bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒08‒21
nine papers selected by
Sofia Costa

  1. Anal Methods. 2022 Aug 18.
      Heterogeneity in metabolite structure and charge state complicates their analysis in electrospray mass spectrometry (ESI-MS). Complications such as diminished signal response and quantitation can be reduced by sequential dual-stage derivatization and capillary RP LC-ESI-MS analysis. Our sequential dual-stage chemical derivatization reacts analyte primary amine and hydroxyl groups with a linear acyl chloride head containing a tertiary amine moiety. Analyte carboxylate groups are then coupled to a linear amine tag with a tertiary amine moiety. This increase in the number of tags on analytes increases analyte proton affinity and hydrophobicity. We derivatized 250 metabolite standards which on average improved signal to noise by >44-fold, with an average limit of detection of 66 nM and R2 of 0.98. This system detected 107 metabolites from 18 BAECs, 111 metabolites from human urine, and 153 from human serum based on retention time, exact mass, and MS/MS matches from a derivatized standard library. As a proof of concept, aortic endothelial cells were treated with epinephrine and analyzed by the dual-stage derivatization. We observed changes in 32 metabolites with many increases related to energy metabolism, specifically in the TCA cycle. A decrease in lactate levels and corresponding increase in pyruvate levels suggest that epinephrine causes a movement away from glycolytic reliance on energy and a shift towards the more efficient TCA respiration for increasing energy.
  2. Anal Bioanal Chem. 2022 Aug 19.
      Different foods, especially mushrooms, are a valuable source of vitamin D2. However, published concentrations in mushrooms show large variabilities. One reason for this is certainly the high biological variability caused by growth conditions, and another could also be found in the analytical methodology. Therefore, this study aimed to develop a sensitive and highly selective two-dimensional liquid chromatography mass spectrometry (LC-MS/MS) method for vitamin D2 analysis in mushrooms. After validation, the method was applied to four different mushroom species. The developed method with a one-step extraction procedure showed a limit of detection of 0.01 µg vitamin D2/g dry mass (DM), a limit of quantification of 0.05 µg vitamin D2/g DM, and recovery rates between 87.6 and 94.8%. The total run time including the re-equilibration of the columns for the next injection was 7.5 min. After adding increased concentrations of pure substance to Pleurotus ostreatus, Lentinula edodes, and brown and white button mushrooms the standard addition plot showed excellent correlation coefficients (R2) of > 0.9994. Mean vitamin D2 concentrations were observed at 0.122 ± 0.007, 0.074 ± 0.005, 0.099 ± 0.007, and 0.073 ± 0.005 µg/g DM. The coefficient of variation (CV) was between 5.1 and 7.6%. This well-optimized, sensitive LC-MS/MS method, with a fast and simple sample preparation and a short run time, can be applied to future studies especially in different mushroom species with variable growing conditions. This will improve our knowledge about the vitamin D2 content in mushrooms.
    Keywords:  Irradiation; LC–MS/MS; Mushrooms; Standard addition; Vitamin D2
  3. Anal Chem. 2022 Aug 17.
      Simultaneous elemental detection of F and Cl offers quantitation of fluorinated and chlorinated compounds and their transformation products without compound-specific standards. Despite wide-ranging applications, this capability has been hindered by fundamental and technical shortcomings of current inductively coupled plasma (ICP)-MS methods in ion formation and isobaric interference elimination. These hurdles are alleviated here via a chemical ionization method. Fluorine and chlorine in analytes are first converted to HF and HCl by an ICP with post-plasma recombination and subsequently react with barium-containing ions supplied by a nanospray, yielding BaF+ and BaCl+ as elemental reporter ions. Notably, the method is readily interfaced to an Orbitrap MS which eliminates isobaric interferences at resolving powers as low as 35,000, far greater than that of current ICP-MS instruments. Moreover, the instrument is easily reverted to the ESI-MS mode for complementary molecular characterization. To demonstrate analytical capabilities, a workflow for rapid quantitation of compounds separated by reversed-phase liquid chromatography is developed using a species-independent calibration. The independent F and Cl measurements agree with each other, providing recoveries of >90% and LODs of 8-12 pmol Cl and 5-12 pmol F on the column. The workflow along with LC-ESI-MS on the same instrument is then applied to identify and quantify in-vitro drug metabolites, yielding total drug-related material recoveries of >80% and quantitation of minor metabolites summing to 8% of the total drug-related compounds. These results highlight the strengths of simultaneous F and Cl speciation for rapid quantitation with applications in early drug development.
