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Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 provide one of the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI–MS spectrum, BFD allowed very high sequence coverage (≥ 99%) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2–8 amino acids), and the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, homocysteinylation, glutathionylation, truncated glutathionylation, and cyanylation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80–90%) and did not detect peptides carrying most of the above-mentioned PTMs. The two C-terminal peptides of a dimer [RBD(319–541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD, low-abundance scrambling variants, free cysteine residues, O-glycoforms, and incomplete processing of the N-terminal end, if present. Artifacts generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented; it has been applied to the characterization of the active pharmaceutical ingredient of two RBD-based vaccines, and we foresee that it can be also helpful to the characterization of mutated RBDs. Graphical abstract: [Figure not available: see fulltext.]

The global manufacturing of clothing is usually composed of multistep processes, which include a large number of chemicals. However, there is generally no information regarding the chemical content remaining in the finished clothes. Clothes in close and prolonged skin contact may thus be a significant source of daily human exposure to hazardous compounds depending on their ability to migrate from the textiles and be absorbed by the skin. In the present study, twenty-four imported garments on the Swedish market were investigated with respect to their content of organic compounds, using a screening workflow. Reversed-phase liquid chromatography coupled to electrospray ionization/high-resolution mass spectrometry was used for both suspect and non-target screening. The most frequently detected compound was benzothiazole followed by quinoline. Nitroanilines with suspected mutagenic and possible skin sensitization properties, and quinoline, a carcinogenic compound, were among the compounds occurring at the highest concentrations. In some garments, the level of quinoline was estimated to be close to or higher than 50,000 ng/g, the limit set by the REACH regulation. Other detected compounds were acridine, benzotriazoles, benzothiazoles, phthalates, nitrophenols, and organophosphates. Several of the identified compounds have logP and molecular weight values enabling skin uptake. This pilot study indicates which chemicals and compound classes should be prioritized for future quantitative surveys and control of the chemical content in clothing as well as research on skin transfer, skin absorption, and systemic exposure. The results also show that the current control and prevention from chemicals in imported garments on the Swedish market is insufficient. Graphical abstract

Antiplatelet and anticoagulant drugs are classified antithrombotic agents with the purpose to reduce blood clot formation. For a successful treatment of many known complex cardiovascular diseases driven by platelet and/or coagulation activity, the need of more than one antithrombotic agent is inevitable. However, combining drugs with different mechanisms of action enhances risk of bleeding. Dual anticoagulant and antiplatelet (APAC), a novel semisynthetic antithrombotic molecule, provides both anticoagulant and antiplatelet properties in preclinical studies. APAC is entering clinical studies with this new exciting approach to manage cardiovascular diseases. For a better understanding of the biological function of APAC, comprehensive knowledge of its structure is essential. In this study, atomic force microscopy (AFM) was used to characterize APAC according to its structure and to investigate the molecular interaction of APAC with von Willebrand factor (VWF), since specific binding of APAC to VWF could reduce platelet accumulation at vascular injury sites. By the optimization of drop-casting experiments, we were able to determine the volume of an individual APAC molecule at around 600 nm3, and confirm that APAC forms multimers, especially dimers and trimers under the experimental conditions. By studying the drop-casting behavior of APAC and VWF individually, we depictured their interaction by using an indirect approach. Moreover, in vitro and in vivo conducted experiments in pigs supported the AFM results further. Finally, the successful adsorption of APAC to a flat gold surface was confirmed by using photothermal-induced resonance, whereby attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) served as a reference method. Graphical abstract

Biopolymers based on polylactic acid (PLA) and starch have numerous advantages, such as coming from renewable sources or being compostable, though they can have deficiencies in mechanical properties, and for this reason, polyester resins are occasionally added to them in order to improve their properties. In this work, migration from a PLA sample and from another starch-based biopolymer to three different food simulants was studied. Attention was focused on the determination of oligomers. The analysis was first performed by ultraperformance liquid chromatography quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF–MS), which allowed the identification of the oligomers present in migration. Then, the samples were analyzed by two ambient desorption/ionization techniques directly coupled to mass spectrometry (ADI), direct analysis in real-time coupled to standardized voltage and pressure (DART-MS) and atmospheric pressure solids analysis probe (ASAP-MS). These methodologies were able to detect simultaneously the main oligomers migrants and their adducts in a very rapid and effective way. Nineteen different polyester oligomers, fourteen linear and five cyclic, composed of different combinations of adipic acid [AA], propylene glycol [PG], dipropylene glycol [DPG], 2,2-dibutyl-1,3-propanediol [DBPG], or isobutanol [i-BuOH] were detected in migration samples from PLA. In migration samples from starch-based biopolymer, fourteen oligomers from poly(butylene adipate co-terephthalate) polyester (PBAT) were identified, twelve cyclic and two linear. The results from ADI techniques showed that they are a very promising alternative tool to assess the safety and legal compliance of food packaging materials. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03755-0.

Graphical abstract Among the deadliest pandemics in history, coronavirus disease 2019 (COVID-19) has wreaked havoc on human lives, economies and public health systems worldwide. To temper its effects, diagnostic methods that are simple, rapid, inexpensive, accurate, selective and sensitive continue to be necessary. In our study, we developed an electrochemical biosensing platform based on gold clusters, mercaptoethanol, the spike protein of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antigen and bovine serum albumin-modified glassy carbon electrode able to detect the SARS-CoV-2 spike antibody. Moreover, during the detection of the SARS-CoV-2 spike antibody in spiked-real samples, the anodic signal of the produced biosensor at 0.85 V decreased as the amount of the SARS-CoV-2 spike antibody increased. Meanwhile, the recovery and relative standard deviation values for saliva and oropharyngeal swab samples were 97.73% and 3.35% and 102.43% and 4.63%, respectively. In 35 min, the biosensing platform could detect 0.03 fg/mL of the SARS-CoV-2 spike antibody in synthetic media and spiked-saliva or -oropharyngeal swab samples. The method thus issues a linear response to the SARS-CoV-2 spike antibody from 0.1 fg/mL to 10 pg/mL. The cross-reactivity studies with spike antigens of Middle East respiratory syndrome-coronavirus and influenza A and the antigen of pneumonia confirmed the excellent selectivity of the proposed method. The developed method was compared with the lateral flow immunoassay method in terms of sensitivity and it was found to be approximately 109 times more sensitive. Biosensing mechanism of the platform to the SARS-CoV-2 spike antibody Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03752-3.

LGD-4033 (ligandrol) is a selective androgen receptor modulator (SARM), which is prohibited in sports by the World Anti-Doping Agency (WADA) and led to 62 adverse analytical findings (AAFs) in 2019. But not only deliberate doping with LGD-4033 constitutes a problem. In the past years, some AAFs that concerned SARMs can be attributed to contaminated dietary supplements (DS). Thus, the urgency to develop methods to differentiate between inadvertent doping and abuse of SARMs to benefit from the performance-enhancing effect of the compound in sports is growing. To gain a better understanding of the metabolism and excretion patterns of LGD-4033, human micro-dose excretion studies at 1, 10, and 50 µg LGD-4033 were conducted. Collected urine samples were prepared for analysis using enzymatic hydrolysis followed by solid-phase extraction and analyzed via LC-HRMS/MS. Including isomers, a total of 15 phase I metabolites were detected in the urine samples. The LC-HRMS/MS method was validated for qualitative detection of LGD-4033, allowing for a limit of detection (LOD) of 8 pg/mL. The metabolite M1, representing the epimer of LGD-4033, was synthesized and the structure elucidated by NMR spectroscopy. As the M1/LGD-4033 ratio changes over time, the ratio and the approximate LGD-4033 concentration can contribute to estimating the time point of drug intake and dose of LGD-4033 in doping control urine samples, which is particularly relevant in anti-doping result management. Graphical abstract

The application of contaminated paper sludge on arable land in southwest Germany caused the occurrence of a broad range of poly- and perfluoroalkyl substances (PFASs) on soil. Recently, the dead-end transformation products (TPs) perfluorooctanoic acid and perfluorooctanesulfonic acid were detected in groundwater and drinking water. The precursors and other transformation products mostly remained unknown. Therefore, HRMS screening by Kendrick mass analysis and assignment of homologous series in combination with suspect screening were applied to identify original PFASs and their TPs in four different soil samples from sites where contaminated paper sludge was applied. In total, twelve compound classes comprising more than 61 PFASs could be fully or tentatively identified. The data reveal that contamination mainly originates from polyfluorinated dialkylated phosphate esters (from 4:2/6:2 to 12:2/14:2), N-ethyl perfluorooctane sulfonamide ethanol–based phosphate diesters (only C8/C8) and transformation products of these precursors. Contamination patterns can be attributed to PFASs used for paper impregnation and can vary slightly from site to site. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-019-02358-0) contains supplementary material, which is available to authorized users.

