bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2023‒04‒16
twenty-six papers selected by
Sofia Costa

  1. Assay Drug Dev Technol. 2023 Apr;21(3): 110-125
      A highly selective, sensitive, rugged, and rapid ultra high-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) is optimized and validated for reliable quantification of atorvastatin (ATR) and its active metabolites, 2-hydroxy atorvastatin (2-ATR) and 4-hydroxy atorvastatin (4-ATR) in human plasma using atorvastatin-D5 (ATR-D5), 2-hydroxy atorvastatin-D5 (2-ATR-D5), and 4-hydroxy atorvastatin-D5 (4-ATR-D5) as deuterium-labeled internal standards (ISTDs), respectively. Isocratic mode chromatographic separation was used with a reverse-phase C18 Symmetry Shield (150 × 4.6 mm, 5.0 μm) column and a mobile phase of acetonitrile:2 mM ammonium formate (pH-3.0) [65:35%v/v] at a flow rate of 0.7 mL/min. Electrospray ionization technique with positive ion mode polarity was applied to achieve the best signal intensity and stable response. Solid-phase extraction by direct elution method was applied to extract the drugs from the plasma sample. The calibration curve range was validated from a concentration range of 0.500-250 ng/mL for ATR and 2-ATR and 0.200-20 ng/mL for 4-ATR. The within-batch and between-batch precision and accuracy were found to be consistent and reproducible for all the analytes across the validation. Extraction recoveries were >80% for all analytes and ISTDs. All peaks of analytes and the respective ISTDs were eluted within 5.2 min. In this validated method, selective multivariate analytical approaches were utilized such as best fit linearity range for different strength formulations, preventive measures for in vivo and ex vivo autodegradation of metabolites, and shorter analysis time. This validated method can be useful for challenging quantification of ATR and its active metabolites for therapeutic drug monitoring and in high-throughput clinical study sample analysis.
    Keywords:  active metabolites; atorvastatin; bioanalytical; method validation (UPLC-MS/MS); ultra high-performance liquid chromatography-liquid chromatography/mass spectrometry
  2. Bioinform Adv. 2023 ;3(1): vbad044
      Motivation: Isotopic labeling is an essential relative quantification strategy in mass spectrometry-based metabolomics, ideal for studying large cohorts by minimizing common sources of variations in quantitation. MS-DIAL is a free and popular general metabolomics platform that has isotopic labeling data processing capabilities but lacks features provided by other software specialized for isotopic labeling data analysis, such as isotopic pair validation and tabular light-to-heavy peak ratio reporting.Results: We developed Peak Pair Pruner (PPP), a standalone Python program for post-processing of MS-DIAL alignment matrixes. PPP provides these missing features and innovation including isotopic overlap subtraction based on a light-tagged pool sample as quality control. The MS-DIAL+PPP workflow for isotopic labeling-based metabolomics data processing was validated using light and heavy dansylated amino acid standard mixture and metabolite extract from human plasma.
    Availability and implementation: Peak Pair Pruner is freely available on Github: Raw MS data and .ibf files analyzed are on Metabolomics Workbench with Study ID ST002427.
    Supplementary information: Supplementary data are available at Bioinformatics Advances online.
  3. J Mass Spectrom. 2023 May;58(5): e4918
      Mass spectrometry imaging (MSI) of volatile metabolites is challenging, especially in matrix-assisted laser desorption/ionization (MALDI). Most MALDI ion sources operate in vacuum, which leads to the vaporization of volatile metabolites during analysis. In addition, tissue samples are often dried during sample preparation, leading to the loss of volatile metabolites even for other MSI techniques. On-tissue chemical derivatization can dramatically reduce the volatility of analytes. Herein, a derivatization method is proposed utilizing N,N,N-trimethyl-2-(piperazin-1-yl)ethan-1-aminium iodide to chemically modify short-chain fatty acids in chicken cecum, ileum, and jejunum tissue sections before sample preparation for MSI visualization.
    Keywords:  MALDI; mass spectrometry imaging; on-tissue chemical derivatization; sample preparation; volatile metabolites
  4. Anal Chem. 2023 Apr 12.
      Liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics provides comprehensive and quantitative profiling of metabolites in clinical investigations. The use of whole metabolome profiles is a promising strategy for disease diagnosis but technically challenging. Here, we developed an approach, namely MetImage, to encode LC-MS-based untargeted metabolomics data into multi-channel digital images. Then, the images that represent the comprehensive metabolome profiles can be employed for developing deep learning-based AI models toward clinical diagnosis. In this work, we demonstrated the application of MetImage for clinical screening of esophageal squamous cell carcinoma (ESCC) in a clinical cohort with 1104 participants. A convolutional neuronal network-based AI model was trained to distinguish ESCC screening positive and negative subjects using their serum metabolomics data. Superior performances such as sensitivity (85%), specificity (92%), and area under curve (0.95) were validated in an independent testing cohort (N = 442). Importantly, we demonstrated that our AI-based ESCC screening model is not a "black box". The encoded images reserved the characteristics of mass spectra from the raw LC-MS data; therefore, metabolite identifications in key image features were readily achieved. Altogether, MetImage is a unique approach that encodes raw LC-MS-based untargeted metabolomics data into images and facilitates the utilization of whole metabolome profiles for AI-based clinical applications with improved interpretability.
