bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒02‒27
forty-one papers selected by
Sofia Costa
Icahn School of Medicine at Mount Sinai

  1. Metabolites. 2022 Feb 09. pii: 165. [Epub ahead of print]12(2):
      Polar hydrophilic metabolites have been identified as important actors in many biochemical pathways. Despite continuous improvement and refinement of hydrophilic interaction liquid chromatography (HILIC) platforms, its application in global polar metabolomics has been underutilized. In this study, we aimed to systematically evaluate polar stationary phases for untargeted metabolomics by using HILIC columns (neutral and zwitterionic) that have been exploited widely in targeted approaches. To do so, high-resolution mass spectrometry was applied to thoroughly investigate selectivity, repeatability and matrix effect at three pH conditions for 9 classes of polar compounds using 54 authentic standards and plasma matrix. The column performance for utilization in untargeted metabolomics was assessed using plasma samples with diverse phenotypes. Our results indicate that the ZIC-c HILIC column operated at neutral pH exhibited several advantages, including superior performance for different classes of compounds, better isomer separation, repeatability and high metabolic coverage. Regardless of the column type, the retention of inorganic ions in plasma leads to extensive adduct formation and co-elution with analytes, which results in ion-suppression as part of the overall plasma matrix effect. In ZIC-c HILIC, the sodium chloride ion effect was particularly observed for amino acids and amine classes. Successful performance of HILIC for separation of plasma samples with different phenotypes highlights this mode of separation as a valuable approach in global profiling of plasma sample and discovering the metabolic changes associated with health and disease.
    Keywords:  HILIC; liquid chromatography; plasma metabolomics; polar metabolites; untargeted metabolomics
  2. Metabolites. 2022 Feb 02. pii: 137. [Epub ahead of print]12(2):
      LC-MS-based untargeted metabolomics is heavily dependent on algorithms for automated peak detection and data preprocessing due to the complexity and size of the raw data generated. These algorithms are generally designed to be as inclusive as possible in order to minimize the number of missed peaks. This is known to result in an abundance of false positive peaks that further complicate downstream data processing and analysis. As a consequence, considerable effort is spent identifying features of interest that might represent peak detection artifacts. Here, we present the CPC algorithm, which allows automated characterization of detected peaks with subsequent filtering of low quality peaks using quality criteria familiar to analytical chemists. We provide a thorough description of the methods in addition to applying the algorithms to authentic metabolomics data. In the example presented, the algorithm removed about 35% of the peaks detected by XCMS, a majority of which exhibited a low signal-to-noise ratio. The algorithm is made available as an R-package and can be fully integrated into a standard XCMS workflow.
    Keywords:  XCMS; algorithm; data processing; data quality; false peaks; metabolomics; peak characterization; peak detection; peak filtering; untargeted
  3. Anal Methods. 2022 Feb 23.
      Carboxylic acids are crucial metabolites in the tricarboxylic acid (TCA) cycle and thus participate in central carbon metabolism (CCM). Research dependent on the analysis of metabolites involved in central carbon metabolism requires fast separation and sensitive detection of carboxylic acids using liquid chromatography-mass spectrometry (LC-MS). However, successful separation of all carboxylic acids from the TCA cycle by liquid chromatography remains a challenging task because of their high polarity and thus low retention on the conventional reversed-phase columns. In this study, we tested a reversed-phase/anion exchange mixed-mode stationary phase (Waters BEH C18 AX) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We developed and optimized a method that enables a 10 minute separation of all carboxylic acids from the TCA cycle and lactic acid without prior derivatization or addition of ion-pair reagents in the mobile phase. The developed method was validated for quantification of 8 acids in murine brown preadipocytes, 5 acids in human plasma and 6 acids in Arabidopsis thaliana leaves with limits of quantification ranging from 0.1 μM for malic acid to 10 μM for isocitric acid. Moreover, the mixed-mode chromatography enabled untargeted screening of medium- to long-chain fatty acids in murine brown preadipocytes, Arabidopsis thaliana, and human plasma, where 23 fatty acids were identified by using liquid chromatography with high-resolution mass spectrometry (HRMS).
  4. Metabolites. 2022 Feb 11. pii: 173. [Epub ahead of print]12(2):
      Liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics experiments have become increasingly popular because of the wide range of metabolites that can be analyzed and the possibility to measure novel compounds. LC-MS instrumentation and analysis conditions can differ substantially among laboratories and experiments, thus resulting in non-standardized datasets demanding customized annotation workflows. We present an ecosystem of R packages, centered around the MetaboCoreUtils, MetaboAnnotation and CompoundDb packages that together provide a modular infrastructure for the annotation of untargeted metabolomics data. Initial annotation can be performed based on MS1 properties such as m/z and retention times, followed by an MS2-based annotation in which experimental fragment spectra are compared against a reference library. Such reference databases can be created and managed with the CompoundDb package. The ecosystem supports data from a variety of formats, including, but not limited to, MSP, MGF, mzML, mzXML, netCDF as well as MassBank text files and SQL databases. Through its highly customizable functionality, the presented infrastructure allows to build reproducible annotation workflows tailored for and adapted to most untargeted LC-MS-based datasets. All core functionality, which supports base R data types, is exported, also facilitating its re-use in other R packages. Finally, all packages are thoroughly unit-tested and documented and are available on GitHub and through Bioconductor.
    Keywords:  R programming; annotation; metabolomics; reproducible research; small-compound databases; untargeted analysis
  5. Metabolites. 2022 Feb 10. pii: 168. [Epub ahead of print]12(2):
      Dysregulation of cellular metabolism is now a well-recognized hallmark of cancer. Studies investigating the metabolic features of cancer cells have shed new light onto processes in cancer cell biology and have identified many potential novel treatment options. The advancement of mass spectrometry-based metabolomics has improved the ability to monitor multiple metabolic pathways simultaneously in various experimental settings. However, questions still remain as to how certain steps in the metabolite extraction process affect the metabolic profiles of cancer cells. Here, we use ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) untargeted metabolomics to investigate the effects of different detachment and lysis methods on the types and abundances of metabolites extracted from MDA-MB-231 cells through the use of in-house standards libraries and pathway analysis software. Results indicate that detachment methods (trypsinization vs. scraping) had the greatest effect on metabolic profiles whereas lysis methods (homogenizer beads vs. freeze-thaw cycling) had a lesser, though still significant, effect. No singular method was clearly superior over others, with certain metabolite classes giving higher abundances or lower variation for each detachment-lysis combination. These results indicate the importance of carefully selecting sample preparation methods for cell-based metabolomics to optimize the extraction performance for certain compound classes.
