bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2023‒08‒06
38 papers selected by
Sofia Costa

  1. Adv Food Nutr Res. 2023 ;pii: S1043-4526(22)00093-6. [Epub ahead of print]105 97-172
      Lipids represent one out of three major macronutrient classes in the human diet. It is estimated to account for about 15-20% of the total dietary intake. Triacylglycerides comprise the majority of them, estimated 90-95%. Other lipid classes include free fatty acids, phospholipids, cholesterol, and plant sterols as minor components. Various methods are used for the characterization of nutritional lipids, however, lipidomics approaches become increasingly attractive for this purpose due to their wide coverage, comprehensiveness and holistic view on composition. In this chapter, analytical methodologies and workflows utilized for lipidomics profiling of food samples are outlined with focus on mass spectrometry-based assays. The chapter describes common lipid extraction protocols, the distinct instrumental mass-spectrometry based analytical platforms for data acquisition, chromatographic and ion-mobility spectrometry methods for lipid separation, briefly mentions alternative methods such as gas chromatography for fatty acid profiling and mass spectrometry imaging. Critical issues of important steps of lipidomics workflows such as structural annotation and identification, quantification and quality assurance are discussed as well. Applications reported over the period of the last 5years are summarized covering the discovery of new lipids in foodstuff, differential profiling approaches for comparing samples from different origin, species, varieties, cultivars and breeds, and for food processing quality control. Lipidomics as a powerful tool for personalized nutrition and nutritional intervention studies is briefly discussed as well. It is expected that this field is significantly growing in the near future and this chapter gives a short insight into the power of nutritional lipidomics approaches.
    Keywords:  Adulteration; Authentication; Differential lipidomics; Foodomics; High-resolution mass spectrometry; Lipid class separation; Lipid species separation; Nutritional intervention studies; Personalized nutrition; Shotgun lipidomics; Targeted lipidomics; UHPLC-MS/MS; Untargeted lipidomics
  2. Food Nutr Res. 2023 ;67
      Background: Recent studies from targeted and untargeted metabolomics have consistently revealed that diet-related metabolites, including carnitine (C0), several species of acylcarnitines (AcyCNs), amino acids, ceramides, and lysophosphatidylcholines (LPCs) may serve as potential multiple myeloma (MM) biomarkers. However, most of these approaches had some intrinsic limitations, namely low reproducibility and compromising the accuracy of the results.Objective: This study developed and validated a precise, efficient, and reliable liquid chromatography tandem mass spectrometric (LC-MS/MS) method for measuring these 28 metabolic risk factors in human serum.
    Design: This method employed isopropanol to extract the metabolites from serum, gradient elution on a hydrophilic interaction liquid chromatographic column (HILIC) for chromatographic separation, and multiple reaction monitor (MRM) mode with positive electrospray ionization (ESI) for mass spectrometric detection.
    Results: The correlation coefficients of linear response for this method were more than 0.9984. Analytical recoveries ranged from 91.3 to 106.3%, averaging 99.5%. The intra-run and total coefficients of variation were 1.1-5.9% and 2.0-9.6%, respectively. We have simultaneously determined the serological levels of C0, several subclasses of AcyCNs, amino acids, ceramides, and LPCs within 15 min for the first time.
    Conclusion: The established LC-MS/MS method was accurate, sensitive, efficient, and could be valuable in providing insights into the association between diet patterns and MM disease and added value in further clinical research.
    Keywords:  diet; liquid chromatography tandem mass spectrometry; metabolites; multiple myeloma
  3. J Chromatogr A. 2023 Jul 22. pii: S0021-9673(23)00464-8. [Epub ahead of print]1706 464239
      Cationic, anionic, zwitterionic and, partially polar metabolites are very important constituents of blood serum. Several of these metabolites underpin the core metabolism of cells (e.g., Krebs cycle, urea cycle, proteins synthesis, etc.), while others might be considered ancillary but still important to grasp the status of any organism through blood serum analysis. Due to its wide chemical diversity, modern metabolomics analysis of serum is still struggling to provide a complete and comprehensive picture of the polar metabolome, due to the limitations of each specific analytical method. In this study, two metabolomics-based analytical methods using the most successful techniques for polar compounds separation in human serum samples, namely hydrophilic interaction liquid chromatography (HILIC) and capillary electrophoresis (CE), are evaluated, both coupled to a high-resolution time-of-flight mass spectrometer via electrospray ionization (ESI-Q-TOF-MS). The performance of the two methods have been compared using five terms of comparison, three of which are specific to metabolomics, such as (1) compounds' detectability (2) Pezzatti score (Pezzatti et al. 2018), (3) intra-day precision (repeatability), (4) ease of automatic analysis of the data (through a common deconvolution alignment and extrapolation software, MS-DIAL, and (5) time & cost analysis. From this study, HILIC-MS proved to be a better tool for polar metabolome analysis, while CE-MS helped identify some interesting variables that gave it interest in completing metabolome coverage in metabolomics studies. Finally, in this framework, MS-DIAL demonstrates for the first time its ability to process CE data for metabolomics, although it is not designed for it.
    Keywords:  CE-MS; HILIC-MS; MS-DIAL; Migration time correction; Polar metabolomics analysis
  4. J Pharm Biomed Anal. 2023 Jul 23. pii: S0731-7085(23)00368-0. [Epub ahead of print]235 115599
      Short-chain fatty acids (SCFAs), the end products of gut microbial fermentation of dietary fibers and non-digestible polysaccharides, act as a link between the microbiome, immune system, and inflammatory processes. The importance of accurately quantifying SCFAs in plasma has recently emerged to understand their biological role. In this work, a sensitive and reproducible LC-MS/MS method is reported for SCFAs quantification in three different matrices such as human, rat and mouse plasma via derivatization, using as derivatizing agent O-benzylhydroxylamine (O-BHA), coupled with liquid-liquid extraction. First, the instrumental parameters of the mass spectrometer and then the chromatographic conditions were optimized using previously SCFAs derivatives synthetized and used as standards. After that, the best conditions for derivatization and extraction from plasma were studied and a series of determinations were performed on human, rat, and mouse plasma aliquots to validate the overall method (derivatization, extraction, and LC-MS/MS determination). The method showed good performance in terms of recovery (> 80%), precision (RSD <14%), accuracy (RE < ± 10%) and sensitivity (LOQ of 0.01 µM for acetic, butyric, propionic and isobutyric acid) in all plasma samples. The method thus developed and validated was applied to the quantification of major SCFAs in adult and aged mice, germ-free mice and in germ-free recipient mice subjected to fecal transplant from adult and aged donors. Results highlighted how plasma concentrations of SCFAs are correlated with age further highlighting the importance of developing a method that is reliable for the quantification of SCFAs to study their biological role.
