bims-metlip 2023-05-07 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2023–05–07
twenty-one papers selected by
Sofia Costa, Matterworks

A validated HPLC-MS/MS method for simultaneously analyzing curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetra-hydrocurcumin and piperine in human plasma, urine or feces.
A Rapid Derivatization for Quantitation of Perfluorinated Carboxylic Acids from Aqueous Matrices by Gas Chromatography-Mass Spectrometry.
Performance comparison of narrow-bore and capillary liquid-chromatography for non-targeted metabolomics profiling by HILIC-QTOF-MS.
Separation and identification of monoglucuronides of vitamin D3 metabolites in urine by derivatization-assisted LC/ESI-MS/MS using a new Cookson-type reagent.
LC-HRMS based metabolomics workflow: an alternative strategy for metabolite identification in the anti-doping field.
Standard addition method (SAM) in LC-MS/MS to quantify gluten-derived metabolites in urine samples.
Regions of Interest Multivariate Curve Resolution Liquid Chromatography with Data-Independent Acquisition Tandem Mass Spectrometry.
Statistical Framework for Identifying Differences in Similar Mass Spectra: Expanding Possibilities for Isomer Identification.
A simple ion chromatography mass spectrometry method for amino acid analysis in beer.
A new method based on dispersive liquid-liquid microextraction for fat soluble vitamins determination in serum by LC-MS/MS.
Liquid Chromatography-Tandem Mass Spectrometry Analysis of Aminoglycosides in Foods Using an Ethylene-Bridged Hybrid Zwitterionic Stationary Phase and Hydrophilic-Lipophilic-Balanced Solid-Phase Extraction Cartridges.
LC-MS/MS-based multiplex antibacterial platform for therapeutic drug monitoring in intensive care unit patients.
Simultaneous determination of 24 antiepileptic drugs and their active metabolites in human plasma by UHPLC-MS/MS.
Evaluating Retention Index Score Assumptions to Refine GC-MS Metabolite Identification.
Development and Validation of an Ultrasensitive LC-MS/MS Method for the Quantification of Melatonin in Human Saliva.
A high-throughput metabolomics in vitro platform for the characterization of hepatotoxicity.
Development and application of a data processing method for food metabolomics analysis.
New Approach for the Identification of Isobaric and Isomeric Metabolites.
Metabolomics Analysis of Urinary Extracellular Vesicles by Nuclear Magnetic Resonance and Liquid Chromatography-Mass Spectrometry.
Quantitative NMR spectroscopy of complex mixtures.
Practical considerations for rapid and quantitative NMR-based metabolomics.

Heliyon. 2023 May;9(5): e15540

A validated HPLC-MS/MS method for simultaneously analyzing curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetra-hydrocurcumin and piperine in human plasma, urine or feces.

M A G M Kroon, H W M van Laarhoven, E L Swart, E M Kemper, O van Tellingen.

   Background: The spice curcumin is supposed to have many different beneficial health effects. To understand the complete pharmacokinetics of curcumin we need an analytical method to determine curcumin and its metabolites in human plasma, urine or feces. We have developed an HPLC-MS/MS method for the simultaneous analysis of curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin and piperine in human plasma, urine or feces.
Methods: Sample pretreatment involved a simple liquid-liquid extraction with tert-butyl methyl ether. Conjugated curcumin and analogs can be measured after enzymatic hydrolysis. Reversed-phase chromatography with a linear gradient of 50-95% methanol in 0.1% formic acid was used. Total run time is 15 min. The method was validated with regards to stability, specificity, sensitivity, linearity, accuracy, repeatability and reproducibility. The applicability of the method was tested using actual patients samples.
Results: The LLOQ in plasma, urine and feces for curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin and piperine ranged from 1 to 5 nM. Whereas all compounds could be quantified on a linear range between 2 and 400 nM. Plasma and feces recovery of curcumin was 97.1 ± 3.7% and 99.4 ± 16.2%, whereas urine showed a recovery of 57.1 ± 9.3%. All compounds had acceptable in-between day or between day variability in the different matrixes.
Conclusion: A HPLC-MS/MS method was developed and validated for the simultaneous quantification of curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin and piperine in human plasma, urine or feces. This method will aid in critically verifying the pharmacokinetics of curcumin made by supplement manufacturers and help us to provide insight in the claimed bioavailability of curcumin supplements.

Keywords:  Curcumin; Curcuminoids; HPLC-MS/MS; Human feces; Human plasma; Human urine; Piperine; Tetrahydrocurcumin; β-glucuronidase

DOI:  https://doi.org/10.1016/j.heliyon.2023.e15540
Anal Chem. 2023 May 03.