    Keywords:  breast cancer; cell metabolomics; detachment; lysis; mass spectrometry; pathway analysis
  6. Metabolites. 2022 Feb 01. pii: 135. [Epub ahead of print]12(2):
      In mass spectrometry-based metabolomics, the differences in the analytical results from different laboratories/machines are an issue to be considered because various types of machines are used in each laboratory. Moreover, the analytical methods are unique to each laboratory. It is important to understand the reality of inter-laboratory differences in metabolomics. Therefore, we have evaluated whether the differences in analytical methods, with the exception sample pretreatment and including metabolite extraction, are involved in the inter-laboratory differences or not. In this study, nine facilities are evaluated for inter-laboratory comparisons of metabolomic analysis. Identical dried samples prepared from human and mouse plasma are distributed to each laboratory, and the metabolites are measured without the pretreatment that is unique to each laboratory. In these measurements, hydrophilic and hydrophobic metabolites are analyzed using 11 and 7 analytical methods, respectively. The metabolomic data acquired at each laboratory are integrated, and the differences in the metabolomic data from the laboratories are evaluated. No substantial difference in the relative quantitative data (human/mouse) for a little less than 50% of the detected metabolites is observed, and the hydrophilic metabolites have fewer differences between the laboratories compared with hydrophobic metabolites. From evaluating selected quantitatively guaranteed metabolites, the proportion of metabolites without the inter-laboratory differences is observed to be slightly high. It is difficult to resolve the inter-laboratory differences in metabolomics because all laboratories cannot prepare the same analytical environments. However, the results from this study indicate that the inter-laboratory differences in metabolomic data are due to measurement and data analysis rather than sample preparation, which will facilitate the understanding of the problems in metabolomics studies involving multiple laboratories.
    Keywords:  hydrophilic metabolite; hydrophobic metabolite; inter-laboratory comparison; mass spectrometry; metabolomics; relative quantification
  7. Anal Sci Adv. 2020 Jun;1(1): 70-80
      Archived metabolomics data represent a broad resource for the scientific community. However, the absence of tools for the meta-analysis of heterogeneous data types makes it challenging to perform direct comparisons in a single and cohesive workflow. Here we present a framework for the meta-analysis of metabolic pathways and interpretation with proteomic and transcriptomic data. This framework facilitates the comparison of heterogeneous types of metabolomics data from online repositories (e.g., XCMS Online, Metabolomics Workbench, GNPS, and MetaboLights) representing tens of thousands of studies, as well as locally acquired data. As a proof of concept, we apply the workflow for the meta-analysis of i) independent colon cancer studies, further interpreted with proteomics and transcriptomics data, ii) multimodal data from Alzheimer's disease and mild cognitive impairment studies, demonstrating its high-throughput capability for the systems level interpretation of metabolic pathways. Moreover, the platform has been modified for improved knowledge dissemination through a collaboration with Metabolomics Workbench and LIPID MAPS. We envision that this meta-analysis tool will help overcome the primary bottleneck in analyzing diverse datasets and facilitate the full exploitation of archival metabolomics data for addressing a broad array of questions in metabolism research and systems biology.
    Keywords:  Archived data; meta-analysis; metabolic pathways; metabolomics; proteomics; systems biology; transcriptomics
  8. Antioxidants (Basel). 2022 Jan 25. pii: 229. [Epub ahead of print]11(2):
      Lipid hydroperoxides (LOOH) are the initial products of the peroxidation of unsaturated lipids and play a crucial role in lipid oxidation due to their ability to decompose into free radicals and cause adverse effects on human health. Thus, LOOHs are commonly considered biomarkers of oxidative stress-associated pathological conditions. Despite their importance, the sensitive and selective analytical method for determination is limited, due to their low abundance, poor stability, and low ionizing efficiency. To overcome these limitations, in this study, we chemically synthesized eight fatty acid hydroperoxides (FAOOH), including FA 18:1-OOH, FA 18:2-OOH, FA 18:3-OOH, FA 20:4-OOH, FA 20:5-OOH, FA 22:1-OOH, FA 22:6-OOH as analytes, and FA 19:1-OOH as internal standard. Then, they were chemically labeled with 2-methoxypropene (2-MxP) to obtain FAOOMxP by one-step derivatization (for 10 min). A selected reaction monitoring assisted targeted analytical method was developed using liquid chromatography/tandem mass spectrometry (LC-MS/MS). The MxP-labelling improved the stability and enhanced the ionization efficiency in positive mode. Application of reverse-phase chromatography allowed coelution of analytes and internal standards with a short analysis time of 6 min. The limit of detection and quantification for FAOOH ranged from 0.1-1 pmol/µL and 1-2.5 pmol/µL, respectively. The method was applied to profile total FAOOHs in chemically oxidized human serum samples (n = 5) and their fractions of low and high-density lipoproteins (n = 4). The linoleic acid hydroperoxide (FA 18:2-OOH) and oleic acid hydroperoxide (FA 18:1-OOH) were the most abundant FAOOHs in human serum and lipoproteins. Overall, our validated LC-MS/MS methodology features enhanced detection and rapid separation that enables facile quantitation of multiple FAOOHs, therefore providing a valuable tool for determining the level of lipid peroxidation with potential diagnostic applications.
    Keywords:  2-methoxypropene; chemical derivatization; human serum; lipid hydroperoxide; lipoprotein oxidation; liquid chromatography; mass spectrometry; unsaturated fatty acids
  9. J Chromatogr A. 2022 Feb 10. pii: S0021-9673(22)00089-9. [Epub ahead of print]1667 462891
      In this work, a stable isotope labelling-flow injection analysis-tandem mass spectrometry (SIL-FIA-MS/MS) with simultaneous monitoring [M+H]+ and [M+Cl]- method was developed for very specific and high throughput screening of anabolic-androgenic steroids (AAS) illegally added to healthy foods. Initially, a simple centrifugation step was carried out for liquid samples, and for solid samples, a solid-liquid extraction step was conducted. Afterwards, batch chemical derivatization was carried out. After adding a certain amount of 13C6-3-NPH labelled AAS standards as the internal standards, it can be directly transferred for FIA-MS/MS analysis based on the no MS response characteristics of 3-NPH. The 3-NPH labelled AAS showed dual-polarity property, observing chloride adduct ion ([M+Cl]-) in negative ion mode and proton adduct ion ([M+H]+) in positive ion mode. The average time cost for pretreatment of each sample was less than 1 min by carrying out batch processing. The subsequent FIA-MS/MS detection enabled rapid and high throughput detection. The addition of 13C6-3-NPH-labelled AAS as internal standards can correct the matrix effect to achieve accurate quantitative analysis. The detection sensitivity was also improved by 2-5 folds after 3-NPH labelling. The limits of detection (LODs) in positive MRM mode were in ranges of 0.1-0.3 ng/mL. The validated method with simultaneous monitoring [M+H]+ and [M+Cl]- was validated in the range of 6.0-1000 ng/mL with the linear coefficient (R2) greater than 0.997. Satisfactory recoveries were found to be in ranges of 93.0-108.7%. The intra-day and inter-day RSDs were in the range of 3.5-9.9% and 5.1-14.1%, respectively. No changes in detection sensitivity of the mass spectrometry and no carry-over effects were found after numerous consecutive injections of AAS derivates. Compared with previously reported methods, the proposed method proved accurate, very specific, high throughput with good sensitivity.
