bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒04‒26
forty-one papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Anal Chem. 2020 Apr 22.
      The nonlinear signal response of electrospray ionization (ESI) presents a critical limitation for mass spectrometry (MS)-based quantitative analysis. In the field of metabolomics research, this issue has largely remained unaddressed - MS signal intensities are usually directly used to calculate fold changes for quantitative comparison. In this work, we demonstrate that, due to the nonlinear ESI response, signal intensity ratios of a metabolic feature calculated between two samples may not reflect their real metabolic concentration ratios (i.e., fold-change compression), implicating that conventional fold change calculations directly using MS signal intensities can be misleading. In this regard, we developed a quality control (QC) sample-based signal calibration workflow to overcome the quantitative bias caused by the nonlinear ESI response. In this workflow, calibration curves for every metabolic feature are first established using QC sample injected in serial injection volumes. The MS signals of each metabolic feature are then calibrated to their equivalent QC injection volumes for comparative analysis. We demonstrated this novel workflow in a targeted metabolite analysis, showing that the accuracy of fold change calculations can be significantly improved. Furthermore, in a metabolomic comparison of the bone marrow interstitial fluid samples from leukemia patients before and after chemotherapy, an additional 59 significant metabolic features were found with fold change larger than 1.5 and an additional 97 significant metabolic features had fold changes corrected by more than 0.1. This work enables high-quality quantitative analysis in untargeted metabolomics, thus providing more confident biological hypotheses generation.
  2. Rapid Commun Mass Spectrom. 2020 Apr 19. e8814
      RATIONALE: Electrospray ionization mass spectrometry (ESI-MS) methodology often shows poor ionization reproducibility in the analysis of biological samples. Therefore, the normalization of the measured peak intensities is essential. It is believed that quantitative data with high reproducibility can be obtained by adding a constant amount of an internal standard material (IS) labeled with stable isotopes to each sample, thus allowing the correction of the quantitative value of the target compound by that of the IS. We investigated whether the presence or absence of a labeled IS improves the accuracy of these quantitative values.METHODS: Triple quadrupole MS coupled with liquid chromatography (LC) was used to analyze fatty acid metabolites in biological samples as target compounds. Two independent systems were used to provide a measure of reproducibility in two different laboratories.
    RESULTS: Data having poor reproducibility in the raw peak areas were efficiently normalized using the IS, but, crucially, the IS method using stable isotopes was not always necessary. In some cases, the reproducibility was relatively good even without using the IS. In a contaminant matrix, the MS response behavior of the target compound and its stable isotope-labeled material was complicated. Since the ion suppression by matrix contaminants was dependent on the concentration of the target compound, the added amounts of the ISs were also important, Furthermore, an equivalent normalization effect was obtained by using a pooled quality control sample as an external standard method, thus obviating the need for labeled IS samples, which are often expensive and sometimes not commercially available.
    CONCLUSIONS: Our results raise the question whether the quantitative method using stable-isotope-labeled IS labeled is always necessary and beneficial. However, the results obtained in this study cannot be generalized because only fatty acid metabolites were examined by ESI-MS and only highly substituted deuterium labeled IS was used.
  3. J Chromatogr A. 2020 Apr 18. pii: S0021-9673(20)30309-5. [Epub ahead of print] 461088
      In this study, a novel and straightforward analytical methodology was proposed for the determination of cocaine (COC) and its main metabolites benzoylecgonine (BZE) cocaethylene (CE) and hydroxy‑cocaine (COCOH) in urine samples. This approach consisted of a high-throughput and semiautomated configuration based on hollow-fiber renewal liquid membrane extraction (HFRLM) coupled to a 96-well plate system, which was proposed for the first time to analyze complex biological samples such as urine. The analytical determinations were performed using ultra-high performance liquid chromatography coupled to quadrupole time-of-flight-mass spectrometry (LC-ESI-QTOF-MS). The analytical methodology was fully optimized through Doehlert and simplex-centroid designs, and univariate approaches. Polypropylene membranes of 1 cm length were inserted in the pins of an extraction blade combined with a 96-well plate system and its pores were filled with hexane:dichloromethane:ethyl acetate (1:1:1 v/v/v) for 180 s; moreover, 20 µL of this mixture was added to the sample to allow for a renewable liquid membrane. The extraction step was carried out by keeping the blades immersed in vials containing 1.5 mL of diluted urine adjusted at pH 10 with 10% (w/v) of Na2CO3 during 20 min, followed by liquid desorption with 100 µL of acetonitrile. Finally, the extract was dried under N2 stream and resuspended with 20 µL of ultrapure water. Satisfactory analytical performance was obtained with coefficients of determination ranging from 0.9875 for BZE to 0.9986 for CE; intra-day precision ranged from 1.6 to 13.5%, and inter-day precision varied from 2.2 to 17.5%. Limits of detection ranged from 1.5 to 15.1 ng mL-1, and limits of quantification varied from 5 to 50 ng mL-1, with relative recoveries varied from 70.7 to 124.1%.
    Keywords:  Cocaine; High-throughput; Microextraction; Sample preparation; Urine
  4. J Sep Sci. 2020 Apr 22.
      The constant emergence of New Psychoactive Substances is a challenge to clinical and forensic toxicologists that need to constantly update analytical techniques to detecting them. A large portion of these substances are synthetic cannabinoids. The aim of this study was to develop a rapid and simple method for the determination of synthetic cannabinoids and their metabolites in urine and blood using GC-MS. The method involves an Ultrasound-Assisted Dispersive Liquid-Liquid Microextraction which implies a rapid procedure, giving excellent extraction efficiencies with minimal use of toxic solvents. This is followed by silylation and analysis with GC-MS. The chromatographic method allows for the separation and identification of 29 selected synthetic cannabinoids and some metabolites. The method was validated on urine and blood samples with the ability to detect and quantify all analytes with satisfactory limits of detection (from 1 to 5 ng/mL), limits of quantification (5 ng/mL), selectivity and linearity (in the range 5 - 200 ng/mL). The developed assay is highly applicable to laboratories with limited instrumental availability, thanks to the use of efficient and low-cost sample preparation and instrumental equipment. The latter may contribute to enhance the detection of NPS in clinical and forensic toxicology laboratories. This article is protected by copyright. All rights reserved.
    Keywords:  Clinical Toxicology; Dispersive Liquid/Liquid Microextraction; Forensic Toxicology; Gas Chromatography-Mass Spectrometry; Synthetic Cannabinoids
  5. Metabolites. 2020 Apr 17. pii: E156. [Epub ahead of print]10(4):
      Genomics-based metabolic models of microorganisms currently have no easy way of corroborating predicted biomass with the actual metabolites being produced. This study uses untargeted mass spectrometry-based metabolomics data to generate a list of accurate metabolite masses produced from the human commensal bacteria Citrobacter sedlakii grown in the presence of a simple glucose carbon source. A genomics-based flux balance metabolic model of this bacterium was previously generated using the bioinformatics tool PyFBA and phenotypic growth curve data. The high-resolution mass spectrometry data obtained through timed metabolic extractions were integrated with the predicted metabolic model through a program called MS_FBA. This program correlated untargeted metabolomics features from C. sedlakii with 218 of the 699 metabolites in the model using an exact mass match, with 51 metabolites further confirmed using predicted isotope ratios. Over 1400 metabolites were matched with additional metabolites in the ModelSEED database, indicating the need to incorporate more specific gene annotations into the predictive model through metabolomics-guided gap filling.