A Rapid Derivatization for Quantitation of Perfluorinated Carboxylic Acids from Aqueous Matrices by Gas Chromatography-Mass Spectrometry.

RenXi Ye, Robert A Di Lorenzo, Jessica T Clouthier, Cora J Young, Trevor C VandenBoer.

Ultrashort-chain perfluorinated carboxylic acids (PFCAs) are receiving more attention due to their ever-increasing presence in the environment. Methods have been established for the analysis of short- and long-chain PFCAs, while robust quantitation of ultrashort-chain species is scarce. Here, we develop a novel derivatization method using diphenyl diazomethane for quantitation of C2-C14 PFCAs in aqueous matrices. The method is highlighted by rapid completion of derivatization (<1 min) and retention and separation of ultrashort-chain (C2/C3) PFCA derivatives using H2 carrier gas (R > 1.5). A weak anion exchange solid-phase extraction procedure for analyte recovery from representative aqueous samples was developed and validated by spike and recovery from ultrapure water, synthetic ocean water, and simulated denuder extracts used for collecting gaseous PFCAs. Recoveries for PFCAs ranged from 83 to 130% for the majority of analytes and matrices. The instrument detection limits (IDLs) range from 8 to 220 fg per injection, and method detection limits (MDLs) range from 0.06 to 14.6 pg/mL for 500 mL aqueous samples, which are within an order of magnitude to conventional LC-MS/MS methods. The method was applied to the analysis of real samples of tap water, rainwater, ocean water, and annular denuder extracts. The overall method provides a cost-effective alternative to conventional LC-MS/MS methods, overcoming the typical GC-MS drawbacks of high detection limits and long sample preparation times while being able to simultaneously analyze the complete spectrum of environmentally relevant PFCAs.

DOI: https://doi.org/10.1021/acs.analchem.3c00593
Talanta. 2023 Apr 22. pii: S0039-9140(23)00329-6. [Epub ahead of print]260 124578

Performance comparison of narrow-bore and capillary liquid-chromatography for non-targeted metabolomics profiling by HILIC-QTOF-MS.

Sylvia K Neef, Ute Hofmann, Thomas E Mürdter, Matthias Schwab, Mathias Haag.

  Clinical metabolomics studies often have to cope with limited sample amounts, thus miniaturized liquid chromatography (LC) systems are a promising alternative. Their applicability has already been demonstrated in various fields, including a few metabolomics studies that mainly used reversed-phase chromatography. However, hydrophilic interaction chromatography (HILIC), which is widely used in metabolomics due to its particular suitability for the analysis of polar molecules, has rarely been tested for miniaturized LC-MS analysis of small molecules. In the present work, the suitability of a capillary HILIC (CapHILIC)-QTOF-MS system for non-targeted metabolomics was evaluated based on extracts of porcine formalin-fixed, paraffin-embedded (FFPE) tissue samples. The performance was assessed with respect to the number and retention time span of metabolic features as well as the analytical repeatability, the signal-to-noise ratio and the signal intensity of 16 annotated metabolites from different compound classes. The results were compared with a well established narrow-bore HILIC-QTOF-MS system. Both platforms have detected a similar number of features and performed excellent with respect to retention time stability (median RT span <0.05 min) and analytical repeatability (>75% of features with CV < 20%). The signal areas of all metabolites assessed were increased up to 18-fold by the use of CapHILIC, although the signal-to-noise ratio was only improved for 50% of the metabolites. An even better reproducibility (median CV = 5.2%) and up to 80-fold increase in signal intensity were observed after optimization of CapHILIC conditions for analysis of bile acid standard solutions. Even though the observed improvement for specific bile acids (e.g. taurocholic acid) in biological matrix needs to be evaluated, the platform comparison indicates, that the tested CapHILIC system is particularly suitable for analyses of a less broad metabolite spectrum, and specifically optimized chromatography.

Keywords:  Hydrophilic interaction chromatography; LC miniaturization; LC-MS; Metabolomics; Platform comparison; Q-TOF

DOI:  https://doi.org/10.1016/j.talanta.2023.124578
Anal Sci. 2023 May 02.

Separation and identification of monoglucuronides of vitamin D3 metabolites in urine by derivatization-assisted LC/ESI-MS/MS using a new Cookson-type reagent.

Kazunari Takada, Shoujiro Ogawa, Natsuki Yoshida, Tatsuya Higashi.