    Keywords:  3-nitrophenylhydrazine; Anabolic-androgenic steroids; Flow injection analysis-mass spectrometry; Stable isotope labelling
  10. Metabolites. 2022 Feb 11. pii: 170. [Epub ahead of print]12(2):
      Short Chain Fatty Acids (SCFAs) are produced by the gut microbiota and are present in varying concentrations in the intestinal lumen, in feces but also in the circulatory system. By interacting with different cell types in the body, they have a great impact on host metabolism and their exact quantification is indispensable. Here, we present a derivatization-free method for the gas chromatography mass spectrometry (GC-MS) based quantification of SCFAs in plasma, feces, cecum, liver and adipose tissue. SCFAs were extracted using ethanol and concentrated by alkaline vacuum centrifugation. To allow volatility for separation by GC, samples were acidified with succinic acid. Analytes were detected in selected ion monitoring (SIM) mode and quantified using deuterated internal standards and external calibration curves. Method validation rendered excellent linearity (R2 > 0.99 for most analytes), good recovery rates (95-117%), and good reproducibility (RSD: 1-4.5%). Matrix effects were ruled out in plasma, feces, cecum, liver and fat tissues where most abundant SCFAs were detected and accurately quantified. Finally, applicability of the method was assessed using samples derived from conventionally raised versus germ-free mice or mice treated with antibiotics. Altogether, a reliable, fast, derivatization-free GC-MS method for the quantification of SCFAs in different biological matrices was developed allowing for the study of the (patho)physiological role of SCFAs in metabolic health.
    Keywords:  GC-MS; SCFA; feces; fermentation; gut bacteria
  11. Talanta. 2022 Feb 09. pii: S0039-9140(22)00094-7. [Epub ahead of print]242 123298
      Recently, there has been growing interest in short-chain fatty acids (SCFA) and ketone bodies (KB) due to their potential use as biomarkers of health and disease. For instance, these diet-related metabolites can be used to monitor and reduce the risk of immune response, diabetes, or cardiovascular diseases. Given the interest in these metabolites, different targeted metabolomic methods based on UPLC-MS/MS have been developed in recent years to detect and quantify SCFA and KB. In this case study, we discovered that applying an existing validated, targeted UPLC-MS/MS method to mouse plasma, resulted in a fragment ion (194 m/z) being originally misidentified as acetic acid (a SCFA), when its original source was 3-hydroxybutyric acid (a KB). Therefore, we report a modified, optimized LC method that can separate both signals. In addition, the metabolite coverage was expanded in this method to detect up to eight SCFA: acetic, propanoic, butyric, isobutyric, 2-methylbutyric, valeric, isovaleric, and hexanoic acids, two KB: 3-hydroxybutyric, and acetoacetic acids, and one related metabolite: 3-hydroxy-3-methylbutyric acid. The optimization of this method increased the selectivity of the UPLC-MS/MS method towards the misidentified compound. These findings encourage the scientific community to increase efforts in validating the original precursor of small molecule fragments in targeted methods.
    Keywords:  Analytical validation; In-source fragmentation; Ketone bodies; Quality assurance; Short-chain fatty acids
  12. Anal Chim Acta. 2022 Mar 15. pii: S0003-2670(22)00083-6. [Epub ahead of print]1198 339512
      There are several challenges associated with LC-MS/MS bioanalytical method development and validation. Low and variable recovery of some analytes, especially the more hydrophobic ones, is often challenging. Analytes can be lost to various extents throughout the process of sample collection, storage, before, during, and/or after sample preparation and analysis. The calculation of overall extraction recovery can detect problems of low recovery during sample preparation but does not identify the source(s) of analyte losses. Low overall analyte recovery is the net result of losses that can happen for multiple reasons at all steps of sample preparation and analysis. Therefore, identifying the source(s) of analyte loss during sample preparation can help guide the optimization the bioanalysis conditions to minimize these losses. In this article we propose a practical protocol to systematically identify and quantify the sources of low analyte recovery. This allows the proper choice of strategies to optimize the relevant bioanalytical conditions to minimize analyte losses and improve overall recovery.
    Keywords:  Bioanalytical method; LC-MS/MS; Matrix effect; Recovery
  13. J Mass Spectrom Adv Clin Lab. 2022 Apr;24 15-21
      Background: Abemaciclib is a new oral targeted treatment option for patients with advanced breast cancer. The emerging field of oral antitumor therapeutics presents challenges for both patients and healthcare teams; non-adherence and high inter-individual pharmacokinetic variability can influence response rates.Methods: For monitoring abemaciclib in human sera, a rapid novel ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed and fully validated. Sample preparation was based on a protein precipitation step followed by on-line solid phase extraction. Chromatographic separation was achieved using a biphenyl column and the isotope labeled standard abemaciclib-d8 was used for quantification.
    Results: The method showed linearity over a wide calibration range from 20.0 to 2500 ng/mL. With accuracies and precisions of ≤13.9% and ≤4.42%, respectively, the validation results were within the criteria of acceptance. The fitness of the method was tested by monitoring abemaciclib levels under compassionate use for a single individual.
    Conclusions: The novelty of the presented two dimensional isotope dilution UHPLC-MS/MS method is in the semi-automated sample preparation, which results in negligible matrix effects, thereby allowing the introduction of abemaciclib into robust routine therapeutic drug monitoring (TDM). This method provides an efficient tool to verify the usefulness of personalized anticancer therapy in clinical practice.
    Keywords:  Abemaciclib; Kinase inhibitor; Oral tumor therapy; Therapeutic drug monitoring; Two dimensional-chromatography; isotope dilution UHPLC-MS/MS
  14. Drug Test Anal. 2022 Feb 23.
      Metabolomics is a multidisciplinary field providing workflows for complementary approaches to conventional analytical determinations. It allows for the study of metabolically related groups of compounds or even the study of novel pathways within the biological system. The procedural stages of metabolomics; experimental design, sample preparation, analytical determinations, data processing and statistical analysis, compound identification and validation strategies, are explored in this review. The selected approach will depend on the type of study being conducted. Experimental design influences the whole metabolomics workflow and thus needs to be properly assessed to ensure sufficient sample size, minimal introduced and biological variation and appropriate statistical power. Sample preparation needs to be simple, yet potentially global in order to detect as many compounds as possible. Analytical determinations need to be optimised either for the list of targeted compounds or a universal approach. Data processing and statistical analysis approaches vary widely and need to be better harmonised for review and interpretation. This includes validation strategies which are currently deficient in many presented workflows. Common compound identification approaches have been explored in this review. Metabolomics applications are discussed for clinical and forensic toxicology, human and equine sports anti-doping and veterinary residues.
  15. J Am Soc Mass Spectrom. 2022 Feb 24.
      Microbial mass spectrometry imaging (MSI) is a powerful tool used to generate biological hypotheses about the roles of metabolites in microbial competition based upon their two-dimensional spatial distribution. The most commonly used ionization method for microbial MSI is matrix-assisted laser desorption ionization (MALDI). However, medium components and microbial metabolites influence the adhesion of agar samples to the MALDI target, limiting the applicability of MALDI MSI to microbes grown on specific media. Here, we describe a three-step process using a robotic sprayer for a matrix application that improves the adherence of agar samples to the MALDI target, enabling the use of different media for microbial growth and an MSI analysis of larger sample surface areas.
