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Kratom is a herbal substance belonging to the group of new psychoactive substances. It contains psychoactive indole alkaloids mitragynine and 7-hydroxymitragynine. At low doses, they act as psychostimulants and at higher doses they mediate an opioid-like effect. The increasing misuse of kratom requires the development of analytical methods that will accurately and reliably identify and quantify its psychoactive alkaloids in biological samples. Therefore, the development of effective, precise, and reliable green analytical methods that are easy to implement in practice is of great importance. On-line combination of capillary zone electrophoresis with tandem mass spectrometry (CZE-MS/MS) seems to be a promising solution. We present a novel green approach based on capillary zone electrophoresis – tandem mass spectrometry (CZE-MS/MS) method with on-line dynamic pH junction sample pretreatment to identify and determine mitragynine and 7-hydroxymitragynine in urine samples. The separation was performed in a background electrolyte composed of 100 mM formic acid (pH 2.39). The dynamic pH junction was ensured by injection of a short plug of 12.5 % NH 4 OH before the sample. Under optimal conditions, the developed method was validated and parameters such as linearity (r2 > 0.99), precision (2.2–8.7 %), accuracy (89.2–102.5 %) or stability of the sample (86.6–114.7 %) met the defined FDA guideline criteria (%RSD and %RE values where within ±15 %). Introduction of a simple in-capillary preconcentration strategy based on dynamic pH junction enabled significant improvement in analytical signal intensity and also the applicability of the method. Applying the presented approach, high sensitivity was achieved as indicated by limit of detection values, which were 0.5 ng mL−1 and 2 ng mL−1 for mitragynine and 7-hydroxymitragynine, respectively. Greenness of the proposed approach was confirmed by the AGREE metrics (score 0.63). The application potential of the developed method was successfully verified using blinded urine model samples. For the first time a fully validated CZE-MS/MS method for kratom alkaloids determination was introduced. The presented novel method is a cheaper and more ecological alternative to conventionally used chromatographic techniques what was clearly confirmed by its greenness evaluation and comparison with previously published liquid chromatography (LC) approaches. In-capillary sample pretreatment (dynamic pH junction) has been demonstrated to be an effective and fast tool in bioanalysis, minimizing the number of pretreatment steps and the manipulation with the sample. Moreover, LOD values comparable to those obtained by LC methods were recorded. High potential for the implementation of this approach into the toxicology environment in the near future is expected. [Display omitted] • The first validated CZE-MS/MS method for the determination of mitragynine and 7-hydroxymitragynine in biological samples. • Effective in-capillary sample pretreatment based on dynamic pH junction. • a suitable tool to prove the presence of kratom alkaloids in urine for toxicological purposes. • Green analytical method in toxicology. [ABSTRACT FROM AUTHOR]

New psychoactive substances (NPS) are of public health concern due to their sporadic proliferation and the dearth of information on toxicity when consumed. In addition to seized data from forensic and toxicology reporting, wastewater analysis serves as a complimentary tool for NPS surveillance. A method to detect 71 NPS by simple filtration followed by liquid-chromatography tandem mass spectrometry was developed to detect multiclass NPS consisting of arylcyclohexylamines, designer benzodiazepines, synthetic cannabinoids, synthetic opioids, phenethylamines, synthetic cathinones, tryptamines, and indole alkaloids. In this work, the influential factors for electrospray ionisation were identified and optimised using the fractional factorial design and face-centred central composite design, respectively. The filtration loss during sample clean-up was assessed for all compounds. The final method was validated and applied to wastewater collected from a music festival held in Queensland in 2022. The validated method had linearity between 0.5 ng L−1 and 5000 ng L−1, the limit of quantification (LOQ) ranges from 0.6 ng L−1 to 70 ng L−1, precision within ±20 %, accuracy ranges from 70 % to 120 %, and matrix effect ranges from soft (0 %–20 %) to medium (20 %–50 %) for the majority of the compounds. NPS detected in the festival were 2-fluorodeschloroketamine, 7-hydroxymitragynine, mitragynine, N,N -dimethylpentylone, pentylone, phenibut, and O- desmethyltramadol. Systematic electrospray ionisation optimisation using the design of experiment for a large method is practical and provides in-depth chemical information on studied compounds. The optimised method demonstrated the applicability of analysing samples collected from a festival in this work. [Display omitted] • A high throughput method was developed for 71 new psychoactive substances. • Important variables for optimisation were temperature, ion source gas, and ion source voltage. • Seven compounds were detected in samples collected from a festival. [ABSTRACT FROM AUTHOR]

Synthetic cathinones (SCs) are a large category of new psychoactive substances (NPS), which pose a serious threat to public health due to limited information about their toxicology and pharmacology. Many SCs are closely related in their chemical structures, with some substances being positional isomers. In this study, we propose a new workflow for the identification of SC isomers using liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS2) combined with electron activated dissociation (EAD) and chemometrics. Differentiation between isomeric SCs is essential for both legislative and public safety reasons, since minor differences in their molecular structures may change their legal status and pharmacological profiles. The workflow was optimized using ring-substituted isomers of methylmethcathinones, methylethcathinones, and chloromethcathinones. The kinetic energy in the EAD cell was investigated at three levels (i.e., 15, 18, and 20 eV) for each group. Two data analysis methods (i.e., t-distributed stochastic neighbor embedding [t-SNE] and a Random Forest [RF] algorithm) were applied using the obtained EAD mass spectral data. The three sets of ring-substituted SCs were clearly distinguished using t-SNE and an RF algorithm. Moreover, the RF approach resulted in a 97 % classification accuracy for isomer identification using various combinations of compounds, isomers, and electron kinetic energies. This workflow was subsequentially applied to the analysis of 26 blind street samples, resulting in a 92 % classification accuracy for isomer identification. However, the accuracy varied based on the kinetic electron energy. A subset of the original data set, focusing on 15-eV data only, was used, resulting in a classification accuracy of 100 %. This study presents the first LC-HRMS2 workflow based on EAD and chemometrics, which resulted in a classification accuracy of 100 % of authentic street samples. The developed LC-HRMS2 workflow demonstrates that EAD product ions and their characteristic ion ratios can be successfully used to identify ring-substituted positional isomers of SCs. [Display omitted] • Novel workflow developed for the identification of synthetic cathinone (SC) isomers using LC-HRMS2. • LC-HRMS2 combined with electron-activated dissociation (EAD) and chemometrics for identification. • t-distributed stochastic neighbor embedding and a Random Forest algorithm used for classification. • Workflow successfully applied to authentic street samples with classification accuracy of 100 %. [ABSTRACT FROM AUTHOR]

The abuse of the Phencyclidine-type substances, especially ketamine is a serious problem worldwide, and retrospective analysis are important for both the analysis and the identification of forms of drug abuse. The current major analytical methods, while all excellent in terms of accuracy, are time- and reagent-consuming. This depletion is made even more unfortunate by the fact that a large number of samples are negative in retrospective analyses. It is clear that a set of methods that can be analyzed both accurately and quickly need to be developed and applied to the screening and analysis of large quantities of samples. We described a urine test based on acoustic ejection mass spectrometry, which allows precise injection at very low volumes and near 1 ejection s−1 and data acquisition. The confidence in identification was increased by the characterization of the abundance ratio of the two pairs of ions. Urine samples could be diluted with water and loaded into a 384-well plate for sampling without complicated sample preparation. The sample in the transparent 384-well plate was pre-scanned by the laser, and then 20 nL droplets were ejected into the ion source for targeted analysis of 2 ion transitions per droplet totaling 9 targeted analytes in the sequence of acquisition methods. It took 90 min to screen 250 samples in this approach, yielding 10 ng mL−1 detection limits. Positive samples were further analyzed by UHPLC-MS/MS for confirmation and quantification of up to 36 analytes. This was the first fast screening method for phencyclidine-type substances based on acoustic ejection mass spectrometry, which greatly reduces the analytical time, and can accomplish in 1.5 h what UHPLC-MS/MS needs 3 days to complete. And the samples can be analyzed without complicated sample preparation, and also can obtain good detectability. It was applied to a short-term retrospective analysis in Shanghai, and its accuracy was also extremely high. [Display omitted] • First application of AEMS technology to phencyclidine-type substance abuse screening. • AEMS is efficient and low-consumption, with 1 ejection s−1 and 20 nL ejection volume. • Sensitive UHPLC-MS/MS method developed for phencyclidine-type substances. • Authentic urine samples were tested using AEMS and UHPLC-MS/MS. • Compares the advantages and disadvantages of Echo and UHPLC. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs) represent a diverse group of organic compounds characterized by the fusion of two or more benzene rings arranged in various structural forms. Due to their harmful effects on human health, it is essential to implement monitoring systems and preventive measures to regulate human exposure. Given the affinity of PAHs for lipids, extensive research has been focused on their presence in vegetable oils. This study aimed to develop an on-line liquid-gas chromatography (LC-GC) method (using tandem mass spectrometry) with minimized solvent consumption for the determination of 16 PAHs in extra-virgin olive oil (EVOO). A side-by-side comparison of the selected-ion-monitoring and the pseudo multiple-reaction-monitoring (p -MRM) acquisition modes was performed, in terms of specificity and detectability. The results obtained using the p -MRM mode were superior, and for this reason it was selected. The method was linear over the concentration range 1–200 μg kg−1 (except in five cases, over 2–200 and 5–200 μg kg−1 ranges). Accuracy (at the 2 μg kg−1 and 20 μg kg−1 concentration levels) was in the 86.9–109.3 % range, with an RSD <10 %. Intra-day and inter-day precision (at 2 μg kg−1 and 20 μg kg−1 concentration levels) were in the 1.2–9.7 % and 3.2–10.8 % ranges, respectively. For all the PAHs, a negative matrix effect was observed. Three out of sixteen PAHs were detected in three EVOOs (among ten samples), albeit at the low ppb level. Limits of quantification were satisfactory in relation to EU legislation on the presence of PAHs in vegetable oils. A dilute-and-inject LC-GC-tandem mass spectrometry method is herein proposed fulfilling EU legislation requirements; sample preparation was very simple, inasmuch that it involved only a dilution step, thus avoiding extraction, clean-up, and thus a high consumption of organic solvents. In fact, considering both oil dilution and the LC mobile phase, less than 8 mL of solvents were used. [Display omitted] • An LC-GC method was developed for polycyclic aromatic hydrocarbon determination. • Sixteen polycyclic aromatic hydrocarbons were monitored in extra-virgin olive oils. • Sample preparation consisted in a simple and low solvent-consumption oil dilution. • Both the specificity and detectability of pseudo-MRM and SIM modes were compared. • The pseudo-MRM mode was characterized by a superior performance. [ABSTRACT FROM AUTHOR]