  5. Molecules. 2023 Mar 23. pii: 2900. [Epub ahead of print]28(7):
      Dopamine, adrenaline and octopamine are small polar molecules that play a vital role in regulatory systems. In this paper, phthalylglycyl chloride was proposed as a derivatization agent for octopamine, adrenaline and dopamine determination in urine for the first time. The derivatization procedure facilitated the use of reversed-phase liquid chromatography with positive electrospray ionization-high-resolution mass spectrometry. An LC-HRMS method was developed that provided quantification limits of 5 ng/mL and detection limits of 1.5 ng/mL for all analytes. The 95-97% yield of derivates was observed after a 10 min derivatization with phthalylglycyl chloride at pH 6.5 and 30 °C. The proposed method was successfully applied to the analysis of human urine samples. The obtained results were compared with those of conventional derivatization procedures with 9-fluorenyl-methoxycarbonyl chloride and dansyl chloride.
    Keywords:  adrenaline; catecholamines; derivatization; dopamine; octopamine; phthalylglycyl chloride
  6. J Chromatogr A. 2023 Apr 05. pii: S0021-9673(23)00192-9. [Epub ahead of print]1696 463966
      Mobile phase selection is of critical importance in liquid chromatography - mass spectrometry (LC-MS) based studies, since it affects retention, chromatographic selectivity, ionization, limits of detection and quantification, and linear dynamic range. Generalized LC-MS mobile phase selection criteria, suitable for a broad class of chemical compounds, do not exist thus far. Here we have performed a large-scale qualitative assessment of the effect of solvent composition used for reversed-phase LC separations on electrospray ionization (ESI) response for 240 small molecular weight drugs, representing various chemical compound classes. Of these 240 analytes 224 were detectable using ESI. The main chemical structural features affecting ESI response were found to all be surface area or surface charge-related. Mobile phase composition was found to be less differentiating, although for some compounds a pH effect was noted. Unsurprisingly, chemical structure was found to be the dominant factor for ESI response for the majority of the investigated analytes, representing about 85% of the replicating detectable complement of the sample data set. A weak correlation between ESI response and structure complexity was observed. Solvents based on isopropanol, and those containing phosphoric or di- and trifluoracetic acids, performed relatively poorly in terms of chromatographic or ESI response, whilst the best performing 'generic' LC solvents were based on methanol, acetonitrile using formic acid and ammonium acetate as buffer components, consistent with current practice in many laboratories.
    Keywords:  Bioanalysis; ESI; Electrospray ionization; LC-MS; MRM; Mass spectrometry; Method development; Multiple reaction monitoring; liquid chromatography
  7. Front Immunol. 2023 ;14 1116760
      Introduction: Immunometabolism examines the links between immune cell function and metabolism. Dysregulation of immune cell metabolism is now an established feature of innate immune cell activation. Advances in liquid chromatography mass spectrometry (LC-MS) technologies have allowed discovery of unique insights into cellular metabolomics. Here we have studied and compared different sample preparation techniques and data normalisation methods described in the literature when applied to metabolomic profiling of human monocytes.Methods: Primary monocytes stimulated with lipopolysaccharide (LPS) for four hours was used as a study model. Monocytes (n=24) were freshly isolated from whole blood and stimulated for four hours with lipopolysaccharide (LPS). A methanol-based extraction protocol was developed and metabolomic profiling carried out using a Hydrophilic Interaction Liquid Chromatography (HILIC) LC-MS method. Data analysis pipelines used both targeted and untargeted approaches, and over 40 different data normalisation techniques to account for technical and biological variation were examined. Cytokine levels in supernatants were measured by ELISA.
    Results: This method provided broad coverage of the monocyte metabolome. The most efficient and consistent normalisation method was measurement of residual protein in the metabolite fraction, which was further validated and optimised using a commercial kit. Alterations to the monocyte metabolome in response to LPS can be detected as early as four hours post stimulation. Broad and profound changes in monocyte metabolism were seen, in line with increased cytokine production. Elevated levels of amino acids and Krebs cycle metabolites were noted and decreases in aspartate and β-alanine are also reported for the first time. In the untargeted analysis, 154 metabolite entities were significantly altered compared to unstimulated cells. Pathway analysis revealed the most prominent changes occurred to (phospho-) inositol metabolism, glycolysis, and the pentose phosphate pathway.
