bims-metlip 2024-06-30 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–06–30
thirty-six papers selected by
Sofia Costa, Matterworks

Quantification of gut microbiome metabolites using chemical isotope derivatization strategy combined with LC-MS/MS: Application in neonatal hypoxic-ischemic encephalopathy rat model.
Quantitation of thiorphan in human plasma using LC-MS/MS and its application to a bioequivalence study.
Development and validation of a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of eight Xiakucao Oral liquid-related compounds in rat plasma and its application in pharmacokinetic study.
Application of the Hydrophilic Interaction Liquid Chromatography (HILIC-MS) Novel Protocol to Study the Metabolic Heterogeneity of Glioblastoma Cells.
Cohort-based strategies as an in-house tool to evaluate and improve phenotyping robustness of LC-MS/MS lipidomics platforms.
High-resolution ion mobility based on traveling wave structures for lossless ion manipulation resolves hidden lipid features.
Characterization of Electrospray Ionization Complexity in Untargeted Metabolomic Studies.
Lipidomics Study of Type 1 Diabetic Rats Using Online Phase Transition Trapping-Supercritical Fluid Extraction-Chromatography Coupled with Quadrupole Time-of-Flight Tandem Mass Spectrometry.
Modification of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method targeting lysergic acid diethylamide (LSD) and its primary metabolite (OH-LSD) to include nine LSD analogs.
Simultaneous multi-targeted forensic toxicological screening in biological matrices by MRM-IDA-EPI mode.
Determination of Patulin in Apple Juice and Apple-Derived Products Using a Robotic Sample Preparation System and LC-APCI-MS/MS.
Designing optimal experiments in metabolomics.
A ng/L Level LC-MS Method Using Membrane SPE as Sampling Technology: Determination of Nine Halobenzoquinones in Potable Water.
Characterizing Lomerizine metabolites in camel urine: High-resolution mass spectrometry method development and validation for enhanced doping control.
Internal Standard Addition System for Online Breath Analysis.
Communicating Mass Spectrometry Quality Information in mzQC with Python, R, and Java.
Mass spectrometry imaging of N-linked glycans: Fundamentals and recent advances.
Liquid Chromatography-Mass Spectrometry Analytical Methods for the Quantitation of p-Cresol Sulfate and Indoxyl Sulfate in Human Matrices: Biological Applications and Diagnostic Potentials.
Development, validation and application of a UPLC-MS/MS method for simultaneous quantification of OPC-61815 and its metabolites tolvaptan, DM-4103 and DM-4107 in human plasma.
Development and validation of an LC-MS/MS method for the quantification of oral-sugar probes in plasma to test small intestinal permeability and absorptive capacity in the domestic cat (Felis catus).
Determination of six short-chain fatty acids in rat feces using headspace solid-phase dynamic extraction coupled with GC-MS.
Development of a simultaneous LC-MS/MS analytical method for plasma: 16 antipsychotics approved in Japan and 4 drug metabolites.
Dried blood spot sampling coupled with liquid chromatography-tandem mass for simultaneous quantitative analysis of multiple cardiovascular drugs.
Determination and Identification of Antibiotic Residues in Fruits Using Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS).
Application of molecular networking to improve the compound annotation in liquid chromatography-mass spectrometry-based metabolomics analysis: A case study of Bupleuri radix.
Explainable AI to Facilitate Understanding of Neural Network-Based Metabolite Profiling Using NMR Spectroscopy.
Scaling up a targeted exposome LC-MS/MS biomonitoring method by incorporating veterinary drugs and pesticides.
Quantification and analyses of seven tyrosine kinase inhibitors targeting hepatocellular carcinoma in human plasma by QuEChERS and UPLC-MS/MS.
Tetrodotoxin Derivatization with a Newly Designed Boron Reagent Leads to Conventional Reversed-Phase Liquid Chromatography.
Single ion mobility monitoring (SIM2) stitching method for high-throughput and high ion mobility resolution chiral analysis.
Liquid chromatography-tandem mass spectrometry for determination of fingolimod and its active metabolite fingolimod phosphate in whole blood of patients with multiple sclerosis.
Quantification of Rhodojaponin II and Rhodojaponin III in Rat Plasma by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.
Problems in Commercial Kits of 25-Hydroxy Vitamin D and the Development of Simple, Robust and Faster HPLC Method.
Differentiation of Cis/trans-geometrical isomers in long-chain unsaturated fatty acids based on ion mobility and theoretical calculations.
Ultrasensitive enantiomeric barbiturate analysis in body fluids through capillary electrophoresis with large volume sample stacking and ultrasound assisted dispersive liquid liquid microextraction.
Determination of ertapenem in plasma and ascitic fluid by UHPLC-MS/MS in cirrhotic patients with spontaneous bacterial peritonitis.

J Pharm Biomed Anal. 2024 Jun 12. pii: S0731-7085(24)00352-2. [Epub ahead of print]248 116312

Quantification of gut microbiome metabolites using chemical isotope derivatization strategy combined with LC-MS/MS: Application in neonatal hypoxic-ischemic encephalopathy rat model.

Fangbo Xia, Peng Cui, Ling Liu, Junhe Chen, Qiqi Zhou, Qian Wang, Hongwei Zhou.

  The gut microbiome plays pivotal roles in various physiological and pathological processes, with key metabolites including short chain fatty acids (SCFAs), bile acids (BAs), and tryptophan (TRP) derivatives gaining significant attention for their diverse physiological roles. However, quantifying these metabolites presents challenges due to structural similarity, low abundance, and inherent technical limitations in traditional detection methods. In this study, we developed a precise and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method utilizing a chemical isotope derivatization technique employing 4-(aminomethyl)-N,N-dimethylaniline-d0/d6 (4-AND-d0/d6) reagents to quantify 37 typical gut microbiome-derived metabolites. This method achieved an impressive 1500-fold enhancement in sensitivity for detecting metabolites, compared to methods using non-derivatized, intact molecules. Moreover, the quantitative accuracy of our chemical isotope derivatization strategy proved comparable to the stable isotope labeled internal standards (SIL-IS) method. Subsequently, we successfully applied this newly developed method to quantify target metabolites in plasma, brain, and fecal samples obtained from a neonatal hypoxic-ischemic encephalopathy (HIE) rat model. The aim was to identify crucial metabolites associated with the progression of HIE. Overall, our sensitive and reliable quantification method holds promise in elucidating the role of gut microbiome metabolites in the pathogenesis of various diseases.

Keywords:  Chemical isotope derivatization Strategy; Gut Microbiome Metabolites; LC-MS/MS; Quantitative Analysis

DOI:  https://doi.org/10.1016/j.jpba.2024.116312
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jun 15. pii: S1570-0232(24)00199-5. [Epub ahead of print]1243 124190

Quantitation of thiorphan in human plasma using LC-MS/MS and its application to a bioequivalence study.

Rana Said, Basel Arafat, Tawfiq Arafat.

