bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒11‒15
seventeen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Anal Methods. 2020 Nov 11.
      Vitamin D metabolites are fat-soluble vitamins that regulate broad spectrum of physiological and pathological processes. Accurate and high-throughput methods for the detection of vitamin D metabolites are essential to elucidate body functions. In this study, a sensitive and high throughput ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method was proposed for the accurate quantification of six vitamin D metabolites, including vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D2, and 1,25-dihydroxyvitamin D3. Through the optimization of chromatographic and mass spectrometric conditions, only 20 μL serum or plasma could satisfy the quantification of six vitamin D metabolites. The limit of detection (LOD) was determined to be 0.02-0.05 pg mL-1. The validation method was carried out following the International Council for Harmonization (ICH) guidelines. All quantification performances, including linearity, accuracy, precision, extraction recovery and matrix effect, were investigated and were satisfactory for the accurate detection of vitamin D metabolites. A disease of the liver or kidney, the main organs for vitamin D metabolism, could lead to abnormal levels of vitamin D. Here, the established UHPLC-MS/MS method was further used for determination of vitamin D levels in plasma samples of patients after liver or kidney transplantation. Thirty-three liver transplant recipients (LTRs) and 63 kidney transplant recipients (KTRs) were included in this study. Vitamin D deficiency or insufficiency is common in KTRs and LTRs, with a prevalence of more than 99%.
  2. Nat Biotechnol. 2020 Nov 09.
      We engineered a machine learning approach, MSHub, to enable auto-deconvolution of gas chromatography-mass spectrometry (GC-MS) data. We then designed workflows to enable the community to store, process, share, annotate, compare and perform molecular networking of GC-MS data within the Global Natural Product Social (GNPS) Molecular Networking analysis platform. MSHub/GNPS performs auto-deconvolution of compound fragmentation patterns via unsupervised non-negative matrix factorization and quantifies the reproducibility of fragmentation patterns across samples.
  3. Methods Mol Biol. 2021 ;2234 271-295
      A method based on reversed phase high-performance liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry (RP-HPLC-ESI-HRMS) for the comprehensive and reliable detection of secondary metabolites of Trichoderma reesei cultured in synthetic minimal liquid medium is presented. A stable isotope-assisted (SIA) workflow is used, which allows the automated, comprehensive extraction of truly fungal metabolite-derived LC-MS signals from the acquired chromatographic data. The subsequent statistical data analysis and a typical outcome of such a metabolomics data evaluation are shown by way of example in a previously published study on the influence of the pleiotropic regulator transcription factor Xylanase promoter binding protein 1 (Xpp1) in T. reesei on secondary metabolism.
    Keywords:  Liquid chromatography–high-resolution mass spectrometry; Metabolomics; Stable isotope-assisted workflow; Stable isotopic labeling
  4. Biomed Chromatogr. 2020 Nov 11. e5027
      The quantification of the circulating dehydroepiandrosterone sulfate (DHEAS) might be of diagnostic help for several diseases. For the DHEAS quantification, LC/ESI-MS/MS has the advantage of a high specificity compared to immunoassay, whereas LC/ESI-MS/MS has room to improve the analysis throughput. One of the promising solutions to enhance the analysis throughput is sample-multiplexing in the same injection, which can reduce the total LC/ESI-MS/MS run time. In this study, a quadruplex LC/ESI-MS/MS method was developed to quantify DHEAS in four different serum samples in a single run. After the four samples were separately deproteinized and derivatized with one of four Girard reagents (Girard reagent T, P and their isotopologues), the resulting samples were mixed, then injected into the LC/ESI-MS/MS. The applicability and advantage of the developed method were evaluated based on the analysis of 9 batches of serum samples from healthy subjects (total 36 samples). The limit of quantitation was 0.050 μg/mL, which was sensitive enough for clinical laboratory use. The method was precise (intra- and inter-assay RSDs ≤ 3.6%), accurate (94.4-108.1%) and robust for the matrix effects. The analysis time was also shortened by about 60% for 36 samples by the introduced method compared to the conventional method.