    Discussion: These data report the emergent changes to monocyte metabolism in response to LPS, in line with reports from later time points. A number of these metabolites are reported to alter inflammatory gene expression, which may facilitate the increases in cytokine production. Further validation is needed to confirm the link between metabolic activation and upregulation of inflammatory responses.
    Keywords:  LC-MS; LPS; data normalization; metabolomics; monocyte
  8. Curr Protoc. 2023 Apr;3(4): e730
      Mycophenolic acid (MPA) is an immunosuppressant that is used as an adjunct therapy in renal, liver, and heart transplantation. Due to its narrow therapeutic range, monitoring MPA levels is essential to avoid toxicity and organ rejection. Although immunoassays are available for the determination of MPA, mass spectrometry methods are preferred due to their higher specificity. Herein, we describe a liquid chromatography tandem mass spectrometry (LC-MS/MS) method utilizing positive ionization electrospray and multiple reaction monitoring (MRM) for the quantification of MPA levels and its conjugate, MPA glucuronide (MPAG). Blood collected in a plain, EDTA, or heparin-containing tube is centrifuged to separate the serum or plasma. Proteins are precipitated using a zinc sulfate solution and acetonitrile containing deuterated internal standards (MPA-d3 and MPAG-d3). The resulting protein-free supernatant is injected into the LC-MS/MS system for analysis. The chromatography involves the use of a C18 column and ammonium acetate/water/formic acid and ammonium acetate/methanol/formic acid mobile phases. Quantification of MPA and MPAG levels is achieved by comparing the MRM peak area ratios of analytes and internal standards, consisting of specific precursor/product pairs, with those of calibrators at various concentrations. Calibration curves are constructed from the MRM peak area ratios of calibrators and internal standards versus concentration. © 2023 Wiley Periodicals LLC. Basic Protocol: Quantitation of mycophenolic acid and mycophenolic acid glucuronide in serum or plasma by LC-MS/MS.
    Keywords:  MPA; MPAG; mass spectrometry; mycophenolic acid; mycophenolic acid glucuronide; therapeutic drug monitoring
  9. J Chromatogr A. 2023 Apr 06. pii: S0021-9673(23)00202-9. [Epub ahead of print]1696 463976
      While the most sensitive LC-MS methods for oligonucleotide analysis contain ion-pairs in the mobile phase, these modifiers have been associated with instrument contamination and ion suppression. Typically, entire LC-MS systems are reserved for oligonucleotide LC-MS when using ion-pairing buffers. To overcome these limitations, numerous HILIC methods, liberated from ion-pairs, have been recently developed. Since ion-pairs play a role in analyte desorption from ESI droplets, their removal from mobile phases tend to impact method sensitivity. An effective way to recover MS sensitivity is to reduce the LC flow rate and therefore reduce ESI droplet size. With a focus on MS sensitivity, this study investigates the applicability of a microflow LC- nanoelectrospray MS platform in oligonucleotide ion-pair RP and HILIC LC-MS methods. The platform is effective and substantially increased the MS sensitivity of HILIC methods. Furthermore, LC method development for both types of separations provide insight into microflow chromatography of oligonucleotides, an under investigated chromatographic scale.
  10. Fa Yi Xue Za Zhi. 2023 Feb 25. pii: 1004-5619(2023)01-0034-06. [Epub ahead of print]39(1): 34-39
      OBJECTIVES: To establish a method for the detection of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS).METHODS: The blood samples were treated with 1-butyl-3-methylimidazolium hexafluorophosphate as an extraction solvent. The samples were extracted by ultrasound-assisted extraction and separated by ZORBAX Eclipse Plus C18, 95Å column. The mobile phase A aqueous solution containing 0.1% formic acid and 10 mmol/L ammonium acetate, and mobile phase B mixed organic solvent containing acetonitrile/methanol (Vacetonitrile∶Vmethanol=2∶3) were used for gradient elution at the flow rate of 1.00 mL/min. An electrospray ion source in positive mode was used for detection in the multiple reaction monitoring.
    RESULTS: The linearities of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples were good within the corresponding range, with correlation coefficients (r) greater than 0.995 6. The limits of detection were 3.00, 0.40 and 1.30 ng/mL, respectively. The limit of quantitation were 8.00, 1.00 and 5.00 ng/mL, respectively. The extraction recoveries ranged from 76.00% to 106.44%. The relative standard deviations of the intra-day and inter-day precisions were less than 16%. Carbamazepine and its main metabolite 10,11-dihydro-10,11-epoxycarbamazepine were detected in blood samples of death cases with a mass concentration of 2.71 μg/mL and 252.14 ng/mL, respectively.
    CONCLUSIONS: This method has high sensitivity and good selectivity, which is suitable for the detection of carbamazepine and its metabolites in blood samples, and can be used for carbamazepine-related forensic identifications.
