bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒06‒19
29 papers selected by
Sofia Costa

  1. Anal Chem. 2022 Jun 15.
      On-tissue chemical derivatization is a valuable tool for expanding compound coverage in untargeted metabolomic studies with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Applying multiple derivatization agents in parallel increases metabolite coverage even further but results in large and more complex datasets that can be challenging to analyze. In this work, we present a pipeline to provide rigorous annotations for on-tissue derivatized MSI data using Metaspace. To test and validate the pipeline, maize roots were used as a model system to obtain MSI datasets after chemical derivatization with four different reagents, Girard's T and P for carbonyl groups, coniferyl aldehyde for primary amines, and 2-picolylamine for carboxylic acids. Using this pipeline helped us annotate 631 unique metabolites from the CornCyc/BraChem database compared to 256 in the underivatized dataset, yet, at the same time, shortening the processing time compared to manual processing and providing robust and systematic scoring and annotation. We have also developed a method to remove false derivatized annotations, which can clean 5-25% of false derivatized annotations from the derivatized data, depending on the reagent. Taken together, our pipeline facilitates the use of broadly targeted spatial metabolomics using multiple derivatization reagents.
  2. Metabolomics. 2022 Jun 16. 18(7): 41
      INTRODUCTION: Stable isotope tracer studies are increasingly applied to explore metabolism from the detailed analysis of tracer incorporation into metabolites. Untargeted LC/MS approaches have recently emerged and provide potent methods for expanding the dimension and complexity of the metabolic networks that can be investigated. A number of software tools have been developed to process the highly complex MS data collected in such studies; however, a method to optimize the extraction of valuable isotopic data is lacking.OBJECTIVES: To develop and validate a method to optimize automated data processing for untargeted MS-based isotopic tracing investigations of metabolism.
    METHODS: The method is based on the application of a suitable reference material to rationally perform parameter optimization throughout the complete data processing workflow. It was applied in the context of 13C-labelling experiments and with two different software, namely geoRge and X13CMS. It was illustrated with the study of a E. coli mutant impaired for central metabolism.
    RESULTS: The optimization methodology provided significant gain in the number and quality of extracted isotopic data, independently of the software considered. Pascal triangle samples are well suited for such purpose since they allow both the identification of analytical issues and optimization of data processing at the same time.
    CONCLUSION: The proposed method maximizes the biological value of untargeted MS-based isotopic tracing investigations by revealing the full metabolic information that is encoded in the labelling patterns of metabolites.
    Keywords:  Isotope labelling experiments; LC/MS; Parameter optimization; Untargeted analysis
  3. Front Pharmacol. 2022 ;13 894099
      Discovery of disease biomarker based on untargeted metabolomics is informative for pathological mechanism studies and facilitates disease early diagnosis. Numerous of metabolomic strategies emerge due to different sample properties or experimental purposes, thus, methodological evaluation before sample analysis is essential and necessary. In this study, sample preparation, data processing procedure and metabolite identification strategy were assessed aiming at the discovery of biomarker of breast cancer. First, metabolite extraction by different solvents, as well as the necessity of vacuum-dried and re-dissolution, was investigated. The extraction efficiency was assessed based on the number of eligible components (components with MS/MS data acquired), which was more reasonable for metabolite identification. In addition, a simplified data processing procedure was proposed involving the OPLS-DA, primary screening for eligible components, and secondary screening with constraints including VIP, fold change and p value. Such procedure ensured that only differential candidates were subjected to data interpretation, which greatly reduced the data volume for database search and improved analysis efficiency. Furthermore, metabolite identification and annotation confidence were enhanced by comprehensive consideration of mass and MS/MS errors, isotope similarity, fragmentation match, and biological source confirmation. On this basis, the optimized strategy was applied for the analysis of serum samples of breast cancer, according to which the discovery of differential metabolites highly encouraged the independent biomarkers/indicators used for disease diagnosis and chemotherapy evaluation clinically. Therefore, the optimized strategy simplified the process of differential metabolite exploration, which laid a foundation for biomarker discovery and studies of disease mechanism.
    Keywords:  UPLC-MS; biomarker discovery; breast cancer; strategy evaluation; untargeted metabolomics
  4. Anal Chem. 2022 Jun 14.
      Metabolomics is a mainstream approach for investigating the metabolic underpinnings of complex biological phenomena and is increasingly being applied to large-scale studies involving hundreds or thousands of samples. Although metabolomics methods are robust in smaller-scale studies, they can be challenging to apply to larger cohorts due to the inherent variability of liquid chromatography mass spectrometry (LC-MS). Much of this difficulty results from the time-dependent changes in the LC-MS system, which affects both the qualitative and quantitative performances of the instrument. Herein, we introduce an analytical strategy for addressing this problem in large-scale microbial studies. Our approach quantifies microbial boundary fluxes using two zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) columns that are plumbed to enable offline column equilibration. Using this strategy, we show that over 397 common metabolites can be resolved in 4.5 min per sample and that metabolites can be quantified with a median coefficient of variation of 0.127 across 1100 technical replicates. We illustrate the utility of this strategy via an analysis of 960 strains of Staphylococcus aureus isolated from bloodstream infections. These data capture the diversity of metabolic phenotypes observed in clinical isolates and provide an example of how large-scale investigations can leverage our novel analytical strategy.
