bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒05‒24
33 papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Metabolomics. 2020 May 18. 16(6): 66
      BACKGROUND: Human urine gives evidence of the metabolism in the body and contains different metabolites at various concentrations. A number of analytical techniques including mass spectrometry (MS) and nuclear magnetic resonance (NMR) have been used to obtain metabolites levels in urine samples. However, gas chromatography-mass spectrometry (GC-MS) is one of the most widely used techniques for urinary metabolomics studies due to its higher sensitivity, resolution, reproducibility, reliability, relatively low cost and ease of operation compared to liquid chromatography-mass spectrometry and NMR.AIM OF REVIEW: This review looks at various aspects of urine preparation prior to analysis by GC-MS including sample storage, urease pretreatment, derivatization, use of internal standard and quality control samples for data correction. In addition, most common types of inlet liners, ionization techniques and columns are discussed and a summary of mass analyzers are also highlighted. Lastly, the role of retention index in metabolite identification and data normalization methods are presented.
    KEY SCIENTIFIC CONCEPTS OF REVIEW: The purpose of this review is summarizing methods of sample storage, pretreatment, and GC-MS analysis that are mostly used in urine metabolomics studies. Specific emphasis is given to the critical steps within the GC-MS urine metabolomics that those new to this field need to be aware of and the remaining challenges that require further attention and studies.
    Keywords:  Derivatization; Gas chromatography; Mass spectrometry; Metabolomics; Urine
  2. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Jun 15. pii: S1570-0232(19)31519-3. [Epub ahead of print]1147 122129
      A simple and rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and used to determine eight antipsychotics (aripiprazole, clozapine, haloperidol, olanzapine, paliperidone, quetiapine, risperidone, and ziprasidone) in human serum for practical clinical usage. Stable isotope-labeled internal standards were used for all drugs to compensate for method variability, including matrix effects, ion extraction and ionization variations. Samples were prepared by simple protein precipitation with methanol. Chromatographic separation was accomplished in less than 3.3 min on a KINTEX C18 column (50 mm × 3.0 mm, 5 μm) using a gradient elution of 2 mM aqueous ammonium formate and methanol at a flow rate of 0.5 mL/min. Quantification was performed by multiple reaction monitoring (MRM) in the positive mode. The method was fully validated according to the latest recommendations of international guidelines. The correlation coefficients of calibration curves were all greater than 0.9945. Internal standard-normalized matrix effects ranged from 96.3% to 115%, and extraction recoveries were between 88.1% and 107%. Coefficients of variation ranged from 1.82 - 13.5% for intra-day precision, 5.69-14.7% for inter-day precision, and the relative error for accuracy did not exceed ± 13.5% for any analyte. The method was successfully applied to routine clinical therapeutic drug monitoring for 2,173 samples.
    Keywords:  Antipsychotics; Clinical application; LC-MS/MS; Therapeutic drug monitoring; Validation
  3. Int J Mol Sci. 2020 May 20. pii: E3611. [Epub ahead of print]21(10):
      Plasma and tissue from breast cancer patients are valuable for diagnostic/prognostic purposes and are accessible by multiple mass spectrometry (MS) tools. Liquid chromatography-mass spectrometry (LC-MS) and ambient mass spectrometry imaging (MSI) were shown to be robust and reproducible technologies for breast cancer diagnosis. Here, we investigated whether there is a correspondence between lipid cancer features observed by desorption electrospray ionization (DESI)-MSI in tissue and those detected by LC-MS in plasma samples. The study included 28 tissues and 20 plasma samples from 24 women with ductal breast carcinomas of both special and no special type (NST) along with 22 plasma samples from healthy women. The comparison of plasma and tissue lipid signatures revealed that each one of the studied matrices (i.e., blood or tumor) has its own specific molecular signature and the full interposition of their discriminant ions is not possible. This comparison also revealed that the molecular indicators of tissue injury, characteristic of the breast cancer tissue profile obtained by DESI-MSI, do not persist as cancer discriminators in peripheral blood even though some of them could be found in plasma samples.
    Keywords:  breast cancer; desorption-electrospray-ionization—mass spectrometry imaging; lipidomics; liquid chromatography-mass spectrometry; plasma; tumor tissue
  4. Clin Chim Acta. 2020 May 14. pii: S0009-8981(20)30217-5. [Epub ahead of print]508 146-153
      N-acetylaspartate is a human endogenous compound synthesized by neurons, which is involved in neuronal metabolism. It is used as a marker in brain magnetic resonance spectroscopy to investigate several neurological and metabolic disorders, that can be related to a variation of its concentration with respect to reference values. N-acetylaspartate is present also in biological fluids, such as plasma, urine, and cerebrospinal fluid, where it can be quantified. Here we describe the development and validation, in compliance with the EMA guidelines, of a novel assay method for the quantification of N-acetylaspartate in plasma based on tandem mass spectrometry coupled to liquid chromatography. Its peculiarity lies in the fact that sample preparation includes an esterification step, which significantly improves the chromatographic performances and, consequently, also the method sensitivity, reproducibility and accuracy. Instrumental LLOQ is 0.06 ng/mL, i.e. at least 300 times lower than the medium N-acetylaspartate concentration in samples, accuracy is in the range 98-103%, while precision lies between 1 and 3%. The method robustness was tested in about 1000 injections of plasma samples, 96 of which were used also to assess the reference ranges in control subjects (16.46-63.40 ng/mL). Controls were then compared to plasma samples from type 2 diabetic patients. Contrary to brain magnetic resonance spectroscopy, which demonstrated a decrease in the N-acetylaspartate levels in right frontal and parieto-temporal region of type 2 diabetic patients, plasma analysis showed no statistical difference with respect to controls. However, the method here described can be profitably used in studies concerning different disorders with CNS involvement, as confirmed by reports available in the literature.