Graphical abstract Parent and alkylated polycyclic aromatic hydrocarbons (PAHs) are present in a number of different sources in varying proportions depending on the source material and weathering. This range of PAH sources can make it difficult to determine the origin of exposure(s). Ratios of alkylated and parent PAHs have been applied as a forensic tool to distinguish between different sources. However, few studies have examined PAH ratios comprehensively as indicators for sourcing beyond a single study area or matrix type. In this paper, we introduce an expanded analytical method based on ASTM D7363-13a which we adapted for a gas chromatography triple quadrupole mass spectrometry instrument. The modifications increase selectivity and sensitivity compared to the ASTM method. We added five alkylated series to the method. This method has then been applied to 22 independent forensic ratios. We evaluated the method and the forensic ratios with certified reference materials and known environmental samples. This analytical method and thirteen PAH ratios were found to accurately predict sources of PAHs. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-020-03127-0.

Intense label-free surface-enhanced Raman scattering (SERS) spectra of serum samples were rapidly obtained on Ag plasmonic paper substrates upon 785 nm excitation. Spectra from the hepatocellular carcinoma (HCC) patients showed consistent differences with respect to those of the control group. In particular, uric acid was found to be relatively more abundant in patients, while hypoxanthine, ergothioneine, and glutathione were found as relatively more abundant in the control group. A repeated double cross-validation (RDCV) strategy was applied to optimize and validate principal component analysis-linear discriminant analysis (PCA-LDA) models. An analysis of the RDCV results indicated that a PCA-LDA model using up to the first four principal components has a good classification performance (average accuracy was 81%). The analysis also allowed confidence intervals to be calculated for the figures of merit, and the principal components used by the LDA to be interpreted in terms of metabolites, confirming that bands of uric acid, hypoxanthine, ergothioneine, and glutathione were indeed used by the PCA-LDA algorithm to classify the spectra. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-020-03093-7.

Recycling will be of increasing importance in the future, especially for plastic packaging waste mainly consisting of polyolefins. One major problem of recyclates comprises impurities which can have a significant negative impact on future product properties. Polyamide 6 can be found widely as contaminant in recycled polyolefins, leading to a need of quantification methods thereof. In this paper, a method development for the quantitative analysis of polyamide 6 is presented based on analysing ε-caprolactam and related cyclic oligomers as marker compounds in model recyclates of high- and low-density polyethylene and polypropylene compounded with low amounts of polyamide 6. For the method development and tentative identification of the different cyclic compounds, a HPLC-QTOF-MS was used and it was possible to detect six different compounds, ε-caprolactam and the corresponding cyclic di- to hexamer. The quantification was performed with a HPLC-QQQ-MS, equipped with a HILIC column, after sample preparation via microwave-assisted extraction. It could be shown that a good linearity from 0.2 up to 5 wt% polyamide 6 in the different polyolefins can be achieved. The cyclic trimer and tetramer show a low limit of quantification and are therefore well-suited for the quantification, whereas the other cyclic compounds can be then used as qualifiers to avoid false positives. To guarantee the applicability of the method, six real recyclate materials were analysed, whereby in three of them low amounts of polyamide 6 could be detected. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-020-03071-z.

Since 1 January 2019 a maximum content of 0.6 mg kg−1 cadmium (Cd) in cocoa powder sold to the final consumer or as an ingredient in sweetened cocoa powder sold to the final consumer (drinking chocolate) is set by the Commission Regulation (EU) No. 488/2014. Monitoring compliance with the specified limit value requires analytical measuring methods and reference materials for quality control. However, suitable certified reference materials intended for quality assurance and quality control purposes are still lacking. Therefore, three cocoa reference materials (ERM®-BD513, ERM®-514 and ERM®-515) were developed according to the requirements of ISO 17034 and the recommendations of ISO Guide 35. The whole process of reference material development, including material preparation, assessment of homogeneity and stability, characterisation and value assignment is presented. The assignment of the certified mass fractions was based upon an interlaboratory comparison study involving 19 expert laboratories for Cd and 12 laboratories for acrylamide. The certified mass fractions and expanded uncertainties (k = 2) of the reference materials were (0.181 ± 0.009) mg kg−1 Cd (ERM®-BD513), (0.541 ± 0.024) mg kg−1 Cd (ERM®-BD514) and (0.690 ± 0.029) mg kg−1 Cd (ERM®-BD515). Acrylamide contents are given for information.

Nanolayer and nanolayer by nanolayer deposition of nanofilms of Ag and C using cold plasma in sequences (Ag, Ag-C, Ag-Ag-C), on porous paper, were used to design three disposable stochastic sensors for the assay of amyloid polypeptide from whole blood. The nanofilms were modified with α-cyclodextrin. The test developed using the nanofilm-based disposable stochastic sensors is used for early detection of diabetes. The wider linear concentration range (1.00 × 10−6–1.00 ng mL−1) and the lower limit of quantification (1.00 × 10−6ng mL−1) were obtained using the disposable stochastic sensors based on Ag-C and Ag-Ag-C, while the highest sensitivity (3.19 × 104 s−1/μg mL−1) was recorded using the disposable stochastic sensor based on Ag-Ag-C. The screening methods were fully validated using whole blood samples from confirmed patients, when the recovery of the islet amyloid polypeptide was higher than 98.00%.