    Keywords:  bioinformatics; flux balance analysis; mass spectrometry; metabolomics; microbiome; multiomics
  6. Analyst. 2020 Apr 24.
      Much evidence suggested that cholesterol, eicosanoid and phospholipid metabolism plays crucial roles in inflammation, atherosclerosis, carcinogenesis, etc. Therefore, fast and accurate quantification of the metabolites in these metabolic pathways is necessary for discovering the molecular mechanisms and biomarkers of related diseases. In this assay, ultra-high performance liquid chromatography combined with triple quadrupole mass spectrometry platform (UPLC-QqQ-MS) based protocols were developed to simultaneously quantify a total of 104 key metabolites including 32 phospholipids (PLs), 44 eicosanoids (EAs), 28 oxysterols and bile acids (BAs), within 15 minutes. Validation results showed that this method is stable, sensitive and accurate for analyzing different matrix samples. Next, this method was used to characterize the metabolic phenotype of a CCl4-induced liver injury model. The results showed that polyunsaturated fatty acids (PUFA) and PUFA acyl-phospholipids (PFA-PLs) were down-regulated and the levels of saturated fatty acyl-phospholipids (SFA-PLs) and EAs were up-regulated in both the liver tissue and plasma of the CCl4-injury group. BAs were up-regulated in plasma, but down-regulated in the liver tissue of the CCl4-injury group. Immunohistochemistry assay demonstrated that the expression levels of cytosolic phospholipase A2 (cPLA2), phosphorylated cytosolic phospholipase A2 (p-cPLA2), secreted phospholipase A2-IIA (sPLA2-IIA) and lysophosphatidylcholine acyltransferase 1 (LPCAT1) were all up-regulated. According to our results, we drew a diagram of the CCl4-induced acute liver injury molecular mechanism. Moreover, we found that the area under the receiver operating characteristic curve (AUC) of 7α-hydroxycholesterol and 7β-hydroxycholesterol was 1.0, which indicates that the two metabolites have significant potential for the diagnosis of acute liver injury. The outstanding performance of this analytical method proves its further usefulness for mechanism studies and biomarker screening of related diseases.
  7. Biomed Chromatogr. 2020 Apr 19. e4859
      A rapid, selective, and sensitive liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the quantitation of the novel CDK5 inhibitor "20-223" in mouse plasma. Separation of analytes was achieved using by a reverse phase column ACE Excel C18 column (1.7μm, 100 X 2.1 mm) with gradient elution using 0.1% formic acid in methanol and 0.1% formic acid as the mobile phase. Analytes were monitored by using MS/MS with an electrospray ionization source in positive multiple reaction monitoring mode. The MS/MS response was linear over the concentration range from 0.2-500 ng/mL for compound 20-223. The within and between batch precision were within the acceptable limits as per FDA guidelines. The validated method was successfully applied to plasma protein binding and in vitro metabolism studies. 20-223 was highly bound to mouse plasma proteins (>95% bound). Utilizing mouse S9 fractions, in vitro intrinsic clearance (CLint ) was 24.68 ± 0.99 μL/min/mg protein. A total of twelve phase I and II metabolites were identified with hydroxylation found to be the major metabolic pathway. The validate method required a low sample volume, was linear from 0.2 to 500 ng/mL and had acceptable accuracy and precision.
    Keywords:  20-223; CDK5 Inhibitor; LC-MS/MS
  8. Bioinformatics. 2020 Apr 23. pii: btaa251. [Epub ahead of print]
      SUMMARY: MIAMI (Mass Isotopolome Analysis for Mode of action Identification) combines the strengths of targeted and non-targeted approaches to detect metabolic flux changes in gas chromatography/mass spectrometry datasets. Based on stable isotope labeling experiments, MIAMI determines a mass isotopomer distribution (MID) based similarity network and incorporates the data into metabolic reference networks. By identifying MID variations of all labeled conpounds between different conditions, targets of metabolic changes can be detected.AVAILABILITY AND IMPLEMENTATION: We implemented the data processing in C ++17 with Qt5 back-end using MetaboliteDetector and NTFD libraries. The data visualization is implemented as web application. Executable binaries and visualization are freely available for Linux operating systems, the source code is licensed under General Public License version 3.
    SUPPLEMENTARY INFORMATION: Source code, documentation and sample data are available at
  9. Metabolites. 2020 Apr 18. pii: E158. [Epub ahead of print]10(4):
      Constant improvements to the Orbitrap mass analyzer, such as acquisition speed, resolution, dynamic range and sensitivity have strengthened its value for the large-scale identification and quantification of metabolites in complex biological matrices. Here, we report the development and optimization of Data Dependent Acquisition (DDA) and Sequential Window Acquisition of all THeoretical fragment ions (SWATH-type) Data Independent Acquisition (DIA) workflows on a high-field Orbitrap FusionTM TribridTM instrument for the robust identification and quantification of metabolites in human plasma. By using a set of 47 exogenous and 72 endogenous molecules, we compared the efficiency and complementarity of both approaches. We exploited the versatility of this mass spectrometer to collect meaningful MS/MS spectra at both high- and low-mass resolution and various low-energy collision-induced dissociation conditions under optimized DDA conditions. We also observed that complex and composite DIA-MS/MS spectra can be efficiently exploited to identify metabolites in plasma thanks to a reference tandem spectral library made from authentic standards while also providing a valuable data resource for further identification of unknown metabolites. Finally, we found that adding multi-event MS/MS acquisition did not degrade the ability to use survey MS scans from DDA and DIA workflows for the reliable absolute quantification of metabolites down to 0.05 ng/mL in human plasma.
    Keywords:  DDA; DIA; Orbitrap Fusion; high resolution mass spectrometry; metabolomics
  10. J Am Soc Mass Spectrom. 2020 Apr 24.
      Imaging mass spectrometry (IMS) has proven to be a useful tool when investigating the spatial distributions of metabolites and proteins in a biological system. One of the biggest advantages of IMS is the ability to maintain the 3D chemical composition of a sample and analyze in a label free manner. However, acquiring the spatial information leads to an increase in data size. Due to the increased availability of commercial mass spectrometers capable of IMS, there has been an exciting development of different statistical tools that can help decipher the spatial relevance of an analyte in a biological sample. To address this need, software packages like SCiLS and the open source R package Cardinal have been designed to perform unbiased spectral grouping based on the similarity of spectra in an IMS data set. In this note we evaluate SCiLS and Cardinal compatibility with MALDI-TOF IMS data sets of the Gram-negative pathogen Pseudomonas aeruginosa PA14. Both software were able to perform unsupervised segmentation with similar performance. There were a few notable differences which are discussed related to the identification of statistically significant features which required optimization of preprocessing steps, region of interest, and manual analysis.