  5. Steroids. 2022 Jun 08. pii: S0039-128X(22)00098-8. [Epub ahead of print]185 109060
      Androgens are endogenous hormones that play a crucial role in the paracrine and intracrine hormone system to perform and maintain vital physiological functions. Altered levels of androgens are implicated in many diseases such as sexual dysregulation, breast cancer, prostate cancer, and heart diseases etc. In this manuscript we describe a liquid chromatography-mass spectrometry (LC-MS) method using multiple reaction monitoring (MRM) for quantitatively measuring specific androgens such as dehydroepiandrosterone, testosterone, androsterone sulphate, androstenedione, and dihydrotestosterone in serum and urine samples. Serum acquired from nine different subjects (three pre-menopausal women, three postmenopausal women, and three healthy males) were used to evaluate the developed methods. In the sample preparation methods for serum either protein precipitation or liquid-liquid extraction (LLE) was used while the analysis of urinary androgens used LLE. The extracted androgens were quantitatively measured using LC-MRM-MS to which known amounts of stable isotope labeled standards were added. This manuscript also presents a LC-MRM-MS method mode for the analysis of oxime derivatized androgens potentially to enhance the sensitivity of the assay if required, from urine and venous-drawn serum samples.
    Keywords:  Androgens; Isotopic dilution; Liquid chromatography mass spectrometry; Method development; Plasma; Postmenopausal; Premenopausal; Urine
  6. Anal Chim Acta. 2022 Jul 11. pii: S0003-2670(22)00548-7. [Epub ahead of print]1216 339977
      Establishing a method for human biomonitoring (HBM) of polyphenols enables the assessment of internal concentrations of these food bio-actives and the correlation with potential health effects such as antioxidant or anti-inflammatory properties. Thus, a targeted LC-MS/MS method for quantifying up to 90 analytes, representing the main polyphenol classes including flavanones, isoflavones, stilbenes, and phenolic acids, was developed for human urine, serum, and plasma. The method was established for low sample volumes and with a cost and time efficient sample preparation protocol for high-throughput, which is critical for its application in large cohort and exposome-wide association studies. On average, the sample preparation yielded extraction efficiencies of 98% for urine, 98% for serum, and 87% for plasma. Limits of detection were between 0.11 ng mL-1 and 300 ng mL-1 for urine, 0.12 ng mL-1 and 190 ng mL-1 for serum, and 0.12 ng mL-1 and 340 ng mL-1 for plasma, excluding one analyte. In-house validation revealed that 66, 49, and 64 analytes for urine, serum, and plasma, respectively, fulfilled all stringent requirements, that are usually utilized for tailored single analyte methods, at all evaluated concentration levels. After validation, this method was applied in a proof-of-principle study that detected 39 polyphenols in urine. Changes in the concentrations of the analytes after the ingestion of a high polyphenol smoothie was examined over 24 h. The study further confirmed that the majority of polyphenols detected were phenolic acids, and phase II conjugated metabolites were more abundant than their respective non-conjugated forms.
    Keywords:  Exposome; Human biomonitoring (HBM); Plasma; Serum; Urine
  7. J Chromatogr Sci. 2022 Jun 16. pii: bmac047. [Epub ahead of print]
      Arginine and its metabolites play important roles in pain and analgesia. This study aimed to develop a comprehensive quantification method for amino acids and metabolites related to arginine metabolism in rat plasma by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS). Rat plasma was diluted to reduce the matrix effect and deproteinized with acetonitrile. The analytes were separated on a Syncronis HILIC column with a gradient elution. MS analysis was performed in positive ion mode with an electrospray ionization source using multiple reaction monitoring technology. All calibration curves for the 10 analytes showed good linear regression (R2 > 0.99). The limits of detection (LODs) were in the range of 0.9-13.4 μg/L. The established method was validated for intra-day and inter-day precisions (relative standard deviation [RSDs] < 6.21%) and accuracy (average recovery ranged from 87.34% to 100.35% with the RSD values less than 11.41%). This method was successfully applied to characterize dynamic alterations in the plasma of rats with neuropathic pain and thus provide service to explore the mechanism of action between metabolite changes and clinical disease.
  8. Bioinformatics. 2022 Jun 13. pii: btac388. [Epub ahead of print]
      MOTIVATION: 1H-NMR metabolomics is rapidly becoming a standard resource in large epidemiological studies to acquire metabolic profiles in large numbers of samples in a relatively low-priced and standardized manner. Concomitantly, metabolomics-based models are increasingly developed that capture disease risk or clinical risk factors. These developments raise the need for user-friendly toolbox to inspect new 1H-NMR metabolomics data and project a wide array of previously established risk models.RESULTS: We present MiMIR (Metabolomics-based Models for Imputing Risk), a graphical user interface that provides an intuitive framework for ad-hoc statistical analysis of Nightingale Health's 1H-NMR metabolomics data and allows for the projection and calibration of 24 pre-trained metabolomics-based models, without any pre-required programming knowledge.
    AVAILABILITY: The R-shiny package is available in CRAN or downloadable at, together with an extensive user manual (also available as Supplementary Documents to the paper).