    Keywords:  Derivatization; N-acetylaspartate; Plasma; Quantification; Tandem mass spectrometry; Validation
  5. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 May 05. pii: S1570-0232(19)31685-X. [Epub ahead of print]1148 122145
      Changes in cellular metabolism accompany tumor therapeutic resistance. Metabolite concentrations specifically reflect the cellular state. Glutathione (GSH) metabolism maintains the redox homeostasis while also confers therapeutic resistance to cancer cells. However, analytical methods for studying GSH metabolism rely on high-resolution-based untargeted metabolomics. Since the aim of untargeted metabolomics studies is covering as much metabolites as possible, these methods lack sensitivity for simultaneous analysis of intracellular GSH-related metabolites with different polarities and structures. In this study, based on cultured lung cancer cells, we described a rapid, robust and sensitive ultra-performance liquid chromatography-triple quadrupole tandem mass spectrographic method (UPLC-QQQ-MS/MS) to simultaneously quantify a repertoire of GSH-related metabolites, including GSH, GSSG, glycine, cysteine, glutamine, glutamate, cystine, γ-glutamyl-cysteine and cysteinyl-glycine. This method avoided the use of derivatization and/or ion-pairing reagents and was validated according to United States Food and Drug Administration (US FDA) criteria. The lower limit of quantification was determined to be 0.5-100 ng/mL with lower limits of detection at 0.14-10.07 ng/mL. The intra- and inter-day precision values for all the analytes were <15% CV, and the accuracy ranged from 85.4% to 114% at three levels of quality control. This method combined simple preparation with rapid analytical procedure (8 min), allowed for high-throughput analysis of GSH metabolism in basic and therapeutic treatment conditions within cultured cells. Our data showed a significant alteration of GSH metabolism in two independent resistant cells compared to sensitive cells. This method monitored the impact of molecularly targeted drugs on GSH metabolism within lung cancer cells and therefore helped identifying potential metabolic vulnerability for the therapeutic resistance in lung cancer.
    Keywords:  EGFR-TKI resistance; Glutathione metabolism; LC-MS/MS; Lung cancer; Metabolic change
  6. J Pharm Biomed Anal. 2020 Apr 02. pii: S0731-7085(19)32696-2. [Epub ahead of print]186 113289
      A high performance hydrophilic interaction chromatography method combined with tandem-mass spectrometry for the quantification of cefepime, meropenem and imipenem in plasma and cerebrospinal fluid is presented. A solution of 0.5 M 3-Morpholinopropanesulfonic acid and ethylene glycol (1:1) was added to the samples before analysis to ensure stability of analytes during work up and storage. Deuterated forms of cefepime and meropenem were used as internal standards. Protein precipitation prior to injection into the LC-MS/MS system provided a fast and easy sample preparation. For online extraction, a Turboflow Cyclone-MCX column was used and the chromatographic separation was carried out on a Hypersil GOLD HILIC column. Linear calibration curves were obtained in the concentration range of 0.4-40 mg/l, 0.6-60 mg/l and 1-100 mg/l for meropenem, imipenem and cefepime, respectively. The intra- and interday imprecision and inaccuracy values were below 10 % for plasma and 13 % for cerebrospinal fluid using a calibration in plasma. The method was employed for therapeutic drug measurements in a university hospital.
    Keywords:  Antibiotics; High pressure liquid chromatography; Mass spectrometry; Online extraction; Protein precipitation; Therapeutic drug monitoring
  7. Spectrochim Acta A Mol Biomol Spectrosc. 2020 May 05. pii: S1386-1425(20)30372-3. [Epub ahead of print]239 118394
      Nowadays, it is well known that early diagnosis directly affects the success of treatment. Biomarkers play a crucial role in early diagnosis of diseases or explanation of pathological condition. The investigation of new biomarkers depends on the reliable quantification of analytes in biological matrices. Regarding to the critical roles of amino acids in metabolism, functions and nutrition of human body, the careful monitoring of their levels in biological samples is required to evaluate their potential in biomarker studies for clinical research. In this study, a reliable and accurate analytical strategy was developed for the simultaneous determination of glycine, methionine and homocysteine using LC-quadrupole-time of flight-tandem MS system. The method detection limit was found to be 0.73 μg/mL, 0.017 μg/mL and 0.019 μg/mL for glycine, methionine and homocysteine, respectively. The calibration curves were obtained with great linearity (R2 ≥ 0.9993) and low relative standard deviation values showed the repeatability of proposed method. The method applicability was determined using human plasma and urine samples, and high percent recoveries demonstrated the accuracy of method developed. Each measurement was taken less than 4.0 min indicating a promising strategy for the fast and reliable quantification of target amino acids in clinical laboratories.
    Keywords:  Amino acids; Biomarkers; Human plasma; Human urine; Tandem mass spectrometry
  8. J Neurosci Methods. 2020 May 16. pii: S0165-0270(20)30183-7. [Epub ahead of print] 108760
      BACKGROUND: So far, analytical investigation of neuroactive molecules in cerebrospinal fluid (CSF) of rodent models has been limited to rats, given the intrinsic anatomic difficulties related to mice sampling and the corresponding tiny amounts of CSF obtained. This poses a challenge for the research in neuroscience, where many, if not most, animal models for neuronal disorders rely on mice.NEW METHOD: We introduce a new, sensitive and robust LC-MS/MS method to analyze a panel of twelve neuroactive molecules (NM) from mouse CSF (aspartic acid, serine, glycine, glutamate, γ-aminobutyric acid, norepinephrine, epinephrine, acetylcholine, dopamine, serotonin, histamine and its metabolite 1-metylhistamine. The paper describes the sampling procedure that allows the collection of 1-2 microliters of pure CSF from individual mouse specimens.
    RESULTS: To test its applicability, we challenged our method on the field, by sampling 37 individual animals, thus demonstrating its strength and reliability.
    COMPARISON WITH EXISTING METHOD(S): Compared to other methods, our procedure does not involve any extraction nor derivatization steps: samples are simply diluted and analyzed as such by LC-MS/MS, using a dedicated ion pairing agent in the chromatographic setup. The panel of neuroactive molecules that is analyzed in a single run is also significantly higher compared to other methods.