    Keywords:  MALDI; agar adherence; microbial interactions; spray-based matrix application
  16. Anal Biochem. 2022 Feb 22. pii: S0003-2697(22)00060-4. [Epub ahead of print] 114604
      Low molecular-mass aliphatic carboxylic acids are critically important for intermediate metabolism and may serve as important biomarkers for metabolic homeostasis. Here in, we focused on multiplexed method development of aliphatic carboxylic analytes, including methylsuccinic acid (MSA), ethylmalonic acid (EMA), and glutaric acid (GA). Also assessed was their utility in a population's health as well as metabolic disease screening in both plasma and urine matrices. MSA, EMA, and GA are constitutional isomers of dicarboxylic acid with high polarity and poor ionization efficiency, resulting in such challenges as poor signal intensity and retention, particularly in reversed-phase liquid chromatography with electrospray mass spectrometry (RP-LC-ESI-MS/MS). Derivatization using n-butanol was performed in the sample preparation to enhance the signal intensity accompanied with a positive charge from ionization in complicated biomatrices as well as to improve the separation of these isomers with optimal retention. Fit-for-purpose method validation results demonstrated quantitative ranges for MSA/EMA/GA from 5/10/20 ng/mL to 400 ng/mL in plasma analysis, and 100/200/100 ng/mL to 5000/10000/5000 ng/mL in urine analysis. This validated method demonstrates future utility when exploring population health analysis and biomarker development in metabolic diseases.
    Keywords:  Ethylmalonic encephalopathy; Glutaric acid; Glutaric acidemia; Methylsuccinic acid; Short-chain acyl-CoA dehydrogenase deficiency
  17. Anal Chem. 2022 Feb 21.
      Many metabolites, including amino acids, neurotransmitters, and pharmaceuticals, contain primary amine functional groups. The analysis of these molecules by mass spectrometry (MS) plays an important role in the study of cancers and psychogenic diseases. However, the MS-based detection and visualization of these bioactive metabolites directly from real biological systems still suffer from challenges such as low ionization efficiency and/or matrix interference effects. Here, we introduce a simple and efficient strategy, the nanosecond photochemical reaction (nsPCR)-enabled fast chemical derivatization, enabling direct MS analysis of primary amine-containing metabolites, with enhanced detection sensitivity for numerous metabolites from cell culture medium and rat brain sections. Furthermore, this nsPCR-based chemical derivatization strategy was demonstrated to be a useful visualizing tool that could provide improved spatial information for these metabolites, potentially offering alternative tools for gaining novel insights into metabolic events.
  18. Bioanalysis. 2022 Mar;14(5): 279-287
      Background: Currently, no regulatory guidelines are available for parallelism assessment for LC-MS biomarker quantification. Spike recovery, standard addition and dilutional linearity are recommended with no mention of the implications of applying these approaches. Results: Here, using human urine creatinine, the authors compared spike recovery and standard addition in LC-MS biomarker quantification, and evaluated a new hybrid approach: parallelism QCs. The authors drew different conclusions based on which approach was used (<15% cutoff). Conclusion: Current recommended approaches may lead to different conclusions and are not equivalent and interchangeable. The authors recommend that standard addition should be the universal 'go-to' method for LC-MS biomarker parallelism assessment; parallelism QCs, which consider the total concentration as the theoretical value, can be used if the authentic matrix is limited.
    Keywords:  LC-MS; biomarker; creatinine; endogenous compound; human urine; method validation; parallelism; parallelism QCs; quantification
  19. Toxins (Basel). 2022 Feb 09. pii: 131. [Epub ahead of print]14(2):
      An accurate, reliable, and specific method was developed for the quantitative determination of fumonisins B1, B2, B3, and their hydrolyzed metabolites, HFB1, HFB2, and HFB3, in broiler chicken feed and excreta using ultra-performance liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS). The samples were extracted and diluted for the determination of parent fumonisins. Another portion of the extracted samples was alkaline-hydrolyzed and cleaned using a strong anionic exchange adsorbent (MAX) for the determination of hydrolyzed fumonisins. Chromatographic separation was performed on a CORTECS C18 column (2.1 mm × 100 mm, 1.6 μm) using 0.2% formic acid aqueous solution and methanol with 0.2% formic acid as the mobile phase under gradient elution. The six fumonisins, FB1, FB2, FB3, HFB1, HFB2, and HFB3, were analyzed by tandem mass spectrometry using multiple-reaction monitoring (MRM) mode. The six fumonisins showed good linearity, with relative coefficients of r > 0.99. The limits of quantitation (LOQs) were 160 μg/kg. At the low, medium, and high spiked levels, the recovery of fumonisins in chicken feed and excreta ranged from 82.6 to 115.8%, with a precision (RSD) of 3.9-18.9%. This method was successfully applied to investigate the migration and transformation of fumonisins in broiler chickens.
    Keywords:  broiler chicken; excreta; feed; fumonisin; hydrolyzed fumonisin; mycotoxins
  20. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Feb 17. pii: S1570-0232(22)00087-3. [Epub ahead of print]1193 123183
      An analytical method of ultra-high performance liquid chromatography coupled to tandem mass spectrometry detection was developed and validated for the simultaneous quantification in plasma of four selective serotonin reuptake inhibitor antidepressants: sertraline, escitalopram, paroxetine, fluoxetine, and its metabolite norfluoxetine. A simple protein precipitation was performed with acetonitrile containing 100 ng/mL of indomethacin, which was used as internal standard. Chromatographic separation was carried out on an Acquity BEH C18 column with isocratic elution of the mobile phase consisting of 5 mmol/L ammonium acetate with 0.1% formic acid (A) and acetonitrile (B) at a 60:40 proportion, respectively. The flow rate was 0.4 mL/min with a run time of 5 min. A positive electrospray ionization source was used for detection. The method was linear in a range of 5-800 ng/mL, with determination coefficients greater than 0.991. The accuracy ranged from 91% to 112% for intra-assay and from 89% to 112% for inter-assay. The variation coefficients ranged from 3.1% to 14.88% for intra-assay and from 3.60% to 14.74% for inter-assay precision. The method was successfully applied for the analysis of 73 samples from patients under treatment with these antidepressants; 36.9% of the samples had concentrations outside therapeutic ranges. This method can be applied for routine analysis in clinical practice, simplifying sample processing, reducing analysis time and consequently the costs associated with it.
    Keywords:  Antidepressants; Isocratic elution; Method validation; SSRI; Therapeutic drug monitoring; UPLC-MS/MS
  21. Curr Opin Biotechnol. 2022 Feb 16. pii: S0958-1669(22)00021-0. [Epub ahead of print]75 102695
      Single-carbon (C1, or one-carbon) substrates are promising feedstocks for sustainable biofuel and biochemical production. Crucial to the goal of engineering C1-utilizing strains for improved production is a quantitative understanding of the organization, regulation and rates of the reactions that underpin C1 metabolism. 13C Metabolic flux analysis (MFA) is a well-established platform for interrogating these questions with multi-carbon substrates, and uses the differential labeling of metabolites that results from feeding a substrate with position-specific incorporation of 13C in order to infer quantitative fluxes and pathway topology. Adapting isotopic tracer approaches to C1 metabolism, where position-specific substrate labeling is impossible, requires additional experimental considerations. Here we review recent studies that have developed isotopic tracer approaches to overcome the challenge of uniform metabolite labeling and provide quantitative insight into C1 metabolism.