  11. Anal Bioanal Chem. 2020 Apr 24.
      Lipidomics aims to characterize lipid alteration in response to internal or external subtle perturbations in complex biological samples. Lipid abnormality is a major risk factor for many diseases. Large-scale lipidomic studies may offer new insights into the pathophysiological mechanisms of diseases, new opportunities in systems biology, functional biology, and personalized medicine. To this end, a highly efficient and stable lipidomic method is highly in demand. We herein present a rapid and relatively high coverage lipidomic profiling approach based on ultra-high performance liquid chromatography-mass spectrometry by comparing the performance of different chromatographic columns, optimizing the elution gradient and selecting an appropriate data acquisition mode of mass spectra. As a result, a total of 481 lipids were detected from 40 μL serum sample within 13 min, covering 20 common lipid (sub)classes. The developed method was well validated with satisfactory analytical characteristics in linearity, repeatability, stability, and lipid coverage. To show the usefulness, the method was employed to investigate serum lipid profiling of 43 subjects with mild diabetic retinopathy and 44 normal controls, and successfully defined the differential lipids related to diabetic retinopathy. We believe that this rapid method will be beneficial for lipidomic analysis of large-scale clinical samples.
    Keywords:  Diabetic retinopathy; Differential lipids; Lipidome; Lipidomic profiling; Liquid chromatography–mass spectrometry
  12. J Chromatogr A. 2020 Apr 18. pii: S0021-9673(20)30307-1. [Epub ahead of print] 461086
      Mass spectrometry imaging (MSI) has been used for localization of various biomolecules in tissues, but it is still challenging to have absolute pixel-to-pixel quantitation of analytes and differentiation of isobaric ions by MSI. In this proof-of-concept study, we present a quantitative MSI method for amino acids with distinguishing their constitutional isomers by exhaustive liquid microjunction surface sampling (LMJSS)-tandem mass tags (TMT) labeling-ultra performance liquid chromatography (UPLC)-MS. TMT6 reagents were used to differentially isotopically label amino acids in extracts from 6 pixels of brain section, resulting in multiplexed analysis of 6 pixels and largely shortening the LC-MSI time. From the results, with TMT labeling, the MS signals of amino acids in brain tissue extract were enhanced by (3-141)-fold. The calibration curves of TMT-labeled amino acids had good linearity in both MS1 and MS2 detection, which is essential for quantitation. A new extraction solvent for LMJSS (10% hexafluoroisopropanol-40% methanol-0.5% acetic acid-water) was developed to improve the extraction efficiencies of polar amino acids from brain tissue. The results showed that the extraction efficiencies of amino acids from different tissue regions were in the range of 75-110% with the new solvent, which made LMJSS an exhaustive sampling. Due to the complete extraction of amino acids from tissue, TMT0-labeled amino acid standards were directly added into the extracts for absolute quantitation. Finally, UPLC-MS was coupled with LMJSS to successfully separate the isobaric labeled amino acids in each pixel, allowing separate imaging of them. The imaging results of amino acid standard pattern demonstrate 500 µm spatial resolution of the MSI method. The brain tissue imaging results showed that the new method enabled quantitative MSI of 11 amino acids including three pairs of isomers, and the quantitation results were highly comparable and correlated with that by traditional bulk extraction-LC-MS method (correlation coefficient = 0.97, the slope of the correlation curve = 0.96).
    Keywords:  Amino acids; Brain tissue; Liquid microjunction surface sampling; Quantitative mass spectrometry imaging; Tandem mass tags
  13. Metabolites. 2020 Apr 16. pii: E155. [Epub ahead of print]10(4):
      Metabolomics techniques are now applied in numerous fields, with the ability to provide information concerning a large number of metabolites from a single sample in a short timeframe. Although high-frequency (HF) nuclear magnetic resonance (NMR) analysis represents a common method of choice to perform such studies, few investigations employing low-frequency (LF) NMR spectrometers have yet been published. Herein, we apply and contrast LF and HF 1H-NMR metabolomics approaches to the study of urine samples collected from type 2 diabetic patients (T2D), and apply a comparative investigation with healthy controls. Additionally, we explore the capabilities of LF 1H-1H 2D correlation spectroscopy (COSY) experiments regarding the determination of metabolites, their resolution and associated analyses in human urine samples. T2D samples were readily distinguishable from controls, with several metabolites, particularly glucose, being associated with this distinction. Comparable results were obtained with HF and LF spectrometers. Linear correlation analyses were performed to derive relationships between the intensities of 1D and 2D resonances of several metabolites, and R2 values obtained were able to confirm these, an observation attesting to the validity of employing 2D LF experiments for future applications in metabolomics studies. Our data suggest that LF spectrometers may prove to be easy-to-use, compact and inexpensive tools to perform routine metabolomics analyses in laboratories and 'point-of-care' sites. Furthermore, the quality of 2D spectra obtained from these instruments in half an hour would broaden the horizon of their potential applications.
    Keywords:  1H-1H COSY; benchtop spectrometers; low-frequency NMR; metabolomics; type 2 diabetes
  14. J Chromatogr A. 2020 Apr 11. pii: S0021-9673(20)30313-7. [Epub ahead of print] 461092
      Dried blood spot (DBS) sampling demonstrates multiple advantages over traditional venous blood collection in terms of quantifying biomarkers for clinical applications. The process is more convenient, less invasive and requires smaller sample size. More importantly, it lowers risk of infection and allows easier sample transportation and storage. In this study, an automated high-throughput DBS-LC-MS/MS method was developed for quantifying endogenous biomarkers in DBS (or 20 μL whole blood) and later applied in riboflavin (i.e. vitamin B2) quantification. The method consists of four steps, including internal standard spraying, high pressure sample extraction, LC-MS/MS sample analysis and automatic extraction module cleaning. The last two steps overlap, thus reducing sample preparation time and shorten the sample analysis cycle to five minutes per sample. The method was validated to be selective and sensitive (LLOQ=2 ng/mL) over a range of 2-120 ng/mL. Matrix effect was compensated by the application of internal standard, while within-run precision, between-run precision, accuracy, stability and ruggedness of the developed method were all assessed to be satisfactory. Quantitative analysis of riboflavin in 133 whole blood samples using the developed method demonstrated strong correlation compared with those quantified using traditional manual sample preparation followed by LC-MS/MS analysis (R = 0.9774). In conclusion, an automated high-throughput DBS-LC-MS/MS method was developed and validated to be sensitive, accurate and robust, suggesting great potential in the quantification of endogenous biomarkers in blood or other biofluids.
    Keywords:  Dried blood spot; Liquid chromatography tandem mass spectrometry; Nutritional biomarker; Online sample preparation; Vitamin b2
  15. Biomed Chromatogr. 2020 Apr 23. e4863
      Ziritaxestat is a first-in-class autotoxin inhibitor. The purpose of this study was to develop a liquid chromatography/electrospray ionization tandem mass spectrometric (LC-MS/MS) method for the determination of ziritaxestat in rat plasma. The plasma sample was deproteinated by using acetonitrile and then separated on an ACQUITY BEH C18 column with water containing 0.1% formic acid and acetonitrile as mobile phase, which was delivered at 0.4 mL/min. Ziritaxestat and internal standard (crizotinib) were quantitatively monitored with precursor-to-product transitions of m/z 589.3 > 262.2 and m/z 450.1 > 260.2, respectively. The total running time was 2.5 min. The method showed excellent linearity over the concentration range of 0.5-2000 ng/mL, with correlation coefficient greater than 0.9987. The extraction recovery was more than 82.09% and the matrix effect was not significant. Inter- and intra-day precisions (RSD%) were below 11.20% and accuracies were in the range of -8.50-7.45%. Ziritaxestat was demonstrated to be stable in rat plasma under the tested conditions. The validated LC-MS/MS method was successfully applied to study the pharmacokinetic profiles of ziritaxestat in rat plasma after intravenous and oral administration. Pharmacokinetic results demonstrated that ziritaxestat displayed short half-life (~3 h) and low bioavailability (20.52%).