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
  9. J Pharm Biomed Anal. 2022 Jun 11. pii: S0731-7085(22)00307-7. [Epub ahead of print] 114886
      Purine and pyrimidine metabolism are vital metabolic pathways in the development, proliferation or repairment of cells or tissues associated with various diseases. Here, a simple, all-in-one injection hydrophilic interaction liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of 20 metabolites: adenine, adenosine, deoxyadenosine, adenosine 5'-monophosphate, cyclic adenosine monophosphate, hypoxanthine, xanthine, inosine, deoxyinosine, xanthosine, xanthosine 5'-monophosphate and uric acid, which are products of purine metabolism; uridine, deoxyuridine, uridine 5'-monophosphate and uracil, are products of pyrimidine metabolism; and corticosterone, methionine, acetylcholine and serotonin. To minimize interference of endogenous molecules in sample matrixes, a combination of activated carbon adsorption and a serum substitute matrix (5% bovine serum albumin in phosphate buffered saline) was utilized and jointly applied. The sensitivity, linearity, stability, precision, accuracy and extraction recovery were evaluated, and the method was demonstrated to be accurate, sensitive and reliable. An analytical strategy was successfully applied to quantitatively determine 20 metabolite levels in the serum and hippocampus of mice with chronic social defeat stress-induced depression. The results showed greatly perturbed purine metabolism in the depressed mice, which was primarily characterized by dramatic increases in hypoxanthine, xanthine and inosine in serum and reduced levels of adenine, adenosine and adenosine 5'-monophosphate in the hippocampus. These findings suggest that this novel strategy can facilitate the quantitative analysis of adenine and other purine and pyrimidine metabolites in tissue and serum and exhibits great potential in the exploration of metabolism-related mechanisms of relevant diseases.
    Keywords:  Depression; HILIC-MS/MS; Purines and pyrimidines; Serum and hippocampus
  10. Nat Protoc. 2022 Jun 17.
      Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) has become a workhorse in global metabolomics studies with growing applications across biomedical and environmental sciences. However, outstanding bioinformatics challenges in terms of data processing, statistical analysis and functional interpretation remain critical barriers to the wider adoption of this technology. To help the user community overcome these barriers, we have made major updates to the well-established MetaboAnalyst platform ( ). This protocol extends the previous 2011 Nature Protocol by providing stepwise instructions on how to use MetaboAnalyst 5.0 to: optimize parameters for LC-HRMS spectra processing; obtain functional insights from peak list data; integrate metabolomics data with transcriptomics data or combine multiple metabolomics datasets; conduct exploratory statistical analysis with complex metadata. Parameter optimization may take ~2 h to complete depending on the server load, and the remaining three stages may be executed in ~60 min.
  11. J Sep Sci. 2022 Jun 18.
      Lipidomics analysis of zebrafish tissues has shown promising results to understand disease-related outcomes of exposure to toxic substances at molecular level. However, knowledge about their lipidome is limited, as most untargeted studies only identify the lipids that are statistically significant in their setup. In this work, liquid chromatography-high resolution mass spectrometry was used to study different aspects of the analytical workflow, i.e., extraction solvents (methanol/chloroform/water (3/2/2, v/v/v), methanol/dichloromethane/water (2/3/2, v/v/v) and methanol/methyl-tert-butyl ether/water (3/10/2.5, v/v/v), instrumental response, and strategies used for lipid annotation. The number of high-quality features (relative standard deviation of the intensity values ≤ 10% in the range 103 -107 counts) was affected by the dilution of lipid extracts, indicating that it is an important parameter for developing untargeted methods. The workflows used allowed the selection of a dilution factor to annotate 712 lipid species (507 bulk lipids) in zebrafish liver using four software (LipidMatch, LipidHunter, MS-DIAL and Lipostar). Retention time mapping was a valuable tool to filter lipid annotations obtained from automatic software annotations. The lipid profiling of zebrafish livers will help in a better understanding of the true constitution of their lipidome at the species level, as well as in the use of zebrafish in toxicological studies. This article is protected by copyright. All rights reserved.
    Keywords:  Danio rerio; Lipid extraction; Model organism; Sample preparation; Tandem mass spectrometry
  12. Anal Chim Acta. 2022 Jul 11. pii: S0003-2670(22)00552-9. [Epub ahead of print]1216 339981
      2/3-Hydroxy fatty acids (2/3-OHFAs) are a class of important biological molecules closely related to many diseases, of which the unsaturated ones (2/3-OHUFAs) obtain special recognition for their bioactivities. However, the comprehensive identification of 2/3-OHFAs has been a daunting task due to the similarity of isomeric structures and lack of authentic standards. Herein, we report a strategy for the 2/3-OHUFA identification by using an in-house synthesized derivatization reagent, 4-amino-1,1-dimethylpiperidin-1-ium iodide hydrochloride (ADMI). Through ADMI derivatization, the diagnostic ion m/z 155.1 or 171.1 were produced by liquid chromatography-mass spectrometry (LC-MS) analysis, which could distinguish the 2-OH or 3-OH group, respectively. Then the meta-chloroperoxybenzoic acid (mcpba) was used to resolve the locations of double bonds in 2/3-OHUFAs by the characteristic cleavage of the newly formed epoxides. Thus, the identification of 2/3-OHUFAs was enabled by all these characteristics. Moreover, in order to simplify the whole analysis, an isotope-labeled ADMI was designed to quickly pick out the low-abundant 2/3-OHFAs from complex biological matrices. Finally, a combined derivatization strategy was established to analyze mouse lung tissues with melanoma metastasis. Different long-chain 2-OHFAs and 3-OHFAs were identified, including FA 12:0-3OH, FA 12:1(Δ9)-3OH, FA 14:0-3OH, FA 14:1(Δ5)-3OH, FA 16:0-2/3OH, and FA 18:0-2/3OH. The results showed that the contents of most identified 2/3-OHFAs were lower in the cancer group than in the control group, suggesting the plausible role of 2/3-OHFAs in cancer development. In summary, the new derivatization method provides a powerful analysis strategy for 2/3-OHUFAs, which sheds light on a better understanding of the biological functions of 2/3-OHUFAs.
