bims-metlip 2023-03-26 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2023–03–26
twenty-two papers selected by
Sofia Costa, Matterworks

Highly Sensitive and Ultrafast Liquid Chromatography-tandem Mass Spectrometry Internal Standard Method for Determination of Alpha-Tocopherol in Serum and Application of Validated Method on Patient Serum Samples in Pakistan.
Simulated-to-real benchmarking of acquisition methods in untargeted metabolomics.
An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of aldosterone in human serum and plasma.
Mass spectrometry quantitation of immunosuppressive drugs in clinical specimens using online solid-phase extraction and accurate-mass full scan-single ion monitoring.
Large-Scale metabolomics: Predicting biological age using 10,133 routine untargeted LC-MS measurements.
Sensitive and rapid method for the quantitation of amoxicillin in minipig plasma and milk by LC-MS/MS: A contribution from the IMI ConcePTION project.
Pickaxe: a Python library for the prediction of novel metabolic reactions.
Development and validation of an HPLC-MS/MS method to simultaneously quantify brigatinib, lorlatinib, pralsetinib and selpercatinib in human K2-EDTA plasma.
Development of the thin film solid phase microextraction (TF-SPME) method for metabolomics profiling of steroidal hormones from urine samples using LC-QTOF/MS.
Online hyphenation of in-capillary aptamer-functionalized solid-phase microextraction and extraction nanoelectrospray ionization for miniature mass spectrometry analysis.
Optimizing transfer and dilution processes when using active solvent modulation in on-line two-dimensional liquid chromatography.
1H-NMR protocol for rapid diagnosis of purine and pyrimidine metabolic disorders in urine.
Quantitation of Total PFAS Including Trifluoroacetic Acid with Fluorine Nuclear Magnetic Resonance Spectroscopy.
Rules for mass spectrometry applications in the clinical laboratory.
Liquid chromatography-mass spectrometry-based metabolomic profiling reveals sex differences of lipid metabolism among the elderly from Southwest China.
Rapid skin biomarker discovery using hydrogel-phase sampling followed by semi-automated liquid-phase re-extraction high-resolution mass spectrometry.
[LC-MS/MS as a method for the determination of isoprostanes: a brief review].
HPLC-MS-MS quantification of short-chain fatty acids actively secreted by probiotic strains.
Bayesian kinetic modeling for tracer-based metabolomic data.
Exploratory data fusion of untargeted multimodal LC-HRMS with annotation by LCMS-TOF-ion mobility: White wine case study.
Development of an Equilibrium Dialysis ID-UPLC-MS/MS Candidate Reference Measurement Procedure for Free Thyroxine in Human Serum.
Inadequate Definition of the Limit of Quantification Applied for the Quantification of Perfluoroalkyl Substances in Food by LC-MS/MS May Compromise the Reliability of the Data Requested by the European Regulation.

J Coll Physicians Surg Pak. 2023 Mar;33(3): 254-260

Highly Sensitive and Ultrafast Liquid Chromatography-tandem Mass Spectrometry Internal Standard Method for Determination of Alpha-Tocopherol in Serum and Application of Validated Method on Patient Serum Samples in Pakistan.

Humera Shafi Makhdoom, Mohammad Dilawar Khan, Omar Rasheed Chughtai, Zeerak Abbas, Areeba Azad, Hijab Batool.

OBJECTIVE: To optimize and validate a specific, sensitive and fast liquid Chromatography coupled to triple quadrupole Mass Spectrometric (LC-MS / MS) technique for accurate detection of serum α-tocopherol (Vitamin E) levels.
STUDY DESIGN: An experimental based study.
PLACE AND DURATION OF STUDY: The Clinical and Forensic Toxicology section of Chughtai Lab, Jail Road Lahore, from April to September 2022.
METHODOLOGY: Methanol was used to deproteinize serum samples. The chromatographic separation was achieved using an Agilent Infinity-Lab Poroshell 120EC-C18 column, Agilent 6470 LC-MS/MS (equipped with an Electron Spray Ionization source) in gradient elution mode using 0.1% LCMS grade formic acid in water and LCMS-grade methanol as mobile phases. Hexa-deuterated α-tocopherol was employed as internal standard to minimise matrix interferences.
RESULTS: The retention time of α-tocopherol was 3.0 ± 0.1 minutes. The linear concentrations obtained were ranged from 0.05-2 mg/dL with ≥0.985% coefficient of linearity. Detection and lower quantification limits determined were 0.025mg/dL and 0.05mg/dL, respectively. Recovery ranged from 96.5 to 99.8% and ionization suppression was -15.2% and -15.9% at high and low concentrations of α-tocopherol in serum. Intra-day and inter-day coefficient variation values were 4.2-4.9% and 5.0-5.9%, respectively.
CONCLUSION: An efficient and reliable tandem mass spectrometric technique for vitamin E analysis in serum was optimized, validated, and applied to 80 patient samples. This method has usefulness in clinical application for the accurate determination of vitamin E without potential matrix interferences.
KEY WORDS: Vitamin E, LC-MS/MS, Tocopherol, Internal standard, Validation.

DOI: https://doi.org/10.29271/jcpsp.2023.03.254
Front Mol Biosci. 2023 ;10 1130781

Simulated-to-real benchmarking of acquisition methods in untargeted metabolomics.

Joe Wandy, Ross McBride, Simon Rogers, Nikolaos Terzis, Stefan Weidt, Justin J J van der Hooft, Kevin Bryson, Rónán Daly, Vinny Davies.