Background: Protein phosphorylation by protein kinases plays a pivotal role in increasing protein diversity, thereby influencing various cellular functions. However, due to the relatively low abundance of phosphopeptides in a mixture of peptides and the ion-suppression effect of non-phosphorylated peptides, the detection of phosphopeptides is not straightforward., Results: Herein, a quantitative high-throughput platform was developed for assessing multikinase activity using nano-LC-MS/MS with a data-independent acquisition (DIA) approach. This platform was evaluated by studying the kinase activity in Taxol-treated SKOV3 cells. A library containing 38 peptide substrates was designed and analyzed to determine the activities of major kinases involved in cancer development. Twenty-three synthetic peptide substrates showed significant phosphorylation changes in triplicate biological experiments, as further verified by western blotting. Our findings reveal that Taxol suppressed SKOV3 cell survival by activating AMPK and suppressing the PI3K-Akt-dependent pathway, ultimately leading to mTOR inhibition. Furthermore, in combination with ERK, Akt, SGK, CK1, and ErbB2 inhibitors, Taxol enhanced the inhibitory effect on ovarian cancer., Significance: This platform can be an attractive approach for large-scale kinase activity studies to comprehensively uncover the mechanisms of drug-disease treatment and to investigate a more effective therapy strategy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Chao-Jung Chen reports financial support was provided by National Science and Technology Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The study of glycopeptides is associated with challenges regarding the microheterogeneity of different isomeric glycans occupying the same glycosylation sites in glycoproteins. It is immensely valuable to perform both qualitative and quantitative site-specific glycosylation analysis of glycopeptide isomers due to their link to several diseases. Achieving isomeric separation of glycopeptides is particularly challenging due to the low abundance of glycopeptides as well as inefficient ionization. Although some methods have demonstrated the isomeric separation of glycopeptides, a more efficient nanoflow-based stationary phase is needed for the isomeric separation of both N- and O-glycopeptides., Results: In this study, the separation of N- and O-glycopeptide isomers at 75 °C was achieved with an in-house packed 1 cm long mesoporous graphitized carbon (MGC) column. Different gradient compositions of the optimized mobile phase for separating permethylated glycans on MGC column were tested, and we observed efficient separation of N- and O-glycopeptide isomers at a gradient elution time of 120 min. After achieving the isomeric separation of sialylated glycopeptides from model glycoproteins derived from bovine fetuin, the separation of isomeric glycopeptides derived from asialofetuin, α-1 glycoprotein and human blood serum were also demonstrated. Furthermore, the developed method for the separation of isomeric N- and O-glycopeptide on MGC column showed high reproducibility over three months. We observed an average retention time shift of 1 min and consistent resolution of separated peaks throughout three months., Significance and Novelty: MGC column can serve as an efficient tool for obtaining the isomeric separation of N- and O-glycopeptide from complex biological samples in future studies. This will enable a more profound understanding of the roles played by isomeric N- and O-glycopeptide in important biological processes and their correlations to various disease progressions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Small Extracellular Vesicles (sEVs) are nano-sized vesicles that are present in all biofluids including human milk (HM) playing a crucial role in cell-to-cell communication and the stimulation of the neonatal immune system. Oxylipins, which are bioactive lipids formed from polyunsaturated fatty acids, have gained considerable attention due to their potential role in mitigating disease progression and modulating the inflammatory status of breastfed infants. This study aims at an in-depth characterization of the oxylipin profiles of HM and, for the first time, of HM-derived sEVs (HMEVs) employing an ad-hoc developed and validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method., Results: The UPLC-MS/MS method covered a panel of 13 oxylipins for quantitation and 93 oxylipins for semi-quantitation. In 200 μL of HM and HMEV isolates of 15 individuals, 42 out of 106 oxylipins were detected in either HM or HMEVs, with 38 oxylipins being detected in both matrices. Oxylipins presented distinct profiles in HM and HMEVs, suggesting specific mechanisms responsible for the encapsulation of target molecules in HMEVs. Ten and eight oxylipins were quantified with ranges between 0.03 - 73 nM and 0.30 pM-0.07 nM in HM and HMEVs, respectively. The most abundant oxylipins found in HMEVs were docosahexaenoic acid derivatives (17-HDHA and 14-HDHA) with known anti-inflammatory properties, and linoleic acid derivatives (9-10-DiHOME and 12,13-DiHOME) in HM samples., Significance and Novelty: This is the first time a selective, relative enrichment of anti-inflammatory oxylipins in HMEVs has been described. Future studies will focus on the anti-inflammatory and pro-healing capacity of oxylipins encapsulated in HMEVs, with potential clinical applications in the field of preterm infant care, specifically the prevention of severe intestinal complications including necrotizing enterocolitis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Julia Kuligowski reports financial support was provided by Carlos III Health Institute. Abel Albiach-Delgado reports financial support was provided by Carlos III Health Institute. Pilar Sepulveda reports financial support was provided by Carlos III Health Institute. Jose L Moreno-Casillas reports financial support was provided by Government of Valencia. Julia Kuligowski reports financial support was provided by Government of Valencia. Julia Kuligowski reports financial support was provided by Valencia Innovation Agency. Pilar Sepulveda reports financial support was provided by Valencia Innovation Agency. Julia Kuligowski reports financial support was provided by Spain Ministry of Science and Innovation. Isabel Ten-Doménech reports financial support was provided by Valencia Innovation Agency. Guillermo Quintas reports financial report was provided by Spain Ministry of Science and Innovation. Abel Albiach-Delgado, Isabel Ten-Domenech, Pilar Sepulveda, and Marta Gomez-Ferrer have patent Composition comprising oxylipins present in human milk derived small extracellular vesicles and its use in the prevention and treatment of intestinal diseases (EP23382313) (pending). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

DIA (Data-Independent Acquisition) is a powerful technique in Liquid Chromatography coupled with high-resolution tandem Mass Spectrometry (LC-MS/MS) initially developed for proteomics studies and recently emerging in metabolomics and lipidomics. It provides a comprehensive and unbiased coverage of molecules with improved reproducibility and quantitative accuracy compared to Data-Dependent Acquisition (DDA). Combined with the Zeno trap and Electron-Activated Dissociation (EAD), DIA enhances data quality and structural elucidation compared to conventional fragmentation under CID. These tools were applied to study the lipidome and metabolome of the freshwater amphipod Gammarus fossarum , successfully discriminating stages and highlighting significant biological features. Despite being underused, DIA, along with the Zeno trap and EAD, holds great potential for advancing research in the omics field. DIA combined with the Zeno trap enhances detection reproducibility compared to conventional DDA, improving fragmentation spectra quality and putative identifications. LC coupled with Zeno-SWATH-DIA methods were used to characterize molecular changes in reproductive cycle of female gammarids. Multivariate data analysis including Principal Component Analysis and Partial Least Square Discriminant Analysis successfully identified significant features. EAD fragmentation helped to identify unknown features and to confirm their molecular structure using fragmentation spectra database annotation or machine learning. EAD database matching accurately annotated five glycerophospholipids, including the position of double bonds on fatty acid chain moieties. SIRIUS database predicted structures of unknown features based on experimental fragmentation spectra to compensate for database incompleteness. Reproducible detection of features and confident identification of putative compounds are pivotal stages within analytical pipelines. The DIA approach combined with Zeno pulsing enhances detection sensitivity and targeted fragmentation with EAD in positive polarity provides orthogonal fragmentation information. In our study, Zeno-DIA and EAD thereby facilitated a comprehensive and insightful exploration of pertinent biological molecules associated with the reproductive cycle of gammarids. The developed methodology holds great promises for identifying informative biomarkers on the health status of an environmental sentinel species. [Display omitted] • Advancements in Data-Independent-Acquisition methods for lipidomics and metabolomics applied to an ecotoxicologically relevant species. • Zeno pulsing improves sensitivity in fragment detection and compound identification. • Comprehensive structure elucidation of lipids and metabolites using EAD fragmentation. [ABSTRACT FROM AUTHOR]

The use of simple and hybrid fragmentation techniques for the identification of molecules in tandem mass spectrometry provides different and complementary information on the structure of molecules. Nevertheless, these techniques have not been as widely explored for oligonucleotides as for peptides or proteins. The analysis of microRNAs (miRNAs) warrants special attention, given their regulatory role and their relationship with several diseases. The application of different fragmentation techniques will be very interesting for their identification. Four synthetic miRNAs and a DNA sequence were fragmented in an ESI-FT-ICR mass spectrometer using both simple and hybrid fragmentation techniques: CID, nETD followed by CID, IRMPD, and, for the first time, nETD in combination with IRMPD. The main fragmentation channel was base loss. The use of nETD-IRMPD resulted in d/z , a/w , and c/y ions at higher intensities. Moreover, nETD-IRMPD provided high sequence coverage and low internal fragmentation. Native MS analysis revealed that only miR159 and the DNA sequence formed stable dimers under physiological ionic strength. The use of organic co-solvents or additives resulted in a lower sequence coverage due to lesser overall ionization efficiency. This work demonstrates that the combination of nETD and IRMPD for miRNA fragmentation constitutes a suitable alternative to common fragmentation methods. This strategy resulted in efficient fragmentation of [miRNA]5- using low irradiation times and fewer internal fragments while ensuring a high sequence coverage. Moreover, given that such low charge states predominate upon spraying in physiological-like conditions, native MS can be applied for obtaining structural information at the same time. [Display omitted] • Oligonucleotide fragmentation by nETD-IRMPD mainly produced c/y and d/z ions. • This technique resulted in low internal fragmentation and high sequence coverage. • MiR159 and DNA formed stable dimers under physiological conditions. • The use of solvents/additives for MS analysis of oligonucleotides reduces sequence coverage. [ABSTRACT FROM AUTHOR]

SARS-CoV-2, the causative agent of COVID-19, has imposed a major public health threat, which needs effective therapeutics and vaccination strategies. Several potential candidate vaccines being rapidly developed are in clinical evaluation and recombinant vaccine has gained much attention thanks to its potential for greater response predictability, improved efficacy, rapid development and reduced side effects. Recombinant vaccines are designed and manufactured using bacterial, yeast cells or mammalian cells. A small piece of DNA is taken from the virus or bacterium against which we want to protect and inserted into the manufacturing cells. Due to the extremely complex heterogeneity of SARS-CoV-2 recombinant vaccine, single technology platform cannot achieve thorough and accurate characterization of such difficult proteins so integrating comprehensive technologies is essential. This study illustrates an innovative workflow employing multiple separation techniques tandem high-resolution mass spectrometry for comprehensive and in-depth characterization of SARS-CoV-2 recombinant vaccine, including ultra-high performance liquid chromatography (UHPLC), ion exchange chromatography (IEX) and imaged capillary isoelectric focusing (icIEF). The integrated methodology focuses on the importance of cutting-edge icIEF-MS online coupling and icIEF fractionation applied to revealing the heterogeneity secret of SARS-CoV-2 recombinant vaccine. [Display omitted] • Comprehensive separation techniques coupled to high-resolution mass spectrometry have been utilized for in-depth heterogeneity characterization of SARS-CoV-2 recombinant vaccine. • As The cutting-edge technologies, icIEF-MS online and icIEF fractionation integrating IEX-MS were successfully explored to investigate their applicability in in-depth characterization of SARS-CoV-2 recombinant vaccine compositions. • The constructed workflow underlined the outstanding importance of comprehensive platforms based on MS technology for the extremely complex heterogeneity of SARS-CoV-2 recombinant vaccine. [ABSTRACT FROM AUTHOR]