    Keywords:  Liquid chromatography tandem mass spectrometry; Ziritaxestat; bioavailability; pharmacokinetics; rat
  16. Molecules. 2020 Apr 17. pii: E1862. [Epub ahead of print]25(8):
      Trimethylamine (TMA) is a gut microbial metabolite-rendered by the enzymatic cleavage of nutrients containing a TMA moiety in their chemical structure. TMA can be oxidized as trimethylamine N-oxide (TMAO) catalyzed by hepatic flavin monooxygenases. Circulating TMAO has been demonstrated to portend a pro-inflammatory state, contributing to chronic diseases such as cardiovascular disease and chronic kidney disease. Consequently, TMAO serves as an excellent candidate biomarker for a variety of chronic inflammatory disorders. The highly positive correlation between plasma TMAO and urine TMAO suggests that urine TMAO has the potential to serve as a less invasive biomarker for chronic disease compared to plasma TMAO. In this study, we validated a method to simultaneously measure urine TMA and TMAO concentrations by liquid chromatography-mass spectrometry (LC/MS). Urine TMA and TMAO can be extracted by hexane/butanol under alkaline pH and transferred to the aqueous phase following acidification for LC/MS quantitation. Importantly, during sample processing, none of the nutrients with a chemical structure containing a TMA moiety were spontaneously cleaved to yield TMA. Moreover, we demonstrated that the acidification of urine prevents an increase of TMA after prolonged storage as was observed in non-acidified urine. Finally, here we demonstrated that TMAO can spontaneously degrade to TMA at a very slow rate.
    Keywords:  biomarker; liquid chromatography–mass spectrometry (LC/MS); trimethylamine (TMA); trimethylamine N-oxide (TMAO); urine
  17. J Ocul Pharmacol Ther. 2020 Apr 22.
      Purpose: Mixed eye drops containing 0.5% tropicamide and 0.5% phenylephrine are commercially available for cycloplegic refraction. Determining the pharmacokinetics (PK) and distribution of tropicamide and phenylephrine simultaneously in ocular tissues is an important but challenging issue. Herein, we developed a sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous determination of tropicamide and phenylephrine concentrations in rabbit ocular tissues and plasma. Methods: The two analytes were extracted with ethyl acetate using etofesalamide as an internal standard and separated using a chromatographic C8 column with isocratic elution. Mass spectrometry analysis was performed with positive electrospray ionization and data were acquired in a multiple reaction monitoring mode. Results: We validated this method over a concentration range of 5-1,600 ng/mL for tropicamide and 1-320 ng/mL for phenylephrine in ocular tissues, as well as 0.5-64 ng/mL for both compounds in plasma. Inter- and intraday precisions in all samples were both <12.9% and the accuracy was within 92.1%-108.4%. The highest concentration of tropicamide was found in aqueous humor (Cmax: 29430 ng/g), while was in cornea for phenylephrine (Cmax: 3465 ng/g). All the ocular tissues concentrations were much higher than those of blood. Conclusion: This UPLC-MS/MS method allowed us to determine the PK and distribution of tropicamide and phenylephrine in rabbit ocular tissue, which may be helpful in the future development and application of mydriatic agents.
    Keywords:  UPLC-MS/MS; ocular tissues; pharmacokinetics/pharmacodynamics; phenylephrine; tropicamide
  18. Talanta. 2020 Aug 01. pii: S0039-9140(20)30158-2. [Epub ahead of print]215 120867
      The increasing need to develop quantitative chromatographic methods with upgradable multi-targeted approach, allowing flexible and reliable application on large daily workload makes the implementation of an efficient strategy of method's validation and maintenance crucial for the quality assurance policy. The expounding case of a gas chromatographic-mass spectrometric method for the urinary endogenous steroid profiling is presented to illustrate a validation strategy that combines rigorous estimation of validation parameters with highly efficient use of the collected data. The analysis of blank urine samples fortified at six concentration levels with 18 targeted steroids was replicated nine times in three working sessions along twelve days. This dataset of 54 analysis formed the groundwork on which the statistical evaluation of several validation parameters was founded, including calibration, intra- and inter-day accuracy and precision, limit of detection (LOD), limit of quantification, ion abundance ratio repeatability, selectivity, specificity, and carry-over. The preliminary comparison of the response variances at different concentration levels provided the evaluation for heteroscedasticity. Then, the most appropriate calibration model was determined for each steroid, in terms of order (linear vs. quadratic) and weighting, allowing to complete their quantitation in each solution. Intra- and inter-day accuracy and precision were calculated therefrom. LOD values were computed with the Hubaux-Vos method from the weighted linear segment of the calibration curves. Only the assessment of recovery and ionization suppression/enhancement required the execution of further independent experiments. The case study demonstrated that the application of adequate statistical testing typically produced non-homogeneous models of calibration curves, mostly arising from heteroscedastic and quadratic distribution of datasets, unlike what is reported in overly simplified approaches. The misleading information obtained from the regression coefficient R2 to evaluate linearity was evidenced. The strong dependence of calculated LOD and accuracy from the selected calibration parameters was highlighted, making the implementation of an adequate calibration maintenance policy highly advisable.
    Keywords:  Androgens; Calibration; Chromatography; Mass spectrometry; Validation
  19. Biomed Chromatogr. 2020 Apr 20. e4862
      A simple and sensitive ultra-high performance liquid chromatography tandem mass spectrometric method was developed and validated for the determination of foretinib in rat plasma. The analyte and internal standard were extracted from the bio-samples with acetonitrile and then separated on ACQUITY UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm) using 0.1% formic acid aqueous and acetonitrile as mobile phase, at a flow rate of 0.4 mL/min. The mass detection was performed in positive selected reaction monitoring mode with precursor-to-product transitions at m/z 317.1 > 128.1 for foretinib and m/z 502.2 > 323.1 for internal standard. The assay was demonstrated to be linear in the concentration range of 0.5-1000 ng/mL, with correlation coefficient > 0.999. The mean extraction recovery of foretinib from rat plasma was within the range of 84.55-88.09%, while the matrix effect was in the range of 88.56-99.21%. The intra- and inter-day precisions were less than 12.95% and the accuracy ranged from -7.55 to 8.57%. Foretinib was stable in rat plasma under the tested storage conditions. The validated assay was successfully applied to the pharmacokinetic study of foretinib in the rats. The results revealed that foretinib showed moderate elimination half-life, low clearance and dose-independent pharmacokinetic profiles in the rats.