  Data-Dependent and Data-Independent Acquisition modes (DDA and DIA, respectively) are both widely used to acquire MS2 spectra in untargeted liquid chromatography tandem mass spectrometry (LC-MS/MS) metabolomics analyses. Despite their wide use, little work has been attempted to systematically compare their MS/MS spectral annotation performance in untargeted settings due to the lack of ground truth and the costs involved in running a large number of acquisitions. Here, we present a systematic in silico comparison of these two acquisition methods in untargeted metabolomics by extending our Virtual Metabolomics Mass Spectrometer (ViMMS) framework with a DIA module. Our results show that the performance of these methods varies with the average number of co-eluting ions as the most important factor. At low numbers, DIA outperforms DDA, but at higher numbers, DDA has an advantage as DIA can no longer deal with the large amount of overlapping ion chromatograms. Results from simulation were further validated on an actual mass spectrometer, demonstrating that using ViMMS we can draw conclusions from simulation that translate well into the real world. The versatility of the Virtual Metabolomics Mass Spectrometer (ViMMS) framework in simulating different parameters of both Data-Dependent and Data-Independent Acquisition (DDA and DIA) modes is a key advantage of this work. Researchers can easily explore and compare the performance of different acquisition methods within the ViMMS framework, without the need for expensive and time-consuming experiments with real experimental data. By identifying the strengths and limitations of each acquisition method, researchers can optimize their choice and obtain more accurate and robust results. Furthermore, the ability to simulate and validate results using the ViMMS framework can save significant time and resources, as it eliminates the need for numerous experiments. This work not only provides valuable insights into the performance of DDA and DIA, but it also opens the door for further advancements in LC-MS/MS data acquisition methods.

Keywords:  data independent acquisition; data-dependent acquisition; digital twin; liquid chromatography tandem mass spectrometry; metabolomics

DOI:  https://doi.org/10.3389/fmolb.2023.1130781
Clin Chem Lab Med. 2023 Mar 24.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of aldosterone in human serum and plasma.

Judith Taibon, Tobias Santner, Neeraj Singh, Sara Cheikh Ibrahim, Galina Babitzki, Daniel Köppl, Alexander Gaudl, Andrea Geistanger, Uta Ceglarek, Manfred Rauh, Christian Geletneky.

   OBJECTIVES: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) for aldosterone quantification in human serum and plasma is presented.
METHODS: The material used in this RMP was characterized by quantitative nuclear magnetic resonance (qNMR) to assure traceability to SI Units. For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis a two-dimensional heart cut LC approach, in combination with an optimal supported liquid extraction protocol, was established for the accurate analysis of aldosterone in human serum and plasma in order to minimize matrix effects and avoid the co-elution of interferences. Assay validation was performed according to current guidelines. Selectivity and specificity were assessed using spiked serum; potential matrix effects were examined by a post column infusion experiment and the comparison of standard line slopes. An extensive protocol over 5 days was applied to determine precision, accuracy and trueness. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM), for which three individual sample preparations were performed on at least two different days.
RESULTS: The RMP allowed aldosterone quantification within the range of 20-1,200 pg/mL without interference from structurally-related compounds and no evidence of matrix effects. Intermediate precision was ≤4.7% and repeatability was 2.8-3.7% for all analyte concentrations. The bias ranged between -2.2 and 0.5% for all levels and matrices. Total measurement uncertainties for target value assignment (n=6) were found to be ≤2.3%; expanded uncertainties were ≤4.6% (k=2) for all levels.
CONCLUSIONS: The RMP showed high analytical performance for aldosterone quantification in human serum and plasma. The traceability to SI units was established by qNMR content determination of aldosterone, which was utilized for direct calibration of the RMP. Thus, this candidate RMP is suitable for routine assay standardization and evaluation of clinical samples.

Keywords:  SI units; aldosterone; isotope dilution-liquid chromatography-tandem mass spectrometry; qNMR; reference measurement procedure; traceability

DOI:  https://doi.org/10.1515/cclm-2022-0996
J Mass Spectrom Adv Clin Lab. 2023 Apr;28 99-104

Mass spectrometry quantitation of immunosuppressive drugs in clinical specimens using online solid-phase extraction and accurate-mass full scan-single ion monitoring.

Priscilla S-W Yeung, Paige Miller, Tran Bao Lai-Nyugen, Phil Cheng, Amira Ibrahim, Run-Zhang Shi, Raffick A R Bowen, Ruben Yiqi Luo.

   Introduction: Therapeutic drug monitoring (TDM) of immunosuppressants is essential for optimal care of transplant patients. Immunoassays and liquid chromatography-mass spectrometry (LC-MS) are the most commonly used methods for TDM. However, immunoassays can suffer from interference from heterophile antibodies and structurally similar drugs and metabolites. Additionally, nominal-mass LC-MS assays can be difficult to optimize and are limited in the number of detectable compounds.
Objectives: The aim of this study was to implement a mass spectrometry-based test for immunosuppressant TDM using online solid-phase extraction (SPE) and accurate-mass full scan-single ion monitoring (FS-SIM) data acquisition mode.
Methods: LC-MS analysis was performed on a TLX-2 multi-channel HPLC with a Q-Exactive Plus mass spectrometer. TurboFlow online SPE was used for sample clean up. The accurate-mass MS was set to positive electrospray ionization mode with FS-SIM for quantitation of tacrolimus, sirolimus, everolimus, and cyclosporine A. MS2 fragmentation pattern was used for compound confirmation.
Results: The method was validated in terms of precision, analytical bias, limit of quantitation, linearity, carryover, sample stability, and interference. Quantitation of tacrolimus, sirolimus, everolimus, and cyclosporine A correlated well with results from an independent reference laboratory (r = 0.926-0.984).
Conclusions: Accurate-mass FS-SIM can be successfully utilized for immunosuppressant TDM with good correlation with results generated by standard methods. TurboFlow online SPE allows for a simple "protein crash and shoot" sample preparation protocol. Compared to traditional MRM, analyte quantitation by FS-SIM facilitates a streamlined assay optimization process.

