bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒02‒06
sixteen papers selected by
Sofia Costa
Icahn School of Medicine at Mount Sinai

  1. Anal Chem. 2022 Feb 04.
      Acyl-coenzyme A derivatives (acyl-CoAs) are core molecules in the fatty acid and energy metabolism across all species. However, in vivo, many other carboxylic acids can form xenobiotic acyl-CoA esters, including drugs. More than 2467 acyl-CoAs are known from the published literature. In addition, more than 300 acyl-CoAs are covered in pathway databases, but as of October 2020, only 53 experimental acyl-CoA tandem mass spectra are present in NIST20 and MoNA libraries to enable annotation of the mass spectra in untargeted metabolomics studies. The experimental spectra originated from low-resolution ion trap and triple quadrupole mass spectrometers as well as high-resolution quadrupole-time of flight and orbital ion trap instruments at various collision energies. We used MassFrontier software and the literature to annotate fragment ions to generate fragmentation rules and intensities for the different instruments and collision energies. These rules were then applied to 1562 unique species based on [M+H]+ and [M-H]- precursor ions to generate two mass spectra per instrument platform and collision energy, amassing an in silico library of 10,934 accurate mass MS/MS spectra that are freely available at The spectra can be imported into a commercial or freely available mass spectral search tool. We used the libraries to annotate 23 acyl-CoA esters in mouse liver, including 8 novel species.
  2. J Pharm Biomed Anal. 2022 Jan 19. pii: S0731-7085(22)00017-6. [Epub ahead of print]211 114596
      Ultra-high-performance liquid chromatography (UHPLC) hyphenated to tandem mass spectrometric methods using the dimethylpentafluorophenylpropyl stationary phase with polar and nonpolar characteristics of a 2-μm fused-core silica particle packing were evaluated to perform efficient separation for the analysis of donepezil in human plasma. The fused-core silica particle design providing the shorter diffusional mass transfer path for the analytes is less affected in resolving power by increasing mobile-phase velocity for faster chromatographic resolution between the administered compound and multiple interfering peaks from the extracted quality control, calibration standard and study samples following simple protein precipitation extraction procedures. In this work, two UHPLC-MS/MS approaches requiring 1.2 min per sample run time and neither expensive ultra-high-pressure instrumentation nor new laboratory protocols were applied and compared for determination of donepezil in human plasma at sub-nanograms per milliliter region to support a randomized, crossover bioequivalence (BE) study in which healthy volunteers each received a single oral dose of the test and reference formulations of 10 mg donepezil hydrochloride. The column performance in terms of chromatographic separation efficiency, peak asymmetry and resolution and retention time reproducibility was found to be sustainable over a thousand extracted plasma injections. The linear dynamic range was detected over a concentration range of 0.2-50 ng/mL. The intra- and inter-day assay accuracy and precision were within 15% for the analyte in individual biological fluids. Positive correlation coefficients (r) greater than 0.98 and 0.99 for donepezil concentrations in study plasma samplers measured by the proposed UHPLC-MS/MS approaches and another validated HPLC-MS/MS method were observed.
    Keywords:  Bioequivalence study; Donepezil; Fused-core; Human plasma; Liquid chromatography-tandem mass spectrometer
  3. Mol Nutr Food Res. 2022 Feb 02. e2100922
      SCOPE: We present a workflow to construct a Dietary Exposome Library (DEL) comprised of phytochemicals and their metabolites derived from host and gut microbiome metabolism for use in peak identification/annotation of untargeted metabolomics datasets.METHODS AND RESULTS: An evidence mapping initiative established target analytes related to the consumption of phytochemical-rich foods. Analytes were confirmed by UPLC-MS(n) analysis of human biospecimens from dietary intervention studies of (poly)phenol-rich diets. One hundred and sixty six verified compounds were subsequently analyzed on an untargeted metabolomics platform to acquire chromatographic and high-resolution mass spectral data for construction of a DEL. The DEL facilitated identification/annotation of 123 metabolites associated with exposure to (poly)phenol enriched diets, which included aromatic ketones, benzoic acids, ellagic acids, caffeoylquinic acids, catecholamines, coumarins, hippuric acid, hydroxytoluenes, phenylamines, stilbenes, urolithins, valerolactones, and xanthonoids, in untargeted metabolomics datasets acquired from human plasma and urine reference materials.