    CONCLUSIONS: Given the number of mouse models used in the neuroscience research, we believe that our work will pave new ways to more advanced research in this field.
    Keywords:  Cerebrospinal fluid; GABA; Liquid chromatography – tandem mass spectrometry; Mice; Neuroactive molecules; Sampling procedure
  9. J Sep Sci. 2020 May 17.
      Switchable-hydrophilicity solvent liquid-liquid microextraction and dispersive liquid-liquid microextraction were compared for the extraction of piperine from Piper nigrum L. prior to its HPLC-UV analysis. Under optimum conditions, limits of detection and quantitation were found as 0.2-0.6 and 0.7-2.0 μg mg-1 with the two methods, respectively. Calibration graphs showed good linearity with coefficients of determination (R2 ) higher than 0.9962 and percentage relative standard deviations lower than 6.8%. Both methods were efficiently used for the extraction of piperine from black and white pepper samples from different origins and percentage relative recoveries ranged between 90.0 and 106.0%. The results showed that switchable-hydrophilicity solvent liquid-liquid microextraction is a better alternative to dispersive liquid-liquid microextraction for the routine analysis of piperine in food samples. A novel scaled-up dispersive liquid-liquid microextraction method was also proposed for the isolation of piperine providing a yield of 102.9 ± 4.9% and purity higher than 98.0% as revealed by NMR analysis. This article is protected by copyright. All rights reserved.
    Keywords:  black pepper; dispersive liquid-liquid microextraction; piperine; switchable-hydrophilicity solvent; white pepper
  10. Food Chem. 2020 May 11. pii: S0308-8146(20)30877-3. [Epub ahead of print]327 127015
      A simple and reliable analytical method was established for the simultaneous determination of tiafenacil and its six metabolites in fruits. The method involves three steps: extraction using acidified acetonitrile, clean-up by octadecylsilane(C18) and graphitized carbon black (GCB), and detection using liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The method was validated on seven matrices spiked at 10, 100 and 1000 μg kg-1. Average recoveries ranged from 73% to 105% with intra-day RSDr (n = 5) of 1.0%-13.0% and inter-day RSDR (n = 15) of 1.1%-14.6%. Good linearities (R2 > 0.9911) were obtained for seven analytes in all matrices. The limit of quantification (LOQs) for tiafenacil and its six metabolites were 10 μg kg-1 in all matrices. This analytical method provides a basis for the establishment of maximum residue limits (MRL) and for the monitoring of tiafenacil residues in fruits.
    Keywords:  Fruits; Metabolites; Residue; Tiafenacil; UHPLC-MS/MS
  11. Mikrochim Acta. 2020 May 19. 187(6): 337
      A sensitive and readily deployable analytical method has been reported for the simultaneous analysis of pirimicarb (PRM) and fenitrothion (FEN) pesticide residues in environmental water samples using fabric phase sorptive extraction (FPSE) followed by high-performance liquid chromatography combined with photodiode array (HPLC-PDA) detector. Both pesticides were successfully determined with a Luna omega C18 column under isocratic elution mode by means of acetonitrile and phosphate buffer (pH 3.0) as the mobile phase. The quantitative data for PRM and FEN were obtained at their maximum wavelengths of 310 nm and 268 nm, respectively. The calibration plots were linear in the range 10.00-750.00 ng mL-1 and 10.00-900.00 ng mL-1 with correlation coefficient of 0.9984 and 0.9992 for PRM and FEN, respectively. Major FPSE experimental variables were investigated in detail, such as contact time with the FPSE membrane, pH and electrolyte concentration, and the volume and type of desorption solvent. Under the optimized conditions, the developed method showed satisfactory reproducibility with relative standard deviations less than 2.5% and low limits of detection of 2.98 and 3.02 ng mL-1 for PRM and FEN, respectively. The combined procedure allows for enhancement factors ranging from 88 to 113, with pre-concentration values of 125 for both analytes. The chromatographic resolutions were approx. 12 for FEN (retention factor of 3.52) and PRM (retention factor of 6.09), respectively, with a selectivity factor of 1.73. Finally, the validated method was successfully applied to real environmental water samples for the determination of these pesticides. Graphical abstract.
    Keywords:  Fabric phase sorptive extraction; Fenitrothion; HPLC-PDA; Pirimicarb; Water samples
  12. Chemosphere. 2020 May 14. pii: S0045-6535(20)31121-8. [Epub ahead of print]256 126928
      Phenols and trans-1,2-dihydro-1,2-diols are metabolites commonly formed in vivo in fish upon exposure to polycyclic aromatic hydrocarbons (PAHs). These metabolites are excreted via the bile and gas chromatography-mass spectrometry (GC-MS) analysis of bile is becoming more frequently used for evaluating PAH exposure levels in fish. Current protocols focus on the detection and quantification of phenols formed during in vivo oxidation of PAHs, leaving out analyses and quantification of other oxidation products such as trans-1,2-dihydro-1,2-diols, potentially underestimating exposure levels. Herein, four trans-1,2-dihydro-1,2-diols, namely trans-1,2-dihydronaphthalene-1,2-diol, trans-6-methyl-1,2-dihydronaphthalene-1,2-diol, trans-5,7-dimethyl-1,2-dihydronaphthalene-1,2-diol, and trans-4,6,7-trimethyl-1,2-dihydronaphthalene-1,2-diol, were successfully prepared and used as standards in the GC-MS analysis, aiming to further develop this qualitative and quantitative analytical method for the determination of PAH exposures. This study shows that the currently used GC-MS analysis, including sample workup, is not suitable for determining the quantity of the corresponding diols derived from naphthalene and methylated naphthalenes. Alternative approaches are needed to provide a correct estimate of PAH exposure levels.