    Keywords:  Gut-brain axis; Human, rat, and mouse plasma; LC-MS/MS; Microbiota; O-benzylhydroxylamine; Quantification; Short-chain fatty acids
  5. J Am Soc Mass Spectrom. 2023 Jul 31.
      Due to its speed, accuracy, and adaptability to various sample types, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become a popular method to identify molecular isotope profiles from biological samples. Often MALDI-MS data do not include tandem MS fragmentation data, and thus the identification of compounds in samples requires external databases so that the accurate mass of detected signals can be matched to known molecular compounds. Most relevant MALDI-MS software tools developed to confirm compound identifications are focused on small molecules (e.g., metabolites, lipids) and cannot be easily adapted to protein data due to their more complex isotopic distributions. Here, we present an R package called IsoMatchMS for the automated annotation of MALDI-MS data for multiple datatypes: intact proteins, peptides, and glycans. This tool accepts already derived molecular formulas or, for proteomics applications, can derive molecular formulas from a list of input peptides or proteins including proteins with post-translational modifications. Visualization of all matched isotopic profiles is provided in a highly accessible HTML format called a trelliscope display, which allows users to filter and sort by several parameters such as match scores and the number of peaks matched. IsoMatchMS simplifies the annotation and visualization of MALDI-MS data for downstream analyses.
    Keywords:  MALDI-MS; R package; isotope profile; mass spectrometry; trelliscope
  6. J Am Soc Mass Spectrom. 2023 Jul 31.
      Feature finding is a common way to process untargeted mass spectrometry (MS) data to obtain a list of chemicals present in a sample. Most feature finding algorithms naïvely search for patterns of unique descriptors (e.g., m/z, retention time, and mobility) and provide a list of unannotated features. There is a need for solutions in processing untargeted MS data, independent of chemical or origin, to assess features based on measurement quality with the aim of improving interpretation. Here, we report the signal response evaluation as a method by which to assess the individual features observed in untargeted MS data. The basis of this method is the ubiquitous relationship between the amount and response in all MS measurements. Three different metrics with user-defined parameters can be used to assess the monotonic or linear relationship of each feature in a dilution series or multiple injection volumes. We demonstrate this approach in metabolomics data obtained from a uniform biological matrix (NIST SRM 1950) and a variable biological matrix (murine kidney tissue). The code is provided to facilitate implementation of this data processing method.
  7. J Am Soc Mass Spectrom. 2023 Aug 04.
      Increased access to cheap and rapid mass spectrometry testing of biofluids is desirable for the analysis of disorders and diseases that may be linked to alterations in metabolite or lipid levels. The objective of this study is to establish an easily customized high-throughput workflow for the analysis of biological samples using desorption electrospray ionization-mass spectrometry (DESI-MS). The guiding principles of this workflow are the use of low-cost, open-source, and readily accessible materials with high-throughput and reproducibility. The design consists of 3 steps: (1) PARAFILM surface customization of size, shape, and depth of features on PARAFILM via 3D printed molds; (2) sample spotting via high-throughput robotics using the relatively inexpensive and open-source Opentrons platform to reduce variability and increase reliability of sample spotting; and (3) an open-source point-and-click graphical user interface (MSI.EAGLE) for data analysis via the R statistical language building on the Cardinal package. Here we describe this workflow and test optimal surface ionization characteristics by comparison of serum extracts spotted on PARAFILM and on PTFE (porous and nonporous). Untargeted analysis across three surfaces suggests that they are all suitable for ionization of a wide range of metabolites and lipids, with 3983 m/z features detected. Differential analysis of polar vs nonpolar serum extracts suggests that ∼80% of ions are desorbed preferentially from different surfaces. PARAFILM is less impacted by the interference of background ions derived from the surface. The developed system allows for a wide range of researchers to access custom surface design workflows and high-throughput analyses in a highly cost-effective manner.
  8. J Am Soc Mass Spectrom. 2023 Jul 31.
      Lipid metabolism is implicated in a variety of diseases, including cancer, cell death, and inflammation, but lipidomics has proven to be challenging due to the vast structural diversity over a narrow range of mass and polarity of lipids. Isotope labeling is often used in metabolomics studies to follow the metabolism of exogenously added labeled compounds because they can be differentiated from endogenous compounds by the mass shift associated with the label. The application of isotope labeling to lipidomics has also been explored as a method to track the metabolism of lipids in various disease states. However, it can be difficult to differentiate a single isotopically labeled lipid from the rest of the lipidome due to the variety of endogenous lipids present over the same mass range. Here we report the development of a dual-isotope deuterium labeling method to track the metabolic fate of exogenous polyunsaturated fatty acids, e.g., arachidonic acid, in the context of ferroptosis using hydrophilic interaction-ion mobility-mass spectrometry (HILIC-IM-MS). Ferroptosis is a type of cell death that is dependent on lipid peroxidation. The use of two isotope labels rather than one enables the identification of labeled species by a signature doublet peak in the resulting mass spectra. A Python-based software, D-Tracer, was developed to efficiently extract metabolites with dual-isotope labels. The labeled species were then identified with LiPydomics based on their retention times, collision cross section, and m/z values. Changes in exogenous AA incorporation in the absence and presence of a ferroptosis inducer were elucidated.
  9. Talanta. 2023 Jul 24. pii: S0039-9140(23)00732-4. [Epub ahead of print]266(Pt 1): 124981
      Comprehensive reference data for steroid hormones are lacking in rat models, particularly for early developmental stages and unconventional matrices as the liver. Therefore, we developed and validated an enzymatic, solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify a panel of 23 steroid hormones in liver and plasma from adult and neonatal rats. Our approach tackles methodological challenges, focusing on undesired byproducts associated with specific enzymatic treatment, and enables a thorough assessment of potential interferences in complex matrices by utilizing unstripped plasma and liver. We propose an optimized enzymatic hydrolysis protocol using a recombinant β-glucuronidase/sulfatase mix (BGS mix) to efficiently deconjugate steroid phase II conjugates. The streamlined sample preparation and high-throughput solid phase extraction in a 96-well plate significantly accelerate sample processing for complex matrices and alarge number of samples. We were able to achieve the necessary sensitivity for accurately measuring the target analytes, particularly estrogens, in small sample sizes of 5-20 mg of liver tissue and 100 μL of plasma. Through the analysis of liver and plasma samples from adult and neonatal rats, including both sexes, our study showed a novel set of steroid hormone reference intervals. This study provides a reliable diagnostic tool for the quantification of steroids in rat models and gives insight in liver and plasma-related steroid hormone dynamics at early developmental stages. In addition, the method covers several pathway intermediates and extend the list of steroid hormones to be investigated.
    Keywords:  Adult and neonatal rats; Liquid chromatography-tandem mass spectrometry; Recombinant enzymes; Steroid hormones
  10. Mass Spectrom (Tokyo). 2023 ;12(1): A0128
      Mass spectrometry imaging (MSI) is a well-known method for the ionization of molecules on tissue sections and the visualization of their localization. Recently, different sample preparation methods and new instruments have been used for MSI, and different molecules are becoming visible. On the other hand, although several quantification methods (q-MSI) have been proposed, there is still room for the development of a simplified procedure. Here, we have attempted to develop a reproducible and reliable quantification method using a calibration curve prepared from tissue debris of a frozen section of a sample when we trim the frozen blocks. We discuss the reproducibility of this method across different sample lots and the effect of the biological matrix (ion suppression) on our results. The quantitative performance was evaluated in terms of accuracy and relative standard deviation, and the reliability of the quantitative values obtained by matrix-assisted laser desorption/ionization-MSI was further evaluated by enzyme-linked immunosorbent assay (ELISA). Our q-MSI method for the quantification of dopamine in mouse brain tissue was found to be highly linear, accurate, and precise. The quantitative values obtained by MSI were found to be highly comparable (>85% similarity) to the results obtained by ELISA from the same tissue extracts.