A standard sample mixture containing thirty-seven fatty acid methyl esters (FAMEs) was measured by femtosecond laser ionization mass spectrometry. FAME molecules with double bonds were efficiently ionized via resonance-enhanced two-photon ionization by absorbing the first photon at 206 nm at the edge of the absorption band of the π→π* transition and subsequently ionized by absorbing the second photon at 257 nm. The intensity of the molecular radical ion was enhanced significantly using this two-color ionization scheme, which minimizes the excess energy in the ionized state, when compared with electron ionization mass spectrometry and vacuum-ultraviolet photoionization mass spectrometry. This approach was then used for the reliable identification of FAMEs contained in an actual sample of biofuel. [Display omitted] • Fatty acid methyl esters (FAMEs) were measured by mass spectrometry. • FAMEs were ionized using a femtosecond laser as the ionization source (fsLIMS). • FAMEs were ionized using a two-color two-photon ionization scheme at 206/257 nm. • The signal intensity of a molecular radical ion was enhanced for unsaturated FAMEs. • FAMEs contained in a real sample of biofuel was determined using this technique. [ABSTRACT FROM AUTHOR]

Background: Catecholamines (CAs) are involved in a wide range of physiological and pathological processes in the body and are progressively being used as important biomarkers for a variety of diseases. It is of great significance for accurate quantification of CAs to the diagnosis and treatment of diseases. However, the separation of CAs from complex biological matrices is still a great challenge due to the trace levels of CAs and the limited selectivity of existing pretreatment methods., Results: In this work, a dual-recognition imprinted membrane (BA-MIM) was developed to utilize the synergistic action of pH-responsive boron affinity and molecular imprinted cavities for highly selective capture and release of CAs. The prepared BA-MIM possessed remarkable adsorption capacity (maximum capacity, 43.3 mg g -1 ), desirable surface hydrophilicity (46.2°), superior selectivity (IF = 6.2, α = 14.3), as well as favorable reusability (number of cycles, 6 times). On this basis, an integrated analytical method based on BA-MIM extraction combined with ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was innovatively developed to highly selective separation, enrichment, and detection of CAs in rat brain tissue. Under the optimum conditions, a low quantitation limits (0.05-0.10 ng mL -1 ), wide linear range (10-1000 ng mL -1 ), good linearity (r 2  > 0.99), and satisfactory recoveries (88.5%-98.5 %) were obtained for CAs. The proven method was further applied to kidney-yang-deficiency-syndrome (KYDS) group rat model, revealed the intrinsic connection between kidney disease and catecholamine metabolism., Significance: This work provides an excellent reference paradigm for the effective construction of dual-recognition functional membrane material to the high-selective analysis of trace targets in complex matrices. Additionally, this integrated analytical strategy demonstrates its efficiency, sustainability, versatility, and convenience, showing remarkable prospect in a variety of applications for biological sample analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Many proteins with thiol groups can bind with trivalent arsenic which are termed as arsenic binding proteins, thus change their physiological functions. Therefore, it is vital to analyze the arsenic binding proteins in cells. The Pull-Down strategy based on biotinylated phenylarsenic acid (Bio-PAO(III)) probes is an effective way for analysis of arsenic binding proteins. In this strategy, streptavidin magnetic beads (SA-MBs) was applied to capture the arsenic binding proteins conjugating with Bio-PAO(III) probe. However, strong interaction between SA and biotin makes the elution of arsenic binding proteins not easy., Results: We developed a novel affinity separation strategy to address the challenge of eluting arsenic binding proteins, a key issue with the existing Bio-PAO(III) Pull-Down method. By employing magnetic beads modified with N α -Bis(carboxymethyl)-l-lysine (NTA-Lys), polyhistidine-tag (His 6 -Tag), and SA (MB-NTA(Ni)-His 6 -SA), we established a more efficient purification process. This innovative approach enables selective capture of arsenic binding proteins in HepG2 cells labeled by Bio-PAO(III) probes, facilitating gentle digestion by trypsin for precise identification through capillary high performance liquid chromatography (Cap HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS). What is more, the magnetic beads can be regenerated by using imidazole as the eluent, and the obtained MB-NTA(Ni) can be reloaded with His 6 -SA for next use. Our method successfully identified 41 arsenic binding proteins, including those involved in cytoskeletal structure, heat shock response, transcriptional regulation, DNA damage repair, redox state regulation, mitochondrial dehydrogenase function, and protein synthesis and structure., Significance: This work contributes to a more comprehensive understanding of the toxic mechanisms of arsenic, potentially providing valuable insights for the prevention or treatment of arsenic-related diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Novel psychoactive substances (NPS) are a group of substances, mainly of synthetic origin, characterized by toxicological properties extremely dangerous. The main difficulty in recognizing NPS in seizures and biological samples lies in their dynamic nature, related to the continuous synthesis and introduction on the market of new drugs, often with very similar structures to existing ones. The aim of this study was the creation of a robust and versatile method for the analysis of traditional drugs and NPS in different matrices., Results: Both target analysis and suspect screening methodologies were developed. The strategy used for suspect screening allowed to collect data through a scheduled multi reaction monitoring (sMRM) survey which triggered the collection of enhanced product ion (EPI) spectra when a compound met information dependent acquisition (IDA) criteria. The retention time of the different drugs, which was crucial to define the sMRM survey scan parameters, was predicted with a Quantitative Structure Retention (Chromatographic) Relationship (QSRR) model by Multiple Linear Regression. The model was validated through the evaluation of training set predictions, an external validation set and a leave-one out strategy; the results showed that the method fit for its purpose. The target method was validated in oral fluid as a testing matrix, with excellent results in term of recovery, accuracy, precision and matrix effect. Finally, the performances of the suspect method were evaluated by analysing a mixture containing 8 reference standards not included in the initial dataset, as well as seizures and real oral fluid samples. Four NPS were putatively identified in the analysed samples., Significance: The advantage of the proposed approach is the possibility of quantifying 65 classical drugs of abuse and NPS and, at the same time, detect and putatively identify 146 additional drugs in one single LC-MS/MS run. This is an innovative strategy for multi analyte detection and enables detection of low concentrations of drugs in complex biological matrices such as oral fluid. Considering the highly dynamic drug market, a strength of this strategy is that the analytical method can be kept up to date through the addition of new compounds based on the last drug monitoring bodies alerts without the need of authentic standards., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Antibody‒drug conjugates (ADCs) are innovative biopharmaceutics consisting of a monoclonal antibody, linkers, and cytotoxic payloads. Monitoring circulating payload concentrations has the potential to identify ADC toxicity; however, accurate quantification faces challenges, including low plasma concentrations, severe matrix effects, and the absence of stable isotope-labeled internal standards (SIL-IS) for payloads and their derivatives. Previous studies used structural analogs as internal standards, but different retention times between structural analogs and target analytes may hinder effective matrix correction. Therefore, a more flexible approach is required for precise payload quantification., Results: We developed an LC‒MS/MS method incorporating a postcolumn-infused internal standard (PCI-IS) strategy for quantifying payloads and their derivatives of trastuzumab emtansine, trastuzumab deruxtecan, and sacituzumab govitecan, including DM1, MCC-DM1, DXd, SN-38, and SN-38G. Structural analogs (maytansine, Lys-MCC-DM1, and exatecan) were selected as PCI-IS candidates, and their accuracy performance was evaluated based on the percentage of samples within 80%-120% quantification accuracy. Compared to the approach without PCI-IS correction, exatecan enhanced the accuracy performance from 30-40%-100% for SN-38 and DXd, while maytansine and Lys-MCC-DM1 showed comparable accuracy for DM1 and MCC-DM1. This validated PCI-IS analytical method showed superior normalization of matrix effect in all analytes compared to the conventional internal standard approach. The clinical application of this approach showed pronounced differences in DXd and SN-38 concentrations before and after PCI-IS correction. Moreover, only DXd concentrations after PCI-IS correction were significantly higher in patients with thrombocytopenia (p = 0.037)., Significance: This approach effectively addressed the issue of unavailability of SIL-IS for novel ADC payloads and provided more accurate quantification, potentially yielding more robust statistical outcomes for understanding the exposure-toxicity relationship in ADCs. It is anticipated that this PCI-IS strategy may be extrapolated to quantify payloads and derivatives in diverse ADCs, thereby providing invaluable insights into drug toxicity and fortifying patient safety in ADC usage., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ching-Hua Kuo reports financial support was provided by National Science and Technology Council of Taiwan. Ching-Hua Kuo reports financial support was provided by Ministry of Education. Ching-Hua Kuo reports financial support was provided by National Taiwan University (NTU) School of Pharmacy Endowment Fund in support of the Platform for Clinical Mass Spectrometry and NMR Structure Elucidation. Chih-Ning Cheng reports financial support was provided by Xin-Miao Education Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Signaling lipids (SLs) play a crucial role in various signaling pathways, featuring diverse lipid backbone structures. Emerging evidence showing the biological significance and biomedical values of SLs has strongly spurred the advancement of analytical approaches aimed at profiling SLs. Nevertheless, the dramatic differences in endogenous abundances across lipid classes as well as multiple isomers within the same lipid class makes the development of a generic analytical method challenging. A better analytical method that combines comprehensive coverage and high sensitivity is needed to enable us to gain a deeper understanding of the biochemistry of these molecules in health and disease. In this study, we developed a fast and comprehensive targeted ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for profiling SLs. The platform enables analyses of 260 metabolites covering oxylipins (isoprostanes, prostaglandins and other oxidized lipids), free fatty acids, lysophospholipids, sphingoid bases (C16, C18), platelet activating factors (C16, C18), endocannabinoids and bile acids. Various validation parameters including linearity, limit of detection, limit of quantification, extraction recovery, matrix effect, intra-day and inter-day precision were used to characterize this method. Metabolite quantitation was successfully achieved in both NIST Standard Reference Material for human plasma (NIST SRM 1950) and pooled human plasma, with 109 and 144 metabolites quantitated. The quantitation results in NIST SRM 1950 plasma demonstrated good correlations with certified or previously reported values in published literature. This study introduced quantitative data for 37 SLs for the first time. Metabolite concentrations measured in NIST SRM 1950 will serve as essential reference data for facilitating interlaboratory comparisons. The methodology established here will be the cornerstone for in-depth profiling of signaling lipids across diverse biological samples and contexts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A method for clinical potency determination of psilocybin and psilocin in hallucinogenic mushroom species Psilocybe cubensis was developed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Five strains of dried, intact mushrooms were obtained and analyzed: Blue Meanie, Creeper, B-Plus, Texas Yellow, and Thai Cubensis. An extraction protocol was developed; this included an evaluation of sample milling technique, extraction solvents, and recovery/stability. Reversed phase chromatography on fused-core particle phases was developed for the determination of the two analytes using internal standard calibration with deuterated isotopologues of each analyte. The separation takes less than 5 min. Matrix effects were investigated by comparing signal response of calibration samples in neat solution and several mushroom matrices; no significant matrix effects were observed. The limit of detection for psilocybin was 1.5 ng/mL (1.5 pg on-column; 300 ng/g mushroom) and for psilocin was 0.15 ng/mL (0.15 pg on-column; 30 ng/g mushroom) using a Shimadzu LCMS-8050 triple quadrupole mass spectrometer. Assessment of the accuracy and precision of the method indicated percent error and RSD were <6 % at all concentration levels. Three whole, intact mushrooms from each strain were analyzed individually to obtain average content differences both between strains and between mushrooms of the same strain. From most to least potent, the study found that the average total psilocybin and psilocin concentrations for the Creeper, Blue Meanie, B+, Texas Yellow, and Thai Cubensis strains were 1.36, 1.221, 1.134, 1.103, and 0.879 % (w/w), respectively. A subset of these mushrooms was also tested in a separate non-affiliated laboratory, and the results were comparable between the two laboratories. Results from the secondary laboratory showed improved precision when multiple mushrooms were homogenized together, prior to extraction. [Display omitted] • Psilocybin and psilocin potency determination method for psilocybin mushrooms. • Dried mushrooms milled and extracted for LC-MS/MS determination. • Potency method results confirmed by an independent laboratory. • Total psilocybin and psilocin concentration varied between 0.85 and 1.45 wt%. [ABSTRACT FROM AUTHOR]

