bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒03‒22
forty papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Metabolites. 2020 Mar 12. pii: E101. [Epub ahead of print]10(3):
      Lipidomics has great promise in various applications; however, a major bottleneck in lipidomics is the accurate and comprehensive annotation of high-resolution tandem mass spectral data. While the number of available lipidomics software has drastically increased over the past five years, the reduction of false positives and the realization of obtaining structurally accurate annotations remains a significant challenge. We introduce Lipid Annotator, which is a user-friendly software for lipidomic analysis of data collected by liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). We validate annotation accuracy against lipid standards and other lipidomics software. Lipid Annotator was integrated into a workflow applying an iterative exclusion MS/MS acquisition strategy to National Institute of Standards and Technology (NIST) SRM 1950 Metabolites in Frozen Human Plasma using reverse phase LC-HRMS/MS. Lipid Annotator, LipidMatch, and MS-DIAL produced consensus annotations at the level of lipid class for 98% and 96% of features detected in positive and negative mode, respectively. Lipid Annotator provides percentages of fatty acyl constituent species and employs scoring algorithms based on probability theory, which is less subjective than the tolerance and weighted match scores commonly used by available software. Lipid Annotator enables analysis of large sample cohorts and improves data-processing throughput as compared to previous lipidomics software.
    Keywords:  automation; ion mobility; lipid annotation; lipidomics; liquid chromatography; metabolomics; software; tandem mass spectrometry; time-of-flight
  2. Metabolites. 2020 Mar 12. pii: E103. [Epub ahead of print]10(3):
      The tricarboxylic acid (TCA) cycle is a central part of carbon and energy metabolism, also connecting to glycolysis, amino acid, and lipid metabolism. The quantitation of the TCA cycle intermediate within one method is lucrative due to the interest in central carbon metabolism profiling in cells and tissues. In addition, TCA cycle intermediates in serum have been discovered to correspond as biomarkers to various underlying pathological conditions. In this work, an Liquid Chromatography-Mass Spectrometry/Mass Spectrometry-based quantification method is developed and validated, which takes advantage of fast, specific, sensitive, and cost-efficient precipitation extraction. Chromatographic separation is achieved while using Atlantis dC18 2.1 mm × 100 mm, particle size 3-μm of Waters column with a gradient elution mobile phase while using formic acid in water (0.1% v/v) and acetonitrile. Linearity was clearly seen over a calibration range of: 6.25 to 6400 ng/mL (r2 > 0.980) for malic acid; 11.72 to 12,000 ng/mL (r2 > 0.980) for cis-aconitic acid and L-aspartic acid; 29.30 to 30,000 ng/mL (r2 > 0.980) for isocitric acid, l-serine, and l-glutamic acid; 122.07 to 125,000 ng/mL (r2 > 0.980) for citric acid, glycine, oxo-glutaric acid, l-alanine, and l-glutamine; 527.34 to 540,000 ng/mL (r2 > 0.980) for l-lactic acid; 976.56 to 1,000,000 ng/mL (r2 > 0.980) for d-glucose; 23.44 to 24,000 ng/mL (r2 > 0.980) for fumaric acid and succinic acid; and, 244.14 to 250,000 ng/mL (r2 > 0.980) for pyruvic acid. Validation was carried out, as per European Medicines Agency (EMA) "guidelines on bioanalytical method validation", for linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LLOQ), recovery, matrix effect, and stability. The recoveries from serum and tissue were 79-119% and 77-223%, respectively. Using this method, we measured TCA intermediates in serum, plasma (NIST 1950 SRM), and in mouse liver samples. The concentration found in NIST SRM 1950 (n = 6) of glycine (246.4 µmol/L), l-alanine (302.4 µmol/L), and serine (92.9 µmol/L).
    Keywords:  bioanalytical method validation; chromatography; mass spectrometry; metabolites; tricarboxylic acid (TCA) cycle
  3. J Chromatogr A. 2020 Mar 07. pii: S0021-9673(20)30237-5. [Epub ahead of print] 461021
      In this work, the impact of biological matrices, such as plasma and urine, was evaluated under SFCHRMS in the field of metabolomics. For this purpose, a representative set of 49 metabolites were selected. The assessment of the matrix effects (ME), the impact of biological fluids on the quality of MS/MS spectra and the robustness of the SFCHRMS method were each taken into consideration. The results have highlighted a limited presence of ME in both plasma and urine, with 30% of the metabolites suffering from ME in plasma and 25% in urine, demonstrating a limited sensitivity loss in the presence of matrices. Subsequently, the MS/MS spectra evaluation was performed for further peak annotation. Their analyses have highlighted three different scenarios: 63% of the tested metabolites did not suffer from any interference regardless of the matrix; 21% were negatively impacted in only one matrix and the remaining 16% showed the presence of matrix-belonging compounds interfering in both urine and plasma. Finally, the assessment of retention times stability in the biological samples, has brought into evidence a remarkable robustness of the SFCHRMS method. Average RSD (%) values of retention times for spiked metabolites were equal or below 0.5%, in the two biological fluids over a period of three weeks. In the second part of the work, the evaluation of the Sigma Mass Spectrometry Metabolite Library of Standards containing 597 metabolites, under SFCHRMS conditions was performed. A total detectability of the commercial library up to 66% was reached. Among the families of detected metabolites, large percentages were met for some of them. Highly polar metabolites such as amino acids (87%), nucleosides (85%) and carbohydrates (71%) have demonstrated important success rates, equally for hydrophobic analytes such as steroids (78%) and lipids (71%). On the negative side, very poor performance was found for phosphorylated metabolites, namely phosphate-containing compounds (14%) and nucleotides (31%).
    Keywords:  Matrix effect; Metabolomics; Retention time variability; Supercritical fluid chromatography; UHPSFC-HRMS
  4. J Clin Med. 2020 Mar 13. pii: E788. [Epub ahead of print]9(3):
      Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disorder caused by mutations in TYMP, leading to a deficiency in thymidine phosphorylase and a subsequent systemic accumulation of thymidine and 2'-deoxyuridine. Erythrocyte-encapsulated thymidine phosphorylase (EE-TP) is under clinical development as an enzyme replacement therapy for MNGIE. Bioanalytical methods were developed according to regulatory guidelines for the quantification of thymidine and 2'-deoxyuridine in plasma and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for supporting the pharmacodynamic evaluation of EE-TP. Samples were deproteinized with 5% perchloric acid (v/v) and the supernatants analyzed using a Hypercarb column (30 × 2.1 mm, 3 µm), with mobile phases of 0.1% formic acid in methanol and 0.1% formic acid in deionized water. Detection was conducted using an ion-spray interface running in positive mode. Isotopically labelled thymidine and 2'-deoxyuridine were used as internal standards. Calibration curves for both metabolites showed linearity (r > 0.99) in the concentration ranges of 10-10,000 ng/mL for plasma, and 1-50 µg/mL for urine, with method analytical performances within the acceptable criteria for quality control samples. The plasma method was successfully applied to the diagnosis of two patients with MNGIE and the quantification of plasma metabolites in three patients treated with EE-TP.
