bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒09‒20
twenty papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Talanta. 2020 Dec 01. pii: S0039-9140(20)30698-6. [Epub ahead of print]220 121407
    Vaudreuil MA, Vo Duy S, Munoz G, Furtos A, Sauvé S.
      With the consumption of chemotherapy agents, residues of anticancer drugs may be increasingly found in hospital and municipal wastewaters. Quantification of these highly polar micropollutants remains challenging due to poor chromatographic retention on typical reversed phases. This study investigated different solid-phase extraction (SPE) materials for automated on-line preconcentration of complex matrices (hospital and municipal wastewaters) and various chromatographic column options. A hyper crosslinked hydroxylated polystyrene-divinylbenzene copolymer SPE sorbent coupled on-line with hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) yielded suitable limits of detection (LOD: 1-2 ng L-1) for 5-fluorouracil (5-FU) and 2',2'-difluorodeoxyuridine (dFdU). Optimization of chromatographic conditions led to a single LC-MS/MS method for the analysis of other cytostatic drugs including cytarabine (CYT), gemcitabine (GEM), methotrexate (MTX), ifosfamide (IFO), cyclophosphamide (CYC) and capecitabine (CAP). The filter membrane for sample pre-treatment, HPLC mobile phase additives, and on-line SPE loading parameters were also investigated. The methods were validated in wastewater matrix with suitable determination coefficients (R2 range: 0.9982-0.9999), LODs (0.5-5 ng L-1), accuracy (78-111%), intraday precision (2.6-12%), and interday precision (2.1-13%). The occurrence of cytostatic drugs was examined in field-collected water samples from hospital effluents and municipal wastewater treatment plants (WWTP) in Canada. CAP (3.7-64 ng L-1), dFdU (6.1-300 ng L-1), and MTX (1.8-68 ng L-1) were frequently detected across both matrix types, while IFO was detected in hospital wastewater (23-140 ng L-1) but not in municipal WWTPs.
    Keywords:  Anticancer drugs; Hospital effluent; Hydrophilic interaction liquid chromatography (HILIC); LC-MS/MS; Municipal wastewater; On-line SPE
  2. Talanta. 2020 Dec 01. pii: S0039-9140(20)30619-6. [Epub ahead of print]220 121328
    Nelis M, Decraecker L, Boeckxstaens G, Augustijns P, Cabooter D.
      An LC-MS/MS method was developed enabling the separation and quantification of histamine and its main metabolites (imidazole acetaldehyde, imidazole acetic acid, methyl imidazole acetic acid, methyl histamine, acetyl histamine) in urine samples. A fast separation was achieved in 10 min on two HILIC columns connected in series by adopting a linear gradient followed by an isocratic hold. The sample preparation consisted of a simple dilution step wherein 10 μL of urine was diluted with acetonitrile (ACN) to a final volume comprising 95% ACN. For methyl imidazole acetic acid, an additional dilution step was incorporated due to its high natural levels. Hereafter, the samples were stored at -20 °C and centrifuged prior to injection. Matrix matched calibrators were unavailable due to the endogenous occurrence of the compounds of interest. The occurrence of matrix effects and the lack of labeled internal standards prompted the use of the standard addition method as a viable alternative to solvent calibration. The validation of the method entailed matrix effects, accuracy and precision and was performed in compliance with the recent guidelines on endogenous compounds issued by the International Conference of Harmonization (ICH). The method was then adopted for the quantification of histamine and its metabolites in human urine samples collected from healthy volunteers and patients suffering from gastrointestinal discomfort.
    Keywords:  Endogenous compounds; HILIC; Histamine; MS/MS; Metabolites; Targeted metabolomics
  3. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Aug 27. pii: S1570-0232(20)30414-1. [Epub ahead of print]1160 122345
    Skiba M, Fatmi S, Elkasri N, Karrout Y, Lahiani-Skiba M.