    Keywords:  10,11-dihydro-10,11-epoxycarbamazepine; 10,11-dihydro-10-hydroxycarbamazepine; LC-MS/MS; carbamazepine; forensic toxicological analysis; metabolites
  11. Nat Methods. 2023 Apr 13.
      A substantial fraction of metabolic features remains undetermined in mass spectrometry (MS)-based metabolomics, and molecular formula annotation is the starting point for unraveling their chemical identities. Here we present bottom-up tandem MS (MS/MS) interrogation, a method for de novo formula annotation. Our approach prioritizes MS/MS-explainable formula candidates, implements machine-learned ranking and offers false discovery rate estimation. Compared with the mathematically exhaustive formula enumeration, our approach shrinks the formula candidate space by 42.8% on average. Method benchmarking on annotation accuracy was systematically carried out on reference MS/MS libraries and real metabolomics datasets. Applied on 155,321 recurrent unidentified spectra, our approach confidently annotated >5,000 novel molecular formulae absent from chemical databases. Beyond the level of individual metabolic features, we combined bottom-up MS/MS interrogation with global optimization to refine formula annotations while revealing peak interrelationships. This approach allowed the systematic annotation of 37 fatty acid amide molecules in human fecal data. All bioinformatics pipelines are available in a standalone software, BUDDY ( ).
  12. Anal Methods. 2023 Apr 14.
      A rapid and accurate ultra-performance liquid chromatography-tandem-mass spectrometry (UPLC-MS/MS) method was developed for the measurement of 19 steroid hormones in dried blood spots and to achieve the highly traceable analysis of steroid hormones in dried blood samples. In this method, a BEH C8 column and UPLC-MS/MS were used for the separation of the steroid hormones. The extraction process was simple and accurate. The distribution ranges of 19 steroid hormones in four healthy individuals (2 males and 2 females) were determined simultaneously by positive ionization mode (ESI+) and negative ionization mode (ESI-) analysis, which were recorded by multiple reaction monitoring (MRM) modes. The linearity of the standard curves of 19 steroid hormones in dried blood spots was good, and the linear correlation coefficients R2 were all ≥0.997. Meanwhile, the matrix effect of the method ranged from 87.1% to 131.3%. Across the analytical range, the inter-assay coefficient of variation (CV) was <12.07% and the intra-assay CV was <18.16%. The spiked recovery was >67.33%. The distribution ranges of 19 steroid hormones in four healthy individuals were in agreement with those in previous reports. A UPLC-MS/MS method for the simultaneous measurement of 19 steroid hormones in dried blood spots was developed to achieve traceable analysis of steroid hormones in dried blood samples.
  13. J Am Soc Mass Spectrom. 2023 Apr 13.
      The visualization of small metabolites by MALDI mass spectrometry imaging in brain tissue sections is challenging due to low detection sensitivity and high background interference. We present an on-tissue chemical derivatization MALDI mass spectrometry imaging approach for the comprehensive mapping of carboxyls and aldehydes in brain tissue sections. In this approach, the AMPP (1-(4-(aminomethyl)phenyl)pyridin-1-ium chloride) derivatization reagent is used for the covalent charge-tagging of molecules containing carboxylic acid (in the presence of peptide coupling reagents) and aldehydes. This includes free fatty acids and the associated metabolites, fatty aldehydes, dipeptides, neurotoxic reactive aldehydes, amino acids, neurotransmitters and associated metabolites, as well as tricarboxylic acid cycle metabolites. We performed sensitive ultrahigh mass resolution MALDI-MS detection and imaging of various carboxyl- and aldehyde-containing endogenous metabolites simultaneously in rodent brain tissue sections. We verified the AMPP-derivatized metabolites by tandem MS for structural elucidation. This approach allowed us to image numerous aldehydes and carboxyls, including certain metabolites which had been undetectable in brain tissue sections. We also demonstrated the application of on-tissue derivatization to carboxyls and aldehydes in coronal brain tissue sections of a nonhuman primate Parkinson's disease model. Our methodology provides a powerful tool for the sensitive, simultaneous spatial molecular imaging of numerous aldehydes and carboxylic acids during pathological states, including neurodegeneration, in brain tissue.
  14. Molecules. 2023 Mar 28. pii: 3022. [Epub ahead of print]28(7):
      The Salicornia genus has great potential in agrifood industries because of its nutritional benefits related to its high content of antioxidant compounds, including flavonoids. A nontargeted method based on reversed-phase liquid chromatography-electrospray orbitrap data-dependent MS2/MS3 and the fragment ion search (FISh) strategy was developed to screen flavonoids in Salicornia plants. An extensive study of fragmentation of a set of flavonoid standards allowed for the definition of 15 characteristic fragment ions for flagging flavonoids in the plant matrix. The nontargeted analysis was applied to Salicornia europaea species and allowed for the annotation of 25 candidate flavonoids, including 14 that had not been reported previously. Structural prediction of two unreported flavonoids and their isomeric forms was based on an advanced data processing method using an in silico approach and in-house databases compiling flavonoid-specific chemical substitution. Finally, the method developed allowed for the optimization of extraction yields of flavonoids from the plant matrix.
