bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒07‒10
28 papers selected by
Sofia Costa

  1. Bioinformatics. 2022 Jul 04. pii: btac441. [Epub ahead of print]
      SUMMARY: We present MobilityTransformR, an R/Bioconductor package for the effective mobility scaling of capillary zone electrophoresis-mass spectrometry (CE-MS) data. It uses functionality from different R packages that are frequently used for data processing and analysis in MS-based metabolomics workflows, allowing the subsequent use of reproducible transformed CE-MS data in existing workflows.AVAILABILITY AND IMPLEMENTATION: MobilityTransformR is implemented in R (Version > = 4.2) and can be downloaded directly from the Bioconductor database ( or GitHub (
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
  2. J Agric Food Chem. 2022 Jul 05.
      Metabolome analysis of high-salt fermented food can be an analytical challenge, as the salts can interfere with the sample processing and analysis. In this work, we describe a four-channel chemical isotope labeling (CIL) LC-MS approach for a comprehensive metabolome analysis of high-salt fermented food. The workflow includes metabolite extraction, chemical labeling of metabolites using dansyl chloride, dansylhydrazine, or p-dimethylaminophenacyl bromide reagents to enhance separation and ionization, LC-UV measurement of the total concentration of dansyl-labeled metabolites in each sample for sample normalization, mixing of 13C- and 12C-reagent-labeled samples, high-resolution LC-MS analysis, and data processing. Metabolome analysis of fermented foods, including fermented red pepper (FRP) sauce, soy sauce, and sufu (a fermented soybean food), showed unprecedented high metabolic coverage. Metabolome comparison of FRP, soy sauce, and sufu, as well as soy sauce and sufu, indicated great diversity of metabolite types and abundances in these foods. In addition, we analyzed two groups of samples of the same type, FRP with 10% (w/w) and 15% (w/w) salt contents, and detected large variations in multiple categories of metabolites belonging to a number of different metabolic pathways. We envisage that this CIL LC-MS approach can be generally used for metabolomic studies of high-salt fermented food. CIL LC-MS allows high-coverage identification and quantification that could not be done using other methods.
    Keywords:  LC−MS; chemical isotope labeling; fermented food; high-salt; metabolomics
  3. BMC Bioinformatics. 2022 Jul 08. 23(1): 267
      BACKGROUND: Modern mass spectrometry has revolutionized the detection and analysis of metabolites but likewise, let the data skyrocket with repositories for metabolomics data filling up with thousands of datasets. While there are many software tools for the analysis of individual experiments with a few to dozens of chromatograms, we see a demand for a contemporary software solution capable of processing and analyzing hundreds or even thousands of experiments in an integrative manner with standardized workflows.RESULTS: Here, we introduce MetHoS as an automated web-based software platform for the processing, storage and analysis of great amounts of mass spectrometry-based metabolomics data sets originating from different metabolomics studies. MetHoS is based on Big Data frameworks to enable parallel processing, distributed storage and distributed analysis of even larger data sets across clusters of computers in a highly scalable manner. It has been designed to allow the processing and analysis of any amount of experiments and samples in an integrative manner. In order to demonstrate the capabilities of MetHoS, thousands of experiments were downloaded from the MetaboLights database and used to perform a large-scale processing, storage and statistical analysis in a proof-of-concept study.
    CONCLUSIONS: MetHoS is suitable for large-scale processing, storage and analysis of metabolomics data aiming at untargeted metabolomic analyses. It is freely available at: . Users interested in analyzing their own data are encouraged to apply for an account.
    Keywords:  Distributed analysis; Distributed storage; Large-scale metabolomics; Mass spectrometry data; Parallel processing
  4. Anal Chem. 2022 Jul 07.
      Mass spectrometry imaging (MSI) encompasses a powerful suit of techniques which provide spatially resolved atomic and molecular information from almost any sample type. MSI is now widely used in preclinical research to provide insight into metabolic phenotypes of disease. Typically, fresh-frozen tissue preparations are considered optimal for biological MSI and other traditional preservation methods such as formalin fixation, alone or with paraffin embedding (FFPE), are considered less optimal or even incompatible. Due to the prevalence of FFPE tissue storage, particularly for rare and therefore high-value tissue samples, there is substantial motivation for optimizing MSI methods for analysis of FFPE tissue. Here, we present a novel modality, atmospheric-pressure infrared laser-ablation plasma postionization (AP-IR-LA-PPI), with the first proof-of-concept examples of MSI for FFPE and fresh-frozen tissues, with no post-sectioning sample preparation. We present ion images from FFPE and fresh tissues in positive and negative ion modes. Molecular annotations (via the Metaspace annotation engine) and on-tissue MS/MS provide additional confidence that the detected ions arise from a broad range of metabolite and lipid classes from both FFPE and fresh-frozen tissues.
  5. Eur J Mass Spectrom (Chichester). 2022 Jul 03. 14690667221111153
      A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantification of procyclidine hydrochloride in human plasma using Procyclidine D11 hydrochloride as internal standard. Liquid-liquid extraction technique with methyl tertiary butyl ether was used for the extraction of plasma samples. Chromatographic separation of the analyte and the internal standard from the endogenous components was done on Zodiac C18 column (50 × 4.6 mm, 5 µm) using a mixture of methanol and 0.1% formic acid in water (70:30, v/v) as mobile phase at a flow rate of 1 mL/min with the run time of 2 min. The detection of the eluents was done using multiple reaction monitoring (MRM) in positive ion mode. Linearity of the method was established in the concentration range of 0.5 to 120 ng/mL. Full validation of the method was done as per USFDA guidelines and the results were well within the acceptance limits. The successful application of the method was done on healthy human subjects under fasting conditions, proving it to be used for bioequivalence and bioavailability (BA/BE) studies of procyclidine.