[EN] Many voltammetry methods have been developed to monitor brain extracellular dopamine levels. Fewer approaches have been successful in detecting serotonin in vivo. No voltammetric techniques are currently available to monitor both neurotransmitters simultaneously across timescales, even though they play integrated roles in modulating behavior. We provide proof-of-concept for rapid pulse voltammetry coupled with partial least squares regression (RPV-PLSR), an approach adapted from multi-electrode systems (i.e., electronic tongues) used to identify multiple components in complex environments. We exploited small differences in analyte redox profiles to select pulse steps for RPV waveforms. Using an intentionally designed pulse strategy combined with custom instrumentation and analysis software, we monitored basal and stimulated levels of dopamine and serotonin. In addition to faradaic currents, capacitive currents were important factors in analyte identification arguing against background subtraction. Compared to fast-scan cyclic voltammetry-principal components regression (FSCV-PCR), RPV-PLSR better differentiated and quantified basal and stimulated dopamine and serotonin associated with striatal recording electrode position, optical stimulation frequency, and serotonin reuptake inhibition. The RPV-PLSR approach can be generalized to other electrochemically active neurotransmitters and provides a feedback pipeline for future optimization of multi-analyte, fit-for-purpose waveforms and machine learning approaches to data analysis., Funding from the National Institute on Drug Abuse (DA045550) and National Institute of Mental Health (MH106806) was received. CSM was supported by the National Science Foundation Graduate Research Fellowship Program (DGE-1650604 and DGE-2034835). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute watery diarrhea and vomiting in unweaned piglets, and is associated with high mortality, thus causing severe economic losses in the pig industry. Currently, although attenuated vaccines are commonly used in commercial pig farms in China, they do not completely protect against all mutated wild-type strains. Existing nucleic acid assays have high sensitivity and specificity, but the complexity of the assay process and expensive instrumentation hinder disease detection. Here, reverse transcription–enzymatic recombinase amplification (RT-ERA) was combined with the CRISPR-Cas12a system to develop a rapid diagnostic method to distinguish PEDV wild-type strains from attenuated vaccine strains. The protocol used crRNA and RT-ERA amplification primers against open reading frame 3 (ORF3), followed by Cas12a/crRNA complex detection of predefined target sequences at 37 °C for 30 min, thus producing results visible to the naked eye under LED blue light. The assay is highly sensitive and specific, detecting as few as two copies of the target gene per test and showing no cross-reactivity with other porcine pathogens. Overall, this integrated RT-ERA pre-amplification and Cas12a/crRNA cleavage assay is a practical tool for reliable and rapid detection of PEDV for diagnostic differentiation. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03716-7.

Graphical abstract The spike protein of SARS-CoV-2, the virus responsible for the global pandemic of COVID-19, is an abundant, heavily glycosylated surface protein that plays a key role in receptor binding and host cell fusion, and is the focus of all current vaccine development efforts. Variants of concern are now circulating worldwide that exhibit mutations in the spike protein. Protein sequence and glycosylation variations of the spike may affect viral fitness, antigenicity, and immune evasion. Global surveillance of the virus currently involves genome sequencing, but tracking emerging variants should include quantitative measurement of changes in site-specific glycosylation as well. In this work, we used data-dependent acquisition (DDA) and data-independent acquisition (DIA) mass spectrometry to quantitatively characterize the five N-linked glycosylation sites of the glycoprotein standard alpha-1-acid glycoprotein (AGP), as well as the 22 sites of the SARS-CoV-2 spike protein. We found that DIA compared favorably to DDA in sensitivity, resulting in more assignments of low-abundance glycopeptides. However, the reproducibility across replicates of DIA-identified glycopeptides was lower than that of DDA, possibly due to the difficulty of reliably assigning low-abundance glycopeptides confidently. The differences in the data acquired between the two methods suggest that DIA outperforms DDA in terms of glycoprotein coverage but that overall performance is a balance of sensitivity, selectivity, and statistical confidence in glycoproteomics. We assert that these analytical and bioinformatics methods for assigning and quantifying glycoforms would benefit the process of tracking viral variants as well as for vaccine development. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03643-7.

In this study, Lba Cas12a (Cpf1) as one of the CRISPR systems from Lachnospiraceae bacterium was coupled with a hybridization chain reaction (HCR) to develop an electrochemical biosensor for detecting the pathogenic bacterium, Salmonella typhimurium. Autonomous cross-opening of functional DNA hairpin structures of HCR yielded polymer double-stranded DNA wires consisting of numerous single-stranded DNAs, which initiated the trans-cleavage activity of CRISPR-Cas12a to indiscriminately cleave random single-stranded DNA labeling electrochemical tags on the surface of the electrode. It led to a variation in the electron transfer of electrochemical tags. The polymer double-stranded DNA of HCR was immobilized on dynabeads (DBs) via the S. typhimurium aptamer and released from DBs. The established method could selectively and sensitively quantify S. typhimurium in samples with detection limits of 20 CFU/mL. Our study provides a novel insight for exploring universal analytical methods for pathogenic bacteria based on CRISPR-Cas12a coupled with HCR. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03733-6.

Graphical abstract The article analyzes experimentally and theoretically the influence of microscope parameters on the pinhole-assisted Raman depth profiles in uniform and composite refractive media. The main objective is the reliable mapping of deep sample regions. The easiest to interpret results are found with low magnification, low aperture, and small pinholes. Here, the intensities and shapes of the Raman signals are independent of the location of the emitter relative to the sample surface. Theoretically, the results can be well described with a simple analytical equation containing the axial depth resolution of the microscope and the position of the emitter. The lower determinable object size is limited to 2–4 μm. If sub-micrometer resolution is desired, high magnification, mostly combined with high aperture, becomes necessary. The signal intensities and shapes depend now in refractive media on the position relative to the sample surface. This aspect is investigated on a number of uniform and stacked polymer layers, 2–160 μm thick, with the best available transparency. The experimental depth profiles are numerically fitted with excellent accuracy by inserting a Gaussian excitation beam of variable waist and fill fraction through the focusing lens area, and by treating the Raman emission with geometric optics as spontaneous isotropic process through the lens and the variable pinhole, respectively. The intersectional area of these two solid angles yields the leading factor in understanding confocal (pinhole-assisted) Raman depth profiles. Spearfishing is a well-known example of the effects of refraction at the boundary between two index-mismatched media. The object Greal is seen, due to refraction, as Gvir from the angle β (without knowing the depth position). The real position is obtained under the angle α. In a microscope (see inset), index mismatch deforms the image point of Greal into an image line. The pinhole substantially reduces deformations and allows the determination of the position of the point emitter G. (Cartoon designed by Sofia Anker)

Sterols are a highly complex group of lipophilic compounds present in the unsaponifiable matter of virtually all living organisms. In this study, we developed a novel gas chromatography with mass spectrometry selected ion monitoring (GC/MS-SIM) method for the comprehensive analysis of sterols after saponification and silylation. A new referencing system was introduced by means of a series of saturated fatty acid pyrrolidides (FAPs) as internal standards. Linked with retention time locking (RTL), the resulting FAP retention indices (RIFAP) of the sterols could be determined with high precision. The GC/MS-SIM method was based on the parallel measurement of 17 SIM ions in four time windows. This set included eight molecular ions and seven diagnostic fragment ions of silylated sterols as well as two abundant ions of FAPs. Altogether, twenty molecular ions of C27- to C31-sterols with 0–3 double bonds were included in the final method. Screening of four common vegetable oils (sunflower oil, hemp oil, rapeseed oil, and corn oil) enabled the detection of 30 different sterols and triterpenes most of which could be identified. Graphical abstract

In order to perform good kinetic experiments, not only the experimental conditions have to be optimized, but the evaluation procedure as well. The focus of this work is the in-depth comparison of different approaches and algorithms to determine kinetic rate constants for biomolecular interaction analysis (BIA). The different algorithms are applied not only to flawless simulated data, but also to real-world measurements. We compare five mathematical approaches for the evaluation of binding curves following pseudo-first-order kinetics with different noise levels. In addition, reflectometric interference spectroscopy (RIfS) measurements of two antibodies are evaluated to determine their binding kinetics. The advantages and disadvantages of the individual approach will be investigated and discussed in detail. In summary, we will raise awareness on how to evaluate and judge results from BIA by using different approaches rather than having to rely on “black box” closed (commercial) software packages. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03623-x.

Cyclic dinucleotides (CDNs) are key secondary messenger molecules produced by cyclic dinucleotide synthases that trigger various cellular signaling cascades from bacteria to vertebrates. In mammals, cyclic GMP-AMP synthase (cGAS) has been shown to bind to intracellular DNA and catalyze the production of the dinucleotide 2′3′ cGAMP, which signals downstream effectors to regulate immune function, interferon signaling, and the antiviral response. Despite the importance of CDNs, sensitive and accurate methods to measure their levels in vivo are lacking. Here, we report a novel LC-MS/MS method to quantify CDNs in vivo. We characterized the mass spectrometric behavior of four different biologically relevant CDNs (c-di-AMP, c-di-GMP, 3′3′ cGAMP, 2′3′ cGAMP) and provided a means of visually representing fragmentation resulting from collision-induced dissociation at different energies using collision energy breakdown graphs. We then validated the method and quantified CDNs in two in vivo systems, the bacteria Escherichia coli OP50 and the killifish Nothobranchius furzeri. We found that optimization of LC-MS/MS parameters is crucial to sensitivity and accuracy. These technical advances should help illuminate physiological and pathological roles of these CDNs in in vivo settings. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03628-6.