The determination of plant hormones is still a very challenging analytical discipline, mainly due to their low concentration in complex plant matrices. Therefore, the involvement of very sensitive high-throughput techniques is required. Cytokinins (CKs) are semi-polar basic plant hormones regulating plant growth and development. Modern methods for CK determination are currently based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), which enables the separation of CK isomeric forms occurring endogenously in plants. Here, ultra-high performance supercritical fluid chromatography coupled with tandem mass spectrometry (UHPSFC-MS/MS) was used for the simultaneous determination of 37 CK metabolites. The chromatographic conditions were tested on three different columns with various retention mechanisms. Hybrid silica modified with 2-picolylamine was selected as the stationary phase. Several parameters such as column temperature, back pressure regulation, mobile phase composition and make-up solvent were investigated to achieve efficient separation of CK isomers and reasonable sensitivity. Compared to UHPLC-MS/MS, a 9-min chromatographic analysis using a mobile phase of supercritical CO 2 and 5 mM ammonia in methanol represents a three-fold acceleration of total run time. The quantification limit of UHPSFC-MS/MS method was in the range of 0.03–0.19 fmol per injection and the method validation showed high accuracy and precision (below 15 % for most analytes). The method was finally applied to the complex plant matrix of the model plant Arabidopsis thaliana and the obtained profiles of CK metabolites were compared with the results from the conventional UHPLC-MS/MS method. The presented work offers a novel approach for quantification of endogenous CKs in plants. Compared to the conventional UHPLC-MS/MS, the total run time is shorter and the matrix effect lower for the key CK metabolites. This approach opens the opportunity to utilize UHPSFC-MS/MS instrumentation for targeted plant hormonomics including other plant hormone families. [Display omitted] • 37 cytokinins analysed in 9 min. • Limits of detection close to 0.03 fmol. • Accuracy and precision close to 15 %. • Low matrix effect for key metabolites. • Endogenous cytokinins determined in Arabidopsis seedlings. [ABSTRACT FROM AUTHOR]

Ceramide is a huge lipid family consisting of diversified structures in which various modifications are seen in the fatty acyl chain and the long chain base (LCB). In this contribution, a higher collision energy (HCD) linear ion-trap mass spectrometric method (LIT MSn) was applied to study the mechanisms underlying the fragmentation processes of ceramide molecules in 12 subclasses, which were desorbed by ESI as the [M + Li]+ ions. Multiple sets of fragment ions reflecting the fatty acyl chain and LCB were observed in the HCD MS2 spectra for all the ceramide classes, resulting in unambiguous definition of the ceramide structures, including the chain length and the modification (α-hydroxy-, β-hydroxy-, ω-hydroxy-FA) of the fatty acyl moiety, and the types of LCB (sphingosine, phytosphigosine, 6-hydroxy-sphingosine). Thereby, this approach permits differentiation of isomeric structures and ceramide species in the biological specimen can be unveiled in detail. By application of sequential MS3, the double bond position along the fatty acyl chain of the molecule can be located, and a complete structural characterization of ceramides can be achieved. Image 1 • High resolution HCD tandem mass spectrometry permits complete structural characterization of ceramides as [M + Li]+ ions. • Multiple sets of fragment ions define the long-chain base and fatty acyl chain, and minor isomer can be identified. • High resolution MS precisely assign the formula of molecular and fragment ions, and facilitates fragmentation mechanism recognition. • The fragmentation pathways of ceramides in 12 subclasses are proposed. [ABSTRACT FROM AUTHOR]

In the last few years, sildenafil and its analogues have emerged as an illegal ingredient in health food and products, causing illness and death. To address this problem, herein, a gold nanoparticle-based immunochromatographic assay for simultaneous detection sildenafil and its analogues was developed by using just one monoclonal antibody. This method can realize qualitative and quantitative analysis of sildenafil and its analogues with a detection range (IC 20 -IC 80) of 0.12–28.05 ng mL−1, the IC 50 was 0.31–8.45 ng mL−1 and the cut-off value was 5.0–80.0 ng mL−1. Meanwhile, this method could also accomplish semi-quantitative analysis of vardenafil with a cut-off value of 0.625 ng mL−1. The detection can be finished in 6 min and showed no cross-reactivity with avanafil and tadalafil. The recoveries of spiked samples ranged from 70.5 to 118.9% with a coefficient variation less than 15%, which confirmed the assay was sensitive, accurate and adapted to the rapid determination of sildenafil and its analogues. Sildenafil adulteration. Image 1 • A sensitive GNPs ICA for 10 sildenafil adulterants was developed based on one broad-spectrum mAb. • The comparison with reported antibodies were also made by molecular simulation to evaluate the better performance. • The test results of real samples have good correlation with UPLC-MS/MS. [ABSTRACT FROM AUTHOR]

Sterols are a class of lipid molecules that include cholesterol, oxysterols, and sterol esters. Sterol lipids play critical functional roles in mammalian biology, including the dynamic regulation of cell membrane fluidity, as precursors for the synthesis of bile acids, steroid hormones and vitamin D, as regulators of gene expression in lipid metabolism, and for cholesterol transport and storage. The most common method employed for sterol analysis is high performance liquid chromatography coupled with tandem mass spectrometry (MS/MS). However, conventional collision induced dissociation (CID) methods used for ion activation during MS/MS typically fail to provide sufficient structural information for unambiguous assignment of sterol species based on their fragmentation behaviour alone. This places a significant burden on the efficiency of the chromatographic separation methods for the effective separation of isomeric sterols. Here, toward developing an improved analysis strategy for sterol lipids, we have explored the novel use of 213 nm photodissociation MS/MS and hybrid multistage-MS/MS (i.e., MSn) data acquisition approaches for the improved structural characterization of cholesterol, representative isomeric oxysterols, and cholesteryl esters. Most notably, UVPD-MS/MS of ammoniated, lithiated and sodiated adducts of cholesterol, several representative oxysterol species, and an oxosterol lipid, are shown to give rise to abundant [M]+. radical cation products, that subsequently fragment during collision induced MS3 to yield extensive structurally informative product ions, similar to those observed by Electron Ionization, and that enable their unambiguously assignment, including isomeric differentiation of oxysterols. For cholesterol esters, a reversed hybrid collision induced-MS/MS and UVPD-MS3 approach is shown to enable assignment of the sterol backbone, and localization of the site(s) of unsaturation within esterified fatty acyl chains. Image 1 • Hybrid UV photodissociation and multistage tandem mass spectrometry is a novel analysis strategy for sterol lipids. • 213 nm UVPD-MS/MS of cholesterol and oxysterols produces abundant [M]+. radical cation product ions. • HCD-MS3 of these products enables unambiguously structural assignment, including for differentiation of isomeric oxysterols. • HCD-MS/MS and UVPD-MS3 enables detailed structural assignment of cholesterol esters, including site(s) of unsaturation. [ABSTRACT FROM AUTHOR]