    Keywords:  LC/ESI-MS/MS; dehydroepiandrosterone sulfate; derivatization; high throughput; sample-multiplexing
  5. J Chromatogr A. 2020 Oct 29. pii: S0021-9673(20)30931-6. [Epub ahead of print]1634 461657
      The 25R-NBOH family is a group of thermally labile compounds that are relevant for forensic sciences and traditionally analyzed by GC-MS after derivatization - a step that is time consuming in a routine work. In this paper, the use of short analytical columns (4 and 10 m) showed to decrease compound degradation in the GC oven during chromatographic separation and to allow the analysis of non-derivatized 25R-NBOH compounds by GC-MS. A shorter column demanded a higher gas flow rate, and both factors decreased residence time of the analytes in the column and their degradation. The inlet temperature (250° C or 280°C) did not impact the response of 25R-NBOH. A 25R-NBOH fragmentation pathway by electron ionization was also presented for the first time. The GC-MS method with a 4 m column was successfully applied to other compounds of forensic interest, and it can be tested in the analysis of biological samples in toxicological investigations.
    Keywords:  25R-NBOH; GC-MS; Short column; Thermal degradation
  6. Clin Chim Acta. 2020 Nov 09. pii: S0009-8981(20)30526-X. [Epub ahead of print]
      INTRODUCTION: The quantitation of glucagon remains challenging immunoassay, mainly due to cross-reactivity. A sensitive, rapid and specific intact glucagon method is therefore necessary for quality routine analysis. A tandem mass spectrometry method to fulfill this objective is described in this work.METHODS: Glucagon was extracted from plasma employing a mixed-mode anion exchange Solid-Phase Extraction. Sample stability was assessed in K2-EDTA and P800 tubes at different temperatures. We compared our method to two different immunoassays. FDA and EMA guidelines were followed for validation. An external quality control program served for comparison with other laboratories.
    RESULTS: Assay imprecision was below 4%. Recoveries were within 95-103%. LoQ was 8.75 pg/mL. Total analytical CV was 2.91%. Samples were found stable at 4°C for less than 4 hours. Diasource RIA disagreed with our method. Mercodia ELISA provided a closer agreement, also proven by external quality control samples.
    CONCLUSIONS: A rapid and specific LC-MS/MS method for glucagon quantitation has been developed, validated and is suitable to routine care. The simplicity and the good performances in terms of time and specificity, could open the possibility to establish a standardized method for glucagon.
    Keywords:  LC-MS/MS; glucagon; liquid chromatography; mass spectrometry
  7. Talanta. 2021 Jan 15. pii: S0039-9140(20)30916-4. [Epub ahead of print]222 121625
      Data-driven engineering of microbes has been demonstrated for the sustainable production of high-performance chemicals. Metabolic profiling analysis is essential to increase the productivity of target compounds. However, improvement of comprehensive analysis methodologies is required for the high demands of metabolic engineering. Therefore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based methodology was designed and applied to cover a wide target range with high precision. Ion-pair free separation of metabolites on a pentafluorophenyl propyl column enabled high-precision quantification of 113 metabolites. The method was further evaluated for high reproducibility and robustness. Target analytes consisted of primary metabolites and intermediate metabolites for microbial production of high-performance chemicals. 95 metabolites could be detected with high reproducibility of peak area (intraday data: CV<15%), and 53 metabolites could be sensitively determined within a wide dynamic linear range (3-4 orders of magnitude). The developed system was further applied to the metabolomic analysis of various prokaryotic and eukaryotic microorganisms. Differences due to culture media and metabolic phenotypes could be observed when comparing the metabolomes of conventional and non-conventional yeast. Furthermore, almost all Kluyveromyces marxianus metabolites could be detected with moderate reproducibility (CV<40%, among independent extractions), where 41 metabolites were detected with very high reproducibility (CV<15%). In addition, the accuracy was validated via a spike-and-recovery test,and 78 metabolites were detected with analyte recovery in the 80-120% range. Together these results establish ion-pair free metabolic profiling as a comprehensive and precise tool for data-driven bioengineering applications.