    Keywords:  Salicornia; flavonoids; fragment ion search; high-resolution tandem mass spectrometry; metabolomics; nontargeted screening
  15. J Pharm Biomed Anal. 2023 Apr 05. pii: S0731-7085(23)00152-8. [Epub ahead of print]230 115383
      Dried blood spot (DBS) samples have been widely used in many fields including newborn screening, with the advantages in transportation, storage and non-invasiveness. The DBS metabolomics research of neonatal congenital diseases will greatly expand the understanding of the disease. In this study, we developed a liquid chromatography-mass spectrometry-based method for neonatal metabolomics analysis of DBS. The influences of blood volume and chromatographic effects on the filter paper on metabolite levels were studied. The levels of 11.11 % metabolites were different between 75 μL and 35 μL of blood volumes used for DBS preparation. Chromatographic effects on the filter paper occurred in DBS prepared with 75 μL whole blood and 6.67 % metabolites had different MS responses when central disks were compared with outer disks. The DBS storage stability study showed that compared with - 80 °C storage, storing at 4 °C for 1 year had obvious influences on more than half metabolites. Storing at 4 °C and - 20 °C for short term (< 14 days) and - 20 °C for longer term (1 year) had less influences on amino acids, acyl-carnitines and sphingomyelins, but greater influences on partial phospholipids. Method validation showed that this method has a good repeatability, intra-day and inter-day precision and linearity. Finally, this method was applied to investigate metabolic disruptions of congenital hypothyroidism (CH), metabolic changes of CH newborns were mainly involved in amino acid metabolism and lipid metabolism.
    Keywords:  Congenital hypothyroidism; Dried blood spots; Metabolite stability; Metabolomics; Newborn screening; Storage
  16. Bioanalysis. 2023 Apr 11.
      Background: The authors present a validated method for the simultaneous quantification of asundexian (BAY 2433334) and its pharmacologically inactive major human metabolite M-10 from human plasma and its application in clinical study sample analysis. Materials & methods: Sample preparation was performed by protein precipitation followed by reverse phase HPLC and positive/negative ESI-MS/MS. Results: Assay working ranges were 0.5-500 ng/ml for asundexian and 5.0-5000 ng/ml for M-10. Validation results met the requirements of pertinent guidelines. In clinical study sample analysis, accuracy and precision acceptance criteria for analyzed quality control samples were met and incurred sample reanalysis was fulfilled. Conclusion: The method proved to be selective, specific, sufficiently sensitive, reproducible and robust for the analysis of samples obtained from clinical trials.
    Keywords:  LC–MS/MS; asundexian; clinical studies; factor XIa inhibitor; quantification; thromboembolic disorders
  17. Biomed Chromatogr. 2023 Apr 13. e5652
      Valproate and lamotrigine are commonly used as antiepileptic drugs even in pregnant and breastfeeding women. The extent and effects of drug exposure on the developing brain of the offspring are not well understood. Animal models can be utilised to investigate the transfer of substances into fetal brain with the ultimate aim of providing insights to aid clinical decisions. In the present study, a LC-MS/MS method was developed and validated for quantification of valproate (VPA), valproate-glucuronide (VPA-Gluc, a major metabolite of valproate) and lamotrigine (LTG) in rat blood plasma, cerebrospinal fluid, and brain tissue. 10 μl of sample was spiked with stable isotope-labelled internal standards and extracted by methanol. An Agilent RRHD Eclipse Plus C18 column (2.1×100 mm, 1.8 μm) was used. The MS/MS transitions were 143.1016 to 143.1016 (VPA), 319.1392 to 143.0978 (VPA-Gluc), and 256.0157 to 210.9826 (LTG). The linear ranges of VPA, VPA-Gluc and LTG were 30-250 μg/ml, 10-140 μg/ml and 0.3-1 μg/ml, respectively. The intra- and inter-day accuracy and precision, carryover, sensitivity, and recovery were evaluated according to the FDA guidance for Bioanalytical method validation. Finally, the validated method was applied to a set of experimental animal samples and produced results highly comparable to an orthogonal analytical method.