A novel solid phase extractant His-rSPG@ZIF-8 was prepared by covalently coupling recombinant streptococcal protein G (His-rSPG) with ZIF-8. The His-rSPG@ZIF-8 composite was characterized by Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy (Raman), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Due to the specific binding between the immunoglobulin binding region of His-rSPG and the Fc region of immunoglobulin G (IgG), the His-rSPG@ZIF-8 composite demonstrated exceptional selectivity in adsorbing IgG. In Britton-Robinson buffer (BR buffer) with a salt concentration of 500 mmol L -1 (0.04 mol L -1 , pH 8.0), the His-rSPG@ZIF-8 composite exhibited a remarkable adsorption efficiency of 99.8 % for 0.05 mg of the composite on 200 μL of IgG solution (100 μg mL -1 ). The adsorption behavior of the His-rSPG@ZIF-8 composite aligns with the Langmuir adsorption model, and the theoretical maximum adsorption capacity is 1428.6 mg g -1 . The adsorbed IgG molecules were successfully eluted using a SDS solution (0.5 %, m/m), resulting in a recovery rate of 91.2 %. Indeed, the His-rSPG@ZIF-8 composite was successfully utilized for the isolation and purification of IgG from human serum samples. The obtained IgG exhibited high purity, as confirmed by SDS-PAGE analysis. Additionally, LC-MS/MS analysis was employed to identify the human serum proteins following the adsorption and elution process using the His-rSPG@ZIF-8 composite material. The results revealed that the recovered solution contained an impressive content of immunoglobulin, accounting for 62.4 % of the total protein content. Furthermore, this process also led to the significant enrichment of low abundance proteins such as Serpin B4 and Cofilin-1. Consequently, the His-rSPG@ZIF-8 composite holds great promise for applications such as IgG purification and immunoassays. At the same time, it expands the application of metal-organic frameworks in the field of proteomics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Sample extraction is one of the most critical steps in most of the analytical processes. Nowadays, there is a demand for simple approaches that can effectively extract and concentrate target analytes from complex matrices, like biofluids, with accurate and reliable results., Results: A porous monolith of poly(methacrylic acid-co-ethylene glycol dimethacrylate) has been immobilized on the inner wall of a 2 mL commercial polypropylene microcentrifuge tube through radical photopolymerization, using bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide as radical initiator and 405 nm laser pointer activation. Photopolymerization parameters were adjusted to obtain a continuous and homogeneous polymer layer (0.16 mm wet polymer thickness and 31 mg dry polymer weight) in the inner tube surface. Extraction efficiency of twelve psychoactive substances was assessed by the evaluation of the effect of sample pH, extraction and desorption times, and desorption solvent volume. Moreover, matrix effect, reusability and stability of monolith-coated microcentrifuge tubes were studied. Sample extracts were measured by liquid chromatography-tandem mass spectrometry, providing limits of quantification (LOQ) from 0.2 to 2.7 μg L -1 , recoveries from 80 to 118 %, relative standard deviations lower than 17 %, and a linear range from LOQ to 500 μg L -1 ., Significance: The proposed device is suitable for the easy and simple extraction of psychoactive substances from urine samples with a high portability, reduced solvent consumption, low cost, and low environmental impact., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Chemical isotope labeling (CIL) LC-MS is a powerful tool for metabolome analysis with high metabolomic coverage and quantification accuracy. In CIL LC-MS, the overall metabolite detection efficiency using Orbitrap MS can be further improved by employing a segment scan method where the full m/z range is divided into multiple segments for spectral acquisition with a significant increase in the in-spectrum dynamic range. Considering the metabolic complexity in different types of biological samples (e.g., feces, urine, serum/plasma, cell/tissue extracts, saliva, etc.), we report the development and evaluation of the segment scan method for metabolome analysis of different sample types., Results: It was found that sample complexity significantly influenced the performance of the segment scan method. In metabolically complex samples such as feces and urine, the method yielded a substantial increase (up to 94 %) in detected peak pairs or metabolites, compared to conventional full scan. Conversely, less complex samples like saliva exhibited more modest gains (approximately 25 %). Based on the observations, a 120-m/z segment scan method was determined as a routine approach for CIL LC-Orbitrap-MS-based metabolomics with good compatibility with different types of biological samples. For this method, a further investigation on relative quantification accuracy was done. The peak area ratios of 12 C-/ 13 -labeled metabolites were slightly reduced with 72%-84 % of peak pairs falling within the ±25 % range of the anticipated peak ratio of 1.0 among different samples, as opposed to 81%-90 % in the full scan, which was attributed to the inclusion of more low-abundance peak pairs within the narrow MS segments. However, the overall peak ratio measurement precision was not significantly affected by the segment scan., Significance and Novelty: The segment scan method was found to be useful for CIL LC-Orbitrap-MS-based metabolome analysis of different types of samples with significant improvement in metabolite detectability (25-94 % increase), compared to the conventional full scan method., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Global profiling of bile acids (BAs) is imperative for understand their function and disease pathogenesis. But it is still a challenging task, as the collision-induced dissociation (CID) fragment ions of unconjugated BAs showed low ion intensities to insufficient analysis. Herein, we developed a highly sensitive method for pseudotargeted profiling of BAs by chemical derivatization. In the developed method, a labeling reagent, 2-dimethylaminoethylamine (DMED), was adopted to label the carboxyl group of BAs. The results demonstrated that the detection sensitivities of unconjugated BAs were increased by 4-200 folds after DMED-labeling. Moreover, to profile other potential BAs not included in the 91 known targets, diverse survey experiments were performed on Qtrap-MS to search BAs for both precursor and fragment ion species, and retention index (RI) strategy was adopted to facilitate the identification of isomers. Finally, MRM-based LC-MS/MS method was validated for the pseudotargeted profiling of the BAs submetabolome with good linearity (r 2  ≥ 0.990 for 89 known BAs) and high sensitivity (0.05-0.5 ng/mL for unconjugated BAs), covering unconjugated, glycine, taurine, sulfuric acid, glucuronic acid, and as well as those doubly-conjugated with above types. With this method, a total of 107 BAs, covering 54 BAs identified by authentic standards and 53 BAs candidates, were successfully determined in human serum of women with intrahepatic cholestasis of pregnancy (ICP). Multivariate analysis revealed deferentially expressed BAs. ICP disease altered the BAs profile with a reduced proportion of unconjugated, sulfate- and doubly-conjugated BAs and an increased proportion of glycine and taurine conjugates. Altered proportion and profile of BAs in ICP groups were gradually recovered during the ursodeoxycholic acid (UDCA) therapy. Overall, the strategy of DMED-labeling technique combined with diverse survey experiments is sufficiently sensitive and robust to comprehensively analysis of metabolic profiling of BAs in human serum., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Ceramides are sphingolipids with a structural function in the cell membrane and are involved in cell differentiation, proliferation and apoptosis. Recently, these chemical species have been pointed out as potential biomarkers in different diseases, due to their abnormal levels in blood. In this research, we present an overall strategy combining data-independent and dependent acquisitions (DIA and DDA, respectively) for identification, confirmation, and quantitative determination of ceramides in human serum. By application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in DIA mode we identified 49 ceramides including d18:1, d18:0, d18:2, d16:1, d17:1 and t18:0 species. Complementary, quantitative determination of ceramides was based on a high-throughput and fully automated method consisting of solid-phase extraction on-line coupled to LC-MS/MS in DDA to improve analytical features avoiding the errors associated to sample processing. Quantitation limits were at pg mL -1 level, the intra-day and between-days variability were below 20 and 25 %, respectively; and the accuracy, expressed as bias, was always within ±25 %. The proposed method was tested with the CORDIOPREV cohort in order to obtain a qualitative and quantitative profiling of ceramides in human serum. This characterization allowed identifying d18:1 ceramides as the most concentrated with 70.8% of total concentration followed by d18:2 and d18:0 with 13.0 % and 8.8 %, respectively. Less concentrated ceramides, d16:1, d17:1 and t18:0, reported a 7.1 % of the total content. Combination of DIA and DDA LC-MS/MS analysis enabled to profile qualitative and quantitatively ceramides in human serum., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Feliciano Priego Capote reports financial support was provided by Spain Ministry of Science and Innovation. Feliciano Priego Capote reports a relationship with University of Cordoba that includes: employment., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: PLS-DA of high-dimensional metabolomics data is frequently employed to capture the most pertinent features to sample classification. But the presence of numerous insignificant input features could distort the PLS-DA model, blow up and scramble the selected differential features. Usually, univariate filtration is subsequently complemented to refine the selected features, but often giving unstable results. Whereas by precluding insignificant features through univariate data prefiltration assessed by FDR adjusted p-value, PLS-DA can generate more stable and reliable differential features. We explored and compared these two data analysis procedures to gain insights into the underlying mechanisms responsible for the disparate results., Results: The effect of univariate data filtration preceding and succeeding PLS-DA analysis on the identified discriminative features/metabolites was investigated using LC-MS data acquired on the samples of human serum and C. elegans extracts, with and without metabolite standards spiked to simulate the treated and control groups of biological samples. It was shown that the univariate data prefiltration before PLS-DA usually gave less but more stable and likely more reliable and meaningful differential features, while PLS-DA applied directly to the original data could be affected by the presence of insignificant features and orthogonal noise. Large number of insignificant variables and orthogonal noise could distort the generated PLS-DA model and affect the p(corr) value, and artificially inflate the calculated VIP values of relevant features due to the increased total number of input features for model construction, thus leading to more false positives selected by the conventional VIP threshold of 1.0., Significance and Novelty: Univariate data filtration preceding PLS-DA was important for the identification of reliable differential features if using a conventional threshold of VIP of 1.0. Presence of insignificant features could distort the PLS-DA model and inflate VIP values. Appropriate VIP threshold is associated with the numbers of input features and the model components. For PLS-DA without univariate prefiltration, threshold of VIP larger than 1.0 is recommended for the selection of discriminative features to reduce the false positives., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Prognosis, diagnosis, and treatment of several diseases strongly rely on the sensitive, selective, and accurate determination of specific biomarkers in relevant biological samples. Free biliverdin and free bilirubin represent important new biomarkers of oxidative stress, however, the lack of suitable analytical methods for their determination has hindered progress in biomedical and clinical research., Results: Here, we introduce a first comprehensive approach for robust and simultaneous determination of these bilins in serum using liquid chromatography - mass spectrometry (LC-MS). The developed analytical method exhibits linearity for both analytes within the concentration range of 0.5-100 nM, with limits of detection and quantitation determined at 0.1 nM and 0.5 nM, respectively. Moreover, several analytical pitfalls related to the intrinsic molecular structures of free bilirubin and free biliverdin and their trace concentration levels in biological samples are discussed here in detail for the first time. We have shown that the solubility, chemical stability, and affinity of these bilins to various materials strongly depend on the solvent, pH, and addition of stabilizing and chelating agents. Finally, the validated LC-MS method was successfully applied to the analysis of both bilins in fetus bovine serums, yielding higher free bilirubin/biliverdin ratios compared with previously reported values for human serum., Significance: Failure to recognize and address the challenges presented here often leads to substantial analytical errors and consequently biased interpretation of the obtained results. This pertains not only to LC-MS, but also to many other analytical platforms due to the compound-derived sources of error., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

A powerful and fast glycopeptide/glycan enrichment method is critical for the efficiency and throughput of mass spectrometry (MS)-based glycoproteomic and glycomic analyses, especially for large-scale sample analysis. Here, we report an ultrafast and effective method for both intact N-glycopeptide and N-glycan enrichment and apply it to human serum samples. In this method, a natural hydrophilic material, bacterial cellulose (BC), was adopted and fully optimized for enrichment. This method offers the following advantages: (i) The enrichment material has natural hydrophilicity and is low-cost, biocompatible, biodegradable and easily accessible; (ii) the whole enrichment procedure is remarkably simple and fast. It takes only 10 min for intact glycopeptides/glycans to be easily purified from mixtures; (iii) the specificity of this method is over 94% for both glycan and glycopeptide enrichment; and (iv) the outstanding specificity of this technique enables high isolation efficiency for the enrichment of both intact glycopeptides and glycans. A total of 36 N-glycans and 31 N-glycopeptides were identified from human immunoglobulin G (IgG). The glycan and glycopeptide absorption capacity of BC was as high as 333 μg/mg and 250 μg/mg (IgG/BC) respectively. The selectivity for glycan and glycopeptide enrichment reached 1:100 (IgG/bovine serum albumin (BSA), molar ratio) and 1:200 (maltoheptaose (DP7)/BSA, molar ratio), respectively. Furthermore, a total of 159 N-glycans and 523 N-glycopeptides were identified in human serum by using this method. Overall, the BC-based enrichment method we present here provides an ultrafast and highly efficient method for the enrichment of both N-glycopeptides and N-glycans in complex samples and shows great potential in large-scale glycoproteomic and glycomic analyses. Image 1 • Bacterial cellulose has natural hydrophilicity and is cost-friendly. • The whole enrichment procedure is remarkably simple and fast (10 min). • This method enables enrichment of both intact N-glycopeptides and N-glycans. • The enrichment specificity is over 94% for both N-glycans and N-glycopeptides. [ABSTRACT FROM AUTHOR]

We present an approach for the elucidation of C=C bond position and cis/trans isomers, which is achieved by the reaction of ambient water radical cations and double bonds, followed by the fragmentation of epoxide radical cations to generate diagnostic ions in tandem mass spectrometry. Hexenol double bond positional isomers and cis/trans isomers which exhibit different properties and biological functions are characterized as a proof of concept. The merits of the approach include the simplicity of experimental setup, rapid derivatization (within seconds), the obviation of organic solvents, as well as easy spectral interpretation. Image 1 • A novel approach for the elucidation of C=C bond position and cis/trans isomers. • In situ epoxidation reaction of ambient water radical cations and C=C bonds. • No need of special instrumentation and organic solvents. [ABSTRACT FROM AUTHOR]