      A rapid, highly sensitive and simple high-performance liquid chromatographic-tandem mass spectrometric (LC-MS) assay is developed and validated for the quantification of leuprolide: a Gonadotropin Releasing Hormone analog (GnRH) in human plasma. Moreover, various parameters of the method stability are determined. After the addition of stable isotope (internal standard), the leuprolide was extracted from human plasma by a C18 solid phase micro extraction (MEPS) cartridge and directly injected into LC-MS/MS system. Chromatographic separation was achieved using a Waters Atlantis HILIC, C18, 150 × 2.1 mm, 5 μ column. Mobile phase was a mixture of acetate buffer (pH 3) and acetonitrile (25/75). Drug detection was performed by MS using electrospray ionization in positive mode. Multiple reaction monitoring (MRM) with a tandem mass spectrometer was used to detect the analytes. Precursor to product ion transitions of: m/z 605.5 → m/z 110.2 and m/z 609.1 → m/z 249.1 were used to quantify leuprolide and leuprolide-13C6-15N, respectively. Sample analysis time was 3 min for each injection. The assay exhibited a linear dynamic range of 0.0500-40 ng/ml for each analyte with a lower limit of quantification (LLOQ) of: 0.0500 ng/ml. Furthermore, a complete analytic validation was carried out, including tests on: The specificity, precision, accuracy, matrix effect and stability under different storage conditions. Importantly, the obtained results established: an acceptable precision and accuracy for concentration over standard curve range. Nevertheless, it is to emphasize the simplicity, rapidity and also the high precision and accuracy of this novel LC-MS method, offering useful information about solution stability. Finally, this work is a good alternative to quantify Leuprolide concentration in human blood, especially on human clinic trials step.
    Keywords:  GnRH analog; Human plasma; LC-MS; Leuprolide; Quantification; Solid phase micro extraction; Validation
  4. Anal Chem. 2020 Sep 15.
    Teo G, Chew WS, Burla B, HErr D, Tai ES, Wenk MR, Torta FT, Choi H.
      MRMkit is an open-source software package designed for automated data processing of large-scale targeted mass spectrometry-based metabolomics data. With increasing automation of sample preparation for LC-MS analysis, a challenging next step is to fully automate the workflow to process raw data and assure the quality of measurements in large-scale analysis settings. MRMkit capitalizes on the richness of large-sample data in capturing peak shapes and interference patterns of individual transitions across many samples and deliver fully automated, reproducible peak integration results in a scalable and time-efficient manner. In addition to fast and accurate peak integration, the tool also provides reliable data normalization functions and quality metrics along with visualizations for fast data quality evaluation. MRMkit learns the retention time offset patterns by user specified compound class and makes recommendations for peak picking in multi-modal ion chromatograms. In summary, MRMkit offers highly consistent and scalable data processing capacity to targeted metabolomics, substantially curtailing the time required to produce the final quantification results after the actual LC-MS analysis.
  5. Methods Mol Biol. 2021 ;2194 291-300
    Tian Y, Cai J, Allman EL, Smith PB, Patterson AD.
      Bile acids are important end products of cholesterol metabolism, having been shown to serve as signaling molecules and intermediates between the host and the gut microbiota. Here we describe a robust and accurate method using ultrahigh-pressure liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) for the quantification of bile acids in stool/cecal and tissue samples.
    Keywords:  Bile acid; Metabolomics; Quantitation; UHPLC-MS/MS
  6. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Sep 02. pii: S1570-0232(20)30802-3. [Epub ahead of print]1160 122355
    Wang Y, Zhou L, Zhou Y, Zhao C, Lu X, Xu G.
      Gas chromatography-mass spectrometry (GC-MS) is an ideal tool for analyzing the intermediates of tricarboxylic acid cycle and glycolysis, sugars, organic acids and amino acids, etc. High-throughput metabolomics methods are required by large-scale clinical researches, and time of flight mass spectrometry (TOF MS) having fast scanning rate is preferable for rapid GC. Quadrupole MS (qMS) instruments have 95% market share, and their potential in rapid metabolomics is worth being studied. In this work, a within 15-min GC program was established and matched by qMS scanning for plasma metabolome analysis after N-methyl-N-(trimethylsilyl)-trifluoroacetamide derivatization. Compared to the longer-time program GC-qMS method, the rapid GC-qMS method had nearly no metabolome information loss, and it had excellent profile performance in repeatability, intra-day and inter-day precision, sampling range, linearity and extraction recovery. Compared to TOF MS, qMS achieved similar results in investigating lung cancer serum metabolic disruptions. Partial least squares-discriminant analysis revealed that the two datasets acquired by qMS and TOF MS had very similar model parameters, and most of top ranked differential metabolites were the same. This study provides a rapid and economical GC-qMS metabolomics method for researchers. Still, MS having faster scanning rate and higher sensitivity are recommended, if possible, to detect more small peaks and some co-eluted peaks.