    Keywords:  2’-deoxyuridine; EE-TP; LC–MS/MS; erythrocyte encapsulated thymidine phosphorylase; method validation; mitochondrial neurogastrointestinal encephalomyopathy; thymidine
  5. Bioinformatics. 2020 Mar 18. pii: btaa200. [Epub ahead of print]
      MOTIVATION: Liquid chromatography-mass spectrometry-based non-targeted metabolomics is routinely performed to qualitatively and quantitatively analyze a tremendous amount of metabolite signals in complex biological samples. However, false-positive peaks in the datasets are commonly detected as metabolite signals by using many popular software, resulting in non-reliable measurement.RESULTS: To reduce false-positive calling, we developed an interactive web tool, termed CPVA, for visualization and accurate annotation of the detected peaks in non-targeted metabolomics data. We used a chromatogram-centric strategy to unfold the characteristics of chromatographic peaks through visualization of peak morphology metrics, with additional functions to annotate adducts, isotopes and contaminants. CPVA is a free, user-friendly tool to help users to identify peak background noises and contaminants, resulting in decrease of false-positive or redundant peak calling, thereby improving the data quality of non-targeted metabolomics studies.
    AVAILABILITY: The CPVA is freely available at Source code and installation instructions are available on GitHub:
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
  6. Biomed Chromatogr. 2020 Mar 17. e4831
      Dual mode heart-cutting two-dimensional liquid chromatography (DMHC 2D-LC) was applied to isotope dilution mass spectrometry (IDMS) to reduce the bias in the quantitative analysis of a target analyte present in a limited quantity in human plasma. Based on a Waters I-Class LC system, the DMHC 2D-LC system was operated in one- and two-dimensional modes to facilitate the determination of heart-cutting time and the efficient trapping of the target LC eluate. Experiments to determine the feasibility of coupling with IDMS was performed with triple quadrupole mass spectrometry using folic acid standards and/or 13 C5 -folic acid. To validate the performance of the DMHC 2D-LC/IDMS system on a complex sample, human plasma was analyzed for folic acid and the result was compared with that obtained using conventional single-column LC. The total run time of the DMHC 2D-LC system was 20 min, the same as that of the single-column LC system. The peak profile of the spiked 13 C5 -folic acid obtained with single-column LC/MS was affected by matrix effects, but resolved with DMHC 2D-LC/MS, thus improving the accuracy of the analysis. The DMHC 2D-LC/IDMS system showed reliable performance in analyzing the target analyte in human plasma eliminating matrix effects and saving analysis time.
    Keywords:  dual-mode heart-cutting two-dimensional liquid chromatography; folic acid; human plasma; isotope dilution mass spectrometry; matrix effects
  7. Bioanalysis. 2020 Mar 18.
      Aim: To investigate the clinical pharmacokinetic profiles of FCN-411, a new EGFR tyrosine kinase inhibitor, an ultra-performance LC-MS/MS method was developed. Methods & results: The method was suitable to determine FCN-411 in plasma due to the fast sample preparation (protein precipitation procedure), a good linear range of 2-500 ng/ml, low amount of sample volume (5 μl) and less run time (4.5 min) for analysis. And it was demonstrated to be acceptable according to the guidelines for bioanalytical assay validation. Conclusion: The method was robust, sensitive and repeatable, and it is ready to be applied to measure FCN-411 in a Phase I clinical pharmacokinetic study.
    Keywords:  FCN-411; UPLC–MS/MS; human plasma; protein precipitation
  8. Res Pharm Sci. 2020 Feb;15(1): 1-13
      Background and purpose: A simple, rapid, and sensitive reversed-phase high performance liquid chromatography (RP-HPLC) method based on liquid-liquid extraction was developed and validated for determination of docetaxel (DTX) in plasma and homogenate tissues of tumor-bearing mice.Experimental approach: Samples were spiked with celecoxib as the internal standard and separation was achieved on a μ-Bondapak C18 HPLC column. The mobile phase consisted of a mixture of acetonitrile/water (40/60 v/v) at flow rate of 1.2 mL/min and the effluent was monitored at 230 nm.
    Results: Calibration curves were linear over the concentration range of 0.1-10 μg/mL of DTX in plasma and 0.25-50 μg/mL in tissue homogenates with acceptable precision and accuracy. The mean recoveries of the drug from plasma extraction was 94.6 ± 1.44% while those of tissue homogenates ranged from 73.5 ± 3.2 to 85.3 ± 2.8% depending on the type of tissues examined. DTX was stable in biological samples with no evidence of degradation during 3 freeze-thaw cycles and two months of storage at -70 ± 15 °C. The developed HPLC method was applied to quantify DTX in the mouse plasma and tissues after intravenous administration of 7.5 mg equivalent DTX/kg dose of DTX-loaded folic acid-polyethylene glycol-heparin-tocopherol (FA-PEG-HEP-CA-TOC) micelle formulation to female Balb/c mice.
    Conclusion: A simple, sensitive, rapid, accurate, and prudent RP-HPLC method was developed, validated, and applied for DTX determination in plasma and tissues.
    Keywords:  Celecoxib; Distribution; Docetaxel; HPLC; Pharmacokinetics; Tissue
  9. MethodsX. 2020 ;7 100812
      A quantification assay based on isotope dilution mass spectrometry to determine the concentration of progesterone in human serum was reported. Incorporated with 13C3-progesterone, serum samples were subjected to progesterone extraction and clean-up by C4 solid-phase-extraction columns and hexane-based liquid/liquid extraction, respectively. The cleaned-up serum samples were then subjected to MALDI-TOF mass spectrometry for the quantification of progesterone. In the study, the recovered progesterone concentration determined by the assay showed good robustness and constancy in comparison to conventional radioimmunologic assay. We concluded that the 13C3-progesterone-based quantification assay is a robust method for the measurement of serum progesterone. Advantages of this technique includes: • This study describes a MALDI-TOF/MS method for the determination of serum progesterone. • The technique is simple and easy to apply on MALDI-TOF/MS for serum progesterone analysis. • The correlation coefficient between MALDI-TOF MS and RIA was 0.981 for serum progesterone.
    Keywords:  Isotope dilution; MALDI-TOF/MS; Progesterone
  10. Int J Anal Chem. 2020 ;2020 6980392
      L-Asparagine (ASN) is the catalyze substrate of L-asparaginase (ASNase), which is an important drug for acute lymphoblastic leukemia (ALL) patients. The ASN level is found to be closely associated with the effectiveness of ASNase treatment. In this study, a hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) method was developed for the determination of ASN in the human serum using a stable isotope-labeled internal standard (ASN-D3). Serum samples were prepared by a one-step precipitation procedure using methanol and separated by an Agilent HILIC Plus column with the mobile phase of methanol-water (95 : 5, v/v, containing 5 mM ammonium formate and 0.1% formic acid), at a constant flow rate of 0.3 mL/min. Mass spectrometric analysis was conducted using multiple-reaction monitoring in the positive electrospray ionization mode. Serum ASN concentrations were determined over a linear calibration curve range of 2-200 μM, with acceptable accuracies and precisions. The validated HILIC-MS/MS method was successfully applied to the quantification of ASN levels in the serum from patients with ALL. Collectively, the research may shed new light on an alternative rapid, simple, and convenient quantitative method for determination of serum ASN in ALL patients treated with ASNase.