  13. Molecules. 2020 May 14. pii: E2310. [Epub ahead of print]25(10):
      Lipidomics suffers from the lack of fast and reproducible tools to obtain both structural information on intact phospholipids (PL) and fatty acyl chain composition. Hydrophilic interaction liquid chromatography with electrospray ionization coupled to an orbital-trap Fourier-transform analyzer operating using all ion fragmentation mode (HILIC-ESI-FTMS-AIF MS) is seemingly a valuable resource in this respect. Here, accurate m/z values, HILIC retention times and AIF MS scan data were combined for PL assignment in standard mixtures or real lipid extracts. AIF scans in both positive and negative ESI mode, achieved using collisional induced dissociation for fragmentation, were applied to identify both the head-group of each PL class and the fatty acyl chains, respectively. An advantage of the AIF approach was the concurrent collection of tandem MS-like data, enabling the identification of linked fatty acyl chains of precursor phospholipids through the corresponding carboxylate anions. To illustrate the ability of AIF in the field of lipidomics, two different types of real samples, i.e., the lipid extracts obtained from human plasma and dermal fibroblasts, were examined. Using AIF scans, a total of 253 intact lipid species and 18 fatty acids across 4 lipid classes were recognized in plasma samples, while FA C20:3 was confirmed as the fatty acyl chain belonging to phosphatidylinositol, PI 38:3, which was found to be down-regulated in fibroblast samples of Parkinson's disease patients.
    Keywords:  AIF scan MS; HILIC separation; collisional induced dissociation; fatty acids; phospholipids
  14. Chem Biol Interact. 2020 May 19. pii: S0009-2797(20)30749-3. [Epub ahead of print] 109140
      A liquid chromatograpy-nanoelectrospray Ionization-high resolution tandem mass spectrometry (LC-NSI-HRMS/MS) method was developed for quantitation of the DNA adducts 7-(2'-carboxyethyl)guanine (7-2'-CEG) and N2-(1'-carboxyethyl)guanine (N2-1'-CEG), as their methyl esters, in human leukocyte DNA from smokers and non-smokers. 7-2'-CEG has been previously identified in all human liver samples analyzed and is formed from an unknown carboxyethylating agent while N2-1'-CEG is formed from the advanced glycation endproduct methyl glyoxal. The method was applied for the analysis of these two DNA adducts in leukocyte DNA from 20 smokers and 20 non-smokers, in part to test the hypothesis that 7-2'-CEG could be formed by endogenous nitrosation, as previously observed in rats treated with nitrosodihydrouracil and nitrite. Levels of 7-2'-CEG (mean ± S.D.) were 0.6 ± 0.2 pmol/μmol dG in smokers and 0.5 ± 0.2 pmol/μmol dG in nonsmokers, while those of N2-1'-CEG were 5.4 ± 1.9 pmol/μmol dG in smokers and 5.6 ± 2 pmol/μmol dG in non-smokers. These results did not support our hypothesis that endogenous nitrosation of dihydrouracil in smokers leads to higher levels of 7-2'-CEG in leukocyte DNA than in non-smokers. However the study provides the first data on levels of these DNA adducts in human leukocyte DNA, and the LC-NSI-HRMS/MS method developed for their quantitation could be important for future studies of DNA damage by methyl glyoxal.
    Keywords:  7-(2′-carboxyethyl)guanine; Advanced glycation end products; Endogenous nitrosation; Liquid chromatograpy-nanoelectrospray ionization-high resolution tandem mass spectrometry; Methyl glyoxal; N(2)-(1′-carboxyethyl)guanine
  15. Anal Chim Acta. 2020 Jun 29. pii: S0003-2670(20)30425-6. [Epub ahead of print]1118 36-43
      Single-cell metabolomics provides information on the biochemical state of an individual cell and its relationship with the surrounding environment. Characterization of metabolic cellular heterogeneity is challenging, in part due to the small amounts of analytes and their wide dynamic concentration ranges within individual cells. CE-ESI-MS is well suited to single-cell assays because of its low sample-volume requirements and low detection limits. While the volume of a cell is in the picoliter range, after isolation, the typical volume of the lysed cell sample is on the order of a microliter; however, only nanoliters are injected into the CE system, with the volume mismatch limiting analytical performance. Here we developed an approach for the detection of intracellular metabolites from a single neuron using field amplified sample injection (FASI) CE-ESI-MS. Through the application of FASI, we achieved 100- to 300-fold detection limit enhancement compared to hydrodynamic injections. We further enhanced the analyte identification and quantification accuracy via introduction of two internal standards. As a result, the relative standard deviations of migration times were reduced to <5%, aiding identification. Finally, we successfully applied FASI CE-ESI-MS to the untargeted profiling of metabolites of Aplysia californica pleural sensory neurons with <50 μm diameter cell somata. As a result, twenty one neurotransmitters and metabolites have been quantified in these neurons.
    Keywords:  Capillary electrophoresis electrospray ionization-mass spectrometry; Field amplified sample injection; Single-cell metabolomics
  16. Sci Rep. 2020 May 18. 10(1): 8198
      Colistin is a polymixin antibiotic (polymixin E) that is produced by Bacillus colistinus bacteria. The aim of the present study was to develop and validate a method to quantify colistin levels in plasma using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique and then apply it in experimental animals (rats) to investigate the pharmacokinetic profile of colistin in this species. Polymyxin B was used as an internal standard (IS) and the quantitation was carried out using ESI + interface and employing multiple reaction monitoring (MRM) mode. A mobile phase consisting of acetonitrile:water:formic acid (30:70:0.1%; v/v/v) was employed and Zorbax eclipse plus C18 (1.8 µm, 2.1 mm i.d. x 50 mm) was the optimal column for this method and utilized at a flow rate of 0.2 mL/min. The full scan mass spectra of precursor/product ions of colistin A were at m/z 585.5 > 100.8, for colistin B at m/z 578.8 > 101 and for the IS at m/z 602.8 > 101. The lower limit of quantification (LLOQ) was 0.5 µg/mL. The method demonstrated acceptable intra-run and inter-run precision and accuracy for both colistin A and colistin B. Colistin was stable when assessed for long-term stability, freeze-thaw stability and autosampler stability. However, it was not stable when stored at room temperature. The matrix effect evaluation showed minimal or no effect. Incurred sample reanalysis findings were within acceptable ranges (<20% of the nominal concentration). The pharmacokinetic parameters of colistin were investigated in rats using the present method. The developed method for colistin demonstrates that it is rapid, sensitive, specific, accurate, precise, and reliable.