    Keywords:  ADMI derivatization; Mcpba oxidation; Melanoma lung metastasis; Stable isotope labeling; Unsaturated 2/3-hydroxyl fatty acid
  13. Res Pract Thromb Haemost. 2022 May;6(4): e12725
      Background: Emicizumab is a new treatment option for people with hemophilia A. Emicizumab was approved with a body-weight-based dosage regimen, without laboratory monitoring requirements. Guidelines, however, recommend measuring emicizumab concentrations when the presence of antidrug antibodies is suspected. Furthermore, drug monitoring can be useful in clinical decision making, in adherence checking, and for research purposes. Therefore, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying emicizumab. We performed a validation study on this LC-MS/MS method quantifying emicizumab in the plasma of people with hemophilia A.Methods: Sample preparation for LC-MS/MS analysis included ammonium sulfate protein precipitation and trypsin digestion. A signature peptide of emicizumab and a matching stable isotope-labeled internal standard were used to quantify emicizumab by LC-MS/MS analysis. Validation was performed in accordance with the "Guideline on Bioanalytical Method Validation" of the European Medicines Agency (EMA). The LC-MS/MS method was cross validated against a modified and calibrated (r 2 Diagnostics) one-stage clotting assay (OSA).
    Conclusions: The LC-MS/MS method demonstrated linearity over a wide range of emicizumab concentrations, far exceeding the concentrations observed in people with hemophilia A. Precision and accuracy were excellent, and all other validation parameters were also within the acceptance EMA criteria. Cross validation showed that the LC-MS/MS method and the OSA-based method can be used interchangeably for drug monitoring of emicizumab without the application of a correction factor.
    Keywords:  drug monitoring; emicizumab; hemophilia A; mass spectrometry; validation study
  14. Analyst. 2022 Jun 16.
      A direct mass spectrometry method utilizing reactive paper spray ionization was developed for sensitive cannabinoid quantitation in biofluid matrices. The ca. 2-minute sample measurements used on-paper derivatization to significantly increase paper spray mass spectrometry (PS-MS) positive ion mode sensitivity while minimizing sample preparation steps. Calibrations demonstrate high linearity, with R2 > 0.99 for (-)-trans-Δ9-tetrahydrocannabinol (THC) in oral fluid and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) in urine. The limit of detection and lower limit of quantitation were 0.78 and 10 ng mL-1 for THC in oral fluid and 1.3 and 10 ng mL-1 for THC-COOH in urine, respectively. THC-COOH levels measured by reactive PS-MS in seven spiked human urine samples showed bias of -9.4 to 5.9%, and percent difference values of -16.8 to 9.8% in comparison with a reference LC-MS method. Based upon the method simplicity, validation experiments, sensitivity, and rapidity, we conclude that reactive PS-MS has potential applications for rapid cannabinoid drug testing in urine and oral fluid.
  15. Metabolomics. 2022 Jun 14. 18(6): 40
      INTRODUCTION: Accuracy of feature annotation and metabolite identification in biological samples is a key element in metabolomics research. However, the annotation process is often hampered by the lack of spectral reference data in experimental conditions, as well as logistical difficulties in the spectral data management and exchange of annotations between laboratories.OBJECTIVES: To design an open-source infrastructure allowing hosting both nuclear magnetic resonance (NMR) and mass spectra (MS), with an ergonomic Web interface and Web services to support metabolite annotation and laboratory data management.
    METHODS: We developed the PeakForest infrastructure, an open-source Java tool with automatic programming interfaces that can be deployed locally to organize spectral data for metabolome annotation in laboratories. Standardized operating procedures and formats were included to ensure data quality and interoperability, in line with international recommendations and FAIR principles.
    RESULTS: PeakForest is able to capture and store experimental spectral MS and NMR metadata as well as collect and display signal annotations. This modular system provides a structured database with inbuilt tools to curate information, browse and reuse spectral information in data treatment. PeakForest offers data formalization and centralization at the laboratory level, facilitating shared spectral data across laboratories and integration into public databases.
    CONCLUSION: PeakForest is a comprehensive resource which addresses a technical bottleneck, namely large-scale spectral data annotation and metabolite identification for metabolomics laboratories with multiple instruments. PeakForest databases can be used in conjunction with bespoke data analysis pipelines in the Galaxy environment, offering the opportunity to meet the evolving needs of metabolomics research. Developed and tested by the French metabolomics community, PeakForest is freely-available at .
    Keywords:  Curation; Database; FAIR; Interoperability; Metabolite identification; Spectral library
  16. Food Chem. 2022 May 28. pii: S0308-8146(22)01298-5. [Epub ahead of print]393 133336
      Polar lipids in milk are receiving increasing interest due to their bioactivities. However, milk polar lipids present a wide range of physical-chemical properties at different concentrations, making their analysis challenging. In this study, we presented a comprehensive lipidomic method using ultraperformance supercritical fluid chromatography (UPSFC)-quadrupole-time of flight-mass spectrometry (Q-TOF-MS), which enabled the separation of 18 lipid classes (including nonpolar lipids, cholesterol, ceramide, glycerophospholipids, sphingomyelin, and gangliosides) within 10 min. The method was used to analyze the polar lipids in seven samples, including human milk, other mammalian milk and milk fat globule membrane ingredients, identifying 14 lipid classes containing 219 lipid molecular species. A mass spectrometry data processing strategy applicable for high-throughput studies was also developed and validated.