    Keywords:  LC-MS/MS; bioanalysis; liquid-liquid extraction; pharmacokinetic study; procyclidine
  6. Int J Mol Sci. 2022 Jun 23. pii: 6985. [Epub ahead of print]23(13):
      In the past two decades, the post-genomic era envisaged high-throughput technologies, resulting in more species with available genome sequences. In-depth multi-omics approaches have evolved to integrate cellular processes at various levels into a systems biology knowledge base. Metabolomics plays a crucial role in molecular networking to bridge the gaps between genotypes and phenotypes. However, the greater complexity of metabolites with diverse chemical and physical properties has limited the advances in plant metabolomics. For several years, applications of liquid/gas chromatography (LC/GC)-mass spectrometry (MS) and nuclear magnetic resonance (NMR) have been constantly developed. Recently, ion mobility spectrometry (IMS)-MS has shown utility in resolving isomeric and isobaric metabolites. Both MS and NMR combined metabolomics significantly increased the identification and quantification of metabolites in an untargeted and targeted manner. Thus, hyphenated metabolomics tools will narrow the gap between the number of metabolite features and the identified metabolites. Metabolites change in response to environmental conditions, including biotic and abiotic stress factors. The spatial distribution of metabolites across different organs, tissues, cells and cellular compartments is a trending research area in metabolomics. Herein, we review recent technological advancements in metabolomics and their applications in understanding plant stress biology and different levels of spatial organization. In addition, we discuss the opportunities and challenges in multiple stress interactions, multi-omics, and single-cell metabolomics.
    Keywords:  abiotic and biotic stresses; metabolomics technologies; multi-omics; single-cell metabolomics; spatial metabolomics
  7. Phytochem Anal. 2022 Jul 05.
      INTRODUCTION: In recent years, LC-MS has become the golden standard for metabolomic studies. Indeed, LC is relatively easy to couple with the soft electrospray ionization. As a consequence, many tools have been developed for the structural annotation of tandem mass spectra. However, it is sometimes difficult to do data-dependent acquisition (DDA), especially when developing new methods that stray from the classical LC-MS workflow.OBJECTIVE: An old tool from petroleomics that has recently gained popularity in metabolomics, the Van Krevelen diagram, is adapted for an overview of the molecular diversity profile in lichens through high-resolution mass spectrometry (HRMS).
    METHODS: A new method is benchmarked against the state-of-the-art classification tool ClassyFire using a database containing most known lichen metabolites (n ≈ 2,000). Four lichens known for their contrasted chemical composition were selected, and extractions with apolar, aprotic polar, and protic polar solvents were performed to cover a wide range of polarities. Extracts were analyzed with direct infusion electrospray ionization mass spectrometry (DI-ESI-MS) and atmospheric solids analysis probe mass spectrometry (ASAP-MS) techniques to be compared with the chemical composition described in the literature.
    RESULTS: The most common lichen metabolites were efficiently classified, with more than 90% of the molecules in some classes being matched with ClassyFire. Results from this method are consistent with the various extraction protocols in the present case study.
    CONCLUSION: This approach is a rapid and efficient tool to gain structural insight regarding lichen metabolites analyzed by HRMS without relying on DDA by LC-MS/MS analysis. It may notably be of use during the development phase of novel MS-based metabolomic approaches.
    Keywords:  extract comparison; lichens; metabolomics; structural classification
  8. Int J Mol Sci. 2022 Jul 04. pii: 7428. [Epub ahead of print]23(13):
      Chiral metabolomics is starting to become a well-defined research field, powered by the recent advances in separation techniques. This review aimed to cover the most relevant advances in indirect enantioseparations of endogenous metabolites that were published over the last 10 years, including improvements and development of new chiral derivatizing agents, along with advances in separation methodologies. Moreover, special emphasis is put on exciting advances in separation techniques combined with mass spectrometry, such as chiral discrimination by ion-mobility mass spectrometry together with untargeted strategies for profiling of chiral metabolites in complex matrices. These advances signify a leap in chiral metabolomics technologies that will surely offer a solid base to better understand the specific roles of enantiomeric metabolites in systems biology.
    Keywords:  chiral derivatization agents; chiral metabolomics; endogenous metabolites; indirect chiral analysis; ion-mobility mass spectrometry; mass spectrometry metabolomics
  9. Front Toxicol. 2022 ;4 932445
      Scientists' ability to detect drug-related metabolites at trace concentrations has improved over recent decades. High-resolution instruments enable collection of large amounts of raw experimental data. In fact, the quantity of data produced has become a challenge due to effort required to convert raw data into useful insights. Various cheminformatics tools have been developed to address these metabolite identification challenges. This article describes the current state of these tools. They can be split into two categories: Pre-experimental metabolite generation and post-experimental data analysis. The former can be subdivided into rule-based, machine learning-based, and docking-based approaches. Post-experimental tools help scientists automatically perform chromatographic deconvolution of LC/MS data and identify metabolites. They can use pre-experimental predictions to improve metabolite identification, but they are not limited to these predictions: unexpected metabolites can also be discovered through fractional mass filtering. In addition to a review of available software tools, we present a description of pre-experimental and post-experimental metabolite structure generation using MetaSense. These software tools improve upon manual techniques, increasing scientist productivity and enabling efficient handling of large datasets. However, the trend of increasingly large datasets and highly data-driven workflows requires a more sophisticated informatics transition in metabolite identification labs. Experimental work has traditionally been separated from the information technology tools that handle our data. We argue that these IT tools can help scientists draw connections via data visualizations and preserve and share results via searchable centralized databases. In addition, data marshalling and homogenization techniques enable future data mining and machine learning.
    Keywords:  analytical data management; computational chemistry; machine learning; metabolite identification; metabolite prediction
  10. Nat Biotechnol. 2022 Jul 07.
      Human untargeted metabolomics studies annotate only ~10% of molecular features. We introduce reference-data-driven analysis to match metabolomics tandem mass spectrometry (MS/MS) data against metadata-annotated source data as a pseudo-MS/MS reference library. Applying this approach to food source data, we show that it increases MS/MS spectral usage 5.1-fold over conventional structural MS/MS library matches and allows empirical assessment of dietary patterns from untargeted data.