    Keywords:  GC-TOF MS; GC-qMS; Lung cancer; Metabolomics; Rapid
  7. J Pharm Biomed Anal. 2020 Sep 01. pii: S0731-7085(20)31482-5. [Epub ahead of print]191 113596
    Patel DP, Pauly GT, Tada T, Parker AL, Toulabi L, Kanke Y, Oike T, Krausz KW, Gonzalez FJ, Harris CC.
      Creatine riboside (CR) is a novel metabolite of cancer metabolism. It is a urinary diagnostic biomarker of lung and liver cancer risk and prognosis. The level of CR is highly positive correlated in tumor and urine indicating that it is derived from human lung and liver cancers. A precise and sensitive ultra-pressure liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated for simultaneous quantification of the noninvasive biomarker CR, along with creatinine riboside (CNR), and their precursors creatine and creatinine, utilizing the labeled internal standard creatine riboside-13C,15N2 (CR-13C,15N2). Chromatography was carried out on a hydrophilic interaction chromatography column under a gradient mobile phase condition. MRM transitions were monitored for CR (264.1 > 132.1, m/z), CNR (246.1 > 113.9, m/z), creatine (132.0 > 72.0, m/z), creatinine (114.0 > 85.8, m/z) and CR-13C,15N2 (267.1 > 134.9, m/z) with a 11.0 min run time in the positive mode ionization. The calibration plot of the method was linear over the concentration range of 4.50-10,000 nM. Method validation was performed according to regulatory guidelines established for sensitivity, selectivity, calibration curve, stability at different storage conditions, reinjection reproducibility, ruggedness with acceptable accuracy, and precision. This assay was applied for the quantification of CR along with CNR, creatine and creatinine in a subset of urine and serum samples from the National Cancer Institute - Maryland (NCI-MD) cohort population controls and lung cancer cases. It can be standardized and used in multiple laboratories for cancer diagnosis and determining the efficacy of cancer therapy and monitoring cancer recurrence.
    Keywords:  Creatine; Creatine ribose; Creatine riboside-(13)C,(15)N(2); Creatinine; Creatinine riboside; UPLC-ESI-MS/MS; Urinary cancer biomarker
  8. ACS Omega. 2020 Sep 08. 5(35): 22289-22298
    Broughton R, Tocher DR, Betancor MB.
      Lipidomics methodologies traditionally utilize either reverse phase- or hydrophilic interaction liquid chromatography-type separations; however, supercritical fluid chromatography can offer a rapid normal phase type separation while reducing the dependence on organic solvents. However, normal phase type lipid separations typically lack pronounced intraclass separation, which is problematic for complex lipidomes containing very-long-chain polyunsaturated fatty acids, especially those from genetically modified organisms. A high-strength silica C18 method was developed, which benefitted from discrete class separation, as well as displaying intraclass selectivity sufficient for profiling flesh of salmon fed with a diet supplemented with oil from the genetically engineered oilseed Camelina sativa, a terrestrial oilseed with a fish oil-type profile. Salmon fed a diet containing this Camelina oil were found to have flesh enriched in triacylglycerols and phospholipids containing 18:3, 20:5, and 22:6, whereas salmon fed the control diet were differentiated by shorter chain plant-type fatty acids integrated within complex lipids. Coupled with active scanning quadrupole technology, data acquisition was enhanced, allowing for fragmentation data to be acquired in a data independent fashion, permitting acyl chain identification of resolved isomers. Therefore, we have developed a method, which is amenable for lipidomics studies of complex lipidomes, specifically those altered by synthetic biology approaches.
  9. Anal Methods. 2020 May 28. 12(20): 2555-2559
    Fujiwara T, Hattori A, Ito T, Funatsu T, Tsunoda M.