    CONCLUSIONS: The DEL focusing on (poly)phenols and their metabolites of dietary exposure facilitated identification/annotation of ingested food components and their associated pathways in untargeted metabolomics datasets acquired from human biospecimens. The DEL continues to expand with the aim to provide evidence-based data for dietary metabolites in exposome research and inform the development of dietary intervention strategies. This article is protected by copyright. All rights reserved.
    Keywords:  dietary exposome; dietary exposome library (DEL); metabolites; polyphenols; untargeted metabolomics
  4. Anal Chem. 2022 Feb 01.
      Chemical isotope labeling liquid chromatography mass spectrometry (LC-MS) is an emerging metabolomic strategy for the quantification and characterization of small molecular compounds in biological samples. However, its subsequent data analysis is not straightforward due to a large amount of data produced and interference of biological matrices. In order to improve the efficiency of searching and identification of target endogenous metabolites, a new software tool for nontargeted metabolomics data processing called MS-IDF was developed based on the principle of a narrow mass defect filter. The developed tool provided two function modules, including IsoFinder and MDFinder. The IsoFinder function module applied a conventional peak extraction method by using a fixed mass differences between the heavy and light labels and by the alignment of chromatographic retention time (RT). On the other hand, MDFinder was designed to incorporate the accurate mass defect differences between or among stable isotopes in the peak extraction process. By setting an appropriate filter interval, the target metabolites can be efficiently screened out while eliminating interference. Notably, the present results showed that the efficiency in compound identification using the new MDFinder module was nearly doubled as compared to the conventional IsoFinder method (an increase from 259 to 423 compounds). The Matlab codes of the developed MS-IDF software are available from github at Based on the MS-IDF software tool, a novel and effective approach from nontargeted to targeted metabolomics research was developed and applied to the exploration of potential primary amine biomarkers in patients with schizophrenia. With this approach, potential biomarkers, including N,N-dimethylglycine, S-adenosine-l-methionine, dl-homocysteine, and spermidine, were discovered.
  5. Anal Chem. 2022 Feb 04.
      Conjugation reactions are of critical significance in human metabolism. Identification of these conjugated metabolites is still challenging. Here, we propose a strategy, post-deconvolution MS/MS spectra extraction with data-independent acquisition (PDMS2E-DIA), to comprehensively profile the glucuronide-conjugated metabolome. PDMS2E-DIA enables the identification of conjugated and unconjugated metabolite pairs through neutral loss filtering combined with a significant change in abundance after the deconjugation reaction. Purified DIA MS/MS spectra were constructed by extracting MS/MS fragments shared between spectra derived from conjugated and unconjugated metabolites. The feasibility of this approach was first demonstrated by the identification of two glucuronide-conjugated metabolite standards spiked in urine samples. For human urine samples, 479 features were structurally annotated as potential glucuronide-conjugated metabolites, resulting in the identification of 211 metabolites. Fragment peaks derived from interferents were found to be removed by PDMS2E-DIA, which increased about 6 times the number of identified urine metabolites compared with those calculated from raw DIA deconvoluted MS/MS spectra. This approach was found to have great potential for identifying glucuronide-conjugated metabolites, as well as other kinds of chemical conjugations.