  11. Front Pharmacol. 2022 ;13 920436
      A reliable and rapid method employing QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) pretreatment coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was successfully developed and validated for the analysis of nine tyrosine kinase inhibitors (TKIs) in human plasma. Biological samples were extracted with acetonitrile and salted out with 350 mg of anhydrous magnesium sulfate (MgSO4), followed by purification with 40 mg of ethyl enediamine-N-propylsilane (PSA) adsorbents. All analytes and internal standards (IS) were separated on the Hypersil GOLD VANQUISH C18 (2.1 mm × 100 mm, 1.9 μM) column using the mobile phases composed of acetonitrile (phase A) and 0.1% formic acid in water (phase B) for 8.0 min. Detection was performed by selection reaction monitoring (SRM) in the positive ion electrospray mode. Lenvatinib, sorafenib, cabozantinib, apatinib, gefitinib, regorafenib, and anlotinib rendered good linearity over the range of 0.1-10 ng/ml, and 1-100 ng/ml for tivantinib and galunisertib. All linear correlation coefficients for all standard curves were ≥ 0.9966. The limits of detection (LOD) and the limits of quantitation (LOQ) ranged from 0.003 to 0.11 ng/ml and 0.01-0.37 ng/ml, respectively. The method was deemed satisfactory with an accuracy of -7.34-6.64%, selectivity, matrix effect (ME) of 90.48-107.77%, recovery, and stability. The proposed method is simple, efficient, reliable, and applicable for the detection of TKIs in human plasma samples as well as for providing a reference for the clinical adjustment of drug administration regimen by monitoring the drug concentrations in the plasma of patients.
    Keywords:  QuEChERS; UPLC-MS/MS; hepatocellular carcinoma; plasma; tyrosine kinase inhibitors
  12. Mol Genet Metab Rep. 2022 Jun;31 100868
      We have developed a fast and accurate method that uses a small volume of sample to determine over 25 of the typically reported amino acids in human plasma. Samples were prepped with a single step using a spin filter to remove proteins, avoiding the decreased sensitivity from dilution in acid precipitation. Using a reverse phase (RP) High Performance Liquid Chromatography (HPLC) system with O-phthaldehyde (OPA) as the pre-column derivatization reagent, and UV detection at 338 nm, we did a direct comparison with the most common ion exchange/ninhydrin method used in clinical labs on the same plasma samples with 95% concurrence, analysis of amino acid standard solutions returned 99% concurrence. With a sample preparation time of 30 min, utilizing less than 25 μl of sample and with a chromatography run of 30 min, this method can substantially increase access to analysis in both clinical and research laboratories using instruments that are more widely available.Synopsis: We describe a rapid and easily deployed method for sensitive amino measurement in biological samples.
    Keywords:  Amino acid; Clinical biochemistry; HPLC; IEM; Inborn error of metabolism
  13. Se Pu. 2022 Jul;40(7): 625-633
      Nitroimidazoles (NMZs) are a crucial group of antibacterial compounds from a historical perspective. In the past, they were used for treating and preventing parasitic infections in fish. Benzodiazepines (BZDs) are second-generation sedative-hypnotics. Some fish farmers or vendors use them illegally to keep aquatic products fresh during the transportation of aquatic animals. Aquatic products are one of the most common food sources of protein and can be contaminated by NMZs and BZDs, which could impact humans through the food chain. Until recently, there was limited information on the simultaneous determination of NMZs and BZDs. Thus, it is critical to accurately quantify NMZs and BZDs for risk assessment and risk monitoring of food safety. For the simultaneous determination of five nitroimidazoles and six benzodiazepines in aquatic products, a new approach based on the dispersive solid-phase extraction (dSPE) coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed. First, the samples were extracted with acetonitrile containing 1% (v/v) ammonium hydroxide, and the extracts were purified using dSPE with C18 and primary secondary amine sorbents. Second, the extracts were collected and dried at 45 ℃ under nitrogen flow. Finally, the extracts were redissolved in 1 mL methanol-water (1∶9, v/v) mixture, filtered through a nylon-66 microfiltration membrane, and analyzed using UHPLC-MS/MS. The separation of compounds was conducted on a Kinetex F5 column (100 mm×3.0 mm, 2.6 μm) using gradient elution with 1% (v/v) formic acid aqueous solution and methanol as the mobile phase. The analytes were detected using MS/MS with positive electrospray ionization (ESI+) source under the multiple reaction monitoring modes. The matrix-matched external standard approach was used for quantitative analysis. The compounds of five nitroimidazoles and six benzodiazepines could be examined within 8.5 min. Under the optimal conditions, the standard curves were linear in the range of 0.5-20 μg/L, with the correlation coefficients exceeding 0.995. The limits of detection and limits of quantification were 0.2-0.5 μg/kg and 0.5-1.0 μg/kg, respectively. The average recoveries at three spiked levels in blank samples (grass carp, large yellow croaker, and prawn) ranged from 73.2% to 110.6%, with relative standard deviations of less than 15%. The developed approach is simple, sensitive, fast, and inexpensive. It can be used for determining five nitroimidazoles and six benzodiazepines in aquatic products.