    Keywords:  Ion-pair free; Liquid chromatography-tandem mass spectrometry; Metabolomics; Microbes; Pentafluorophenyl propyl column
  8. Am J Clin Nutr. 2020 Nov 12. pii: nqaa295. [Epub ahead of print]
      BACKGROUND: Serum 25-hydroxyvitamin D [25(OH)D] concentration is an indicator of vitamin D exposure, but it is also influenced by clinical characteristics that affect 25(OH)D production and clearance. Vitamin D is the precursor to 25(OH)D but is analytically challenging to measure in biological specimens.OBJECTIVES: We aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification of vitamins D3 and D2 in serum and to explore the potential of circulating vitamin D as a biomarker of exposure in supplementation trials.
    METHODS: The method was validated using guideline C62-A from the Clinical and Laboratory Standards Institute and was applied in 2 pilot clinical trials of oral vitamin D3 supplementation. Pilot study 1 included 22 adults randomly assigned to placebo or 2000 IU/d. Blood was collected at baseline, 1, 3, 6, and 12 mo. Pilot study 2 included 15 adults randomly assigned to 2000 or 4000 IU/d. Blood and subcutaneous (SUBQ) adipose tissue were collected at baseline and 3 mo.
    RESULTS: In study 1, mean change (baseline to 3 mo) in serum vitamin D3 was -0.1 ng/mL in the placebo group and 6.8 ng/mL in the 2000 IU/d group (absolute difference: 6.9; 95% CI: 4.5, 9.3 ng/mL). In study 2, mean change (baseline to 3 mo) in serum vitamin D3 was 10.4 ng/mL in the 2000 IU/d group and 22.2 ng/mL in the 4000 IU/d group (fold difference: 2.15; 95% CI: 1.40, 3.37). Serum and adipose tissue vitamin D3 concentrations were correlated, and the dose-response of vitamin D3 in adipose mirrored that in serum.
    CONCLUSIONS: We validated a sensitive, robust, and high-throughput LC-MS/MS method to quantify vitamins D3 and D2 in serum. Serum and SUBQ adipose tissue vitamin D3 concentrations increased proportionally to dose with 3 mo of daily supplementation.These trials were registered at as NCT00552409 (pilot study 1) and NCT01477034 (pilot study 2).
    Keywords:  Clinical and Laboratory Standards Institute; cholecalciferol; ergocalciferol; liquid chromatography–tandem mass spectrometry; liquid-liquid extraction; validation; vitamin D
  9. Talanta. 2021 Jan 15. pii: S0039-9140(20)30871-7. [Epub ahead of print]222 121580
      Feature detection is a crucial pre-processing step for high-resolution liquid chromatography-mass spectrometry (LC-MS) data analysis. Typical practices based on thresholds or rigid mathematical assumptions can cause ineffective performance in detecting low abundance and non-ideal distributed compounds. We herein introduce a novel feature detection method based on deep learning named SeA-M2Net that considers feature detection as an image-based object detection task. By fully employing raw data directly, and integrating all related factors (e.g., LC elution, charge state, and isotope distribution) with two-dimensional pseudo color images to calculate the probability of the presence of the compound, low abundance compounds can be well preserved and observed. More importantly, SeA-M2Net, with deep multilevel and multiscale structures focuses on compound pattern detection in a learned method instead of assuming a mathematical parametric model. All parameters in SeA-M2Net are learned from data in the training procedure, thus allowing for maximum flexibility of pattern distribution deformation. The algorithm is tested on several LC-MS datasets of multiple biological samples obtained from different instruments with varied experimental settings. We demonstrate the superiority of the new approach in handling complex compound patterns (e.g., low abundance, overlapping regions, LC shifts, and missing values). Our experiments indicate that SeA-M2Net outperforms widely used detection methods in terms of detection accuracy.