    Keywords:  Human milk; Membrane lipids; Polar lipid; Sphingomyelin; Ultraperformance supercritical fluid chromatography
  17. Sci Rep. 2022 Jun 17. 12(1): 10208
      Although nereistoxin insecticides (NIs) are banned for animal husbandry operations, they are still used because of their high insecticidal activities. Therefore, a reliable residue analysis method for the simultaneous detection of cartap, bensultap, thiocyclam, and nereistoxin in foods of animal origins, including beef, pork, chicken, milk, and eggs, was developed using hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC-MS/MS). The NIs were extracted with an acidic cysteine and formate buffer solution and hydrolyzed to nereistoxin. The molarity and pH of the buffer were optimized at 20 mM and 3, respectively, to keep the pH of the extracts at 4-5. pH-dependent acid-base partitioning coupled with salting-out-assisted liquid-liquid extraction using acetonitrile was performed for purification and for the direct introduction of the extracts to LC. The optimal pH values were 5 and 9 for the acid-base partitioning. Nereistoxin quantitation was achieved with consistent column retention (RSD < 0.6%) and a high degree of separation (N > 106). The matrix-dependent method limit of quantitation was 2 μg nereistoxin/kg, and the calibration curve showed good linearity (R2 > 0.998). The recovery efficiencies were in the range of 89.2-109.9% with relative standard deviations less than 10%, and matrix effects did not exceed ± 10%, which satisfied the criteria outlined in the European SANTE/12682/2019 guidelines.
  18. Clin Chem. 2022 Jun 14. pii: hvac070. [Epub ahead of print]
      BACKGROUND: Newborn screening (NBS) laboratories in the United Kingdom adhere to common protocols based on single analyte cutoff values (COVs); therefore, interlaboratory harmonization is of paramount importance. Interlaboratory variation for screening analytes in UK NBS laboratories ranges from 17% to 59%. While using common stable isotope internal standards has been shown to significantly reduce interlaboratory variation, instrument set-up, sample extraction, and calibration approach are also key factors.METHODS: Dried blood spot (DBS) extraction processes, instrument set-up, mobile-phase composition, sample introduction technique, and calibration approach of flow injection analysis-tandem mass spectrometry (FIA-MS/MS) methods were optimized. Inter- and intralaboratory variation of methionine, leucine, phenylalanine, tyrosine, isovaleryl-carnitine, glutaryl-carnitine, octanoyl-carnitine, and decanoyl-carnitine were determined pre- and postoptimization, using 3 different calibration approaches.
    RESULTS: Optimal recovery of analytes from DBS was achieved with a 35-min extraction time and 80% methanol (150 μL). Optimized methodology decreased the mean intralaboratory percentage relative SD (%RSD) for the 8 analytes from 20.7% (range 4.1-46.0) to 5.4% (range 3.0-8.5). The alternative calibration approach reduced the mean interlaboratory %RSD for all analytes from 16.8% (range 4.1-25.0) to 7.1% (range 4.1-11.0). Nuclear magnetic resonance analysis of the calibration material highlighted the need for standardization. The purities of isovaleryl-carnitine and glutaryl-carnitine were 85.13% and 69.94% respectively, below the manufacturer's stated values of ≥98%.
    CONCLUSIONS: For NBS programs provided by multiple laboratories using single analyte COVs, harmonization and standardization of results can be achieved by optimizing legacy FIA-MS/MS methods, adopting a common analytical protocol, and using standardized calibration material rather than internal calibration.
    Keywords:  MS/MS; inherited disorders; interlaboratory performance; laboratory methods and tools; mass spectrometry; newborns; proficiency testing
  19. Chem Res Toxicol. 2022 Jun 13.
      Per- and poly-fluorinated substances (PFASs) are organic pollutants that have been linked to numerous health effects, including diabetes, cancers, and dysregulation of the endocrine system. This study aims to develop a liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay to measure changes in 17 hormones in H295R cell line (a steroid producing adrenocortical cells) upon exposure to PFASs. Due to the challenges in the analysis of steroid hormones using electrospray ionization MS, a chemical derivatization method was employed to achieve 0.07-2 μg/L detection limits in LC-MS/MS. Furthermore, a 10-fold concentration factor through solid-phase extraction (SPE) allows for consistent sub-parts per billion detections. Optimization of the derivatization conditions showed doubly-derivatized products in some hormone analytes, including progesterone, corticosterone, and cortisol, and gave improved ionization efficiency up to 20-fold higher signal than the singly-derivatized product. The use of SPE for sample cleanup to analyze hormones from cellular media using weak anion exchange sorbent yielded 80-100% recovery for the 17 targeted hormones. The method was validated by exposing H295R cells to two known endocrine disruptors, forskolin and prochloraz, which showed expected changes in hormones. An initial exposure of H295R cells with various PFAS standards and their mixtures at 1 μM showed significant increases in progestogens with some PFAS treatments, which include PFBS, PFHxA, PFOS, PFDA, and PFDS. In addition, modest changes in hormone levels were observed in cells treated with other sulfonated or carboxylated headgroup PFASs. This sensitive LC-MS/MS method for hormone analysis in H295R cells will allow for the investigations of the alterations in the hormone production caused by exposure to various environmental insults in cell-based assays and other in vitro models.