  A liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method was developed using a new Cookson-type reagent, 4-[4-(1-pipelidinyl)phenyl]-1,2,4-triazoline-3,5-dione (PIPTAD), to analyze the monoglucuronides (Gs) of vitamin D3 metabolites in human urine. The G of 23S,25-dihydroxyvitamin D3 [23,25(OH)2D3] was previously found as a major metabolite of vitamin D3 in the urine, but its conjugation position remained undetermined. Determination of the position was an important research issue to clarify the whole picture of the excretion of surplus 25-hydroxyvitamin D3 [25(OH)D3, the circulating form of vitamin D3] in humans. After the pretreated urine sample was derivatized with PIPTAD, the peak corresponding to the G of 23,25(OH)2D3 was satisfactorily separated from the urine-derived interfering substances on reversed-phase LC, which could not be achieved by using the previous analogous reagent, DAPTAD. The PIPTAD-derivatized Gs of the vitamin D3 metabolites provided characteristic product ions useful for identifying the conjugation positions during the MS/MS. Accordingly, we successfully determined the glucuronidated position of 23,25(OH)2D3 to be the C23-hydroxy group. The developed method also enabled the simultaneous detection of Gs of 25(OH)D3 and 24R,25-dihydroxyvitamin D3 as well as 23,25(OH)2D3-23-G without interference from the urine components.

Keywords:  Cookson-type reagent; Derivatization; LC/ESI–MS/MS; Monoglucuronide; Urine; Vitamin D3 metabolite

DOI:  https://doi.org/10.1007/s44211-023-00350-4
Rapid Commun Mass Spectrom. 2023 May 05. e9532

LC-HRMS based metabolomics workflow: an alternative strategy for metabolite identification in the anti-doping field.

P Leogrande, D Jardines, D Martinez-Brito, X de la Torre, M K Parr, F Botrè.

RATIONALE: The proposed metabolomic workflow, based on coupling high-resolution mass spectrometry with computational tools, can be an alternative strategy for metabolite detection and identification. This approach allows the extension of the investigation field to chemically different compounds, maximizing the information obtainable from the data and minimizing the time and resources required.
METHODS: Urine samples were collected from 5 healthy volunteers before and after oral administration of 3β-hydroxyandrost-5-ene-7,17-dione as a model compound and defining three excretion time intervals. Raw data were acquired in both positive and negative ionization modes using an Agilent Technologies 1290 Infinity II series HPLC coupled to a 6545 Accurate-Mass Quadrupole Time-of-Flight. They were then processed to align peak retention times with the same accurate mass, and the resulting data matrix was subjected to multivariate analysis.
RESULTS: Multivariate analysis (PCA and PLS-DA models) demonstrated high similarity between samples belonging to the same collection time interval and clear discrimination between different excretion intervals. The blank and long excretion groups were distinguished suggesting the presence of long excretion markers, which are of remarkable interest in anti-doping analyses. The correspondence of some significant features with metabolites reported in the literature confirmed the rationale and usefulness of the proposed metabolomic approach.
CONCLUSIONS: The presented study proposes a metabolomics workflow for the early detection and characterization of drug metabolites by untargeted urinary analysis to reduce the range of substances still excluded from routine screening. Its application has detected minor steroid metabolites, as well as unexpected endogenous alterations, proving to be an alternative strategy that can allow gathering a more complete range of information in the antidoping field.

DOI: https://doi.org/10.1002/rcm.9532
J Pharm Biomed Anal. 2023 Apr 24. pii: S0731-7085(23)00185-1. [Epub ahead of print]232 115416

Standard addition method (SAM) in LC-MS/MS to quantify gluten-derived metabolites in urine samples.

Albino Coglianese, Bruno Charlier, Francesca Mensitieri, Amelia Filippelli, Viviana Izzo, Fabrizio Dal Piaz.

  A tight adherence to a gluten-free diet (GFD), the most effective treatment currently available for celiac disease, is important to reduce symptoms, avoid nutritional deficiencies and improve quality of life in celiac patients. The development of analytical methods allowing detecting gluten exposure due to occasional or involuntary food transgressions could represent a useful tool to monitor patient habits and conditions and prevent long-term complications. The aim of this work was to develop and validate an approach based on the standard addition methodology (SAM) for the detection and quantification of two main metabolites of alkylresorcinols, 3,5-dihydroxybenzoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-propanoic acid (DHPPA), whose presence in urine samples is related to the intake of gluten-containing foods. Analytically, the method consisted of a protein precipitation step followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. The chromatographic method involved the use of a hydrophilic interaction liquid chromatography (HILIC) in a direct phase approach; LC-MS/MS analyses were performed in selected reaction monitoring (SRM) mode. Manipulation and instrumental errors were normalised using stable isotopic standards (ISs). The SAM approach here described requires less than 1 mL of urine per sample, thus greatly reducing the sample volume needed. Noteworthy, despite the small cohort of samples analysed, our data allowed to identify a potential "threshold" value, around 200 ng/mL for DHBA and 400 ng/mL for DHPPA, to discriminate between a GFD and a gluten rich diet (GRD).