Keywords:  Accurate-mass; CAP, College of American Pathologists; CLSI, Clinical & Laboratory Standards Institute; CV, coefficient of variation; ESI, electrospray ionization; FS-SIM, full scan-single ion monitoring; Full scan single-ion monitoring; HCD, high-energy C-trap dissociation; IRB, Institutional Review Board; Immunosuppressive drugs; LC-MS, liquid chromatography-mass spectrometry; LDT, laboratory developed test; MRM, multiple reaction monitoring; Mass spectrometry; Online solid-phase extraction; SD, standard deviation; SPE, solid-phase extraction; TDM, therapeutic drug monitoring; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.jmsacl.2023.03.002
Aging Cell. 2023 Mar 19. e13813

Large-Scale metabolomics: Predicting biological age using 10,133 routine untargeted LC-MS measurements.

Johan K Lassen, Tingting Wang, Kirstine L Nielsen, Jørgen B Hasselstrøm, Mogens Johannsen, Palle Villesen.

  Untargeted metabolomics is the study of all detectable small molecules, and in geroscience, metabolomics has shown great potential to describe the biological age-a complex trait impacted by many factors. Unfortunately, the sample sizes are often insufficient to achieve sufficient power and minimize potential biases caused by, for example, demographic factors. In this study, we present the analysis of biological age in ~10,000 toxicologic routine blood measurements. The untargeted screening samples obtained from ultra-high pressure liquid chromatography-quadruple time of flight mass spectrometry (UHPLC- QTOF) cover + 300 batches and + 30 months, lack pooled quality controls, lack controlled sample collection, and has previously only been used in small-scale studies. To overcome experimental effects, we developed and tested a custom neural network model and compared it with existing prediction methods. Overall, the neural network was able to predict the chronological age with an rmse of 5.88 years (r2 = 0.63) improving upon the 6.15 years achieved by existing normalization methods. We used the feature importance algorithm, Shapley Additive exPlanations (SHAP), to identify compounds related to the biological age. Most importantly, the model returned known aging markers such as kynurenine, indole-3-aldehyde, and acylcarnitines along with a potential novel aging marker, cyclo (leu-pro). Our results validate the association of tryptophan and acylcarnitine metabolism to aging in a highly uncontrolled large-s cale sample. Also, we have shown that by using robust computational methods it is possible to deploy large LC-MS datasets for metabolomics studies to reduce the risk of bias and empower aging studies.

Keywords:  accelerated aging; big data; inflammaging; machine learning; metabolomics; molecular biology of aging; tryptophan metabolism

DOI:  https://doi.org/10.1111/acel.13813
J Pharmacol Toxicol Methods. 2023 Mar 21. pii: S1056-8719(23)00015-1. [Epub ahead of print] 107264

Sensitive and rapid method for the quantitation of amoxicillin in minipig plasma and milk by LC-MS/MS: A contribution from the IMI ConcePTION project.

Yeghig Armoudjian, Qi Lin, Bart Lammens, Johan Van Daele, Pieter Annaert.

  The IMI project ConcePTION was launched to fill the knowledge gap of using medicines during pregnancy and lactation. To achieve this goal, several studies are being conducted, including the bioanalysis of amoxicillin in minipig plasma and milk. A high-throughput, robust and reliable liquid chromatography tandem mass spectrometry method was developed and validated according to FDA and EMA guidelines to determine the concentrations of amoxicillin in a large number of minipig plasma and milk samples. Chromatographic separation was achieved on a Luna® Omega Polar C18, 1.6 μm, 100 × 2.1 mm column, with a mobile phase consisting of 0.1% formic acid in water and acetonitrile. Mass spectrometry used in a positive ionization mode and the transitions m/z 366.1 → 349.2 was selected to monitor amoxicillin, while m/z 370.1 → 114.15 was selected for the stable isotope labelled internal standard. This method features a linear quantification range of 10 ng/mL - 10 μg/mL, recovery of not less than 94.1%, a single sample extraction method for both plasma and milk matrices, and an analysis runtime of 5 min.

Keywords:  Amoxicillin; LC-MS/MS; Minipig milk; Minipig plasma; Pharmacokinetics

DOI:  https://doi.org/10.1016/j.vascn.2023.107264
BMC Bioinformatics. 2023 Mar 22. 24(1): 106

Pickaxe: a Python library for the prediction of novel metabolic reactions.

Kevin M Shebek, Jonathan Strutz, Linda J Broadbelt, Keith E J Tyo.