  11. J Pharm Biomed Anal. 2020 Mar 09. pii: S0731-7085(19)33009-2. [Epub ahead of print]185 113246
      The simultaneous quantitative estimation of tryptophan (TRP) and its metabolites represents a great challenge because of their diverse chemical properties, e.g., presence of acidic, basic, and nonpolar functional groups and their immensely different concentrations in biological matrices. A short ultra high-performance liquid chromatography (UHPLC)-tandem mass spectrometry (MS/MS) method was validated for targeted analysis of TRP and its 11 most important metabolites derived via both kynurenine (KYN) and serotonin (SERO) pathways in human serum and cerebrospinal fluid (CSF): SERO, KYN, 3-hydroxyanthranilic acid, 5-hydroxyindoleacetic acid, anthranilic acid, kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), xanthurenic acid, melatonin, picolinic acid (PICA), and quinolinic acid (QUIN). After selecting the "best" reversed-phase column and organic modifier, DryLab®4 was used to optimize the gradient time and temperature in chromatographic separation. To achieve absolute quantification, deuterium-labeled internal standards were used. Among all compounds, 3 were analyzed in derivatized (butyl ester) forms (3-HK, PICA, and QUIN) and the remaining 9 in underivatized forms. Validation was performed in accordance with the ICH and FDA guidelines to determine the intraday and interday precision, accuracy, sensitivity, and recovery. To demonstrate the applicability of the developed UHPLC-MS/MS method, the aforementioned metabolites were analyzed in serum and CSF samples from patients with multiple sclerosis (multiple sclerosis group) and those with symptomatic or noninflammatory neurological diseases (control group). The concentration of QUIN dramatically increased, whereas that of KYNA slightly decreased in the multiple sclerosis group, resulting in a significantly increased QUIN/KYNA ratio and significantly decreased PICA/QUIN ratio.
    Keywords:  Derivatization; DryLab®4; Liquid chromatography-mass spectrometry; Multiple sclerosis; Tryptophan metabolism; Validation
  12. Foods. 2020 Mar 09. pii: E309. [Epub ahead of print]9(3):
      Determination of the L- and D-amino acid composition in proteins is important for monitoring process-induced racemization, and thereby protein quality loss, in food and feed. Such analysis has so far been challenging due to the need for sample hydrolysis, which generates racemization, thereby leading to an overestimation of D-amino acids. Here, validation of an LC-MS/MS-based method for the simultaneous determination of L- and D-amino acids in complex biological matrixes, like food and feed, was performed in combination with deuterated HCl hydrolysis. This approach eliminated a racemization-induced bias in the L- and D-amino acid ratios. The LC-MS/MS method was applied for the analysis of 18 free amino acids, with a quantification limit of either 12.5 or 62 ng/mL, except for D-phenylalanine, for which quantification was impaired by background interference from the derivatization agent. For hydrolyzed samples, the composition of 10 L- and D-amino acids pairs could be determined in protein. The average relative standard deviation was 5.5% and 6.1%, depending on the type of hydrolysis tubes. The method was applied on a green protein isolate (lucerne), which contained an average of 0.3% D-amino acids. In conclusion, this method allows for an unbiased analysis of L- and D-amino acid ratios in complex protein samples, such as food and feed.
    Keywords:  D-amino acid; L-amino acid; LC–MS/MS; enantiomer; feed; food; hydrolysis; processing; protein quality; racemization
  13. Metabolomics. 2020 Mar 18. 16(4): 42
      INTRODUCTION: The use of 2D NMR data sources (COSY in this paper) allows to reach general metabolomics results which are at least as good as the results obtained with 1D NMR data, and this with a less advanced and less complex level of pre-processing. But a major issue still exists and can largely slow down a generalized use of 2D data sources in metabolomics: the experiment duration.OBJECTIVE: The goal of this paper is to overcome the experiment duration issue in our recently published MIC strategy by considering faster 2D COSY acquisition techniques: a conventional COSY with a reduced number of transients and the use of the Non-Uniform Sampling (NUS) method. These faster alternatives are all submitted to novel 2D pre-processing workflows and to Metabolomic Informative Content analyses. Eventually, results are compared to those obtained with conventional COSY spectra.
    METHODS: To pre-process the 2D data sources, the Global Peak List (GPL) workflow and the Vectorization workflow are used. To compare this data sources and to detect the more informative one(s), MIC (Metabolomic Informative Content) indexes are used, based on clustering and inertia measures of quality.
    RESULTS: Results are discussed according to a multi-factor experimental design (which is unsupervised and based on human urine samples). Descriptive PCA results and MIC indexes are shown, leading to the direct and objective comparison of the different data sets.
    CONCLUSION: In conclusion, it is demonstrated that conventional COSY spectra recorded with only one transient per increment and COSY spectra recorded with 50% of non-uniform sampling provide very similar MIC results as the initial COSY recorded with four transients, but in a much shorter time. Consequently, using techniques like the reduction of the number of transients or NUS can really open the door to a potential high-throughput use of 2D COSY spectra in metabolomics.
    Keywords:  2D NMR; 2D pre-processing workflows; COSY spectra; Global peak list; Metabolomic informative content (MIC); Non-uniform sampling (NUS); Vectorization
  14. J Genet Genomics. 2019 Dec 18. pii: S1673-8527(19)30200-0. [Epub ahead of print]
      Mass spectrometry (MS)-based omics technologies are now widely used to profile small molecules in multiple matrices to confer comprehensive snapshots of cellular metabolic phenotypes. The metabolomes of cells, tissues, and organisms comprise a variety of molecules including lipids, amino acids, sugars, organic acids, and so on. Metabolomics mainly focus on the hydrophilic classes, while lipidomics has emerged as an independent omics owing to the complexities of the organismal lipidomes. The potential roles of lipids and small metabolites in disease pathogenesis have been widely investigated in various human diseases, but system-level understanding is largely lacking, which could be partly attributed to the insufficiency in terms of metabolite coverage and quantitation accuracy in current analytical technologies. While scientists are continuously striving to develop high-coverage omics approaches, integration of metabolomics and lipidomics is becoming an emerging approach to mechanistic investigation. Integration of metabolome and lipidome offers a complete atlas of the metabolic landscape, enabling comprehensive network analysis to identify critical metabolic drivers in disease pathology, facilitating the study of interconnection between lipids and other metabolites in disease progression. In this review, we summarize omics-based findings on the roles of lipids and metabolites in the pathogenesis of selected major diseases threatening public health. We also discuss the advantages of integrating lipidomics and metabolomics for in-depth understanding of molecular mechanism in disease pathogenesis.
    Keywords:  Disease progression; Integration; Lipidomics; Metabolomics
  15. Biochimie. 2020 Mar 13. pii: S0300-9084(20)30056-0. [Epub ahead of print]
      Lipid mediators such as eicosanoids maintain various physiological processes, and their alterations are involved in the development of numerous cardiovascular diseases. Therefore, the reliable assessment of their profile could be helpful in diagnosis as well as in eicosanoid biomarker-based treatment. Hence, the presented study aimed to develop and validate a new rapid, specific and sensitive LC-MS/MS method for quantification of arachidonic acid-derived eicosanoids in plasma, including lipid mediators generated via COX-, LOX- and CYP450-dependent pathways. The developed method features high sensitivity because the lower limit of quantification ranged from 0.05-0.5 ng mL-1 as well as an accuracy and precision estimated within 88.88-111.25% and 1.03-11.82%, respectively. An application of a simple and fast liquid-liquid extraction procedure for sample cleaning resulted in a highly satisfactory recovery of the analytes (>88.30%). Additionally, the method was validated using artificial plasma, an approach that enabled elimination of the matrix effect caused by endogenous concentration of studied lipid mediators. Importantly, the presented LC-MS/MS method allowed for simultaneous quantitative and qualitative [quan/qual] analysis of the selected eicosanoids, leading to an additional improvement of the method specificity. Moreover, the validated method was successfully applied for eicosanoid profiling in rat, mouse and human plasma samples, clearly demonstrating the heterogeneity of studied lipid mediators between those species.