      Branched-chain keto acids and branched-chain amino acids are metabolites of branched-chain amino acid aminotransferases (BCATs), which catalyzes reversible transamination between them. We found that BCAT1 plays an important role in the progression of myeloid leukaemia, and a method for the analysis of intracellular α-keto acids including branched-chain keto acids was necessary to further investigate their role. In this study, we developed a method to analyze six α-keto acids (α-ketoglutaric acid (KG), pyruvic acid, α-ketobutyric acid, α-ketoisovaleric acid, α-ketoisocaproic acid, and α-keto-β-methylvaleric acid) in K562 cells by HPLC with fluorescence detection, using 1,2-diamino-4,5-methylenedioxybenzene (DMB) as a derivatization reagent. Because split peaks of DMB-KG were observed when injection samples were too acidic, the derivatization solution was diluted with NaOH solution to obtain a single peak. Limits of detection and limits of quantification were 1.3-5.4 nM and 4.2-18 nM, respectively. Intracellular concentrations of α-keto acids were 1.55-316 pmol/1 × 106 K562 cells. The developed method realized reproducible and sensitive analysis of intracellular α-keto acids. Thus, the method could be used to elucidate the role of BCAT in myeloid leukaemia.
  10. J Am Soc Mass Spectrom. 2020 Sep 15.
    Rodrigues MF, Pereira I, Lima Morais R, Sanz Lobon G, Gil E, Vaz BG.
      A new approach using paper spray ionization mass spectrometry (PSI-MS) for the analysis of steroid hormones in wastewater samples has been demonstrated. Triangular papers containing paraffin barriers as microfluidic channels were used to direct the sample solution to the paper tip, preventing the sample from spreading over the corners of the paper. The method was used to analyze the hormones levonorgestrel and algestone acetophenide in industrial wastewaters. Analytical curves presented a correlation coefficient (R2) above 0.99. Limits of quantification were below 2.3 ppm, and limits of detection below 0.7 ppm. Values of precision (coefficient of variation) and accuracy (relative error) were less than 15% for all analyses. Recovery results ranged from 82% to 102%. Levonorgestrel was also analyzed by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) in order to compare the analytical performance with PSI-MS. No statistically significant differences were found between both methods. This study demonstrates the usefulness of PSI-MS for the rapid analysis of hormones in industrial wastewater samples and also indicates its potential to be employed as a simple and reliable analytical method in environmental sciences.
  11. Talanta. 2020 Dec 01. pii: S0039-9140(20)30706-2. [Epub ahead of print]220 121415
    Luque-Córdoba D, López-Bascón MA, Priego-Capote F.
      Sex steroids are involved in biological functions that encompass from the complete sexual development of individuals up to the deregulation of metabolic pathways leading to some pathologies. Steroids are present in blood at low concentration levels from pg mL-1 to ng mL-1. For this reason, a high sensitive and selective method based on gas chromatography-negative chemical ionization-tandem mass spectrometry (GC-NCI-MS/MS) is here proposed to quantify either androgens (androstenedione, dehydroepiandrosterone, dihydrotestosterone and testosterone), estrogens (estrone and estradiol) and a progestogen (progesterone) in human plasma. The sample preparation steps, protein precipitation and solid phase extraction, were optimized to ensure the sample matrix removal and to extract steroids with high efficiency. The NCI-MS/MS detection approach was compared with that based on electron impact to evaluate the incidence of the ionization source in the determination of steroids. The quantification limits for determination of these analytes were in a range from 10 pg mL-1 to 5 ng mL-1, with a high sensitivity for estrogens, typically found at low concentrations. The proposed method was tested for the determination of steroids in male blood samples, in which 6 out of 7 steroids were detected and quantified to report concentration values in agreement with those described in the literature.
    Keywords:  GC–MS/MS; Negative chemical ionization; Plasma; Sample preparation; Sex steroids
  12. Methods Mol Biol. 2021 ;2194 301-313
    Mal TK, Tian Y, Patterson AD.
      NMR spectroscopy has become one of the preferred analytical techniques for metabolomics studies due to its inherent nondestructive nature, ability to identify and quantify metabolites simultaneously in a complex mixture, minimal sample preparation requirement, and high degree of experimental reproducibility. NMR-based metabolomics studies involve the measurement and multivariate statistical analysis of metabolites present in biological samples such as biofluids, stool/feces, intestinal content, tissue, and cell extracts by high-resolution NMR spectroscopy-the goal then is to identify and quantify metabolites and evaluate changes of metabolite concentrations in response to some perturbation. Here we describe methodologies for NMR sample preparation of biofluids (serum, saliva, and urine) and stool/feces, intestinal content, and tissues for NMR experiments including extraction of polar metabolites and application of NMR in metabolomics studies. One dimensional (1D) 1H NMR experiments with different variations such as pre-saturation, relaxation-edited, and diffusion-edited are routinely acquired for profiling and metabolite identification and quantification. 2D homonuclear 1H-1H TOCSY and COSY, 2D J-resolved, and heteronuclear 1H-13C HSQC and HMBC are also performed to assist with metabolite identification and quantification. The NMR data are then subjected to targeted and/or untargeted multivariate statistical analysis for biomarker discovery, clinical diagnosis, toxicological studies, molecular phenotyping, and functional genomics.
