bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒03‒06
38 papers selected by
Sofia Costa
Icahn School of Medicine at Mount Sinai

  1. Metabolomics. 2022 Feb 28. 18(3): 16
      INTRODUCTION: Recent advances in high-throughput methodologies in the 'omics' and synthetic biology fields call for rapid and sensitive workflows in the metabolic phenotyping of complex biological samples.OBJECTIVE: The objective of this research was to evaluate a straightforward to implement LC-MS metabolomics method using a commercially available chromatography column that provides increased throughput. Reducing run time can potentially impact chromatography and therefore the effects of ion mobility spectrometry to expand peak capacity were also evaluated. Additional confidence provided via collision cross section measurements for detected features was also explored.
    METHODS: A rapid untargeted metabolomics workflow was developed with broad metabolome coverage, combining zwitterionic-phase hydrophilic interaction chromatography (HILIC-Z) with drift tube ion mobility-quadrupole time-of-flight (DTIM-qTOF) mass spectrometry. The analytical performance of our method was explored using extracts from complex biological samples, including a reproducibility study on chicken serum and a simple comparative study on a bacterial metabolome.
    RESULTS: The method is acronymised RHIMMS for rapid HILIC-Z ion mobility mass spectrometry. We present the RHIMMS workflow starting with data acquisition, followed by data processing and analysis. RHIMMS demonstrates improved chromatographic separation for a selection of metabolites with wide physicochemical properties while maintaining reproducibility at better than 20% over 200 injections at 3.5 min per sample for the selected metabolites, and a mean of 13.9% for the top 50 metabolites by intensity. Additionally, the combination of rapid chromatographic separation with ion mobility allows improved annotation and the ability to distinguish isobaric compounds.
    CONCLUSION: Our results demonstrate RHIMMS to be a rapid, reproducible, sensitive and high-resolution analytical platform that is highly applicable to the untargeted metabolomics analysis of complex samples.
    Keywords:  HILIC; High throughput; Ion mobility; Untargeted metabolomics
  2. Anal Chem. 2022 Mar 04.
      Choosing appropriate data processing parameters is critical in processing liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics data. The conventional design of experiments (DOE) approach is time-consuming and provides no intuitive explanation why the selected parameters generate the best results. After studying commonly used metabolomics data processing software, this work summarized a set of universal parameters, including mass tolerance, peak height, peak width, and instrumental shift. These universal parameters are shared among different feature extraction programs and are critical to metabolic feature extraction. We then developed Paramounter, an R program that automatically measures these universal parameters from raw LC-MS-based metabolomics data prior to metabolic feature extraction. This is made possible through novel concepts of rank-based intensity sorting, zone of interest, and many others. Paramounter also translates universal parameters to software-specific parameters for data processing in different programs. Applying Paramounter is demonstrated to provide a threefold increase in the extracted metabolites compared to using default parameters in MS-DIAL-based feature extraction. Furthermore, the comparison between Paramounter, AutoTuner, and IPO showed that Paramounter generates 3.7- and 1.6-fold more true positive features than AutoTuner and IPO, respectively. Further validation of Paramounter on 11 datasets covering different sample types, data acquisition modes, and MS vendors proved that Paramounter is a convenient and robust program. Overall, the proposed universal parameters and the development of Paramounter address a critical need in metabolomics data processing, transforming metabolomics feature extraction from a "black box" to a "white box." Paramounter is freely available on GitHub (
  3. Rapid Commun Mass Spectrom. 2022 Mar 01. e9280
      RATIONALE: Saturated fatty acids (SFAs) are associated with many diseases in humans. Developing a reliable analytical method to analyze SFAs in plasma is essential to understand their biological activities. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the quantification of medium and long chain saturated fatty acids (M/LCSFAs) in hamster plasma.METHODS: We compared three methods (DOLE, Folch, and MTBE) for extracting M/LCSFAs from plasma. The M/LCSFAs derivatives were separated on a C18 column. The method was validated and applied to analyze M/LCSFAs concentrations in normal-fat diet (NFD) and high-fat diet (HFD) hamster plasma.
    RESULTS: Among the three extraction methods, the DOLE method had the highest extraction recovery and was simple to operate with a short incubation time. All of the calibration curves exhibited good linear relationships (r ≥0.9958). The results for selectivity, accuracy, precision, matrix effects, and recovery were all within the acceptance criteria. In HFD hamster plasma, the concentration of M/LCSFAs with even-carbon was significantly increased.
    CONCLUSIONS: A simple, robust, and reproducible method for the simultaneous quantification of M/LCSFAs by UPLC-MS/MS was developed and validated. The method had successfully quantification of M/LCSFAs in plasma samples from NFD and HFD hamsters.
  4. Anal Chem. 2022 Feb 28.
      Dry-state microsampling techniques are convenient and advantageous for sample collection in resource-limited settings, including healthcare systems designed for the underserved population. In this work, a microsampling platform based on an embossed hydrophobic paper substrate is introduced together with three-dimensional (3D) printed cartridges that offer opportunities for rapid (<30 min) drying of the collected samples while also preserving sample integrity when the embossed paper chip is shipped at room temperature. More importantly, a new pinhole paper spray ionization method was developed that facilitates direct mass spectrometry (MS) analysis of the dried blood samples without prior sample preparation. We compared the direct pinhole paper spray MS method with a liquid chromatographic (LC) MS approach that relied upon electrospray ionization (ESI) after analytes present in the blood sample were extracted through liquid-liquid extraction. Limits of detection as low as 0.12 and 0.49 ng/mL were calculated for cocaine and its metabolite benzoylecgonine, respectively, when using the direct pinhole paper spray MS method. Analytical merits such as precision and accuracy, recovery, carryover effects, and analyte stability were all quantified for this new paper spray method and compared to the traditional LC-ESI-MS. Although LC-ESI-MS was observed to be 10× more sensitive, the linear dynamic range for both methods was determined to be the same, in the range of 1-500 ng/mL for both cocaine and benzoylecgonine analytes. When fully developed, the current microsampling strategy could offer an easy-to-use kit that can enable a more effective MS analysis of 20 μL dried blood samples delivered by mail. Both sensitivity (10×) and sample stability are found to be more superior for blood prepared in the embossed hydrophobic paper compared to samples prepared in the planar hydrophilic paper.
  5. Rapid Commun Mass Spectrom. 2022 Mar 01. e9282
      RATIONALE: In this study, a derivatization-switchable solvent liquid-liquid microextraction-quadruple isotope dilution-gas chromatography-mass spectrometry (D-SS-LLME-ID4 -GC-MS) method was presented for the determination of hydroxychloroquine sulfate in human biofluids.METHODS: While mixing type/period and concentration of NaOH were optimized via univariate optimization approach, multivariate optimization approach was used to determine optimum values for relatively more important parameters such as volumes of derivatization agent (acetic anhydride), NaOH and switchable solvent.
    RESULTS: Under the optimum experimental conditions, limit of detection (LOD) and limit of quantification (LOQ) were calculated as 0.03 mg/kg and 0.09 mg/kg (mass based), respectively. An isotopically labelled material (hydroxychloroquine methyl acetate-d3 ) was firstly synthesized to be used in quadruple isotope dilution (ID4 ) experiments which have highly accurate and precise recovery results. After the application of D-SS-LLME- ID4 , superior percent recovery results were recorded as 99.9 ± 1.6 - 101.3 ± 1.2 for human serum, 99.9 ± 1.7 - 99.8 ± 1.8 for urine and 99.6 ± 1.5 - 101.0 ± 1.1 for saliva samples.
    CONCLUSION: Developed D-SS-LLME-ID4 -GC-MS method compensates the complicated matrix effects of human biofluids and provides highly accurate quantification of the analyte with precise results.