  Racecadotril, an anti-secretory medication, has been used as an adjuvant in an oral rehydration therapy for children experiencing severe diarrhea. Racecadotril is quickly converted to thiorphan, an active metabolite, after oral treatment, which mediates all subsequent activities. An efficient and rapid liquid chromatography-tandem mass spectrometry method was developed and fully validated to measure thiorphan in human plasma, using thiorphan-d7 as an internal standard. The extraction method used was protein precipitation while chromatographic separation was achieved using InertSil CN-3 (50 × 2.1 mm, 5 µm column). The assay was linear over the concentration range of 1-200 ng/ml with correlation coefficients of ≥0.9991. The intra- and inter-day precisions were less than 10.0 % for all concentrations investigated. 0.02 % aqueous formic acid and methanol (30:70 v: v) were used as mobile phases, with an analysis time of less than 1 min. This method proved stable under several conditions. The developed method worked well in a three-period pharmacokinetic bioequivalence study after a single oral administration of 100 mg racecadotril to 15 healthy Jordanian volunteers under fasting conditions.

Keywords:  Bioequivalence; HPLC; LC MS/MS; Protein precipitation; Racecadotril; Thiorphan metabolite; Validation

DOI:  https://doi.org/10.1016/j.jchromb.2024.124190
Biomed Chromatogr. 2024 Jun 23. e5902

Development and validation of a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of eight Xiakucao Oral liquid-related compounds in rat plasma and its application in pharmacokinetic study.

Jingyi Jin, Xiaoqing Xu, Xinwei Wang, Biao Chen, Yingying Miao, Zhongguo Chen, Dongming Yan, Furong Qiu.

  Xiakucao Oral Liquid (XKCOL) has been widely used for treating mammary gland hyperplasia and goiter in China. However, its pharmacokinetic data have been missing to date. To conduct its pharmacokinetic study, we established an LC-tandem mass spectrometry method for the simultaneous determination of eight XKCOL-related compounds in rat plasma. Liquid-liquid extraction was used for the sampling process. Chromatographic separation was performed on a Phenomenon Luna C18 column with a mobile phase of methanol and 2 mM ammonium acetate, using gradient elution at a flow rate of 0.8 mL/min. Detection was performed in the multiple reaction monitoring mode using negative electrospray ionization (ESI-) with optimized MS parameters. Endogenous substances and carryover did not interfere in the detection of analytes. The calibration curves showed a good linear relationship within the linear ranges. The intra- and inter-batch accuracy and precision were 94.8%-110.0% and ≤11.2%, respectively. There was no significant matrix effect and the recovery was reproducible. The dilution of samples did not affect the accuracy and precision. The solution and plasma samples were stable under the various test conditions. The major components of XKCOL absorbed into the blood were salvianic acid A and rosmarinic acid. They demonstrated linear kinetics over the dose range used in this study.

Keywords:  LC–MS/MS method; Xiakucao Oral liquid; pharmacokinetic; rat plasma

DOI:  https://doi.org/10.1002/bmc.5902
Metabolites. 2024 May 23. pii: 297. [Epub ahead of print]14(6):

Application of the Hydrophilic Interaction Liquid Chromatography (HILIC-MS) Novel Protocol to Study the Metabolic Heterogeneity of Glioblastoma Cells.

Jakub Šofranko, Eduard Gondáš, Radovan Murín.

  Glioblastoma is a highly malignant brain tumor consisting of a heterogeneous cellular population. The transformed metabolism of glioblastoma cells supports their growth and division on the background of their milieu. One might hypothesize that the transformed metabolism of a primary glioblastoma could be well adapted to limitations in the variety and number of substrates imported into the brain parenchyma and present it their microenvironment. Additionally, the phenotypic heterogeneity of cancer cells could promote the variations among their metabolic capabilities regarding the utilization of available substrates and release of metabolic intermediates. With the aim to identify the putative metabolic footprint of different types of glioblastoma cells, we exploited the possibility for separation of polar and ionic molecules present in culture media or cell lysates by hydrophilic interaction liquid chromatography (HILIC). The mass spectrometry (MS) was then used to identify and quantify the eluted compounds. The introduced method allows the detection and quantification of more than 150 polar and ionic metabolites in a single run, which may be present either in culture media or cell lysates and provide data for polaromic studies within metabolomics. The method was applied to analyze the culture media and cell lysates derived from two types of glioblastoma cells, T98G and U118. The analysis revealed that even both types of glioblastoma cells share several common metabolic aspects, and they also exhibit differences in their metabolic capability. This finding agrees with the hypothesis about metabolic heterogeneity of glioblastoma cells. Furthermore, the combination of both analytical methods, HILIC-MS, provides a valuable tool for metabolomic studies based on the simultaneous identification and quantification of a wide range of polar and ionic metabolites-polaromics.

Keywords:  HILIC; LC-MS; amino acid; glioblastoma; metabolic heterogeneity; metabolomics

DOI:  https://doi.org/10.3390/metabo14060297
Anal Bioanal Chem. 2024 Jun 28.

Cohort-based strategies as an in-house tool to evaluate and improve phenotyping robustness of LC-MS/MS lipidomics platforms.

Benedikt Zöhrer, Cristina Gómez, Joaquim Jaumot, Helena Idborg, Signe S Torekov, Åsa M Wheelock, Craig E Wheelock, Antonio Checa.

  In recent years, instrumental improvements have enabled the spread of mass spectrometry-based lipidomics platforms in biomedical research. In mass spectrometry, the reliability of generated data varies for each compound, contingent on, among other factors, the availability of labeled internal standards. It is challenging to evaluate the data for lipids without specific labeled internal standards, especially when dozens to hundreds of lipids are measured simultaneously. Thus, evaluation of the performance of these platforms at the individual lipid level in interlaboratory studies is generally not feasible in a time-effective manner. Herein, using a focused subset of sphingolipids, we present an in-house validation methodology for individual lipid reliability assessment, tailored to the statistical analysis to be applied. Moreover, this approach enables the evaluation of various methodological aspects, including discerning coelutions sharing identical selected reaction monitoring transitions, pinpointing optimal labeled internal standards and their concentrations, and evaluating different extraction techniques. While the full validation according to analytical guidelines for all lipids included in a lipidomics method is currently not possible, this process shows areas to focus on for subsequent method development iterations as well as the robustness of data generated across diverse methodologies.

Keywords:  Bioanalytical methods; LC-MS/MS; Lipidomics; Sphingolipids

DOI:  https://doi.org/10.1007/s00216-024-05404-8
Anal Bioanal Chem. 2024 Jun 27.

High-resolution ion mobility based on traveling wave structures for lossless ion manipulation resolves hidden lipid features.

Allison R Reardon, Jody C May, Katrina L Leaptrot, John A McLean.

  High-resolution ion mobility (resolving power > 200) coupled with mass spectrometry (MS) is a powerful analytical tool for resolving isobars and isomers in complex samples. High-resolution ion mobility is capable of discerning additional structurally distinct features, which are not observed with conventional resolving power ion mobility (IM, resolving power ~ 50) techniques such as traveling wave IM and drift tube ion mobility (DTIM). DTIM in particular is considered to be the "gold standard" IM technique since collision cross section (CCS) values are directly obtained through a first-principles relationship, whereas traveling wave IM techniques require an additional calibration strategy to determine accurate CCS values. In this study, we aim to evaluate the separation capabilities of a traveling wave ion mobility structures for lossless ion manipulation platform integrated with mass spectrometry analysis (SLIM IM-MS) for both lipid isomer standards and complex lipid samples. A cross-platform investigation of seven subclass-specific lipid extracts examined by both DTIM-MS and SLIM IM-MS showed additional features were observed for all lipid extracts when examined under high resolving power IM conditions, with the number of CCS-aligned features that resolve into additional peaks from DTIM-MS to SLIM IM-MS analysis varying between 5 and 50%, depending on the specific lipid sub-class investigated. Lipid CCS values are obtained from SLIM IM (TW(SLIM)CCS) through a two-step calibration procedure to align these measurements to within 2% average bias to reference values obtained via DTIM (DTCCS). A total of 225 lipid features from seven lipid extracts are subsequently identified in the high resolving power IM analysis by a combination of accurate mass-to-charge, CCS, retention time, and linear mobility-mass correlations to curate a high-resolution IM lipid structural atlas. These results emphasize the high isomeric complexity present in lipidomic samples and underscore the need for multiple analytical stages of separation operated at high resolution.