    Keywords:  LC-MS/MS; arachidonic acid; eicosanoids; method validation
  16. PLoS One. 2020 ;15(3): e0230072
      In the urinary metabolomics for finding biomarkers in urine, owing to high concentrations of urea, for chromatography-based metabolomic analysis, urea needed to be degraded by urease. This urease pretreatment has been the key step of sample preparation for standard urinary metabolomics until today even for mass spectrometry-based analysis. The urease pretreatment involving incubation of urine with urease contradicts the concept of metabolome sampling, which should immediately arrest metabolic reactions to prevent alterations of a metabolite profile. Nonetheless, the impact of urease pretreatment has not been clearly elucidated yet. We found that activities of urease and endogenous urinary enzymes and metabolite contaminants from the urease preparations introduce artefacts into metabolite profiles, thus leading to misinterpretation.
  17. Food Chem. 2020 Mar 10. pii: S0308-8146(20)30445-3. [Epub ahead of print]319 126583
      This study established a simple and rapid method for determination of short-chain chlorinated paraffins (SCCPs) and medium-chain chlorinated paraffins (MCCPs) by dispersive liquid-liquid micro-extraction coupled with high performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOF/MS) in white and red wines. Affecting variables, including extraction solvent, salt concentration, ultrasound-vortex conditions and ethanol content, were evaluated. Under optimized conditions, the limit of detection (LODs) for SCCPs and MCCPs were in the range of 0.15-3.00 ng mL-1 and 0.08-2.50 ng mL-1, respectively. The spiked recoveries of SCCPs and MCCPs from white and red wine ranged from 63.2% to 127%. The method is precise with intra- and inter-day variations within 14.0% and 17.0%, respectively. SCCPs and MCCPs in wines from china varied widely, from <LOD to 415 ng mL-1 and <LOD to 153 ng mL-1, respectively.
    Keywords:  Chlorinated paraffins; Dispersive liquid–liquid micro-extraction; High performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry; Red wine; White wine
  18. OMICS. 2020 Mar;24(3): 140-147
      Acute T cell-mediated rejection (TCMR) is a major complication after renal transplantation. TCMR diagnosis is very challenging and currently depends on invasive renal biopsy and nonspecific markers such as serum creatinine. A noninvasive metabolomics panel could allow early diagnosis and improved accuracy and specificity. We report, in this study, on urine metabolome changes in renal transplant recipients diagnosed with TCMR, with a view to future metabolomics-based diagnostics in transplant medicine. We performed urine metabolomic analyses in three study groups: (1) 7 kidney transplant recipients with acute TCMR, (2) 15 kidney transplant recipients without rejection but with impaired kidney function, and (3) 6 kidney transplant recipients with stable renal function, using 1H-nuclear magnetic resonance. Multivariate modeling of metabolites suggested a diagnostic panel where the diagnostic accuracy of each metabolite was calculated by receiver operating characteristic curve analysis. The impaired metabolic pathways associated with TCMR were identified by pathway analysis. In all, a panel of nine differential metabolites encompassing nicotinamide adenine dinucleotide, 1-methylnicotinamide, cholesterol sulfate, gamma-aminobutyric acid (GABA), nicotinic acid, nicotinamide adenine dinucleotide phosphate, proline, spermidine, and alpha-hydroxyhippuric acid were identified as novel potential metabolite biomarkers of TCMR. Proline, spermidine, and GABA had the highest area under the curve (>0.7) and were overrepresented in the TCMR group. Nicotinate and nicotinamide metabolism was the most important pathway in TCMR. These findings call for clinical validation in larger study samples and suggest that urinary metabolomics warrants future consideration as a noninvasive research tool for TCMR diagnostic innovation.
    Keywords:  biomarkers; kidney disease; nuclear magnetic resonance; transplant medicine; transplant rejection; urinary metabolomics
  19. J Chromatogr A. 2020 Feb 27. pii: S0021-9673(20)30206-5. [Epub ahead of print] 461000
      This work focused on the development and validation of a method based on hollow fiber-based solid-phase microextraction coupled to ultra-performance liquid chromatography tandem mass spectrometry (HF-based-SPME-UPLC-MS/MS) for the determination of five antipsychotics at a pg mL-1 level in human whole blood and urine. Four types of hollow fiber membrane materials, including polyether sulfone, polypropylene, polyvinyl chloride and polyvinylidene fluoride were investigated. Finally, polyether sulfone hollow fiber without any modification was selected as the adsorption medium for solid-phase microextraction (SPME) with the following extraction procedure: the analytes were adsorbed onto the hollow fiber in the sample bottle with application of ultrasonication. Subsequently, the hollow fiber was transferred into a slim glass tube containing an appropriate solvent, and the analytes were desorbed by ultrasound treatment before detection by UPLC-MS/MS. In order to obtain satisfactory extraction efficiency, extraction parameters such as hollow fiber membrane material, pH, hollow fiber length, extraction time, desorption solvent and desorption time were investigated. Under the optimum experimental conditions, this method allowed for determination of five antipsychotics in human whole blood with excellent limits of quantification (LOQs) (25.0, 12.5, 25.0, 25.0 and 12.5 pg mL-1 for perphenazine, chlorpromazine, chlorprothixene, promethazine and trifluoperazine, respectively). The corresponding LOQs in human urine were 25.0, 12.5, 12.5, 12.5 and 12.5 pg mL-1 for the respective antipsychotics. The precision (RSD) was no more than 13.3%. The extraction recoveries for human whole blood and urine were in the range of 46.4-96.6% and 65.2-101.9%, respectively. The proposed method was compared with other methods from the literature and the results demonstrate that it is a simple, sensitive, efficient and green technique. It is suitable for analyzing trace target analytes in complex matrices such as biological samples and can provide a reliable tool for drug monitoring especially in forensic analysis and case of drug abuse.
    Keywords:  Antipsychotic; Hollow fiber-based solid-phase microextraction; UPLC-MS/MS; Urine; Whole blood
  20. Anal Chem. 2020 Mar 20.
      Rapid and efficient determination of contaminants at trace levels in tissue samples has become an unmet need around the globe. Coated Blade Spray (CBS) extraction/ionization is a technology capable of performing, with a single device, enrichment of analytes present in complex matrices, as well as the direct interface and introduction of said analytes into the mass spectrometer via electrospray ionization. To facilitate the challenging rapid tissue screening, we describe for the first time the use of a very thin layer of biocompatible polyacrylonitrile as a CBS device undercoating to make metal surface biocompatible. This add-on is meant to protect the portion of the uncoated stainless-steel of the blade that is normally exposed to the matrix, consequently becoming susceptible to adhesion of matrix macromolecules, cells, and fat. In addition, we present for the first time the use of CBS in negative ionization mode. The optimized CBS workflow allows for rapid and high-throughput screening and quantitation of 105 veterinary drugs in homogenized bovine tissue in both negative and positive ionization mode in one single run using a single CBS device with analysis times as short as 1 min per sample when 96 extractions are simultaneously conducted. While only two internal standards were used for correction, one per ionization mode, excellent accuracy and precision were achieved, with more than 90% of analytes falling within the 70-120% range of their true concentrations and yielding RSD ≤25% at three validation levels. The majority of analytes achieved linear correlation coefficients > 0.99, and all 105 analytes were able to meet both Canadian and US regulatory levels.