  6. Calcif Tissue Int. 2022 Mar 03.
      Recent research activities have provided new insights in vitamin D metabolism in various conditions. Furthermore, substantial progress has been made in the analysis of vitamin D metabolites and related biomarkers, such as vitamin D binding protein. Liquid chromatography tandem mass spectrometric (LC-MS/MS) methods are capable of accurately measuring multiple vitamin D metabolites in parallel. Nevertheless, only 25(OH)D and the biologically active form 1,25(OH)2D are routinely measured in clinical practice. While 25(OH)D remains the analyte of choice for the diagnosis of vitamin D deficiency, 1,25(OH)2D is only recommended in a few conditions with a dysregulated D metabolism. 24,25(OH)2D, free and bioavailable 25(OH)D, and the vitamin D metabolite ratio (VMR) have shown promising results, but technical pitfalls in their quantification, limited clinical data and the lack of reference values, impede their use in clinical practice. LC-MS/MS is the preferred method for the measurement of all vitamin D related analytes as it offers high sensitivity and specificity. In particular, 25(OH)D and 24,25(OH)2D can accurately be measured with this technology. When interpreted together, they seem to provide a functional measure of vitamin D metabolism beyond the analysis of 25(OH)D alone. The determination of VDBP, free and bioavailable 25(OH)D is compromised by unresolved analytical issues, lacking reference intervals and insufficient clinical data. Therefore, future research activities should focus on analytical standardization and exploration of their clinical value. This review provides an overview on established and new vitamin D related biomarkers including their pathophysiological role, preanalytical and analytical aspects, expected values, indications and influencing conditions.
    Keywords:  Bone health; Chronic kidney disease; Vitamin D; Vitamin D metabolites
  7. Se Pu. 2022 Mar 08. 40(3): 253-265
      An accurate mass database and a method based on ultra high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry (UHPLC-Orbitrap HRMS) were developed. These were applied in the screening and identification of illegally added medicines in herbal tea. Based on investigations, 167 medicines were selected to build an accurate MS database; these medicines included antipyretic analgesics, glucocorticoids, antibiotics, and antihistamines, among other categories. The database was established using Orbitrap HRMS and TraceFinder software. The database carried information on all selected compounds, including the molecular formula, accurate mass of precursor ions and fragment ions, retention time, and mass spectra. The samples were ultrasonically extracted with a 50% (v/v) methanol aqueous solution. The extracted solutions were separated using a Waters XBrigde BEH C18 column (100 mm×2.1 mm, 2.5 μm). As the mobile phases, 0.1% (v/v) formic acid aqueous solution and acetonitrile containing 0.1% (v/v) formic acid were used, with gradient elution. The sample solutions were analyzed by Orbitrap HRMS in the full-scan MS and data-dependent MS/MS acquisition modes (Full MS/dd-MS2). Positive and negative polarity data were simultaneously acquired. Some parameters were optimized to increase the peak intensity and sensitivity of all compounds. The resolutions in the full-MS scan and dd-MS2 scan were set to 70000 and 17500, respectively. In the full-MS mode, scanning was performed in the range of m/z 100 to 1000. In the MS/MS mode, the normalized collision energy (NEC) was set to 20%, 40%, and 60% for each compound. The inclusion list was not used during the measurement, and the dynamic exclusion time was set to 10.0 s. The loop count was set to 5. After acquiring the sample data with these conditions using Orbitrap MS, they were imported into TraceFinder software, through which the sample information was extracted and automatically matched with the information on compounds in the MS database. Screening and identification were conducted by comparing the retention times as well as the exact masses of precursor ions and fragment ions that were experimentally measured. If the errors between the experimentally and theoretically obtained masses of the precursor ions were below 5×10-6 and the deviations in retention times were less than 20 s, then suspicious positive compounds might be identified. Furthermore, if such compounds possess more than one similar fragment ion with a mass tolerance below 5×10-6, and exhibit similar ion distributions in the MS/MS profiles (compared to those in the database), they could be confirmed to be the same. The validation result showed that all compounds had good linear relationships, with correlation coefficients (r) greater than 0.99. Because pefloxacin, norfloxacin, desloratadine, astemizole and clindamycin had background interference, the method was not suitable for their quantification. Following experiments using three spiked concentrations, the recoveries of the rest 162 compounds were found to be in the range of 66.4%-118.1%, and the relative standard deviations (RSDs, n=6), in the range of 0.1%-16.1%. When the limit of detection (LOD) was 0.2 mg/kg, 83 compounds were detected, while when the LOD was 1.0 mg/kg, 167 compounds were detected. All compounds were matched successfully to the standard added sample with the MS database in TraceFinder software. To lower the likelihood of false positive and false negative results, a quality control method was recommended. The method was applied to analyze 245 herbal tea samples, among which 12 positive samples were detected. Thirteen positive compounds were found, including acetaminophen, diclofenac sodium, chlorpheniramine, brompheniramine, dexamethasone, dexamethasone 21-acetate, prednisone, prednisone 21-acetate, metronidazole, erythromycin, ciprofloxacin, amantadine, and dextromethorphan. In particular, amantadine, dextromethorphan, brompheniramine, and ciprofloxacin were newly detected, compared to standard methods. The developed method is rapid and accurate, and will be useful in the high-throughput screening of illegally added medicines in herbal tea.
    Keywords:  electrostatic field orbitrap high-resolution mass spectrometry (Orbitrap HRMS); herbal tea; illegally added medicines; ultra high performance liquid chromatography (UHPLC)
  8. Front Chem. 2022 ;10 808226
      Background: 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) can cause serious toxicity problems in humans and animals, but direct analyses of RDX and HMX in biological samples are very limited. A rapid and efficient liquid chromatography-electrospray quadrupole linear ion trap mass spectrometry (LC-MS/MS) method suitable for the simultaneous determination of RDX and HMX in rat plasma after intravenous administration of two nitramine compound mixed solutions has been developed. Methods: Plasma samples were pretreated with one-step protein precipitation, the plasma consumption is as low as 100 μl. RDX, HMX, and internal standard mycophenolic acid were eluted for 8.0 min on a reversed-phase C18 analytical column with a water/acetonitrile mixture as the mobile phase. An electrospray ionization (ESI) source was applied and operated in negative ion mode. The optimized mass transition ion pairs (m/z) monitored for RDX, HMX, and internal standard mycophenolic acid were m/z 284.1→61.7, m/z 331.0→108.8, and m/z 319.2→191.1, respectively. Results: The detection ranges of both RDX and HMX in plasma were 5.00-200.00 ng⋅ml-1 with an LOD of 1.00 ng⋅ml-1. The extraction recoveries of RDX and HMX were 60.04 ± 4.18% and 79.57 ± 3.35%, respectively. The precision and accuracy met the requirements, and the method was stable under all tested conditions. Conclusion: The present method is miniaturized, effective, portable, rapid and can be easily used for simultaneous quantification of RDX and HMX in rat plasma.
    Keywords:  HMX; LC-MS/MS; RDX; plasma; quantitative analysis
  9. J Nat Prod. 2022 Mar 02.
      The comprehensive chemical characterization of biological samples remains a central challenge in the field of natural products. Conventional workflows using liquid chromatography (LC)-coupled high-resolution tandem mass spectrometry (MS/MS or MS2) allow the detection of relevant small molecules while providing diagnostic fragment ions for their structural assignment. Still, many natural product extracts are of a molecular complexity that challenges the resolving power of modern LC-MS2 pipelines. In this study, we examined the effect of integrating ion mobility spectrometry (IMS) to our LC-MS2 platform for the characterization of natural product mixtures. IMS provides an additional axis of separation in the gas phase as well as experimental collision cross-sectional (CCS) values. We analyzed a mixture of 20 commercial standards at 2 concentration ranges, either solubilized in solvent or spiked into an actinobacterial extract. Data were acquired in positive ion mode using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) MS2 fragmentation approaches and assessed for both chemical coverage and spectral quality. IMS-DIA identified the largest number of standards in the spiked extract at the lower concentration of standards (17), followed by IMS-DDA (10), DDA (8), and DIA (6). In addition, we examined how these data sets performed in the Global Natural Products Social Molecular Networking (GNPS) platform. Overall, integrating IMS increased both metabolite detection and the quality of MS2 spectra, particularly for samples analyzed in DIA mode.