Keywords:  High-resolution ion mobility-mass spectrometry; Ion mobility-mass spectrometry; Lipid structure; Lipidomics; Structures for lossless ion manipulation

DOI:  https://doi.org/10.1007/s00216-024-05385-8
Anal Chem. 2024 Jun 25.

Characterization of Electrospray Ionization Complexity in Untargeted Metabolomic Studies.

William J Nash, Judith B Ngere, Lukas Najdekr, Warwick B Dunn.

The annotation of metabolites detected in LC-MS-based untargeted metabolomics studies routinely applies accurate m/z of the intact metabolite (MS1) as well as chromatographic retention time and MS/MS data. Electrospray ionization and transfer of ions through the mass spectrometer can result in the generation of multiple "features" derived from the same metabolite with different m/z values but the same retention time. The complexity of the different charged and neutral adducts, in-source fragments, and charge states has not been previously and deeply characterized. In this paper, we report the first large-scale characterization using publicly available data sets derived from different research groups, instrument manufacturers, LC assays, sample types, and ion modes. 271 m/z differences relating to different metabolite feature pairs were reported, and 209 were annotated. The results show a wide range of different features being observed with only a core 32 m/z differences reported in >50% of the data sets investigated. There were no patterns reporting specific m/z differences that were observed in relation to ion mode, instrument manufacturer, LC assay type, and mammalian sample type, although some m/z differences were related to study group (mammal, microbe, plant) and mobile phase composition. The results provide the metabolomics community with recommendations of adducts, in-source fragments, and charge states to apply in metabolite annotation workflows.

DOI: https://doi.org/10.1021/acs.analchem.4c00966
J Proteome Res. 2024 Jun 23.

Lipidomics Study of Type 1 Diabetic Rats Using Online Phase Transition Trapping-Supercritical Fluid Extraction-Chromatography Coupled with Quadrupole Time-of-Flight Tandem Mass Spectrometry.

Binhong He, Ting Zhou, Jiaqi Liu.

  Chromatography-mass spectrometry-based lipidomics represents an essential tool for elucidating lipid dysfunction mechanisms and is extensively employed in investigating disease mechanisms and identifying biomarkers. However, the detection of low-abundance lipids in biological matrices, along with cumbersome operational procedures, complicates comprehensive lipidomic analyses, necessitating the development of highly sensitive, environmentally friendly, and automated methods. In this study, an online phase transition trapping-supercritical fluid extraction-chromatography-mass spectrometry (PTT-SFEC-MS/MS) method was developed and successfully applied to plasma lipidomics analysis in Type 1 diabetes (T1D) rats. The PTT strategy captured entire extracts at the column head by converting CO2 from a supercritical state to a gaseous state, thereby preventing peak spreading, enhancing peak shape for precise quantification, and boosting sensitivity without any sample loss. This method utilized only 5 μL of plasma and accomplished sample extraction, separation, and detection within 27 min. Ultimately, 77 differential lipids were identified, including glycerophospholipids, sphingolipids, and glycerolipids, in T1D rat plasma. The results indicated that the progression of the disease might be linked to alterations in glycerophospholipid and sphingolipid metabolism. Our findings demonstrated a green, highly efficient, and automated method for the lipidomics analysis of biological samples, providing a scientific foundation for understanding the pathogenesis and diagnosis of T1D.

Keywords:  Type 1 diabetes; lipidomics; online supercritical fluid extraction-chromatography; phase transition trapping

DOI:  https://doi.org/10.1021/acs.jproteome.4c00337
J Forensic Sci. 2024 Jun 27.

Modification of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method targeting lysergic acid diethylamide (LSD) and its primary metabolite (OH-LSD) to include nine LSD analogs.

Amy L Patton, Erin L Karschner, Jeffrey P Walterscheid, Jason M Garcia.

  A variety of LSD analogs have emerged in recent years with dual purposes of avoiding prosecution from possession while providing new options for those willing to experiment with hallucinogenic drugs. In this study, a previously published automated sample preparation method for LSD and its primary metabolite (OH-LSD) was utilized to extract LSD, OH-LSD, and nine LSD analogs from urine. The liquid chromatography tandem mass spectrometry (LC-MS/MS) method was modified from the previously published LC conditions to utilize a different analytical column and gradient elution program. Mobile phases of 10 mM ammonium formate with 0.1% formic acid in deionized water (mobile phase A) and 0.1% formic acid in methanol (mobile phase B) were employed. The method was validated to ANSI/ASB Standard 036 with a 0.1 ng/mL limit of detection for all analytes and was utilized for the analysis of 325 urine specimens. Although no LSD analogs were observed in the samples analyzed, this validated method was demonstrated to be suitable for the analysis of these compounds in laboratories seeking to expand their testing scope. Automated sample preparation allows for the efficient analysis of these analytically challenging compounds with minimal manual handling. Additionally, there was no increased analytical time burden when the LC column and gradient were modified to target nine additional analytes. Detection may improve as new reference standards are developed to allow laboratories to focus on the metabolic products of these analogs. For now, this validated procedure can assist with the routine analysis and surveillance of these emerging substances.

Keywords:  Hamilton; LC–MS/MS; LSD; LSD analogs; automation; forensic toxicology; urine analysis

DOI:  https://doi.org/10.1111/1556-4029.15572
Arch Toxicol. 2024 Jun 25.

Simultaneous multi-targeted forensic toxicological screening in biological matrices by MRM-IDA-EPI mode.

Martina Franzin, Rebecca Di Lenardo, Rachele Ruoso, Paolo Dossetto, Stefano D'Errico, Riccardo Addobbati.

  The toxicologist ascertains drug assumptions in case of paediatric intoxications and death for overdose. The analytical approach consists of initially screening and consequently confirming drug positivity. We developed a toxicological screening method and validated its use comparing the results with a LC-MS/MS analysis. The method identifies 751 drugs and metabolites (704 in positive and 47 in negative mode). Chromatographic separation was achieved eluting mobile phase A (10 mM ammonium formate) and B (0.05% formic acid in methanol) in gradient on Kinetex Phenyl-Hexyl (50 × 4.6 mm, 2.6 μm) with 0.7 mL/min flow rate for 11 min. Multiple Reaction Monitoring (MRM) was adopted as survey scan and, after an Information-Dependent Analysis (IDA) (threshold of 30,000 for positive and 1000 cps for negative mode), the Enhanced Product Ion (scan range: 50-700 amu) was triggered. The MS/MS spectrum generated was compared with one of the libraries for identification. Data processing was optimised through creation of rules. Sample preparation, mainly consisting of deproteinization and enzymatic hydrolysis, was set up for different matrices (blood, urine, vitreous humor, synovial fluid, cadaveric tissues and larvae). Cut-off for most analytes resulted in the lowest concentration tested. When the results from the screening and LC-MS/MS analysis were compared, an optimal percentage of agreement (100%) was assessed for all matrices. Method applicability was evaluated on real paediatric intoxications and forensic cases. In conclusion, we proposed a multi-targeted, fast, sensitive and specific MRM-IDA-EPI screening having an extensive use in different toxicological fields.