    Keywords:  aquatic products; benzodiazepines; dispersive solid-phase extraction (dSPE); nitromidazoles; ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)
  14. Se Pu. 2022 Jul;40(7): 644-652
      Polychlorinated naphthalenes (PCNs) have a structure similar to that of polychlorinated biphenyls (PCBs) and represent a new type of persistent organic pollutants (POPs) that are widely present in the environment and biological communities. PCNs can migrate and transform via different environmental media, which severely affects the health of humans and organisms. Researchers have devoted considerable focus on ambient air pollution. Although the current ambient air quality has not yet limited the concentration of PCNs, the Stockholm Convention has required parties to prohibit and eliminate their production and use. As one of the contracting parties, China is obligated to improve its environmental monitoring. In other words, the development of a method for monitoring PCNs in ambient air is important for understanding ambient air quality and safeguarding human health. PCNs are generally present at trace levels (pg/m3) in ambient air. To achieve accurate quantification of PCNs, high demands are raised on the methods for extraction, purification, and instrumental analysis, which can directly affect the efficiency, accuracy, and sensitivity of a method. Considering the trace-level presence of PCNs in ambient air and the high efficiency and accuracy of the analytical method, accelerated solvent extraction (ASE), combined with column chromatography using a multilayer silica gel column and a neutral alumina column, was established for the extraction and purification of PCNs in ambient air. The important parameters involved in the aforementioned steps, such as the type of extraction and volume of elution solvent, were optimized. The results indicated that dichloromethane-hexane (1∶1, v/v) was the best extraction solvent for the recovery of PCNs. Hexane and dichloromethane-hexane (5∶95, v/v) were used as the elution solvents for the multi-silica gel column and neutral alumina column, respectively. Isotope dilution gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) was used to quantify the target compounds. Gas chromatographic parameters, such as temperature program conditions and inlet temperature, were also optimized. The oven temperature program was as follows: 80 ℃ for 1 min, 80 ℃ to 160 ℃ at 15 ℃/min, 160 ℃ to 265 ℃ at 3 ℃/min, and 265 ℃ to 280 ℃ at 5 ℃/min, followed by holding the temperature at 280 ℃ for 10 min. The inlet temperature was set at 260 ℃. The optimal characteristics of ion pair, collision energy, and ion source temperature were determined by optimizing the key mass spectrometry parameters. The developed instrumental method, combined with suitable sample preparation techniques, was used to determine the concentrations of PCNs in ambient air samples. Quality control (QC) and quality assurance (QA) were performed by adding isotope internal standards before sampling, extraction, and injection analysis to monitor the entire analysis process. The relative standard deviations (RSDs) of the relative response factors (RRFs) for trichloronaphthalene to octachloronaphthalene were less than 16% in the concentration range of 2-100 ng/mL. The method detection limits (MDLs) for PCN homologues were in the range of 1-3 pg/m3(calculated using a sample volume of 288 m3). The precision and accuracy of this method for determining PCNs in ambient air samples were evaluated using a spiked matrix. The average spiked recoveries of trichloronaphthalene to octachloronaphthalene were 89.0%-119.4%, 98.6%-122.5% and 93.7%-124.5% at low, medium, and high spiked concentrations (20, 50, and 90 ng/mL), respectively. The RSDs of the assay results were 1.9%-7.0%, 1.6%-6.6%, and 1.0%-4.8%, respectively. During the entire analysis process, the average recoveries of the sampling and extracted internal standards were 136.2%-146.0% and 42.4%-78.1%, respectively, and the corresponding RSDs were 5.6%-7.5% and 2.7%-17.5%. Thus, this method meets the requirements of trace analysis and exhibits good parallelism, high sensitivity, high accuracy, and good precision, and it is suitable for the accurate quantitative determination of trichloronaphthalene to octachloronaphthalene in ambient air.
    Keywords:  ambient air; gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS); isotope dilution; polychlorinated naphthalenes (PCNs)
  15. Biomed Chromatogr. 2022 Jul 05. e5443
      A sensitive, specific and rapid liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed and validated to quantify azithromycin concentrations in human plasma. Azithromycin (AZI) is the most common outpatient prescribed antibiotic in the US and clinical studies have demonstrated the efficacy and safety of AZI in many bacterial infections. To support a clinical study, we developed a high throughput LC-MS/MS method to process up to 250 samples per day to quantify AZI in human plasma. Samples were prepared by solid phase extraction. Separation was achieved with an ACE C18 column (2.1 x 100 mm, 1.7 μm) equipped with a C18 guard column. The mobile phase consisted of 0.1% formic acid and methanol/acetonitrile (1:1, v/v) at a flow rate of 0.25 mL/min. The ionization was optimized with positive electrospray source using multiple reaction monitoring transition, m/z 749.50>591.45 for AZI and m/z 754.50>596.45 for AZI-d5. Extraction recoveries were approximately 90% for AZI. The assay was linear from 0.5 to 2000 ng/mL and required only 100 μL of plasma with total analysis time of 4.5 minutes. The method was successfully applied to pharmacokinetic studies of a weight-based dosing protocol for AZI.
    Keywords:  Azithromycin; HPLC; human plasma; tandem mass spectrometry; yaws disease
  16. Anal Bioanal Chem. 2022 Jul 04.
      A common method to quantify chronic stress is the analysis of stress markers in keratinized matrices such as hair or nail. In this study, we aimed to validate a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the combined quantification of steroid hormones and endocannabinoids (eCBs) in the keratinized matrix nail. Furthermore, we aimed to investigate the suitability of the nail matrix for the detection of these stress markers in a pilot study. An LC-MS/MS method was used for the simultaneous identification and quantification of four eCBs (2-arachidonoylglycerol (2-AG), anandamide (AEA), oleoylethanolamide (OEA), palmitoylethanolamide (PEA)) and five steroid hormones (cortisol, cortisone, androstenedione, progesterone, testosterone) in human nails using a surrogate analyte method for each analyte. The method was validated in terms of selectivity, response factor, linearity, limit of quantification (LOQ), precision, accuracy, matrix effect, recovery, robustness, and autosampler stability. Nail samples were extracted for 1 h with methanol following a clean-up with a fully automated supported liquid extraction (SLE). The influence of nail weight on the quantification was investigated by using 0.5-20 mg of nail sample. As a proof of concept, nail samples (N = 57) were analyzed from a cohort representing newborns (1 month old), children (between 1 and 10 years), and adults (up to 43 years). It could be shown that the established workflow using a 1 hour extraction and clean-up by SLE was very robust and resulted in a short sample preparation time. The LC-MS/MS method was successfully validated. Matrix effects with ion enhancement occurred mainly for 2-AG. Sample weights below 5 mg showed variations in quantification for some analytes. Certain analytes such as PEA and progesterone could be accurately quantified at a sample weight lower than 5 mg. This is the first study where steroids and eCBs could be simultaneously detected and quantified in infant and adult nails. These results show that nails may serve as an alternative keratinized matrix (compared to hair) for the retrospective monitoring of cumulative eCB and steroid hormone levels. The combined assessment of eCBs and steroids from nails could provide a new approach to gain new insights into stress exposure in newborns and adults.