  10. Bioanalysis. 2022 Mar 02.
      Aim: To report the development and validation of an LC-MS/MS method for the simultaneous determination of unconjugated payload DM4 and its metabolite S-methyl-DM4 in human plasma. Methodology: A workflow of protein precipitation followed by reduction and solid phase extraction was employed to remove antibody-maytansinoid conjugates from plasma matrix, release DM4 from endogenous conjugates, and generate a clean sample extract for analysis, respectively. Sodium adduct species of both analytes were selected for multiple reaction monitoring to meet the assay sensitivity requirement in liquid chromatography with tandem mass spectrometry. Conclusion: The method was fully validated for a dynamic range of 0.100-50.0 ng/ml for both analytes along with desired stability and acceptable incurred sample reanalysis.
    Keywords:  LC-MS/MS; S-methyl-DM4; antibody–maytansinoid conjugate; bioanalysis; maytansinoid DM4; protein precipitation; reduction
  11. J Chromatogr A. 2022 Feb 10. pii: S0021-9673(22)00088-7. [Epub ahead of print]1668 462890
      An approach is described for determining if there is an intrinsic advantage, from a selectivity and resolution perspective, of using two different UHPLC/HPLC reversed-phase columns in tandem for a separation of a given sample compared to a single U/HPLC reversed-phase column that provides the same plate number. Retention data for 16 compounds extracted directly from the hydrophobic subtraction model (HSM) database at are used to simulate and then compare the critical resolution of those compounds obtained using HSM conditions (isocratic elution at 35°C using 50% acetonitrile, 50% aqueous phosphate buffer at pH 2.8 or 7) for each of 662 U/HPLC single columns or 218,791 combinations of tandem columns and assuming a modest plate number of 8000. The critical resolution obtained for 16 additional "n-1" samples created by the systematic removal of one of the original 16 compounds was also compared using single- and tandem-column LC, as was the critical resolution obtained for thousands of synthetic samples generated by randomly varying HSM solute descriptors for each synthetic compound. When all possible single-column or tandem-column results were compared, a significant advantage was observed with tandem-column liquid chromatography (TC-LC), with an average increase in critical resolution of 0.63 (pH 2.8) or 0.75 (pH 7) units observed for the synthetic samples with the smallest number of components (m = 5). As the number of components in a sample increased, the average improvement in critical resolution (∆Rs,crit) using TC-LC gradually decreased from about 0.70 for m = 5 to 0.18 for m = 32 components. The average improvement in critical resolution achieved by switching from SC-LC to TC-LC was also lower when a smaller number of columns and column combinations were available to explore, as would be the case for a finite column inventory in a real laboratory. Nevertheless, on average there does appear to be an intrinsic advantage of tandem-column liquid chromatography, however small, which can be amplified by using high efficiency columns.
    Keywords:  Critical resolution; Hydrophobic subtraction model; Selectivity; Serially-coupled columns; Simulated UHPLC/HPLC separations; Tandem-column liquid chromatography
  12. Se Pu. 2022 Mar 08. 40(3): 296-301
      The drug budesonide exists as 22R and 22S enantiomers. However, the drug activity of 22R-budesonide is 2-3 times stronger than that of 22S-budesonide. The development of enantiomeric separation and quantitative analysis methods for budesonide can provide an important basis for its drug development and quality control. At present, the enantiomers of budesonide are separated on a reversed C18 solid phase column. However, chiral stationary phases are rarely reported for the separation of the enantiomers of budesonide. In this study, a high performance liquid chromatography (HPLC) method with a chiral stationary phase was developed for the rapid separation and determination of budesonide enantiomers. The effects of the type of chiral stationary phase, mobile phase additives, and column temperature on the resolution of the budesonide enantiomers were also investigated. The results showed that the chiral stationary phase amylose-tris-[(S)-1-phenylethyl carbamate] was more suitable for the separation of budesonide enantiomers. The mobile phase additives used in the experiment had no significant effect on the chromatographic parameters (peak height, peak width, and resolution) of the budesonide enantiomers. However, with an increase in the column temperature, the peak width of the budesonide enantiomers decreased, while the peak height and resolution increased. The optimized HPLC conditions were as follows: column, Chiralpak AS-RH (150 mm×4.6 mm, 5.0 μm); mobile phase, acetonitrile-water (45∶55, v/v); column temperature, 40 ℃; flow rate, 1.0 mL/min; detector, diode array detector (DAD); detection wavelength, 246 nm; injection volume, 10 μL. The external standard method was used to quantify the budesonide enantiomers. Under the optimized conditions, the enantiomers were well separated, and the retention times of 22R-budesonide and 22S-budesonide were 6.40 min and 7.77 min, respectively. The resolution of the enantiomers was 4.64. The linear ranges of 22R-budesonide and 22S-budesonide were 0.16-1000 μg/mL and 0.20-1000 μg/mL, respectively. The peak area of the enantiomers showed a good linear relationship with the corresponding concentration, and the correlation coefficients (R2) were 0.9999. The limits of detection (LODs) of 22R-budesonide and 22S-budesonide were 0.05 μg/mL and 0.07 μg/mL, respectively, based on a signal-to-noise ratio of 3. The limits of quantification (LOQs) were calculated to be 0.16 μg/mL and 0.20 μg/mL, respectively, based on a signal-to-noise ratio of 10. The recoveries at four spiked levels were in the range of 102.63% to 104.17%, with the relative standard deviations (RSDs) of 0.08% to 0.57% (n=6). The budesonide solution was stored in dark at 4 ℃ for 24 h, and no obvious degradation was observed. Finally, the method was applied to determine four actual samples of budesonide suspension for inhalation in a batch. The samples were dissolved in methanol, filtered through a 0.45 μm microporous membrane, and then analyzed. The amounts of 22R-budesonide and 22S-budesonide in the samples were in the ranges of 283.15-284.63 μg/mL and 259.86-261.51 μg/mL, respectively. This method is simple and rapid, in addition to having good repeatability and high accuracy. It can be used for the resolution of budesonide enantiomers and for quality control in budesonide preparations.
    Keywords:  budesonide; chiral stationary phase; enantiomers; high performance liquid chromatography (HPLC)
  13. Mass Spectrom Rev. 2022 Mar 03.
      Metabolomics is an area of intriguing and growing interest. Since the late 1990s, when the first Omic applications appeared to study metabolite's pool ("metabolome"), to understand new aspects of the global regulation of cellular metabolism in biology, there have been many evolutions. Currently, there are many applications in different fields such as clinical, medical, agricultural and food. In our opinion, it is clear that developments in metabolomics analysis have also been driven by advances in mass spectrometry (MS) technology. As natural complex products (NCPs) are increasingly used around the world as medicines, food supplements, and substance-based medical devices, their analysis using metabolomic approaches will help to bring more and more rigor to scientific studies and industrial production monitoring. This review is intended to emphasize the importance of metabolomics as a powerful tool for studying NCPs, by which significant advantages can be obtained in terms of elucidation of their composition, biological effects and quality control. The different approaches of metabolomic analysis, the main and basic techniques of multivariate statistical analysis are also briefly illustrated, in order to allow an overview of the workflow associated with the metabolomic studies of NCPs. Therefore, various articles and reviews are illustrated and commented as examples of the application of MS-based metabolomics to NCPs. This article is protected by copyright. All rights reserved.