Keywords:  Biological matrices; Forensic cases; Overdose; Paediatric intoxications; Toxicological screening

DOI:  https://doi.org/10.1007/s00204-024-03806-2
Toxins (Basel). 2024 May 23. pii: 238. [Epub ahead of print]16(6):

Determination of Patulin in Apple Juice and Apple-Derived Products Using a Robotic Sample Preparation System and LC-APCI-MS/MS.

Kai Zhang, Lauren Zhang.

  Patulin, a toxic mycotoxin, can contaminate apple-derived products. The FDA has established an action level of 50 ppb (ng/g) for patulin in apple juice and apple juice products. To effectively monitor this mycotoxin, there is a need for adequate analytical methods that can reliably and efficiently determine patulin levels. In this work, we developed an automated sample preparation workflow followed by liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS) detection to identify and quantify patulin in a single method, further expanding testing capabilities for monitoring patulin in foods compared to traditional optical methods. Using a robotic sample preparation system, apple juice, apple cider, apple puree, apple-based baby food, applesauce, fruit rolls, and fruit jam were fortified with 13C-patulin and extracted using dichloromethane (DCM) without human intervention, followed by an LC-APCI-MS/MS analysis in negative ionization mode. The method achieved a limit of quantification of 4.0 ng/g and linearity ranging from 2 to 1000 ng/mL (r2 > 0.99). Quantitation was performed with isotope dilution using 13C-patulin as an internal standard and solvent calibration standards. Average recoveries (relative standard deviations, RSD%) in seven spike matrices were 95% (9%) at 10 ng/g, 110% (5%) at 50 ng/g, 101% (7%) at 200 ng/g, and 104% (4%) at 1000 ng/g (n = 28). The ranges of within-matrix and between-matrix variability (RSD) were 3-8% and 4-9%, respectively. In incurred samples, the identity of patulin was further confirmed with a comparison of the information-dependent acquisition-enhanced product ion (IDA-EPI) MS/MS spectra to a reference standard. The metrological traceability of the patulin measurements in an incurred apple cider (21.1 ± 8.0 µg/g) and apple juice concentrate (56.6 ± 15.6 µg/g) was established using a certified reference material and calibration data to demonstrate data confidence intervals (k = 2, 95% confidence interval).

Keywords:  LC-APCI-MS/MS; automated sample preparation; patulin

DOI:  https://doi.org/10.3390/toxins16060238
Metabolomics. 2024 Jun 28. 20(4): 69

Designing optimal experiments in metabolomics.

Mathies Brinks Sørensen, Jan Kloppenborg Møller, Mikael Lenz Strube, Charlotte Held Gotfredsen.

   BACKGROUND: Metabolomics data is often complex due to the high number of metabolites, chemical diversity, and dependence on sample preparation. This makes it challenging to detect significant differences between factor levels and to obtain accurate and reliable data. To address these challenges, the use of Design of Experiments (DoE) techniques in the setup of metabolomic experiments is crucial. DoE techniques can be used to optimize the experimental design space, ensuring that the maximum amount of information is obtained from a limited sample space.
AIM OF REVIEW: This review aims at providing a baseline workflow for applying DoE when generating metabolomics data.
KEY SCIENTIFIC CONCEPTS OF REVIEW: The review provides insights into the theory of DoE. The review showcases the theory being put into practice by highlighting different examples DoE being applied in metabolomics throughout the literature, considering both targeted and untargeted metabolomic studies in which the data was acquired using both nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry techniques. In addition, the review presents DoE concepts not currently being applied in metabolomics, highlighting these as potential future prospects.

Keywords:  Design of Experiments; Mass spectrometry; Metabolomics; Nuclear magnetic resonance

DOI:  https://doi.org/10.1007/s11306-024-02122-1
Molecules. 2024 Jun 15. pii: 2856. [Epub ahead of print]29(12):

A ng/L Level LC-MS Method Using Membrane SPE as Sampling Technology: Determination of Nine Halobenzoquinones in Potable Water.

Qin Huang, Hua Zhou, Xianglun Wu, Jiaqi Jiang, Bingdong Zhan, Pinggu Wu.

  A promising method was established for the determination of nine halobenzoquinones (HBQs) in potable water by membrane solid-phase extraction (MSPE) pretreatment and the liquid chromatography-mass spectrometry (LC-MS) method. A 500 mL water sample was taken for enrichment by the SDB-RPS membrane, which was previously activated by methanol and ultrapure water. The sample was eluted with methanol and re-dissolved with the initial mobile phase after nitrogen blowing. Then, it was detected in negative ion mode using the working curve, and HBQs were quantified by the external standard method. The linearity was satisfactory in the concentration range of 4-1000 ng/L, with correlation coefficients of 0.9963~0.9994. The recoveries were 73.5~126.6% at three spiked levels, with relative standard deviations (RSDs) of 6.8~15.5%. The limits of detection (LOD, S/N = 3) values were 0.1~0.7 ng/L. The results demonstrate that the MSPE-LC-MS method is reliable, rapid, and sensitive for the simultaneous analysis of nine HBPs in potable water.

Keywords:  halobenzoquinones (HBQs); liquid chromatography–mass spectrometry (LC-MS); membrane solid-phase extraction (MSPE); potable water

DOI:  https://doi.org/10.3390/molecules29122856
Rapid Commun Mass Spectrom. 2024 Sep 15. 38(17): e9846

Characterizing Lomerizine metabolites in camel urine: High-resolution mass spectrometry method development and validation for enhanced doping control.

Jahfar Nalakath, Rasik Puzhithinipra Rasik, Ahmed Kadry, Ansar Babu, Ibrahim Waseem, Praseen Ok, Christiana Hebel, Narayanan Selvapalam, Erumaipatty Rajagounder Nagarajan.

RATIONALE: Lomerizine (LMZ) is an antimigraine drug that works as a calcium channel blocker and has selective effects on the central nervous system. It is metabolized into trimetazidine (TMZ), which is a prohibited substance owing to its performance-enhancing effects in both human and animal sports. Effective doping control measures are imperative to distinguish the source of TMZ in samples to ensure integrity and fairness of the sport, therefore a comprehensive analysis of LMZ metabolites is essential to identify potential biomarkers in camel urine for effective doping control.
METHODS: Camel urine samples were collected from four healthy animals following a single oral administration of LMZ at a dosage of 1 mg/kg body weight. In vitro studies were conducted using homogenized camel liver samples. Lomerizine and its metabolites were extracted using solid-phase extraction and analyzed with a Thermo Fisher Orbitrap Exploris liquid chromatography mass spectrometry system. The acquired data was processed with the Compound Discoverer software.
RESULTS: The study conducted a comprehensive analysis of LMZ metabolites in camels and identified 10 phase I and one phase II metabolites. The primary pathway for the formation of phase I metabolites was de-alkylation, while phase II metabolite was formed through alkylation of the parent drug. The study provided valuable insights into the unique metabolic pathways of LMZ in camels under specific experimental conditions.
CONCLUSION: The developed method enables the detection and characterization of LMZ and its metabolites in camels. The identified metabolites has the potential to act as marker metabolites for the distinctive detection of LMZ in camel urine to ensure efficient analytical strategies for routine doping control applications.