    Keywords:  dopamine; mass spectrometry imaging; mimic model; q-MSI; quantitation
  11. Anal Chem. 2023 Aug 02.
      High-throughput chemical analysis of natural product mixtures lags behind developments in genome sequencing technologies and laboratory automation, leading to a disconnect between library-scale chemical and biological profiling that limits new molecule discovery. Here, we report a new orthogonal sample multiplexing strategy that can increase mass spectrometry-based profiling up to 30-fold over traditional methods. Profiled pooled samples undergo subsequent computational deconvolution to reconstruct peak lists for each sample in the set. We validated this approach using in silico experiments and demonstrated a high assignment precision (>97%) for large, pooled samples (r = 30), particularly for infrequently occurring metabolites of relevance in drug discovery applications. Requiring only 5% of the previously required MS acquisition time, this approach was repeated in a recent biological activity profiling study on 925 natural product extracts, leading to the rediscovery of all previously reported bioactive metabolites. This new method is compatible with MS data from any instrument vendor and is supported by an open-source software package:
  12. Anal Chem. 2023 Aug 04.
      The inability to identify the structures of most metabolites detected in environmental or biological samples limits the utility of nontargeted metabolomics. The most widely used analytical approaches combine mass spectrometry and machine learning methods to rank candidate structures contained in large chemical databases. Given the large chemical space typically searched, the use of additional orthogonal data may improve the identification rates and reliability. Here, we present results of combining experimental and computational mass and IR spectral data for high-throughput nontargeted chemical structure identification. Experimental MS/MS and gas-phase IR data for 148 test compounds were obtained from NIST. Candidate structures for each of the test compounds were obtained from PubChem (mean = 4444 candidate structures per test compound). Our workflow used CSI:FingerID to initially score and rank the candidate structures. The top 1000 ranked candidates were subsequently used for IR spectra prediction, scoring, and ranking using density functional theory (DFT-IR). Final ranking of the candidates was based on a composite score calculated as the average of the CSI:FingerID and DFT-IR rankings. This approach resulted in the correct identification of 88 of the 148 test compounds (59%). 129 of the 148 test compounds (87%) were ranked within the top 20 candidates. These identification rates are the highest yet reported when candidate structures are used from PubChem. Combining experimental and computational MS/MS and IR spectral data is a potentially powerful option for prioritizing candidates for final structure verification.
  13. J Am Soc Mass Spectrom. 2023 Aug 01.
      Increasing the spatial resolution of a mass spectrometry imaging (MSI) method results in a more defined heatmap of the spatial distribution of molecules across a sample, but it is also associated with the disadvantage of increased acquisition time. Decreasing the area of the region of interest to achieve shorter durations results in the loss of potentially valuable information in larger specimens. This work presents a novel MSI method to reduce the time of MSI data acquisition with variable step size imaging: nested regions of interest (nROIs). Using nROIs, a small ROI may be imaged at a higher spatial resolution while nested inside a lower-spatial-resolution peripheral ROI. This conserves the maximal spatial and chemical information generated from target regions while also decreasing the necessary acquisition time. In this work, the nROI method was characterized on mouse liver and applied to top-hat MSI of zebrafish using a novel optical train, which resulted in a significant improvement in both acquisition time and spatial detail of the zebrafish. The nROI method can be employed with any step size pairing and adapted to any method in which the acquisition time of larger high-resolution ROIs poses a practical challenge.
    Keywords:  IR-MALDESI; mass spectrometry imaging; nested region of interest; spatial resolution; zebrafish
  14. Anal Bioanal Chem. 2023 Aug 03.
      An automated microextraction by packed sorbent followed by liquid chromatography-tandem mass spectrometry (MEPS-LC-MS/MS) method was developed for the determination of four endocrine disruptors-parabens, benzophenones, and synthetic phenolic antioxidants-in wastewater samples. The method utilizes a lab-made repackable MEPS device and a multi-syringe robotic platform that provides flexibility to test small quantities (2 mg) of multiple extraction phases and enables high-throughput capabilities for efficient method development. The overall performance of the MEPS procedure, including the investigation of influencing variables and the optimization of operational parameters for the robotic platform, was comprehensively studied through univariate and multivariate experiments. Under optimized conditions, the target analytes were effectively extracted from a small sample volume of 1.5 mL, with competitive detectability and analytical confidence. The limits of detection ranged from 0.15 to 0.30 ng L-1, and the intra-day and inter-day relative standard deviations were between 3 and 21%. The method's applicability was successfully demonstrated by determining methylparaben, propylparaben, butylated hydroxyanisole, and oxybenzone in wastewater samples collected from the São Carlos (SP, Brazil) river. Overall, the developed method proved to be a fast, sensitive, reliable, and environmentally friendly analytical tool for water quality monitoring.
    Keywords:  Automated sample preparation; Liquid chromatography; Mass spectrometry; Microextraction by packed sorbent; Organic pollutants; Wastewater analysis
  15. Sci Rep. 2023 08 02. 13(1): 12554
      Tryptophan breakdown metabolites formed along the kynurenine pathway play a significant role in pregnancy and fetal development. To understand their involvement, it is crucial to quantify the levels of tryptophan (TRP), kynurenine (KYN), and kynurenic acid (KYNA) in relevant biological samples such as the placenta, fetal membranes, and umbilical cord. This study used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine TRP, KYN, and KYNA levels. The LC-MS/MS method was optimized for high sensitivity and specificity, demonstrating good reproducibility with a precision of < 10% CV and an accuracy of 85-115%. The lower limit of quantification for both TRP and KYN was 0.5 µg/ml, while for KYNA, it was 0.5 ng/mL. The method exhibited linearity within the examined range of concentrations in the homogenate, ranging from 0.5 to 30 µg/ml for TRP and KYN and from 0.5 to 25 ng/ml for KYNA. Using this method, we found significant differences in the concentrations of these substances in investigated maternal-fetal compartments. Placenta samples exhibited higher KYN and lower KYNA concentrations than the umbilical cord and fetal membrane, indicating a potentially important role for kynurenines in late pregnancy. Collectively, this finding may facilitate further research and provide inside into the involvement of the kynurenine pathway of TRP metabolism in fetal development.
  16. J Steroid Biochem Mol Biol. 2023 Jul 28. pii: S0960-0760(23)00122-X. [Epub ahead of print] 106367
      Many assays are currently being developed to measure the levels of vitamin D metabolites in various samples (such as blood, urine, and saliva). This study focused on the measurement of vitamin D metabolites in serum and urine using the NLucVDR assay system, which consists of a split-type nanoluciferase and ligand-binding domain (LBD) of the human vitamin D receptor. Blood and urine samples were collected from 23 participants to validate the NLucVDR assay. The 25(OH)D3 levels in the serum and urine determined by the NLucVDR assay showed good correlations with those determined by standard analytical methods (ECLIA for serum and LC-MS/MS for urine), with correlation coefficients of 0.923 and 0.844 for serum and urine samples, respectively. In the case of serum samples, 25(OH)D3 levels determined by the NLucVDR assay were in good agreement with those determined by ECLIA. Therefore, the NLucVDR assay is a useful tool for measuring serum 25(OH)D3 levels. The contribution of each vitamin D metabolite to the luminescence intensity obtained during the NLucVDR assay depends on its concentration and affinity for NLucVDR. Thus, the contribution of 25(OH)D3 in serum appears to be much higher than that of the other metabolites. In contrast, the 25(OH)D3 levels in the urine determined by the NLucVDR assay were more than 20-fold higher than those determined by a standard analytical method (LC-MS/MS), suggesting that some vitamin D metabolite(s) in the urine remarkably increased the luminescence intensity of the NLucVDR assay. Notably, the 25(OH)D3 concentration in the urine determined by the NLucVDR assay and the serum 25(OH)D3 concentration determined by standard analytical methods showed a significant positive correlation (r = 0.568). These results suggest that the analysis of a small amount of urine using the NLucVDR assay may be useful for predicting the serum 25(OH)D3 levels.