   BACKGROUND: Biochemical reaction prediction tools leverage enzymatic promiscuity rules to generate reaction networks containing novel compounds and reactions. The resulting reaction networks can be used for multiple applications such as designing novel biosynthetic pathways and annotating untargeted metabolomics data. It is vital for these tools to provide a robust, user-friendly method to generate networks for a given application. However, existing tools lack the flexibility to easily generate networks that are tailor-fit for a user's application due to lack of exhaustive reaction rules, restriction to pre-computed networks, and difficulty in using the software due to lack of documentation.
RESULTS: Here we present Pickaxe, an open-source, flexible software that provides a user-friendly method to generate novel reaction networks. This software iteratively applies reaction rules to a set of metabolites to generate novel reactions. Users can select rules from the prepackaged JN1224min ruleset, derived from MetaCyc, or define their own custom rules. Additionally, filters are provided which allow for the pruning of a network on-the-fly based on compound and reaction properties. The filters include chemical similarity to target molecules, metabolomics, thermodynamics, and reaction feasibility filters. Example applications are given to highlight the capabilities of Pickaxe: the expansion of common biological databases with novel reactions, the generation of industrially useful chemicals from a yeast metabolome database, and the annotation of untargeted metabolomics peaks from an E. coli dataset.
CONCLUSION: Pickaxe predicts novel metabolic reactions and compounds, which can be used for a variety of applications. This software is open-source and available as part of the MINE Database python package ( https://pypi.org/project/minedatabase/ ) or on GitHub ( https://github.com/tyo-nu/MINE-Database ). Documentation and examples can be found on Read the Docs ( https://mine-database.readthedocs.io/en/latest/ ). Through its documentation, pre-packaged features, and customizable nature, Pickaxe allows users to generate novel reaction networks tailored to their application.

Keywords:  Biosynthetic design; Enzyme promiscuity; Metabolite identification; Network generation; Retrobiosynthesis

DOI:  https://doi.org/10.1186/s12859-023-05149-8
Biomed Chromatogr. 2023 Mar 20. e5628

Development and validation of an HPLC-MS/MS method to simultaneously quantify brigatinib, lorlatinib, pralsetinib and selpercatinib in human K2-EDTA plasma.

J L Gulikers, A J van Veelen, E M J Sinkiewicz, Y M de Beer, M Slikkerveer, L M L Stolk, V C G Tjan-Heijnen, L E L Hendriks, S Croes, R M J M van Geel.

A liquid chromatography-tandem mass spectrometry method was developed and validated to quantify the small molecule inhibitors (SMIs) brigatinib, lorlatinib, pralsetinib and selpercatinib, which are used in patients with oncogenic driven non-small cell lung cancer. Chromatographic separation was performed on a HyPURITY® C18 analytical column with a gradient elution using ammonium acetate in water and in methanol, both acidified with formic acid 0,1%. Detection and quantification were performed by a triple quad mass spectrometer with an electrospray ionization interface. The assay was validated over a linear range of 50 - 2,500 ng/mL for brigatinib, 25 - 1,000 ng/mL for lorlatinib, 100 - 10,000 ng/mL for pralsetinib, and 50 - 5,000 ng/mL for selpercatinib. All four SMIs were stable for at least seven days at cooling conditions (2 - 8 °C), and at least 24 hours at room temperature (15 - 25 °C) in K2-EDTA plasma. At freezing conditions (-20°C), all SMIs were stable for at least 30 days, except for the lowest quality control (QCLOW ) of pralsetinib. The QCLOW of pralsetinib was stable for at least 7 days at -20°C. This method provides an efficient and simple way to quantify four SMIs with a single assay in clinical practice.

DOI: https://doi.org/10.1002/bmc.5628
Front Mol Biosci. 2023 ;10 1074263

Development of the thin film solid phase microextraction (TF-SPME) method for metabolomics profiling of steroidal hormones from urine samples using LC-QTOF/MS.

Wiktoria Struck-Lewicka, Beata Karpińska, Wojciech Rodzaj, Antoni Nasal, Bartosz Wielgomas, Michał Jan Markuszewski, Danuta Siluk.

  In the present study, the development and optimization of a thin film solid phase microextraction method (TF-SPME) was conducted for metabolomics profiling of eight steroid compounds (androsterone, dihydrotestosterone, dihydroepiandrosterone, estradiol, hydroxyprogesterone, pregnenolone, progesterone and testosterone) from urine samples. For optimization of extraction method, two extraction sorbents (PAN-C18 and PS-DVB) were used as they are known to be effective for isolation of low-polarity analytes. The stages of sample extraction and analyte desorption were considered as the most crucial steps in the process. Regarding the selection of the most suitable desorption solution, six different mixtures were analyzed. As a result, the mixture of ACN: MeOH (1:1, v/v) was chosen in terms of the highest analytes' abundances that were achieved using the chosen solvent. Besides other factors were examined such as the volume of desorption solvent and the time of both extraction and desorption processes. The analytical determination was carried out using the ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometry detection in electrospray ionization and positive polarity in a scan mode (UHPLC-ESI-QTOF/MS). The developed and optimized TF-SPME method was validated in terms of such parameters as extraction efficiency, recovery as well as matrix effect. As a result, the extraction efficiency and recovery were in a range from 79.3% to 99.2% and from 88.8% to 111.8%, respectively. Matrix effect, calculated as coefficient of variation was less than 15% and was in a range from 1.4% to 11.1%. The values of both validation parameters (recovery and matrix effect) were acceptable in terms of EMA criteria. The proposed TF-SPME method was used successfully for isolation of steroids hormones from pooled urine samples before and after enzymatic hydrolysis of analytes.

Keywords:  LC-QTOF/MS; endogenous steroid hormones; thin-film solid phase microextraction TF-SPME; untargeted metabolomics profiling; urine

DOI:  https://doi.org/10.3389/fmolb.2023.1074263
Analyst. 2023 Mar 20.

Online hyphenation of in-capillary aptamer-functionalized solid-phase microextraction and extraction nanoelectrospray ionization for miniature mass spectrometry analysis.

Yueguang Lv, Yuhan Shang, Linsen Li, Ying Zhang, Qiang Ma.