The present study reports on a streamlined analytical platform for rapid, straightforward, and on-site sample analysis using a miniature mass spectrometer with adequate tandem mass spectrometry (MS/MS) capability. An "all-in-one" workflow is developed combining accelerated air-assisted in-syringe extraction, sorbent-packed membrane clean-up, and needle spray ionization with an in-house built syringe assembly. A custom-made metal needle with a sharp, thin, and conical tip of micrometer-scale served as an integral component of the syringe assembly. This needle was capable of both aspirating a high-speed airflow for a highly efficient in-syringe extraction and performing stable electrospray for direct miniature mass spectrometry analysis. As a proof-of-concept demonstration, the developed protocol was implemented for the identification of 40 adulterated cosmetic ingredients and validated in terms of sensitivity, linearity, matrix effect, and recovery. A custom expandable mass spectral library was established in-house for smart suspect screening. Compound search and identification were conducted following a two-stage strategy using the full-scan MS and MS/MS data. The methodology demonstrates excellent potential for outside-the-laboratory chemical analysis by reducing the high time and labor costs associated with traditional sample preparation and chromatographic separation procedures, which would meet product quality and safety inspection requirements for on-site compliance check and regulatory enforcement at the testing sites such as supermarkets, department stores, wholesale markets, and roadside stalls. Image 1 • A streamlined analytical platform was established for rapid on-site sample analysis using a miniature mass spectrometer. • An "all-in-one" workflow combined sample extraction, clean-up, and ionization with a syringe assembly built in-house. • A pulled micrometer-scale needle enabled a high-speed airflow to be aspirated for highly efficient in-syringe extraction. • A custom expandable mass spectral library was established for smart suspect screening. [ABSTRACT FROM AUTHOR]

This paper describes a simple and reliable method for the sensitive and selective determination of the pesticides Glyphosate and Glufosinate and their main metabolites aminomethylphosphonic acid (AMPA) and 3-[hydroxy(methyl)phosphinoyl]propionic acid (MPPA) in surface water. The developed method is based on ion chromatography hyphenated to electrospray tandem mass spectrometry and does not require derivatization. A membrane suppressor, regenerated at pH 9, has been used in this work to strongly improve the peak shape of AMPA, which suffered from huge tailing due to the interaction of this compound with acidic membrane suppressors. With this modified suppressor the sensitivity for AMPA improved about 100 times. Moreover, addition of 40% methanol to the hydroxy eluent improved MS sensitivity for all compounds by 1.3–2.8 times. The separation is performed on a strong anion exchange column. Glyphosate and AMPA are detected in the negative ion ESI mode, whereas Glufosinate and MPPA show much better sensitivity in the positive ion ESI mode. Surface water samples were spiked with the labelled parent compounds. Sample preparation comprised a filtering step over coupled solid phase extraction Ba, Ag and Na cartridges to remove chloride and sulphate ions. The performance characteristics of the method were determined with real surface water samples. Samples containing 12.6 g L−1 chloride were also used in the validation process and showed no problem. Nitrate is not removed from the samples and may give ion-suppression for Glyphosate. Linearity of the method was established over at least three orders of magnitude in the measuring range 10–2000 ng L−1 for surface water. The reliability of the results was ensured by the application of the criteria from the Dutch Technical Agreement (NTA 8379) concerning "identification", "indication", and "absence" of substances. The limit of quantitation with regard to identification was 10 ng L−1 for all compounds and the limits of detection with regard to indication were 6.5–9.6 ng L−1. The recovery (94–104%) and reproducibility variance (5.5–6.2%) were excellent. The suitability of the developed method was demonstrated by the analysis of 172 surface water samples of low to moderate salinity from different parts of The Netherlands. AMPA was identified in 99% of the samples and always exceeded the maximum allowable concentration of 100 ng L−1. The maximal concentration found in surface water was 9900 ng L−1. Glyphosate was identified in 82% of the samples and in only 6% Glyphosate exceeded the maximum allowable concentration of 100 ng L−1. MPPA was identified in about 75% of the samples whereas Glufosinate was rarely detected and never exceeded the limit of quantitation. Image 1 [ABSTRACT FROM AUTHOR]

Bile acids (BAs), as crucial endogenous metabolites, are closely related to cholestasis, metabolic disorders, and cancer. To better understand their function and disease pathogenesis, global profiling of BAs is necessary. Here, multidimensional data mining was developed for the discovery and identification of potentially unknown BAs in cholestasis rats. Based on an in-house theoretical BA database and using a newly established liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS/MS) method, four-dimensional (4D) data including the retention times (RT), abundances, HRMS, and HRMS/MS spectra were acquired and elucidated. And 491 BAs were totally profiled. Then, the relationships between RT with different conjugation types, different positions and configurations of hydroxyl/ketone groups as well as fragmentation rules of hydroxyl, ortho-hydroxyl, ketone, and conjugated groups of BAs were summarized to assist BA identification for the first time. Finally, 292 BAs were assigned with molecular formulas, 201 of which were putatively identified by integrating the 4D data, applying structure-driven relative retention time rules, and a comparison with synthetic BAs. The estimated concentrations of 201 BAs, including 93 reported and 108 newly identified BAs, were quantified by using surrogate standards with similar structure. Among 201 BAs, 38 BAs were detected in both humans and rats for the first time. Our strategy has expanded the scope of BAs and provides a way to identify a class of metabolites. Compared to normal rats, the significantly increased sulfated and glucuronide conjugated BAs in urine and feces from experimentally cholestatic rats may reveal a way to diagnose intrahepatic cholestasis. Image 1 • An in-house BA database and novel UHPLC-MS/MS method were established for global profiling of 491 BAs in rats. • Multidimensional data mining was developed to putatively identify 201 BAs in bio-samples. • The change in sulfo-BAs and glucu-BAs suggested new ways to diagnose and treat BA metabolic disorders. [ABSTRACT FROM AUTHOR]

Aberrant expression of glycosphingolipids has been implicated in a myriad of diseases, but our understanding of the strucural diversity, spatial distribution, and biological function of this class of biomolecules remains limited. These challenges partly stem from a lack of sensitive tools that can detect, identify, and quantify glycosphingolipids at the molecular level. Mass spectrometry has emerged as a powerful tool poised to address most of these challenges. Here, we review the recent developments in analytical glycosphingolipidomics with an emphasis on sample preparation, mass spectrometry and tandem mass spectrometry-based structural characterization, label-free and labeling-based quantification. We also discuss the nomenclature of glycosphingolipids, and emerging technologies like ion mobility spectrometry in differentiation of glycosphingolipid isomers. The intrinsic advantages and shortcomings of each method are carefully critiqued in line with an individual's research goals. Finally, future perspectives on analytical sphingolipidomics are stated, including a need for novel and more sensive methods in isomer separation, low abundance species detection, and profiling the spatial distribution of glycosphingolipid molecular species in cells and tissues using imaging mass spectrometry. Image 1 • A critical review of recent advances in MS analysis of glycosphingolipidome. • Nomenclature and biological significance of glycosphingolipids are covered. • Comprehensive coverage of novel enrichment strategies for glycosphingolipids. • Extensive coverage of fragmentation techniques in glycosphingolipid analysis. • Labeling approaches for quantitation and perspectives on future directions. [ABSTRACT FROM AUTHOR]

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous bioactive lipids with anti-diabetic and anti-inflammatory effects. Identification of FAHFAs is challenging due to both the relatively low abundance of these metabolites in most biological samples and the significant structural diversity arising from the co-occurrence of numerous regioisomers. Ultimately, development of sensitive analytical techniques that enable rapid and unambiguous identification of FAHFAs is integral to understanding their diverse physiological functions in health and disease. While a battery of mass spectrometry (MS) based methods for complex lipid analysis has been developed, FAHFA identification presents specific challenges to conventional approaches. Notably, while the MS2 product ion spectra of [FAHFA – H]¯ anions afford the assignment of fatty acid (FA) and hydroxy fatty acid (HFA) constituents, FAHFA regioisomers are usually indistinguishable by this approach. Here, we report the development of a novel MS-based technique employing charge inversion ion/ion reactions with tris-phenanthroline magnesium complex dications, Mg(Phen) 3 2+, to selectively and efficiently derivatize [FAHFA – H]¯ anions in the gas phase, yielding fixed-charge cations. Subsequent activation of [FAHFA – H + MgPhen 2 ]+ cations yield product ions that facilitate the assignment of FA and HFA constituents, pinpoints unsaturation sites within the FA moiety, and elucidates ester linkage regiochemistry. Collectively, the presented approach represents a rapid, entirely gas-phase method for near-complete FAHFA structural elucidation and confident isomer discrimination without the requirement for authentic FAHFA standards. Image 1 • Selective gas-phase charge switching of anions of fatty acid esters of hydroxyl fatty acids (FAHFAs). • Charge switching and CID enables the elucidation of ester linkage regiochemistry. • Charge switching and CID enables the elucidation of site(s) of unsaturation in the FA moiety. • Charge switching and CID facilitates the assignment of the FA and HFA consituents of a given FAHFA. [ABSTRACT FROM AUTHOR]

With the merits of non-destructive, high penetration ability and minimizing autofluorescence, near-infrared (NIR) fluorescent probes have attracted much attention. In this paper, a NIR emission fluorescent turn-on probe THQ-L for H 2 S was synthesized by the knoevenagel condensation between tetrahydroquinoxaline-6- formaldehyde derivative and 2-benzothiazoleacetonitrile. THQ-L can recognize H 2 S through tandem reaction triggered by HS− to construct 1,4-diethylpiperazine-modified iminocoumarin-benzothiazole, which produces a strong red fluorescent signal. THQ-L displayed an excellent selectivity toward H 2 S, a large stokes shift (126 nm), a high signal-to-noise ratio (200-fold), the detection limits of 38.3 nM in PBS (10 mM, pH 7.4, 30% THF). The application study indicates that THQ-L can sensitively detect H 2 S in red wine, natural waters, living cells and can be prepared for a test paper strip for the qualitative detection of H 2 S. Image 1 • A novel NIR fluorescent probe for H 2 S based on tandem reaction to construct iminocoumarin-benzothiazole. • TCF-DNs detects H 2 S with a large Stokes shift, a high signal-to-noise ratio, a low detection limit. • TCF-DNs can detect H 2 S in red wine, natural waters, and image H 2 S living cells. • TCF-DNs was prepared for test strip and was used for the qualitative detection of H 2 S. [ABSTRACT FROM AUTHOR]