  17. Commun Biol. 2020 May 20. 3(1): 247
      Altered central carbon metabolism is a hallmark of many diseases including diabetes, obesity, heart disease and cancer. Identifying metabolic changes will open opportunities for better understanding aetiological processes and identifying new diagnostic, prognostic, and therapeutic targets. Comprehensive and robust analysis of primary metabolic pathways in cells, tissues and bio-fluids, remains technically challenging. We report on the development and validation of a highly reproducible and robust untargeted method using anion-exchange tandem mass spectrometry (IC-MS) that enables analysis of 431 metabolites, providing detailed coverage of central carbon metabolism. We apply the method in an untargeted, discovery-driven workflow to investigate the metabolic effects of isocitrate dehydrogenase 1 (IDH1) mutations in glioblastoma cells. IC-MS provides comprehensive coverage of central metabolic pathways revealing significant elevation of 2-hydroxyglutarate and depletion of 2-oxoglutarate. Further analysis of the data reveals depletion in additional metabolites including previously unrecognised changes in lysine and tryptophan metabolism.
  18. Curr Protoc Toxicol. 2020 Jun;84(1): e92
      Immunosuppressant medications help suppress the immune system response through inhibition of various checkpoints in the regulatory biochemical pathway. This is useful in prevention of organ rejection in transplantation or in the treatment of autoimmune diseases such as lupus or rheumatoid arthritis. Quantification of immunosuppressive drugs in blood is needed clinically for optimization of treatment and to avoid toxicity or unwanted side effects. Here, we describe a quantitative method to determine the concentration of cyclosprine A, tacrolimus, sirolimus, and everolimus in whole blood. This method has been used for many years clinically to support patient care. © 2020 by John Wiley & Sons, Inc.
    Keywords:  HPLC; immunosuppressant drugs; tandem mass spectrometry; transplantation
  19. Metabolites. 2020 May 16. pii: E203. [Epub ahead of print]10(5):
      Metabolomics is the comprehensive study of metabolism, the biochemical processes that sustain life. By comparing metabolites between healthy and disease states, new insights into disease mechanisms can be uncovered. NMR is a powerful analytical method to detect and quantify metabolites. Standard one-dimensional (1D) 1H-NMR metabolite profiling is informative but challenged by significant chemical shift overlap. Multi-dimensional NMR can increase resolution, but the required long acquisition times lead to limited throughput. Non-uniform sampling (NUS) is a well-accepted mode of acquiring multi-dimensional NMR data, enabling either reduced acquisition times or increased sensitivity in equivalent time. Despite these advantages, the technique is not widely applied to metabolomics. In this study, we evaluated the utility of NUS 1H-13C heteronuclear single quantum coherence (HSQC) for semi-quantitative metabolomics. We demonstrated that NUS improved sensitivity compared to uniform sampling (US). We verified that the NUS measurement maintains linearity, making it possible to detect metabolite changes across samples and studies. Furthermore, we calculated the lower limit of detection and quantification (LOD/LOQ) of common metabolites. Finally, we demonstrate that the measurements are repeatable on the same system and across different systems. In conclusion, our results detail the analytical capability of NUS and, in doing so, empower the future use of NUS 1H-13C HSQC in metabolomic studies.
    Keywords:  NUS NMR; metabolomics; reproducibility
  20. Int J Mol Sci. 2020 May 18. pii: E3548. [Epub ahead of print]21(10):
      It is well-established that aminothiols, to which cysteine (Cys) belongs, are highly reactive towards aldehydes in an aqueous environment, forming substituted thiazolidine carboxylic acids. This report provides evidence that formation of the product containing a thiazolidine ring through non-enzymatic condensation of Cys and an active form of vitamin B6 pyridoxal 5'-phosphate (PLP) occurs in vivo in humans. To prove this point, a new method, based on a gas chromatography coupled with mass spectrometry (GC-MS), has been designed to identify and quantify Cys and PLP adduct, 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA) in human plasma. The GC-MS assay relies on sample deproteinization by ultrafiltration over cut-off membranes and preconcentration by drying under vacuum, followed by treatment of the residue with derivatization mixture containing anhydrous pyridine, N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS). The method quantifies HPPTCA in a linear range from 1 to 20 µmol L-1, where the lowest standard on the calibration curve refers to the limit of quantification (LOQ). The validity of the method was demonstrated. Furthermore, the method was successfully applied to plasma samples donated by apparently healthy volunteers and breast cancer patients. The GC-MS assay provides a new tool that will hopefully facilitate studies on the role of HPPTCA in living systems.