  6. Biomed Chromatogr. 2022 Feb 01. e5351
      In recent years, tobacco smoking is a risk factor for a series of diseases including cardiovascular diseases, cerebrovascular diseases, and cancers. Nicotine, the primary component of tobacco smoke, is mainly transformed to its active metabolite cotinine, which is often used as biomarker for tobacco exposure for its higher blood concentration and longer residence time than nicotine. Various analytical methods have been developed for the determination of nicotine and cotinine in biological matrices. This article reviewed the HPLC-MS based methods for nicotine and/or cotinine analysis in various biological matrices. The sample preparation, mass and chromatographic conditions and method validation results of these methods have been summarized and analyzed. Sample was mainly pretreated by protein precipitation and/or extraction. Separation was achieved using methanol and/or acetonitrile:water (with or without ammonium acetate) on C18 columns, and acetonitrile:water (with formic acid, ammonium acetate/formate) on HILIC columns. Nicotine-d3, nicotine-d4 and cotinine-d3 were commonly used internal standards. Other non-deuterated IS were also used such as ritonavir, N-ethylnorcotinine, and milrinone. For both nicotine and cotinine, the calibration range was 0.005-35000 ng/mL, the matrix effect was 75.96% - 126.8% and the recovery was 53% - 124.5%. The two analytes were stable at room temperature for 1-10 days, at -80 °C for up to 6 months, and after 3-6 freeze-thaw cycles. Comedications did not affect nicotine and cotinine analysis.
    Keywords:  Analysis; Cotinine; HPLC-MS; Method development; Method validation; Nicotine; Review
  7. J Pharm Biomed Anal. 2022 Jan 11. pii: S0731-7085(22)00003-6. [Epub ahead of print]211 114582
      In this study, a sensitive and rapid ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous analysis of cytarabine (ara-C), cytarabine monophosphate (ara-CMP), cytarabine diphosphate (ara-CDP) and cytarabine triphosphate (ara-CTP) in the cytosol and nucleus. The separation of analytes and endogenous interferents was achieved in 8 min on a hypercarb column (2.1 mm × 100 mm, 3 µm) by using a gradient elution with 95% acetonitrile and aqueous 5 mM hexylamine with 0.4% (v/v) diethylamine adjusted to pH 10. The analytes were detected with both negative and positive electrospray ionization in multiple reaction monitoring (MRM) mode. The calibration curve demonstrated good linearity ranging from 5 to 750 nM for ara-C, 50-7500 nM for ara-CMP, 20-3000 nM for ara-CDP and 1-150 nM for ara-CTP in the cytosol. In the nucleus, good linearity was achieved over a concentration range of 1-100 nM for ara-C, 5-500 nM for ara-CMP, 2.5-250 nM for ara-CDP and 0.5-50 nM for ara-CTP. Intra- and interbatch accuracies and precisions met the standards of validation. The matrix effect, recovery and stability were also within acceptable ranges. After incubation with 10 μM ara-C for 3 h, the levels of ara-C, ara-CMP, ara-CDP and ara-CTP in the cytosol and nucleus of HL-60 cells and HL-60/ara-C cells were determined. Most of the metabolites were found within the quantitation range. The results showed that the nuclear ara-CTP level was significantly different than the intracellular ara-CTP level between HL-60 and HL-60/ara-C cells.
    Keywords:  Ara-C; Ara-CTP; Cellular pharmacokinetics; Centrifugal ultrafiltration; Intranuclear level; UHPLC-MS/MS
  8. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Jan 26. pii: S1570-0232(22)00042-3. [Epub ahead of print]1192 123138
      Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are synthetic chemicals that have been used in various industries and household products. These can easily accumulate in the human body, causing adverse effects on human health. In this study, a high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous analysis of PFOA and linear PFOS in human serum. Owing to a lack of PFOA- and PFOS-free human serum, 13C8-PFOA and 13C8-PFOS were used as surrogate analytes for quantification. A sensitive and selective sample preparation method was developed and optimized by combining solid-phase extraction and protein precipitation method. The lower limit of quantification was 0.05 ng/mL, and the analytical response was linear up to 10 ng/mL for both PFOA and linear PFOS. Chromatographic separation of the linear PFOS from branched isomers was achieved within 5.5 min. The method was validated at various concentrations and afforded acceptable accuracy and precision values. After validation, the method was successfully applied to evaluate the exposure levels of PFOA and linear PFOS in the Korean population. The serum concentrations of PFOA and linear PFOS were 0.42-28.3 ng/mL and 0.81-57.6 ng/mL, respectively. The median concentration of linear PFOS was approximately 2.6-fold higher than that of PFOA. The concentration of PFOA was higher in women than men (p < 0.05) and that of linear PFOS was not significantly different between men and women. Therefore, a sensitive, selective, and reliable bioanalytical method was developed and validated. This method can potentially be applied to biomonitoring studies involving PFOA and linear PFOS.