    Keywords:  Endocannabinoids; LC–MS/MS; Long-term monitoring; Nails; Steroids; Stress
  17. Biomed Chromatogr. 2022 Jul 07. e5445
      Caffeine is one of the naturally occurring alkaloids and it is metabolized to paraxanthine, theophylline, and theobromine. The analyses of caffeine and its metabolites are challenging since the metabolites theophylline and paraxanthine generate similar product and precursor ions. In this study, a new method was developed for simultaneous analysis of caffeine, paraxanthine, theobromine and theophylline in horse urine using Gas chromatography-Mass Spectrometry (GC-MS). Urine samples were treated using solid phase extraction followed by the elution with dichloromethane/isopropanol (90/10) after pH was adjusted to 6, and then derivatization with MSTFA-1%TMCS before analysis by GC-MS. Sample preparation and derivatization steps were optimized and the method permitted elution all of these analytes within 13 min. The method was fully validated according to Commission Decision 2002/657/EC guidelines. The calibration curves were linear with a correlation coefficient of >0.99. Precision and accuracy were well within the 15% acceptance range and the method was robust. The validation results demonstrated that the method is highly reproducible, easily applicable and selective. The method was applied to urine samples collected from racehorses to demonstrate its applicability.
    Keywords:  Caffeine; GC-MS; paraxanthine; theobromine; theophylline
  18. Se Pu. 2022 Jul;40(7): 669-676
      Due to the harmful effects of estrogens and their prevalence in animal foods, accurate analysis of estrogen levels in animal foods is imperative in order to effectively assess food safety risks and ensure consumer safety. Therefore, a rapid and accurate method based on PRiME HLB solid phase extraction (SPE) cartridge purification and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to determine nine estrogen residues in bullfrogs. The nine estrogens included estriol (E3), 17β-estradiol (β-E), 17α-estradiol (α-E), 17α-ethinylestradiol (EE2), estrone (EI), diethylstilbestrol (DES), dienestrol (DE), hexestrol (HEX), and dienestrol diacetate (DD). This study optimized the mobile phase system, extraction solvent, and SPE cartridges. Because estrogens present weak alkalinity, adding a small amount of alkaline substance to the mobile phase benefits estrogen ionization into the ionic state, eliminates the peak trailing phenomenon, and enhances the signal response of estrogens to improve sensitivity. Estrogens have one or more hydroxyl groups in their chemical structures. According to the principle of similar solubility, polar solvents are chosen as extraction solvents. Based on the complex matrix composition of meat samples, SPE is required for purification to reduce matrix effects. The liquid chromatographic conditions were optimized, and the 0.5 mmol/L ammonium fluoride aqueous solution-acetonitrile system as mobile phases showed better sensitivity than the ammonium acetate aqueous solution-acetonitrile system and the ammonia-acetonitrile system for the nine estrogens. When acetonitrile was used as the extraction solvent, the extraction rates of all nine estrogens exceeded those of methanol and ethyl acetate and increased by 15%-40%. By focusing on the matrix purification effect of four different SPE cartridges, the results showed that the matrix purification ability of the PRiME HLB cartridge outperformed that of the HLB, C18, and Silica SPE cartridges. After purification by the PRiME HLB cartridge, the recoveries of all compounds were in the range of 70%-125%, and the DD recovery was increased from 47% to 74%, whereas the HEX recovery was reduced from 180% to 123%. Therefore, the PRiME HLB SPE cartridge was selected as the cleanup material for this experiment. Finally, the sample was extracted using acetonitrile, purified by PRiME HLB SPE cartridge, and separated on a Waters Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm) with a mobile phase of 0.5 mmol/L ammonium fluoride aqueous acetonitrile solution at a flow rate of 0.3 mL/min. The detection was conducted in positive and negative ion switching mode (ESI+/ESI-) and multiple reaction monitoring (MRM) scanning, and it was quantified using a matrix-matched external standard method. Under the optimal experimental conditions, the linear ranges were 0.5-100.0 μg/L for E3, β-E, α-E, EI, DE, HEX, and DD, and 1.0-100.0 μg/L for EE2 and DES. The nine estrogens showed good linearity in all linear ranges, with correlation coefficients of 0.9953-0.9994. The limits of detection were 0.17-0.33 μg/kg, and the limits of quantification were 0.5-1.0 μg/kg. The recoveries of the nine estrogens spiked at the three spiked levels of low (2.0 μg/kg), medium (10.0 μg/kg), and high (80.0 μg/kg) were 107.4%-125.3%, 67.0%-123.3%, and 65.1%-128.2%, respectively. The relative standard deviations were 1.9%-17.6%. The method established in this study was applied to detect nine estrogen residues in 50 commercially available bullfrog samples, and the results showed that HEX, EI, and DES were detected in few samples. The method is simple, rapid, sensitive, and reproducible, and can be used for the simultaneous, rapid and accurate determination of large quantities of samples.
    Keywords:  bullfrog; estrogen; solid phase extraction (SPE); ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)
  19. J Am Soc Mass Spectrom. 2022 Jul 06. 33(7): 1276-1281
      The identification and confirmation of steroid sulfate metabolites in biological samples are essential to various fields, including anti-doping analysis and clinical sciences. Ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) is the leading method for the detection of intact steroid conjugates in biofluids, but because of the inherent complexity of biological samples and the low concentration of many targets of interest, metabolite identification based solely on mass spectrometry remains a major challenge. The confirmation of new metabolites typically depends on a comparison with synthetically derived reference materials that encompass a range of possible conjugation sites and stereochemistries. Herein, energy-resolved collision-induced dissociation (CID) is used as part of UHPLC-HRMS/MS analysis to distinguish between regio- and stereo-isomeric steroid sulfate compounds. This wholly MS-based approach was employed to guide the synthesis of reference materials to unambiguously confirm the identity of an equine steroid sulfate biomarker of testosterone propionate administration.