    Keywords:  Metabolism; NanoBiT; serum; split-luciferase; urine; vitamin D
  17. Front Plant Sci. 2023 ;14 1248781
    Keywords:  GC-MS; LC-MS; NMR; metabolomics; multivariate data analysis; natural products; plant secondary metabolites; technological advances
  18. Chemosphere. 2023 Aug 02. pii: S0045-6535(23)01957-4. [Epub ahead of print] 139690
      The use of suspect and non-target screening (SNTS) for the characterization of the chemical exposome employing human biofluids is gaining attention. Among the biofluids, urine is one of the preferred matrices since organic xenobiotics are excreted through it after metabolization. However, achieving a consensus between selectivity (i.e. preserving as many compounds as possible) and sensitivity (i.e. minimizing matrix effects by removing interferences) at the sample preparation step is challenging. Within this context, several sample preparation approaches, including solid-phase extraction (SPE), liquid-liquid extraction (LLE), salt-assisted LLE (SALLE) and dilute-and-shoot (DS) were tested to screen not only exogenous compounds in human urine but also their phase II metabolites using liquid-chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS). Additionally, enzymatic hydrolysis of phase II metabolites was evaluated. Under optimal conditions, SPE resulted in the best sample preparation approach in terms of the number of detected xenobiotics and metabolites since 97.1% of the total annotated suspects were present in samples extracted by SPE. In LLE and SALLE, pure ethyl acetate turned out to be the best extractant but fewer suspects than with SPE (80.7%) were screened. Lastly, only 52.5% of the suspects were annotated in the DS approach, showing that it could only be used to detect compounds at high concentration levels. Using pure standards, the presence of diverse xenobiotics such as parabens, industrial chemicals (benzophenone-3, caprolactam and mono-2-ethyl-5-hydroxyhexyl phthalate) and chemicals related to daily habits (caffeine, cotinine or triclosan) was confirmed. Regarding enzymatic hydrolysis, only 10 parent compounds of the 44 glucuronides were successfully annotated in the hydrolysed samples. Therefore, the screening of metabolites in non-hydrolysed samples through SNTS is the most suitable approach for exposome characterization.
    Keywords:  Exposome; Liquid-chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS); Phase II metabolites; Sample preparation; Suspect and non-target screening (SNTS); Urine
  19. Nat Commun. 2023 Aug 03. 14(1): 4653
      Untargeted metabolomics is an established approach in toxicology for characterising endogenous metabolic responses to xenobiotic exposure. Detecting the xenobiotic and its biotransformation products as part of the metabolomics analysis provides an opportunity to simultaneously gain deep insights into its fate and metabolism, and to associate the internal relative dose directly with endogenous metabolic responses. This integration of untargeted exposure and response measurements into a single assay has yet to be fully demonstrated. Here we assemble a workflow to discover and analyse pharmaceutical-related measurements from routine untargeted UHPLC-MS metabolomics datasets, derived from in vivo (rat plasma and cardiac tissue, and human plasma) and in vitro (human cardiomyocytes) studies that were principally designed to investigate endogenous metabolic responses to drug exposure. Our findings clearly demonstrate how untargeted metabolomics can discover extensive biotransformation maps, temporally-changing relative systemic exposure, and direct associations of endogenous biochemical responses to the internal dose.
  20. NMR Biomed. 2023 Aug 02. e5010
      Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for quantitative metabolomics; however, quantification of metabolites from NMR data is often a slow and tedious process requiring user input and expertise. In this study, we propose a neural network approach for rapid, automated lipid identification and quantification from NMR data. Multilayered perceptron (MLP) networks were developed with NMR spectra as the input and lipid concentrations as output. Three large synthetic datasets were generated, each with 55,000 spectra from an original 30 scans of reference standards, by using linear combinations of standards and simulating experimental-like modifications (line broadening, noise, peak shifts, baseline shifts) and common interference signals (water, tetramethylsilane, extraction solvent), and were used to train MLPs for robust prediction of lipid concentrations. The performances of MLPS were first validated on various synthetic datasets to assess the effect of incorporating different modifications on their accuracy. The MLPs were then evaluated on experimentally acquired data from complex lipid mixtures. The MLP-derived lipid concentrations showed high correlations and slopes close to unity for most of the quantified lipid metabolites in experimental mixtures compared with ground-truth concentrations. The most accurate, robust MLP was used to profile lipids in lipophilic hepatic extracts from a rat metabolomics study. The MLP lipid results analyzed by two-way ANOVA for dietary and sex differences were similar to those obtained with a conventional NMR quantification method. In conclusion, this study demonstrates the potential and feasibility of a neural network approach for improving speed and automation in NMR lipid profiling and this approach can be easily tailored to other quantitative, targeted spectroscopic analyses in academia or industry.
    Keywords:  experimental mixtures, lipid, liver samples, neural network, NMR metabolomics, synthetic data
  21. Mass Spectrom Rev. 2023 Aug 02.
      Mass spectrometry (MS) has been proven as an excellent tool in ocular drug research allowing analyzes from small samples and low concentrations. This review begins with a short introduction to eye physiology and ocular pharmacokinetics and the relevance of advancing ophthalmic treatments. The second part of the review consists of an introduction to ocular proteomics, with special emphasis on targeted absolute quantitation of membrane transporters and metabolizing enzymes. The third part of the review deals with liquid chromatography-MS (LC-MS) and MS imaging (MSI) methods used in the analysis of drugs and metabolites in ocular samples. The sensitivity and speed of LC-MS make simultaneous quantitation of various drugs and metabolites possible in minute tissue samples, even though ocular sample preparation requires careful handling. The MSI methodology is on the verge of becoming as important as LC-MS in ocular pharmacokinetic studies, since the spatial resolution has reached the level, where cell layers can be separated, and quantitation with isotope-labeled standards has come more reliable. MS will remain in the foreseeable future as the main analytical method that will progress our understanding of ocular pharmacokinetics.