DOI: https://doi.org/10.1002/rcm.9846
Anal Chem. 2024 Jun 27.

Internal Standard Addition System for Online Breath Analysis.

Cedric Wüthrich, Timon Käser, Renato Zenobi, Stamatios Giannoukos.

Breath analysis with secondary electrospray ionization (SESI) coupled to mass spectrometry (MS) is a sensitive method for breath metabolomics. To enable quantitative assessments using SESI-MS, a system was developed to introduce controlled amounts of gases into breath samples and carry out standard addition experiments. The system combines gas standard generation through controlled evaporation, humidification, breath dilution, and standard injection with the help of mass-flow controllers. The system can also dilute breath, which affects the signal of the detected components. This response can be used to filter out contaminating compounds in an untargeted metabolomics workflow. The system's quantitative capabilities have been shown through standard addition of pyridine and butyric acid into breath in real time. This system can improve the quality and robustness of breath data.

DOI: https://doi.org/10.1021/acs.analchem.4c01924
J Am Soc Mass Spectrom. 2024 Jun 25.

Communicating Mass Spectrometry Quality Information in mzQC with Python, R, and Java.

Chris Bielow, Nils Hoffmann, David Jimenez-Morales, Tim Van Den Bossche, Juan Antonio Vizcaíno, David L Tabb, Wout Bittremieux, Mathias Walzer.

Mass spectrometry is a powerful technique for analyzing molecules in complex biological samples. However, inter- and intralaboratory variability and bias can affect the data due to various factors, including sample handling and preparation, instrument calibration and performance, and data acquisition and processing. To address this issue, the Quality Control (QC) working group of the Human Proteome Organization's Proteomics Standards Initiative has established the standard mzQC file format for reporting and exchanging information relating to data quality. mzQC is based on the JavaScript Object Notation (JSON) format and provides a lightweight yet versatile file format that can be easily implemented in software. Here, we present open-source software libraries to process mzQC data in three programming languages: Python, using pymzqc; R, using rmzqc; and Java, using jmzqc. The libraries follow a common data model and provide shared functionalities, including the (de)serialization and validation of mzQC files. We demonstrate use of the software libraries in a workflow for extracting, analyzing, and visualizing QC metrics from different sources. Additionally, we show how these libraries can be integrated with each other, with existing software tools, and in automated workflows for the QC of mass spectrometry data. All software libraries are available as open source under the MS-Quality-Hub organization on GitHub (https://github.com/MS-Quality-Hub).

DOI: https://doi.org/10.1021/jasms.4c00174
Mass Spectrom Rev. 2024 Jun 27.

Mass spectrometry imaging of N-linked glycans: Fundamentals and recent advances.

Tana V Palomino, David C Muddiman.

  With implications in several medical conditions, N-linked glycosylation is one of the most important posttranslation modifications present in all living organisms. Due to their nontemplate synthesis, glycan structures are extraordinarily complex and require multiple analytical techniques for complete structural elucidation. Mass spectrometry is the most common way to investigate N-linked glycans; however, with techniques such as liquid-chromatography mass spectrometry, there is complete loss of spatial information. Mass spectrometry imaging is a transformative analytical technique that can visualize the spatial distribution of ions within a biological sample and has been shown to be a powerful tool to investigate N-linked glycosylation. This review covers the fundamentals of mass spectrometry imaging and N-linked glycosylation and highlights important findings of recent key studies aimed at expanding and improving the glycomics imaging field.

Keywords:  N‐linked glycans; glycomics; mass spectrometry imaging

DOI:  https://doi.org/10.1002/mas.21895
Pharmaceutics. 2024 May 30. pii: 743. [Epub ahead of print]16(6):

Liquid Chromatography-Mass Spectrometry Analytical Methods for the Quantitation of p-Cresol Sulfate and Indoxyl Sulfate in Human Matrices: Biological Applications and Diagnostic Potentials.

Ala'a R Al-Dajani, Qi Kun Hou, Tony K L Kiang.

  Indoxyl sulfate (IxS) and p-cresyl sulfate (pCS) are toxic uremic compounds with documented pathological outcomes. This review critically and comprehensively analyzes the available liquid chromatography-mass spectrometry methods quantifying IxS and pCS in human matrices and the biological applications of these validated assays. Embase, Medline, PubMed, Scopus, and Web of Science were searched until December 2023 to identify assays with complete analytical and validation data (N = 23). Subsequently, citation analysis with PubMed and Scopus was utilized to identify the biological applications for these assays (N = 45). The extraction methods, mobile phase compositions, chromatography, and ionization methods were evaluated with respect to overall assay performance (e.g., sensitivity, separation, interference). Most of the assays focused on human serum/plasma, utilizing acetonitrile or methanol (with ammonium acetate/formate or formic/acetic acid), liquid-liquid extraction, reverse phase (e.g., C18) chromatography, and gradient elution for analyte separation. Mass spectrometry conditions were also consistent in the identified papers, with negative electrospray ionization, select multiple reaction monitoring transitions and deuterated internal standards being the most common approaches. The validated biological applications indicated IxS and/or pCS were correlated with renal disease progression and cardiovascular outcomes, with limited data on central nervous system disorders. Methods for reducing IxS and/or pCS concentrations were also identified (e.g., drugs, natural products, diet, dialysis, transplantation) where inconsistent findings have been reported. The clinical monitoring of IxS and pCS is gaining significant interest, and this review will serve as a useful compendium for scientists and clinicians.

Keywords:  indoxyl sulfate; kidney disease; liquid chromatography-mass spectrometry; p-Cresyl sulfate; protein-bound uremic toxins

DOI:  https://doi.org/10.3390/pharmaceutics16060743
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jun 19. pii: S1570-0232(24)00222-8. [Epub ahead of print]1242 124213

Development, validation and application of a UPLC-MS/MS method for simultaneous quantification of OPC-61815 and its metabolites tolvaptan, DM-4103 and DM-4107 in human plasma.

Yufang Ma, Mengyang Yu, Hongyun Wang.

  OPC-61815 is an intravenous formulation vasopressin antagonist designed to treat heart failure patients, especially who have difficulty in oral intake. Tolvaptan together with DM-4103 and DM-4107 are considered as the major metabolites of OPC-61815 biotransformed in the liver via cytochrome P450 (CYP) 3A. An efficient and robust ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of OPC-61815 and its three metabolites in human plasma was developed and fully validated. To our best knowledge, it was the first published method that simultaneously quantified all of these four analytes in only one run. Simple and rapid sample preparation procedure and very short UPLC-MS/MS run time (3.5 min) offered OPC-61815 and its metabolites relatively high throughput detection, which was greatly beneficial to further clinical bio-sample analysis. The method showed good linearity and sufficient sensitivity in the range of 2.00-1000 ng/mL with a low limit of quantitation (2.00 ng/mL) for each analyte. For samples with concentrations above 1000 ng/mL, 100-fold dilution with blank plasma before sample preparation was accepted. High precision and accuracy, high selectivity and satisfactory recovery of this method were demonstrated. For all of the four analytes, no significant matrix effect or carry-over was observed. The stability of analytes and internal standards under different conditions were evaluated to ensure they were stable during the whole period of storage, preparation and detection. Also, re-injection reproducibility was investigated. In addition, the conversion test showed that almost no OPC-61815 converted into DM-4103 and DM-4107 during sample processing, while attention should be paid to the concentration difference between OPC-61815 and tolvaptan in bioanalysis. The developed UPLC-MS/MS method was successfully applied to an open, single and multiple dose administration phase I trial for monitoring the pharmacokinetics of OPC-61815. This work provided a promising way for further pharmacokinetic study of OPC-61815.