This manuscript describes a new multiresidue method utilising ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) via multiple reaction monitoring (MRM), for the identification and quantification of 58 antibiotics and their 26 metabolites, in various solid and liquid environmental matrices. The method was designed with a ‘one health’ approach in mind requiring multidisciplinary and multisectoral collaborative efforts. It enables comprehensive evaluation of antibiotic usage in surveyed communities via wastewater-based epidemiology, as well as allowing for the assessment of potential environmental impacts. The instrumental performance was very good, demonstrating linearity up to 3000 μg L−1, and high accuracy and precision. The method accuracy in several compounds was significantly improved by dividing calibration curves into separate ranges. This was accompanied by applying a weighting factor (1/x). Microwave-assisted and/or solid-phase extraction of analytes from liquid and solid matrices provided good recoveries for most compounds, with only a few analytes underperforming. Method quantification limits were determined as low as 0.017 ng L−1 in river water, 0.044 ng L−1 in wastewater, 0.008 ng g−1 in river sediment, and 0.009 ng g−1 in suspended solids. Overall, the method was successfully validated for the quantification of 64 analytes extracted from aqueous samples, and 45 from solids. The analytes that underperformed are considered on a semi-quantitative basis, including aminoglycosides and carbapenems. The method was applied to both solid and liquid environmental matrices, whereby several antibiotics and their metabolites were quantified. The most notable antibiotic-metabolite pairs are three sulfonamides and their N-acetyl metabolites; four macrolides/lincomycins and their N-desmethyl metabolites; and five quinolone metabolites. Graphical abstract

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is widely used as a crosslinker for fluorescence labeling of protein in the fields of biochemistry and food analysis. Many natural polysaccharides often contain some proteins or peptides that are very low in content but play a vital role in their biological function as well as technical applications. Determination of these low-content proteinaceous matters requires a highly sensitive and selective method. In this study, a methodological approach for investigations of the presence of proteinaceous material over the molar mass distribution (MD) of polysaccharides was developed using gum acacia (GA) as a model polysaccharide. EDC fluorescence-labeling method was modified by changing the pH (7, 9, and 11) of the solution for the analysis of low-content protein in food materials. Fluorescence spectroscopy and asymmetrical flow field-flow fractionation (AF4) were employed for characterizing the labeling efficiency and physiochemical properties of unlabeled and fluorescence-labeled GA. AF4 provided molar mass (M) and the radius of gyration (rG) of arabinogalactan (AG) and arabinogalactan protein complex (AGP) and determined the presence of proteinaceous matter over the MD. The labeling efficiencies of GA at pH 7, 9, and 11 determined by fluorescence spectroscopy were 56.5, 68.4, and 72.0%, respectively, with an increment of 15.5% when pH was increased from 7 to 11. The modified EDC fluorescence-labeling method allows highly sensitive and selective analysis of low-content proteinaceous matters and their distribution in natural polysaccharides. Graphical abstract

Graphical abstract The use of magnetic beads bio-functionalized by antibodies (Ab) is constantly increasing with a wide range of biomedical applications. However, despite an urgent need for current methods to monitor Ab’s grafting process and orientation, existing methods are still either cumbersome and/or limited. In this work, we propose a new simple and rapid analytical approach to evaluate antibody orientation and density on magnetic beads. This approach relies on the cleavage by IdeS, a highly specific protease for human immunoglobulin G (hIgG), of immobilized antibodies. The F(ab)2 and Fc fragments could be then accurately quantified by size exclusion chromatography (SEC)-coupled to fluorescent detection (FLD), and the ratio of these fragments was used to give insight on the IgG orientation at the bead surface. Four different commercially available magnetic beads, bearing carboxyl groups, tosyl groups, streptavidin, or protein G on their surface have been used in this study. Results obtained showed that this approach ensures reliable information on hIgG orientation and bead surface coverage. Protein G magnetic beads demonstrated an optimal orientation of antibodies for antigen capture (75% of accessible F(ab)2 fragment) compared to tosylactivated, carboxylated, and streptavidin ones. Capture efficiency of the different functionalized beads towards human TNF-α immunocapture, a biomarker of inflammation, has been also compared. Protein G beads provided a more efficient capture compared to other beads. In the future, this approach could be applied to any type of surface and beads to assess hIgG coverage and orientation after any type of immobilization. A rapid and simple approach to evaluate orientation and density of antibodies immobilized on magnetic beads Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03608-w.

Short-chain fatty acids (SCFAs) are increasingly being monitored to elucidate the link between gut health and disease. These metabolites are routinely measured in faeces, but their determination in serum is more challenging due to their low concentrations. A method for the determination of eight SCFAs in serum is described here. High-resolution mass spectrometry and gas chromatography were used to identify the presence of isomeric interferences, which were then overcome through a combination of chromatographic separation and judicious choice of MS fragment ion. The SCFAs were derivatised to form 3-nitrophenylhydrazones before being separated on a reversed-phase column and then detected using liquid chromatography tandem mass spectrometry (LC-QQQ-MS). The LODs and LOQs of SCFAs using this method were in the range 1 to 7 ng mL−1 and 3 to 19 ng mL−1, respectively. The recovery of the SCFAs in serum ranged from 94 to 114% over the three concentration ranges tested. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03589-w.

Over the past few years, laser-induced breakdown spectroscopy (LIBS) has earned a lot of attention in the field of online polymer identification. Unlike the well-established near-infrared spectroscopy (NIR), LIBS analysis is not limited by the sample thickness or color and therefore seems to be a promising candidate for this task. Nevertheless, the similar elemental composition of most polymers results in high similarity of their LIBS spectra, which makes their discrimination challenging. To address this problem, we developed a novel chemometric strategy based on a systematic optimization of two factors influencing the discrimination ability: the set of experimental conditions (laser energy, gate delay, and atmosphere) employed for the LIBS analysis and the set of spectral variables used as a basis for the polymer discrimination. In the process, a novel concept of spectral descriptors was used to extract chemically relevant information from the polymer spectra, cluster purity based on the k-nearest neighbors (k-NN) was established as a suitable tool for monitoring the extent of cluster overlaps and an in-house designed random forest (RDF) experiment combined with a cluster purity–governed forward selection algorithm was employed to identify spectral variables with the greatest relevance for polymer identification. Using this approach, it was possible to discriminate among 20 virgin polymer types, which is the highest number reported in the literature so far. Additionally, using the optimized experimental conditions and data evaluation, robust discrimination performance could be achieved even with polymer samples containing carbon black or other common additives, which hints at an applicability of the developed approach to real-life samples.Graphical abstract

Graphical abstract Abundant studies have been published evaluating different parameters of reverse-phase liquid chromatography (LC) and supercritical fluid chromatography (SFC), both coupled to electrospray (ESI)/mass spectrometry (MS) for pesticide residue analysis. However, there is a lack of a comprehensive comparative study that facilitates deep knowledge about the benefits of using each technique. In the present study, the same mass spectrometer was used coupled to both liquid and supercritical fluid chromatographies with a multiresidue method of 215 compounds, for the analysis of pesticide residues in food samples. Through the injection of the spiked extracts, separate experiments were conducted. A study of the optimum ion source temperature using the different chromatography modes was performed. The results were evaluated in terms of sensitivity with tomato, leek, onion, and orange as representative fruit and vegetable matrices. The compounds which reported the highest area values in each chromatography were evaluated through their substance groups and polarity values. The impact of matrix effects obtained in tomato matrix was similar for both cases; however, SFC clearly showed better results in analyzing matrices with a higher number of natural co-extracted compounds. This can be explained by the combination of two effects: (i) chromatography separation and (ii) ion source efficiency. The chromatographic elution presented different profiles of matrix components, which had diverse impact on the coelution with the analytes, being more beneficial when SFC was used in the matrices studied. The data showed that the best results obtained in SFC are also related to a higher ionization efficiency even when the ESI emitter tip was not optimized for SFC flow. In the present study a comprehensive evaluation of the benefits and drawbacks of these chromatography modes for routine pesticide residue analysis related to target compounds/commodities is provided. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03565-4.