    Keywords:  HPLC-MS/MS; Human serum; Korean population; Perfluorooctanesulfonic acid; Perfluorooctanoic acid; Surrogated analyte
  9. J Sep Sci. 2022 Feb 02.
      Trans-fatty acids are unsaturated fatty acids that are considered to have health risks. 1,3,5,7-Tetramethyl-8-butyrethylenediamine-difluoroboradiaza-s-indacene is a highly-sensitive fluorescent labeling reagent for carboxylic acids developed by our lab. In this study, using this pre-column fluorescent derivatization reagent, a rapid and accurate high-performance liquid chromatography-fluorescence detection method was developed for the determination of two trans-fatty acids in food samples. Under the optimized derivative conditions, two trans-fatty acids were tagged with the fluorescent labeling reagent in the presence of 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide at 25 °C for 30 min. Then, the baseline separation of trans- and cis-fatty acids and their saturated fatty acid with similar structures was achieved with less interference using a reversed-phased C18 column with isocratic elution in 14 min. With fluorescence detection at λex /λem = 490 nm/510 nm, the linear range of the trans-fatty acids was 1.0-200 nM with low detection limits in the range of 0.1-0.2 nM (signal-to-noise ratio = 3). In addition, the proposed approach was successfully applied for the detection of trans-fatty acids in food samples, and the recoveries using this method ranged from 96.02% to 109.22% with low relative standard deviations of 1.2-4.3% (n = 6). This article is protected by copyright. All rights reserved.
    Keywords:  derivatization; fluorescence detection; high-performance liquid chromatography; trans-fatty acids
  10. J Chem Ecol. 2022 Jan 31.
      In the study of chiral biologically active compounds such as pheromones, the analysis of the stereoisomer composition is essential to gain more insight into their stereochemical diversity, which affects the pheromone communication channels and therefore the diversification of species. This mini-review summarizes the development of fluorescence derivatization reagents for high-performance liquid chromatographic (HPLC) determination of the absolute configuration and stereoisomer composition of natural products with a chiral branched alkyl chain. The diastereomeric separation of anteiso fatty acids bearing a branched methyl group up to the C-26 position was achieved by reversed-phase HPLC under very low column temperature conditions using (1S,2S)-2-(2,3-anthracenedicarboximido)cyclohexanol as a derivatization reagent, enabling fluorescent detection of these compounds at femtomole levels. This method was also applicable to chiral alcohols and amines with chiral branched methyl groups using similar reagents containing a carboxyl group. These reagents were successfully applied to determine the absolute configurations and stereoisomer composition of the chiral alkyl chain of natural compounds including some insect pheromones, miyakosyne A, and plakoside A. The combination of these reagents and two-dimensional HPLC constitutes a very powerful tool for the analysis of the stereoisomers of natural crude samples. Furthermore, the analysis of some natural bioactive substances using this method demonstrated that natural substances are not always optically pure, consisting instead of stereoisomer mixtures exhibiting stronger activity than optically pure enantiomers. These results cast doubts on the concept of biological homochirality and demonstrate that natural pheromones do not always show the highest activity among all stereoisomers.
    Keywords:  chiral branched alkyl chain; chiral derivatization reagents; determination of absolute configuration; determination of stereoisomer composition; high-performance liquid chromatography (HPLC)
  11. Front Nutr. 2021 ;8 812803
      The accumulation of antimicrobial residues in edible animal products and aquaculture products could pose health concerns to unsuspecting consumers. Hence, this study aimed to develop a validated method for simultaneous quantification of chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in beef, pork, chicken, shrimp, eel, and flatfish using a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Primary-secondary amine (PSA) and MgSO4 were used for sample purification. The analytes were separated on a reversed-phase analytical column. The coefficients of determination for the linear matrix-matched calibration curves were ≥0.9941. Recovery rates ranged between 64.26 and 116.51% for the four analytes with relative standard deviations (RSDs) ≤ 18.05%. The calculated limits of detection (LODs) and limits of quantification (LOQs) were 0.005-3.1 and 0.02-10.4 μg/kg, respectively. The developed method was successfully applied for monitoring samples obtained from local markets in Seoul, Republic of Korea. The target residues were not detected in any tested matrix. The designed method was versatile, sensitive, and proved suitable for quantifying residues in animal-derived products.
