptide in real serum samples. Graphical abstract ᅟ
Keywords: Molecularly imprinted polymers; Nanoparticle; MALDI; Mass spectrometry; Peptide; Serum

Pre-analytic evaluation of volumetric absorptive microsampling and integration in a mass spectrometry-based metabolomics workflow by Chiara Volani; Giulia Caprioli; Giovanni Calderisi; Baldur B. Sigurdsson; Johannes Rainer; Ivo Gentilini; Andrew A. Hicks; Peter P. Pramstaller; Guenter Weiss; Sigurdur V. Smarason; Giuseppe Paglia (6263-6276).
Volumetric absorptive microsampling (VAMS) is a novel approach that allows single-drop (10 μL) blood collection. Integration of VAMS with mass spectrometry (MS)-based untargeted metabolomics is an attractive solution for both human and animal studies. However, to boost the use of VAMS in metabolomics, key pre-analytical questions need to be addressed. Therefore, in this work, we integrated VAMS in a MS-based untargeted metabolomics workflow and investigated pre-analytical strategies such as sample extraction procedures and metabolome stability at different storage conditions. We first evaluated the best extraction procedure for the polar metabolome and found that the highest number and amount of metabolites were recovered upon extraction with acetonitrile/water (70:30). In contrast, basic conditions (pH 9) resulted in divergent metabolite profiles mainly resulting from the extraction of intracellular metabolites originating from red blood cells. In addition, the prolonged storage of blood samples at room temperature caused significant changes in metabolome composition, but once the VAMS devices were stored at − 80 °C, the metabolome remained stable for up to 6 months. The time used for drying the sample did also affect the metabolome. In fact, some metabolites were rapidly degraded or accumulated in the sample during the first 48 h at room temperature, indicating that a longer drying step will significantly change the concentration in the sample. Graphical abstract Volumetric absorptive microsampling (VAMS) is a novel technology that allows single-drop blood collection and, in combination with mass spectrometry (MS)-based untargeted metabolomics, represents an attractive solution for both human and animal studies. In this work, we integrated VAMS in a MS-based untargeted metabolomics workflow and investigated pre-analytical strategies such as sample extraction procedures and metabolome stability at different storage conditions. The latter revealed that prolonged storage of blood samples at room temperature caused significant changes in metabolome composition, but if VAMS devices were stored at − 80 °C, the metabolome remained stable for up to 6 months
Keywords: Metabolomics; Volumetric absorptive microsampling; Mass spectrometry