    Keywords:  Compounds pattern; Deep learning; Feature detection; Liquid chromatography-mass spectrometry; Probability; Pseudo color images
  10. Talanta. 2021 Jan 15. pii: S0039-9140(20)30790-6. [Epub ahead of print]222 121499
      Endogenous steroid hormones and endocannabinoids (ECs) are important regulators in the stress response of the human body. For the measurement of chronic stress, hair analysis has been established as method of choice for long-term and retrospective determination of endogenous stress markers. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of five steroid hormones (cortisone, cortisol, androstenedione, testosterone, progesterone) and four endocannabinoids (anandamide, palmitoylethanolamide, 2-arachidonylglycerol, oleoylethanolamide) in hair was developed and validated. The hair samples were extracted with methanol and cleaned up with a fully automated supported liquid extraction (SLE) before analysis. Special attention was paid to the difficulties accompanying the quantification of endogenous analytes in hair. Five different strategies for endogenous compound quantification in hair (surrogate analyte, standard addition, background correction, stripped matrix and solvent calibration) were tested and compared. As a result, the approach of the surrogate analyte was used for the quantification of steroid hormones whereas background correction was used for endocannabinoids. The measurement of 58 samples from healthy young adults allowed insights into endocannabinoid ranges in hair and the correlation to steroid hormones. No significant differences in steroid and EC concentration levels of male and female in hair were found, except for testosterone (p < 0.001) and androstenedione (p < 0.0001). Cortisol to cortisone and testosterone to androstenedione concentrations were significantly and positively correlated. There were significant intercorrelations between endocannabinoids.
    Keywords:  Endocannabinoids; Endogenous substance quantification; Hair matrix; Liquid chromatography-tandem mass spectrometry; Steroid hormones
  11. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2020 Nov 10. 1-14
      A holistic strategy for discovering structural analogs was established using characteristic structural fragments filtering by high-resolution Orbitrap mass spectrometry and successfully employed for discovering potential hazards in meat. The mass spectrometry fragmentation mechanisms of 113 compounds (including sulphonamides, tetracyclines, benzimidazoles, steroid hormones, cephalosporins, β-blockers) were investigated and a new strategy for screening of characteristic fragment ions was proposed. To process the data acquired by two scan modes, firstly an integrated filtering strategy was conducted to facilitate the characterisation of multi-class drugs. The integrated filtering strategy was applied to reduce interference in the raw data, which could help extracting the MS1 characteristics of the homolog-type chemical substances and expand the screening of the compounds as effectively as possible. This strategy was based on a combination of nitrogen rule, neutral loss and multiple characteristic fragment ions filtering. The method was validated by rapid screening and identification of targeted compounds in spiked samples. Particularly, the successful detection of several new compounds indicated that this strategy had significant advantages over individual filtration methods and could be a promising method for screening and identifying newly homolog-type drug residues in complex samples.