    Keywords:  LC-MS/MS; chloramphenicol; florfenicol; florfenicol amine; method development; residue analysis; thiamphenicol
  12. Ther Drug Monit. 2022 Jan 28.
      INTRODUCTION: Although unbound ciprofloxacin is responsible for antibacterial effects, assays measuring the unbound drug plasma concentrations are scarce. This study aimed to develop and validate a rapid, reproducible, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the determination of total and unbound ciprofloxacin plasma concentrations.METHODS: The determination of total ciprofloxacin concentrations required a 10 μL sample, while for unbound ciprofloxacin concentrations it was 100μL. Unbound ciprofloxacin was separated from protein-bound ciprofloxacin via ultrafiltration. A deuterated internal standard was used, and the sample preparation involved protein precipitation. The method was fully validated over a concentration range of 0.02-5.0 mg/L, according to the U.S. Food and Drug Administration guidelines. In addition, its clinical application was demonstrated.
    RESULTS: The total run time was 1.5 minutes. For total ciprofloxacin plasma concentrations, the mean accuracy ranged from 94.5%-105.0% across the validated range, the intra-day imprecision was ≤7.6%, and the inter-day imprecision was ≤9.8%. For unbound ciprofloxacin plasma concentrations, the mean accuracy ranged from 92.8%-102.1% across the validated range, the intra-day imprecision was ≤7.0%, and the inter-day imprecision was ≤9.6%. Ciprofloxacin in plasma and ultrafiltrate remained stable for at least 96 h at room temperature, at least four years at -80 °C, and at least three freeze/thaw cycles (-80 °C), with a minimum interval of 24 hours.
    CONCLUSION: The presented method is precise and accurate. It has been implemented in clinical care and research projects at a university hospital, permitting rapid determination of total and unbound ciprofloxacin.
  13. J Proteome Res. 2022 Feb 01.
      N-Acyl-l-homoserine lactones (AHLs) are a large family of signaling molecules in "quorum sensing" communication. This mechanism is present in a number of bacterial physiological phenomena, including pathogenic phenomena. In this study, we described a simple and accessible way to detect, annotate, and quantify these compounds from bacterial culture media. Analytical standards and ethyl acetate bacterial extracts containing AHLs were analyzed by an ultra-high-performance liquid chromatography system coupled to a mass spectrometer using a nontargeted FullMS data-dependent MS2 method. The results were processed in MZmine2 and then analyzed by a Feature-Based Molecular Networking (FBMN) workflow in the Global Natural Products Social Networking (GNPS) platform for the discovery and annotation of known and unknown AHLs. Our group analyzed 31 AHL standards and included the MS2 spectra in the spectral library of the GNPS platform. We also provide the 31 standard AHL spectrum list for inclusion in molecular networking analyses. FBMN analysis annotated 30 out of 31 standards correctly. Then, as an example, a set of five bacterial extracts was prepared for AHL annotation. Following the method described in this Article, 5 known and 11 unknown AHLs were properly annotated using the FBMN-based molecular network approach. This study offers the possibility for the automatic annotation of known AHLs and the search for nonreferenced AHLs in bacterial extracts in a somewhat straightforward approach even without acquiring analytical standards. The method also provides relative quantification information.