  20. J Chromatogr A. 2022 Jun 21. pii: S0021-9673(22)00385-5. [Epub ahead of print]1673 463192
      Effective purification and enrichment of polypeptide antibiotics in animal tissues is always a challenge, due to the co-extraction of other endogenous peptides which usually interfere their final determination. In this study, a molecularly imprinted column was prepared by packing polymyxin E-imprinted particles into a 100 mm × 4.6 mm i.d. HPLC column. The as-prepared imprinted columns were able to tolerate 100% aqueous phase and exhibited good stability and high column efficiency. Polypeptides antibiotics with similar molecular size or spatial structure to polymyxin E were well retained by the imprinted column, suggesting class selectivity. After optimization of mobile phase conditions of imprinted column, polypeptide antibiotics in animal tissue extracts were enriched and cleaned up by in-line molecularly imprinted solid-phase extraction, allowing the screening of target analytes in complex samples at low concentration levels by UV detection. Eluate fraction from the imprinted column was collected, and further dried and re-dissolved with methanol-0.5% formic acid aqueous solution (80:20, v/v) for final LC-MS/MS analysis. Analysis was accomplished using multiple reaction monitoring (MRM) in positive electrospray ionization mode and analytes quantified using the matrix-matched external calibration curves. The results showed high correlation coefficients for target analytes in the linear range of 2 ∼ 200 μg kg-1. At four different concentration levels (limit of quantification, 50, 100 and 200 μg kg-1), recoveries of four polypeptide antibiotics in swine, cattle and chicken muscles ranged from 66.7 to 94.5% with relative standard deviations lower than 16.0%. The limits of detection (LOD) were 2.0 ∼ 4.0 μg/kg, depending upon the analyte and sample. Compared with a conventional pretreatment method, the imprinted column was able to remove more impurities and to significantly reduce matrix effects, allowing the accurate analysis of polypeptide antibiotics.
    Keywords:  Animal tissue; Imprinted stationary phase; Molecular imprinting; Polypeptide antibiotics; Sample preparation
  21. Rapid Commun Mass Spectrom. 2022 Jun 15. e9335
      RATIONALE: In 2012, arterolane (ART) in combination with piperaquine received approval in India for the treatment of plasmodium induced malaria, however, till date, the detailed metabolite identification study of ART has not been reported. Being polar in nature, ART shows early elution on reversed phase columns which might be the rate-limiting factor of its systematic analytical studies. We have utilized hydrophilic interaction liquid chromatography (HILIC) to separate in vitro and in vivo metabolites of ART.METHODS: The possible sites of metabolism were predicted by XenoSite software to get an initial assessment. In vitro studies were conducted by incubating the drug with liver microsomes such as human, rat and human S9 fractions. Later, in vivo studies were performed to check the metabolites in urine, faeces and plasma. The samples were pooled and subjected to the protein precipitation method before LC-QTOF analysis.
    RESULTS: We have observed 15 metabolites in this study which were phase I metabolites formed due to hydroxylation, dihydroxylation, peroxide bond scission and oxidation. Here, we report 11 metabolites of ART for the first time. The metabolic pathways and plausible structures were proposed according to accurate mass measurements and its MS/MS data.
    CONCLUSION: The present study comprehensively reports in vitro and in vivo metabolism of ART mentioning 11 novel metabolites. Here, extensive use of HILIC chromatography has helped to efficiently separate various metabolites. These findings would help prospects of ART disposition and its congeners.
  22. Front Chem. 2022 ;10 908572
      The exposure of human DNA to genotoxic compounds induces the formation of covalent DNA adducts, which may contribute to the initiation of carcinogenesis. Liquid chromatography (LC) coupled with high-resolution mass spectrometry (HRMS) is a powerful tool for DNA adductomics, a new research field aiming at screening known and unknown DNA adducts in biological samples. The lack of databases and bioinformatics tool in this field limits the applicability of DNA adductomics. Establishing a comprehensive database will make the identification process faster and more efficient and will provide new insight into the occurrence of DNA modification from a wide range of genotoxicants. In this paper, we present a four-step approach used to compile and curate a database for the annotation of DNA adducts in biological samples. The first step included a literature search, selecting only DNA adducts that were unequivocally identified by either comparison with reference standards or with nuclear magnetic resonance (NMR), and tentatively identified by tandem HRMS/MS. The second step consisted in harmonizing structures, molecular formulas, and names, for building a systematic database of 279 DNA adducts. The source, the study design and the technique used for DNA adduct identification were reported. The third step consisted in implementing the database with 303 new potential DNA adducts coming from different combinations of genotoxicants with nucleobases, and reporting monoisotopic masses, chemical formulas, .cdxml files, .mol files, SMILES, InChI, InChIKey and IUPAC nomenclature. In the fourth step, a preliminary spectral library was built by acquiring experimental MS/MS spectra of 15 reference standards, generating in silico MS/MS fragments for all the adducts, and reporting both experimental and predicted fragments into interactive web datatables. The database, including 582 entries, is publicly available ( This database is a powerful tool for the annotation of DNA adducts measured in (HR)MS. The inclusion of metadata indicating the source of DNA adducts, the study design and technique used, allows for prioritization of the DNA adducts of interests and/or to enhance the annotation confidence. DNA adducts identification can be further improved by integrating the present database with the generation of authentic MS/MS spectra, and with user-friendly bioinformatics tools.