    Keywords:  MS feature filtering; New structural analogs; UHPLC Q-Orbitrap; characteristic fragment ions; neutral loss
  12. J Steroid Biochem Mol Biol. 2020 Oct 22. pii: S0960-0760(20)30299-5. [Epub ahead of print]205 105774
      Conjugation with glucuronic acid is one of the major metabolic reactions in human steroid hormone catabolism. Recently, increasing interest has been raised concerning the biological roles of steroid glucuronides. We have therefore developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 15 urinary steroid hormone glucuronides in human urine: androsterone glucuronide (An-G), etiocholanolone glucuronide (Etio-G), epiandrosterone glucuronide (epiAn-G), dihydrotestosterone glucuronide (DHT-G), dehydroepiandrosterone glucuronide (DHEA-G), testosterone glucuronide (T-G), epitestosterone glucuronide (epiT-G), estrone glucuronide (E1-3 G), 17β-estradiol 17-glucuronide (E2-17 G), 17β-estradiol 3-glucuronide (E2-3 G), estriol 16-glucuronide (E3-16 G), pregnenolone glucuronide (Preg-G), tetrahydro-11-deoxycorticosterone 3-glucuronide (THDOC-3 G), cortisol 21-glucuronide (F-G) and pregnanediol glucuronide (PD-G). Sample workup included protein precipitation and solid phase extraction. Internal standards were used to correct for the loss of analytes during sample preparation and analysis. The method showed good linearity (R2≥0.99) and recovery ranged from 89.6 % to 113.8 %. Limit of quantification ranged from 1.9 nmol/L for F-G to 21.4 nmol/L for An-G. Intra-day and inter-day accuracy and precision were below 15 % for all quality controls. The method was successfully applied to 67 urine samples from children and adolescents in whom total concentrations of free and conjugated steroids had been previously determined by GC-MS after enzymatic hydrolysis. Free and sulfated steroids were also measured by LC-MS/MS. In general, the sums of the respective glucuronidated, sulfated and free forms of an analyte corresponded well with its total amount determined after enzymatic hydrolysis by GC-MS. Regarding the most prominent steroid metabolites, the total mean levels of androsterone and etiocholanolone showed an increase up to 5820.0 nmol/L and 4017.8 nmol/L in the group of 15-20 year-old children, respectively. Glucuronide conjugates (4374.3 nmol/L and 3588.5 nmol/L, respectively) dominated. DHEA was excreted mostly as sulfate (0-1 month of age: 184.5 nmol/L; 15-20 years of age: 1618.4 nmol/L) in all age groups. Cortisol was present predominantly as sulfate (mean: 173.8 nmol/L) in newborns. Levels of sulfated cortisol decreased with age, its glucuronidated form increased. The levels of free cortisol were relatively constant throughout childhood. Sex hormones were preferably excreted as glucuronides. In general, steroid hormone metabolites were conjugated to various extents with glucuronic acid or sulfuric acid and their ratio changed over lifetime.
    Keywords:  Liquid chromatography; Mass spectrometry; Steroid; Steroid glucuronide; Urine
  13. Lipids. 2020 Nov 09.
      Bovine milk is a complex mixture of lipids, proteins, carbohydrates, and other factors of which lipids comprise 3-5% of the total mass. Rapid analysis and characterization of the triacylglycerols (TAG) that comprise about 95% of the total lipid is daunting given the numerous TAG species. In the attached methods paper, we demonstrate an improved method for identifying and quantifying TAG species by infusion-based "shotgun" lipidomics. Because of the broad range of TAG species in milk, a single internal standard was insufficient for the analysis and required sectioning the spectrum into three portions based upon mass range to provide accurate quantitation of TAG species. Isobaric phospholipid interferences were removed using a simple dispersive solid-phase extraction step. Using this method, > 100 TAG species were quantitated by acyl carbon number and desaturation level in a sample of commercially purchased bovine milk.
    Keywords:  Infusion; Lipidomics; Milk; Triacylglycerols
  14. Anal Chem. 2020 Nov 11.
      Atmospheric pressure ionization methods confer a number of advantages over more traditional vacuum based techniques, in particular ease of hyphenation to a range of mass spectrometers. For atmospheric pressure matrix assisted desorption/ionization (AP-MALDI), several ion sources, operating in a range of geometries have been reported. Most of these platforms have, to date, generally demonstrated relatively low ion yields and/or poor ion transmission compared to vacuum sources. To improve the detection of certain ions, we have developed a second-generation transmission mode (TM) AP-MALDI imaging platform with in-line plasma postionization using the commercially available SICRIT device, replacing the previously used low temperature plasma probe from our developmental AP-TM-MALDI stage. Both plasma devices produce a significant ionization enhancement for a range of compounds, but the overall higher enhancement obtained by the SICRIT device in addition to the ease of installation and the minimal need for optimization presents this commercially available tool as an attractive method for simple postionization in AP-MALDI MSI.