Direct mass spectrometry (MS) analysis is vital to chemical and biological investigations. However, measuring complex samples is challenging due to matrix interference, resulting in compromised MS performance. In this study, an integrated experimental protocol has been developed, combining in-capillary aptamer-functionalized solid-phase microextraction (SPME), extraction nanoelectrospray ionization (nanoESI), and miniature MS analysis. The established method was applied to analyze caffeine in electronic cigarette liquid and beverage samples as proof-of-concept demonstrations. A custom SPME strip fabricated with caffeine-binding aptamers was prepared with an immobilization density of up to 0.812 nmol cm-2. Critical parameters affecting the effects of extraction, desorption, and ionization were optimized. A novel transition ion ratio-based strategy with enhanced quantitation accuracy was developed. The analytical performance of the proposed method was evaluated under optimized conditions. Acceptable recoveries of 87.5-111.5% with relative standard deviations of 3.1-6.1% and satisfactory sensitivity with limits of detection of 1.5 and 3 ng mL-1 and limits of quantitation of 5 and 10 ng mL-1 were obtained, respectively. The developed approach demonstrates a promising potential for rapid on-site applications with appealing analytical performance and efficiency.

DOI: https://doi.org/10.1039/d3an00111c
Anal Chim Acta. 2023 Apr 29. pii: S0003-2670(23)00261-1. [Epub ahead of print]1252 341040

Optimizing transfer and dilution processes when using active solvent modulation in on-line two-dimensional liquid chromatography.

Marie Pardon, Soraya Chapel, Peter de Witte, Deirdre Cabooter.

  Two-dimensional liquid chromatography (2D-LC) is becoming increasingly popular for the analysis of complex samples, which is partly due to the recent introduction of commercial 2D-LC systems. To deal with the mobile phase incompatibility between highly orthogonal retention mechanisms, such as hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC (RPLC), several strategies have been introduced over the years. One of these strategies is active solvent modulation (ASM), a valve-based approach allowing the on-line dilution of the effluent eluting from the first dimension before transfer to the second dimension. This strategy has gained a lot of attention and holds great potential, however, no clear guidelines are currently in place for its use. Therefore, this study aims to investigate how the ASM process can be optimized when using highly incompatible LC combinations, such as HILIC and RPLC, in a simplified selective comprehensive 2D-LC set-up (sHILIC x RPLC) to suggest guidelines for future users. Using a representative sample, the dilution factor (DF), the duration of the ASM phase, the filling percentage of the sample loops, and their unloading configuration are investigated and optimized. It is observed that a DF of 10 with an optimal ASM phase duration, a sample loop filling of maximum 25%, and an unloading configuration in backflush mode, result in the best peak shapes, intensities, and recoveries for early eluting compounds, while keeping the total analysis time minimal. Based on these results, some general recommendations are made that could also be applied in other 2D-LC modes, such as comprehensive 2D-LC (LC x LC), heart-cutting 2D-LC (LC-LC), and other chromatographic combinations with mobile phase incompatibility issues.

Keywords:  Mobile phase incompatibility; On-line dilution; Solvent strength mismatch; sHILIC x RPLC

DOI:  https://doi.org/10.1016/j.aca.2023.341040
STAR Protoc. 2023 Mar 22. pii: S2666-1667(23)00139-9. [Epub ahead of print]4(2): 102181

1H-NMR protocol for rapid diagnosis of purine and pyrimidine metabolic disorders in urine.

Elmarie Davoren, Shayne Mason.

  Purine and pyrimidine disorders are often difficult to diagnose. Here, we present a 1H-NMR analysis protocol for the quantification of purines and pyrimidines in urine to diagnose associated disorders. We describe steps for pH adjustment, sample preparation, and 1H-NMR analysis and data analysis. The use of 1H-NMR requires a relatively small sample volume (1 mL) and minimal sample preparation. Analysis time produces accurate and reproducible data within 2 h.

Keywords:  Metabolism; Metabolomics; NMR

DOI:  https://doi.org/10.1016/j.xpro.2023.102181
Anal Chem. 2023 Mar 22.

Quantitation of Total PFAS Including Trifluoroacetic Acid with Fluorine Nuclear Magnetic Resonance Spectroscopy.

Dino Camdzic, Rebecca A Dickman, Abigail S Joyce, Joshua S Wallace, P Lee Ferguson, Diana S Aga.

Fluorine nuclear magnetic resonance (19F-NMR) spectroscopy has been shown to be a powerful tool capable of quantifying the total per- and polyfluoroalkyl substances (PFAS) in a complex sample. The technique relies on the characteristic terminal -CF3 shift (-82.4 ppm) in the alkyl chain for quantification and does not introduce bias due to sample preparation or matrix effects. Traditional quantitative analytical techniques for PFAS, such as liquid chromatography-mass spectrometry (LC-MS) and combustion ion chromatography (CIC), contain inherent limitations that make total fluorine analysis challenging. Here, we report a sensitive 19F-NMR method for the analysis of total PFAS, with a limit of detection of 99.97 nM, or 50 μg/L perfluorosulfonic acid. To demonstrate the capabilities of 19F-NMR, the technique was compared to two commonly used methods for PFAS analysis: total oxidizable precursor (TOP) assay and LC-high resolution MS analysis for targeted quantification and suspect screening. In both cases, the 19F-NMR analyses detected higher total PFAS quantities than either the TOP assay (63%) or LC-MS analyses (65%), suggesting that LC-MS and TOP assays can lead to underreporting of PFAS. Importantly, the 19F-NMR detected trifluoroacetic acid at a concentration more than five times the total PFAS concentration quantified using LC-MS in the wastewater sample. Therefore, the use of 19F-NMR to quantify the total PFAS in highly complex samples can be used to complement classic TOP or LC-MS approaches for more accurate reporting of PFAS contamination in the environment.