  21. Clin Chem Lab Med. 2020 Mar 19. pii: /j/cclm.ahead-of-print/cclm-2019-0723/cclm-2019-0723.xml. [Epub ahead of print]
      The diffusion of laboratory automation, initiated nearly 50 years ago with consolidation of preanalytical, clinical chemistry and immunochemistry workstations, is now also gradually embracing mass spectrometry (MS). As for other diagnostic disciplines, the automation of MS carries many advantages, such as efficient personnel management (i.e. improving working atmosphere by decreasing manual activities, lowering health risks, simplifying staff training), better organization (i.e. reducing workloads, improving inventory handling, increasing analytical process standardization) and the possibility to reduce the number of platforms. The development and integration of different technologies into automated MS analyzers will also generate technical and practical advantages, such as prepackaged and ready-to-use reagents, automated dispensing, incubation and measurement, automated sample processing (e.g. system fit for many models of laboratory automation, bar code readers), multiplex testing, automatic data processing, also including quality control assessment, and automated validation/interpretation (e.g. autoverification). A new generation of preanalytical workstations, which can be directly connected to MS systems, will allow the automation of manual extraction and elimination of time-consuming activities, such as tube labeling and capping/decapping. The use of automated liquid-handling platform for pipetting samples, along with addition of internal standards, may then enable the optimization of some steps of extraction and protein precipitation, thus decreasing turnaround time and increasing throughput in MS testing. Therefore, this focused review is aimed at providing a brief update on the importance of consolidation and integration of MS platforms in laboratory automation.
    Keywords:  LC-MS/MS; blood tube; mass spectrometry; preanalytical phase
  22. Anal Bioanal Chem. 2020 Mar 21.
      The intestinal microbiome plays an important role in human health and disease and fecal materials reflect the microbial activity. Thus, analysis of fecal metabolites provides insight in metabolic interactions between gut microbiota and host organism. In this work, we applied flow injection analysis coupled to Fourier transform mass spectrometry (FIA-FTMS) to identify and quantify lipid species in human fecal samples. Fecal homogenates were subjected to lipid extraction and analyzed by FIA-FTMS. The analysis of different subjects revealed a vast heterogeneity of lipid species abundance. The majority of samples displayed prominent signals of triacylglycerol (TG) and diacylglycerol (DG) species that could be verified by MS2 spectra. Therefore, we focused on the quantification of TG and DG. Method validation included limit of quantification, linearity, evaluation of matrix effects, recovery, and reproducibility. The validation experiments demonstrated the suitability of the method, with exception for approximately 10% of samples, where we observed coefficients of variation higher than 15%. Impaired reproducibility was related to sample inhomogeneity and could not be improved by additional sample preparation steps. Additionally, these experiments demonstrated that compared with aqueous samples, samples containing isopropanol showed higher amounts of DG, presumably due to lysis of bacteria and increased TG lipolysis. These effects were sample-specific and substantiate the high heterogeneity of fecal materials as well as the need for further evaluation of pre-analytic conditions. In summary, FIA-FTMS offers a fast and accurate tool to quantify DG and TG species and is suitable to provide insight into the fecal lipidome and its role in health and disease.
    Keywords:  Diglyceride; Feces; High-resolution mass spectrometry; Lipidomics; Microbiome; Triglyceride
  23. Biochem Biophys Res Commun. 2020 Mar 11. pii: S0006-291X(20)30475-7. [Epub ahead of print]
      Evidences suggest that dietary docosahexaenoic acid (DHA) supplementation may have pleiotropic beneficial effects on health. However, the underlying mechanisms and crucial targets that are involved in achieving these benefits remain to be clarified. In this study, we employed biochemical analysis and liquid chromatography-mass spectrometry (LC-MS) based untargeted metabolomics coupled with multivariate statistical analysis to identify potential metabolic targets of DHA in adult rats at 48 h post-feeding. Blood biochemical analysis showed a significant decrease in triglyceride level of DHA diet group, the untargeted metabolomic analysis revealed that some metabolites were significantly different between the DHA diet group and the basal diet group, including fatty acids (16:0, 18:1, 20:5n3, 22:2n6 and 24:0), diglyceride (20:0/18:2n6, 18:3n6/22:6n3, 20:4n3/20:4n3, and 22:0/24:0), PIP2 (18:2/20:3), phytol, lysoSM (d18:1), 12-hydroxyheptadecatrienoic acid, dihydrocorticosterone and N1-acetylspermine, which are mainly involved in fat mobilization and triglyceride hydrolysis, arachidonic acid, steroid hormone, and polyamine metabolism. To our knowledge, this is the first report that links the metabolic effects of DHA with arachidonic acid, steroid, and polyamine metabolism. Our finding suggests that the beneficial effects of DHA, may not directly require its own metabolic derivatives, but could be achieved by metabolic regulation.
    Keywords:  Docosahexaenoic acid; LC-MS; Metabolic target; Metabolomics
  24. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Jan 13. pii: S1570-0232(19)31427-8. [Epub ahead of print]1143 121972
      Short-chain fatty acids (SCFAs) are gut microbiota metabolites recognized for their beneficial effects on the host organism. In this study, a simple and rapid sample preparation method combined to SCFAs analysis by direct injection and gas chromatography coupled with flame ionization detection (GC-FID), for the determination and quantification of eight SCFAs (acetic, propionic, i-butyric, butyric, i-valeric, valeric, i-caproic and caproic acids) in rat, mice and human faeces and in fermentation fluids samples, has been developed and validated. The method consists of extraction of the SCFAs by ethyl ether after acidification of the samples. The effect of the number of extractions has been assessed in order to optimize the procedure and to obtain a satisfactory yield for all the analyzed SCFAs. The increase of the extracted analytes quantity was significant passing from 1 to 2 and from 2 to 3 extractions (P < 0.05), while no significant differences were found performing 3, 4 or 5 extractions (P > 0.05). The SCFAs extracted are directly analyzed by GC-FID without derivatization and separated on a polyethylene glycol nitroterephthalic acid modified coated capillary column, with a chromatographic run time of 13 min. The proposed method showed good sensitivity, with limits of quantifications in the range 0.14-0.48 µM for SCFAs from propionic to caproic acids and 2.12 µM for acetic acid; recovery was between 80.8 and 108.8% and intraday and interday repeatability in the range 0.6-5.0% of precision (RSD, %) The optimized method is suitable for the quantitative analysis of SCFAs in real samples of rat, mouse and human faeces and in fermentation fluids, and it can be applied also to very small amount of faecal sample (20 mg).