    Keywords:  LC-MS/MS; MSI; drug analysis; mass spectrometry; ocular pharmacokinetics; proteomics; quantitation; spatial distribution
  22. Anal Bioanal Chem. 2023 Aug 02.
      Azaspiracids (AZAs) are a group of polyether marine algal toxins known to accumulate in shellfish, posing a risk to human health and the seafood industry. Analysis of AZAs is typically performed using LC-MS, which can suffer from matrix effects that significantly impact the accuracy of measurement results. While the use of isotopic internal standards is an effective approach to correct for these effects, isotopically labelled standards for AZAs are not currently available. In this study, 18O-labelled AZA1, AZA2, and AZA3 were prepared by reaction with H218O under acidic conditions, and the reaction kinetics and sites of incorporation were studied using LC-HRMS/MS aided by mathematical analysis of their isotope patterns. Analysis of the isotopic incorporation in AZA1 and AZA3 indicated the presence of four exchangeable oxygen atoms. Excessive isomerization occurred during preparation of 18O-labelled AZA2, suggesting a role for the 8-methyl group in the thermodynamic stability of AZAs. Neutralized mixtures of 18O-labelled AZA1 and AZA3 were found to maintain their isotopic and isomeric integrities when stored at -20 °C and were used to develop an isotope-dilution LC-MS method which was applied to reference materials of shellfish matrices containing AZAs, demonstrating high accuracy and excellent reproducibility. Preparation of isotopically labelled compounds using the isotopic exchange method, combined with the kinetic analysis, offers a feasible way to obtain isotopically labelled internal standards for a wide variety of biomolecules to support reliable quantitation.
    Keywords:  Azaspiracid; CRM-AZA-Mus; CRM-FDMT1; Isotopic labelling; LC–MS; Matrix effect; Oxygen-18
  23. J Agric Food Chem. 2023 Aug 02.
      Veterinary drug residues present in foods can pose severe health threats to the population. The present study aims to develop a high-resolution mass spectral library of 158 veterinary drugs of 16 different classes for their rapid identification in food samples through liquid chromatography-high-resolution electrospray ionization-tandem mass spectrometry (LC-HR-ESI-MS/MS). Standard drugs were pooled according to their log P values and exact masses before analysis. Spectra were collected at system automated collision energy, i.e., of 25-60 eV and four predetermined collision energies (10, 20, 30, and 40 eV) for each compound using a schedule precursor list of [M + H]+, [M + Na]+, and [M + NH4]+ ions. The utility of the developed database was checked by analyzing food samples. A total of 17 veterinary drugs based on the reference standard retention times (RTs), HR-MS spectra, and MS/MS spectra were identified in the analyzed samples. Moreover, five veterinary drugs were selected for quantitative analysis, including doxycycline hyclate, lincomycin, sulfasalazine, moxifloxacin, and diphenoxylate, using liquid chromatography-ion trap mass-spectrometry (LC-IT-MS). Concentrations of the drug were obtained to vary from 0.0805 to 0.9731 mg/kg in food samples and were found to be exceeded in most of the cases as per the maximum residue levels described by Food and Agriculture Organization (FAO)/World Health Organization (WHO). The MS data were submitted to the MetaboLights online database (MTBLS2914). This study will help in the high-throughput screening of multiclass veterinary drugs in foodstuffs.
    Keywords:  food samples; identification; quantification; spectral library; veterinary drugs
  24. J Anal Toxicol. 2023 Jul 27. pii: bkad048. [Epub ahead of print]
      Confirmation of cannabinoid use by forensic toxicology testing in urine has been traditionally focused on ∆9-tetrahydrocannabinol (∆9-THC) with analysis of its major metabolite, 11-nor-9-carboxy-∆9-THC (∆9-cTHC), in free and conjugated forms. Legalization of hemp, however, has led to the widespread production and sale of cannabidiol (CBD) derivatives with psycho-activity, including ∆8-THC and ∆10-THC isomers. The increasing availability and growing use of isomer derivatives necessitate an expanded scope of cannabinoid confirmation test protocols. We report a quantitative, isomer-selective method of cannabinoid confirmation by liquid chromatography-tandem mass spectrometry determination of parent drug isomers (∆8-THC, ∆9-THC, ∆10-THC and CBD) as well as isomeric metabolites (∆8-cTHC and ∆9-cTHC). An efficient C18 phase chromatography on 1.6-µm solid core particles was used with a step gradient for near isocratic separation of both early-eluting THC metabolite isomers and later-eluting CBD and THC isomers. A rapid method of hydrolysis, dilution and analysis was employed for the quantitative co-determination of free and conjugated analytes, using stable isotope internal standardization. Method validation is reported, along with interference assessment from a prior confirmation method. Casework experience with the isomer-selective method revealed a 14% prevalence of ∆8-cTHC positive cases with a pattern of concomitant ∆8-THC and ∆9-THC use. A comparison of ∆8-cTHC and ∆9-cTHC phase two metabolism is also reported.
  25. Clin Chem Lab Med. 2023 Aug 04.
      OBJECTIVES: To develop a sensitive liquid chromatography-tandem mass spectrometry method capable of measuring serum methotrexate in patients with rheumatoid arthritis to assess adherence to drug treatment.METHODS: Isotopically labelled internal standard and deionised water were added to sample prior to solid phase extraction using a Waters Oasis Max ion-exchange 96-well plate. Following extraction, samples were analysed by LC-MS/MS on a TQS-micro mass spectrometer.
    RESULTS: Mean recovery was 107 % for four different concentrations of methotrexate spiked into seven patient samples, whilst post extraction spiking gave a mean recovery of 100 %. Between-batch and within-batch CVs were ≤6 % at three different concentrations of methotrexate in fresh frozen plasma. Mean bias was <5 % for between-batch and within batch analysis at three different weighed in concentrations of methotrexate certified reference material. The lower limit of quantification of the assay was 0.1 nmol/L with linearity up to approximately 100 nmol/L. Dilution linearity studies were used to validate the dilution of patient samples prior to analysis. There was no significant interference in the method from lipaemia, haemolysis or icterus.
    CONCLUSIONS: A sensitive LC-MS/MS assay for methotrexate has been developed and validated. The method has been used to measure methotrexate adherence in patient samples from clinical trials and could be used in future research to assess the ability of the assay as a biofeedback intervention to improve adherence to methotrexate therapy.
    Keywords:  adherence; mass spectrometry; methotrexate; rheumatoid arthritis
  26. Anal Bioanal Chem. 2023 Aug 03.
      Antimicrobial resistance is a major threat to human health as resistant pathogens spread globally, and the development of new antimicrobials is slow. Since many antimicrobials function by targeting cell wall and membrane components, high-throughput lipidomics for bacterial phenotyping is of high interest for researchers to unveil lipid-mediated pathways when dealing with a large number of lab-selected or clinical strains. However, current practice for lipidomic analysis requires the cultivation of bacteria on a large scale, which does not replicate the growth conditions for high-throughput bioassays that are normally carried out in 96-well plates, such as susceptibility tests, growth curve measurements, and biofilm quantitation. Analysis of bacteria grown under the same condition as other bioassays would better inform the differences in susceptibility and other biological metrics. In this work, a high-throughput method for cultivation and lipidomic analysis of antimicrobial-resistant bacteria was developed for standard 96-well plates exemplified by methicillin-resistant Staphylococcus aureus (MRSA). By combining a 30-mm liquid chromatography (LC) column with ion mobility (IM) separation, elution time could be dramatically shortened to 3.6 min for a single LC run without losing major lipid features. Peak capacity was largely rescued by the addition of the IM dimension. Through multi-linear calibration, the deviation of retention time can be limited to within 5%, making database-based automatic lipid identification feasible. This high-throughput method was further validated by characterizing the lipidomic phenotypes of antimicrobial-resistant mutants derived from the MRSA strain, W308, grown in a 96-well plate.