Keywords:  Bioanalytical methods validation; Celiac disease; Endogenous quantification; Gluten free diet monitoring; LC-MS/MS; Standard addition method (SAM)

DOI:  https://doi.org/10.1016/j.jpba.2023.115416
Anal Chem. 2023 May 05.

Regions of Interest Multivariate Curve Resolution Liquid Chromatography with Data-Independent Acquisition Tandem Mass Spectrometry.

Carlos Pérez-López, Bernat Oró-Nolla, Silvia Lacorte, Romà Tauler.

New data-independent acquisition (DIA) modes coupled to chromatographic separations are opening new perspectives in the processing of massive mass spectrometric (MS) data using chemometric methods. In this work, the application of the regions of interest multivariate curve resolution (ROIMCR) method is shown for the simultaneous analysis of MS1 and MS2 DIA raw data obtained by liquid chromatography coupled to quadrupole-time-of-flight MS analysis. The ROIMCR method proposed in this work relies on the intrinsic bilinear structure of the MS1 and MS2 experimental data which allows us for the fast direct resolution of the elution and spectral profiles of all sample constituents giving measurable MS signals, without needing any further data pretreatment such as peak matching, alignment, or modeling. Compound annotation and identification can be achieved directly by the comparison of the ROIMCR-resolved MS1 and MS2 spectra with those from standards or from mass spectral libraries. ROIMCR elution profiles of the resolved components can be used to build calibration curves for the prediction of their concentrations in complex unknown samples. The application of the proposed procedure is shown for the analysis of mixtures of per- and polyfluoroalkyl substances in standard mixtures, spiked hen eggs, and gull egg samples, where these compounds tend to accumulate.

DOI: https://doi.org/10.1021/acs.analchem.2c05704
Anal Chem. 2023 05 02. 95(17): 6996-7005

Statistical Framework for Identifying Differences in Similar Mass Spectra: Expanding Possibilities for Isomer Identification.

Hoi-Ting Wu, Dylan L Riggs, Yana A Lyon, Ryan R Julian.

Isomeric molecules are important analytes in many biological and chemical arenas, yet their similarity poses challenges for many analytical methods, including mass spectrometry (MS). Tandem-MS provides significantly more information about isomers than intact mass analysis, but highly similar fragmentation patterns are common and include cases where no unique m/z peaks are generated between isomeric pairs. However, even in such situations, differences in peak intensity can exist and potentially contain additional information. Herein, we present a framework for comparing mass spectra that differ only in terms of peak intensity and include calculation of a statistical probability that the spectra derive from different analytes. This framework allows for confident identification of peptide isomers by collision-induced dissociation, higher-energy collisional dissociation, electron-transfer dissociation, and radical-directed dissociation. The method successfully identified many types of isomers including various d/l amino acid substitutions, Leu/Ile, and Asp/IsoAsp. The method can accommodate a wide range of changes in instrumental settings including source voltages, isolation widths, and resolution without influencing the analysis. It is shown that quantification of the composition of isomeric mixtures can be enabled with calibration curves, which were found to be highly linear and reproducible. The analysis can be implemented with data collected by either direct infusion or liquid-chromatography MS. Although this framework is presented in the context of isomer characterization, it should also prove useful in many other contexts where similar mass spectra are generated.

DOI: https://doi.org/10.1021/acs.analchem.3c00495
J Sep Sci. 2023 May 06. e2300106

A simple ion chromatography mass spectrometry method for amino acid analysis in beer.

Matthias Schmitt, Florian Fillsack, Andreas Seubert.

  The amino acid footprint of different beer samples was analyzed using ion chromatography coupled to electrospray ionization mass spectrometry. A tailor-made polymer-based cation-exchange resin was operated with a mass spectrometry-compatible eluent under isocratic conditions on a standard HPLC system coupled to a single quadrupole mass spectrometer using formic acid as a volatile eluent ion source. The partially separated peaks of the isomeric pair isoleucine/leucine were processed according to their area response ratio using vertical peak splitting or Gaussian fit. Additionally, the chromatographic resolution of the isomers was optimized with an adjusted, solely aqueous mobile phase from 0.85 to 2.92. Ion suppression in the electrospray ion source was investigated for the derivatization-free method and found to be insignificant (recovery value 100 ± 15%) for 15 out of the 20 analytes. Quantitative results for various beer and mixed-beer beverages were found to be in high agreement with existing methods. Simultaneous photometric detection demonstrated the method's ability to successfully remove most of interfering matrix compounds. This article is protected by copyright. All rights reserved.