    Keywords:  Faeces; Fermentation fluids; Gas chromatography; Gut microbiota metabolite; Short-chain fatty acids (SCFAs)
  25. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Mar 05. pii: S1570-0232(20)30048-9. [Epub ahead of print]1143 122054
      Glyoxylic acid (GA) is the intermediate metabolite in various mammalian metabolic pathways. GA showed high reactivity towards formation of advanced glycation end-products (AGEs); the main cause of pathogenesis and complications of many diseases. The presented study aimed to detect GA in healthy and cardiovascular patients' (CV) sera; however analysis of GA in biological fluid is a challenge and requires chemical derivatization. Hence, a new, highly sensitive, time saving and reproducible pre-column fluorescence derivatization procedure coupled with high performance liquid chromatography (HPLC) method was developed. The derivatization method was based on reaction of 2-aminobenzenthiol (2-ABT), a fluorogenic reagent, with GA in acidic medium to form highly fluorescent thiazine derivative (290 and 390 nm for excitation and emission wavelengths respectively). The fluorescent derivative was separated within 6 min on a reversed-phase ODS column using an isocratic elution with a mixture of methanol-water (70:30, v/v%). The proposed method parameters were optimized and the method was validated. A good linearity in the concentration range (0.05-5.0 µM) was obtained with detection limit (LOD) of 10 nM (200 fmol/injection), which is more sensitive than several previous methods. Moreover, the recovery results were within the range of 85.0-95.5 % and the intra- and inter-day precision results were ≤3.5%. It should be emphasized that this method is the first one for monitoring of GA in CV patients; to investigate its role for diagnosis and monitoring the severity and complications of this disease in clinical laboratory.
    Keywords:  2-Aminobenzenthiol; Glyoxylic acid; High Performance Liquid Chromatography (HPLC); Pre-column fluorescence derivatization; Serum analysis
  26. J Proteome Res. 2020 Mar 16.
      In metabolomics, identification of metabolic pathways altered by disease, genetics or environmental perturbations is crucial to uncover the underlying biological mechanisms. A number of pathway analysis methods are currently available, which are generally based on equal-probability, topological-centrality or model-separability methods. In brief, prior identification of significant metabolites is needed for the first two types of methods, while each pathway is modeled separately in the model-separability based methods. In these methods, interactions between metabolic pathways are not taken into consideration. The current study aims to develop a novel metabolic pathway identification method based on multi-block partial least squares (MB-PLS) analysis, by including all pathways into a global model to facilitate biological interpretation. The detected metabolites are first assigned to pathway blocks based on their roles in metabolism as defined by the KEGG pathway database. The metabolite intensity or concentration data matrix is then reconstructed as data blocks according to the metabolite subsets. Then, a MB-PLS model is built on these data blocks. A new metric, named the pathway importance in projection (PIP), is proposed for evaluation of the significance of each metabolic pathway for group separation. A simulated dataset was generated by imposing artificial perturbation on four pre-defined pathways of the healthy control group of a colorectal cancer study. Performance of the proposed method was evaluated and compared with seven other commonly used methods using both an actual metabolomics dataset and the simulated dataset. For the real metabolomics dataset, most of the significant pathways identified by the proposed method were found to be consistent with the published literature. For the simulated dataset, the significant pathways identified by the proposed method are highly consistent with the pre-defined pathways. The experimental results demonstrate that the proposed method is effective for identification of significant metabolic pathways which may facilitate biological interpretation of metabolomics data.
  27. Anal Bioanal Chem. 2020 Mar 18.
      GC/MS coupled metabolomics analysis, using a simplified and much less expensive silylation process with trimethylsilyl cyanide (TMSCN), was conducted to investigate metabolic abnormalities in stomach cancer cells. Under optimized conditions for derivatization by TMSCN and methanol extraction, 228 metabolites were detected using GC/MS spectrometry analysis, and 89 metabolites were identified using standard compounds and the NIST database. Ten metabolite levels were found to be lower in stomach cancer cells relative to normal cells. Among those ten metabolites, four metabolites-ribose, proline, pyroglutamic acid, and glucose-were known to be linked to cancers. In particular, pyroglutamic acid level showed a drastic reduction of 22-fold in stomach cancer cells. Since glutamine and glutamic acid are known to undergo cyclization to pyroglutamic acid, the 22-fold reduction might be the actual reduction in the levels of glutamine and/or glutamic acid-both known to be cancer-related. Hence, the marked reduction in pyroglutamic acid level might serve as a biomarker to aid early detection of stomach cancer. Graphical abstract.
    Keywords:  GC/MS; Metabolomics; Sample treatment; Stomach cancer cells
  28. Iran J Pharm Res. 2019 ;18(4): 1735-1741
      Therapeutic Drug Monitoring (TDM) of first-line anti-tuberculosis (TB) drugs is a decisive tool, allowing the clinician to successfully treat TB patients. The objective of the study was to develop and optimize a simple, sensitive, and reliable high-performance liquid chromatography (HPLC) method for the simultaneous determination of isoniazid (INH), pyrazinamide (PZA), and rifampin (RIF) levels in human plasma. Nicotinamide was used as the internal standard and the samples were prepared after protein precipitation using acetonitrile and zinc sulfate. The separation was achieved using a C18 reversed-phase applying gradient elution. The mobile phase was a combination of water-methanol solution with a ratio of 95:05 (v/v) at the initial phase. All calibration curves had good linearity (r2 > 0.99) and the inter- and intra-day RSDs were lower than 15%. The limit of detection with a signal-to-noise ratio (S/N) of 3:1 was 0.16, 0.5, and 0.33 μg mL-1 for INH, PZA, and RIF, respectively. The method presented here was selective, sensitive, and reproducible, and could be used for‌ therapeutic drug monitoring in the patients who were under treatment with these drugs.
    Keywords:  Anti-TB drugs; HPLC-UV; Isoniazid; Pyrazinamide; Rifampin; Therapeutic drug monitoring
  29. Anal Biochem. 2020 Mar 16. pii: S0003-2697(20)30220-7. [Epub ahead of print] 113688
      Glutaric acid is a precursor of a plasticizer that can be used for the production of polyester amides, ester plasticizer, corrosion inhibitor, and others. Glutaric acid can be produced either via bioconversion or chemical synthesis, and some metabolites and intermediates are produced during the reaction. To ensure reaction efficiency, the substrates, intermediates, and products, especially in the bioconversion system, should be closely monitored. Until now, high performance liquid chromatography (HPLC) has generally been used to analyze the glutaric acid-related metabolites, although it demands separate time-consuming derivatization and non-derivatization analyses. To substitute for this unreasonable analytical method, we applied herein a gas chromatography - mass spectrometry (GC-MS) method with ethyl chloroformate (ECF) derivatization to simultaneously monitor the major metabolites. We determined the suitability of GC-MS analysis using defined concentrations of six metabolites (L-lysine, cadaverine, 5-aminovaleric acid, 2-oxoglutaric acid, glutamate, and glutaric acid) and their mass chromatograms, regression equations, regression coefficient values (R2), dynamic ranges (mM), and retention times (RT). This method successfully monitored the production process in complex fermentation broth.
    Keywords:  Analytical method; Ethyl chloroformate; Gas chromatography-mass spectrometry; Glutaric acid; Mass chromatograms; Metabolites
  30. PLoS Comput Biol. 2020 Mar 16. 16(3): e1007654
      The high-resolution and mass accuracy of Fourier transform mass spectrometry (FT-MS) has made it an increasingly popular technique for discerning the composition of soil, plant and aquatic samples containing complex mixtures of proteins, carbohydrates, lipids, lignins, hydrocarbons, phytochemicals and other compounds. Thus, there is a growing demand for informatics tools to analyze FT-MS data that will aid investigators seeking to understand the availability of carbon compounds to biotic and abiotic oxidation and to compare fundamental chemical properties of complex samples across groups. We present ftmsRanalysis, an R package which provides an extensive collection of data formatting and processing, filtering, visualization, and sample and group comparison functionalities. The package provides a suite of plotting methods and enables expedient, flexible and interactive visualization of complex datasets through functions which link to a powerful and interactive visualization user interface, Trelliscope. Example analysis using FT-MS data from a soil microbiology study demonstrates the core functionality of the package and highlights the capabilities for producing interactive visualizations.