DOI: https://doi.org/10.1021/acs.analchem.2c05354
Anal Bioanal Chem. 2023 Mar 25.

Rules for mass spectrometry applications in the clinical laboratory.

Michael Vogeser, Katharina Habler.

  MS-based analytical methods now play an important role in medical laboratory analysis. Predominantly triple-stage mass spectrometry is used for the quantification of small molecule biomarkers and xenobiotics in blood and urine. The spectrum of applications ranges from completely in-house developed analytical methods, to industrially manufactured kit solutions used on generic equipment, to the first closed MS-based analysis systems. It is to be expected that the weights will shift in the coming years. Thus, operation and evaluation for most applications will remain very challenging and very different from the far more user-friendly and fully automated systems - mainly photometry-based - which are commonly used in clinical laboratories. General regulatory requirements for medical analysis differ significantly between countries globally. General requirements for in-house developed assay methods are valid in some countries, but concrete and methodology-specific rules for operation and quantification when using MS methods in the medical diagnostic laboratory are not applied. This differs significantly from other bio-analytical areas such as food monitoring, pharmaceutical research, or forensics, where legally binding, detailed rules exist in some cases, e.g., for substance identification. Internationally used relevant and helpful general standards with regard to mass spectrometric examination procedures in the clinical laboratory are in particular CLSI 62A and ISO 15189, while the IVDR in the EU primarily regulates the manufacture of diagnostic articles and not their application. In addition, from many years of application experience, some general advice can be recommended as rules that can contribute to robustness and patient safety in the clinical application of MS procedures; with emphasis on: reasonable method description, batch release, competence management, maintenance, and continuity management. This article also proposes some procedural basic requirements for the application of MS procedures in the clinical laboratory.

Keywords:  Diagnostics; Laboratory medicine; Liquid chromatography; Mass spectrometry; Quality assurance; Regulation

DOI:  https://doi.org/10.1007/s00216-023-04648-0
BMC Geriatr. 2023 03 21. 23(1): 156

Liquid chromatography-mass spectrometry-based metabolomic profiling reveals sex differences of lipid metabolism among the elderly from Southwest China.

Yuan-Jun Huang, Wei Ke, Ling Hu, You-Dong Wei, Mei-Xue Dong.

   BACKGROUND: The sexual dimorphism represents one of the triggers of the metabolic disparities while the identification of sex-specific metabolites in the elderly has not been achieved.
METHODS: A group of aged healthy population from Southwest China were recruited and clinical characteristics were collected. Fasting plasma samples were obtained and untargeted liquid chromatography-mass spectrometry-based metabolomic analyses were performed. Differentially expressed metabolites between males and females were identified from the metabolomic analysis and metabolite sets enrichment analysis was employed.
RESULTS: Sixteen males and fifteen females were finally enrolled. According to clinical characteristics, no significant differences can be found except for smoking history. There were thirty-six differentially expressed metabolites between different sexes, most of which were lipids and lipid-like molecules. Twenty-three metabolites of males were increased while thirteen were decreased compared with females. The top four classes of metabolites were fatty acids and conjugates (30.6%), glycerophosphocholines (22.2%), sphingomyelins (11.1%), and flavonoids (8.3%). Fatty acids and conjugates, glycerophosphocholines, and sphingomyelins were significantly enriched in metabolite sets enrichment analysis.
CONCLUSIONS: Significant lipid metabolic differences were found between males and females among the elderly. Fatty acids and conjugates, glycerophosphocholines, and sphingomyelins may partly account for sex differences and can be potential treatment targets for sex-specific diseases.

Keywords:  Lipid metabolism; Metabolite sets enrichment analysis; Metabolomic analysis; Sex difference; The elderly

DOI:  https://doi.org/10.1186/s12877-023-03897-z
Anal Chim Acta. 2023 Apr 29. pii: S0003-2670(23)00249-0. [Epub ahead of print]1252 341028

Rapid skin biomarker discovery using hydrogel-phase sampling followed by semi-automated liquid-phase re-extraction high-resolution mass spectrometry.

Decibel P Elpa, Chamarthi Maheswar Raju, Hsien-Yi Chiu, Shu-Pao Wu, Pawel L Urban.

  A facile and rapid skin metabolomics protocol is proposed. The liquid microjunction-surface sampling probe system has been partly automated, and used in conjunction with hydrogel probes for skin metabolite analysis. A control device was built to precisely control the segmented solvent flow and analyte re-extraction into the liquid microjunction. This mode provides rapid online re-extraction of the analytes from hydrogel probes. Humectant was added to the hydrogel, and moist heat treatment was used to make the hydrogel probes rugged for sampling in the clinical setting. The developed method was validated for the analysis of choline - a putative biomarker of psoriasis. A linear relationship over six calibration levels from 3.18 × 10-5 to 3.18 × 10-4 mol m-2 has been obtained. The limit of detection was 6.6 × 10-6 mol m-2, while the recoveries range from 92 to 109%. The within-run and between-run precision were evaluated and the coefficients of variation range from 1.84 to 7.13%. Furthermore, the developed method has been used to screen patients (n = 10) and healthy participants (control group; n = 10) for psoriasis-related skin metabolites. Metabolomic profiling of the skin excretion-related signals identified potential biomarkers of psoriasis: choline, pipecolic acid, ornithine, urocanic acid, and methionine.