Keywords:  Cation exchanger; Ion suppression; Isomer resolution; Liquid chromatography mass spectrometry; Stationary phase

DOI:  https://doi.org/10.1002/jssc.202300106
Biomed Chromatogr. 2023 May 03. e5666

A new method based on dispersive liquid-liquid microextraction for fat soluble vitamins determination in serum by LC-MS/MS.

Jufang Li, Congyan Chen, Xingchao Sun, Zhineng Hu, Chaochao Wu, Qiang Gao, Guoqing Ying.

  A green and inexpensive pretreatment known as dispersive liquid-liquid microextraction (DLLME) was developed in this assay coupled with the LC-MS/MS method for routine analysis of fat soluble vitamins (FSVs). The technique was performed with methanol as dispersive solvent and dichloromethane as extraction solvent. The extraction phase containing FSVs was evaporated to dryness and reconstituted in a mixture of acetonitrile and water. The influence variables concerning DLLME procedure were optimized. After then, the method was investigated its applicability in LC-MS/MS analysis. As a result, the parameters were settled in the optimal condition during the DLLME process. A cheap and lipid-free substance was founded as an alternative to serum to eliminate the matrix effect while preparing the calibrators. The method validation indicated that it was suitable to determine FSVs in serum. Moreover, this method was applied successfully to determine serum samples which was consisitantly with the literature. In summary, the DLLME method developed in this report was reliable and more cost-effective than the traditional method adopted in LC-MS/MS method, which could be applied in the future.

Keywords:  LC-MS/MS; dispersive liquid-liquid microextraction; fat soluble vitamins; serum

DOI:  https://doi.org/10.1002/bmc.5666
J Agric Food Chem. 2023 May 04.

Liquid Chromatography-Tandem Mass Spectrometry Analysis of Aminoglycosides in Foods Using an Ethylene-Bridged Hybrid Zwitterionic Stationary Phase and Hydrophilic-Lipophilic-Balanced Solid-Phase Extraction Cartridges.

Jinchuan Yang, Paul Rainville.

  This work aimed to develop an analytical method for the screening of multiple aminoglycoside residues in foods of animal origin using an ethylene-bridged hybrid (BEH) particle-based sulfoalkylbetaine stationary phase. The effects of chromatographic conditions on the separation of 17 aminoglycosides have been systematically investigated. Sample preparation and mass spectrometry detection have also been investigated and optimized. In contrast to high buffer concentrations in the mobile phase required for silica-based sulfoalkylbetaine stationary phases, a moderate buffer concentration (20 mM) provided the optimal separation of 17 aminoglycosides with the BEH sulfoalkylbetaine stationary phase. The developed method has been evaluated in milk, beef, pork, liver, and honey samples with good performance for retention, selectivity, sensitivity, linearity, precision, and accuracy. The majority of the limit of quantitation estimated with the matrix was less than 25 μg/kg. The overall accuracy across five matrices was in the range from 96 to 111%, with standard deviations of less than 19%.

Keywords:  HILIC; HLB; aminoglycoside; beef; honey; liver; milk; pork; zwitterionic stationary phase

DOI:  https://doi.org/10.1021/acs.jafc.3c01175
Front Pharmacol. 2023 ;14 1116071

LC-MS/MS-based multiplex antibacterial platform for therapeutic drug monitoring in intensive care unit patients.

Liang Liu, Liu Zhang, Xiangyi Zheng, Xing Liu, Wei Liu, Jianhua Wu.

  Empirically prescribed standard dosing regimens of antibacterial agents may result in insufficient or excess plasma concentrations with persistently poor clinical outcomes, especially for patients in intensive care units (ICUs). Therapeutic drug monitoring (TDM) of antibacterial agents can guide dose adjustments to benefit patients. In this study, we developed a robust, sensitive, and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform for the quantification of 14 antibacterial and antifungal agents (beta-lactams piperacillin, cefoperazone, and meropenem; beta-lactamase inhibitors tazobactam and sulbactam; antifungal agents fluconazole, caspofungin, posaconazole, and voriconazole; and daptomycin, vancomycin, teicoplanin, linezolid, and tigecycline) that can be used for patients with severe infection. This assay requires only 100 µL of serum with rapid protein precipitation. Chromatographic analysis was performed using a Waters Acquity UPLC C8 column. Three stable isotope-labeled antibacterial agents and one analogue were used as internal standards. Calibration curves ranged from 0.1-100 μg/mL, 0.1-50 μg/mL, and 0.3-100 μg/mL for different drugs, and all correlation coefficients were greater than 0.9085. Intra- and inter-day imprecision and inaccuracy values were below 15%. After validation, this new method was successfully employed for TDM in routine practice.