    Keywords:  Mass Spectrometry; Medical Devices; Medicinal plants; Metabolomic
  14. Leg Med (Tokyo). 2022 Feb 19. pii: S1344-6223(22)00035-9. [Epub ahead of print]56 102047
      Direct detection and accurate quantification of chlorine in autopsy samples are difficult because of the volatility and rapid metabolism of chlorine. Here, we developed and validated a method for quantitative analysis of 3-chloro-l-tyrosine (Cl-Tyr) and 3,5-dichloro-l-tyrosine (DiCl-Tyr) as stable markers of chlorine exposure. Chemical derivatization followed by liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) enabled us to simultaneously analyze both Cl-Tyr and DiCl-Tyr in an autopsy sample from the victim of chlorine exposure. Cl-Tyr was detected in the heart blood (53.6 ng/mL), urine (9.5 ng/mL), and lung tissue (211.1 ng/g); however, DiCl-Tyr was detected only in the lung tissue (10.3 ng/g). In contrast, in autopsy samples obtained from cases without exposure to chlorine, DiCl-Tyr was not detected in any matrixes. Our result suggested that the simultaneous detection of Cl-Tyr and DiCl-Ty may provide a better appreciation of chlorine exposure. To our knowledge, this is the first time Cl-Tyr and DiCl-Tyr have been determined simultaneously in a real human autopsy sample from a victim of chlorine exposure.
    Keywords:  3,5-Dichloro-l-tyrosine; 3-Chloro-l-tyrosine; Chlorine exposure; Derivatization; LC/ESI–MS/MS
  15. Anal Chem. 2022 Mar 01.
      The analysis of (trace) contaminants in environmental samples represents an important tool for exposure assessment and for the evaluation of potential risks to human health. Currently, mass spectrometric detection using triple quadrupole (TQMS) systems is the established method of choice. However, screening methods using high resolution mass spectrometry (HRMS) find increasing application as they provide advantages such as enhanced selectivity. A complex composition of environmental samples is known to have enormous effects on mass analyzers. The present work therefore compares the impact of a highly matrix-loaded sample material like house-dust on the performance of mass spectrometric detection of the emerging indoor contaminant group of mycotoxins by quadrupole time-of-flight (QTOF) and TQMS after ultrahigh-performance liquid chromatographic separation. Furthermore, the role of ionization efficiencies of different ion sources in instrument sensitivity was compared using an electrospray ionization source and a newly developed heated electrospray ion source (Bruker VIP-HESI) during QTOF experiments. Finally, it was evaluated whether an additional dimension of separation enables increased sensitivity in QTOF-HRMS detection by applying mycotoxins in house-dust to an (trapped) ion mobility spectrometry instrument. The sensitivity of the QTOF detection was positively influenced by the application of the VIP-HESI ion source, and overall HRMS instruments provided enhanced selectivity resulting in simplified data evaluation compared to the TQMS. However, all performed experiments revealed strong signal suppression due to matrix components. QTOF results showed more severe effects, enabling a more sensitive detection of mycotoxins in house-dust by applying TQMS detection.
  16. Drug Metab Lett. 2022 Mar 02.
      OBJECTIVE: To explore the human in vivo metabolism of SEP-227900 (4H-furo[3, 2-b]pyrrole-carboxylic acid, m.w 151.03), a D-amino-acid oxidase (DAAO) inhibitor by using plasma and urine samples from first-in-human study.METHODS: The human plasma and urine samples were from a single dose cohort that consisted of 9 healthy male volunteers each received 80-mg dose of SEP-227900 orally. The pooled pre-dose urine and the pooled 0-24 h urine sample were created across 9 subjects by equal volume. Plasma samples were pooled by equal volume across 9 subjects to obtain 0-12 h plasma for metabolite searching, and also pooled by timepoints across 9 subjects to obtain 0.5-, 5-, and 12-h plasma for semi-quantitation. The plasma was de-proteinized by acetonitrile (1:3 v/v plasma-acetonitrile) then the supernatant was dried down, reconstituted and injected for LC-HRMS/UV analysis. The urine sample was just simply centrifuged before analysis. LC-HRMS/UV was utilized to search predictable and unknown metabolites and estimate their relative abundances. Accurate mass measurement by Orbitrap-MS and MS/MS were used for metabolite identification. Chromatographic separation was achieved on a MACMOD AQ C8 column (250 × 4.6 mm, 5-µm) with a gradient mobile phase (A: 10 mM NH4Ac; B: acetonitrile; flowrate: 0.700 ml/min) for a total run-time of 65 min. The definite position in the molecule for the glucuronidation metabolism was characterized by detected migration phenomenon, methylation with diazomethane (CH2N2), and NMR.
    RESULTS: Unchanged parent drug and four metabolite peaks were detected in humans: M1 was a mono-oxidative metabolite of SEP-227900; M2 was a glucuronide conjugate of SEP-227900; M3 was a glycine conjugate of SEP-227900; and M4 was a glycine conjugate of M1. The specific position of the oxidation in M1 solely based on the mass spectral (MS and MS/MS) data was not identified. However, for the major metabolite M2, the acyl glucuronidation was unambiguously determined through multiple pieces of experimental evidence such as the observation of a migration pattern, mono-methylation by diazomethane, and NMR measurement. This determination is of significance related to the safety evaluation of an investigational new drug development. The glycine conjugate of SEP-227900, i.e. M3 was found to be the most abundant metabolite in human urine (approximately 3-fold higher level as the glucuronide level). All together (mainly glycine-conjugate and glucuronide), it resulted in greater than 80% of the dosed amount in urine excretion (a separate measurement showed 23% of the dosed amount in urine excretion as the glucuronide).
    CONCLUSION: Four metabolites were found in humans: SEP-227900-glycine conjugate, SEP227900-glucuronide, mono-oxidative metabolite and its consequent glycine conjugate. The glucuronide metabolite was identified as the acyl glucuronide. Greater than 80% of the dosed amount of SEP-227900 was excreted in urine mainly in the forms of glycine- and glucuronide- conjugates.
    Keywords:  D-amino-acid oxidase (DAAO) inhibitor; LC-UV/HRMS; NMR; SEP-227900; acyl glucuronide; drug metabolism
  17. ACS Omega. 2022 Feb 22. 7(7): 6403-6411
      Glycosphingolipids (GSLs), including gangliosides, are essential components of the cell membrane. Because of their vital biological functions, a facile method for the analysis and comparison of GSLs in biological issues is desired. To this end, a new method for GSL analysis was developed based on two-stage matching of the carbohydrate and glycolipid product ions of experimental and reference MS/MS spectra of GSLs. The applicability of this method to the analysis of gangliosides in biological tissues was verified using human plasma and mouse brains spiked with standards. The method was then used to characterize endogenous gangliosides in mouse and human brains. It was shown that each endogenous ganglioside species had varied lipid forms and that mouse and human brains had different compositions of ganglioside species and lipid forms. Moreover, a 36-carbon ceramide is found to represent the major lipid form for mouse brain gangliosides, while the major lipid form for most human brain gangliosides is a 38-carbon ceramide. This study has verified that the two-stage MS/MS spectral matching method could be used to study gangliosides or GSLs and their lipid forms in complex biological samples, thereby having a broad application.
  18. J Sep Sci. 2022 Mar 02.
      Tolperisone and etodolac were proven to have synergistic effect for patients of acute low back pain associated with musculoskeletal spasm. In this work, a specific, highly sensitive and reproducible analytical method was developed and validated for the simultaneous determination of tolperisone and etodolac in human plasma using liquid chromatography-tandem mass spectrometric technique. Liquid-liquid extraction was optimized for sample preparation. Zorbax C8 column (3.5 μm, 50 × 4.6 mm) was used, carrying a mobile phase mixture of 10.0 mM ammonium formate: acetonitrile (40:60, v/v) pH 3.8, running in an isocratic mode. Chlorzoxazone acted as an internal standard. Sample volume of injection was 5.0 μL, analysis was achieved within 2.5 min. Detection and quantitation were performed by electrospray ionization mass spectrometry using the multiple-reaction monitoring mode. The proposed method could determine the analytes in the range of concentration 0.5 - 200.0 ng mL-1 for tolperisone and 0.05 - 20.0 μg mL-1 for etodolac. Findings of inter- and intra-day precisions were ≤ 12.3% with accuracy of ± 5.0%. Pharmacokinetics study for the two drugs after oral administration of healthy human volunteers was achieved with the aid of application of the developed study. This article is protected by copyright. All rights reserved.