Keywords:  Metabolite; OPC-61815; Pharmacokinetics; Quantification; Tolvaptan; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2024.124213
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jun 22. pii: S1570-0232(24)00225-3. [Epub ahead of print]1242 124216

Development and validation of an LC-MS/MS method for the quantification of oral-sugar probes in plasma to test small intestinal permeability and absorptive capacity in the domestic cat (Felis catus).

Keely Patterson, Karl Fraser, Daniel Bernstein, Emma N Bermingham, Karin Weidgraaf, Anna Kate Shoveller, David Thomas.

  A novel method for quantifying the concentration of lactulose, rhamnose, xylose, and 3-O-methylglucose (3-OMG) in cat plasma using liquid chromatography-mass spectrometry (LC-MS) was developed. Domestic male cats (n = 13) were orally dosed with a solution containing the four sugars to test the permeability and absorptive capacity of their intestinal barrier. Plasma samples were taken 3 h later and were prepared with acetonitrile (ACN), dried under N2, and reconstituted in 90 % ACN with 1 mM ammonium formate. Stable isotope labelled 13C standards for each analyte were used as internal standards. Chromatographic separation was conducted using a Phenomenex Luna NH2 column with a gradient elution system of deionized water and 90 % ACN with 1 mM ammonium formate at 300 µL/min for 13 min total analysis time. Recovery trials were conducted in triplicate over three days with RSD values (%) for each day ranging from 1.2 to 1.4 for lactulose, 5.4 - 6.0 for rhamnose, 3.3 - 5.5 for xylose, and 2.6 - 5.6 for 3-OMG. Inter-day variations for each analyte were not different (p > 0.05). Limit of detection and quantification were 0.2 and 0.7 µg/mL for lactulose, 0.8 and 2.4 µg/mL for rhamnose, 0.6 and 1.8 µg/mL for xylose, and 0.3 and 1.1 µg/mL for 3-OMG, respectively. Plasma sugar concentrations recovered from cats were above the limit of quantification and below the highest calibration standard, validating the use of this method to test intestinal permeability and absorptive capacity in cats.

Keywords:  Absorptive capacity; Differential sugar absorption test; Intestinal permeability; LC-MS/MS; Plasma; Tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.jchromb.2024.124216
J Sep Sci. 2024 Jun;47(12): e2400032

Determination of six short-chain fatty acids in rat feces using headspace solid-phase dynamic extraction coupled with GC-MS.

Lihua Xing, Qiang Zhang, Juan Liu, Nianjun Yu, Yiqun Jia.

  Short-chain fatty acids (SCFAs) are organic acids with carbon atoms less than six, released through fermentation products by intestinal microbiome, having multiple physiological activities. Considering weak acidity and high volatility, derivatization or liquid-liquid extraction is essential, which is time consuming. Headspace-solid-phase dynamic extraction (HS-SPDE) coupled with gas chromatography-mass spectrometry is automated and effortless to determine SCFAs in rat feces. The extraction procedure is performed by aspirating and discharging the headspace cyclically through a steel needle, coated with an inner polyethylene glycol sorbent. The key parameters of SPDE were optimized including coating type, incubation time and temperature, and number of extraction strokes. Besides, salting-out was conducted. Then, a method by HS-SPDE-GC-MS was established and validated. It only took 3-min incubation time, 4.5 min extraction time, and 13 min chromatographic separation in a run. The recovery, linearity, limit of quantification, and stability were evaluated. Then, the proposed method was applied to analyze rat feces including 18 rats with liver injury and 23 normal controls. Mann-Whitney U test indicated that the concentrations of six SCFAs in normal rat feces were higher than those with liver injury. This method provides a choice for fast, solvent-free, automated, and high-throughput analysis of SCFAs.

Keywords:  gas chromatography‐mass spectrometry; liver injury; rat feces; short‐chain fatty acids; solid‐phase dynamic microextraction

DOI:  https://doi.org/10.1002/jssc.202400032
Anal Sci. 2024 Jun 25.

Development of a simultaneous LC-MS/MS analytical method for plasma: 16 antipsychotics approved in Japan and 4 drug metabolites.

Masamitsu Maekawa, Maki Yokota, Toshihiro Sato, Yu Sato, Masaki Kumondai, Yuji Sato, Masato Suzuka, Daisuke Kobayashi, Kotaro Sakamoto, Masaki Matsuura, Masafumi Kikuchi, Hiroshi Komatsu, Kumiko Fujii, Yuji Ozeki, Hiroaki Tomita, Nariyasu Mano.

  The increased risk of adverse drug reactions due to the concomitant use of antipsychotics is problematic in the treatment of schizophrenia. Therefore, the simultaneous analysis of their plasma concentrations is required. In this study, we developed a simultaneous liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for analyzing plasma antipsychotics approved in Japan for therapeutic drug monitoring (TDM) applications. First, we counted the prescriptions for 16 antipsychotics and concomitant drugs used at the Tohoku University Hospital. LC-MS/MS was used for the simultaneous analysis of 16 antipsychotics and four drug metabolites. This analysis was conducted using a combination of selected reaction monitoring mode and reversed-phase chromatography. Following the examination of the MS/MS and LC conditions, an analytical method validation test was conducted. The developed method was used to analyze plasma antipsychotic levels in patients with schizophrenia. One-third of the patients received treatment with multiple antipsychotics. Under LC-MS/MS conditions, LC separation was performed using a combination of a C18 column and ammonium formate-based mobile phases with a gradient flow. The calibration curves were optimized by adjusting the ion abundance, and 11 compounds met the criteria for intra- and inter-day reproducibility tests. Some stability test results did not meet these criteria; therefore, further investigation is required. The developed method permitted the measurement of all the plasma parameters, including concentrations above the therapeutic range. Therefore, this method may be useful in the daily TDM practice of antipsychotics.

Keywords:  Antipsychotics; LC–MS/MS; Plasma drug concentrations; Schizophrenia; Simultaneous analysis

DOI:  https://doi.org/10.1007/s44211-024-00619-2
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jun 22. pii: S1570-0232(24)00224-1. [Epub ahead of print]1242 124215

Dried blood spot sampling coupled with liquid chromatography-tandem mass for simultaneous quantitative analysis of multiple cardiovascular drugs.

Mariam M Abady, Ji-Seon Jeong, Ha-Jeong Kwon.