Analysis of fatty acids (FA) in food and biological samples such as blood is indispensable in modern life sciences. We developed a rapid, sensitive and comprehensive method for the quantification of 41 saturated and unsaturated fatty acids by means of LC-MS. Optimized chromatographic separation of isobaric analytes was carried out on a C8 reversed phase analytical column (100 × 2.1 mm, 2.6 μm core–shell particle) with a total run time of 15 min with back pressure lower than 300 bar. On an old triple quadrupole instrument (3200, AB Sciex), pseudo selected reaction monitoring mode was used for quantification of the poorly fragmenting FA, yielding limits of detection of 5–100 nM. Sample preparation was carried out by removal of phospholipids and triglycerides by solid-phase extraction (non-esterified fatty acids in oils) or saponification in iso-propanol (fatty acyls). This is not only a rapid strategy for quantification of fatty acyls, but allows the direct combination with the LC-MS-based analysis of fatty acid oxidation products (eicosanoids and other oxylipins) from the same sample. The concentrations of fatty acyls determined by means of LC-MS were consistent with those from GC-FID analysis demonstrating the accuracy of the developed method. Moreover, the method shows high precisions with a low intra-day (≤ 10% for almost all fatty acids in plasma and ≤ 15% in oils) and inter-day as well as inter-operator variability (< 20%). The method was successfully applied on human plasma and edible oils. The possibility to quantify non-esterified fatty acids in samples containing an excess of triacylglycerols and phospholipids is a major strength of the described approach allowing to gain new insights in the composition of biological samples. Graphical abstract

Trimethylamine-N-oxide (TMAO), a microbiome-derived metabolite from the metabolism of choline, betaine, and carnitines, is associated to adverse cardiovascular outcomes. A method suitable for routine quantification of TMAO and its precursors (trimethylamine (TMA), choline, betaine, creatinine, and propionyl-, acetyl-, and l-carnitine) in clinical and food samples has been developed based on LC-MS. TMA was successfully derivatized using iodoacetonitrile, and no cross-reactions with TMAO or the other methylamines were detected. Extraction from clinical samples (plasma and urine) was performed after protein precipitation using acetonitrile:methanol. For food samples (meatballs and eggs), water extraction was shown to be sufficient, but acid hydrolysis was required to release bound choline before extraction. Baseline separation of the methylamines was achieved using a neutral HILIC column and a mobile phase consisting of 25 mmol/L ammonium formate in water:ACN (30:70). Quantification was performed by MS using external calibration and isotopic labelled internal standards. The assay proved suitable for both clinical and food samples and was linear from ≈ 0.1 up to 200 μmol/L for all methylamines except for TMA and TMAO, which were linear up to 100 μmol/L. Recoveries were 91–107% in clinical samples and 76–98% in food samples. The interday (n=8, four duplicate analysis) CVs were below 9% for all metabolites in clinical and food samples. The method was applied successfully to determine the methylamine concentrations in plasma and urine from the subjects participating in an intervention trial (n=10) to determine the effect of animal food ingestion on methylamine concentrations. Graphical abstract

Detailed molecular analysis is of increasing importance in research into the regulation of biochemical pathways, organismal growth and disease. Lipidomics in particular is increasingly sought after as it provides insight into molecular species involved in energy storage, signalling and fundamental cellular structures. This has led to the use of a range of tools and techniques to acquire lipidomics data. 31P NMR for lipidomics offers well-resolved head group/lipid class analysis, structural data that can be used to inform and strengthen interpretation of mass spectrometry data and part of a priori structural determination. In the present study, we codify the use of 31P NMR for lipidomics studies to make the technique more accessible to new users and more useful for a wider range of questions. The technique can be used in isolation (phospholipidomics) or as a part of determining lipid composition (lipidomics). We describe the process from sample extraction to data processing and analysis. This pipeline is important because it allows greater thoroughness in lipidomics studies and increases scope for answering scientific questions about lipid-containing systems. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03430-4.

Raman spectroscopy is an analytical method to identify medical samples of bacteria. Because Raman spectroscopy detects the biochemical properties of a cell, there are many factors that can influence and modify the Raman spectra of bacteria. One possible influence is a proper method for isolation of the bacteria. Medical samples in particular never occur in purified form, so a Raman-compatible isolation method is needed which does not affect the bacteria and thus the resulting spectra. In this study, we present a Raman-compatible method for isolation of bacteria from bronchoalveolar lavage (BAL) fluid using density gradient centrifugation. In addition to measuring the bacteria from a patient sample, the yield and the spectral influence of the isolation on the bacteria were investigated. Bacteria isolated from BAL fluid show additional peaks in comparison to pure culture bacteria, which can be attributed to components in the BAL sample. The isolation gradient itself has no effect on the spectra, and with a yield of 63% and 78%, the method is suitable for isolation of low concentrations of bacteria from a complex matrix. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03488-0.

Electrospray ionization (ESI) generates bare analyte ions from charged droplets, which result from spraying a liquid in a strong electric field. Experimental observations available in the literature suggest that at least a significant fraction of the initially generated droplets remain large, have long lifetimes, and can thus aspirate into the inlet system of an atmospheric pressure ionization mass spectrometer (API-MS). We report on the observation of fragment signatures from charged droplets penetrating deeply the vacuum stages of three commercial mass spectrometer systems with largely different ion source and spray configurations. Charged droplets can pass through the ion source and pressure reduction stages and even into the mass analyzer region. Since droplet signatures were found in all investigated instruments, the incorporation of charged droplets is considered a general phenomenon occurring with common spray conditions in ESI sources. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03452-y.

Sulfur mustard (SM) is a banned chemical warfare agent recently used in the Syrian Arab Republic conflict causing erythema and blisters characterized by complicated and delayed wound healing. For medical and legal reasons, the proof of exposure to SM is of high toxicological and forensic relevance. SM reacts with endogenous human serum albumin (HSA adducts) alkylating the thiol group of the cysteine residue C34, thus causing the addition of the hydroxyethylthioethyl (HETE) moiety. Following proteolysis with pronase, the biomarker dipeptide C(-HETE)P is produced. To expand the possibilities for verification of exposure, we herein introduce a novel biomarker produced from that alkylated dipeptide by derivatization with propionic anhydride inducing the selective propionylation of the N-terminus yielding PA-C(-HETE)P. Quantitative derivatization is carried out at room temperature in aqueous buffer within 10 s. The biomarker was found to be stable in the autosampler at 15 °C for at least 24 h, thus documenting its suitability even for larger sets of samples. Selective and sensitive detection is done by micro liquid chromatography-electrospray ionization tandem-mass spectrometry (μLC-ESI MS/MS) operating in the selected reaction monitoring (SRM) mode detecting product ions of the single protonated PA-C(-HETE)P (m/z 379.1) at m/z 116.1, m/z 137.0, and m/z 105.0. The lower limit of detection corresponds to 32 nM SM in plasma in vitro and the limit of identification to 160 nM. The applicability to real exposure scenarios was proven by analyzing samples from the Middle East confirming poisoning with SM. Graphical abstract Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659