    Keywords:  2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid; N-trimethylsilyl-N-methyl trifluoroacetamide; cysteine; gas chromatography–mass spectrometry; human plasma; pyridoxal 5′-phosphate
  21. Front Pharmacol. 2020 ;11 508
      Acetylglutamine (NAG) is the derivative of glutamine, which is the richest free amino acid in the human body. In this work, a novel reliable method of the combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and microdialysis (MD) technique for the evaluation of NAG and its metabolites γ-aminobutyric acid (GABA) and glutamic acid (Glu) in rat blood and brain was proposed. A Zorbax SB-C18 column (2.1 × 100 mm, 3.5 μM) was applied to separate the analytes. The mobile phase was acetonitrile-water (70:30, v/v) containing 5 mM ammonium acetate and the flow rate was 0.3 ml/min. Based on the multiple reaction monitoring (MRM) mode of positive ion, the precursors of product ions chosen for NAG, Glu, GABA, and N-carbamyl-L-glutamic (NCG, IS) were (m/z) 189.1→130.0, 148.0→84.1, 104→87.1, and 191.0→130.1, respectively. All the validation data, including precision, accuracy, inter-day repeatability, matrix effect, and stability, were within the acceptable ranges according to the reference of Bioanalytical Method Validation Guidance for Industry (2018). Rats with microdialysis probes inserted into jugular vein and hippocampus were administered the low (75 mg/kg, NAG-L), medium (150 mg/kg, NAG-M), and high (300 mg/kg, NAG-H) doses of NAG and 10 ml/kg Guhong injection (GHI) by tail vein, respectively. In the blood test, the Cmax values of NAG-L group were markedly lower (P < 0.01) than those of NAG-M, NAG-H, and GHI groups, respectively. No differences were observed between NAG-M and GHI groups, while the Cmax values in GHI group were significantly upgraded compared with NAG-H group. There were notable differences in the Cmax values of NAG in brain dialysate after administration of NAG and GHI. The drug distribution coefficients of NAG, Glu, GABA in brain and blood at low, medium, high doses of NAG and GHI groups were 13.99, 27.43, 34.81, 31.37; 11.04, 59.07, 21.69, 2.69%; 212.88, 234.92, 157.59, and 102.65%, respectively. Our investigation demonstrates that NAG and its related metabolites in rat blood and brain can be simultaneously measured according to the above proposed method. Meanwhile, NAG has easy and dose-dependently access to the blood-brain barrier and exhibits a medium retention time in rat.
    Keywords:  acetylglutamine; blood-brain barrier; glutamic acid; liquid chromatography-tandem mass spectrometry; microdialysis; γ-aminobutyric acid
  22. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 May 08. pii: S1570-0232(20)30079-9. [Epub ahead of print]1148 122148
      Screening for potential drug-drug interaction (DDI) or herb-drug interaction (HDI) using in vitro cytochrome P450 inhibition (IVCI) assays requires robust analytical methods with high sensitivity and reproducibility. Utilization of liquid chromatography-mass spectrometry (LC-MS) for analyte quantification is often hampered by the presence of non-volatile IVCI sample buffer constituents that often results in ion suppression. In this study, to enable screening of drug interactions involving tamoxifen (TAM) metabolism using IVCI-LC-MS/MS, a liquid-liquid extraction (LLE) method was developed and optimized for sample clean-up. Utilization of chloroform as extraction solvent and adjustment of sample pH to 11 was found to result in satisfactory recovery (>70%) and low ion suppression (<19%). A LC-MS/MS method was subsequently developed and validated for simultaneous quantification of major TAM metabolites, such as N-desmethyltamoxifen (NDT), endoxifen (EDF) and 4-hydroxytamoxifen (HTF) to enable IVCI sample analysis. Satisfactory separation of E-/Z-isomers of endoxifen with peak resolution (Rs) of 1.9 was achieved. Accuracy and precision of the method was verified within the linear range of 0-50 ng/mL for NDT, 0-25 ng/mL for HTF and 0-25 ng/mL for EDF (E/Z isomers). Inhibitory potency (IC50, Ki and mode of inhibition) of known CYP inhibitors and Strobilanthes crispus extract was then evaluated using the validated method. In summary, the results demonstrated applicability of the developed LLE and validated LC-MS/MS method for in vitro screening of DDI and HDI involving TAM metabolism.
    Keywords:  Cancer; Cytochrome P450; Drug-drug interaction; LC-MS/MS; Liquid-liquid extraction; Method development and validation; Tamoxifen
  23. Bioinformatics. 2020 May 21. pii: btaa529. [Epub ahead of print]
      SUMMARY: Biological pathways are fundamental for learning about healthy and disease states. Many existing formats support automatic software analysis of biological pathways, for examples BioPAX (Biological Pathway Exchange). Although some algorithms are available as web application or standalone tools, no general graphical application for the parsing of BioPAX pathway data exists. Also, very few tools can perform Pathway Enrichment Analysis (PEA) using pathway encoded in the BioPAX format. To fill this gap we introduce BiP, an automatic and graphical software tool aimed at performing the parsing and accessing of BioPAX pathway data, along with pathway enrichment analysis by using information coming from pathways encoded in BioPAX.AVAILABILITY: BiP is freely available for academic and non-profit organizations at under the LGPL 2.1, the GNU Lesser General Public License.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
  24. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Jun 15. pii: S1570-0232(20)30584-5. [Epub ahead of print]1147 122150
      One of the most common techniques for assessing the intestinal absorption characteristics of drugs is single-pass intestinal perfusion (SPIP) method. Metoprolol tartrate (MT, reference standard) and phenol red (PR, zero permeability marker) are the compounds that are normally used in SPIP studies. The aim of this study was to develop a reverse phase high-performance liquid chromatography (RP-HPLC) method combined with UV-detection for the simultaneous determination of MT and PR in the perfusion medium used in SPIP experiments. Elution was performed using a Restek Raptor C18 column (5 μm, 4.6 mm × 250) at a temperature of 25 °C. The mixture of the mobile phase consisted of (MeOH):(Phosphate buffer solution, PBS), (20 mM, pH 3.0 adjusted with ortho-phosphoric acid),(55:45, v/v). Flow rate and column temperature were set at 1.2 mL min-1 and 25 °C, respectively. MT and PR were injected as 20 µL into the HPLC system. UV detection was performed at 227 nm. The obtained retention times were reported as 2.89 and 3.80 min for MT and PR, respectively. The developed RP-HPLC method was validated according to Q2(R1) guideline of The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). The method was linear within the range of 2-50 μg mL-1 for PR and 10-75 μg mL-1 for MT. The developed RP-HPLC method was successfully applied on determination of MT and PR in perfusion medium. The developed method could be helpful for researchers working on in-situ rat intestinal permeability studies and it could be easily modified on further studies.