    Keywords:  Antimicrobial resistance; Bacteria; High throughput; Ion mobility-mass spectrometry; Lipidomics
  27. Anal Sci. 2023 Aug 01.
      The combination of laser ablation and inductively coupled plasma mass spectrometry (LA-ICP-MS) offers a powerful tool for directly analyzing solid samples. However, LA-ICP-MS has a limitation in quantitative analyses owing to a requirement for matrix-matched standard materials. In this study, we have developed a sample preparation method that facilitates quantitative analyses by LA-ICP-MS. The sample powder is dispersed in a liquid resin and film-like samples are prepared from the resulting paste by a screen-printing technique. The sample includes the analyte spiked with internal standards and is prepared by mixing standard solutions in the sample paste. Because all reagents except for the sample powder are liquids, homogeneous samples can be easily obtained. The internal standard and concentration of the spiked analyte can be tailored for each sample, which is a requirement for accurate quantitative analyses. The amount of sample and concentration of the spiked analyte are controlled against an internal standard, enabling internal standardization without the need to have an element of known concentration in the sample. The accuracy of this method was evaluated by analyzing impurity elements in TiO2 powder; however, it is expected that other materials could also be analyzed. The versatility and flexibility of this method suggest great potential for quantitative analyses by LA-ICP-MS, for which reliable matrix-matched standard materials are required.
    Keywords:  ICP-MS; Inductively coupled plasma mass spectrometry; LA; Laser ablation; Screen printing
  28. Curr Protoc. 2023 Aug;3(8): e862
      This protocol describes a high-throughput absolute quantification protocol for the aromatic essential amino acid, tryptophan (Trp). This procedure consists of a milligram-scale alkaline hydrolysis followed by an absolute quantification step using a multiple reaction monitoring tandem mass spectrometric (LC-MS/MS) detection method. The approach facilitates the analysis of a few hundred samples per week by using a 96-well plate extraction setup. Importantly, the method uses only ∼4 mg of tissue per sample and uses the common alkaline hydrolysis protocol, followed by water extraction that includes L-Trp-d5 as an internal standard to enable the quantification of the absolute level of the bound Trp with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples for Arabidopsis thaliana, Glycine max, and Zea mays but could be applied to other plant tissues. © 2023 Wiley Periodicals LLC. Basic Protocol: Analysis of protein-bound tryptophan from seeds.
    Keywords:  LC-MS/MS; alkaline hydrolysis; amino acids; high-throughput method; seeds; tryptophan
  29. Anal Chem. 2023 Aug 02.
      Proton-transfer-reaction mass spectrometry (PTR-MS) is widely used for measuring organic trace gases in air. In traditional PTR-MS, both nonpolar and polar analytes are ionized with unit efficiency, as predicted from ion-molecule collision theories. This well-defined ion chemistry allows for direct quantification of analytes without prior calibration and therefore is an important characteristic of PTR-MS. In an effort to further increase the sensitivity, recently developed ultrahigh sensitivity chemical ionization mass spectrometry (CIMS) analyzers have, however, been reported to have sacrificed unit ionization efficiency for selected analytes or classes of analytes. We herein report on the development of a novel ultrasensitive PTR-MS instrument, the FUSION PTR-TOF 10k, which exhibits the same universal unit response as conventional PTR-MS analyzers. The core component of this analyzer is the newly designed FUSION ion-molecule reactor, which is a stack of concentric ring electrodes generating a static longitudinal electric field superimposed by a focusing transversal radiofrequency (RF) field. The FUSION PTR-TOF 10k instrument is equipped with an improved ion source, capable of switching between different reagent ions (H3O+, O2+, NO+, NH4+) in less than one second. The improved time-of-flight mass spectrometer analyzes m/z signals with a mass resolution in the 10000-15000 range. FUSION PTR-TOF 10k achieves sensitivities up to 80000 cps ppbV-1 and detection limits down to 0.5 pptV for a 1 s measurement time. We show time-series of naphthalene and 13C-napthalene as measured in ambient air in Innsbruck for demonstrating the sub-pptV detection capability of this novel FUSION PTR-TOF 10k.
  30. Anal Chem. 2023 Aug 03.
      Lung cancer (LC) has the highest mortality rate among various cancer diseases. Developing an early screening method for LC with high classification accuracy is essential. Herein, 2-hydrazinoquinoline (2-HQ) is utilized as a dual-mode reactive matrix for metabolic fingerprint analysis and LC screening via matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Metabolites in both positive mode and negative mode can be detected using 2-HQ as the matrix, and derivative analysis of aldehyde and ketone compounds can be achieved simultaneously. Hundreds of serum and urine samples from LC patients and healthy volunteers were analyzed. Combined with machine learning, LC patients and healthy volunteers were successfully distinguished with a high area under the curve value (0.996 for blind serum samples and 0.938 for urine). The MS signal was identified for metabolic profiling, and dysregulated metabolites of the LC group were analyzed. The above results showed that this method has great potential for rapid screening of LC.
  31. Clin Chem Lab Med. 2023 Aug 07.
      OBJECTIVES: In laboratory medicine, external quality assessment (EQA) schemes have become versatile tools for detecting analytical flaws. However, EQA schemes are lacking for pediatric sex steroid levels. We aimed to investigate the suitability of different estradiol and testosterone immunoassays in a pediatric setting in comparison with clinical liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays.METHODS: The study was conducted by staff and the advisory group on endocrinology at Equalis, the Swedish provider of EQA schemes for laboratory medicine. The test material consisted of five pooled serum samples from children who were either prepubertal or in puberty. Clinical laboratories enrolled in Equalis EQA schemes for estradiol and testosterone were invited to participate, as were clinical laboratories using LC-MS/MS-assays. Samples were analyzed by either routine immunoassays (n=18) or in-house LC-MS/MS assays (n=3).
    RESULTS: For estradiol, LC-MS/MS assays showed a high degree of conformity with interlaboratory coefficients of variation (CV) below 24.2 %. Reported levels were between 4.9 ± 1.2 and 33.9 ± 1.6 pmol/L (group mean ± standard deviation). The direct immunoassays had lower precision; their CVs were up to 81.4 %. Reported concentrations were between 25.3 ± 18.1 and 45.7 ± 19.4 pmol/L, an overestimation compared to LC-MS/MS. Testosterone LC-MS/MS also showed a high degree of conformity, CVs were below 13.4 %, and reported concentrations were from 0.06 ± 0.00 to 1.00 ± 0.11 nmol/L. The direct immunoassays had a larger discrepancy between results; CVs were up to 95.8 %. Concentrations were between 0.12 ± 0.11 and 0.85 ± 0.23 nmol/L.
    CONCLUSIONS: For the safe diagnosis and determination of sex steroids in children, analysis with mass spectrometry-based methods is recommended.