Remdesivir is a nucleotide analog prodrug that has received much attention since the outbreak of the COVID-19 pandemic in December 2019. GS-441524 (Nuc) is the active metabolite of remdesivir and plays a pivotal role in the clinical treatment of COVID-19. Here, a robust HPLC-MS/MS method was developed to determine Nuc concentrations in rat plasma samples after a one-step protein precipitation process. Chromatographic separation was accomplished on Waters XBrige C18 column (50 × 2.1 mm, 3.5 μm) under gradient elution conditions. Multiple reaction monitoring transitions in electrospray positive ion mode were m/z 292.2 → 163.2 for Nuc and 237.1 → 194.1 for the internal standard (carbamazepine). The quantitative analysis method was fully validated in line with the United States Food and Drug Administration guidelines. The linearity, accuracy and precision, matrix effect, recovery, and stability results met the requirements of the guidelines. Uncertainty of measurement and incurred sample reanalysis were analyzed to further ensure the robustness and reproducibility of the method. This optimized method was successfully applied in a rat pharmacokinetics study of remdesivir (intravenously administration, 5 mg kg−1). The method can act as a basis for further pharmacokinetic and clinical efficacy investigations in patients with COVID-19. Graphical abstract

The understanding of the initial cell adhesion to biomaterials is crucial for the survival of implants. The manifold possibilities to tailor an implant surface and the diverse requirements for different implant applications necessitate a timesaving and highly parallelized analytical methodology. Due to its intrinsic advantages (label-free, time-resolved, robust against temperature fluctuations, and particularly the multiplexing possibilities), single colour reflectometry (SCORE) is used for the first time to investigate cell adhesion to different extracellular matrix protein–coated surfaces. The excellent correlation between the novel SCORE technology and well-established reference methods proves that the results obtained by using this direct optical method are able to reflect the cell binding processes at the transducer surface. Additionally, the high time resolution of SCORE revealed the differences in the adhesion behaviour of the cells on the different extracellular matrix protein–coated glass slides during the initial adsorption phase and during the spreading of the cells on the surfaces. Therefore, we conclude that SCORE is a perfectly suited methodology for studying the entire cell adsorption process, including morphological changes, and shows great potential for other cell-based sensing applications.Graphical abstract

Plant viruses are major contributors to crop losses and induce high economic costs worldwide. For reliable, on-site and early detection of plant viral diseases, portable biosensors are of great interest. In this study, a field-effect SiO2-gate electrolyte-insulator-semiconductor (EIS) sensor was utilized for the label-free electrostatic detection of tobacco mosaic virus (TMV) particles as a model plant pathogen. The capacitive EIS sensor has been characterized regarding its TMV sensitivity by means of constant-capacitance method. The EIS sensor was able to detect biotinylated TMV particles from a solution with a TMV concentration as low as 0.025 nM. A good correlation between the registered EIS sensor signal and the density of adsorbed TMV particles assessed from scanning electron microscopy images of the SiO2-gate chip surface was observed. Additionally, the isoelectric point of the biotinylated TMV particles was determined via zeta potential measurements and the influence of ionic strength of the measurement solution on the TMV-modified EIS sensor signal has been studied., Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), Projekt DEAL

Human activities have greatly increased the input of reactive nitrogen species into the environment and disturbed the balance of the global N cycle. This imbalance may be offset by bacterial denitrification, an important process in maintaining the ecological balance of nitrogen. However, our understanding of the activity of mixotrophic denitrifying bacteria is not complete, as most research has focused on heterotrophic denitrification. The aim of this study was to investigate substrate preferences for two mixotrophic denitrifying bacterial strains, Acidovorax delafieldii and Hydrogenophaga taeniospiralis, under heterotrophic, autotrophic or mixotrophic conditions. This complex analysis was achieved by simultaneous identification and quantification of H2, O2, CO2, 14N2, 15N2 and 15N2O in course of the denitrification process with help of cavity-enhanced Raman spectroscopic (CERS) multi-gas analysis. To disentangle electron donor preferences for both bacterial strains, microcosm-based incubation experiments under varying substrate conditions were conducted. We found that Acidovorax delafieldii preferentially performed heterotrophic denitrification in the mixotrophic sub-experiments, while Hydrogenophaga taeniospiralis preferred autotrophic denitrification in the mixotrophic incubation. These observations were supported by stoichiometric calculations. The results demonstrate the prowess of advanced Raman multi-gas analysis to study substrate use and electron donor preferences in denitrification, based on the comprehensive quantification of complex microbial gas exchange processes.

Graphical abstract Ovarian cancer remains the most lethal gynaecological malignancy, as its timely detection at early stages remains elusive. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy of biofluids has been previously applied in pilot studies for ovarian cancer diagnosis, with promising results. Herein, these initial findings were further investigated by application of ATR-FTIR spectroscopy in a large patient cohort. Spectra were obtained by measurements of blood plasma and serum, as well as urine, from 116 patients with ovarian cancer and 307 patients with benign gynaecological conditions. A preliminary chemometric analysis revealed significant spectral differences in ovarian cancer patients without previous chemotherapy (n = 71) and those who had received neo-adjuvant chemotherapy—NACT (n = 45), so these groups were compared separately with benign controls. Classification algorithms with blind predictive model validation demonstrated that serum was the best biofluid, achieving 76% sensitivity and 98% specificity for ovarian cancer detection, whereas urine exhibited poor performance. A drop in sensitivities for the NACT ovarian cancer group in plasma and serum indicates the potential of ATR-FTIR spectroscopy to identify chemotherapy-related spectral changes. Comparisons of regression coefficient plots for identification of biomarkers suggest that glycoproteins (such as CA125) are the main classifiers for ovarian cancer detection and responsible for smaller differences in spectra between NACT patients and benign controls. This study confirms the capacity of biofluids’ ATR-FTIR spectroscopy (mainly blood serum) to diagnose ovarian cancer with high accuracy and demonstrates its potential in monitoring response to chemotherapy, which is reported for the first time. ATR-FTIR spectroscopy of blood serum achieves good segregation of ovarian cancers from benign controls, with attenuation of differences following neo-adjuvant chemotherapy. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03472-8.

Graphical abstract Due to growing concern about organic micropollutants and their transformation products (TP) in surface and drinking water, reliable identification of unknowns is required. Here, we demonstrate how non-target liquid chromatography (LC)-high-resolution tandem mass spectrometry (MS/MS) and the feature-based molecular networking (FBMN) workflow provide insight into water samples from four riverbank filtration sites with different redox conditions. First, FBMN prioritized and connected drinking water relevant and seasonally dependent compounds based on a modification-aware MS/MS cosine similarity. Within the resulting molecular networks, forty-three compounds were annotated. Here, carbamazepine, sartans, and their respective TP were investigated exemplarily. With chromatographic information and spectral similarity, four additional TP (dealkylated valsartan, dealkylated irbesartan, two oxygenated irbesartan isomers) and olmesartan were identified and partly verified with an authentic standard. In this study, sartans and TP were investigated and grouped regarding their removal behavior under different redox conditions and seasons for the first time. Antihypertensives were grouped into compounds being well removed during riverbank filtration, those primarily removed under anoxic conditions, and rather persistent compounds. Observed seasonal variations were mainly limited to varying river water concentrations. FBMN is a powerful tool for identifying previously unknown or unexpected compounds and their TP in water samples by non-target analysis. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03500-7.