  20. Emerg Contam. 2022 Jul 01.
      Quaternary ammonium compounds (QACs) are a class of antimicrobial disinfectants whose use in cleaning products increased during the COVID-19 pandemic. Chemically, their low vapor pressure indicates a proclivity to persist on surfaces, and their presence suggests a level of protection against microorganisms. The widespread application of QACs in response to the SARS CoV-2 virus created a need to evaluate their longevity on surfaces, for both efficacy and possible health risks. There are however, no standardized analytical methods for QAC surface sampling and analysis, and no published studies quantifying their concentrations on mass transportation vehicles-a high occupancy, close-contact microenvironment documented to facilitate the spread the SARS CoV-2 virus. Here, we describe a robust liquid chromatography mass spectrometry (LC-MS) method for the analysis of QACs and simultaneous development of a direct surface sampling and extraction protocol. We demonstrate the applicability of the method through the analysis of surface samples collected from in-service public transportation buses. The rapid, sensitive LC-MS method included 8 target QACs quantified on a Q-Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer using an electrospray ionization source and Dionex UltiMate 3000 UHPLC system for analyte separation. QAC standard mixtures at concentrations between 0.1 ng mL-1 and 2000 ng mL-1 were analyzed, and chromatographic separation of all analytes was achieved in less than 10 minutes. All correlation coefficients were reported at r > 0.986, and LODs ranged from 0.007-2.103 ng mL-1 for all compounds, confirming the method's sensitivity. A previously reported surface sampling and extraction protocol was modified to further simplify the procedure and expand the number of target compounds. The new sampling protocol was optimized from 10 commercially available wipes and 4 solvent types by quantifying recovery from the surface. Band-Aid brand small gauze pads saturated with isopropanol had the highest recovery efficiencies, ranging from 61.5-102.9% across all analytes. To test the real-world applicability, wipe samples were collected from 4 in-circulation New Jersey Transit buses on 5 separate days over the course of a month to assess the occurrence and longevity of QACs on sanitized mass transportation vehicles. Concentrations of QACs were detected on every wipe sample taken, and at all sampled time points, confirming their persistence on hard surfaces. QACs have the potential to form polymers, and detection of the polymer might serve as a secondary indication of their effectiveness on surfaces. None of the polymers detected however, were unique to QACs from this study. The polymers detected were already present in the wipe and used as an internal standard to demonstrate the efficacy of extraction and analysis of polymeric QACs.
    Keywords:  Alkyl (ethyl benzyl) dimethyl ammonium chloride; Alkyl dimethyl benzyl ammonium chloride; COVID-19; Orbitrap mass spectrometry; Public transportation; Surface sampling
  21. Prog Lipid Res. 2022 Jun 30. pii: S0163-7827(22)00032-7. [Epub ahead of print] 101177
      Large 'omics studies are of particular interest to population and clinical research as they allow elucidation of biological pathways that are often out of reach of other methodologies. Typically, these information rich datasets are produced from multiple coordinated profiling studies that may include lipidomics, metabolomics, proteomics or other strategies to generate high dimensional data. In lipidomics, the generation of such data presents a series of unique technological and logistical challenges; to maximize the power (number of samples) and coverage (number of analytes) of the dataset while minimizing the sources of unwanted variation. Technological advances in analytical platforms, as well as computational approaches, have led to improvement of data quality - especially with regard to instrumental variation. In the small scale, it is possible to control systematic bias from beginning to end. However, as the size and complexity of datasets grow, it is inevitable that unwanted variation arises from multiple sources, some potentially unknown and out of the investigators control. Increases in cohort sizes and complexity has led to new challenges in sample collection, handling, storage, and preparation stages. If not considered and dealt with appropriately, this unwanted variation may undermine the quality of the data and reliability of any subsequent analysis. Here we review the various experimental phases where unwanted variation may be introduced and review general strategies and approaches to handle this variation, specifically addressing issues relevant to lipidomics studies.
    Keywords:  Data analysis; High-throughput; Lipidomics; Normalization; Preprocessing; Unwanted variation
  22. Steroids. 2022 Jul 01. pii: S0039-128X(22)00115-5. [Epub ahead of print] 109077
      In epidemiological studies, blood levels of 17β-estradiol (E2) are associated with hormone-dependent diseases. The lack of specific methods impedes studies on the role of E2 metabolites and their conjugates in the etiology of hormone-dependent diseases. Stable-isotope dilution tandem mass spectrometry methods (coupled to gas chromatography and liquid chromatography systems) for the analysis of 22 endogenous estrogens, including both oxidative metabolites, as well as sulfates and glucuronides, was validated and the method applied to plasma of women with no breast cancer. No changes in estrogen profile during sample cleanup were observed and values for limit of detection (7fmol/ml - 2 pmol/ml), accuracies (80-122%) as well as intra- and inter-day precision (below 28%) at levels near the limit of quantification were acceptable. In human plasma only seven estrogens were detected and estrone conjugates contributed most to the estrogen profile.