  4. Ther Drug Monit. 2022 Aug 15.
      BACKGROUND: Optimization of antimicrobial therapy is a challenge in critically ill patients who develop extreme inter- and intra-individual pharmacokinetic variability. Therapeutic drug monitoring (TDM) is a valuable tool for maximizing the effect of a drug and minimizing its adverse and unwanted effects. The aim of the current work was to develop and validate an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to determine multiple antibiotics in clinical plasma samples from critically ill patients; low sample volume and rapid processing of samples were considered the main criteria.METHODS: A separation method based on an on-line combination of ultra-high-performance UHPLC-MS/MS was developed for the simultaneous determination of four β-lactam antibiotics (cefepime, meropenem, cefotaxime, and piperacillin), tazobactam, and linezolid in human plasma samples. The volume of plasma sample used for analysis was 20 µL. The developed method was validated according to FDA guideline.
    RESULTS: The chromatographic run time was 8 min. Calibration curves were linear for concentration ranges of 0.1-100 µg/mL (r2 > 0.99) for tazobactam, meropenem, cefotaxime, linezolid, and piperacillin, and 1-100 µg/mL (r2 > 0.99) for cefepime. The intra-day and inter-day accuracy of the method ranged from 92.4% to 110.7% and 93.6% to 113.3%, respectively. The intra- and inter-day precision values were ≤17.3% and ≤17.4%, respectively. No interfering and carryover analytes were observed.
    CONCLUSIONS: The developed UHPLC-MS/MS method is an appropriate and practical tool for TDM of the selected antibiotics. Owing to its rapidity, requirement of low sample volume, and high selectivity, sensitivity, and reliability, it can be effectively implemented in routine clinical laboratory tests for critically ill patients.
  5. Anal Bioanal Chem. 2022 Aug 17.
      The effect of LC mobile phase composition and flow rate (2-50 µL/min) on mobility behavior in vacuum differential mobility spectrometry (vDMS) was investigated for electrosprayed isobaric antidepressant drugs (AD); amitriptyline, maprotiline, venlafaxine; and structurally related antidepressants nortriptyline, imipramine, and desipramine. While at 2 µL/min, no difference in compensation voltage was observed with methanol and acetonitrile, at 50 µL/min, acetonitrile used for LC elution of analytes enabled the selectivity of the mobility separation to be improved. An accurate and sensitive method could be developed for the quantification of six AD drugs in human plasma using trap/elute micro-LC setup hyphenated to vDMS with mass spectrometric detection in the selected ion monitoring mode. The assay was found to be linear over three orders of magnitude, and the limit of quantification was of 25 ng/mL for all analytes. The LC-vDMS-SIM/MS method was compared to a LC-MRM/MS method, and in both cases, inter-assay precisions were lower than 12.5 and accuracies were in the range 91.5-110%, but with a four times reduced analysis time (2 min) for the LC-vDMS-SIM/MS method. This work illustrates that with vDMS, the LC mobile phase composition can be used to tune the ion mobility separation and to improve assay selectivity without additional hardware.
    Keywords:  Antidepressants; Differential mobility spectrometry; FAIMS; Isobaric drugs; Plasma; Quantification; Trap-elute LC
  6. J Pharm Biomed Anal. 2022 Aug 01. pii: S0731-7085(22)00401-0. [Epub ahead of print]220 114980
      Mescaline is a psychedelic phenethylamine found in different species of cacti. Currently, mescaline's acute subjective effects and pharmacokinetics are investigated in several modern clinical studies. Therefore, we developed a bioanalytical method for the rapid quantification of mescaline and its metabolites in human plasma. Mescaline and its metabolites 3,4,5-trimethoxyphenylacetic acid (TMPAA), N-acetyl mescaline (NAM), and 3,5-dimethoxy-4-hydroxyphenethylamine (4-desmethyl mescaline) were simultaneously analyzed by ultra-high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). Optimal chromatographic separation was achieved with an Acquity Premier HSS T3 C18 column. The analytes were detected in positive ionization mode using scheduled multiple reaction monitoring. A single step extraction method was implemented to enable fast and automatable plasma sample preparation. An intra-assay accuracy between 84.9% and 106% and a precision of ≤ 7.33% was observed in three validation runs. Plasma was extracted by simple protein precipitation, resulting in a complete recovery (≥ 98.3%) and minor matrix effects (≤ 7.58%). No interference with endogenous matrix components could be detected in human plasma samples (n = 7). Importantly, method sensitivity sufficed for assessing pharmacokinetic parameters of mescaline in clinical study samples with lower limits of quantification of 12.5, 12.5, and 1.25 ng/mL for mescaline, TMPAA, and NAM, respectively. Nonetheless, 4-desmethyl mescaline could not be selectively quantified in pharmacokinetic samples due to interference with another mescaline metabolite. Overall, we developed and validated a reliable and very easy-to-use method for forensic applications as well as investigating the clinical pharmacokinetics of mescaline.