    Keywords:  LC-MS/MS; Lamotrigine; Rat plasma CSF & brain; Valproate; Valproate-glucuronide
  18. Chembiochem. 2023 Apr 14. e202300170
      Mass spectrometry-based high-throughput screening methods combine the advantages of photometric or fluorometric assays and analytical chromatography, as they are reasonably fast (throughput ≥1 sample/min) and broadly applicable, with no need for labelled substrates or products. However, the established MS-based screening approaches require specialised and expensive hardware, which limits their broad use throughout the research community. We show that a more common instrumental platform, a single-quadrupole HPLC-MS, can be used to rapidly analyse diverse biotransformations by flow-injection mass spectrometry (FIA-MS), that is, by automated infusion of samples to the ESI-MS detector without prior chromatographic separation. Common organic buffers can be employed as internal standard for quantification, and the method provides readily validated activity and selectivity information with an analytical run time of one minute per sample. We report four application examples that cover a broad range of analyte structures and concentrations (0.1-50 mM before dilution) and diverse biocatalyst preparations (crude cell lysates and whole microbial cells). Our results establish FIA-MS as a versatile and reliable alternative to more traditional methods for screening enzymatic reactions.
    Keywords:  Biocatalysis; Flow injection analysis; High-throughput screening; Mass spectrometry
  19. Gigascience. 2022 12 28. pii: giad021. [Epub ahead of print]12
      Mass spectrometry imaging (MSI), which localizes molecules in a tag-free, spatially resolved manner, is a powerful tool for the understanding of underlying biochemical mechanisms of biological phenomena. When analyzing MSI data, it is essential to delineate regions of interest (ROIs) that correspond to tissue areas of different anatomical or pathological labels. Spatial segmentation, obtained by clustering MSI pixels according to their mass spectral similarities, is a popular approach to automate ROI definition. However, how to select the number of clusters (#Clusters), which determines the granularity of segmentation, remains to be resolved, and an inappropriate #Clusters may lead to ROIs not biologically real. Here we report a multimodal fusion strategy to enable an objective and trustworthy selection of #Clusters by utilizing additional information from corresponding histology images. A deep learning-based algorithm is proposed to extract "histomorphological feature spectra" across an entire hematoxylin and eosin image. Clustering is then similarly performed to produce histology segmentation. Since ROIs originating from instrumental noise or artifacts would not be reproduced cross-modally, the consistency between histology and MSI segmentation becomes an effective measure of the biological validity of the results. So, #Clusters that maximize the consistency is deemed as most probable. We validated our strategy on mouse kidney and renal tumor specimens by producing multimodally corroborated ROIs that agreed excellently with ground truths. Downstream analysis based on the said ROIs revealed lipid molecules highly specific to tissue anatomy or pathology. Our work will greatly facilitate MSI-mediated spatial lipidomics, metabolomics, and proteomics research by providing intelligent software to automatically and reliably generate ROIs.
    Keywords:  mass spectrometry imaging; multimodal data fusion; spatial segmentation
  20. J Pharm Biomed Anal. 2023 Mar 31. pii: S0731-7085(23)00153-X. [Epub ahead of print]230 115384
      In the present work an isocratic enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the separation and quantitative determination of dextro - and levo -methorphan and their pharmacologically relevant metabolites, dextrorphan and levorphanol, respectively, in human blood samples. The separation of enantiomers of methorphan and metabolites was performed on the polysaccharide-based chiral column Lux AMP in combination with acetonitrile and 5 mM aqueous ammonium bicarbonate pH 11 in the ratio 50:50 (%, v/v) as mobile phase with the flow rate 1 mL/min. The mass spectrometer was operated in scheduled multiple reaction monitoring (MRM) mode, with four transitions for each dextromethorpan, levomethorphan, dextrorphan and dextromethorphan-d3 and two transitions for each levorphanol, levorphanol-d3 and dextrorphan-d3. Application of this method to human post-mortem blood samples confirmed cases of severe overdosing with dextromethorphan, levomethorphan, and less commonly with both.
    Keywords:  Dextromethorphan; Dextrorphan; Enantioselective analysis; Levomethorphan; Levorphanol
  21. Biomed Chromatogr. 2023 Apr 11. e5643
      A sensitive and selective liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of dual PI3K/BRD4 inhibitor SF2523 in mouse plasma. The analysis was performed on a UPLC system connected to a Shimadzu 8060 mass spectrometer by electrospray in positive multiple reaction monitoring mode. Chromatographic separation was carried out on an ACE Excel C18 column with a gradient elution containing 0.1% formic acid and methanol as the mobile phase. The linearity was conducted in the concentration range of 0.1-500 ng/mL for SF2523 in 100μL plasma. The inter- and intra-batch precision (% RSD) were both lower than 13.5 %, with the accuracy (%Bias) ranged from varied from -10.03% to 11.56%. The validated method was successfully applied to plasma protein binding and in vitro metabolism studies. SF2523 was highly bound to mouse plasma proteins (>95% bound). Utilizing mouse S9 fractions, a total of seven phase I and II metabolites were identified with hydroxylation found to be the major metabolic pathway. Metabolites identification included analysis of retention behaviors, molecular weight changes and MS/MS fragment patterns of SF2523 and the metabolites. This newly developed and validated method allows the rapid and easy determination of the SF2523 concentration with high sensitivity in low sample volume and can be applied to future pre-clinical studies.