  31. J Chromatogr A. 2020 Mar 13. pii: S0021-9673(20)30259-4. [Epub ahead of print] 461047
      The extracellular vesicles (EVs) released by plant pathogens of the Pectobacterium genus were investigated. The isolates were obtained using differential centrifugation followed by filtration and were characterized in terms of total protein content and particle size distribution. The transmission electron microscopy (TEM) analysis revealed the presence of two morphologically differentiated subpopulations of vesicles in the obtained isolates. The proteomic analysis using matrix-assisted laser desorption ionization mass spectrometry with time of flight detector (MALDI-TOF/TOF-MS) enabled to identify 62 proteomic markers commonly found in EVs of Gram-negative rods from the Enterobacteriaceae family. Capillary electrophoresis (CE) was proposed as a novel tool for the characterization of EVs. The method allowed for automated and fast (<15 min per sample) separation of vesicles from macromolecular aggregates with low sample consumption (about 10 nL per analysis). The approach required simple background electrolyte (BGE) composed of 50 mM BTP and 75 mM glycine (pH 9.5) and standard UV detection. The report presents a new opportunity for quality control of samples containing EVs.
    Keywords:  Capillary electrophoresis; Extracellular vesicles; Mass spectrometry; Outer membrane vesicles; Pectobacterium; Soft rot bacteria
  32. Biomed Chromatogr. 2020 Mar 21. e4835
      Bear bile is a valuable medicinal material used in traditional Chinese medicine for over 2000 years. However, developing a substitute has become exceptionally urgent because of endangered species protection measures. In vitro cultured bear bile powder (CBBP) has ingredients, including TUDCA, TCDCA, UDCA, and CDCA, and pharmacological properties that are similar to those of natural bear bile powder (NBBP). In this study, pharmacokinetic parameters of both CBBP and NBBP were measured in rats with a new surrogate analyte LC-MS method using stable isotopes as surrogate analytes (D4-TUDCA, D4-TCDCA, D4-UDCA, and D4-CDCA) with response factors validated in authentic matrix (plasma) for simultaneously monitoring the authentic analytes (TUDCA, TCDCA, UDCA, and CDCA). The method validation was satisfactory for the linear regression (r, 0.9975-0.9994), precision (RSD intra-day, 0.72%-9.35%; inter-day, 3.82%-9.02%), accuracy (RE, -12.42%-5.67%), and matrix effect (95.53%-99.80%), along with analyte recovery (95.90%-98.82%) and stability (89.48%-101.81%) of surrogate analytes, and precision (RSD intra-day, 1.06%- 11.51%; inter-day, 2.23%- 11.38%), accuracy (RE, -7.40%-10.76%) and stability (87.37%-111.70%) of authentic analytes. We successfully applied this method to evaluate the pharmacokinetics of CBBP and NBBP in rats, which revealed critical in vivo properties of both bear bile preparations.
    Keywords:  UPLC-MS/MS; bile acid; cultured bear bile powder; pharmacokinetics; rat plasma; surrogate analyte
  33. J Sep Sci. 2020 Mar 16.
      Two HPLC-MS/MS methods were developed and validated for the quantification of edaravone (method A) or taurine (method B) in human plasma. After protein precipitation, separations were achieved on an Ultimate XB-C8 (2.1 × 50 mm, 3.0 μm) column for edaravone and a ZORBAX SB-Aq column (2.1 × 100 mm, 3.5 μm) for taurine, respectively. The detection used electrospray ionization source via multiple reaction monitoring in positive-ion mode for edaravone and negative-ion mode for taurine, respectively. The lower limits of quantification were 10.0 ng/mL for edaravone and 3.00 μg/mL for taurine, respectively. The selectivity, accuracy and precision of the methods were all within acceptable limits. Two methods were successfully applied to a drug-drug interaction study and a pharmacokinetic study of edaravone and taurine in healthy Chinese volunteers after intravenous infusion of single or compound injection. The results showed that co-administration of edaravone with taurine increased the Cmax and AUC0-24 of taurine in human plasma while taurine did not affect the systemic exposure of edaravone. Edaravone and taurine have the dose-dependent pharmacokinetic profiles in human. This article is protected by copyright. All rights reserved.
    Keywords:  drug interaction; edaravone; mass spectrometry; pharmacokinetics; taurine
  34. Anal Bioanal Chem. 2020 Mar 20.
      Homoserine lactones (HSLs) are signaling molecules synthesized by Gram-negative bacteria in order to communicate in a process termed "quorum sensing." Until recently, only the L-stereoisomers of HSLs were thought to be produced and able to incite quorum sensing. However, recent studies have shown that select Gram-negative bacteria additionally produce non-trivial amounts of D-HSLs which may also play a role in quorum sensing. Current methods for the separation of HSL enantiomers cannot effectively separate all classes of HSLs and its enantiomers. More robust methods of separation and detection of D-HSLs are necessary. We have developed rapid and selective methods using liquid chromatography (LC) and gas chromatography (GC) coupled with mass spectrometry (MS) which can simultaneously enantiomerically separate all classes of HSLs. The advantages of these methods are in the MS compatibility as well as the ability to enantiomerically separate all classes of HSLs in a single run. The first enantiomeric separations of oxo- and hydroxy-HSLs by GC-MS, through the use of N,O-bis(trimethylsilyl)trifluoroacetamide-derivatizing reagents are discussed. Graphical Abstract.
    Keywords:  Enantiomeric separation; Homoserine lactone; Mass spectrometry; Quorum sensing
  35. Clin Biochem. 2020 Mar 15. pii: S0009-9120(20)30105-3. [Epub ahead of print]
      Tandem mass spectrometry - especially in combination with liquid chromatography (LC-MS/MS) - is applied in a multitude of important diagnostic niches of laboratory medicine. It is unquestioned in its routine use and is often unreplaceable by alternative technologies. This overview illustrates the development in the past decade (2009-2019) and intends to provide insight into the current standing and future directions of the field. The instrumentation matured significantly, the applications are well understood, and the in vitro diagnostics (IVD) industry is shaping the market by providing assay kits, certified instruments, and the first laboratory automated LC-MS/MS instruments as an analytical core. In many settings the application of LC-MS/MS is still burdensome with locally lab developed test (LDT) designs relying on highly specialized staff. The current routine applications cover a wide range of analytes in therapeutic drug monitoring, endocrinology including newborn screening, and toxicology. The tasks that remain to be mastered are, for example, the quantification of proteins by means of LC-MS/MS and the transition from targeted to untargeted omics approaches relying on pattern recognition/pattern discrimination as a key technology for the establishment of diagnostic decisions.