    Keywords:  N-acyl-l-homoserine lactones; automatic annotation; molecular network; quorum sensing
  14. Anal Chem. 2022 Feb 02.
      Compound-specific stable isotope analysis (CSIA) is a unique analytical technique for determining small variations in isotope ratios of light isotopes in analytes from complex mixtures. A problem of CSIA using gas chromatography (GC) and liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) is that any structural information of the analytes is lost due to the processes involved in determining the isotope ratio. To obtain the isotopic composition of, for example, carbon from organic compounds, all carbon in each analyte is quantitatively converted to CO2. For GC-IRMS, open split GC-IRMS-MS couplings have been described that allow additional acquisition of structural information of analytes and interferences. Structural analysis using LC-IRMS is more difficult and requires additional technical and instrumental efforts. In this study, LC was combined for the first time with simultaneous analysis by IRMS and high-resolution mass spectrometry (HRMS), enabling the direct identification of unknown or coeluting species. We have thoroughly investigated and optimized the coupling and showed how technical problems, arising from instrumental conditions, can be overcome. To this end, it was successfully demonstrated that a consistent split ratio between IRMS and HRMS could be obtained using a variable postcolumn flow splitter. This coupling provided reproducible results in terms of resulting peak areas, isotope values, and retention time differences for the two mass spectrometer systems. To demonstrate the applicability of the coupling, we chose to address an important question regarding the purity of international isotope standards. In this context, we were able to confirm that the USGS41 reference material indeed contains substantial amounts of pyroglutamic acid as suggested previously in the literature. Moreover, the replacement material, USGS41a, still has significant amounts of pyroglutamic acid as impurity, rendering some caution necessary when using this material for isotopic calibration.
  15. Drug Test Anal. 2022 Feb 01.
      N-(2-Substituted benzyl)-2,5-dimethoxyphenethylamines often cause severe poisonings which has led to their legal prohibition in many countries. At the same time, their positional isomers can be studied as potential therapeutic drugs. In this regard, the search for various approaches to differentiate these isomers is an important practical task, the solution of which would guarantee from identification errors during laboratory analysis. In this paper, the possibilities of differentiation of isomers varying in the position of two methoxy groups in the phenylethyl part of the molecule are considered on the example of compounds of NBF, NBCl and NBBr series by chromatography-mass spectrometry methods. Gas or liquid reverse-phase chromatography in the proposed chromatographic separation modes has demonstrated their ability to resolve this problem reliably. Data on retention indices of isomeric compounds and their derivatives can serve as an additional identification criterion for gas chromatography-mass spectrometry (GC-MS) analysis. Differentiation of NBF and NBCl isomers using electron ionization (EI) mass spectra is feasible only if both the spectrum of the compound and its N-trifluoroacetyl derivative are registered; differentiation of NBBr positional isomers under these conditions does not require obtaining the derivatives. Using electrospray ion source, the compounds can easily be differentiated based on the distinctive features of their collision induced dissociation (CID) spectra recorded at low energy values, which also does not require the synthesis of derivatives. The data presented in current paper will be useful for analysis in laboratories providing the determination of narcotic drugs.
    Keywords:  N-benzylphenethylamines; chromatography; differentiation; mass spectrometry; positional isomers
  16. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Jan 12. pii: S1570-0232(21)00567-5. [Epub ahead of print]1192 123086
      Monosaccharides are important players in cell metabolism and potential biomarkers. An effective tool to quantify monosaccharides is required in basic research and healthcare. In this study, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that could simultaneously quantify 14 free monosaccharides and evaluated its performance according to clinical guidance. The LC-MS/MS step separated and quantified 14 monosaccharides with 6 min. The coefficient of variation at the lower limit of quantification was less than 20% for each analyte. The R square values from linear regression analyses were all greater than 0.995. The validated assay was employed to profile free monosaccharides in conditioned media from cell culture and patient sera from glucose tolerance test. Both LO2 cells and HEK293 cells consumed D-glucosamine, D-glucose and produced D-glucuronic acid, N-acetyl-D-glucosamine. Additionally, LO2 cells produced D-mannose and L-fucose, whereas HEK293 cells consumed D-mannose. In patient sera from glucose tolerance test, the level of D-glucose increased significantly after glucose intake, but the levels of other monosaccharides didn't. In conclusion, the LC-MS/MS assay we developed for 14-monosaccharide profiling met clinical requirements. The monosaccharide profiling results showed the distinct monosaccharide metabolism between liver and kidney cells, and the ignorable diet effect on 6 serum monosaccharides.
    Keywords:  Biofluid; Biomarker; Free monosaccharide; LC-MS/MS; Profiling