    Keywords:  GC-MS/MS; LC-MS/MS; endogenous estrogens; human plasma; metabolism
  23. Se Pu. 2022 Jul;40(7): 653-660
      Sanzi San, a Mongolian medicine, comprises three herbs: Terminalia chebula, Melia toosendan, and Gardenia jasminoides. Clinically, Sanzi San is administered orally and distributed via blood to the action site, which implies that the absorption, distribution, metabolism, and excretion (ADME) are closely related to the pharmacological action and curative effect. Therefore, possible explanations for the material basis of Sanzi San were explored in this study preliminarily. A strategy based on serum pharmacochemistry was first applied to explore the absorbed bioactive components and metabolites of Sanzi San. Wistar rats were randomly divided into normal and dosing groups, which were provided with the Sanzi San's water extract for three days. Then, the rat's blood samples were obtained from their abdomiral aorta using a sterile blood collection tube after administering the medicine. The blood samples were then centrifuged at 3500 r/min for 10 min to obtain the serum samples. A practical method based on high performance liquid chromatography coupled with quadrupole and electrostatic field orbitrap high resolution mass spectrometry (HPLC-Q/Orbitrap HRMS) was developed to screen and analyze numerous bioactive components and metabolites adsorbed in the serum of the dosing rats after oral administration of the Sanzi San's water extract. Chromatographic separation was achieved on a SHIMADZU GIST C18 chromatographic column (150 mm×4.6 mm, 5 μm). The temperature of the column was maintained at 30 ℃. The flow rate was 0.5 mL/min, and the injection volume was 10 μL. The mobile phase comprised an aqueous solution of 0.1% formic acid and methanol under gradient elution. A heated electrospray ion (HESI) source was used with positive and negative ion scanning modes. To rapidly screen out and identify the absorbed bioactive components and metabolites of Sanzi San in the rat serum samples, a simple three-step approach was developed. First, the known components in Sanzi San were listed systematically by exploring various databases, such as the Web of Science, PubMed, and Chinese National Knowledge Infrastructure. In addition, relevant information on drug biotransformation and the characteristic fragmentation patterns of parent compounds were summarized. Second, the absorbed components and metabolites were ascertained using the Xcalibur 3.0 software. Based on the information related to the parent compound's structure, the software could be used to identify the unique peaks by comparing the chromatograms of the normal and dosing samples. Consequently, the total ion chromatograms of serum samples were established. Finally, the Compound Discover 3.0 software was used to predict the metabolic pathways and fragmentation of the absorbed compounds. Using this approach, 55 compounds were characterized, including 41 prototype components and 14 metabolites. The main prototype components in the serum sample were tannins, iridoids, and phenolic acids. The details of these compounds have been summarized and presented. Regarding the absorbed bioactive components and metabolites in the serum samples of rats administered with Sanzi San, phase Ⅰ and phase Ⅱ biochemical reactions were involved in the biotransformation pathways. The phase Ⅰ reaction modified the components and created sites for the phase Ⅱ reaction, involving reduction and hydrolysis. The phase Ⅱ reaction coupled groups to existing conjugation sites, including glucuronide to glucuronic acid, sulfate, and methyl. MS/MS spectra indicated that methylation, demethylation, and dehydroxylation are the metabolic pathways of procyanidins. Additionally, glucuronidation, deglucosidation, hydration, and demethylation are the metabolic pathways of iridoids in Sanzi San. This study comprehensively analyzed the components of the Sanzi San's water extract absorbed in the rat's serum. Our results revealed information regarding the pharmacodynamic substances and the major pathways involved in the ADME of Sanzi San. Further, potential medicinal ingredients for the pharmacological effects and clinical use of Sanzi San were explored at the serum pharmacochemistry level.
    Keywords:  Sanzi San; high performance liguid chromatography (HPLC); quadrupole/electrostatic field orbitrap high resolution mass spectroscopy (Q/Orbitrap HRMS); serum pharmacochemistry
  24. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Jun 26. pii: S1570-0232(22)00255-0. [Epub ahead of print]1206 123351
      ATP and its degradation products are essential metabolic and signaling molecules. Traditionally, they have been quantified via high-performance liquid chromatography (HPLC) with UV-Vis detection while utilizing phosphate buffer mobile phase, but this approach is incompatible with modern mass detection. The goal of this study was to develop an ultra-performance liquid chromatography (UPLC) method free of phosphate buffer, to allow for analysis of adenine nucleotides with UV-Vis and mass spectrometry (MS) simultaneously. The final conditions used an Acquity HSS T3 premier column with a volatile ammonium acetate buffer to successfully separate and quantify ATP-related analytes in a standard mixture and in extracts from non-contracted and contracted mouse hindlimb muscles. Baseline resolution was achieved with all 10 metabolites, and a lower limit of quantification down to 1 pmol per inject was observed for most metabolites using UV-Vis. Therefore, this method allows for the reliable quantification of adenine nucleotides and their degradation products via UV-Vis and their confirmation and/or identification of unknown peaks via MS.
    Keywords:  ATP; Adenine nucleotides; Mouse; Muscle; UPLC/MS
  25. eNeuro. 2022 Jun 30. pii: ENEURO.0037-22.2022. [Epub ahead of print]
      Neuroestrogens are synthesized within the brain and regulate social behavior, learning and memory, and cognition. In song sparrows, Melospiza melodia, 17β-estradiol (17β-E2) promotes aggressive behavior, including during the non-breeding season when circulating steroid levels are low. Estrogens are challenging to measure because they are present at very low levels, and current techniques often lack the sensitivity required. Furthermore, current methods often focus on 17β-E2 and disregard other estrogens. Here, we developed and validated a method to measure four estrogens (estrone, 17β-E2, 17α-estradiol, estriol) simultaneously in microdissected songbird brain, with high specificity, sensitivity, accuracy, and precision. We used liquid chromatography tandem mass spectrometry (LC-MS/MS), and to improve sensitivity, we derivatized estrogens using 1,2-dimethylimidazole-5-sulfonyl-chloride (DMIS). The straightforward protocol improved sensitivity by 10-fold for some analytes. There is substantial regional variation in neuroestrogen levels in brain areas that regulate social behavior in male song sparrows. For example, the auditory area NCM, which has high aromatase levels, has the highest estrone and 17β-E2 levels. In contrast, estrogen levels in blood are very low. Estrogen levels in both brain and circulation are lower in the non-breeding season than in the breeding season. This technique will be useful for estrogen measurement in songbirds and potentially other animal models.SIGNIFICANCE STATEMENTEstrogens are locally synthesized within the brain and play important roles in neural function, but measurement of estrogens in specific brain regions is extremely challenging. We developed a method using liquid chromatography tandem mass spectrometry for the measurement of estrone, 17β-estradiol, 17α-estradiol, and estriol in microdissected brain, plasma, and blood in a songbird model. There are robust regional differences and seasonal changes in estrogen levels. This technique will be useful for measurement of estrogens and will facilitate studies of neuroestrogens and their functions.