    Keywords:  Foretinib; bioavailability; liquid chromatography tandem mass spectrometry; pharmacokinetics
  20. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Apr 03. pii: S1570-0232(20)30024-6. [Epub ahead of print]1145 122099
      Three normal phase HPLC methods were produced to separate lipid classes on a PVA-Sil stationary phase including: 9 polar lipids (method 1); 13 combined polar and neutral lipids (method 2); and a combined method that further separates the neutral lipids into 2-4 subclasses based on the presence of fatty acids containing a polar functional group (e.g. hydroxyl) for a total of 20 lipid classes and subclasses separated in a single run (method 3). Polar lipids separated include: the phosphoglycerolipids PG, PE, PI, PS, PC and LPC; the galactoglycerolipids MGDG and DGDG; and a sulfoglycerolipid SQDG. Neutral lipids include TAG, DAG, and MAG classes and sub-classes containing 0-3, 0-2, and 0-1 hydroxy fatty acids, respectively. The hexane/isopropanol/methanol/aqueous system separates polar lipids without the use of chloroform such that it is suitable for radioactivity analysis by in-line flow scintillation counting. Each method was optimized using the natural lipid standards comprised of diverse molecular species that were detected by ELSD. All molecular species of each lipid class eluted together as single peak detected by ELSD. The methods were demonstrated to be suitable for resolving lipid extracts from animal, microbial, and plant sources as well as application to 14C based metabolic tracing of lipid metabolism in leaves and seeds.
    Keywords:  Galactolipids; Hydroxy fatty acids; Metabolic flux; Phospholipids; Radioactivity; Triglycerides
  21. Chimia (Aarau). 2020 Apr 29. 74(4): 220-224
      Microfluidic autosamplers for electrospray ionization mass spectrometry (ESI-MS) are of major importance when using ESI-MS as a high-throughput and low sample consumption analytical method. In this article, microfluidic ESI-MS autosampler designs are overviewed and a group-owned prototype is discussed. The socalled gap sampler is a pin-based sampler for miniaturized flow injection (FI) analysis. To date, it has been used in various applications. Following proof of concept applications with FI of small molecules, pin modifications were implemented for unspecific and specific extraction for the analysis of complex samples. Most recently, further optimization allowed the study of non-covalent protein-ligand interactions for bioaffinity screenings, which constitutes a major milestone in the development of this novel high-throughput autosampler.
  22. Toxins (Basel). 2020 Apr 19. pii: E263. [Epub ahead of print]12(4):
      A method was developed to extract and quantify microcystins (MCs) from mouse liver with limits of quantification (LOQs) lower than previously reported. MCs were extracted from 40-mg liver samples using 85:15 (v:v) CH3CN:H2O containing 200 mM ZnSO4 and 1% formic acid. Solid-phase extraction with a C18 cartridge was used for sample cleanup. MCs were detected and quantified using HPLC-orbitrap-MS with simultaneous MS/MS detection of the 135.08 m/z fragment from the conserved Adda amino acid for structural confirmation. The method was used to extract six MCs (MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, and MC-LW) from spiked liver tissue and the MC-LR cysteine adduct (MC-LR-Cys) created by the glutathione detoxification pathway. Matrix-matched internal standard calibration curves were constructed for each MC (R2 ≥ 0.993), with LOQs between 0.25 ng per g of liver tissue (ng/g) and 0.75 ng/g for MC-LR, MC-RR, MC-YR, MC-LA, and MC-LR-Cys, and 2.5 ng/g for MC-LF and MC-LW. The protocol was applied to extract and quantify MC-LR and MC-LR-Cys from the liver of mice that had been gavaged with 50 µg or 100 µg of MC-LR per kg bodyweight and were euthanized 2 h, 4 h, or 48 h after final gavage. C57Bl/6J (wild type, control) and Leprdb/J (experiment) mice were used as a model to study non-alcoholic fatty liver disease. The Leprdb/J mice were relatively inefficient in metabolizing MC-LR into MC-LR-Cys, which is an important defense mechanism against MC-LR exposure. Trends were also observed as a function of MC-LR gavage amount and time between final MC-LR gavage and euthanasia/organ harvest.
    Keywords:  liquid chromatography-mass spectrometry; liver; microcystins; quantification; solid-phase extraction
  23. J Chromatogr A. 2020 Apr 17. pii: S0021-9673(20)30299-5. [Epub ahead of print] 461078
      A new derivatization reagent, N-(naphthalen-1-yl)-2-oxopropanehydrazonoyl chloride (UOSA54), was prepared and coupled with four drugs, bearing primary amino, secondary amino or mercapto functional groups. Glucosamine sulfate (GLU), cysteine (CYS), captopril (CAP), and vildagliptin (VIL), were used as representative reactive analytes. The prepared reagent was successfully coupled with the targeted analytes in the presence of triethylamine (TEA) as hydrochloride acceptor and acetonitrile as solvent. The resulting reaction products were separated by high-performance liquid chromatography and monitored simultaneously by diode array and triple quad mass spectrometry detectors. Enhanced DAD and electrospray ionization-MS (ESI-MS) responses were observed for the derivatized products. Complete derivatization of VIL was achieved after heating at 65 ± 3 °C for 4 min, while other analytes were derivatized instantaneously at room temperature. Both, the ESI-ionization suppression, due to the excess reagent, and matrix effect, due to co-eluted biogenic plasma constituents, were negligible. The derivatized GLU, CYS, CAP, and VIL showed a maximum absorption wavelength at 376, 417, 340, and 376 nm, with MS-limit of quantification value of 250.0, 2.0, 2.5, and 3.0 pg/μL, respectively. The relative ESI-MS response of UOSA54 derivatization products was within the range of 0.6-4.1 compared with dansylated products. The method was optimized and validated for optimal reaction product stability, sensitivity, linearity, range, precision, and accuracy. The percentage recovery was exceeding 97.2%, with an RSD value of less than 4.0%. The limit of quantification of targeted analytes was ranged from 80.0 to 0.7 pg/μL.
    Keywords:  Derivatization reagent; Ionization suppression; Mass spectrometry; Matrix effect; Method sensitivity
  24. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Apr 06. pii: S1570-0232(20)30298-1. [Epub ahead of print]1145 122107
      Ureteral stents are the most widely used surgical implant in urology. However, they may cause adverse effects to patients, including pain, discomfort, and inflammation. In this work, the inflammatory effect of stent placement and the associated elevation of cyclooxygenase-2 (COX-2) expression were observed. Furthermore, a capillary electrophoresis mass spectrometry (CE-MS) based approach was subsequently developed to quantify urinary prostaglandin E2 (PGE2), a COX-2 metabolite known to contribute to inflammatory renal diseases, to further interrogate the role of this pathway. Urine samples were cleaned and preconcentrated by solid-phase extraction (SPE), and an on-line sample stacking method was used for the enrichment of analytes. The accuracy, precision, and specificity of this method were validated. Standard addition methods were performed to assess the reliability of using deuterated internal standards (IS) in compensating the remaining matrix effect after SPE as well as the detector fluctuation. Through the analysis of 32 pig urine samples, a statistically significant increase of PGE2 was observed in the stented group compared to the unstented (P = 0.01) and the recovered (P = 0.004) groups. This work determined that stent placement may contribute to COX-2-dependent inflammation and developed a reliable CE-MS based methodology to quantify PGE2 in stented individuals that may further understand the biology of stent-associated inflammation and inform urologic patient management.