    Keywords:  DNA adduct; carcinogenesis; database; identification; mass spectrometry; toxicology
  23. Anal Chem. 2022 Jun 13.
      The importance of multi-omic-based approaches to better understand diverse pathological mechanisms including neurodegenerative diseases has emerged. Spatial information can be of great help in understanding how biomolecules interact pathologically and in elucidating target biomarkers for developing therapeutics. While various analytical methods have been attempted for imaging-based biomolecule analysis, a multi-omic approach to imaging remains challenging due to the different characteristics of biomolecules. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful tool due to its sensitivity, chemical specificity, and high spatial resolution in visualizing chemical information in cells and tissues. In this paper, we suggest a new strategy to simultaneously obtain the spatial information of various kinds of biomolecules that includes both labeled and label-free approaches using ToF-SIMS. The enzyme-assisted labeling strategy for the targets of interest enables the sensitive and specific imaging of large molecules such as peptides, proteins, and mRNA, a task that has been, to date, difficult for any MS analysis. Together with the strength of the analytical performance of ToF-SIMS in the label-free tissue imaging of small biomolecules, the proposed strategy allows one to simultaneously obtain integrated information of spatial distribution of metabolites, lipids, peptides, proteins, and mRNA at a high resolution in a single measurement. As part of the suggested strategy, we present a sample preparation method suitable for MS imaging. Because a comprehensive method to examine the spatial distribution of multiple biomolecules in tissues has remained elusive, our strategy can be a useful tool to support the understanding of the interactions of biomolecules in tissues as well as pathological mechanisms.
  24. J Mass Spectrom Adv Clin Lab. 2022 Aug;25 12-18
      Introduction: Advances in liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) have enabled the quantification of immunosuppressants using microsampling techniques. In this context, dried matrix on paper discs (DMPD) could be a useful alternative to conventional venipuncture. Although analytical validation is necessary to establish the suitability of method performance, it is not sufficient to proceed with its implementation into routine clinical practice. Also necessary is that equivalence between sampling methods be demonstrated in a clinical validation study.Objetives: To clinically validate a LC-MS/MS method for the quantification of tacrolimus, sirolimus, everolimus and cyclosporin A using DMPD.
    Methods: According to the recommendations of international guidelines, at least 40 whole blood (WB) and DMPD paired samples for each analyte were collected by skilled technicians and analyzed using LC-MS/MS. Results were evaluated in terms of statistical agreement and bias values at medical decision points.
    Results: For all analytes, Passing-Bablok regression analysis revealed that confidence intervals (CIs) for slopes and intercepts included 1 and 0, respectively. It also showed that biases at medical decision points were not clinically relevant. No statistically significant differences between DMPD and WB were found using difference plots and agreement analysis. In this regard, CIs for bias estimators included 0, and more than 95% of the results fell within the limits of agreement.
    Conclusion: The feasibility of the clinical application of simultaneous quantification of tacrolimus, sirolimus, everolimus and cyclosporin A in DMPD was demonstrated. Results showed that this microsampling technique is interchangeable with conventional WB sampling when specimens are collected by trained personnel.
    Keywords:  Clinical validation; Dried matrix on paper discs (DMPD); Immunosuppressants; Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS); Method comparison
  25. Biomed Chromatogr. 2022 Jun 16. e5428
      Unconjugated bile acids (BAs) have gained more attention than conjugated BAs in the association studies among diet, gut microbiota and diseases. Gas chromatography-mass spectrometry (GC-MS) is probably a good choice for specialized analysis of unconjugated BAs due to high separation capacity and identification convenience. However, few reports have focused on the rodent unconjugated BAs by GC-MS, and the main library for identification has not included rodent-specific BAs. We developed a GC-MS method for targeted profiling of eight main unconjugated BAs in rodent models, which showed excellent performance on sensitivity, reproducibility and accuracy. Quantitative reproducibility in terms of relative standard deviation (RSD) was in the range of 2.05%-2.91%, with detection limits of 3-55 ng/mL, quantitation limits of 9-182 ng/mL and the recovery rate of 72%-115%. All the calibration curves displayed good linearity with correlation coefficient values (R2 ) more than 0.99. Using the established method, the influence of high-fat diet on the metabolism of unconjugated BAs were revealed. Significant increasing of fecal unconjugated BAs induced by high-fat diet, would be a potential risk to gut inflammation and cancer. The study provides a convenient and targeted GC-MS method for specialized profiling of rodent unconjugated BAs in physiological and pathological studies.
    Keywords:  GC-MS; High-fat diet; Targeted analysis; Unconjugated BAs
  26. J Food Drug Anal. 2021 Sep 15. 29(3): 502-509
      Cosmetic products containing hemp seed oil as permitted raw materials required the specific compound delta-9-tetrahydrocannabinol (THC) below 10 μg/g. THC was the main psychoactive constituent of cannabis. Since hemp seed oil became an increasingly popular ingredient in cosmetics over the last few years, an efficient and reliable analytical method for THC and other cannabinoids in cannabis-infused cosmetic products was in need. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) in hemp seed oil based cosmetic products was developed. Method validation was performed by fertilizing blank samples with analytes and internal standards (THC-d3, CBD-d3, and CBN-d3). Chromatographic method utilized a Xbridge BEH Shield RP18 column with gradient elution containing 10 mM ammonium formate in water and methanol provided successful separation of THC, CBD, and CBN in cosmetic matrix. The combination of MS detection in positive electrospray ionization (ESI) and multiple reaction monitoring (MRM) mode offered rapid run time 13 minutes with limit of quantification (LOQ) of 0.05 μg/g. The intra- and inter-day recoveries were 79.23-114.04% and 83.55-111.61% with spiking levels ranged between 0.05 μg/g and 0.5 μg/g, respectively. Surveillance results of 90 cosmetic products showed 22, 34, and 5 products containing THC (0.06-1777 μg/g), CBD (0.47-37217 μg/g), and CBN (2.2-25.2 μg/g), respectively. This validated method offered accurate, reliable, and fast way for the determination of drug contaminations including THC, CBD, and CBN in cosmetics. The surveillance results for commercial cosmetic products purchased in Taiwan between 2018-2020 provided valuable background references for THC, CBD, and CBN in hemp seed oil based cosmetic products, and could be used for administration purpose.