The specific determination of L -hydroxyproline (Hyp) can serve as a potential indicator for early clinical diagnosis of liver fibrosis. In this work, an integrated strategy based on 4-plex stable isotope labeling derivatization combined with dummy magnetic molecularly imprinted polymers (QSILD-DMMIPs) was developed for specific extraction and rapid determination of Hyp in human serum by ultra high performance liquid chromatography tandem mass spectrometry. A new series of QSILD reagents d 0 /d 1 /d 2 /d 3 -6- N -methyl-rhodamine 6G- N -hydroxysuccinimidyl formate (d 0 /d 1 /d 2 /d 3 -MRSF) were designed, synthesized and applied for the high-throughput labeling of Hyp in serum samples. The structural analogue derivative of Hyp with 6- N -ethyl-rhodamine 6G- N -hydroxysuccinimidyl formate (ERSF-Hyp) was synthesized and used as a novel dummy template to prepare DMMIPs. The DMMIPs were well characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), fourier transform infrared spectroscopy (FTIR), Brunner Emmet Teller (BET) measurements, thermogravimetric analysis (TGA), X-ray diffraction (XRD), zeta potential and adsorption experiments. All d 0 /d 1 /d 2 /d 3 -MRSF-Hyp derivatives were conveniently and specifically adsorbed by DMMIPs in magnetic dispersive solid phase extraction procedure before injection. Method validation results including linearity (0.2–100 ng mL−1), limits of detection and quantitation (0.05 and 0.2 ng mL−1), accuracy, precision, stability, matrix effect and derivatization efficiency were satisfactory. The analytical performances benefited from efficient integration of QSILD and specific DMMIPs extraction. The proposed strategy was successfully applied for Hyp determination in human serum of liver fibrosis patients and healthy controls, which was of great significance to early diagnosis. Image 1 • Derivatization-based dummy template ERSF-Hyp was proposed for DMMIPs synthesis. • 4-Plex stable isotope labeling derivatization reagents of d 0-3 -MRSF were synthesized. • An integrated strategy of QSILD-MDSPE with UHPLC-MS/MS for Hyp was developed. • This method was specific, high-throughput, sensitive, accurate and low matrix effect. • Measured serum Hyp can be used as potential biomarker for liver fibrosis diagnosis. [ABSTRACT FROM AUTHOR]

Coupling supercritical fluid extraction (SFE) on-line with supercritical fluid chromatography (SFC) – tandem mass spectrometry (MS/MS) provides a single platform for efficient extraction, separation, and detection in a chemical analysis. SFE–SFC–MS/MS requires consideration of many extraction and chromatographic variables to not only provide the most efficient extraction, but also to analytically transfer the extracted analytes to the column for separation. There is a fundamental lack of understanding of how the variables in SFE affect those in SFC. Typically, a univariate approach is taken in on-line SFE–SFC–MS/MS method development, but this provides little insight into the relative importance of variables and their potential interactions. Here, a multivariate approach was used to develop a better understanding of the synergistic relationship between the extraction and separation processes by focusing on the optimization of extraction parameters for target analytes with a wide range of physicochemical properties in matrices of variable retentivity. The methodology used a set of optimal on-line SFE–SFC–MS/MS extraction parameters for 18 analytes of variable physicochemical properties in three different silica gel-based sample matrices are presented. Image 1 • Multivariate on-line SFE–SFC–MS/MS method development. • Two-level half factorial determination of most significant extraction parameters. • Significant parameters are dependent on type of sample. • Central composite design determination of optimal extraction parameters for range of analytes. • Enabled compromise in optimal extraction parameters for simultaneous extraction of different analytes. [ABSTRACT FROM AUTHOR]

Glucosylsphingosine (GlcS) in plasma is considered to be a reliable biomarker of Gaucher disease. The detection difficulty of GlcS is that it is difficult to achieve simultaneous separation and quantification with its isomer galactosylsphingosine (GalS), a biomarker of Krabbe disease. In this work, a multiplexed stable isotope labeling absolute quantization strategy coupled with magnetic dispersive solid phase extraction using new prepared dummy magnetic molecularly imprinted polymers (DMMIPs) has been developed for this purpose by ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). 8-Plex Amine-reactive Mass Difference Tags (M360/361/362/363/373/375/376/378-AMDTs), were designed, synthesized and used to label GalS and GlcS in different 8 plasma samples, respectively. Synchronously, M359-AMDTs was prepared and used to label mixed standards of GalS and GlcS, which served as internal standards in UHPLC-MS/MS quantitation. Then DMMIPs possessing dual recognition function were applied for specific enrichment and purification of all GlcS and GalS derivatives from a combined solution of labeled 8-plex plasma samples and mixed standards before UHPLC-MS/MS injection. The labeling efficiency, chromatographic retention and mass spectrometry responses of all the 9 AMDTs reagents were consistent for GlcS and GalS. The established and validated method enabled 8-plex plasma samples quantification in a single UHPLC-MS/MS run (<2.0 min). Good linearity of AMDTs-GlcS/GalS derivatives was obtained in the range of 0.02–800 nM. LODs of GlcS and GalS were both 0.005 nM. The recoveries were in the range of 96.1–107.2%. The method was successfully applied for multiplex quantitative analysis of GlcS and GalS in human plasma samples. The results indicated that this method was capable of better realizing the simultaneous separation and quantification of GalS and GlcS compared to reported methods. A new workflow of multiplexed stable isotope labeling absolute quantitation (MSILAQ) strategy combined with magnetic dispersive solid phase extraction (MDSPE) has been developed for specific extraction and simultaneous determination of Glucosylsphingosine (GlcS) and Galactosylsphingosine (GalS) in plasma by UHPLC-MS/MS with high analysis-throughput, sensitivity, accuracy, selectivity and unbiasedness. The developed and validated method was successfully applied for the simultaneous quantification of GlcS and GalS in human plasma. Image 1 • An analytical method was developed for glucosylsphingosine and galactosylsphingosine. • Multi-plex reagents M359/360/361/362/363/373/375/376/378-AMDTs were firstly reported. • Dual recognizing MIPs were prepared based on derivative template and boron affinity. • This method enabled 8-plex real samples quantitation in a single LC-MS/MS run. • The method was high-throughput, sensitive, accurate, selective and unbiased. [ABSTRACT FROM AUTHOR]

Disulfide-cyclized peptides, including the somatostatin receptor agonist octreotide, are usually resistant against collision-induced dissociation (CID) complicating their bioanalysis by MS/MS. Post-extraction reductive cleavage of the disulfide bridge of such peptides utilizing tris(2-carboxyethyl)phosphine generates the corresponding linear dithiol-peptides. This procedure enables monitoring of larger, specific fragments in CID which usually avoid matrix interference present for single amino acid iminium ions abundant in CID of the intact peptides. To assist formulation development for oral administration of the cystine-cyclized therapeutic peptide octreotide, we applied this methodology to the development of an ultra-sensitive UPLC-MS/MS assay for plasma octreotide and validated it according to FDA's and EMA's pertinent guidelines. Octreotide was extracted from plasma by fast and simple protein precipitation with acetonitrile and subsequently reduced to linear dithiol-octreotide for the monitoring of specific fragments in selected reaction monitoring. The calibrated concentration range of 10 (9.8 pM) to 20,000 pg mL−1 was linear with correlation coefficients > 0.99. Interday accuracy ranged between 100.0 and 108.9% with corresponding precision of <8.1%. The assay was successfully applied to the quantification of octreotide plasma concentrations after intravenous administration in beagle dogs. The presented procedure of disrupting the cyclic structure of cystine-cyclized peptides by reductive cleavage of the intramolecular disulfide bond enables the generation of more specific and intense fragments in CID can serve as a general methodology for the sensitive bioanalysis of cystine-bearing cyclic peptides. • Optimized methodology for the sensitive bioanalysis of cystine-cyclized peptides with MS/MS. • Fast and efficient procedure for generating linear peptide analogues after extraction. • Application to the development of an ultra-sensitive quantification assay for octreotide. [ABSTRACT FROM AUTHOR]

Ingested drugs can be taken up into nails and hairs from the bloodstream and these drugs can be stably retained over several months or more. Free edges of nails can often be used as a specimen to prove drug intake. However, the mechanism of drug uptake into the nails is unclear. Although it is presumed that there are horizontal uptake routes at the nail root and vertical uptake routes at the nail bed against the direction of growth, three-dimensional distribution analysis is required to verify this phenomenon. Herein, we first developed a method to measure the three-dimensional distribution of drugs in the nails using the combination of micro-segmentation of nails (0.2 × 1.5 × 0.06 mm size) and highly sensitive quantification of drugs by LC-MS/MS. Carbinoxamine was administered as a model compound to a subject in a single dose, and then the free edges of big toenails were collected every several weeks over a year. The three-dimensional distribution of carbinoxamine in each free edge was visualized and arranged along the collection period. Carbinoxamine was localized in the lower layer during the early collection period (up to 78 days after drug ingestion) and in the upper and middle layers later (134 days or later). The changes in the drug distribution in the nails along the collection period implied that two-way drug uptake takes place in the whole nail through both the nail bed and the nail root simultaneously. The developed analytical method could be useful to elucidate the mechanism of drug uptake into the nails. Image 1 • We first developed a method to measure 3D distribution of drugs in nails. • Big toenails were collected continuously over a year after drug administration. • Each free edge of nail was segmented at 0.2 mm long × 1.5 mm wide × 0.06 mm thick. • Drugs in each segment were quantified by highly sensitive LC-MS/MS. • 3D drug distribution in each free edge was visualized based on the segment position. [ABSTRACT FROM AUTHOR]

A sensitive, rapid, precise and specific analytical method of hydrophilic interaction ultra-performance liquid chromatography coupled with triple-quadrupole linear ion-trap tandem mass spectrometry (HILIC-UHPLC–QTRAP®/MS2) combined with a high-efficiency and easy sample preparation technology of ultrasound-assisted ionic liquid dispersive liquid–liquid microextraction (UA-IL-DLLME) was developed to investigate neurotransmitters (NTs) in mild cognitive impairment, mild dementia and moderate dementia patients' urine samples. Firstly, the UA-IL-DLLME parameters were optimized using Plackett–Burman screening and rotatable central composite design, and the main optimal conditions were obtained: ultrasound power of 307 W, ultrasound time of 4.3 min and agitation time of 4.8 min. Secondly, HILIC-UHPLC–QTRAP®/MS2 method was developed to simultaneously determine 15 underivatized NTs in urine samples. The analysis results of clinical samples showed that some NTs such as γ-aminobutyric acid (GABA), acetylcholine (Ach) and glutamic acid (Glu) presented significant differences in different dementia stages. Finally, multivariate analysis based on the combination of principal component analysis and supervised counter propagation artificial neural network was developed for comprehensive analysis of the obtained clinical data sets. As a result, GABA and Glu were simultaneously presented meaningful contribution for classification of samples, and might be considered as potential differential compounds to the urine samples from cluster patients with different dementia stages. In summary, the presented strategy of preparation, analysis and statistics might be used to investigate NTs in different clinical biological fluids. Image 1 • Ultrasound ionic liquid microextraction was used to extract polar neurotransmitters. • The ultrasound assisted microextraction method was optimized by design experiment. • Hydrophilic interaction chromatography was used to underivatized neurotransmitters. • Dementia patients' urine samples were firstly used to study neurotransmitters. • Glutamic acid and γ-aminobutyric acid were biomarkers to diagnose dementia patients. [ABSTRACT FROM AUTHOR]