  Dried Blood Spots (DBS) revolutionize therapeutic drug monitoring using LC-MS for the precise quantification of cardiovascular drugs (CDs), enabling personalized treatment adapted to patient-specific pharmacokinetics with minimal invasiveness. This study aims to achieve simultaneous quantification of eight CDs in DBS, overcoming physicochemical challenges. A two-step protein precipitation method was used for simple and precise sample preparation. The drugs were analyzed using LC-MS/MS in ESI positive-ion mode, showing high sensitivity and linearity, with a correlation coefficient (r2) exceeding 0.999, after being separated on a reversed-phase chromatography by gradient elution of DW-acetonitrile containing 0.1 % formic acid + 2 mM ammonium formate. The validation results indicate good selectivity, with no observed matrix effect and carry-over. The intra- and inter-day accuracy and precision were within 6 % for most drugs, except for digoxin and deslanoside at low therapeutic levels where the variation was within 20 %. Stability tests confirmed suitable DBS handling and storage conditions, indicating drug stability for at least 30 days at room temperature. The analysis of whole spot has demonstrated remarkable precision and reliability in all target drugs. The analysis of 3 mm internal diameter discs, punched in and out of DBS, presumed to contain 3 µL of blood, showed acceptable accuracy for most drugs, with less polar drugs like digoxin and deslanoside showing lower accuracy, indicating a need for further correction due to non-uniform drug distribution. Consequently, the developed LC-MS/MS method enables the quantification of multiple CDs in a single DBS analysis, while suggesting the potential for accuracy-based analysis.

Keywords:  Cardiovascular drugs; Dried blood spots; LC-MS/MS; Method validation; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.jchromb.2024.124215
J Agric Food Chem. 2024 Jun 27.

Determination and Identification of Antibiotic Residues in Fruits Using Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS).

Sherri B Turnipseed, Jessica P Rafson, Christine R Casey.

  Antibiotic residues may be present in fruit products from trees that were treated to combat bacterial diseases such as citrus greening or blight. A liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was developed for the simultaneous determination and identification of streptomycin, kasugamycin, penicillin, and oxytetracycline residues in fruit. Samples were extracted with acidic methanol and separation was optimized for a hydrophilic interaction LC column. A Q-Exactive HRMS instrument was used to obtain product ion spectra for analyte identification. Quantitation was performed with matrix-extracted calibration curves and internal standard correction. The method was tested on many different types of fruit. In general, fortified samples demonstrated acceptable recoveries (82-116%) and reproducibility (<15% RSD). Method detection limits for these analytes were well below the established US EPA tolerance levels. It was also possible to analyze the fruit extracts prepared using this method for additional chemical contaminants using LC-HRMS.

Keywords:  HILIC; LC-HRMS; QuPPe; antibiotic residues; fruit

DOI:  https://doi.org/10.1021/acs.jafc.4c01792
Phytochem Anal. 2024 Jun 24.

Application of molecular networking to improve the compound annotation in liquid chromatography-mass spectrometry-based metabolomics analysis: A case study of Bupleuri radix.

Weibo Qin, Yi Wu, Wenyi Gao, Yang Wang.

   INTRODUCTION: Compound annotation is always a challenging step in metabolomics studies. The molecular networking strategy has been developed recently to organize the relationship between compounds as a network based on their tandem mass (MS2) spectra similarity, which can be used to improve compound annotation in metabolomics analysis.
OBJECTIVE: This study used Bupleuri Radix from different geographic areas to evaluate the performance of molecular networking strategy for compound annotation in liquid chromatography-mass spectrometry (LC-MS)-based metabolomics.
METHODOLOGY: The Bupleuri Radix extract was analyzed by LC-quadrupole time-of-flight MS under MSe acquisition mode. After raw data preprocessing, the resulting dataset was used for statistical analysis, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). The chemical makers related to the sample growth place were selected using variable importance in projection (VIP) > 2, fold change (FC) > 2, and p < 0.05. The molecular networking analysis was applied to conduct the compound annotation.
RESULTS: The score plots of PCA showed that the samples were classified into two clusters depending on their growth place. Then, the PLS-DA model was constructed to explore the chemical changes of the samples further. Sixteen compounds were selected as chemical makers and tentatively annotated by the feature-based molecular networking (FBMN) analysis.
CONCLUSION: The results showed that the molecular networking method fully exploits the MS information and is a promising tool for facilitating compound annotation in metabolomics studies. However, the software used for feature extraction influenced the results of library searching and molecular network construction, which need to be taken into account in future studies.

Keywords:  compound annotation; data‐independent acquisition; liquid chromatography–mass spectrometry; metabolomics; molecular networking

DOI:  https://doi.org/10.1002/pca.3412
Metabolites. 2024 Jun 14. pii: 332. [Epub ahead of print]14(6):

Explainable AI to Facilitate Understanding of Neural Network-Based Metabolite Profiling Using NMR Spectroscopy.

Hayden Johnson, Aaryani Tipirneni-Sajja.

  Neural networks (NNs) are emerging as a rapid and scalable method for quantifying metabolites directly from nuclear magnetic resonance (NMR) spectra, but the nonlinear nature of NNs precludes understanding of how a model makes predictions. This study implements an explainable artificial intelligence algorithm called integrated gradients (IG) to elucidate which regions of input spectra are the most important for the quantification of specific analytes. The approach is first validated in simulated mixture spectra of eight aqueous metabolites and then investigated in experimentally acquired lipid spectra of a reference standard mixture and a murine hepatic extract. The IG method revealed that, like a human spectroscopist, NNs recognize and quantify analytes based on an analyte's respective resonance line-shapes, amplitudes, and frequencies. NNs can compensate for peak overlap and prioritize specific resonances most important for concentration determination. Further, we show how modifying a NN training dataset can affect how a model makes decisions, and we provide examples of how this approach can be used to de-bug issues with model performance. Overall, results show that the IG technique facilitates a visual and quantitative understanding of how model inputs relate to model outputs, potentially making NNs a more attractive option for targeted and automated NMR-based metabolomics.

Keywords:  NMR metabolomics; NMR spectroscopy; explainable AI; integrated gradients; machine learning; metabolite profiling; neural networks

DOI:  https://doi.org/10.3390/metabo14060332
Anal Bioanal Chem. 2024 Jun 28.

Scaling up a targeted exposome LC-MS/MS biomonitoring method by incorporating veterinary drugs and pesticides.

Md Zakir Hossain, Max L Feuerstein, Yunyun Gu, Benedikt Warth.

  Humans are exposed to a cocktail of food-related and environmental contaminants, potentially contributing to the etiology of chronic diseases. Better characterizing the "exposome" is a challenging task and requires broad human biomonitoring (HBM). Veterinary drugs (VDs)/antibiotics, widely used and regulated in food and animal production, however, are typically not yet included in exposomics workflows. Therefore, in this work, a previously established multianalyte liquid chromatography-tandem mass spectrometry (LC-MS/MS) method covering >80 diverse xenobiotics was expanded by >40 VDs/antibiotics and pesticides. It was investigated if the generic workflow allowed for the successful integration of a high number of new analytes in a proof-of-principle study. The expanded method was successfully in-house validated and specificity, matrix effects, linearity, intra- and inter-day precision, accuracy, limits of quantification, and detection were evaluated. The optimized method demonstrated satisfactory recovery (81-120%) for most of the added analytes with acceptable RSDs (<20%) at three spiking levels. The majority of VDs/antibiotics and pesticides (69%) showed matrix effects within a range of 50-140%. Moreover, sensitivity was excellent with median LODs and LOQs of 0.10 ng/mL and 0.31 ng/mL, respectively. In total, the expanded method can be used to detect and quantify more than 120 highly diverse analytes in a single analytical run. To the best of the authors' knowledge, this work represents the first targeted biomonitoring method integrating VDs with various other classes of pollutants including plasticizers, PFAS, bisphenols, mycotoxins, and personal care products. It demonstrates the potential to expand targeted multianalyte methods towards additional groups of potentially toxic chemicals.