  15. Rapid Commun Mass Spectrom. 2020 Nov 09. e9003
      RATIONALE: Proxalutamide is a novel drug for the treatment of prostate cancer. However, to date, there are almost no reports on the pharmacokinetics of proxalutamide in vivo. Herein, a liquid chromatography tandem-mass spectrometry (LC/MS/MS) method was developed to determine the concentrations of proxalutamide in biological samples for pharmacokinetic studies.METHODS: Chromatographic separation was achieved on a Kromasil 100-5C8 column, followed by gradient elution with a Shimadzu HPLC system. Mass spectrometry was performed in positive ion electrospray ionization mode using a SCIEX API 4000 triple quadrupole system. A simple and rapid one-step protein precipitation method was used for sample processing, and a low sample volume of 10 μL was used for processing and analysis.
    RESULTS: The method was validated to show good selectivity, sensitivity, precision, and accuracy. Good linearity (r2 > 0.99) was observed for rat plasma (range: 2-5000 ng/mL) and rat tissue homogenates (range: 2-2000 ng/mL). The extraction recovery was above 98 %, and no significant matrix effect was observed. This method was successfully applied to investigate the pharmacokinetics and tissue distribution of proxalutamide in rats.
    CONCLUSIONS: A rapid and sensitive LC/MS/MS method was developed and validated to determine the quantity of proxalutamide in rat plasma and tissue homogenates and to further study the pharmacokinetic parameters of proxalutamide in a rat model. The results showed that proxalutamide had good oral bioavailability and wide tissue distribution in vivo.
  16. Bioanalysis. 2020 Nov 13.
      Aim: Numerous guideline studies required for regulatory toxicity testing now include the measurement of the thyroid hormones 3,3',5-triiodo-L-thyronine (T3) and L-thyroxine (T4) in blood serum from rodents. A rapid, high-throughput method for the determination of the thyroid hormones T4 and T3 is reported. Materials & methods: Sample preparation is done using a 96-well microtiter plate format. Stable isotope analogs of both hormones are used as internal standards for study and quality control samples. Results & conclusion: The validated quantification levels are T3: 10 pg/ml and T4: 1 ng/ml, with CVs of <10% at the limit of quantification and up to 50*limit of quantification. The use of isotope analog internal standards eliminates the need for quantitative transfers and complete evaporations.
    Keywords:  LC–MS/MS; high-throughput; quantitation; serum; thyroid hormones
  17. Metabolites. 2020 Nov 06. pii: E447. [Epub ahead of print]10(11):
      Metabolic flux analysis (MFA) is an increasingly important tool to study metabolism quantitatively. Unlike the concentrations of metabolites, the fluxes, which are the rates at which intracellular metabolites interconvert, are not directly measurable. MFA uses stable isotope labeled tracers to reveal information related to the fluxes. The conceptual idea of MFA is that in tracer experiments the isotope labeling patterns of intracellular metabolites are determined by the fluxes, therefore by measuring the labeling patterns we can infer the fluxes in the network. In this review, we will discuss the basic concept of MFA using a simplified upper glycolysis network as an example. We will show how the fluxes are reflected in the isotope labeling patterns. The central idea we wish to deliver is that under metabolic and isotopic steady-state the labeling pattern of a metabolite is the flux-weighted average of the substrates' labeling patterns. As a result, MFA can tell the relative contributions of converging metabolic pathways only when these pathways make substrates in different labeling patterns for the shared product. This is the fundamental principle guiding the design of isotope labeling experiment for MFA including tracer selection. In addition, we will also discuss the basic biochemical assumptions of MFA, and we will show the flux-solving procedure and result evaluation. Finally, we will highlight the link between isotopically stationary and nonstationary flux analysis.
    Keywords:  MFA assumptions; metabolic flux analysis; non-steady-state versus steady-state; tracer selection