    Keywords:  Electrospray ionization; Etodolac; Liquid chromatography; Pharmacokinetics; Tolperisone
  19. Anal Chem. 2022 Mar 02.
      Mass spectrometry (MS) allows for automated analysis of complex samples at high resolution without the need for labeling/derivatization. Liquid atmospheric pressure matrix-assisted laser desorption/ionization (LAP-MALDI) enables rapid sample preparation and MS analysis using microtiter-plate formats and high-performing mass spectrometers. We present a step change in high-speed, large-scale MS sample analysis of peptides at 20 samples/s and an enzymatic assay at 40 samples/s, i.e., an order of magnitude faster than current MS platforms. LAP-MALDI requires only low amounts of sample volume (<2 μL), of which only a fraction (<1%) is typically consumed, and allows for multiplexing and high-speed MS/MS analysis, demonstrated at ∼10 samples/s. Its high ion signal stability and similarity to electrospray ionization enables CVs below 10% and the analysis of multiply charged peptide ions at these extreme speeds. LAP-MALDI MS fulfills the speed requirements for large-scale population diagnostics and compound screening with the potential of analyzing >1 million samples per day.
  20. Se Pu. 2022 Mar 08. 40(3): 273-280
      Glyphosate (GLY) and glufosinate (GLUF) are non-selective translocated herbicides that are used in agricultural and non-agricultural land worldwide. The extensive use of GLY and GLUF may lead to their accumulation in soil, which causes soil pollution and affects the soil micro-ecological environment; the accumulated GLY and GLUF also migrate to groundwater via leaching. However, GLY, GLUF, and their metabolites are highly water-soluble and lack chromogenic and fluorescent groups, making them difficult to analyze. Currently, derivatization methods are mostly used to detect GLY, GLUF, and their metabolites. However, these methods also have some drawbacks, such as complex operation, long time consumption, and poor stability. In addition, these compounds are easily passivated and made inactive in soil; they also react with organic matter, humic acid, metal oxides, and heavy metal ions, making their extraction from soil difficult. To date, the method for the determination of GLY, GLUF, and their metabolites in soil is limited. Therefore, it is necessary to establish a quick and sensitive method to determine the residues of GLY, GLUF, and their metabolites in soil. In this study, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of GLY, GLUF, and their metabolites in soil. Parameters like extraction solvent, extraction temperature, extraction time, and adsorbents, which affected the extraction efficiencies, were optimized. Finally, the soil samples were extracted with 0.5 mol/L ammonia solution in a bath shaker at 50 ℃, and then centrifuged at 10000 r/min for 5 min. The supernatant was filtered through 0.2-μm syringe filters and then determined by HPLC-MS/MS. A Dikma Polyamino HILIC column (150 mm×2.0 mm, 5 μm) was used for chromatographic separation with good peak shape and high response of the target compounds. Ammonium acetate (0.2 mmol/L) with 0.07% ammonia aqueous solution and acetonitrile were used as the mobile phase. The flow rate of the elute was 0.25 mL/min. MS/MS was conducted under multiple reaction monitoring (MRM) mode using an electrospray ionization (ESI) source, and was quantified by the external standard method using matrix-matched calibration curves. All the target compounds were ionized in the negative ionization mode. The linear ranges of GLY and its metabolites were between 5.0 and 500 μg/L, and those of GLUF and its metabolites were between 2.0 and 500 μg/L. Linear correlation coefficients were greater than 0.99. The limit of detection (LOD) and limit of quantification (LOQ) were assessed using signal-to-noise (S/N) ratios of 3 and 10, respectively. The LOD and LOQ values of both GLY and (aminomethyl)phosphonic acid (AMPA) were 4.0 and 13.3 μg/kg, respectively. The LOD and LOQ values of GLUF, MPP, and N-acetyl glufosinate (NAG) were 2.0 and 6.7 μg/kg, respectively. Method accuracy was acquired by recovery test at three spiked levels (0.02, 0.05, 0.2 mg/kg). The average recoveries of five targets spiked in soil with low organic matter content were 74.2%-101%, and the relative standard deviation (RSD) was 0.93%-6.8%; the average recoveries of the five targets spiked in soil with high organic matter content were 90.8%-116%, and the RSD was 0.40%-7.1%. The established method was used to determine 20 soil samples in peach orchard, and the detection rates of AMPA, GLY, MPP, GLUF and NAG were 45%, 25%, 10%, 5% and 5%, respectively. The maximum residues were 147, 35.2, 154, 21.6 and 11.0 μg/kg, respectively. This method is simple, rapid, green, inexpensive, allows pretreatment without organic reagents, and affords high accuracy, high sensitivity, and good reproducibility. The method is suitable for testing a large number of soil samples with different organic matter contents. It can provide reliable technical support for the study of residue status and environmental behavior in soil.
    Keywords:  glufosinate; glyphosate; high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS); metabolites; non-derivatization; soil
  21. Environ Res. 2022 Feb 28. pii: S0013-9351(22)00380-2. [Epub ahead of print] 113053
      Environmental pollutants (EPOLs), such as phthalates, volatile organic compounds, phenols, parabens, polycyclic aromatic hydrocarbons, pyrethroids, and environmental tobacco smoke, are highly heterogeneous compounds. Recently, attention has been drawn to the assessment of the combinatory effects of multiple EPs. To correlate multiple exposures with potential health implications, advanced comprehensive analytical methods covering multiclass EPOLs are essential. However, because of several technical problems associated with enzyme hydrolysis, simultaneous extraction, and multiresidue liquid chromatography-tandem mass spectrometry analysis, it is difficult to establish a comprehensive method covering a number of EPOLs in a single sample preparation and analytical run. We developed tandem hybrid hydrolysis, modified direct injection, and a comprehensive mobile phase to overcome these technical problems and established a comprehensive analytical method for simultaneous biomonitoring of multiclass EPOLs. Tandem hybrid hydrolysis using β-glucuronidase and consecutive acid hydrolysis allowed selective hydrolysis of glucuronide- and sulfate-conjugated metabolites without phthalate degradation. The comprehensive mobile phase composed of 0.01% acetic acid and acetonitrile enabled us to simultaneously analyze 86 EPOLs, with good chromatographic behavior and ionization efficiency. Modified direct injection allowed a small amount of sample and simultaneous urinary extraction. The method was validated and applied to 39 urine samples from 19 mother-newborn pairs for multiple exposure assessment. Results showed that BP-3, a general component in sunblock products, and monoethyl phthalate, a metabolite of diethyl phthalate, exhibit a clear positive correlation between mothers and newborns. Therefore, the developed method has potential as a novel analytical tool for long-term, large-scale, and data-rich human biomonitoring of EPOLs.
    Keywords:  Comprehensive mobile phase; Environmental pollutants (EPOLs); Liquid chromatography-mass spectrometry (LC-MS); Tandem hybrid hydrolysis
  22. J Pharm Biomed Anal. 2022 Feb 22. pii: S0731-7085(22)00108-X. [Epub ahead of print]213 114687
      Sorafenib (SOR) is a multikinase inhibitor with a mild activity against colorectal cancer cells due to multi-drug resistance mechanisms. Potentiated SOR activity was expected upon combination with some ginger derived compounds due to their interference with intracellular drug metabolism. Studying such combination necessitates the development of a sensitive validated LC-MS/MS method for the determination of intra and extracellular concentration of SOR and its N-oxide metabolite (SNX) in colorectal cancer cells. SOR, SNX and the internal standard (diclofenac sodium) were efficiently separated on Eclipse plus C18 column (3.0 ×150 mm, 5 µm) using isocratic elution with acetonitrile and 0.01 M ammonium formate aqueous solution containing 0.1% formic acid (69:31, v/v). Sample pretreatment using solid phase extraction was optimized and the mean percent recoveries were more than 97.01% for both analytes. Detection was conducted at positive ion multiple reaction monitoring (MRM) mode and the monitored mass transitions were 465.2 → 252.2 for SOR and 481.1 → 286.0 for SNX. The method was linear over the range 0.25 - 200.00 ng/mL (r2 ≥ 0.9992) for SOR and 0.10 - 125.00 ng/mL (r2 ≥ 0.9990) for SNX in both intra and extracellular matrices. The lower limits of quantification (LLOQ) were 0.25 and 0.10 ng/mL for SOR and SNX, respectively. Accuracies were within 94.25 - 109.45% and precision CV values did not exceed 7.63%. The method was able to monitor the cellular uptake and entrapment of both analytes and to prove the positive effect of the ginger derived compounds on SOR activity.