  22. J Pharm Biomed Anal. 2022 Feb 19. pii: S0731-7085(22)00102-9. [Epub ahead of print]212 114681
      Short-chain fatty acids are metabolites widely presented in many natural sources, including human feces and blood. Estimation of their composition is a common procedure, usually performed using nuclear magnetic resonance or gas chromatography with a flame ionization detector. However, the commonly used methods often depend on specific sample preparation, such as filtration and homogenization. The gas-chromatography/mass-spectrometry (GC/MS) method with headspace extraction allows sample preparation to be kept to a minimum regardless of the physical state of the sample, which can be potentially useful in metabolomics research of complex natural samples such as blood or feces. In this work, we have demonstrated the applicability of Headspace GC-MS for estimating short chain fatty acid (SCFA) composition. The main problem here is the complex, non-linear dependence between the composition of the compounds in the source phase and the relative pressures in the vapor phase, which are directly measured by this method. We have implemented a thermodynamic model that performs the reverse transformation of relative abundances in the vapor phase to relative concentrations in the liquid phase, and have tested it on some synthetic SCFA mixtures. The developed method is available as a pip package called UniqPy and can be used to describe liquid-vapor equilibrium for any multicomponent system if a sufficient amount of training data is provided. The gas chromatography method with headspace extraction in conjunction with the UniqPy data transformation showed satisfactory quantification accuracy for propionic acid, butyric acid, isobutyric acid, and valeric acid (R-squared > 0.96). The applicability of the method was additionally demonstrated on a series of fecal samples.
    Keywords:  Headspace GC-MS; Short chain fatty acids; UNIQUAC
  23. J Biosci Bioeng. 2022 Feb 17. pii: S1389-1723(22)00011-1. [Epub ahead of print]
      Metabolomics is a tool used for quantitative assessment of metabolites that has been applied extensively in the field of food science. Recently, metabolomics-based gas chromatography-mass spectrometry (GC/MS) is becoming a common tool for analyzing, not only volatile compounds, but also non-volatile compounds due to the development of various derivatization methods. Although several studies have reviewed the application of metabolomics in food science, this present review article specifically focuses on metabolomics research using GC/MS for analysis of non-volatile compounds such as sugars, amino acids, and organic acids. From exhaustive literature research, the application of GC/MS-based metabolomics for non-volatile compounds in food science includes discriminating food samples based on cultivars and authentication of food samples to prevent food fraud, characterizing the profile of food samples to provide a general overview of the sample, evaluating stress-response, optimizing postharvest processes based on metabolic changes, monitoring changes during growth and food processing, evaluating and predicting food quality, and evaluating food shelf-life. GC/MS-based analysis of non-volatile compounds has been proven to be extremely valuable in food science, and might open new avenues for future researchers and engineers to develop instruments or improving production process in food industry.
    Keywords:  Food science; Gas chromatography-mass spectrometry; Metabolomics; Non-volatile
  24. Steroids. 2022 Feb 17. pii: S0039-128X(22)00017-4. [Epub ahead of print]180 108979
      Dehydrochloromethyltestosterone (DHCMT) is one of the most detected illicit used anabolic-androgenic steroids in professional sports. Therefore, a fast and accurate analysis of this substance is of great importance for a constructive fight against doping abuse. The conventional method for the analysis of this drug, GC-MSMS, is very sensitive and selective but also very time- and resource-consuming. With the presented work, a new approach for simple detection with LC-HRMSMS without any sample preparation is introduced. The method is based on the direct analysis of two newly described phase-II metabolites of the DHCMT long-term metabolite 4-chloro-18-nor-17β-hydroxymethyl-17α-methyl-5β-androst-13-en-3α-ol (M3). LC-HRMSMS, GC-MSMS, fractionation and derivatization experiments are combined to identify and characterize for the first time two different glucuronide-acid conjugates of this metabolite in positive human urine samples. In addition, a third glucuronide metabolite was identified, however without isomeric structure determination. The detection of these metabolites is particularly interesting for confirmation analyses, as the method is rapid and requires little sample material.
    Keywords:  Anabolic androgenic steroids; Anti-doping; Dehydrochloromethyltestosterone; Glucuronide; High-resolution mass spectrometry; Phase-II metabolite
  25. Eur J Endocrinol. 2022 Feb 01. pii: EJE-22-0081. [Epub ahead of print]
      OBJECTIVE: Endogenous hormones regulate numerous physiological processes in humans. Some of them are routinely measured in blood, saliva and/or urine for the diagnosis of disorders. The analysis of fluids may however require multiple samples collected at different time points to avoid the high variability in the concentration of some hormones. In contrast, hair analysis has been proposed as an interesting alternative to reveal average hormone levels over longer period. In this work, we developed and validated an analytical method for analyzing 36 endogenous steroid and thyroid hormones and one pineal hormone in human hair using ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS).METHODS: Sample preparation involved hair decontamination, pulverization, methanol extraction, and purification with C18-solid phase extraction. Extracts were then divided into two portions, respectively injected into an UPLC-MS/MS system, and analyzed using two different instrumental methods. The method was applied to a healthy female population aged 25-45 years.
    RESULTS: The method was validated on supplemented hair samples for the 37 targeted hormones, and its application to the population under study allowed to detect 32 compounds in 2 to 100% of the samples. Complete reference intervals (2.5th-97.5th percentiles) were established for estrone, 17β-estradiol, androstenedione, dehydroepiandrosterone, progesterone, 17α-hydroxyprogesterone, cortisone, cortisol and 3,3',5-triiodo-L-thyronine. Hair cortisone, cortisol, tetrahydrocortisone and tetrahydrocortisol concentrations were highly correlated with each other, with Kendall's τ correlation coefficients ranging from 0.52 to 0.68.
    CONCLUSION: Allowing the detection of 32 hormones from different chemical classes, the present method will allow to broaden hormonal profiling for better identifying endocrine disorders.
  26. Bioanalysis. 2022 Mar;14(5): 267-278
      Background: The degree of human hepatocyte replacement in chimeric mice with humanized liver has previously been shown to correlate with human plasma albumin measurements. However, there are no reports that directly compare the remaining endogenous mouse albumin with the newly expressed human albumin following engraftment. To better understand the disposition of serum albumin in PXB-mice, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to simultaneously quantitate both human and mouse albumin from plasma. Results: A robust correlation was observed between the serum human albumin levels measured by LC-MS/MS and the estimated replacement index of PXB-mice. Conclusion: All data were shown to be specific and suitable to accurately quantify both human and mouse albumin from plasma of chimeric mice with humanized livers.
    Keywords:  LC-MS/MS; PXB-mouse; albumin; chimera; hepatocytes; humanized; liver; mice; plasma; quantification
  27. Molecules. 2022 Feb 11. pii: 1223. [Epub ahead of print]27(4):
      Gungha-tang (GHT), a traditional herbal medicine, consists of nine medicinal herbs (Cnidii Rhizoma, Pinelliae Tuber, Poria Sclerotium, Citri Unshius Pericarpium, Citri Unshius Pericarpium Immaturus, Aurantii Fructus Immaturus, Atracylodis Rhizoma Alba, Glycyrrhizae Radix et Rhizoma, and Zingiberis Rhizoma Recens). It has been used for various diseases caused by phlegm. This study aimed to develop and verify the simultaneous liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis method, using nine marker components (liquiritin apioside, neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, liquiritigenin, glycyrrhizin, and 6-shogaol) for quality control of GHT. LC-MS/MS analysis was conducted using a Waters TQ-XS system. All marker analytes were separated on a Waters Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) using gradient elution with a distilled water solution (containing 5 mM ammonium formate and 0.1% [v/v] formic acid)-acetonitrile mobile phase. LC-MS/MS multiple reaction monitoring (MRM) analysis was carried out in negative and positive ion modes of an electrospray ionization source. The developed LC-MS/MS MRM method was validated by examining the linearity, limits of detection and quantification, recovery, and precision. LOD and LOQ values of nine markers were calculated as 0.02-8.33 ng/mL and 0.05-25.00 ng/mL. The recovery was determined to be 89.00-118.08% and precision was assessed with a coefficient of variation value of 1.74-8.64%. In the established LC-MS/MS MRM method, all markers in GHT samples were detected at 0.003-16.157 mg/g. Information gathered during the development and verification of the LC-MS/MS method will be useful for the quality assessment of GHT and other herbal medicines.