An ambient ionization tandem mass spectrometric approach was developed to rapidly screen multiresidue pesticides on fruits and vegetables without sample preparation and chromatographic separation. The residual pesticides on fruits and vegetables were collected by sweeping a metallic probe across the sample surface for 2 cm. The analytes collected on the probe were desorbed and ionized in a thermal desorption electrospray ionization (TD-ESI) source, after which analyte ions were detected by a triple quadruple mass analyzer (QqQ) operated in multiple reaction monitoring (MRM) mode. With this TD-ESI/MS/MS approach, 308 pesticides were monitored, where a mixture containing 15 pesticide standards was successfully identified to demonstrate the capability of this approach to screen trace multiresidue pesticides. The approach had reasonable detection limits (<50 ppb) and reproducibility (RSD: 8.43%, n = 9) from the analysis of a benthiazole standard solution. Real samples including a tomato and bell pepper were analyzed using this TD-ESI/MS/MS approach. After TD-ESI/MS/MS analysis, the organic solvent extracts from the same samples were subjected to TD-ESI/MS/MS, gas chromatography mass spectrometry (GC-MS), and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for validation. Image 1 • TD-ESI/MS/MS was developed to rapidly screen 308 residual pesticides on fruits and vegetables within 1 min. • The samples were directly characterized without sample preparation and chromatographic separation. • The identity of pesticides on bell peppers and tomatoes revealed by TD-ESI/MS/MS was confirmed with GC-MS and LC-MS/MS. [ABSTRACT FROM AUTHOR]

There is an increasing need for rapid and on-site detection of emerging drugs of abuse. In this work, we developed a method using a miniature dual-LIT (linear ion trap) mass spectrometer recently developed with comprehensive tandem mass spectrometry analysis capability, for qualitative and quantitative analysis of multiple drugs of abuse. Paper-capillary spray cartridges were used with related workflow established to simplify overall analysis procedure. Quantitation of ketamine and methamphetamine was achieved by beam-type collision-induced dissociation on the miniature dual-LIT mass spectrometer and a linear concentration range of 100–5000 ng/mL was obtained. The system has been applied in analysis of real urine samples from individuals addicted to morphine and methamphetamine use. The changes of the ratio of cocaine to its metabolite benzoylecgonine were also explored to estimate the time of cocaine intaking. Image 1 • Miniature dual-LIT mass spectrometry system was developed for quantitative analysis. • On-site testing of drugs of abuse in urine samples. • Testing of real urine samples from morphine and methamphetamine users. • Exploration of the time of cocaine intaking by the changes of cocaine/benzoylecgonine ratios in urine. [ABSTRACT FROM AUTHOR]

An automatic online solid-phase dehydrate extraction (SPDE)-ultra-high performance supercritical fluid chromatography (UHPSFC)-MS/MS system was developed in this study, in which the automatic SPDE procedure was coupled with UHPSFC to allow UHPSFC to analyze aqueous samples directly. Moreover, a pre-column dilution strategy was employed, which focused the analytes in strong desorption solvent on the column head and helped to obtain narrow and symmetric peaks. The online SPDE-UHPSFC-MS/MS system was firstly applied to the screening of 45 prohibited substances in human urine for doping control, during which all the mechanisms and features of the online system were fully studied. The majority (91%) of the target compounds achieved weak matrix effects (80–120%), indicating that the online method was accurate and reliable thanks to the SPDE procedure and efficient UHPSFC separation. Owing to the reduction of the matrix effects, large volume injection and the pre-column dilution, the online system could achieve high sensitivity with the LODs ranging from 0.0380 ng L−1 to 1.24 μg L−1. Under the optimized conditions, the extraction recoveries of 66% target analytes were more than 50%. All the target compounds showed good linearity with linear correlation coefficients higher than 0.9928. The accuracy values of all the spiked prohibited substances were within 80.8–119.7%, while the RSDs% for the intra-/inter-day precision were within 10.8% and 15.4%. Compared with the dilute-and-shoot-ultra-high performance liquid chromatography-MS/MS method, in which the urine samples were simply diluted before analyzing, this online method was superior in sensitivity and reducing matrix effects, which demonstrated its utility in doping control. Compared with the previously reported online SPE-SFC system, the online SPDE-UHPSFC-MS/MS system showed advantages in automation, efficiency, sensitivity and chromatographic performance. In summary, the online SPDE-UHPSFC-MS/MS system is capable of analyzing complex aqueous samples. Image 1 • A novel online SPDE-UHPSFC-MS/MS system was developed. • The online system enabled the direct analysis of aqueous samples by UHPSFC. • A pre-column dilution was adapted to improve the chromatographic performance. • Advantages: high sensitivity and weak matrix interferences. [ABSTRACT FROM AUTHOR]

A robust, portable and miniature battery powered gradient capillary liquid chromatograph (total weight ∼2.7 kg, without battery ∼2.0 kg), with integrated microfluidic injection, column heating and high sensitivity low-UV absorbance detection is presented. The portable capillary chromatograph, was applied with a packed reversed-phase capillary column (100 mm × 300 μm I.D., 5 μm ODS), housed within an integrated capillary column heater controlled by a proportional-integral-derivative (PID) chip module. The system delivered retention time and peak area relative standard deviation in isocratic mode of <0.7% (n = 10) and <3.3% (n = 10), respectively, and <0.1% (n = 10) and <2.3% (n = 10) respectively, for gradient elution mode. Detection was based upon a 255 nm light-emitting diode (LED) using one of two commercial capillary flow-cell options, namely a high sensitivity 12 nL Agilent capillary z-cell (HSDC) and a 45 nL Thermo Fisher Scientific UZ-View™ flow cell (UZFC). The HSDC, housed within a 3D printed detector arrangement, gave an effective pathlength of 1.01 mm (84% of nominal pathlength) and stray light of only 0.2%. Limits of detection for four test small molecule pharmaceuticals ranged from 65 to 101 μg L−1 based upon a 316 nL injection volume, with separation efficiencies of between 18,000 and 29,700 N m−1, with sub-4 min run times. The portable capillary LC system was successfully coupled to a small footprint portable mass spectrometer (Microsaic 4500 MiD) to demonstrate compatibility and 'point-of-need' miniaturised LC-MS capability. Image 1 • A hand-portable (2.7 kg) and battery-operated liquid chromatograph is presented. • Highly repeatable gradient capillary chromatography with UV absorbance detection. • Developed UV-LED based z-cell provides linearity up to 1.6 AU with low stray light of ∼0.2%. • Demonstrated coupled with miniature single quadrupole mass spectrometer. [ABSTRACT FROM AUTHOR]

Short-chain fatty acids (SCFAs) and hydroxylated short-chain fatty acids (OH–SCFAs) are crucial intermediates related to a variety of diseases, such as bowel disease, cardiovascular disease, renal disease and cancer. A global profiling method to screen SCFAs and OH–SCFAs was developed by tagging these analytes with d 0 /d 6 -N, N-dimethyl-6,7-dihydro-5H-pyrrolo [3,4-d] pyrimidine-2-amine (d 0 /d 6 -DHPP) and using ultra-high performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-MS/MS) in parallel reaction monitoring (PRM) mode. The derivatization procedure was simple and rapid. The targeted compounds could be derivatized within 3 min under mild condition and analyzed without the need of further purification. The derivatization significantly improved the chromatographic performance and mass spectrometry response. The d 6 -DHPP tagged standards were used as internal standards, which remarkably reduced the matrix effects. The use of high resolution PRM mode made it possible to locate unknown SCFA and OH-SCFA species, and greatly reduced the false positive identification results. The developed method was successfully applied to the analysis of mouse fecal, serum, and liver tissue samples harvested from the breast cancer nude mice that had been exposed with 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47). Results showed that 40 analytes (10 SCFAs and 30 OH–SCFAs) were characterized. Semi-quantitative analysis indicated that the exposure of BDE-47 to the mice altered the SCFA and OH-SCFA metabolism, especially in the high dose group. This study provides a high-throughput method to characterize SCFAs and OH–SCFAs in mouse samples. Image 1 • A pair of novel derivatization reagents d 0 /d 6 -DHPP was reported. • The derivatization procedure was simple and rapid in mild conditions. • The application of parallel reaction monitoring reduced false positive identification results. • 2,2′,4,4′-Tetrabromodiphenyl ether significantly altered un-/hydroxylated short-chain fatty acid metabolism in nude mice. [ABSTRACT FROM AUTHOR]

A selective and sensitive method that uses automated in-tube solid-phase microextraction coupled to ultra-performance liquid chromatography–tandem mass spectrometry (in-tube SPME/UHPLC–MS/MS) was developed to determine cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC) in plasma samples. A new dummy molecularly imprinted monolithic capillary (MIP monolith) for in-tube SPME was prepared by in situ polymerization in a fused silica capillary; hydrogenated cannabidiol was employed as dummy template. Fourier Transform Infrared Spectroscopy (FTIR) confirmed that the synthesis reagents were incorporated into the polymer chain. On the basis of the microscopy images (scanning electron microscopy – SEM and transmission electron microscopy – TEM), the MIP monolithic phase presented larger pores than the non-imprinted monolithic phase (NIP monolith), as well as a skeleton comprising clusters consisting of microspheres. By optimizing the polymerization conditions, the MIP monolith specifically recognized CBD and Δ9-THC. The MIP monolith had CBD and Δ9-THC sorption capacity of 148.05 and 44.49 ng cm−3, respectively. The capillary was reused over fifty times without significant changes in its extraction efficiency. For both CBD and Δ9-THC, in-tube SPME/UHPLC–MS/MS presented linear range from 10 to 300 ng mL−1, precision with coefficient of variation (CV) values ranging from 0.2% to 19.1% (LLOQ), and accuracy with relative standard deviation (RSD) values spanning from −9.3% to 19.6% (LLOQ). The developed method was successfully applied to determine cannabinoid levels in plasma samples from volunteer patients in treatment with CBD. Image 1 • In-tube SPME-UHPLC-MS/MS method was developed to determine CBD and Δ9-THC in plasma samples. • Using hydrogenated cannabidiol as template, MIP monolith capillary was synthesized by in situ polymerization. • The method was successfully applied to determine cannabinoids in plasma samples from Parkinson's patients. [ABSTRACT FROM AUTHOR]

A streamlined analytical workflow was developed for the analysis of infant drink samples using a miniature mass spectrometry system preceded by solid-phase microextraction (SPME) and extraction nano-electrospray ionization. Potential chemical contaminants in infant drinks (milk, lactic acid bacteria beverage, and fruit juice) were extracted and enriched using a custom-made stainless-steel SPME probe, which was coated with a thin layer of polyaniline and multi-walled carbon nanotubes nanocomposites (PANI/MWCNTs) by electrochemical deposition. The resulting porous microstructure has a larger surface area and enhanced microextraction efficiency, with enrichment factors ranging from 3055 to 8695 for exemplary analytes of antibiotics, bisphenol A, and perfluorinated compounds. The enriched analytes on the electrochemically fabricated SPME probe were simultaneously desorbed and ionized within a pulled glass capillary by extraction nano-electrospray ionization. The ionized species were subjected to instrumental analysis on a miniature ion trap mass spectrometer with adequate tandem mass spectrometry capability. The developed method was optimized and validated in terms of sensitivity, linearity, repeatability, and recovery. The integrated experimental protocol combining SPME, ambient ionization, and miniature mass spectrometry is promising for rapid, on-site screening of hazardous substances in food to ensure consumer health. Image 1 • A streamlined protocol combining solid-phase microextraction and miniature mass spectrometry analysis. • A custom-made solid-phase microextraction probe coated with polyaniline and multi-walled carbon nanotubes nanocomposites. • Direct, on-site identification of potential chemical pollutants in infant drinks. [ABSTRACT FROM AUTHOR]