Keywords:  High-resolution mass spectrometry; Liquid microjunction; Metabolic profiling; Skin biomarker; Skin metabolomics; Sweat

DOI:  https://doi.org/10.1016/j.aca.2023.341028
Med Pr. 2023 Mar 22. pii: 161304. [Epub ahead of print]

[LC-MS/MS as a method for the determination of isoprostanes: a brief review].

Magdalena Beata Król, Sławomir Brzeźnicki.

  Isoprostanes are a large group of compounds formed as products of free radical oxidation of polyunsaturated fatty acids, which are isomers of prostaglandin. They are present in all body tissues and biological fluids in quantifiable concentrations. Since 2018, the determination of isoprostanes by chromatographic technique with mass spectrometry is the golden standard of the oxidative stress markers determination in relation to oxidative damage to lipids. The publication is a synthetic review of recently published articles on the use of isoprostanes as a marker of lipid peroxidation determined with the liquid chromatography with tandem mass spectrometry technique. It presents the results of research using isoprostanes as a marker in medicine, in monitoring people working in exposure to harmful substances and in lifestyle research. Med Pr. 2023;74(2).

Keywords:  LC-MS/MS; application of isoprostanes; isoprostanes; lipid peroxidation; oxidative stress markers; prostaglandin isomers

DOI:  https://doi.org/10.13075/mp.5893.01336
Front Microbiol. 2023 ;14 1124144

HPLC-MS-MS quantification of short-chain fatty acids actively secreted by probiotic strains.

Marco Calvigioni, Andrea Bertolini, Simone Codini, Diletta Mazzantini, Adelaide Panattoni, Mariacristina Massimino, Francesco Celandroni, Riccardo Zucchi, Alessandro Saba, Emilia Ghelardi.

   Introduction: Short-chain fatty acids (SCFAs) are the main by-products of microbial fermentations occurring in the human intestine and are directly involved in the host's physiological balance. As impaired gut concentrations of acetic, propionic, and butyric acids are often associated with systemic disorders, the administration of SCFA-producing microorganisms has been suggested as attractive approach to solve symptoms related to SCFA deficiency.
Methods: In this research, nine probiotic strains (Bacillus clausii NR, OC, SIN, and T, Bacillus coagulans ATCC 7050, Bifidobacterium breve DSM 16604, Limosilactobacillus reuteri DSM 17938, Lacticaseibacillus rhamnosus ATCC 53103, and Saccharomyces boulardii CNCM I-745) commonly included in commercial formulations were tested for their ability to secrete SCFAs by using an improved protocol in high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS-MS).
Results: The developed method was highly sensitive and specific, showing excellent limits of detection and quantification of secreted SCFAs. All tested microorganisms were shown to secrete acetic acid, with only B. clausii and S. boulardii additionally able to produce propionic and butyric acids. Quantitative differences in the secretion of SCFAs were also evidenced.
Discussion: The experimental approach described in this study may contribute to the characterization of probiotics as SCFA-producing organisms, a crucial stage toward their application to improve SCFA deficiency.

Keywords:  HPLC-MS-MS; SCFAs; acetic acid; butyric acid; probiotics; propionic acid; secretion; short-chain fatty acids

DOI:  https://doi.org/10.3389/fmicb.2023.1124144
BMC Bioinformatics. 2023 Mar 22. 24(1): 108

Bayesian kinetic modeling for tracer-based metabolomic data.

Xu Zhang, Ya Su, Andrew N Lane, Arnold J Stromberg, Teresa W M Fan, Chi Wang.

   BACKGROUND: Stable Isotope Resolved Metabolomics (SIRM) is a new biological approach that uses stable isotope tracers such as uniformly [Formula: see text]-enriched glucose ([Formula: see text]-Glc) to trace metabolic pathways or networks at the atomic level in complex biological systems. Non-steady-state kinetic modeling based on SIRM data uses sets of simultaneous ordinary differential equations (ODEs) to quantitatively characterize the dynamic behavior of metabolic networks. It has been increasingly used to understand the regulation of normal metabolism and dysregulation in the development of diseases. However, fitting a kinetic model is challenging because there are usually multiple sets of parameter values that fit the data equally well, especially for large-scale kinetic models. In addition, there is a lack of statistically rigorous methods to compare kinetic model parameters between different experimental groups.
RESULTS: We propose a new Bayesian statistical framework to enhance parameter estimation and hypothesis testing for non-steady-state kinetic modeling of SIRM data. For estimating kinetic model parameters, we leverage the prior distribution not only to allow incorporation of experts' knowledge but also to provide robust parameter estimation. We also introduce a shrinkage approach for borrowing information across the ensemble of metabolites to stably estimate the variance of an individual isotopomer. In addition, we use a component-wise adaptive Metropolis algorithm with delayed rejection to perform efficient Monte Carlo sampling of the posterior distribution over high-dimensional parameter space. For comparing kinetic model parameters between experimental groups, we propose a new reparameterization method that converts the complex hypothesis testing problem into a more tractable parameter estimation problem. We also propose an inference procedure based on credible interval and credible value. Our method is freely available for academic use at https://github.com/xuzhang0131/MCMCFlux .
CONCLUSIONS: Our new Bayesian framework provides robust estimation of kinetic model parameters and enables rigorous comparison of model parameters between experimental groups. Simulation studies and application to a lung cancer study demonstrate that our framework performs well for non-steady-state kinetic modeling of SIRM data.