    Keywords:  Dual PI3K/BRD4 inhibitor, LC-MS/MS; SF2523
  22. Anal Chim Acta. 2023 May 15. pii: S0003-2670(23)00352-5. [Epub ahead of print]1255 341131
      A method was developed for the analysis of four ceramide species; namely C16:0, C18:0, C24:0 and C24:1 in quantitative Dried Blood Samples (qDBS) by LC-MS/MS and validated with the aim to give prominence to an interesting application of at-home blood microsampling for health monitoring. Ceramides, being key-role metabolites implicated in regulation of diverse cellular processes have been considered as emerging biomarkers for different disease states, such as cardiovascular diseases, type 2 diabetes and others. Here, Capitainer device was utilized to provide accurate and consistent volumes of samples, ideal for accurate determinations. The method requires a 10 μL sample offering duplicate analysis by device, is quick and enables the sample collection by distance as it was proved that ceramides under study were stable at various conditions, including RT. Intra and inter-day accuracy of the determination were estimated between 87.6% - 113% and 90.6% -113%, respectively, while intra- and inter-day precision were calculated from 0.2% to 9.9% %RSD and 0.1% - 8.0% %RSD, respectively. The data acquired by ten healthy individuals indicated that circulating ceramides are at higher levels in whole blood taken from the fingertip in comparison to the reported values in plasma or serum.
    Keywords:  Blood microsampling; Dried blood spot; LC-MS/MS; qDBS
  23. Clin Chem Lab Med. 2023 Apr 11.
      OBJECTIVES: Monitoring estradiol (E2) is important for determining the onset of pubertal development as well as in the evaluation of girls with precocious puberty. However, E2 measurement remains an analytical challenge in children, who have lower circulating levels. We developed and evaluated a simple and sensitive LC-MS/MS procedure for serum E2 quantification in pediatric populations and established age- and sex-specific pediatric reference intervals.METHODS: Residual patient serum samples were used to evaluate the analytical performance of our in-house LC-MS/MS E2 assay. The evaluation included accuracy, precision, linearity, functional sensitivity (LLoQ), and method comparison. Age- and sex-specific pediatric E2 reference intervals were also established from a cohort of 405 healthy children (birth to 18 years) recruited with informed consent. Age- and sex-specific differences were assessed, and outliers were removed. Reference intervals were established using the robust method.
    RESULTS: The assay imprecision was <5.3 %. Assay linearity ranged from 13.7 to 1923.3 pmol/L. The LLoQ corresponding to a CV of 20 % was determined to be 8.9 pmol/L. Bland-Altman analysis revealed a mean bias of 29.3 pmol/L or 9.1 % between our LC-MS/MS E2 assay and an external reference laboratory measuring E2 by LC-MS/MS.
    CONCLUSIONS: Our LC-MS/MS E2 assay shows acceptable accuracy, precision, functional sensitivity (LLoQ), and linearity for E2 quantification. Our LC-MS/MS E2 assay also showed good agreement with an external reference laboratory measuring E2 by LC-MS/MS. In addition, using CALIPER samples, we established robust age- and sex-specific pediatric E2 reference intervals to improve accuracy of test result interpretation and clinical decision making.
    Keywords:  LC-MS/MS; estradiol; immunoassay; pediatric; reference intervals
  24. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Apr 01. pii: S1570-0232(23)00099-5. [Epub ahead of print]1222 123689
      Since neonicotinoid insecticides are now the most extensively used insecticides worldwide, there are increasing cases of neonicotinoid poisoning. A rapid and sensitive method was developed for the determination of ten neonicotinoid insecticides and a metabolite 6-chloronicotinic acid in human whole blood. The types and amounts of extraction solvent, salting-out agent, and adsorbent in the QuEChERS method were optimized by comparing the absolute recoveries of 11 analytes. The separation was performed on an Agilent EC18 column with the gradient elution with 0.1% formic acid in water and acetonitrile as the mobile phase. The quantification was achieved by Q Exactive orbitrap high-resolution mass spectrometry under parallel reaction monitoring scan mode. The 11 analytes showed good linearity with R2 ≥ 0.9950, LODs ranging from 0.01 μg/L to 0.30 μg/L, and LOQs from 0.05 μg/L to 1.00 μg/L. The recoveries ranged from 78.3% to 119.9% at low, medium, and high spiked concentrations of blank blood, with matrix effects ranging from 80.9% to 117.8%, inter-day RSDs from 0.7% to 6.7%, and intra-day RSDs from 2.7% to 9.8%. The method was furthermore applied to a real case of neonicotinoid insecticide poisoning to demonstrate its feasibility. The proposed method is suitable for the rapid screening of neonicotinoid insecticides in poisoned human blood in the field of forensic science, as well as monitoring of neonicotinoid insecticide residues in humans in the field of environmental safety, compensating for a lack of studies on neonicotinoid insecticide determination in biological samples.