Keywords:  ICU; LC-MS/MS; MRM; antibacterial agents; multiplex analysis; therapeutic drug monitoring

DOI:  https://doi.org/10.3389/fphar.2023.1116071
J Pharm Biomed Anal. 2023 May 02. pii: S0731-7085(23)00206-6. [Epub ahead of print]232 115437

Simultaneous determination of 24 antiepileptic drugs and their active metabolites in human plasma by UHPLC-MS/MS.

Ruiqi Jiang, Dongjie Zhang, Zhigang Zhao, Shenghui Mei.

  Antiepileptic drugs (AEDs) have narrow therapeutic ranges with large individual variability. Routine therapeutic drug monitoring of AEDs was useful for dose optimization, but the common immunoassays could not meet the detection requirements of AEDs, especially for new generation AEDs. The aim of this study was to validate an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for simultaneously quantification of 24 AEDs and their active metabolites in human plasma and comparison with a chemiluminescent immunoassay (Simens ADVIA Centaur). The method validation was performed according to FDA and EMEA guidelines. A one-step protein precipitation by acetonitrile followed a five-fold dilution was performed for sample pretreatment. A 5.2 min gradient separation by methanol and 10 mM ammonium acetate was used for separation at 0.6 mL/min under 45 °C. Both positive and negative electrospray ionization were used. Isotopic internal standard was used for all analytes. The inter-day (36 days) accuracy and precision of quality control samples were - 1.07-13.69% and < 6.70% for all analytes. The stability was acceptable for all analytes under routine storing conditions. A total of 436 valproic acid, 118 carbamazepine, and 65 phenobarbital samples were determined twice by each of the UHPLC-MS/MS and immunoassay. Evaluated by Bland-Altman plot, the mean overestimation of the immunoassay compared to UHPLC-MS/MS was 16.5% for valproic acid, 5.6% for carbamazepine, and 40.3% for phenobarbital.

Keywords:  Antiepileptic drugs; Carbamazepine; Phenobarbital; The ADVIA Centaur assay; UHPLC-MS/MS; Valproic acid

DOI:  https://doi.org/10.1016/j.jpba.2023.115437
Anal Chem. 2023 May 02.

Evaluating Retention Index Score Assumptions to Refine GC-MS Metabolite Identification.

David J Degnan, Lisa M Bramer, Javier E Flores, Vanessa L Paurus, Yuri E Corilo, Chaevien S Clendinen.

As metabolomics grows into a high-throughput and high demand research field, current metrics for the identification of small molecules in gas chromatography-mass spectrometry (GC-MS) still require manual verification. Though steps have been taken to improve scoring metrics by combining spectral similarity (SS) and retention index (RI), the problem persists. A large body of literature has analyzed and refined SS scores, but few studies have explicitly studied improvements to RI scores. Here, we examined whether uninvestigated assumptions of the RI score are valid and propose ways to improve them. Query RIs were matched to library RI with a generous window of ±35 to avoid unintentional removal of valid compound identifications. Each match was manually verified as a true positive (TP), true negative, or unknown. Metabolites with at least 30 TP identifications were included in downstream analyses, resulting in a total of 87 metabolites from samples of varying complexity and type (e.g., amino acid mixtures, human urine, fungal species, and so on.). Our results showed that the RI score assumptions of normality, consistent variance across metabolites, and a mean error centered at 0 are often violated. We demonstrated through a cross-validation analysis that modifying these underlying assumptions according to empirical metabolite-specific distributions improved the TP and negative rankings. Further, we statistically determined the minimum number of samples required to estimate distributional parameters for scoring metrics. Overall, this work proposes a robust statistical pipeline to reduce the time bottleneck of metabolite identification by improving RI scores and thus minimize the effort to complete manual verification.

DOI: https://doi.org/10.1021/acs.analchem.2c05783
J Am Soc Mass Spectrom. 2023 May 02.

Development and Validation of an Ultrasensitive LC-MS/MS Method for the Quantification of Melatonin in Human Saliva.

Justine J Demeuse, Chiara Calaprice, Loreen C Huyghebaert, Marwa Rechchad, Stéphanie Peeters, Etienne Cavalier, Caroline Le Goff.

  A growing body of literature describes the potential effects of circadian disruption on human health. Indeed, psychiatric diseases, metabolic syndrome, and cancers may be linked to disturbance of the circadian rhythm. Currently, the best practice to assess circadian rhythm is the measurement of melatonin levels. Our goal was thus to develop and validate a highly sensitive LC-MS/MS method to follow salivary melatonin levels throughout the day and night. Our method reached a lower limit of the measuring interval (LLMI) of 0.8 pg/mL. To our knowledge, it is the most sensitive method allowing quantitation of melatonin in saliva. Saliva, obtained from passive drooling or salivette, was extracted by an efficient and quick liquid-liquid extraction with no further cleanup needed. The method was validated according to the European Medicines Agency (EMA) guidelines and provided excellent results regarding accuracy, precision, linearity, selectivity, and specificity. Comparison between radioimmunoassay and our method was performed and showed differences at low levels, most likely due to cross-reactivity with other indols. To assess daytime melatonin levels in humans, salivary melatonin levels of ten volunteers were monitored throughout the day and showed lower daytime levels than reported in previous studies.