    Keywords:  Drug metabolism; Forensics; Liquid chromatography; Pharmacokinetics; Psychedelics; Tandem Mass spectrometry
  7. J Forensic Sci. 2022 Aug 18.
      Etomidate, with efficacy similar to that of propofol, has been used as a propofol substitute because propofol is a designated narcotic drug, and an increase in the frequency of illegal distribution and misuse has been reported in Korea. Previous analytical studies on etomidate used blood and urine. For long-term use and timing estimation, a method for etomidate analysis using hair should be developed. Therefore, in this study, an analytical method using LC-MS/MS was developed to determine etomidate and its major metabolite in hair. Human hair samples were segmented after washing to eliminate possible contaminants on the hair and stirred with methanol. The LC-MS/MS conditions were optimized, and the chromatographic separation time was 10 min. Selectivity, linearity, limit of detection, limit of quantification, precision, accuracy, recovery, process efficiency, matrix effect, and stability were evaluated to validate the analytical method. The calibration curves ranged from 0.25 to 50 pg/mg for etomidate and 2-250 pg/mg for etomidate acid; the coefficients of determination were higher than 0.997. The intra- and inter-assay precision results for all the compounds were <15% and satisfied at recovery, process efficiency, matrix effect, and stability. In addition, this method was applied to the hair of 4 rats which are administered with etomidate to evaluate. The etomidate concentrations in the rat hair ranged from 2.60 to 8.50 pg/mg, and the etomidate acid concentrations were 2.06-7.13 pg/mg. Thus, this method can be used as basic data for monitoring etomidate in hair.
    Keywords:  LC-MS/MS; etomidate; etomidate acid; forensic toxicology; hair analysis; method validation
  8. Metabolomics. 2022 Aug 17. 18(9): 69
      BACKGROUND & AIMS: A metabolomic study of hepatolithiasis has yet to be performed. The purpose of the present study was to characterize the metabolite profile and identify potential biomarkers of hepatolithiasis using a metabolomic approach.METHODS: We comprehensively analyzed the serum metabolites from 30 patients with hepatolithiasis and 20 healthy individuals using ultra-high performance liquid chromatography-tandem mass spectrometry operated in negative and positive ionization modes. Statistical analyses were performed using univariate (Student's t-test) and multivariate (orthogonal partial least-squares discriminant analysis) statistics and R language. Receiver operator characteristic (ROC) curve analysis was performed to identify potential predictors of hepatolithiasis.
    RESULTS: We identified 277 metabolites that were significantly different between hepatolithiasis serum group and healthy control serum group. These metabolites were principally lipids and lipid-like molecules and amino acid metabolites. The steroid hormone biosynthesis pathway was enriched in hepatolithiasis serum group. In all specific metabolites, 75 metabolites were over-expressed in hepatolithiasis serum group. The AUC values for 60 metabolites exceeded 0.70, 4 metabolites including 18-β-Glycyrrhetinic acid, FMH, Rifampicin and PC (4:0/16:2) exceeded 0.90.
    CONCLUSIONS: We have identified serum metabolites that are associated with hepatolithiasis for the first time. 60 potential metabolic biomarkers were identified, 18-β-Glycyrrhetinic acid, FMH, Rifampicin and PC (4:0/16:2) may have the potential clinical utility in hepatolithiasis.
    Keywords:  Amino acid metabolism; Biomarker; Hepatolithiasis; Lipid metabolism; Metabolomics; Ultra-high performance liquid chromatography-tandem mass spectrometry
  9. Anal Bioanal Chem. 2022 Aug 15.
      To assess chocolate quality and authenticity comprehensively, a combination of various analytical procedures is involved, thereby making the process time-consuming and costly. Thus, we investigated the potential of ultra-high performance supercritical fluid chromatography coupled to quadrupole-time of flight mass spectrometry (UHPSFC-QTOF-MS) as an alternative to "classic" methods. By combining hexane and aqueous extracts from sequential extraction, a single 8-min analytical run enabled us (i) to determine cocoa butter equivalents (CBEs) and milk fat content based on the detection of selected triacylglycerols, (ii) to calculate dry non-fat cocoa solids based on determined theobromine and caffeine content, and (iii) to profile contained sugars. To obtain the most comprehensive information about sample composition, the MS method comprised a full MS scan for non-target screening and several time-scheduled targeted MS/MS functions ("parallel reaction monitoring") optimized according to the possible concentration ranges of the analytes. For 40 different chocolate samples, our results and those obtained by using standard methods (LC-UV for non-fat cocoa solids, and GC-FID for CBEs) were in good agreement. Compared to the conventional approach for chocolate quality and authenticity control, the presented SFC-MS method is a fast, cost-effective, and efficient alternative, and only samples suspicious for the presence of CBE should be referred to the standard GC-FID method for exact CBE quantification. In the study, also some challenges offered by SFC-MS have been addressed.
    Keywords:  Cocoa butter equivalents; SFC-HRMS; Sugars; Theobromine; Triacylglycerols; UPC2