    Keywords:  Cell line; LC-MS/MS; Solid phase extraction; Sorafenib; Sorafenib N-oxide
  23. Drug Metab Dispos. 2022 Mar 03. pii: DMD-AR-2022-000832. [Epub ahead of print]
      Glucuronidation is the most common phase II metabolic pathway to eliminate small molecule drugs from the body. However, determination of glucuronide structure is quite challenging by mass spectrometry due to its inability to generate structure informative fragments about the site of glucuronidation. Herein we described a simple method to differentiate acyl-, O-, and N-glucuronides using chemical derivatization. The idea is that derivatization of acyl-, O- or N-glucuronide of a molecule results in predictable and different numbers of derivatized functional groups, which can be determined by the mass shift using mass spectrometry. The following two reactions were applied to specifically derivatize carboxyl and hydroxyl groups that are present on the aglycone and its glucuronide metabolite. Carboxyl groups were activated by thionyl chloride followed by esterification with ethanol. Hydroxyl groups were derivatized via silylation by 1-(trimethylsilyl)imidazole. The mass shift per derivatized carboxyl and hydroxyl group was +28.031 Da and +72.040 Da, respectively. This approach was successfully validated using commercial glucuronide standards including benazepril acyl-glucuronide, raloxifene O-glucuronides, and silodosin O-glucuronide. In addition, this approach was applied to determine the type of glucuronide metabolites that were isolated from liver microsomal incubation, where alvimopan and diclofenac acyl-glucuronides, darunavir, haloperidol, and propranolol O-glucuronides, and darunavir N-glucuronide were identified. Lastly, this approach was successfully utilized to elucidate the definitive structure of a clinically observed metabolite, soticlestat O-glucuronide. In conclusion, a novel efficient and cost-effective approach was developed to determine acyl-, O-, and N-glucuronide using chemical derivatization coupled with liquid chromatography-high resolution mass spectrometry. Significance Statement The method described in this study can differentiate acyl-, O-, and N-glucuronides and allow for elucidation of glucuronide structures when multiple possibilities of glucuronidation exist. The type of glucuronidation information is particularly useful for a drug candidate containing carboxyl group(s), which can form reactive acyl-glucuronides. Additionally, the method can potentially be used for the definitive structure elucidation for a compound containing a single carboxyl, hydroxyl, or amino group even when multiple types of functional groups are present for glucuronidation.
    Keywords:  Glucuronidation; High performance liquid chromatography (HPLC); Mass spectrometry (MS); Structure determinations
  24. ACS Cent Sci. 2022 Feb 23. 8(2): 223-234
      Few tools exist in natural products discovery to integrate biological screening and untargeted mass spectrometry data at the library scale. Previously, we reported Compound Activity Mapping as a strategy for predicting compound bioactivity profiles directly from primary screening results on extract libraries. We now present NP Analyst, an open online platform for Compound Activity Mapping that accepts bioassay data of almost any type, and is compatible with mass spectrometry data from major instrument manufacturers via the mzML format. In addition, NP Analyst will accept processed mass spectrometry data from the MZmine 2 and GNPS open-source platforms, making it a versatile tool for integration with existing discovery workflows. We demonstrate the utility of this new tool for both the dereplication of known compounds and the discovery of novel bioactive natural products using a challenging low-resolution antimicrobial bioassay data set. This new platform is available at
  25. Appl Biochem Biotechnol. 2022 Mar 02.
      Betel leaf is consumed as a mouth freshener due to its characteristic flavor, aromaticity, and medicinal values. Abundance of phytochemicals in betel leaf contributes towards unique qualitative features. Screening of metabolites is quintessential for identifying flavoring betel leaves and their origin. Metabolomics presently lays emphasis on the cumulative application of gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopic approaches. Here we adopted different protocols based on the above-mentioned analytical metabolomics platform for untargeted plant metabolite profiling followed by multivariate analysis methods and a phytochemical characterization of Piper betel leaf cultivars endemic to coastal Odisha, India. Based on variation in the solvent composition, concentration of solvent, extraction temperature, and incubation periods, five extraction methods were followed in GC-MS and NMR spectroscopy of betel leaf extracts. Phytochemical similarities and differences among the species were characterized through multivariate analysis approaches. Principal component analysis, based on the relative abundance of phytochemicals, indicated that the betel cultivars could be grouped into three groups. Our results of FTIR-, GC-MS-, and NMR-based profiling combined with multivariate analyses suggest that untargeted metabolomics can play a crucial role in documenting metabolic signatures of endemic betel leaf varieties.
    Keywords:  FTIR; Flavor; GCMS; Metabolites; NMR; Piper betle; Variations
  26. Anal Chem. 2022 Mar 02.
      Sensitive analysis of metabolites in a single cell is of fundamental significance for the better understanding of biological variability, differential susceptibility in disease therapy, and cell-to-cell heterogeneity as well. Herein, polarity-specific profiling of metabolites in a single cell was implemented by probe electrophoresis mass spectrometry (PEMS), which combined electrophoresis sampling of metabolites from a single cell and nanoelectrospray ionization-mass spectrometry (nanoESI-MS) analysis of the sampled metabolites. Enhanced extraction of either negatively or positively charged metabolites from a single cell was achieved by applying a DC voltage offset of +2.0 and -2.0 V to the probe, respectively. The experimental data demonstrated that PEMS features high throughput (≥200 peaks) and high sensitivity (≥10-times signal enhancement for [Choline + H]+, [Glutamine + H]+, [Arginine + H]+, etc.) in comparison with direct nanoESI-MS analysis. The biological effects of CdSe quantum dots (QDs) and γ-radiation on Allium cepa cells were investigated by PEMS, which revealed that CdSe QDs lead to the increase of intracellular amines while γ-radiation causes the decrease of intracellular acids. Therefore, this work provides an alternative platform to probe novel insights of cells by sensitive analysis of polarity-specific metabolites in a single cell.
  27. Anal Chem. 2022 Mar 03.
      The structural diversity of phospholipids plays a critical role in cellular membrane dynamics, energy storage, and cellular signaling. Despite its importance, the extent of this diversity has only recently come into focus, largely owing to advances in separation science and mass spectrometry methodology and instrumentation. Characterization of glycerophospholipid (GP) isomers differing only in their acyl chain configurations and locations of carbon-carbon double bonds (C═C) remains challenging due to the need for both effective separation of isomers and advanced tandem mass spectrometry (MS/MS) technologies capable of double-bond localization. Drift tube ion mobility spectrometry (DTIMS) coupled with MS can provide both fast separation and accurate determination of collision cross section (CCS) of molecules but typically lacks the resolving power needed to separate phospholipid isomers. Ultraviolet photodissociation (UVPD) can provide unambiguous double-bond localization but is challenging to implement on the timescales of modern commercial drift tube time-of-flight mass spectrometers. Here, we present a novel method for coupling DTIMS with a UVPD-enabled Orbitrap mass spectrometer using absorption mode Fourier transform multiplexing that affords simultaneous localization of double bonds and accurate CCS measurements even when isomers cannot be fully resolved in the mobility dimension. This method is demonstrated on two- and three-component mixtures and shown to provide CCS measurements that differ from those obtained by individual analysis of each component by less than 1%.
  28. Drug Discov Today. 2022 Feb 24. pii: S1359-6446(22)00076-9. [Epub ahead of print]
      The pharmaceutical industry adapted proteomics and other 'omics technologies for drug research early following their initial introduction. Although metabolomics lacked behind in this development, it has now become an accepted and widely applied approach in early drug development. Over the past few decades, metabolomics has evolved from a pure exploratory tool to a more mature and quantitative biochemical technology. Several metabolomics-based platforms are now applied during the early phases of drug discovery. Metabolomics analysis assists in the definition of the physiological response and target engagement (TE) markers as well as elucidation of the mode of action (MoA) of drug candidates under investigation. In this review, we highlight recent examples and novel developments of metabolomics analyses applied during early drug development.