    Keywords:  COSY; CPMG; HMBC; HRMAS; HSQC; Metabolomics; NMR; TOCSY
  13. Anal Methods. 2020 Jun 11. 12(22): 2865-2871
    Wang T, Zhang X, Ye Y, Shi R, Ma Y.
      A sensitive, specific, and accurate high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the quantification of prostaglandins D2 (PGD2) and E2 (PGE2) in a mouse ear edema model. We used activated charcoal to obtain PG-free ear samples. The chromatographic separation was performed using a Hypersil Gold C18 column. The limit of detection of each PG was 0.4 ng mL-1, and the intra- and inter-assay estimates of precision and accuracy were <14.5 and 94.2-102.9%, respectively. Stability studies showed that all analytes were stable under various storage conditions and analytical processes. The developed and validated method was successfully used to investigate the anti-inflammatory effects of cultured bear bile powder (CBBP) by quantitatively determining PGE2 and PGD2 levels in mouse ear edema samples. These results showed that CBBP significantly inhibited the xylene-induced ear edema in mice and reversed the xylene-induced elevation of PGE2 and PGD2 levels. These results provide useful data about the anti-inflammatory bioactivities in tissues, mediated by the reduction of PGE2 and PGD2 levels, and may further encourage research and development studies of CBBP for its use as an anti-inflammatory agent.
  14. Anal Chem. 2020 Sep 14.
    Wagner A, Zhang J, Liu C, Covey TR, Olah TV, Weller HBN, Shou WZ.
      Bioanalysis of polar analytes using liquid chromatography - tandem mass spectrometry (LC-MS/MS) remains a significant challenge, due to their poor chromatographic retention on the commonly used reversed-phase LC columns, and the resulting severe ionization suppression from co-eluting matrix components. Here we present a novel approach to perform ultrahigh-throughput and chromatography-free bioanalysis of polar compounds, using a prototype acoustic ejection mass spectrome-ter (AEMS) platform. Previously developed for direct analysis of solid or liquid samples by MS, the open port interface (OPI) has recently been modified and coupled to an acoustic nanoliter dispenser to enable high-speed direct MS analysis from 384-well plates with a reported speed as fast as 0.5 second per sample. Ionization suppression was reduced due to the >1000 fold dilution of the original sample by the carrier solvent in the AE-OPI-MS operation. Taking full advantage of the chroma-tography-free and suppression-reducing features of this prototype instrument, we successfully demonstrated the ultrahigh-throughput bioanalysis of metformin, a small polar substrate commonly used in high-throughput in vitro transporter inhibi-tion assays in the early ADME profiling space in drug discovery. The AEMS platform achieved a speed of 2.2 sec-onds/sample using only 10 nL of sample volume. Similar bioanalytical and biological results from actual assay samples were obtained by AEMS when compared to those obtained by the fastest LC-MS/MS method previously reported, along with a 15-fold speed advantage, and a ~500-fold less sample consumption to enable future assay miniaturization. The gen-eral applicability of this novel approach to bioanalysis of several classes of polar analytes including ethambutol, isoniazid, ephedrine and gemcitabine in biological matrices was further demonstrated.
  15. Wei Sheng Yan Jiu. 2020 Jul;49(4): 597-602
    Yin D, Hu J, Zhao J, Qiao H, Zhao Y.
      OBJECTIVE: To establish a method for analysis of 25-hydroxylvitamin D_2(25(OH)D_2), 25-hydroxylvitamin D_3(25(OH)D_3)and vitamin K_1 in serum by ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS), which can be applied in diagnosis of vitamin deficiency and estimation on the nutritional status of people.METHODS: Serum samples mixed with d_6-25(OH)D_3, d_7-vitamin K_1(internal standard)were precipitated with acetonitrile and extracted with n-hexane. The sample solution was separated using BEH C_(18) column(2. 1 mm×100 mm, 1. 7 μm) with gradient elution using methanol-water containing 0. 1% formic acid as mobile phase. The target molecule was detected in positive electrospray ionization(ESI~+) and multiple reaction monitoring.