Graphical abstract Testing multiple biomarkers, as opposed to one, has become a preferred approach for diagnosing many heterogeneous diseases, such as cancer and infectious diseases. However, numerous technologies, including gold standard ELISA and PCR, can detect only one type of biomarker, either protein or nucleic acid (NA), respectively. In this work, we report for the first time simultaneous detection of proteins and NAs in the same solution, using solely functional NA (FNA) molecules. In particular, we combined the thrombin binding aptamer (TBA) and the 10-23 RNA-cleaving DNA enzyme (DNAzyme) in a single aptazyme molecule (Aptazyme1.15-3′), followed by extensive optimization of buffer composition, sequences and component ratios, to establish a competitive bioassay. Subsequently, to establish a multiplex bioassay, we designed a new aptazyme (Aptazyme2.20-5′) by replacing the target recognition and substrate sequences within Aptazyme1.15-3′. This designing process included an in silico study, revealing the impact of the target recognition sequence on the aptazyme secondary structure and its catalytic activity. After proving the functionality of the new aptazyme in a singleplex bioassay, we demonstrated the capability of the two aptazymes to simultaneously detect thrombin and NA target, or two NA targets in a multiplex bioassay. High specificity in target detection was achieved with the limits of detection in the low nanomolar range, comparable to the singleplex bioassays. The presented results deepen the barely explored features of FNA for diagnosing multiple targets of different origins, adding an extra functionality to their catalogue. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03458-6.

Graphical abstract The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a serious threat to human health all over the world. The development of effective vaccines has been focusing on the spike (S) glycoprotein, which mediates viral invasion to human cells through its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor. In this work, we perform analytical characterization of N- and O-linked glycosylation of the SARS-CoV-2 S glycoprotein. We explore the novel use of dual-functionalized titanium (IV)-immobilized metal affinity chromatography (Ti-IMAC) material for simultaneous enrichment and separation of neutral and sialyl glycopeptides of a recombinant SARS-CoV-2 S glycoprotein from HEK293 cells. This strategy helps eliminate signal suppression from neutral glycopeptides for the detection of sialyl glycopeptides and improves the glycoform coverage of the S protein. We profiled 19 of its 22 potential N-glycosylated sites with 398 unique glycoforms using the dual-functional Ti-IMAC approach, which exhibited improvement of coverage by 1.6-fold compared to the conventional hydrophilic interaction chromatography (HILIC) glycopeptide enrichment method. We also identified O-linked glycosylation site that was not found using the conventional HILIC approach. In addition, we reported on the identification of mannose-6-phosphate (M6P) glycosylation, which substantially expands the current knowledge of the spike protein’s glycosylation landscape and enables future investigation into the influence of M6P glycosylation of the spike protein on its cell entry. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03433-1.

In order to detect the misuse of testosterone (T), urinary steroid concentrations and concentration ratios are quantified and monitored in a longitudinal manner to enable the identification of samples exhibiting atypical test results. These suspicious samples are then forwarded to isotope ratio mass spectrometry (IRMS)–based methods for confirmation. Especially concentration ratios like T over epitestosterone (E) or 5α-androstanediol over E proved to be valuable markers. Unfortunately, depending on the UGT2B17 genotype and/or the gender of the athlete, these markers may fail to provide evidence for T administrations when focusing exclusively on urine samples. In recent years, the potential of plasma steroids has been investigated and were found to be suitable to detect T administrations especially in female volunteers. A current drawback of this approach is the missing possibility to confirm that elevated steroid concentrations are solely derived from an administration of T and cannot be attributed to confounding factors. Therefore, an IRMS method for plasma steroids was developed and validated taking into account the comparably limited sample volume. As endogenous reference compounds, unconjugated cholesterol and dehydroepiandrosterone sulfate were found suitable, while androsterone and epiandrosterone (both sulfo-conjugated) were chosen as target analytes. The developed method is based on multi-dimensional gas chromatography coupled to IRMS in order to optimize the overall assay sensitivity. The approach was validated, and a reference population encompassing n = 65 males and females was investigated to calculate population-based thresholds. As proof-of-concept, samples from volunteers receiving T replacement therapies and excretion study samples were investigated. Graphical abstract

Cytotoxic macrocyclic trichothecenes such as satratoxins are produced by chemotype S strains of Stachybotrys chartarum. Diseases such as stachybotryotoxicosis in animals and the sick building syndrome as a multifactorial disease complex in humans have been associated with this mold and its toxins. Less toxic non-chemotype S strains of S. chartarum are morphologically indistinguishable from chemotype S strains, which results in uncertainties in hazard characterization of isolates. To selectively identify macrocyclic trichothecene producing S. chartarum isolates, a set of sat14 gene-specific primers was designed and applied in a loop-mediated isothermal amplification (LAMP) assay using neutral red for visual signal detection. The assay was highly specific for S. chartarum strains of the macrocyclic trichothecene producing chemotype and showed no cross-reaction with non-macrocyclic trichothecene producing S. chartarum strains or 152 strains of 131 other fungal species. The assay’s detection limit was 0.635 pg/rxn (picogram per reaction) with a reaction time of 60 min. Its high specificity and sensitivity as well as the cost-saving properties make the new assay an interesting and powerful diagnostic tool for easy and rapid testing.

Simple and rapid methods are required for screening and analysis of water samples to detect cyanobacterial cyclic peptide hepatotoxins: microcystin/nodularin. Previously, we reported a highly sensitive non-competitive heterogeneous assay for microcystin/nodularin utilizing a generic anti-immunocomplex (anti-IC) single-chain fragment of antibody variable domains (scFv) isolated from a synthetic antibody library together with a generic adda ((2S,3S,4E,6E,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid)-specific monoclonal antibody (Mab) recognizing the common adda part of the microcystin/nodularin. Using the same antibody pair, here we report a homogeneous non-competitive assay for microcystin/nodularin based on TR-FRET (time-resolved Förster resonance energy transfer) measurement. The anti-IC scFv labeled with Alexa Fluor 680 and the Mab labeled with europium enabled the FRET process to occur in the presence of microcystin/nodularin. The TR-FRET signal is proportional to the toxin concentration in the sample. The rapid (15 min) homogeneous assay without requiring any washing step detected all the tested nine toxin variants (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF -LW, and nodularin-R). Very good signal to blank ratio (~13) was achieved using microcystin-LR and the sample detection limit (blank+3SD of blank) for microcystin-LR was ~0.3 μg/L (~0.08 μg/L in 80-μL reaction well). The practical application of the TR-FRET assay was demonstrated with water samples spiked with microcystin-LR as well as with environmental water. The average recoveries of microcystin-LR from spiked water ranged from 65 to 123%. Good correlation (r2 = 0.73 to 0.99) with other methods (liquid chromatography-mass spectrometry and previously reported heterogeneous assay) was found when environmental samples were analyzed. The developed wash-free assay has the potential to play as a quick screening tool to detect microcystin/nodularin from water below the World Health Organization’s guideline limit (1 μg/L of microcystin-LR). Graphical abstract

Graphical abstract The goal of the research was the synthesis and application of an oligonucleotide immobilized stationary phase for the analysis of unmodified and antisense oligonucleotides. The method for attaching these molecules to aminopropyl silica modified with pentanedioic acid was developed. Each step of the synthesis was carefully controlled with the application of spectroscopic, elemental, and chromatographic analyses. The oligonucleotide-based stationary phase was applied for the retention studies. Unmodified oligonucleotides of different complementarity to the molecule attached as a stationary phase, as well as antisense oligonucleotides, were tested. The comparative study upon complex optimization of oligonucleotide analysis in different liquid chromatography modes was performed. Results have shown that this stationary phase may be applied for oligonucleotide analysis in hydrophilic interaction liquid chromatography and ion exchange chromatography, but no unique sequence-based selectivity was obtained. Contrary results were observed for affinity chromatography, which allowed for specific separation of the complementary strands based on hydrogen bonding and stacking interactions, where the temperature was the main factor influencing the selectivity of the separation. Furthermore, the oligonucleotide-based stationary phase may be applied for comparative antisense oligonucleotide hybridization studies to a specific RNA sequence. All of the results have shown that affinity chromatography with oligonucleotide-based stationary phases is a powerful technique for the specific base recognition of polynucleotides. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03473-7.