    Keywords:  Gungha-tang; LC–MS/MS; quality control; simultaneous analysis
  28. SLAS Technol. 2022 Feb 21. pii: S2472-6303(22)00012-7. [Epub ahead of print]
      High-Performance Liquid Chromatography-Tandem Mass Spectrometry has emerged triumphant over the years as a reliable and high throughput clinical instrument to assess different metabolic irregularities. One of the latest applications of LC-MS/MS has been in the field of quantification of glucocorticoids, such as cortisol and cortisone from saliva, that can be an indicator of abnormalities such as Congenital Adrenal Hyperplasia (CAH), Cushing's Syndrome, and Addison's disease. We have developed and validated an LC-MS/MS-based assay for simultaneous detection of cortisol and cortisone in human saliva, which requires only 20 µL of sample, to measure cortisol across a 0.5 - 70 ng/mL range, and cortisone across a 1.2- 100 ng/mL range, respectively. The developed method exhibits linearity of R2>0.99, for both analytes, inclusive of both MRMs, and a percent coefficient of variation that is less than or equal to 20%. Using dilute-and-shoot for sample preparation, we have exhibited sample accuracy of 100±20 for the assay calibrators, and integrated Needle Wash Solvent Chemistry for minimal sample carryover, making it adaptable for crucial for potential diagnostic use. We have exhibited a method that is simplistic, specific, and highly automatable on liquid handling platforms, such as the JANUS® G3 Workstation. With our innovation, we have introduced a potential to test 81-unknown samples in singlicate within an on-deck plating time of fewer than four minutes. We performed additional verification studies, including an accelerated stability study and a freeze-thaw study showcasing the potential long-term usability of our proposed prototype kit. Overall, this work presents an optimized LC-MS/MS method with automated sample preparation that is ready to be utilized for cortisol-cortisone detection for clinical diagnostic contexts related to Cushing's Disease, among other adrenal and endocrine disorders.
  29. Minerva Med. 2022 Feb 22.
      Chronic obstructive pulmonary disease (COPD) is the third cause of death worldwide, presenting poor long-term outcomes and chronic disability. COPD is a condition with a wide spectrum of clinical presentations because its pathophysiological determinants relate to tobacco smoke, genetic factors, alteration of several metabolic pathways, and oxidative stress. As a consequence, patients present different phenotypes even with comparable degrees of airflow limitation. Because of the increasing social and economic costs of COPD, a growing attention is currently payed to "omics" techniques for more personalized treatments and patient-tailored rehabilitation programs. In this regard, the systematic investigation of the metabolome (i.e., the whole set of endogenous molecules) in biomatrices, namely metabolomics, has become indispensable for phenotyping respiratory diseases. The metabolomic profiling of biological samples contains the small molecules produced during biological processes and their identification and quantification help in the diagnosis, comprehension of disease outcome and treatment response. Exhaled breath condensate (EBC), plasma and serum are biofluids readily available, with negligible invasiveness, and, therefore, suitable for metabolomics investigations. In this paper, we describe the latest advances on metabolomic profiling of EBC, plasma and serum in COPD patients.
  30. Drug Test Anal. 2022 Feb 23.
      Oxytetracycline is a broad-spectrum antibiotic, which inhibits protein synthesis and is generally used for the treatment of pneumonia, shipping fever, leptospirosis and wound infections in cattle and swine. The present work proposes a novel LC-MS/MS method for oxytetracycline quantification in bull plasma, seminal plasma and urine, requiring limited sample treatment before analysis. Extraction with trichloroacetic acid followed by dilution of the supernatant in mobile phase proved to be effective in all three matrices, allowing to rapidly process large batches of samples. Sharp and symmetrical peak shape was obtained using a BEH C18 reversed-phase column in a chromatographic run of just 3.5 min. The mass spectrometer operated in positive electrospray ionisation mode and monitored specific transitions for (461.1>425.8) and the internal standard demeclocycline (465.0>447.6). The method was validated over concentration ranges suitable for field concentrations of oxytetracycline found in each matrix, showing good linearity during each day of testing (R2 always >0.99), as also confirmed by ANOVA and lack-of-fit tests. Excellent accuracy and precision were demonstrated by calculated bias always within ±15% and CV% below 10% at all QC levels in the three matrices. Matrix effect and recovery were investigated for both analytes, which showed consistent and comparable behaviour in each matrix. To our knowledge, this is the first validated approach for mass spectrometric determination of oxytetracycline in seminal plasma and urine. The method was successfully applied to samples collected during a pharmacokinetic study in bulls, allowing to assess the oxytetracycline concentration-time profile in plasma, seminal plasma and urine.
    Keywords:  LC-MS; oxytetracycline; pharmacokinetics; plasma; seminal plasma; urine
  31. Anal Chem. 2022 Feb 21.
      Structural characterization of novel metabolites in drug discovery or metabolomics is one of the most challenging tasks. Multilevel fragmentation (MSn) based approaches combined with various dissociation modes are frequently utilized for facilitating structure assignment of unknown compounds. As each of the MS precursors undergoes MSn, the instrument cycle time can limit the total number of precursors analyzed in a single LC run for complex samples. This necessitates splitting data acquisition into several analyses to target lower concentration analytes in successive experiments. Here we present a new LC/MS data acquisition strategy, termed Met-IQ, where the decision to perform an MSn acquisition is automatically made in real time based on the similarity between the experimental MS2 spectrum and a spectrum in a reference spectral library for the known compounds of interest. If similarity to a spectrum in the library is found, the instrument performs a decision-dependent event, such as an MS3 spectrum. Compared to an intensity-based, data-dependent MSn experiment, only a limited number of MS3 are triggered using Met-IQ, increasing the overall MS2 instrument sampling rate. We applied this strategy to an Amprenavir sample incubated with human liver microsomes. The number of MS2 spectra increased 2-fold compared to a data dependent experiment where MS3 was triggered for each precursor, resulting in identification of 14-34% more unique potential metabolites. Furthermore, the MS2 fragments were selected to focus likely sources of useful structural information, specifically higher mass fragments to maximize acquisition of MS3 data relevant for structure assignment. The described Met-IQ strategy is not limited to metabolism experiments and can be applied to analytical samples where the detection of unknown compounds structurally related to a known compound(s) is sought.