  27. Anal Chim Acta. 2022 Aug 01. pii: S0003-2670(22)00554-2. [Epub ahead of print]1219 339983
      As a non-invasive biological matrix, the placenta offers great and novel opportunities to monitor fetal exposure to exogenous chemicals and their biotransformation products (the internal chemical exposome), as well as the biological responses associated, in large-scale epidemiological studies. However, it is first crucial to ensure that analytical methods based on high-resolution mass spectrometry (HRMS) can detect the low abundant components of the internal chemical exposome present in these complex biological matrices. In this study, we aimed to develop a robust analytical method (extraction and sample preparation) sensitive enough to profile the internal chemical exposome and the metabolome of placenta using high-resolution mass spectrometry (HRMS) for future application in mother child cohorts. Several extraction solvents (methanol, methanol/H2O (50/50 v/v), methyl tert-butyl ether/methanol/H2O) were tested and their ability to extract components of the internal exposome and metabolome were compared. Then, sample preparation methods commonly used for metabolomics application (methanolic protein precipitation) were compared with solid phase extraction (SPE), protein and phospholipid removal plates (PPRP) and combination of SPE and PPRP. The methods were compared and validated using qualitative (i.e., numbers of features and chemical classes ID), quantitative parameters adopted from targeted multi-residue analysis (recovery experiments, repeatability and matrix effect) as well as the ability of these methods to be implemented for high-throughput applications. The analytical repeatability of the two most effective methods (methanolic extraction followed by either protein precipitation or PPRP) were tested at the batch level to determine the best concentration factors to be used for improving detection of components of the internal chemical exposome and metabolome without impacting on the analytical response. Finally, these two methods based on protein precipitation and PPRP were tested on 40 placenta samples from the French PELAGIE birth cohort, and annotation was performed on the related datasets to compare the respective impacts of PPT and PPRP. A wide range of exogenous (e.g., biocides, pharmaceuticals, personal care products) and endogenous chemicals (steroids, prostanoids, lipids, carnitins) could be detected and annotated (some of them for the first time in placenta). We show that both methods are complementary but that PPRP allows the injection of more concentrated extracts without impacting the LC repeatability and therefore improve the detection (presence and signal area fold change) of many exogenous and endogenous chemicals.
    Keywords:  High-resolution mass spectrometry; Non-targeted analyses; Placenta; Sample preparation; Suspect screening
  28. J Food Drug Anal. 2021 Sep 15. 29(3): 419-432
      The compliance assessment on the labeling of food additives is a hard job, because there are nearly thousand legal food additives can be used in food, and countless illegal additives must also deal with. This study developed a non-targeted data acquisition screening method based on liquid chromatography high resolution mass spectrometry (HRMS) in which a precursor ion and two product ions of each analyte are able to be recorded. The high throughput screening method worked as foodomics that characterized and identified every food components as long as they were ionized in terms of theory. The data acquisition method called data independent acquisition (DIA) was achieved by a full scan form m/z 70-1050, and then followed wide window fragmentations of product ions recording. A full scan and the followed fragmentations generated 21 spectra in 2.6 s contributed about 6 data points for a typical 0.2-0.3 min width peak in HPLC. A detection database list of 120 additives included 79 colorants, 13 sweeteners, 12 preservatives and 7 antioxidants was established. Thirty-three commercial samples including beverages, candies, and sauces were surveyed for testing additives. Sweeteners (rebaudioside A) and flavoring agents (malic acid and fumaric acid) were found the most under declared additives. HPLC column often do not provide adequate retention for highly polar compounds such as organic acids (flavoring agents). In this study they were coeluted, but were able to be separated and determined by HRMS worked as the secondary separation tool. The surveillance results showed there is still room for food manufacturers to improve the connection between their product information and consumers.
  29. Methods Mol Biol. 2022 ;2528 127-143
      R-loops are three-stranded nucleic acid structures consisting of an RNA-DNA hybrid and an unpaired strand of nontemplate DNA that represent a major source of genomic instability and are involved in regulation of several important biological processes in eukaryotic cells. A growing body of experimental evidence suggests that RNA moieties of RNA-DNA hybrids may convey RNA modifications influencing various aspects of R-loop biology. Here we present a protocol for quantitative analysis of RNA modifications on RNA-DNA hybrids using stable-isotope dilution ultraperformance liquid chromatography coupled with tandem mass spectrometry (SID-UPLC-MS/MS). Supplemented by other techniques, this method can be instrumental in deciphering the roles of RNA modifications in R-loop metabolism.
    Keywords:  N6-methyladenosine; R-loops; RNA modifications; RNA–DNA hybrids; RNase H; Tandem mass spectrometry