    RESULTS: The concentration of 25(OH)D_2, 25(OH)D_3 and vitamin K_1 showed good linearity in the range 5. 0-75. 0 ng/mL, 2. 0-81. 5 ng/mL and 0. 3-12. 0 ng/mL, respectively, with the correlation coefficients greater than 0. 995. The limits of detection(LOD) of 25(OH)D_2, 25(OH)D_3 and vitamin K_1 were 1, 0. 25 and 0. 1 ng/mL, and the limits of quantification(LOQ) for 25(OH)D_2, 25(OH)D_3 and vitamin K_1were 3, 0. 75 and 0. 3 ng/mL, respectively. The recoveries of three levels in the matrix were 98. 5%-104. 3%, the relative standard deviation(RSD) were all less than 5. 0%(n=6).
    CONCLUSION: An UPLC-MS/MS method for analysis of 25(OH)D_2, 25(OH)D_3 and vitamin K_1 in serum is sensitive, rapid, accurate and suitable for the nutritional surveillance of vitamin D and K_1 in the population.
    Keywords:  25-hydroxylvitamin D_2; 25-hydroxylvitamin D_3; UPLC-MS/MS; serum; vitamin K_1
  16. Metabolites. 2020 Sep 15. pii: E371. [Epub ahead of print]10(9):
    Hurst EA, Homer NZ, Mellanby RJ.
      The demand for vitamin D analysis in veterinary species is increasing with the growing knowledge of the extra-skeletal role vitamin D plays in health and disease. The circulating 25-hydroxyvitamin-D (25(OH)D) metabolite is used to assess vitamin D status, and the benefits of analysing other metabolites in the complex vitamin D pathway are being discovered in humans. Profiling of the vitamin D pathway by liquid chromatography tandem mass spectrometry (LC-MS/MS) facilitates simultaneous analysis of multiple metabolites in a single sample and over wide dynamic ranges, and this method is now considered the gold-standard for quantifying vitamin D metabolites. However, very few studies report using LC-MS/MS for the analysis of vitamin D metabolites in veterinary species. Given the complexity of the vitamin D pathway and the similarities in the roles of vitamin D in health and disease between humans and companion animals, there is a clear need to establish a comprehensive, reliable method for veterinary analysis that is comparable to that used in human clinical practice. In this review, we highlight the differences in vitamin D metabolism between veterinary species and the benefits of measuring vitamin D metabolites beyond 25(OH)D. Finally, we discuss the analytical challenges in profiling vitamin D in veterinary species with a focus on LC-MS/MS methods.
    Keywords:  1,25-dihydroxyvitamin-D; 24,25-dihydroxyvitamin-D; 25-hydroxyvitamin-D; C3-epimers; LC-MS/MS; comparative; free vitamin D; profiling; veterinary; vitamin D
  17. Anal Methods. 2020 Jun 11. 12(22): 2881-2892
    McKenzie K, Moffat CF, Petrie B.
      This study proposes a new multi-residue enantioselective method for the determination of emerging drug contaminants in sea water by solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). To achieve satisfactory enantiomeric separation with a vancomycin stationary phase it was essential to limit sodium chloride in extracted samples to <1 μg per injection. This was achieved through a straightforward SPE method using a 50 mL water wash volume and analyte elution in acetonitrile. A Chiral-V enantioselective column (150 × 2.1 mm; 2.7 μm particle size) operated in polar ionic mode enabled simultaneous drug separations in 30 minutes. Analytes with enantioresolution ≥1 were the stimulants amphetamine and methamphetamine, the beta-agonist salbutamol, the beta-blockers propranolol, sotalol and acebutolol, the anti-depressants fluoxetine, venlafaxine, desmethylvenlafaxine and citalopram, and the antihistamine chlorpheniramine. Method quantitation limits were <10 ng L-1 and method trueness was 80-110% for most analytes. The method was applied to samples from the Forth and Clyde estuaries, Scotland. Chiral drugs were present at concentrations in the range 4-159 ng L-1 and several were in non-racemic form (enantiomeric fraction ≠ 0.50) demonstrating enantiomer enrichment. This emphasises the need for further enantiospecific drug exposure and effect studies in the marine environment.