    Keywords:  children; estradiol; immunoassays; mass spectrometry; puberty; testosterone
  32. Anal Chim Acta. 2023 Sep 22. pii: S0003-2670(23)00798-5. [Epub ahead of print]1275 341577
      Volatile phenols possess "smoky, spicy" aromas and are routinely measured in grapes, wines and other foodstuffs for quality control. Routine analyses of volatile phenols rely on gas chromatography - mass spectrometry (GC-MS), but slow throughput of GC-MS can cause challenges during times of surge demand, i.e. following 'smoke taint' events involving forest fires near vineyards. Parallel extraction of headspace volatiles onto sorbent sheets (HS-SPMESH) followed by direct analysis in real time mass spectrometry (DART-MS) is a rapid alternative to conventional GC-MS approaches. However, HS-SPMESH extraction is poorly suited for lower volatility odorants, including volatile phenols. This work reports development and validation of an HS-SPMESH-DART-MS approach for five volatile phenols (4-ethylphenol, 4-ethylguiacol, guaiacol, 4-methylguaiacol, and cresols). Prior to HS-SPMESH extraction, volatile phenols were acetylated to facilitate their extraction. A unique feature of this work was the use of d6-Ac2O as a derivatizing agent to overcome issues with isobaric interferences inherent to chromatography-free MS techniques. The use of alkaline conditions during derivatization resulted in cumulative measurement of both free and bound forms of volatile phenols. The validated HS-SPMESH-DART-MS method achieved a throughput of 24 samples in ∼60 min (including derivatization and extraction time) with low limits of detection (<1 μg L-1) and good repeatability (3-6% RSD) in grape and wine matrices. Validation experiments with smoke-tainted grape samples indicated good correlation between total (free + bound) volatile phenols measured by HS-SPMESH-DART-MS and a gold standard GC-MS method.
    Keywords:  Acetylation; Ambient-ionization; Smoke taint; Volatile phenols; Wine
  33. Se Pu. 2023 Aug;41(8): 673-682
      Malachite green (MG) and its metabolite, leucomalachite green (LMG), exert toxic effects on the human body. The use of these dyes is illegal, but they are still detected in aquatic products. Freshwater fish are aquatic products with the high non-qualified rates. Therefore, the sensitive screening of MG and LMG in freshwater fish is of great importance to ensure the safety of aquatic products. Owing to the low contents of MG and LMG in fish and the complex matrix of actual samples, sample preparation is required before detection to purify impurities and enrich the target compounds. Graphite carbon nitride (GCN), a polymer material composed of C, N, and H, has good chemical and thermal stability, a large specific surface area, and a large number of active sites. It has a wide range of application prospects in adsorption and can be used in food safety testing when compounded with Fe3O4 to form magnetic graphite carbon nitride (MGCN). In this study, sulfonated magnetic graphite carbon nitride (S-MGCN) was prepared by further functionalizing MGCN with sulfonic acid. After characterization by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and vibrating sample magnetometry (VSM), a magnetic solid-phase extraction (MSPE) method based on S-MGCN was established to extract MG and LMG from freshwater fish. The targets were screened using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Following sulfonic acid functionalization, S-MGCN showed increased electrostatic interactions based on the MGCN adsorption mechanism, which includes hydrogen bonds and π-π interactions; thus, its adsorption efficiency was significantly improved. The matrix effects were -42.21% and -33.77% before functionalization, -11.40% and -7.84% after functionalization, thus confirming that S-MGCN has significant matrix removal ability. Given that S-MGCN demonstrated excellent efficiency as an MSPE adsorbent, the adsorption conditions for S-MGCN were optimized. The optimal conditions were as follows: adsorbent dosage, 15 mg; adsorption time, 2 min; solution pH, 5; and ionic strength, not adjusted. Under these conditions, the adsorption efficiency of S-MGCN could reach 94.2%. Different organic solvents were used to elute adsorbed MG and LMG, and the desorption efficiency peaked when 1%(v/v) ammonia acetonitrile was used as the elution solvent. The elution volume was also optimized, and a maximum desorption efficiency of 93.2% was obtained when 1 mL of 1%(v/v) ammonia acetonitrile was added to S-MGCN. The limits of detection (LODs) and quantification (LOQs) of the two targets were determined at signal-to-noise ratios (S/N) of 3 and 10, respectively. The LODs and LOQs were 0.075 μg/kg and 0.25 μg/kg, respectively. The linear ranges of the two target compounds were 0.25-20.0 μg/kg with correlation coefficients (r) greater than 0.998. To assess accuracy and precision, we prepared spiked samples at three levels (low, medium, and high) with six parallel samples per level (n=6). The recoveries ranged from 88.8% to 105.9%. The intra- and inter-day relative standard deviations were 5.4%-13.7% (n=6) and 3.3%-11.1% (n=3), respectively. Compared with the national standard method, the proposed method features simpler sample pretreatment procedures, less use of organic reagents (5 mL), and a shorter extraction time (2 min); moreover, the method does not require complicated elution steps, and the eluent can be directly analyzed by UPLC-MS/MS. The test results of actual samples were consistent with those obtained via the national standard method, thus confirming the practical feasibility of the developed method. The proposed MSPE method based on S-MGCN is an efficient and environmentally friendly method that could provide a new methodological reference for the sensitive screening of MG and LMG in actual samples.
    Keywords:  leucomalachite green (LMG); magnetic solid-phase extraction(MSPE); malachite green (MG); sulfonated magnetic graphite carbon nitride (S-MGCN); ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)
  34. Front Nutr. 2023 ;10 1226530
      Background: The misuse of animal-derived stimulants in food is becoming increasingly common, and mass spectrometry (MS) is used extensively for their detection and analysis. There is a growing demand for abused-substances detection, highlighting the need for systematic studies on the advantages of MS-based methods in detecting animal-derived stimulants.Objective: We reviewed the application of chromatography-mass spectrometry to the screening and detection of food stimulants of animal origin. Specifically, we analyzed four common animal sources of synthetic steroids, β-receptor agonists, zearalenol (ZAL), and glucocorticoids. We also explored the potential of using chromatography-mass spectrometry to detect and analyze animal-derived foods.
    Methods: We searched and screened the Web of Science and Google Scholar databases until April 2023. Our inclusion criteria included a publication year within the last 5 years, publication language of English, and the research fields of food analysis, environmental chemistry, and polymer science. Our keywords were "mass spectrometry," "anabolic androgenic steroids," "β-2agonists," "glucocorticoids," "zearalenone," and "doping."
    Results: Although traditional techniques such as thin-layer chromatography and enzyme-linked immunoassays are simple, fast, and suitable for the initial screening of bulk products, they are limited by their relatively high detection limits. Among the methods based on MS, gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry are the most widely used for detecting food doping agents of animal origin. However, a sensitive method with high repeatability and a short analysis time for a large number of samples is still required. Advances in MS have enabled the detection of extremely low concentrations of these substances. Combining different techniques, such as high-resolution mass spectrometry, ultra-high performance liquid chromatography-tandem mass spectrometry, gas chromatography-combustion-isotope ratio mass spectrometry, ultra-high performance liquid chromatography-high resolution mass spectrometry, and two-dimensional chromatography, offers significant advantages for detecting trace illicit drugs in animal-derived foods. Due to advances in assay technology and sample preparation methods, sample collection and storage methods such as dried blood spots, dried urine spots, and volumetric absorptive microsampling are increasingly accepted because of their increased stability and cost-effectiveness.
    Significance: MS significantly improves the efficiency of detecting doping agents of animal origin. With the continuous development of MS technology, its application in the fields of doping detection and the analysis of doping agents of animal origin is expected to become more extensive.