  32. Front Chem. 2021 ;9 782432
      Compared with conventional optical microscopy techniques, mass spectrometry imaging (MSI) or imaging mass spectrometry (IMS) is a powerful, label-free analytical technique, which can sensitively and simultaneously detect, quantify, and map hundreds of biomolecules, such as peptides, proteins, lipid, and other organic compounds in cells and tissues. So far, although several soft ionization techniques, such as desorption electrospray ionization (DESI) and secondary ion mass spectrometry (SIMS) have been used for imaging biomolecules, matrix-assisted laser desorption/ionization (MALDI) is still the most widespread MSI scanning method. Here, we aim to provide a comprehensive review of MALDI-MSI with an emphasis on its advances of the instrumentation, methods, application, and future directions in single cell and biological tissues.
    Keywords:  imaging mass spectrometry; matrix-assisted laser desorption/ionization (MALDI); proteomics; single-cell metabolomics; spatial distribution; tissue mapping
  33. Ann Clin Biochem. 2022 Feb 24. 45632221077183
      BACKGROUND: Vancomycin, norvancomycin, methotrexate, paclitaxel, and imatinib are five commonly used drugs which are all recommended to therapeutic drug monitoring in clinical settings. However, the blood concentration monitoring of these drugs and the interpretations of the test results are limited to some extent due to the differences of testing instruments and testing methods.METHODS: We established an ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantification of vancomycin, norvancomycin, methotrexate, paclitaxel, and imatinib in human plasma. The method was validated according to the guideline for bioanalytical method validation and applied in clinical therapy.
    RESULTS: The calibration ranges of vancomycin, norvancomycin, methotrexate, paclitaxel, and imatinib were 0.5-100 μg/mL, 0.5-100 μg/mL, 5-1000 ng/mL, 10-2000 ng/mL, and 5-500 ng/mL, respectively. Inaccuracy and imprecision of every drug were less than 15%. The internal standard normalized recovery rates of vancomycin and norvancomycin were about 45%, while which of methotrexate, paclitaxel, and imatinib were almost 100%. No obvious carryover effect was observed. Samples were stable for at least 24 h in the automatic sampler, 72 h at 4°C, and 1 week in -80°C. There were no differences of concentrations between plasma and serum for the five drugs. Moreover, there were positive correlations between methotrexate and vancomycin concentrations and creatinine, as well as positive correlation between imatinib concentration and age of the patient.
    CONCLUSIONS: The UPLC-MS/MS method was competent for the simultaneous monitoring of vancomycin, norvancomycin, methotrexate, paclitaxel, and imatinib because of its short analysis time, high specificity, and accuracy.
    Keywords:  imatinib; methotrexate; norvancomycin; paclitaxel; therapeutic drug monitoring; vancomycin
  34. Drug Test Anal. 2022 Feb 20.
      INTRODUCTION: Cannabis is the most frequently consumed illegal substance worldwide. More recently, an increasing number of legal cannabis-products low in psychoactive Δ9 -tetrahydrocannabinol (THC), but high in non-intoxicating cannabidiol (CBD) are being more widely consumed. While the detection and quantification of THC and its metabolites in biological matrices is an important forensic-toxicological task, additional detection of CBD is also important, for example when examining the plausibility of consumer's statements. This report describes the method validation for the quantitative determination of THC and its two major metabolites, 11-hydroxy-THC (OH-THC) and 11-nor-9-carboxy-THC (THC-COOH), as well as CBD and cannabinol (CBN) in whole blood and urine.METHOD: The method employs automated on-line solid phase extraction coupled to gas chromatography tandem mass spectrometry (GC-MS/MS). The method was fully validated according to guidelines of the Swiss Society of Legal Medicine (SGRM) and the Society of Toxicological and Forensic Chemistry (GTFCh).
    RESULTS: The method fulfilled the validation criteria regarding analytical limits, accuracy and precision, extraction efficacy, and sample stability. Limits of detection (LOD) and quantification (LOQ) in whole blood and urine were 0.15 ng/mL and 0.3 ng/mL, respectively, for THC, OH-THC and CBD, 0.1 ng/mL and 0.2 ng/mL, respectively, for CBN, and 1.0 ng/mL and 3.0 ng/mL, respectively, for THC-COOH.
    CONCLUSION: The fully validated and automated method allows sensitive and robust measurement of cannabinoids in whole blood and urine. Detection of CBD provides additional information regarding consumed products.
    Keywords:  Cannabinoids; GC-MS/MS; driving under the influence of drugs; on-line solid phase extraction
  35. Talanta. 2022 Feb 05. pii: S0039-9140(22)00087-X. [Epub ahead of print]242 123291
      Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and imaging mass spectrometry (IMS) are being increasingly recognized for the detection and visualization of various organic species including lipids and fatty acids. Nevertheless, most MALDI matrices perform optimally in one ionization mode. This study investigates the performance of cyano derivative of graphene (G-CN) as a matrix in two polarities of MALDI MS and IMS for the detection of oil binders and fatty acids in artworks, and compares it with classical MALDI matrices (2,5-dihydroxybenzoic acid, 9-aminoacridine). Results revealed the ability of G-CN to provide high quality positive and negative mass spectra of oils and fatty acids, respectively, with lowest matrix-induced interferences among tested matrices and minimal effects of the presence of inorganic pigments. The newly developed approach makes both oil and fatty acid identifiable in a single spot simply by covering the sample surface with one matrix and switching the polarity in MALDI without any sample manipulation. G-CN offers effective matrix to analyte energy transfer, ability to detect components in less than 100 ng of oil at a MALDI spot and lesser analyte fragmentation than the compared conventional matrices. Furthermore, it enables direct mapping of specific m/z features corresponding to triacylglycerol (TAG), products of TAG oxidation and deprotonated acids using one nanoparticle matrix in MALDI IMS. This research shows potential for technical innovations in the study of art micro-environments and degradation phenomena of historical artworks.
    Keywords:  Artwork analysis; Fatty acids; Graphene derivative; MALDI imaging mass spectrometry; Nanomaterials; Oil binders
  36. Mar Drugs. 2022 Feb 15. pii: 143. [Epub ahead of print]20(2):
      The analysis of marine lipophilic toxins in shellfish products still represents a challenging task due to the complexity and diversity of the sample matrix. Liquid chromatography coupled with mass spectrometry (LC-MS) is the technique of choice for accurate quantitative measurements in complex samples. By combining unambiguous identification with the high selectivity of tandem MS, it provides the required high sensitivity and specificity. However, LC-MS is prone to matrix effects (ME) that need to be evaluated during the development and validation of methods. Furthermore, the large sample-to-sample variability, even between samples of the same species and geographic origin, needs a procedure to evaluate and control ME continuously. Here, we analyzed the toxins okadaic acid (OA), dinophysistoxins (DTX-1 and DTX-2), pectenotoxin (PTX-2), yessotoxin (YTX) and azaspiracid-1 (AZA-1). Samples were mussels (Mytilus galloprovincialis), both fresh and processed, and a toxin-free mussel reference material. We developed an accurate mass-extracted ion chromatogram (AM-XIC) based quantitation method using an Orbitrap instrument, evaluated the ME for different types and extracts of mussel samples, characterized the main compounds co-eluting with the targeted molecules and quantified toxins in samples by following a standard addition method (SAM). An AM-XIC based quantitation of lipophilic toxins in mussel samples using high resolution and accuracy full scan profiles (LC-HR-MS) is a good alternative to multi reaction monitoring (MRM) for instruments with HR capabilities. ME depend on the starting sample matrix and the sample preparation. ME are particularly strong for OA and related toxins, showing values below 50% for fresh mussel samples. Results for other toxins (AZA-1, YTX and PTX-2) are between 75% and 110%. ME in unknown matrices can be evaluated by comparing their full scan LC-HR-MS profiles with those of known samples with known ME. ME can be corrected by following SAM with AM-XIC quantitation if necessary.