  18. J Pharm Biomed Anal. 2020 Sep 09. pii: S0731-7085(20)31501-6. [Epub ahead of print]191 113615
    Körmöczi T, Szabó Í, Farkas E, Penke B, Janáky T, Ilisz I, Berkecz R.
      The orthogonal heart-cutting liquid chromatography (LC) modes coupled to high-resolution tandem mass spectrometry (HRMS/MS) provide a number of possibilities to enhance selectivity and sensitivity for the determination of targeted compounds in complex biological matricies. Here we report the development of a new fast 2D-LC-(HRMS/MS) method and its successful application for quantitative determination of the level of plasma and brain N,N-dimethyltriptamine (DMT) using α-methyltryptamine (AMT) as internal standard in an experimental model of cerebral ischemia/reperfusion using DMT administration. The 2D-LC separation was carried out by a combination of hydrophilic interaction liquid chromatography (HILIC) in the first dimension followed by second-dimensional reversed-phase (RP) chromatography within a total run time of 10 min. The enrichment of HILIC effluent of interest containing DMT was performed using a C18 trapping column. During method development several parameters of sample preparation procedures, chromatographic separation and mass spectrometric detection were optimised to achieve high DMT recovery (plasma: 90 %, brain: 88 %) and sensitivity (plasma: 0.108 ng/mL of LOD, brain: 0.212 ng/g of LOD) applying targeted analytical method with strict LC and HRMSMS confirmatory criteria. Concerning rat plasma sample, the concentration of DMT before hypoxia (49.3-114.3 ng/mL plasma) was generally higher than that after hypoxia (10.6-96.1 ng/mL plasma). After treatment, the concentration of DMT in brain was elevated up to the range of 2-6.1 ng/g. Overall, our analytical approach is suitable to detect and confirm the presence of DMT administered to experimental animals with therapeutic purpose in a reliable manner.
    Keywords:  Heart-cutting 2D-LC-HRMS/MS; N,N-dimethyltryptamine; Rat brain; Rat plasma; α-methyltryptamine
  19. Anal Chem. 2020 Sep 14.
    Delcourt V, Barnabé A, Loup B, Garcia P, André F, Chabot B, Trevisiol S, Moulard Y, Popot MA, Bailly-Chouriberry L.
      With recent advances in analytical chemistry, LC-HRMS/MS has become an essential tool for metabolite discovery and detection. Even if most common drug transformations have already been extensively described, manual search of drug metabolites in LC-HRMS/MS datasets is still a common practice in toxicology laboratories, complicating metabolite discovery. Furthermore, the availability of free open-source software for metabolite discovery is still limited. In this article, we present MetIDfyR, an opensource and cross-platform R package for in-silico drug phase I/II biotransformations prediction and mass-spectrometric data mining. MetIDfyR has proven efficacy for advanced metabolite identification in semi-complex and complex mixtures in in-vitro or in-vivo drug studies and is freely available at
  20. Neurochem Res. 2020 Sep 19.
    Dienel GA.
      Metabolomic technologies including imaging mass spectrometry (IMS; also called mass spectrometry imaging, MSI, or matrix-assisted laser desorption/ionization-mass spectrometry imaging, MALDI MSI) are important methods to evaluate levels of many compounds in brain with high spatial resolution, characterize metabolic phenotypes of brain disorders, and identify disease biomarkers. ATP is central to brain energetics, and reports of its heterogeneous distribution in brain and regional differences in ATP/ADP ratios reported in IMS studies conflict with earlier studies. These discordant data were, therefore, analyzed and compared with biochemical literature that used rigorous methods to preserve labile metabolites. Unequal, very low regional ATP levels and low ATP/ADP ratios are explained by rapid metabolism during postmortem ischemia. A critical aspect of any analysis of brain components is their stability during and after tissue harvest so measured concentrations closely approximate their physiological levels in vivo. Unfortunately, the requirement for inactivation of brain enzymes by freezing or heating is not widely recognized outside the neurochemistry discipline, and procedures that do not prevent postmortem autolysis, including decapitation, brain removal/dissection, and 'snap freezing' are commonly used. Strong emphasis is placed on use of supplementary approaches to calibrate metabolite abundance in units of concentration in IMS studies and comparison of IMS results with biochemical data obtained by different methods to help identify potential artifacts.
    Keywords:  ATP; Glycolysis; Labile metabolites; Postmortem ischemia; TCA cycle