    Keywords:  animal-derived food; chromatography-mass spectrometry; glucocorticoids; synthetic steroids; zearalenol; β-agonists
  35. Anal Methods. 2023 Aug 03.
      The quantification capability of high-resolution mass spectrometry (HRMS) has received increasing interest from analysts. In this study, we present a method for analyzing 37 glucocorticoids in chicken muscle using UHPLC-Q-Orbitrap MS with parallel reaction monitoring (PRM). The analytes were extracted using acetonitrile (ACN) containing 0.1% formic acid and subjected to commercial PRiME HLB solid-phase extraction (SPE) cartridge clean-up. Under optimized conditions, the analytes were separated on an analytical column and subsequently detected using a high-resolution hybrid quadrupole/Orbitrap mass spectrometer coupled with PRM scan mode. The Q-Orbitrap with PRM exhibited remarkable sensitivity, with limits of quantification (LOQs) ranging from 0.08 μg kg-1 to 7.59 μg kg-1. To validate the method, we conducted intra- and inter-day tests using a blank matrix sample at different spiking levels. The achieved results demonstrated satisfactory recovery values (74.1-97.5%) and precise results (RSDs < 15%) for all the studied analytes. In application, we found dexamethasone with 6.5 μg kg-1 and fluorometholone with 3.9 μg kg-1 in two chicken samples. These findings suggest that the UHPLC-Q-Orbitrap system, in conjunction with the SPE sample preparation method, has great potential as a routine quantification approach for multiple glucocorticoid residues in chicken samples.
  36. J Pharm Biomed Anal. 2023 Jul 28. pii: S0731-7085(23)00359-X. [Epub ahead of print]235 115590
      Bevacizumab is a humanized monoclonal antibody used in the treatment of advanced colorectal and non-small cell lung cancer. Our main aim was to establish a simple, economical, and high efficiency liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantifying the content of bevacizumab in various biological fluids (rat, cynomolgus monkey, and human serum). A surrogate peptide of bevacizumab, specifically FTFSLDTSK, was generated through trypsin hydrolysis, and quantified using an isotopically labeled peptide containing two amino acids, FTFSLDTSK[13C6, 15N2]ST, as an internal standard to correct for variations introduced during the enzymatic hydrolysis process and any mass spectrometry variabilities. The pre-treatment process included denaturation, disulfide bond reduction and alkylation, trypsin hydrolysis, and termination of the reaction, with a total duration of approximately 2.5-3 h. The results of the methodological validation showed that the linear range in three different biological matrices was 0.2 µg/mL to300 µg/mL, with an LLOQ of 0.2 µg/mL. The precision and accuracy of the measurements met the required standards. The validated LC-MS/MS method was used to conduct pharmacokinetic analysis in rats administered bevacizumab at a dose of 10 mg/kg intravenously.
    Keywords:  Bevacizumab; LC-MS/MS; Monoclonal antibody; Pharmacokinetics; Serum; Surrogate peptide
  37. Phytochem Anal. 2023 Aug 01.
      INTRODUCTION: Many secondary metabolites isolated from plants have been described in the literature owing to their important biological properties and possible pharmacological applications. However, the identification of compounds present in complex plant extracts has remained a great scientific challenge, is often laborious, and requires a long research time with high financial cost.OBJECTIVES: The aim of this study was to develop a method that allows the identification of secondary metabolites in plant extracts with a high degree of confidence in a short period of time.
    MATERIAL AND METHODS: In this study, an ethanolic extract of Coffea arabica leaves was used to validate the proposed method. Countercurrent chromatography was chosen as the initial step for extraction fractionation using gradient elution. Resulting fractions presented a variation of compounds concentrations, allowing for statistical total correlation spectroscopy (STOCSY) calculations between liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) and NMR across fractions.
    RESULTS: The proposed method allowed the identification of 57 compounds. Of the annotated compounds, 20 were previously described in the literature for the species and 37 were reported for the first time. Among the inedited compounds, we identified flavonoids, alkaloids, phenolic acids, coumarins, and terpenes.
    CONCLUSION: The proposed method presents itself as a valid alternative for the study of complex extracts in an effective, fast, and reliable way that can be reproduced in the study of other extracts.
    Keywords:  Coffea arabica; LC-HRMS/MS; NMR; complex mixtures; countercurrent chromatography; data fusion
  38. Se Pu. 2023 Aug;41(8): 722-729
      Sophorolipids are secondary metabolites produced during fermentation by nonpathogenic yeasts. These molecules are amphiphilic and consist of a hydrophilic sophora sugar moiety and a hydrophobic hydroxylated fatty acid. Based on their degree of esterification, sophorolipids can be divided into the acid and lactone types. Sophorolipids are highly promising biosurfactants with good antibacterial, antiviral, and other biological activities. Moreover, they are characterized by mildness, low toxicity, and environmental friendliness. However, their composition is quite complex, and effective methods for their quality evaluation are lacking. Since sophorolipids do not absorb ultraviolet (UV) light, common UV detectors are unsuitable for fingerprint establishment. In this study, we first selected a charged aerosol detector (CAD) to establish the ultra-high performance liquid chromatography (UHPLC) fingerprint of sophorolipids. The detector had high sensitivity, good reproducibility, and excellent suitability for the detection of substances with no or weak ultraviolet absorption. We then evaluated the similarities between 17 batches of sophorolipid samples. The samples were extracted by ultrasound for 10 min in 80% ethanol aqueous solution at a liquid-solid ratio of 10∶1 (mL/g) and then separated on a Thermo Fisher Scientific Hypersil Gold chromatographic column (150 mm×2.1 mm, 1.9 μm). Separation was performed using acetonitrile-0.01% (v/v) formic acid aqueous solution as the mobile phase via gradient elution. The flow rate was 0.2 mL/min, and the column temperature was 40 ℃. The CAD was used under the following conditions: power function of 1.0, data rate of 5 Hz, filter constant of 3.6, and evaporation temperature of 45 ℃. The chromatograms and retention times of the sophorolipids were compared, and 16 common peaks with strong responses, good resolutions, and stable retention times were selected as characteristic peaks. Oleic acid was chosen as the reference peak because it achieved good separation and a strong chromatographic response in all batches of samples. UHPLC-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) was used to identify chromatographic peaks in the sophorolipid fingerprints. The results were combined with the retention time rule of the sophorolipids, leading to their identification based on matching with the results of the primary database, the precise relative molecular mass and fragmentation rule of secondary fragments, a self-built database, and the PubChem database. Sixteen compounds were identified, including eight acid sophorolipids, six lactone sophorolipids, and two aliphatic acids. The results of precision, repeatability, and 24 h stability tests indicated that the relative standard deviations (RSDs) of the retention times and peak areas of the 15 characteristic peaks relative to the control peak (oleic acid) were less than 3.0% (n=6). Seventeen batches of sophorolipid samples were analyzed, and the similarity values of all fingerprints were found to be 0.965 or higher. Little differences in chemical composition were observed among the different batches of sophorolipid samples, and the quality of the sophorolipids was relatively consistent. The fingerprint established in this study is stable and reliable; it can be used for the quality evaluation of sophorolipids and lays a solid foundation for future research on production technology and the development and utilization of sophorolipids. The successful application of a universal CAD to the fingerprint establishment of sophorolipids also provides a reliable solution for the fingerprint establishment of substances with no or weak ultraviolet absorption.
    Keywords:  charged aerosol detector (CAD); fingerprint; ultra-high performance liquid chromatography (UHPLC)