    Keywords:  aromatase; estrogen profiling; mass spectrometry; microdissected brain; social behavior network; songbird
  26. Se Pu. 2022 Jul;40(7): 677-683
      According to the Report of Drug Situation in China (2020), the growth rate of the number of drug abusers in China has decreased, but the number of drug abusers is still large. An efficient screening method is necessary for controlling drug abuse. As an important type of biological sample, urine is widely used for the rapid screening of drug addicts. However, because of the complex composition, low content, and strong interference from the body's metabolism, the detection of drugs in urine remains a challenge. Traditional rapid screening techniques such as immunocolloidal gold analysis have a high false positive rate and insufficient quantitative capability. In addition, laboratory mass spectrometry methods require complicated time-consuming sample pre-processing and strict environmental conditions, and hence, are unsuitable for on-site rapid analysis. In recent years, various direct ionization mass spectrometry techniques such as direct analysis in real time (DART), desorption electrospray ionization (DESI), and dielectric barrier discharge ionization (DBDI) have advanced rapidly. These techniques have been applied to public safety, food safety, environmental detection, etc. In contrast to traditional ionization mass spectrometry methods, these direct ionization techniques allow for the in situ analysis of samples with simple or no pretreatment; moreover, they have the advantages of high analytical efficiency and sensitivity. In particular, pulsed electrospray ionization has the characteristics of less sample demand, compact, lightweight equipment, and no carrier gas. This paper presents a rapid method based on pulsed electrospray ionization mass spectrometry for the detection of urine samples. A rapid detection platform comprising a probe electrospray ionization source, a portable linear ion trap mass spectrometer (MS), and their coupling interface is adopted. The probe electrospray ion source includes a conducting metal wire, plastic handle, and silica glass capillary, whose tip has an inner diameter of 50 μm. The guide rail at the coupling interface is used to align the probe with the sample inlet of the portable mass spectrometer and maintain a distance of 10 mm between the probe tip and the sample inlet of the MS. The spray voltage of the probe electrospray ion source and the temperature of the MS inlet capillary are optimized at 1.8 kV and 205 ℃, respectively. In addition, rapid and efficient pretreatment techniques for urine samples have been developed. Buffer salts used for pH regulation and liquid-liquid extraction based on ethyl acetate were adopted for the pretreatment process. The linearity of the detection ability and the linear ranges of various drug-spiked solutions were also investigated. The results showed that the correlation coefficients for the quantitative detection of methamphetamine, ketamine, methylenedioxymethamphetamine (MDMA), and cocaine were greater than 0.99 at concentrations ranging from 1 to 100 ng/mL. Moreover, the limits of detection (LODs) for the five conventional drug-spiked urine were 0.5-30 ng/mL. The spiked recoveries ranged from 56.1% to 103.7%, with relative standard deviations (RSDs) of 9.0%-27.8%, implying that the combination of the instruments and the pretreatment method can lead to good accuracy. To validate the performance of the rapid detection method, 40 positive and 110 negative urine samples were tested and analyzed. The overall accuracy was over 99%, and the five conventional drugs in urine samples could be detected within 20 s. The research findings of this work could promote the development of rapid detection technology, accelerate the popularization and application of ambient direct ionization mass spectrometry, and improve the services of on-site law enforcement.
    Keywords:  ambient direct ionization; drug; portable mass spectrometer; rapid detection; urine
  27. Anal Chem. 2022 Jul 05.
      In high-throughput scenarios of targeted metabolomics, it is a significant challenge to process complex NMR spectra with severely overlapping signals produced by metabolites with similar chemical structures. Traditional frequency-domain NMR is ineffective to some degree due to the low sensitivity and poor resolution, and the precision of quantitation is usually affected by poorly or inconsistently phased and baselined spectra. Here, we established a strategy based on time-domain NMR focusing on methyl protons for targeted metabolomics. The targeted metabolomics focusing on bufadienolides for varietal discrimination of three toad venoms was conducted to demonstrate the practicability of time-domain-based methyl proton NMR. It revealed that the signals could be precisely identified and quantitated with an signal-to-noise ratio (SNR) of about 10 and a resolution of about 1.0 Hz. The sensitivity and resolution improvement make it particularly applicable in targeted metabolomics. The precise and absolute quantitation ability confirmed by triple-quadrupole mass spectrometry (QqQ-MS) could further extend its application range. Importantly, the methyl group is common in metabolites with a relatively wide chemical shift range. Time-domain analysis could be conducted in common NMR software. This technique is very easy and convenient for general researchers when employed as a complementary alternative to traditional frequency-domain NMR, especially in the field of targeted metabolomics.
  28. J Pharm Biomed Anal. 2022 Jun 26. pii: S0731-7085(22)00331-4. [Epub ahead of print]219 114910
      In 2018, high levels of the IARC class IIA carcinogen N-nitrosodimethylamine (NDMA) were analytically verified in the active pharmaceutical ingredient (API) valsartan, resulting in extensive regulatory action on angiotensin-II-receptor antagonists and recall of finished drug products by the pharmaceutical industry to ensure patient safety. The root cause of contamination was the unintended reaction of common reagents utilized during drug synthesis. This lead to serious effects on drug quality and immediate regulatory action. Thus, routine analysis of drug product contents are inevitable and necessitate thoroughly performed work up procedures of the product as well as adequate validated analytical methods. The nature of N-nitrosamines (NA), ranging from small, semi-volatile compounds up to highly polar molecules, effort sophisticated requirements in terms of instrumental analysis. Up today, gas as well as liquid chromatographic devices coupled to mass spectrometers are the most widespread systems for analysis. Gas chromatographic - mass spectrometric (GC-MS) systems, obviously superior towards liquid chromatography - mass spectrometry (LC-MS) for detecting small volatile compounds like NDMA, reach their limits for broadly designed studies including polar or acidic NA. In this study, a complementary and highly sensitive approach by means of liquid chromatography - tandem mass spectrometry (LC-MS/MS) is presented, including detection of 13 NA deduced from major classes of secondary amines. Thereby, the fully validated approach was performed in accordance to ICH and European Medicines Agency (EMA) guidelines. Quantitative proof-of-concept measurements with various APIs and market authorized tablets as representative drug formulations conclude applicability for further presumably contaminated substances. The approach employs organic or inorganic extraction steps with solid phase extraction (SPE). The limit of detection for the most prominent NA, NDMA and N-diethylnitrosamine (NDEA), were both 0.025 parts-per-billion (ppb) per matrix, respectively.
    Keywords:  Analysis; Liquid chromatography; Mass spectrometry; NDMA; Nitrosamines; Validation