    Keywords:  azaspiracid-1 (AZA-1); dinophysistoxin-1 (DTX-1); dinophysistoxin-2 (DTX-2); full scan; liquid chromatography-high resolution mass spectrometry (LC-HR-MS); liquid chromatography-mass spectrometry (LC-MS); matrix effects; okadaic acid (OA); pectenotoxin-2 (PTX-2); yessotoxin (YTX)
  37. Metabolites. 2022 Jan 27. pii: 122. [Epub ahead of print]12(2):
      MetAMDB is an open-source metabolic atom mapping database, providing atom mappings for around 43,000 metabolic reactions. Each atom mapping can be inspected and downloaded either as an RXN file or as a graphic in SVG format. In addition, MetAMDB offers the possibility of automatically creating atom mapping models based on user-specified metabolic networks. These models can be of any size (small to genome-scale) and can subsequently be used in standard 13C metabolic flux analysis software.
    Keywords:  13C metabolic flux analysis; AAM; RXN; atom mapping model; atom mappings; atom transitions; database; metabolic model; systems biochemistry
  38. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Feb 10. pii: S1570-0232(22)00070-8. [Epub ahead of print]1193 123166
      Simultaneous purification of fucoxanthin isomers from brown seaweeds by two steps of open-column chromatography (OCC) and reversed-phase (RP)-high-performance liquid chromatography (HPLC) is described. Analysis and identification of fucoxanthin isomers were performed by chromatographic and spectrophotometric properties such as retention time (tR), spectral shape, maximal absorption wavelength (λmax), Q-ratio, and mass spectrometry (MS) data including the ratio of fragment ions. The optimal conditions for a simultaneous separation and purification were examined by changing several parameters of HPLC, i.e., mobile phase composition, equilibration time, and column oven temperature. The purification procedure consisted of the following two steps: first, highly purified fucoxanthin fraction was obtained by a silica-gel OCC. Then, four major fucoxanthin isomers, all-trans, 13'-cis, 13-cis, and 9'-cis, were simultaneously separated and purified by RP-HPLC with an analytical C30 column and gradient elution in a mixture of water, methanol, and methyl tert-butyl ether. The purity of fucoxanthin isomers purified was >95% for all-trans and 9'-cis, 85% for 13'-cis, and >80% for 13-cis. A large-scale purification by RP-HPLC using a preparative C18 column was effective for the purification of all-trans and 9'-cis with a yield of 95%. This developed technique was fully applicable to analyze the enhanced production of fucoxanthin isomers by iodine-catalyzed stereomutation which composed of 9 isomer species including 9-cis.
    Keywords:  Brown seaweeds; C(30) stationary phase; Carotenoids; Fucoxanthin isomers; HPLC; Simultaneous purification
  39. Curr Protoc. 2022 Feb;2(2): e375
      Carotenoid oxidative cleavage products, apocarotenoids (APOs), are a class of important plant secondary metabolites, which include phytohormones abscisic acid (ABA) and strigolactones (SLs), and growth regulators and signaling molecules such as β-cyclocitral, zaxinone, anchorene, β-apo-11-carotenoids, and retinal. Qualitative and quantitative analysis of these bioactive compounds is crucial for understanding their metabolism and may also enable discovering further regulatory APOs. The state-of-the-art mass spectrometry (MS) technology has advanced the detection of plant APOs; however, it is still challenging to perform an accurate analysis of the low-level phytohormones ABA and SL and the structurally diverse APOs from complex plant matrices. Here, we describe ultrahigh-performance liquid chromatography-MS (UHPLC-MS) methods to determine carotenoid-derived hormones and APOs from plants by integrating ultrasound-assisted extraction and solid-phase extraction. These assays enable an accurate quantification of carotenoid-derived hormones and APOs from plant tissues by using an UHPLC hybrid quadrupole-Orbitrap mass spectrometer. © 2022 Wiley Periodicals LLC. Basic Protocol 1: UHPLC-MS analysis of APOs from rice roots Support Protocol: Preparation of dried plant root powder Basic Protocol 2: UHPLC-MS analysis of SLs from rice roots Basic Protocol 3: UHPLC-MS analysis of ABA from rice roots.
    Keywords:  abscisic acid; apocarotenoids; rice root; solid-phase extraction; strigolactones; ultrahigh-performance liquid chromatography-mass spectrometry; ultrasound-assisted extraction
  40. J Clin Med. 2022 Feb 12. pii: 956. [Epub ahead of print]11(4):
      Steroids are present in all animals and plants, from mammals to prokaryotes. In the medical field, steroids are commonly classified as glucocorticoids, mineralocorticoids, and gonadal steroid hormones. Monitoring of hormones is useful in clinical and research fields for the assessment of physiological changes associated with aging, disease risk, and the diagnostic and therapeutic effects of various diseases. Since the discovery and isolation of steroid hormones, measurement methods for steroid hormones in biological samples have advanced substantially. Although immunoassays (IAs) are widely used in daily practice, mass spectrometry (MS)-based methods have been reported to be more specific. Steroid hormone measurement based on MS is desirable in clinical practice; however, there are several drawbacks, including the purchase and maintenance costs of the MS instrument and the need for specialized training of technicians. In this review, we discuss IA- and MS-based methods currently in use and briefly present the history of steroid hormone measurement. In addition, we describe recent advances in IA- and MS-based methods and future applications and considerations.
    Keywords:  gas chromatography tandem mass spectrometry; immunoassay; liquid chromatography tandem mass spectrometry; mass spectrometry; matrix-assisted laser desorption/ionization; radioimmunoassay; steroid hormone
  41. Metabolites. 2022 Feb 08. pii: 156. [Epub ahead of print]12(2):
      Eleven species of lichens of the genus Sticta, ten of which were collected in Colombia (S. pseudosylvatica S. luteocyphellata S. cf. andina S. cf. hypoglabra, S. cordillerana, S. cf. gyalocarpa S. leucoblepharis, S. parahumboldtii S. impressula, S. ocaniensis) and one collected in Chile (S. lineariloba), were analyzed for the first time using hyphenated liquid chromatography with high-resolution mass spectrometry. In the metabolomic analysis, a total of 189 peaks were tentatively detected; the analyses were divided in five (5) groups of compounds comprising lipids, small phenolic compounds, saturated acids, terpenes, and typical phenolic lichen compounds such as depsides, depsidones and anthraquinones. The metabolome profiles of these eleven species are important since some compounds were identified as chemical markers for the fast identification of Sticta lichens for the first time. Finally, the usefulness of chemical compounds in comparison to traditional morphological traits to the study of ancestor-descendant relationships in the genus was assessed. Chemical and morphological consensus trees were not consistent with each other and recovered different relationships between taxa.
    Keywords:  Sticta; UHPLC-MS-MS; chemotaxonomyc; lichens; metabolomics; phylogenetic