    Keywords:  Human whole blood; Neonicotinoid insecticide; QuEChERS; UPLC-Q Exactive orbitrap high-resolution mass spectrometry
  25. J Chromatogr A. 2023 Mar 30. pii: S0021-9673(23)00184-X. [Epub ahead of print]1696 463958
      In in-situ mass spectrometry (MS), different on-tissue derivatization methods have been developed to enhance the signals of poorly ionizable primary amines. However, those chemical derivatization methods are laborious and time-consuming, and are usually limited to detection of high-abundance amino acids which suppress the reaction of low-abundance monoamine neurotransmitters and drugs. Herein, A rapid and selective photocatalytic derivatization technique for alpha-unsubstituted primary amine was developed with 5-hydroxyindole as derivatization reagent and TiO2 as photocatalyst, and was introduced into liquid microjunction surface sampling (LMJSS)-MS system as online derivatization. The results showed that the photocatalytic derivatization method largely enhanced the signals of primary amines by 5-300 fold, and were selective to alpha-unsubstituted primary amines. Thus, the suppression effects from high-abundance amino acids to the reaction of monoamine neurotransmitters and benzylamine drugs proved to be largely reduced in the new method (matrix effect>50%) comparing with those in chemical derivatization method (matrix effect<10%). In addition, the optimal pH of the derivatization reaction was measured to be 7, which indicates the mild and physiologically compatible reaction conditions. By in-situ synthesis of TiO2 monolith in the transfer capillary of the LMJSS-MS system, rapid on-line photocatalytic derivatization was achieved and completed in 5 s during the transfer of sampling extract from the flow-probe to the MS inlet. With the new photocatalytic reactive LMJSS-MS method, detection limits of three primary amines on glass slides were in the range of 0.031-0.17 ng/mm2 with acceptable linearity (r=0.9815-0.9998) and relatively high repeatability (relative standard deviations <22.1%). Finally, endogenous tyramine, serotonin, two dipeptides and one doped benzylamine drug were identified and in-situ analyzed in the mouse cerebrum by the new method with largely enhanced signals comparing with LMJSS-MS without online derivatization. The new method provides a more selective, rapid and automated way to analyze alpha-unsubstituted amine metabolites and drugs in-situ comparing with traditional methods.
    Keywords:  Liquid microjunction surface sampling; Neurotransmitters; Online derivatization; Photocatalysis; Spatially resolved mass spectrometry
  26. Chemosphere. 2023 Apr 12. pii: S0045-6535(23)00934-7. [Epub ahead of print] 138667
      Assessing the impact of human exposure to environmental toxicants is often crucial to biomonitoring the exposed dose. In this work, we report a novel fast urinary metabolites extraction (FaUMEx) technique coupled with UHPLC-MS/MS analysis for the highly sensitive and simultaneous biomonitoring of the five major urinary metabolites (thiodiglycolic acid, s-phenylmercapturic acid, t,t-muconic acid, mandelic acid, and phenyl glyoxylic acid) of common volatile organic compounds' (VOCs) exposure (vinyl chloride, benzene, styrene, and ethylbenzene) in human. FaUMEx technique comprises of two-steps, liquid-liquid microextraction was performed first in an extraction syringe using 1 mL of methanol (pH 3) as an extraction solvent and then, the extractant was passed through a clean-up syringe (pre-packed-with various sorbents including 500 mg anhydrous MgSO4, 50 mg C18, and 50 mg SiO2) to obtain the high order of matrice clean-up and preconcentration efficiency. The developed method displayed excellent linearity, and the correlation coefficients were >0.998 for all the target metabolites with detection and quantification limits of 0.02-0.24 ng mL-1 and 0.05-0.72 ng mL-1, respectively. Furthermore, the matrix effects were < ±5%, and inter and intra-day precision were <9%. Moreover, the presented method was applied and validated to real sample analysis for biomonitoring of VOC's exposure levels. The results showed that the developed FaUMEx-UHPLC-MS/MS method is fast, simple, low-cost, low-solvent consumption, high sensitivity with good accuracy and precision for five targeted urinary VOCs' metabolites. Therefore, the presented dual-syringe mode FaUMEx strategy with UHPLC-MS/MS technique can be applied to biomonitoring of various urinary metabolites to assess human exposure to environmental toxicants.
    Keywords:  Bio-monitoring; Fast urinary metabolites extraction technique; Human urine; LC-MS/MS; Volatile organic compounds' metabolites (mVOCs)