    Keywords:  HPLC; Metoprolol tartrate; Oral absorption; Permeability; Phenol red
  25. Andrologia. 2020 May 22. e13611
      Benign prostate hyperplasia (BPH) is a common disease in elderly men. It has been found that the occurrence of BPH was closely related to dysregulated steroid hormones. Here, a rapid, sensitive, accurate and specific method for the quantitative profiling of five androgens in man serum was developed and validated by the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using this method, dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), androsterone (A), dihydrotestosterone (DHT), oestrone (E1) and oestradiol (E2) were quantified in serum from man with and without BPH. BPH patients were characterised by the decreases in DHEA, A4 and T as well as increases in DHT, E2 and E1 in serum. Meanwhile, DHEA and DHT in serum were screened as sensitive biomarkers of BPH patients. This study will provide a new perspective of dysregulated steroid hormones for the diagnosis and prevention of BPH.
    Keywords:  LC-MS/MS; androgens; benign prostate hyperplasia; clinical samples; hormones homeostasis; oestrogens
  26. Life Sci. 2020 May 13. pii: S0024-3205(20)30532-4. [Epub ahead of print] 117784
      Two-dimensional (2D) cell cultures, in which cells grow in flat layers on plastic surfaces, are considered the standard model for use in drug screening and for biological assays. However, these models do not accurately represent in vivo cell organization due to a lack in cell-cell/matrix interactions and in tissue and microenvironment structure. For that reason, three-dimensional (3D) cell cultures have been introduced as an innovative platform in recent years, allowing cells to grow and interact with each other in all three dimensions thanks to an artificial environment. In a 3D model cells show more interesting aspects from a physiological point of view, demonstrating several improvements in viability, morphology, proliferation and differentiations, response to external and internal stimuli, drug metabolism and efficacy and in vivo relevance. This review explores recent techniques in the development of 3D cell models with a particular focus on their application from a pharmacological point of view, starting from the concept of spheroid models generated by scaffold-free or scaffold-based techniques. Finally, special attention is paid to the concept of organoids, 3D constructs that replicate the 3D architecture of intact organs and the technology involved.
    Keywords:  Cellular pharmacology; Organoid; Scaffold; Three-dimensional (3D) cell cultures; Tissue engineering
  27. Nucleic Acids Res. 2020 May 22. pii: gkaa409. [Epub ahead of print]
      Next-generation sequencing has paved the way for the reconstruction of genome-scale metabolic networks as a powerful tool for understanding metabolic circuits in any organism. However, the visualization and extraction of knowledge from these large networks comprising thousands of reactions and metabolites is a current challenge in need of user-friendly tools. Here we present Fluxer (, a free and open-access novel web application for the computation and visualization of genome-scale metabolic flux networks. Any genome-scale model based on the Systems Biology Markup Language can be uploaded to the tool, which automatically performs Flux Balance Analysis and computes different flux graphs for visualization and analysis. The major metabolic pathways for biomass growth or for biosynthesis of any metabolite can be interactively knocked-out, analyzed and visualized as a spanning tree, dendrogram or complete graph using different layouts. In addition, Fluxer can compute and visualize the k-shortest metabolic paths between any two metabolites or reactions to identify the main metabolic routes between two compounds of interest. The web application includes >80 whole-genome metabolic reconstructions of diverse organisms from bacteria to human, readily available for exploration. Fluxer enables the efficient analysis and visualization of genome-scale metabolic models toward the discovery of key metabolic pathways.
  28. J Appl Lab Med. 2020 Mar 01. 5(2): 300-310
      BACKGROUND: Rapid identification and quantification of toxic alcohols and ethylene glycol is imperative for appropriate treatment. Clinical laboratories frequently rely on direct injection gas chromatography (GC) methods, but these methods require inlet maintenance and multiple GC systems. To overcome these challenges, we developed a single-column headspace GC method for both toxic alcohols and glycols that streamlines patient sample analysis for toxic alcohol ingestion.METHODS: Optimal parameters for nonderivatized (volatile) and derivatized (glycol) plasma samples were determined using a 7890 A headspace sampler, an Agilent 7697 A GC system, a DB-200 column, and a flame ionization detector. Limit of Quantification (LoQ), linearity, imprecision, carry-over, method comparison, and interference studies were performed using quality control materials and prepared plasma samples.
    RESULTS: Our volatile method is linear to 3000 mg/L (ethanol) with LoQ concentrations below 20 mg/L (ethanol). The glycol method is linear to 2000 mg/L (ethylene glycol) with LoQ concentrations below 40 mg/L (ethylene glycol). Total assay impression ranged from 1.7% for ethanol to 13.3% for propylene glycol. Both methods were free of sample carryover and compared favorably with a similar clinical method at an outside laboratory. Propionic acid, an accumulating metabolite in methylmalonic acidemia that interferes with ethylene glycol identification by a different method, did not interfere with the ethylene glycol method reported here.
    CONCLUSIONS: Our single-column headspace GC method provides reliable, robust, and rapid identification and quantification of commonly encountered toxic alcohols. Clinical laboratories relying on direct injection Gas Chromatography (GC) for toxic alcohol analysis face challenges including frequent inlet maintenance, sample carryover, or the need for separate GC systems for volatile and glycol analysis. We summarize our development and optimization of two headspace GC methods for nonderivatized (volatile) and derivatized (glycol) plasma samples that use a single DB-200 analytical column. These methods are comparable to other GC methods, not prone to sample carryover, eliminate the need for multiple GC systems or columns, and are readily applicable to other laboratories that provide toxic alcohol analysis.