Keywords:  LC-MS/MS; circadian rhythms; melatonin; method validation; ultrahigh sensitivity

DOI:  https://doi.org/10.1021/jasms.3c00021
Cell Biol Toxicol. 2023 May 04.

A high-throughput metabolomics in vitro platform for the characterization of hepatotoxicity.

Sabina Ramirez-Hincapie, Barbara Birk, Philipp Ternes, Varun Giri, Volker Haake, Michael Herold, Franziska Maria Zickgraf, Andreas Verlohner, Hans-Albrecht Huener, Hennicke Kamp, Peter Driemert, Robert Landsiedel, Elke Richling, Dorothee Funk-Weyer, Bennard van Ravenzwaay.

  Cell-based metabolomics provides multiparametric physiologically relevant readouts that can be highly advantageous for improved, biologically based decision making in early stages of compound development. Here, we present the development of a 96-well plate LC-MS/MS-based targeted metabolomics screening platform for the classification of liver toxicity modes of action (MoAs) in HepG2 cells. Different parameters of the workflow (cell seeding density, passage number, cytotoxicity testing, sample preparation, metabolite extraction, analytical method, and data processing) were optimized and standardized to increase the efficiency of the testing platform. The applicability of the system was tested with seven substances known to be representative of three different liver toxicity MoAs (peroxisome proliferation, liver enzyme induction, and liver enzyme inhibition). Five concentrations per substance, aimed at covering the complete dose-response curve, were analyzed and 221 uniquely identified metabolites were measured, annotated, and allocated in 12 different metabolite classes such as amino acids, carbohydrates, energy metabolism, nucleobases, vitamins and cofactors, and diverse lipid classes. Multivariate and univariate analyses showed a dose response of the metabolic effects, a clear differentiation between liver toxicity MoAs and resulted in the identification of metabolite patterns specific for each MoA. Key metabolites indicative of both general and mechanistic specific hepatotoxicity were identified. The method presented here offers a multiparametric, mechanistic-based, and cost-effective hepatotoxicity screening that provides MoA classification and sheds light into the pathways involved in the toxicological mechanism. This assay can be implemented as a reliable compound screening platform for improved safety assessment in early compound development pipelines.

Keywords:  Hepatotoxicity; High throughput; Liver toxicity; Metabolomics; Mode of action; Toxicology in vitro; Toxicometabolomics

DOI:  https://doi.org/10.1007/s10565-023-09809-6
Mol Omics. 2023 May 04.

Development and application of a data processing method for food metabolomics analysis.

Yuanluo Lei, Xiaoying Chen, Jiachen Shi, Yuanfa Liu, Yong-Jiang Xu.

Food metabolomics is described as the implementation of metabolomics to food systems such as food materials, food processing, and food nutrition. These applications generally create large amounts of data, and although technologies exist to analyze these data and different tools exist for various ecosystems, downstream analysis is still a challenge and the tools are not integrated into a single method. In this article, we developed a data processing method for untargeted LC-MS data in metabolomics, derived from the integration of computational MS tools from OpenMS into the workflow system Konstanz Information Miner (KNIME). This method can analyze raw MS data and produce high-quality visualization. A MS1 spectra-based identification, two MS2 spectra-based identification workflows and a GNPSExport-GNPS workflow are included in this method. Compared with conventional approaches, the results of MS1&MS2 spectra-based identification workflows are combined in this approach via the tolerance of retention times and mass to charge ratios (m/z), which can greatly reduce the rate of false positives in metabolomics datasets. In our example, filtering with the tolerance removed more than 50% of the possible identifications while retaining 90% of the correct identification. The results demonstrated that the developed method is a rapid and reliable method for food metabolomics data processing.

DOI: https://doi.org/10.1039/d2mo00338d
Anal Chem. 2023 Apr 29.

New Approach for the Identification of Isobaric and Isomeric Metabolites.

Ahmed Ben Faleh, Stephan Warnke, Teun Van Wieringen, Ali H Abikhodr, Thomas R Rizzo.