    Keywords:  Capillary electrophoresis; Cyclooxygenase-2; Mass spectrometry; Prostaglandin E(2); Ureteral stent associated inflammation
  25. Anal Bioanal Chem. 2020 Apr 22.
      In breast cancer, overexpression of human epidermal growth factor receptor 2 (HER2) correlates with overactivation of lipogenesis, mutation of tumor suppressor p53, and increased metastatic potential. The mechanisms through which lipids mediate p53, HER2, and metastatic potential are largely unknown. We have developed a desorption electrospray ionization mass spectrometry (DESI-MS) method to identify lipid biomarkers of HER2/p53 expression, metastatic potential, and disease state (viz. cancer vs. non-cancerous) in monolayer and suspension breast cancer cell cultures (metastatic potential: MCF-7, T-47D, MDA-MB-231; HER2/p53: HCC2218 (HER2+++/p53+), HCC1599 (HER2-/p53-), HCC202 (HER2++/p53-), HCC1419 (HER2+++/p53-) HCC70 (HER2-/p53+++); non-cancerous: MCF-10A). Unsupervised principal component analysis (PCA) of DESI-MS spectra enabled identification of twelve lipid biomarkers of metastatic potential and disease state, as well as ten lipids that distinguish cell lines based on HER2/p53 expression levels (> 200 lipids were identified per cell line). In addition, we developed a DESI-MS imaging (DESI-MSI) method for mapping the spatial distribution of lipids in metastatic spheroids (MDA-MB-231). Of the twelve lipids that correlate with changes in the metastatic potential of monolayer cell cultures, three were localized to the necrotic core of spheroids, indicating a potential role in promoting cancer cell survival in nutrient-deficient environments. One lipid species, which was not detected in monolayer MDA-MB-231 cultures, was spatially localized to the periphery of the spheroid, suggesting a potential role in invasion and/or proliferation. These results demonstrate that combining DESI-MS/PCA of monolayer and suspension cell cultures with DESI-MSI of spheroids is a promising approach for identifying lipid biomarkers of specific genotypes and phenotypes, as well as elucidating the potential function of these biomarkers in breast cancer. Graphical Absract.
    Keywords:  Breast cancer; Desorption electrospray ionization; Lipid; Mass spectrometry imaging; Spheroid
  26. Metabolites. 2020 Apr 22. pii: E162. [Epub ahead of print]10(4):
      Non-targeted mass spectrometry (MS) has become an important method over recent years in the fields of metabolomics and environmental research. While more and more algorithms and workflows become available to process a large number of non-targeted data sets, there still exist few manually evaluated universal test data sets for refining and evaluating these methods. The first step of non-targeted screening, peak detection and refinement of it is arguably the most important step for non-targeted screening. However, the absence of a model data set makes it harder for researchers to evaluate peak detection methods. In this Data Descriptor, we provide a manually checked data set consisting of 255,000 EICs (5000 peaks randomly sampled from across 51 samples) for the evaluation on peak detection and gap-filling algorithms. The data set was created from a previous real-world study, of which a subset was used to extract and manually classify ion chromatograms by three mass spectrometry experts. The data set consists of the converted mass spectrometry files, intermediate processing files and the central file containing a table with all important information for the classified peaks.
    Keywords:  EIC; LC-MS; XIC; peak detection; peak picking
  27. Talanta. 2020 Aug 01. pii: S0039-9140(20)30185-5. [Epub ahead of print]215 120894
      Three dimensional printed open port probe (3DP-OPP) and air displacement based liquid handler, were designed and optimized using fused deposition modeling (FDM) and stereolitography (SLA) 3D printing. The performance of the devices were investigated for the analysis of solid and liquid samples with electrospray ionization mass spectrometry (ESI-MS). Direct analysis in less than 1 min and without any sample preparation, enabled detection of pesticides (azoxtystrobin/imazalil) on fruits peel surface and illegal substances (MDMA/MDEA) in home-made pills. Conjunction of OPP in the overspill mode with a customized autosampler, equipped with disposable pipette tips, enables direct quantitative analysis of drugs of abuse in urine and plasma, with minimized carry-over and reduced matrix effect compared to flow injection analysis.
    Keywords:  3D printing; Direct analysis; Electrospray; Fused deposition modeling; Mass spectrometry; Open port probe; Stereolithography
  28. J Chromatogr A. 2020 Apr 17. pii: S0021-9673(20)30324-1. [Epub ahead of print] 461103
      Triacylglycerols (TAGs) containing cyclofatty acids (cycloFAs) from two oilseeds of Sterculia foetida and Hydnocarpus wightiana were analysed using both reversed-phase (RP18) and chiral phase columns. TAGs were identified using high-resolution electrospray ionization mass spectrometry in the positive ion mode. Fifty-five molecular species of TAGs have been identified in sterculic oil, 27 of which contained at least one cyclopropenyl-FA (e.g., malvalic or sterculic acids). The structures of regioisomers and enantiomers were determined for five major TAGs with cyclopropenyl-FAs. One hundred thirty-six TAGs were identified in chaulmoogra oil, 71 of which contained at least one cyclopentenyl-FA (e.g., gorlic, chaulmoogric, and hydnocarpic acids, etc.). Furthermore, in three molecular species, regioisomers and enantiomers were identified using HPLC on a chiral phase column. Eight molecular species of TAGs were prepared through organic synthesis to facilitate the identification of enantiomers. Retention times of fatty acid-containing triacylglycerols with one ring and one double bond are very similar to triacylglycerols with a dienoic fatty acid, but elution times are shorter. For example, dimalvaloylpalmitate elutes earlier than dilinoleylpalmitate. The order of elution of TAGs on the chiral column differs. In TAGs with 2 degrees of unsaturation (ring and double bond, e.g. PStP-StPP-PPSt), the order of elution is symmetric-asymmetric-asymmetric TAGs. TAGs with 4 degrees of unsaturation (one ring and three double bonds or two rings and two double bonds) present a different pattern. When TAGs contain two rings and two double bonds, the order of elution TAGs is asymmetric-symmetric-asymmetric (StStP-StPSt-PStSt); when TAGs contain a ring and 3 double bonds, the elution order is symmetric-asymmetric-asymmetric TAGs (OStO-StOO-OOSt). In species with a higher degree of unsaturation (e.g., 5), the elution order of the TAGs is asymmetric-asymmetric-symmetric (e.g. CCO-OCC-COC).
    Keywords:  Chaulmoogra oil; Cyclic fatty acids; Enantiomeric separation; Non-aqueous reversed-phase-HPLC; Sterculic seeds; Triacylglycerols
  29. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2020 Mar 20. 38(3): 213-216
      Objective: To describe for the determination of contents of metabolites of benzene compounds in urine sample by high performance liquid chromatography. Methods: After acidification with hydrochloric acid, metabolites in urine were first extracted by acetonitrile and isopropanol (V∶V, 9∶1) with excessive sodium chloride, then gradient separated on a C18 column and then determined by DAD detector. Results: There were good linear relationship between peak areas and injection quality in range of 2.00-100 mg/L (r>0.999). The detection limit and quantitative limit of this method were 4.15-70.7 μg/L and 13.8-235 μg/L respectively. The precision for the analysis of urine was1.78%-8.23% (n =6). The average recovery of metabolites was 85.4%-105.5% at thee spiked levels in the range of 2.00-100 mg/L. Conclusion: The accuracy and reproducibility obtained make this method useful for the biological monitoring of occupational exposure to toluene, xylene, styrene and ethylbenzene.