    Keywords:  Drug discovery; Flux; LC-MS; Lipidomics; Metabolomic; NMR; Target
  29. Anal Bioanal Chem. 2022 Mar 02.
      This work describes the development of an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination of 23 primary aromatic amines (PAAs) that can potentially migrate from food contact materials. The chromatographic separation was performed in a pentafluorophenylpropyl (PFPP) column achieving the separation of all PAAs in less than 6.5 min using water to acetonitrile (0.1% acetic acid in both solvents) as mobile phase and a gradient elution. The feasibility of atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) was evaluated as alternative to electrospray ionization (ESI) for the analysis of PAAs. Results showed that for most of the compounds, better responses were obtained with APCI, which shows the advantage of being less susceptible to matrix effects. Tandem mass spectrometry (MS/MS) fragmentation studies of [M + H]+ allowed for the selection of the two most characteristic and abundant product ions of the 23 PAAs which led to the development of a selective and sensitive UHPLC-APCI-MS/MS method with limits of detection ranging from 0.2 to 2 μg kg-1. Moreover, intra-day and inter-day precisions of the method in terms of relative standard deviation (RSD%) were lower than 10% and 15%, while trueness as relative error was <15% for most of the compounds. The UHPLC-APCI-MS/MS method was applied to the analysis of twenty black Nylon kitchenware samples that were submitted to migration tests using food simulant B (3% acetic acid, w/v), and the presence of PAAs were detected in eighteen samples at concentrations above the legislated limit (2 μg kg-1 of food or food simulants).
    Keywords:  Atmospheric pressure chemical ionization; Atmospheric pressure ionization; Food contact materials; Liquid chromatography-tandem mass spectrometry; Pentafluorophenylpropyl column; Primary aromatic amines
  30. Rapid Commun Mass Spectrom. 2022 Mar 01. e9284
      RATIONALE: The oil crops and products are important food materials in daily life. Pesticide residues in food could directly and indirectly endanger human health. However, the method for detecting multiple pesticides simultaneously is limited. In this study, an easy and efficient method for simultaneously determination of 38 pesticides in oil crops and products was established and validated.METHODS: All samples were treated with modified QuEChERS procedure followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. Mass spectrometry was performed in positive and negative ion electrospray ionization mode. The mobile phase consisted of 0.1% formic acid in water and 0.1% formic acid in MeCN. The column used was Poroshell 120 EC-C18 and the flow rate was 0.3 mL/min.
    RESULTS: The method was validated that the calibration curves for all pesticides were had good linearity in the concentration range of 10-1000 μg/L with correlation coefficients (R2 ) above 0.9945. The recovery rates were between 70.1 and 120.0%, with RSDs (n=6) ≤20.0%. The limits of quantification (LOQ) ranged from 0.5 to 10 μg/kg, limits of detection (LOD) ranged from 2.0 to 30 μg/kg, and the matrix effect (ME) ranged from -18.77 to 19.33%, respectively.
    CONCLUSIONS: The method proved to be accurate, sensitive, and stable. It can be used for rapid screening and confirmation of 38 pesticide residues in oil crops and products which takes 10 minutes in sample extraction and clean-up with less requirement of solvents. This study provides a technical basis for regulatory analysis and quality supervision of the food.
  31. BMC Cancer. 2022 Feb 28. 22(1): 214
      Bladder cancer (BC) is one of the most frequent cancer in the world, and its incidence is rising worldwide, especially in developed countries. Urine metabolomics is a powerful approach to discover potential biomarkers for cancer diagnosis. In this study, we applied an ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) method to profile the metabolites in urine from 29 bladder cancer patients and 15 healthy controls. The differential metabolites were extracted and analyzed by univariate and multivariate analysis methods. Together, 19 metabolites were discovered as differently expressed biomarkers in the two groups, which mainly related to the pathways of phenylacetate metabolism, propanoate metabolism, fatty acid metabolism, pyruvate metabolism, arginine and proline metabolism, glycine and serine metabolism, and bile acid biosynthesis. In addition, a subset of 11 metabolites of those 19 ones were further filtered as potential biomarkers for BC diagnosis by using logistic regression model. The results revealed that the area under the curve (AUC) value, sensitivity and specificity of receiving operator characteristic (ROC) curve were 0.983, 95.3% and 100%, respectively, indicating an excellent discrimination power for BC patients from healthy controls. It was the first time to reveal the potential diagnostic markers of BC by metabolomics, and this will provide a new sight for exploring the biomarkers of the other disease in the future work.
    Keywords:  Bladder cancer; Diagnosis; Potential biomarker; UPLC-MS; Urinary metabolomics
  32. Environ Int. 2022 Feb 26. pii: S0160-4120(22)00085-X. [Epub ahead of print]162 107159
      OBJECTIVE: To summarize the application of non-targeted metabolomics in epidemiological studies that assessed metabolite and metabolic pathway alterations associated with per- and polyfluoroalkyl substances (PFAS) exposure.RECENT FINDINGS: Eleven human studies published before April 1st, 2021 were identified through database searches (PubMed, Dimensions, Web of Science Core Collection, Embase, Scopus), and citation chaining (Citationchaser). The sample sizes of these studies ranged from 40 to 965, involving children and adolescents (n = 3), non-pregnant adults (n = 5), or pregnant women (n = 3). High-resolution liquid chromatography-mass spectrometry was the primary analytical platform to measure both PFAS and metabolome. PFAS were measured in either plasma (n = 6) or serum (n = 5), while metabolomic profiles were assessed using plasma (n = 6), serum (n = 4), or urine (n = 1). Four types of PFAS (perfluorooctane sulfonate(n = 11), perfluorooctanoic acid (n = 10), perfluorohexane sulfonate (n = 9), perfluorononanoic acid (n = 5)) and PFAS mixtures (n = 7) were the most studied. We found that alterations to tryptophan metabolism and the urea cycle were most reported PFAS-associated metabolomic signatures. Numerous lipid metabolites were also suggested to be associated with PFAS exposure, especially key metabolites in glycerophospholipid metabolism which is critical for biological membrane functions, and fatty acids and carnitines which are relevant to the energy supply pathway of fatty acid oxidation. Other important metabolome changes reported included the tricarboxylic acid (TCA) cycle regarding energy generation, and purine and pyrimidine metabolism in cellular energy systems.
    CONCLUSIONS: There is growing interest in using non-targeted metabolomics to study the human physiological changes associated with PFAS exposure. Multiple PFAS were reported to be associated with alterations in amino acid and lipid metabolism, but these results are driven by one predominant type of pathway analysis thus require further confirmation. Standardizing research methods and reporting are recommended to facilitate result comparison. Future studies should consider potential differences in study methodology, use of prospective design, and influence from confounding bias and measurement errors.
    Keywords:  Exposome; Metabolomics; Perfluorinated compounds; Persistent organic pollutants; Polyfluoroalkyl substances
  33. J Environ Sci Health B. 2022 Mar 03. 1-8
      Rapid, sensitive and selective method was studied for the determination of glyphosate in rice and wheat flour using a Raptor Polar column. Samples were extracted with acidified methanol solution. Isotope labeled internal standard was added with the extraction solvent to ensure accurate tracking and quantitation. The glyphosate was analyzed by liquid chromatography tandem mass spectrometry using a Raptor Polar column. Method performance was evaluated through a series of parameters that include accuracy, precision, linearity, limit of detection (LOD), limit of quantitation (LOQ) and matrix effect. Mean recoveries for all matrices were within 96-116% at three fortification levels. Precision for replicates was ≤19% relative standard deviation (RSD%) for wheat and ≤9% RSD% for rice across all fortification levels. Linearity was good for both matrices with a correlation coefficient >0.999 and residual <20% in the concentration range 0.025-1 mg kg-1. LOD was 0.005 mg kg-1for both rice and wheat flour and the LOQ was 0.05 mg kg-1for both matrices. The matrix effect was also studied. A successful method for the analysis of glyphosate in wheat and rice flour assure that the criteria of acceptability of the maximum residue levels (MRLs) are insured.