Keywords:  Bayesian method; Kinetic modeling; SIRM

DOI:  https://doi.org/10.1186/s12859-023-05211-5
Eur J Mass Spectrom (Chichester). 2023 Mar 21. 14690667231164096

Exploratory data fusion of untargeted multimodal LC-HRMS with annotation by LCMS-TOF-ion mobility: White wine case study.

Mpho Mafata, Maria Stander, Keabetswe Masike, Astrid Buica.

  Applied sciences have increased focus on omics studies which merge data science with analytical tools. These studies often result in large amounts of data produced and the objective is to generate meaningful interpretations from them. This can sometimes mean combining and integrating different datasets through data fusion techniques. The most strategic course of action when dealing with products of unknown profile is to use exploratory approaches. For omics, this means using untargeted analytical methods and exploratory data analysis techniques. The current study aimed to perform data fusion on untargeted multimodal (negative and positive mode) liquid chromatography-high-resolution mass spectrometry data using multiple factor analysis. The data fusion results were interpreted using agglomerative hierarchical clustering on biplot projections. The study reduced the thousands of spectral signals processed to less than a hundred features (a primary parameter combination of retention time and mass-to-charge ratios, RT_m/z). The correlations between cluster members (samples and features from) were calculated and the top 10% highly correlated features were identified for each cluster. These features were then tentatively identified using secondary parameters (drift time, ion mobility constant and collision cross-section values) from the ion mobility spectra. These ion mobility (secondary) parameters can be used for future studies in wine chemical analysis and added to the growing list of annotated chemical signals in applied sciences.

Keywords:  Data fusion; agglomerative hierarchical clustering; cluster analysis; exploratory multivariate analysis; high-resolution mass spectrometry; ion mobility; multiple factor analysis; white wine

DOI:  https://doi.org/10.1177/14690667231164096
Clin Biochem. 2023 Mar 18. pii: S0009-9120(23)00069-3. [Epub ahead of print]

Development of an Equilibrium Dialysis ID-UPLC-MS/MS Candidate Reference Measurement Procedure for Free Thyroxine in Human Serum.

Ashley Ribera, Li Zhang, Amonae Dabbs-Brown, Otoe Sugahara, Krista Poynter, Katleen van Uytfanghe, Eri Shimizu, A E van Herwaarden, Julianne C Botelho, Uliana Danilenko, Hubert W Vesper.

   BACKGROUND: Accurate and reliable measurement of human serum free thyroxine (FT4) is critical for the diagnosis and treatment of thyroid diseases. However, concerns have been raised regarding the performance of FT4 measurements in patient care. Centers for Disease Control and Prevention Clinical Standardization Programs (CDC-CSP) address these concerns by creating a FT4 standardization program to standardize FT4 measurements. The study aims to develop a highly accurate and precise candidate Reference Measurement Procedure (cRMP), as one key component of CDC-CSP, for standardization of FT4 measurements.
METHODS: Serum FT4 was separated from protein-bound thyroxine with equilibrium dialysis (ED) following the recommended conditions in the Clinical and Laboratory Standards Institute C45-A guideline and the published RMP [23]. FT4 in dialysate was directly quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. Gravimetric measurements of specimens and calibrator solutions, calibrator bracketing, isotope dilution, enhanced chromatographic resolution, and T4 specific mass transitions were used to ensure the accuracy, precision, and specificity of the cRMP.
RESULTS: The described cRMP agreed well with the established RMP and two other cRMPs in an interlaboratory comparison study. The mean biases of each method to the overall laboratory mean were within ±2.5%. The intra-day, inter-day, and total imprecision for the cRMP were within 4.4%. The limit of detection was 0.90 pmol/L, which was sufficiently sensitive to determine FT4 for patients with hypothyroidism. The structural analogs of T4 and endogenous components in dialysate did not interfere with the measurements.
CONCLUSION: Our ED-LC-MS/MS cRMP provides high accuracy, precision, specificity, and sensitivity for FT4 measurement. The cRMP can serve as a higher-order standard for establishing measurement traceability and provide an accuracy base for the standardization of FT4 assays.

Keywords:  FT4; Free thyroxine; Interlaboratory comparison; LC-MS/MS; Reference Measurement Procedure; equilibrium dialysis

DOI:  https://doi.org/10.1016/j.clinbiochem.2023.03.010
Rapid Commun Mass Spectrom. 2023 Mar 23. e9507

Inadequate Definition of the Limit of Quantification Applied for the Quantification of Perfluoroalkyl Substances in Food by LC-MS/MS May Compromise the Reliability of the Data Requested by the European Regulation.

Thierry Delatour, Xanthippe Theurillat, Bjørn Eriksen, Claudia Mujahid, Pascal Mottier.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a widespread technology used for the quantitative determination of per- and polyfluoroalkyl substances (PFAS) in foodstuffs. Specifically, LC-MS/MS offers attractive performance combining the sensitivity and selectivity required by the European Union for testing perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorohexane sulfonic acid (PFHxS) with maximum limits of quantification (LOQs) in the sub part-per-billion (μg/kg) or the part-per-trillion (ng/kg) domains. In this article we highlight the important diversity in LOQ definitions applied in LC-MS/MS methods described in the literature that raise concerns about the capability of some of those to generate reliable data requested by the European regulation. Here, we point the risk of false response or misquantification if criteria for assessing LOQ suffer from a lack of rigor. We emphasize the need to use PFAS-free samples spiked with the analyte(s) of interest and the application of identification criteria according to official documents for a sound measurement of the LOQ.

DOI: https://doi.org/10.1002/rcm.9507