    Keywords:  Benzene; Creatinine; High performance liquid chromatography; Metabolites; Urine
  30. Anal Chem. 2020 Apr 20.
      Olefin copolymers are complex polymer materials that exhibit multiple distributions in molecular properties such as molar mass, chemical composition and branching. To address the multivariate molecular compositions, in this work chromatographic protocols have been developed that synergistically combine solvent and temperature gradients. As representative examples, blends of olefin copolymers have been fractionated on porous graphitic carbon stationary phases. This is the first study that makes complementary use of solvent and temperature gradient interaction chromatography (SGIC and TGIC, respectively) to capitalize on the advantages of both techniques. In a first experimental setup, solvent and temperature gradients were used simultaneously and complex blends of low molar mass polyethylene and ethylene-co-1-octene copolymers were separated with high efficiency. The separation of oligomers was observed to be significantly better in SGIC as compared to TGIC while comonomer blends could be separated in either TGIC or SGIC mode. In another innovation, a two-column setup was employed where the columns were placed in different temperature zones. It was demonstrated that the separation of both low and high comonomer content blends was improved significantly when the temperatures of the two zones were manipulated reasonably.
  31. Anal Chem. 2020 Apr 21.
      A series of novel anthranilic acid derivatives I-IV, of which COOH-NH2 (I) and COOH-NHMe (IV) are endowed with acid and base bifunctionality, were designed and synthesized for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry applications in dual polarity molecular imaging of biological samples, particularly for lipids. The heat of protonation, deprotonation and proton transfer reaction as well as the capability of analyzing biomol-ecules in both positive and negative ion modes for I-IV were systematically investigated under standard 355-nm laser excitation. The results indicate correlation between dual polarity and acid-base property. Further, COOH-NHMe (IV) showed a unique performance and was successfully applied as the matrix for MALDI-TOF mass spectrometry imaging (MSI) for studying the mouse brain. Our results demonstrate the superiority of COOH-NHMe (IV) in detecting more lipid and protein species compared to commercially available matrices. Moreover, MALDI-TOF MSI results were obtained for lipid distributions, making COOH-NHMe (IV) a potential next generation universal matrix.
  32. Talanta. 2020 Aug 01. pii: S0039-9140(20)30220-4. [Epub ahead of print]215 120929
      The present study reports the first conception to incorporate a non-polar solvent, dichloromethane, as the spray solution in easy ambient sonic-spray ionization (EASI) for mass spectrometry (MS) analysis of hydrophobic compounds. An imidazolium-based dicationic ionic liquid (DIL) at a low concentration of 20 μM was used in combination with dichloromethane. A reactive EASI strategy was implemented, by which the overall positively charged associated complexes of anionized perfluorinated compounds (PFCs) were formed in the presence of the DIL. In positive ionization mode, 19- and 6-fold enhancements in signal intensity were witnessed for the analysis of perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) respectively, compared to that in negative ionization mode with no dicationic ion-pairing reagent. The limits of detection (LODs) and quantitation (LOQs) are 0.5 and 0.8 μg/m2 for PFOA and 0.4 and 0.6 μg/m2 for PFOS, respectively. The developed method allowed direct ambient analysis of samples in their native conditions and was applied to the analysis of PFCs in a variety of real textile, popcorn bucket, and oil-proof hamburger wrapping paper samples.
    Keywords:  Ambient mass spectrometry; Dicationic ionic liquid; Non-polar spray solvent; Reactive easy ambient sonic-spray ionization
  33. Anal Chem. 2020 Apr 20.
      Diversified oxidized-lipid molecules are responsible for inflammation and cell death, including ferroptosis. Lipid radicals are the source of these oxidized lipids, which are the initial key molecules in the lipid peroxidation chain reaction. However, owing to their extremely high reactivity and short half-life, an established detection technique is not available. Here, we pro-posed a high-performance liquid chromatography fluorometry and high-resolution tandem mass spectrometry system combined with a fluorescent probe as a structural analysis method for lipid-derived radicals. We detected 132 lipid-derived radicals, including 111 new species, from five polyunsaturated fatty acids. In addition, a database was constructed, for which the initial fatty acid could be determined using the radical structure. Further, 12 endogenous lipid-derived radicals were identified in carcinogen-induced liver cancer mouse models. Therefore, this method and its corresponding database will provide novel insights into mechanisms underlying the lipid peroxidation, including the associated inflammation and ferroptosis.
  34. Nucleic Acids Res. 2020 Apr 23. pii: gkaa258. [Epub ahead of print]
      Biological processes (like microbial growth & physiological response) are usually dynamic and require the monitoring of metabolic variation at different time-points. Moreover, there is clear shift from case-control (N=2) study to multi-class (N>2) problem in current metabolomics, which is crucial for revealing the mechanisms underlying certain physiological process, disease metastasis, etc. These time-course and multi-class metabolomics have attracted great attention, and data normalization is essential for removing unwanted biological/experimental variations in these studies. However, no tool (including NOREVA 1.0 focusing only on case-control studies) is available for effectively assessing the performance of normalization method on time-course/multi-class metabolomic data. Thus, NOREVA was updated to version 2.0 by (i) realizing normalization and evaluation of both time-course and multi-class metabolomic data, (ii) integrating 144 normalization methods of a recently proposed combination strategy and (iii) identifying the well-performing methods by comprehensively assessing the largest set of normalizations (168 in total, significantly larger than those 24 in NOREVA 1.0). The significance of this update was extensively validated by case studies on benchmark datasets. All in all, NOREVA 2.0 is distinguished for its capability in identifying well-performing normalization method(s) for time-course and multi-class metabolomics, which makes it an indispensable complement to other available tools. NOREVA can be accessed at
  35. Int J Anal Chem. 2020 ;2020 5405184
      An extraction-free method requiring microliter (μL) volumes has been developed for the determination of caffeine in beverages. Using a pyrolysis-gas chromatography mass spectrometry system, the conditions required for the direct thermal desorption-gas chromatography mass spectrometry (TD-GC/MS) determination of caffeine were optimised. A 5 μL aliquot was introduced to the thermal desorption unit, dried, and thermally desorbed to the GC/MS. The response was linear over the range 10 to 500 μg/mL (R 2 = 0.996). The theoretical limit of detection (3 σ) was 0.456 μg/mL. No interferences were recorded from endogenous beverage components or from commonly occurring drugs, such as nicotine, ibuprofen, and paracetamol. Replicate caffeine determinations on fortified latte style white coffee and Pepsi Max® gave mean recoveries of 93.4% (%CV = 4.1%) and 95.0% (%CV = 0.98%), respectively. Good agreement was also obtained with the stated values of caffeine for an energy drink and for Coca-Cola®. These data suggest that the method holds promise for the determination of caffeine in such samples.
  36. Curr Opin Biotechnol. 2020 Apr 15. pii: S0958-1669(20)30035-5. [Epub ahead of print]64 151-160
      Metabolite concentrations, fluxes, and free energies constitute the basis for understanding and controlling metabolism. Mass spectrometry and stable isotopes are integral tools in quantifying these metabolic features. For absolute metabolite concentration and flux measurement, 13C internal standards and tracers have been the gold standard. In contrast, no established methods exist for comprehensive thermodynamic quantitation under physiological environments. Recently, using high-resolution mass spectrometry and multi-isotope tracing, flux quantitation has been increasingly adopted in broader metabolism. The improved flux quantitation led to determination of Gibbs free energy of reaction (ΔG) in central carbon metabolism using a relationship between reaction reversibility and thermodynamic driving force. Here we highlight recent advances in multi-isotope tracing for metabolic flux and free energy analysis.