The structural elucidation of metabolite molecules is important in many branches of the life sciences. However, the isomeric and isobaric complexity of metabolites makes their identification extremely challenging, and analytical standards are often required to confirm the presence of a particular compound in a sample. We present here an approach to overcome these challenges using high-resolution ion mobility spectrometry in combination with cryogenic vibrational spectroscopy for the rapid separation and identification of metabolite isomers and isobars. Ion mobility can separate isomeric metabolites in tens of milliseconds, and cryogenic IR spectroscopy provides highly structured IR fingerprints for unambiguous molecular identification. Moreover, our approach allows one to identify metabolite isomers automatically by comparing their IR fingerprints with those previously recorded in a database, obviating the need for a recurrent introduction of analytical standards. We demonstrate the principle of this approach by constructing a database composed of IR fingerprints of eight isomeric/isobaric metabolites and use it for the identification of these isomers present in mixtures. Moreover, we show how our fast IR fingerprinting technology allows to probe the IR fingerprints of molecules within just a few seconds as they elute from an LC column. This approach has the potential to greatly improve metabolomics workflows in terms of accuracy, speed, and cost.

DOI: https://doi.org/10.1021/acs.analchem.2c04962
Methods Mol Biol. 2023 ;2668 57-68

Metabolomics Analysis of Urinary Extracellular Vesicles by Nuclear Magnetic Resonance and Liquid Chromatography-Mass Spectrometry.

Marta Martin-Lorenzo, Dolores Molero, Gloria Alvarez-Llamas.

  Extracellular vesicle (EV) release and their content are influenced by diverse clinical conditions. EVs participate in inter-cellular communication and have been postulated as reflectors of the pathophysiology of the cells, tissues, organs or the whole system with which they are in contact. Urinary EVs have been proved to reflect pathophysiology not only of renal system related diseases constituting an additional source of potential biomarkers easily accessible in a non-invasive way. The interest in EVs cargo has been mostly focused on proteins and nucleic acids and more recently it has been extended to metabolites. Metabolites represent the downstream changes in the genome, transcriptome, and proteome as a reflection of processes occurring in living organisms. For their study, nuclear magnetic resonance (NMR) and mass spectrometry in tandem (LC-MS/MS) are widely used. NMR is a reproducible and non-destructive technique and we show here methodological protocols for the metabolomics analysis of urinary EVs by NMR. Additionally, we also describe the workflow for a targeted LC-MS/MS analysis that is extensible to untargeted studies.

Keywords:  Extracellular vesicles; LC–MS; Mass spectrometry; Metabolites; Metabolomics; Nuclear magnetic resonance

DOI:  https://doi.org/10.1007/978-1-0716-3203-1_6
Chem Commun (Camb). 2023 May 03.

Quantitative NMR spectroscopy of complex mixtures.

Patrick Giraudeau.

Complex mixtures are ubiquitous in many branches of chemistry, be it a complex pharmaceutical formulation, a collection of biofluids analysed in a metabolomics workflow, or a flowing mixture in a reaction monitoring setting. The accurate quantitative determination of mixture components is one of the toughest challenges posed to analytical chemists, requiring the determination of often heavily overlapped signals from compounds in very diverse concentrations. NMR spectroscopists have developed an impressive variety of approaches to deal with such challenges, including the development of innovative pulse sequences, hyperpolarization methods and processing tools. We describe the most recent advances in the field of quantitative NMR, and the many subsequent application perspectives in fields where the sample complexity is a daily challenge, such as pharmaceutical science, metabolomics, isotopic analysis, and monitoring.

DOI: https://doi.org/10.1039/d3cc01455j
J Magn Reson. 2023 Apr 26. pii: S1090-7807(23)00097-6. [Epub ahead of print]352 107462

Practical considerations for rapid and quantitative NMR-based metabolomics.

Frans A A Mulder, Leonardo Tenori, Cristina Licari, Claudio Luchinat.

  NMR is a key technology for metabolomics because of its robustness and reproducibility. Herein we discuss practical considerations that extend the utility of NMR spectroscopy. First, the long T1 spin relaxation times of small molecules limits high-throughput data acquisition because most experimental time is lost while waiting for signal recovery. In principle, the addition of a small amount of commercially-available paramagnetic gadolinium chelate allows cost-effective and efficient high-throughput mixture analysis with correct concentration determination. However, idle time caused by slow temperature regulation during sample exchanges, poses a next constraint. We show how, with proper care, NMR sample scanning times can be reduced additionally by a factor of two. Lastly, we describe how equidistant bucketing is a simple and fast procedure for metabolomic fingerprinting. The combination of these advancements help to make NMR metabolomics more versatile than it is today.

Keywords:  Metabolomics; NMR data acquisition; Temperature stabilization; bucketing; paramagnetic NMR

DOI:  https://doi.org/10.1016/j.jmr.2023.107462