    Keywords:  LC-MS/MS; Mass spectrometry; TDM; endocrinology; laboratory automation; toxicology
  36. Theranostics. 2020 ;10(6): 2621-2630
      The development of improved or targeted drugs that discriminate between normal and tumor tissues is the key therapeutic issue in cancer research. However, the development of an analytical method with a high accuracy and sensitivity to achieve quantitative assessment of the tumor targeting of anticancer drugs and even intratumor heterogeneous distribution of these drugs at the early stages of drug research and development is a major challenge. Mass spectrometry imaging is a label-free molecular imaging technique that provides spatial-temporal information on the distribution of drugs and metabolites in organisms, and its application in the field of pharmaceutical development is rapidly increasing. Methods: The study presented here accurately quantified the distribution of paclitaxel (PTX) and its prodrug (PTX-R) in whole-body animal sections based on the virtual calibration quantitative mass spectrometry imaging (VC-QMSI) method, which is label-free and does not require internal standards, and then applied this technique to evaluate the tumor targeting efficiency in three treatment groups-the PTX-injection treatment group, PTX-liposome treatment group and PTX-R treatment group-in nude mice bearing subcutaneous A549 xenograft tumors. Results: These results indicated that PTX was widely distributed in multiple organs throughout the dosed body in the PTX-injection group and the PTX-liposome group. Notably, in the PTX-R group, both the prodrug and metabolized PTX were mainly distributed in the tumor tissue, and this group showed a significant difference compared with the PTX-liposome group, the relative targeting efficiency of PTX-R group was increased approximately 50-fold, leading to substantially decreased systemic toxicities. In addition, PTX-R showed a significant and specific accumulation in the poorly differentiated intratumor area and necrotic area. Conclusion: This method was demonstrated to be a reliable, feasible and easy-to-implement strategy to quantitatively map the absorption, distribution, metabolism and excretion (ADME) of a drug in the whole-body and tissue microregions and could therefore evaluate the tumor-targeting efficiency of anticancer drugs to predict drug efficacy and safety and provide key insights into drug disposition and mechanisms of action and resistance. Thus, this strategy could significantly facilitate the design and optimization of drugs at the early stage of drug research and development.
    Keywords:  intratumor heterogeneity; paclitaxel; quantitative mass spectrometry imaging; tumor-targeting efficiency; whole-body animal
  37. Anal Bioanal Chem. 2020 Mar 18.
      The popularity of mass spectrometry-based lipidomics has soared in the past decade. While the majority of the lipidomics work is being performed in mammalian and other eukaryotic systems, there is also a growing rise in the exploration of bacterial lipidomics. The lipids found in bacteria can be substantially different from those in eukaryotic systems, but they are equally important for maintaining the structure of the bacteria and providing protection from the surrounding environment. In this article, recent applications of lipidomics in combination with molecular biology and applications in microbial strain identification and antibiotic susceptibility are highlighted. The authors' perspectives on current challenges facing the field and future directions are also provided.
    Keywords:  Antibiotic resistance; Bacteria; Lipid A; Lipidomics; Lipopolysaccharide; Mass spectrometry
  38. Clin Chim Acta. 2020 Mar 17. pii: S0009-8981(20)30128-5. [Epub ahead of print]
      BACKGROUND AND AIMS: The active coenzymes of the water soluble vitamins B1 and B6 (thiamine pyrophosphate (TPP) and pyridoxal-5-phosphate (P5P) respectively) play an important role in numerous bodily functions. The simultaneous analysis of both these analytes is limited to either mass spectrometry based methods or commercial kit suppliers. In this study we developed a novel method for analysis of both TPP and P5P by fluorescence detection.METHODS: Briefly, whole blood samples are precipitated by trichloroacetic acid, and P5P and TPP are both derivatised before separation on a C18-PFP column. The new assay's performance was compared against a recent cycle from an external quality assurance program (RCPAQAP) and the current only existing commercial kit (n=76).
    RESULTS: Linearity for both analytes was above 0.99 (r2) up to a concentration range of: 4000 nmol/L (P5P) and 2000 nmol/L (TPP). Precision of the method (intra-day and inter-day) compared against commercial quality control material was below 6% (coefficient of variation). Recovery of both compounds exceeded 90%. Accuracy of the protocol displayed satisfactory results in proficiency testing and had an acceptable level of agreement with the existing current kit method.
    CONCLUSIONS: Overall, this method provides an economical alternative in routine clinical diagnostic laboratories wishing to perform P5P and TPP analysis.
    Keywords:  HPLC-FLD; P5P; TPP; Vitamins B1 and B6
  39. Clin Biochem. 2020 Mar 17. pii: S0009-9120(19)31354-2. [Epub ahead of print]
      BACKGROUND: Given that both insufficient and excess maternal iodine have adverse consequences such as poor cognitive performance, delayed physical development and increased fetal and infant mortality, the determination of maternal iodine status is very important. In this study, we established and verified a method involving inductively coupled plasma-mass spectrometry (ICP-MS) technology for the rapid determination of the amniotic fluid iodine concentration (AFIC), breast milk iodine concentration (BMIC) and cerebrospinal fluid (CSF) iodine concentration (CSFIC).METHOD: Amniotic fluid, breast milk and CSF were collected from residual samples at Peking Union Medical College Hospital (PUMCH). The linearity, detection limit, precision, recovery, carryover and matrix effect of the testing method using ICP-MS technology were thoroughly evaluated according to the EP-10-A2 evaluation protocol approved by the Clinical and Laboratory Standards Institute (CLSI) guidelines. Furthermore, we evaluated the AFIC, BMIC and CSFIC distributions among clinical patients from PUMCH.
    RESULTS: The correlation coefficient (r) was higher than 0.99 (0.995-1.000). The limit of detection (LOD) was 0.233 µg/L, and the limit of quantification (LOQ) was 0.778 µg/L. For amniotic fluid, breast milk and CSF, the assay repeatability ranges were 1.5%-1.8%, 1.9%-4.0% and 1.8%-4.0%, respectively, and the within-laboratory coefficient of variation (CV%) ranges over five days were 3.3%-9.2%, 7.2%-8.0% and 3.2%-7.8%, respectively. The recovery rates ranged from 97.7% to 109.8%. Moreover, the median concentrations of iodine in the amniotic fluid, breast milk and CSF of the patients from PUMCH were 176.3 µg/L, 136.0 µg/L, and 81.8 µg/L, respectively.
    CONCLUSION: A rapid, stable and accurate method that incorporates ICP-MS technology for the determination of iodine concentration was established for amniotic fluid, breast milk and CSF in this study.
    Keywords:  CSF; ICP-MS; amniotic fluid; breast milk; iodine
  40. J Mol Graph Model. 2020 Mar 05. pii: S1093-3263(19)30949-0. [Epub ahead of print]97 107576
      Understanding in silico the dynamics of metabolic reactions made by a large number of molecules has led to the development of different tools for visualising molecular interactions. However, most of them are mainly focused on quantitative aspects. We investigate the potentiality of the topological interpretation of the interaction-as-perception at the basis of a multiagent system, to tackle the complexity of visualising the emerging behaviour of a complex system. We model and simulate the glycolysis process as a multiagent system, and we perform topological data analysis of the molecular perceptions graphs, gained during the formation of the enzymatic complexes, to visualise the set of emerging patterns. Identifying expected patterns in terms of simplicial structures allows us to characterise metabolic reactions from a qualitative point of view and conceivably reveal the simulation reactivity trend.
    Keywords:  Interactive computation; Metabolic reactions; Multiagent modelling and simulation; Spatial simulator; Topological data analysis