    Keywords:  Gas chromatography; glycols; methylmalonic acidemia; toxic alcohols; volatiles
  29. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Jun 15. pii: S1570-0232(20)30319-6. [Epub ahead of print]1147 122142
      The metabolic investigation in the drug discovery process is an imperative aspect for selection of drug candidates with excellent therapeutic efficacy and safety profile. Ribociclib (RIBO), an orally active Cyclin dependent kinases inhibitor recently approved by USFDA for its clinical efficacy against human epithelial growth factor receptor negative and hormonal receptor positive advanced breast cancer. Although an in vitro metabolite identification study of RIBO is available in literature, no systematic metabolic investigation including detailed structural characterization and toxicity prediction of the metabolites generated in in vivo system is reported till date. Therefore, in this study, we focused on the characterization of its entire metabolites generated in in vitro as well as in vivo matrices. In vitro study includes incubation of RIBO in rat and human liver microsomes and human S9 fraction, while in vivo study was carried out using plasma, urine and faeces samples of male Sprague Dawley rats. A total of 22 metabolites were successfully separated on Agilent SB C18 (100 × 4.6 mm, 2.7µ) column using ammonium formate (pH 3.5) and acetonitrile as mobile phase. Metabolites were identified with the help of UHPLC-ESI-Q-TOF-MS/MS by accurate mass measurement. RIBO was found to be metabolised by N- dealkylation, sulphation, acetylation, oxidation, hydroxylation, carbonylation, dehydrogenation and by a combination of these reactions. The in silico toxicity profiling of all the metabolites was carried out with the help of ProTox-II software. Ten out of twenty two newly identified metabolites showed to have potential for possessing immunotoxicity. Novelty of this investigation can be justified by the unavailability of any previously published literature on complete in vitro and in vivo metabolite profiling of RIBO. Moreover, in silico toxicity of the metabolites were also not known till date.
    Keywords:  In silico toxicity; In vitro and in vivo metabolites; Metabolite profiling; Ribociclib
  30. Anal Chim Acta. 2020 Jul 04. pii: S0003-2670(20)30403-7. [Epub ahead of print]1119 77-100
      This review is an update of a previous review in 2009 and covers publications from 2009 to 2019. The review focuses on experimental design, referred to as the design of experiments (DoE), used in developing bioanalytical solid-phase microextraction (SPME) methods. Characteristics of different SPME approaches are illustrated and critically discussed. The literature selection evidences that two-level full factorial designs, with a limited number of factors (<5), are most frequently used for preliminary factors screening. When applying the response surface methodology for the quantitative assessment of factorial effects, few quadratic models were used. The most popular were the rotatable central composite and Box-Benkhen designs. Models including more than four factors, such as fractional factorial designs (including the Plackett-Burman and Taguchi designs), were rarely used. Definitive screening and D-Optimal designs were not reported anywhere in the literature selection. When examining the diagnostic criteria used to evaluate different model's quality and validity, it was apparent the researchers relied heavily on commercial software for experimental design, analysis, and reporting of the results.
    Keywords:  Bioanalysis; Experimental design (design of experiments, DoE); Method optimization; Quality by design; SPME; Sample treatment
  31. Metabolites. 2020 May 15. pii: E202. [Epub ahead of print]10(5):
      As researchers are increasingly able to collect data on a large scale from multiple clinical and omics modalities, multi-omics integration is becoming a critical component of metabolomics research. This introduces a need for increased understanding by the metabolomics researcher of computational and statistical analysis methods relevant to multi-omics studies. In this review, we discuss common types of analyses performed in multi-omics studies and the computational and statistical methods that can be used for each type of analysis. We pinpoint the caveats and considerations for analysis methods, including required parameters, sample size and data distribution requirements, sources of a priori knowledge, and techniques for the evaluation of model accuracy. Finally, for the types of analyses discussed, we provide examples of the applications of corresponding methods to clinical and basic research. We intend that our review may be used as a guide for metabolomics researchers to choose effective techniques for multi-omics analyses relevant to their field of study.
    Keywords:  biological pathways; clustering; co-regulation; deep learning; dimensionality reduction; machine learning; multi-omics integration; network analysis; pathway enrichment; visualization
  32. Proteomics. 2020 May 17. e1900353
      Multi-run alignment is widely used in proteomics to establish analyte correspondence across runs. Generally alignment algorithms return a cumulative score, which may not be easily interpretable for each peptide. Here we present a novel tool, DrawAlignR, to visualize each chromatographic alignment for DIA/SWATH data. Furthermore, we have developed a novel C++ based implementation of raw chromatogram alignment which is 35 times faster than the previously published algorithm. This not only enables users to plot alignment interactively by DrawAlignR, but also allows other software platforms to use the algorithm. DrawAlignR is an open-source web application using R Shiny that can be hosted using the source-code available at This article is protected by copyright. All rights reserved.
    Keywords:  data-independent acquisition
  33. J Sep Sci. 2020 May 20.
      Chemotherapeutics are pharmaceutical compounds whose occurrence in the environment is of growing concern because of the increase in treatments against cancer diseases. They can reach the aquatic ecosystems after passing through wastewater treatment plants without complete removal. One of the most frequently used chemotherapeutics is 5-fluorouracil which exhibits a strong cytostatic effect. In this paper, an analytical methodology was developed, validated and applied to determine 5-fluorouracil, its precursor, 5-fluorocytosine, and its major active metabolite, 5-fluorouridine, in hospital wastewater samples. Due to the expected low concentrations after dilution and interferences present in such a complex matrix, a very selective and sensitive detection method is required. Moreover, an extraction method must be implemented prior to the determination in order to purify the sample extract and preconcentrate the target analytes at μg·L-1 concentration levels. Solid phase extraction followed by liquid chromatography with tandem mass spectrometry was the combination of choice and all included parameters were studied. Under optimized conditions for wastewater samples analysis, recoveries from 63 to 108 % were obtained, while intra-day and inter-day relative standard deviations never exceeded 20 and 25%, respectively. Limits of detection between 61 and 620 ng·L-1 were achieved. Finally, the optimized method was applied to samples from hospital wastewater effluents. This article is protected by copyright. All rights reserved.
    Keywords:  cytostatic compounds; sample pretreatment; tandem mass spectrometry; wastewater samples