Cutaneous leishmaniasis (CL) is one of the illnesses caused by Leishmania parasite infection, which can be asymptomatic or severe according to the infecting Leishmania strain. CL is commonly diagnosed by directly detecting the parasites or their DNA in tissue samples. New diagnostic methodologies target specific proteins (biomarkers) secreted by the parasite during the infection process. However, specific bioreceptors for the in vivo or in vitro detection of these novel biomarkers are rather limited in terms of sensitivity and specificity. For this reason, we here introduce three novel peptides as bioreceptors for the highly sensitive and selective identification of acid phosphatase (sAP) and proteophosphoglycan (PPG), which have a crucial role in leishmaniasis infection. These high-affinity peptides have been designed from the conservative domains of the lectin family, holding the ability to interact with the biological target and produce the same effect than the original protein. The synthetic peptides have been characterized and the affinity and kinetic constants for their interaction with the targets (sAP and PPG) have been determined by a surface plasmon resonance biosensor. Values obtained for K are in the nanomolar range, which is comparable to high-affinity antibodies, with the additional advantage of a high biochemical stability and simpler production. Pep2854 exhibited a high affinity for sAP (K = 1.48 nM) while Pep2856 had a good affinity for PPG (K 1.76 nM). This study evidences that these peptidomimetics represent a novel alternative tool to the use of high molecular weight proteins for biorecognition in the diagnostic test and biosensor devices for CL. Graphical abstract: [Figure not available: see fulltext.]., This study was supported by a Doctoral Fellowship from COLCIENCIAS in Colombia. The ICN2 is funded by the CERCA programme/Generalitat de Catalunya. ICN2 is supported by the Severo Ochoa program from Spanish Ministry of Science MICINN (Grant No. SEV-2017-0706). This work has made use of the Biodeposition and Biodetection Unit from ICTS NANBIOSIS (http://www.nanbiosis.es/portfolio/u4-biodeposition-andbiodetection-unit/) partially supported by MICINN/FEDER (FICTS-1420-27).

Graphical abstract COVID-19 is the most disturbing pandemic of the past hundred years. Its causative agent, the SARS-CoV-2 virus, has been the subject of an unprecedented investigation to characterize its molecular structure and intimate functioning. While markers for its detection have been proposed and several diagnostic methodologies developed, its propensity to evolve and evade diagnostic tools and the immune response is of great concern. The recent spread of new variants with increased infectivity requires even more attention. Here, we document how shotgun proteomics can be useful for rapidly monitoring the evolution of the SARS-CoV-2 virus. We evaluated the heterogeneity of purified SARS-CoV-2 virus obtained after culturing in the Vero E6 cell line. We found that cell culture induces significant changes that are translated at the protein level, such changes being detectable by tandem mass spectrometry. Production of viral particles requires careful quality control which can be easily performed by shotgun proteomics. Although considered relatively stable so far, the SARS-CoV-2 genome turns out to be prone to frequent variations. Therefore, the sequencing of SARS-CoV-2 variants from patients reporting only the consensus genome after its amplification would deserve more attention and could benefit from more in-depth analysis of low level but crystal-clear signals, as well as complementary and rapid analysis by shotgun proteomics. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03401-9.

Graphical abstract The position and configuration of carbon-carbon double bonds in unsaturated fatty acids is crucial for their biological functions and influences health and disease. However, double bond isomers are not routinely distinguished by classical mass spectrometry workflows. Instead, they require sophisticated analytical approaches usually based on chemical derivatization and/or instrument modification. In this work, a novel strategy to investigate fatty acid double bond isomers (18:1) without prior chemical treatment or modification of the ion source was implemented by non-covalent adduct formation in the gas phase. Fatty acid adducts with sodium, pyridinium, trimethylammonium, dimethylammonium, and ammonium cations were characterized by a combination of cryogenic gas-phase infrared spectroscopy, ion mobility-mass spectrometry, and computational modeling. The results reveal subtle differences between double bond isomers and confirm three-dimensional geometries constrained by non-covalent ion-molecule interactions. Overall, this study on fatty acid adducts in the gas phase explores new avenues for the distinction of lipid double bond isomers and paves the way for further investigations of coordinating cations to increase resolution. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03334-3.

In this work, a novel standardization strategy for quantitative elemental bioimaging is evaluated. More specifically, multi-element quantification by laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS) is performed by multi-point calibration using gelatin-based micro-droplet standards and validated using in-house produced reference materials. Fully automated deposition of micro-droplets by micro-spotting ensured precise standard volumes of 400 ± 5 pL resulting in droplet sizes of around 200 μm in diameter. The small dimensions of the micro-droplet standards and the use of a low-dispersion laser ablation setup reduced the analysis time required for calibration by LA-ICPMS significantly. Therefore, as a key advance, high-throughput analysis (pixel acquisition rates of more than 200 Hz) enabled to establish imaging measurement sequences with quality control- and standardization samples comparable to solution-based quantification exercises by ICP-MS. Analytical figures of merit such as limit of detection, precision, and accuracy of the calibration approach were assessed for platinum and for elements with biological key functions from the lower mass range (phosphorus, copper, and zinc). As a proof-of-concept application, the tool-set was employed to investigate the accumulation of metal-based anticancer drugs in multicellular tumor spheroid models at clinically relevant concentrations. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03357-w.

Raman spectral data are best described by mathematical functions; however, due to the spectroscopic measurement setup, only discrete points of these functions are measured. Therefore, we investigated the Raman spectral data for the first time in the functional framework. First, we approximated the Raman spectra by using B-spline basis functions. Afterwards, we applied the functional principal component analysis followed by the linear discriminant analysis (FPCA-LDA) and compared the results with those of the classical principal component analysis followed by the linear discriminant analysis (PCA-LDA). In this context, simulation and experimental Raman spectra were used. In the simulated Raman spectra, normal and abnormal spectra were used for a classification model, where the abnormal spectra were built by shifting one peak position. We showed that the mean sensitivities of the FPCA-LDA method were higher than the mean sensitivities of the PCA-LDA method, especially when the signal-to-noise ratio is low and the shift of the peak position is small. However, for a higher signal-to-noise ratio, both methods performed equally. Additionally, a slight improvement of the mean sensitivity could be shown if the FPCA-LDA method was applied to experimental Raman data. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03360-1.

Bulevirtide is a first-in-class entry inhibitor of the hepatitis B and hepatitis delta virus blocking the sodium/bile acid co-transporter NTCP, and was recently approved for the treatment of hepatitis D as a priority medicine (prime) in an accelerated assessment by the European Medicines Agency. It is a very large lipopeptide comprising 47 amino acids in its sequence and a myristoylation at the N-terminus. For support of clinical development, we established highly sensitive plasma quantification assays using 100 μL of plasma, spanning concentrations of 0.1 to 100 ng/mL and 1 to 1000 ng/mL with the option to measure ten-fold diluted samples up to 10,000 ng/mL. Quantification was performed with UPLC-MS/MS measurements after extraction with protein precipitation. Both assays were fully validated according to the pertinent guidelines of the FDA and EMA, including incurred sample reanalyses and cross-validation using clinical study samples. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03384-7.