    Keywords:  Herbicide; glyphosate; liquid chromatography; mass spectrometry; meal; polar column
  34. Nucleic Acid Ther. 2022 Mar 02.
      The relatively large molecular size, diastereoisomeric nature, and complex impurity profiles of therapeutic phosphorothioate oligonucleotides create significant analytical challenges for the quality control laboratory. To overcome the lack of selectivity inherent to traditional chromatographic approaches, an ion pair liquid chromatography-mass spectrometry (LCMS) method combining ultraviolet and mass spectrometry quantification was developed and validated for >35 different oligonucleotide drug substances and products, including several commercialized drugs. The selection of chromatographic and spectrometric conditions, data acquisition and processing, critical aspects of sample and buffer preparation and instrument maintenance, and results from method validation experiments are discussed.
    Keywords:  LCMS; ion pair; oligonucleotide; quality control; validation
  35. J AOAC Int. 2022 Mar 03. pii: qsac026. [Epub ahead of print]
      BACKGROUND: Gas chromatography-mass spectrometry (GC-MS) is a powerful tool for component analysis of unknown compounds, especially in the fields of analytical chemistry or detection of biological samples. To effectively identify compounds in GC-MS, one of the most important ways is to use a matching algorithm to compare the similarity between the reference spectrum and the query spectrum.OBJECTIVE: To propose a novel way to improve compound accuracy.
    METHOD: This article proposes a method based on m/z set match. First of all, select the maximum m/z and the m/z corresponding to the highest peak intensity for pre-search. Next, employ the space vector model to carry on a refined search in the remaining spectra after pre-search. Then, distinguish stereoisomers according to the order of G value.
    RESULTS: Compared with the Ten peaks and the method based on m/z number matching pre-search, the method based on m/z set matching showed higher accuracy and fewer remaining (missing) spectra. Furthermore, the refined search which based on m/z set matching method possesses shorter calculation time compared with no pre-search.
    HIGHLIGHTS: A method based on m/z set match reduces the remaining spectra and speeds up the identification of compounds.
    Keywords:  m/z set matching algorithm; mass spectrometry; refined search; space vector
  36. Anal Bioanal Chem. 2022 Feb 27.
      Due to the absence of chromatographic separation, ambient ionization mass spectrometry had the potential to improve the throughput of control laboratories in the last decades and will soon be an excellent approach for on-site use as well. In this study, an atmospheric solids analysis probe (ASAP) with a single quadrupole mass analyzer has been evaluated to identify anabolic steroid esters rapidly. Sample introduction, applied scan time, and probe temperature were optimized for sensitivity. The in-source fragmentations of seventeen selected steroid esters, commonly found in illicit samples, were determined by applying different cone voltages (12, 20, 30, and 40 V). A spectral library was created for these steroid esters based on the four stages of in-source fragmentation spectra. The applicability of this method was demonstrated for the rapid identification of steroid esters in oily injection solutions, providing test results in less than 2 min.
    Keywords:  Ambient ionization; Atmospheric solids analysis probe; Mass spectrometry; On-site testing; Transportable MS
  37. Se Pu. 2022 Mar 08. 40(3): 281-288
      Understanding the diffusion behavior of particles during chromatographic analysis is critical for optimizing the operation conditions, improving the chromatographic performance, and designing a new separation device. Most of the existing simulations focus on chromatographic thermodynamics, while very few consider the overall diffusion and separation process. Herein, a new simulation method for gas chromatography separation was developed based on the random diffusion theory in microscale restricted space. This method retained the key information for controlling the diffusion behavior, simplified the interaction between the particles to be separated, and enlarged the time scale of each step one molecule walks. Thus, the operational efficiency could be significantly improved due to reduced calculation, and the entire diffusion process in the gas chromatography capillary column could be simulated. In this model, the capillary column was represented by a two-dimensional long and narrow rectangle where the particles to be separated randomly diffused. Besides, a directional velocity along the axis of the chromatographic column exerted on the particle represented the driving force of the mobile phase. If a particle collided with the inner wall of the column, it would remain at the collision position even after some time lapsed. When desorption occured, the particle would flow along with the mobile phase until its next adsorption on the surface. The interaction between the particle and the inner wall was expressed by the equivalent adsorption time. By dynamically tracking the trajectories of particles, the statistical distribution of time for the residence of the particles in the chromatographic column could be obtained, that is, the detection signal (retention time). Based on the previous simulation studies on the separation of n-alkane homologues, combined with the Kovats Retention Index, the functional relationships between adsorption steps and temperature as well as carbon number were established. As a result, the separation parameter systems for various homologues at different temperatures were set up. The separation of alcohol homologues at different temperatures was considered as an example to verify the reliability of the simulation method. The relative errors between the measured and simulated values were within 5% for the retention time and 0.75%-60% for the peak width. The reasons for the large relative errors in the peak width are summarized as follows. On the one hand, parameterization of alcohol homologues was performed on the basis of a previous study on the separation law of n-alkane. Given the limitations of the current computing capability, the insufficient iteration in the parameterized process led to large errors. In addition, the errors at different temperatures further accumulated in extrapolated approximations. On the other hand, the strong hydrogen bond force between the alcohol molecules was simplified with the elastic collision, which increased the magnitude of the errors. Although the simulation method proposed in this paper can accurately predict the retention time and reasonably describe the morphological characteristics of chromatographic peaks, there is still room for improvement. In particular, the description of the detailed interactions between molecules must be improved. For example, the method of molecular mechanics may be assistant with the investigation of the functional relationship between interaction potential and adsorption steps. The interaction potential calculated on the basis of molecular mechanics replaces the parameterized adsorption steps, yielding more accurate simulation results. In general, the simulation method proposed in this study is a valuable reference for the optimization of chromatographic operating conditions and for the development of new chromatographic techniques.
    Keywords:  diffusion; gas chromatography (GC); random simulation; separation
  38. Anal Bioanal Chem. 2022 Mar 02.
      Liquid chromatography tandem mass spectrometry (LC-MS/MS) is used routinely in clinical diagnostics; however, automating the sample pretreatment is challenging. We established and evaluated an automated method based on the magnetic bead extraction principle (MBE) to measure normetanephrine (NMN), metanephrine (MN), and 3-methoxytyramine (3-MT). The target analytes were extracted, purified, and concentrated using different solvents and chemical bond-modified magnetic beads transferred via a magnetic bar. The linearity, recovery, matrix effect, and precision of MBE were evaluated thoroughly, and compared with traditional solid-phase extraction (SPE) using 131 plasma samples. The chromatography peaks of metanephrines and 3-MT, extracted via MBE, are symmetrical, without interfering peaks. The linearity was excellent with correlation coefficient (r) > 0.99. The MBE exhibited good reproducibility with within-run coefficient variations (CVs) of 1.96-2.00%, 4.06-5.75%, and 3.89-4.90% for MN, NMN, and 3-MT, respectively. The total CVs for MN, NMN, and 3-MT were 1.96-2.80%, 5.12-5.75%, and 5.44-6.27%, respectively. The relative recoveries for MN, NMN, and 3-MT varied between 93.5 and 107.4%, whereas their biases were all within 10%. The results for MN, NMN, and 3-MT extracted via MBE compared with SPE exhibited excellent correlation, with r > 0.99; the mean bias% for MN, NMN, and 3-MT were small (-2.9%, -3.2%, and -3.2%, respectively). In conclusion, the automated MBE method for measuring plasma metanephrines and 3-MT can be applied in future routine clinical diagnostics, and the MBE principle may indicate a new era for LC-MS/MS in clinical application.
    Keywords:  Catecholamines; LC/MS; Magnetic bead extraction; Plasma; Sample preparation