  37. Anal Bioanal Chem. 2020 Apr 24.
      In this study, we demonstrated nano-flow injection analysis (nano-FIA) with quadrupole time-of-flight mass spectrometry (Q-TOFMS) for 17 highly polar intermediates produced during glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway (PPP). We optimized the analytical conditions for nano-flow injection/Q-TOFMS, and set the flow rate and ion source temperature to 1000 nL/min and 150 °C, respectively. Under optimal conditions, a single run was finished within 3 min, and the RSD value of 50 sequential injections was 4.2%. The method also showed quantitativity of four stable-isotope-labeled compounds (r2 > 0.99), demonstrating its robustness, high repeatability, and specificity. In addition, we compared three sample-preparation methods for rodent blood samples and found that protein precipitation with threefold methanol was the most effective. Finally, we applied the method to plasma samples from the serotonin syndrome (SS) model and control rats, the results of which were evaluated by principal component analysis (PCA). The two groups showed clearly separated PCA score plots, suggesting that the method could successfully catch the differences in metabolic profiles between SS and control rats. The results obtained from our new method were further validated by using the established gas chromatography/tandem mass spectrometry method, which demonstrated that there were good correlations between the two methods (R = 0.902 and 0.958 for lactic acid and malic acid, respectively, each at p < 0.001), thus proving the validity of our method. The method described here enables high-throughput analysis of metabolites and will be of use for the rapid analysis of metabolic profiles. Graphical abstract.
    Keywords:  High-throughput metabolite analysis; Nano-flow injection analysis; Quadrupole time-of-flight mass spectrometry; Serotonin syndrome model
  38. Chembiochem. 2020 Apr 23.
      The increased interest in (enzymatic) transformations between nucleosides and nucleobases has demanded the development of efficient analytical tools. In this report we present an update and extension of our recently described method for the monitoring of these reactions via spectral unmixing. The presented method uses differences in the UV absorption spectra of nucleosides and nucleobases after alkaline quenching to derive their ratio based on spectral shape by fitting of normalized reference spectra. It is applicable to a broad compound spectrum comprising more than 35 examples, offers HPLC-like accuracy, ease of handling and significant reductions in both cost and data acquisition time compared to other methods. This contribution details the principle of spectral unmixing-based reaction monitoring, gives recommendations regarding solutions to common problems and applications that necessitate special sample treatment. We provide software, workflows and reference spectra that facilitate the straightforward and versatile application of the method.
    Keywords:  UV/Vis spectroscopy; nucleobase; nucleoside; nucleoside phosphorylase; spectral unmixing
  39. J Chromatogr A. 2020 Apr 18. pii: S0021-9673(20)30356-3. [Epub ahead of print] 461127
      Gas chromatographic columns based on ionic liquids (ILs) are very promising since the selectivity of these columns can be tuned by both the cation and the anion chemical nature. In this paper, efficiencies of capillary columns based on four phosphonium ionic liquids were studied. The performance of seven columns containing the cation trihexyl(tetradecyl)phosphonium and the anions bromide, chloride, and bis(trifluoromethylsulfonyl)imide was evaluated by measuring the solute band broadening as a function of gas velocities at three temperatures. Hence, classical height equivalent to a theoretical plate (H) against gas velocity (u) plots corresponding to those columns were generated and the data were fitted to the Golay-Guiochon equation with the aim of seeking the optimum conditions to be operated each of them. Band broadening at practical gas velocities is mainly due to poor mass transfer properties of solutes in the (viscous) liquid phases, which limits the achieved efficiencies. These H/u plots proved to be necessary to characterize the column quality at a given temperature, to interpret the band broadening phenomena and thus, to establish the lower temperature limits and the expected plate counts at that temperature.
    Keywords:  Capillary GC; Golay-Guiochon plots; Solute diffusion in ionic liquids; Trihexyl(tetradecyl) phosphonium cation
  40. Molecules. 2020 Apr 15. pii: E1817. [Epub ahead of print]25(8):
      A simple, quick, easy and cheap tandem mass spectrometry (MS/MS) method for the determination of adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) has been newly developed. This novel MS/MS method was applied for the evaluation of the inhibitory effect of a novel 2-oxo-1,2-dihydropyridine-3-carbonitrile derivative, also named DF492, on PDE3 enzyme activity in comparison to its parent drug milrinone. Molecule DF492, with an IC50 of 409.5 nM, showed an inhibition of PDE3 greater than milrinone (IC50 = 703.1 nM). To explain the inhibitory potential of DF492, molecular docking studies toward the human PDE3A were carried out with the aim of predicting the binding mode of DF492. The presence of different bulkier decorating fragments in DF492 was pursued to shift affinity of this novel molecule toward PDE3A compared to milrinone in accordance with both the theoretical and experimental results. The described mass spectrometric approach could have a wider potential use in kinetic and biomedical studies and could be applied for the determination of other phosphodiesterase inhibitor molecules.
    Keywords:  AMP; cyclic AMP; dihydropyridine; docking studies; phosphodiesterases activity; tandem mass spectrometry
  41. Anal Bioanal Chem. 2020 Apr 22.
      A quantitative analytical method based on laser ablation molecular isotopic spectrometry (LAMIS) and multivariate analysis was developed and evaluated for the determination of the isotopic composition of enriched materials. The method consists preparing a concentrated solution of the enriched material, using small quantities of a sample (125 mg), and ensuring the economic efficiency of the analysis. Standard solutions of known isotopic contents are prepared by employing mixtures of urea highly enriched in 15N and urea of natural isotopic ratio and analyzed by mass spectrometry. A small volume (30 μL) of these solutions is delivered to a filter paper disc (3 cm diameter). After drying, the disc, offering a homogeneously distributed analyte, is presented to a LAMIS equipment to acquire the vibronic emission spectra containing information about the isotopologues of interest. To illustrate the proposed method, the content of 15N is determined in enriched samples of urea. In this case, each spectrum is normalized by the intensity of emission of the CN isotopologues for the electronic (Δν = 0) emission band at 387.1 nm, ensuring better accuracy. Selected regions and single wavelengths of the vibronic emission spectrum (Δν = + 1 or - 1) related to CN species were employed to construct multivariate partial least squares (PLS) and univariate regression models to predict the isotopic content of new samples. Besides, the LAMIS data set was evaluated by multivariate curve resolution (MCR) algorithm. The best MCR and PLS models presented similar results regarding the accuracy to determine 15N content in enriched urea. MCR is capable of identifying spectral interferences and minimizing its effect. The results show that the proposed method based on LAMIS and PLS or MCR multivariate analysis can determine the 15N content in the range 5-50% with a root mean square error of prediction (RMSEP) respectively equal to 0.5 or 0.7% (m/m) in comparison with reference results obtained by mass spectrometry. Graphical abstract.
    Keywords:  15N determination in enriched urea; Isotopic composition of enriched materials; Laser ablation molecular isotopic spectrometry (LAMIS); Multivariate curve resolution (MCR); Multivariate regression; Partial least squares