Keywords:  Biomarkers of exposure; Exposome research; Food safety; Mass spectrometry; Veterinary drugs/antibiotics; Xenobiotics

DOI:  https://doi.org/10.1007/s00216-024-05374-x
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jun 24. pii: S1570-0232(24)00226-5. [Epub ahead of print]1242 124217

Quantification and analyses of seven tyrosine kinase inhibitors targeting hepatocellular carcinoma in human plasma by QuEChERS and UPLC-MS/MS.

Yan Liang, Yilin Li, Li Song, Xiaolan Zhen, Jiangning Peng, Hui Li.

  Tyrosine kinase inhibitors (TKIs) are commonly used to treat various cancers. Literature suggests that the blood concentration of TKIs strongly correlates with their efficacy and adverse effects. Therefore, establishing a Therapeutic Drug Monitoring (TDM) methodology for TKI drugs is crucial to improving their clinical efficacy and minimizing the treatment-related adverse effects. However, quantifying their concentrations in the plasma using existing methods to avoid potential toxicity is challenging. Herein, seven TKIs, namely sorafenib tosylate, axitinib, erlotinib, cediranib, brivanib, linifanib, and golvatinib, were successfully analyzed in human plasma by following a quick, easy, cheap, effective, rugged, and safe (QuEChERS) pretreatment method combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Briefly, biological samples were extracted using 1 mL of methanol, followed by the sequential addition of 250 mg of anhydrous magnesium sulfate and 25 mg of N-propylethylenediamine (PSA) for salinization and purification by adsorption, respectively. In this study, dovitinib was used as the internal standard. The seven TKIs were detected by the gradient elution method for 4 min in the positive ion electrospray mode. The mobile phase comprised methanol (phase A) and 0.1 % aqueous formic acid solution (phase B) on the Agilent Zorbax RRHD Stablebond Aq, (2.1 × 50 mm; 1.8 μm). Brivanib, linifanib, axitinib, sorafenib tosylate, and golvatinib exhibited good linearity in the range of 5-500 ng/mL, and erlotinib and cediranib exhibited good linearity in the range of 10-1000 ng/mL, with linear correlation coefficients (R2) ≥ 0.99. The limits of detection and quantification were 0.60-0.18 ng/mL and 5-10 ng/mL, respectively. The intraday and interday accuracy values ranged from -6.12 % to 7.31 %, with a precision (RSD) of ≤ 10.57 %. The method was rapid, accurate, specific, simple, reproducible, and suitable for the quantitative determination of the seven TKIs in human plasma.

Keywords:  Hepatocellular carcinoma; Human plasma; QuEChERS; TKI; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2024.124217
Toxins (Basel). 2024 Jun 04. pii: 260. [Epub ahead of print]16(6):

Tetrodotoxin Derivatization with a Newly Designed Boron Reagent Leads to Conventional Reversed-Phase Liquid Chromatography.

Shimba Kawasue, Kyoko Kuniyoshi, Masashi Uema, Naomasa Oshiro.

  Tetrodotoxin (TTX) is a representative natural toxin causing pufferfish food poisoning, which is especially prominent in East and Southeast Asia, including Japan. TTX has been analyzed through post-column derivatization high-performance liquid chromatography (HPLC), ion-pair LC-MS(/MS), and hydrophilic interaction liquid chromatography (HILIC)-MS(/MS) as alternatives to the mouse bioassay method. However, post-column derivatization requires a system for online derivatization reactions, and with the ion-pair LC-MS approach, it is difficult to remove residual ion-pair reagents remaining in the equipment. Moreover, HILIC-MS provides poor separation compared to reversed-phase (RP) HPLC and requires a long time to reach equilibration. Therefore, we decided to develop a TTX analytical method using pre-column derivatization and RP HPLC for the rapid assessment of outbreak samples, including food remnants. In this study, we focused on the vic-diol moiety of TTX and designed a new derivatization reagent coded as NBD-H-DAB. This NBD-H-DAB was synthesized from 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H) and 3-fluoro-2-formylphenylboronic acid (FFPBA) with a simple reaction system and rapidly converted to its boronate form, coded NBD-H-PBA, in an aqueous reaction solution. The NBD-H-PBA demonstrated appropriate hydrophobicity to be retained on the RP analytical column and successfully detected with a UV spectrometer. It was easily reacted with the vic-diol moiety of TTX (C6 and C11) to synthesized a boronic ester. The derivatized TTX could be detected using the RP HPLC-UV, and the limit of detection in the fish flesh samples was 0.06 mg/kg. This novel pre-column derivatization of TTX with NBD-H-PBA proves capable for the analysis of TTX.

Keywords:  boronic acid; derivatization; pufferfish; reversed-phase HPLC-UV; tetrodotoxin

DOI:  https://doi.org/10.3390/toxins16060260
Anal Bioanal Chem. 2024 Jun 27.

Single ion mobility monitoring (SIM2) stitching method for high-throughput and high ion mobility resolution chiral analysis.

Clément Chalet, Estelle Rathahao-Paris, Sandra Alves.

  Chiral analysis is of high interest in many fields such as chemistry, pharmaceuticals and metabolomics. Mass spectrometry and ion mobility spectrometry are useful analytical tools, although they cannot be used as stand-alone methods. Here, we propose an efficient strategy for the enantiomer characterization of amino acids (AAs) using non-covalent copper complexes. A single ion mobility monitoring (SIM2) method was applied on a TIMS-ToF mass spectrometer to maximize the detection and mobility separation of isomers. Almost all of the 19 pairs of proteinogenic AA enantiomers could be separated with at least one combination with the chiral references L-Phe and L-Pro. Furthermore, we extended the targeted SIM2 method by stitching several mobility ranges, in order to be able to analyze complex mixtures in a single acquisition while maintaining high mobility resolution. Most of the enantiomeric pairs of AAs separated with the SIM2 method were also detected with this approach. The SIM2 stitching method thus opens the way to a more comprehensive chiral analysis with TIMS-ToF instruments.

Keywords:  Amino acids; Chiral analysis; Ion mobility; Mass spectrometry; TIMS

DOI:  https://doi.org/10.1007/s00216-024-05399-2
Biomed Chromatogr. 2024 Jun 26. e5947

Liquid chromatography-tandem mass spectrometry for determination of fingolimod and its active metabolite fingolimod phosphate in whole blood of patients with multiple sclerosis.

Veronika Pesakova, Hana Brozmanova, Pavel Sistik, Zuzana Kusnirikova, Ivana Kacirova, Milan Grundmann.

  Fingolimod is an oral drug for the escalation of treatment of relapsing-remitting multiple sclerosis in patients with persistent disease activity on first-line drugs or in patients with rapidly progressive severe relapsing-remitting multiple sclerosis. An ultra-high-performance liquid chromatography-tandem mass spectrometry method for determining the concentrations of fingolimod and its active metabolite fingolimod phosphate in whole blood has been developed and validated. The advantages of this method are the easy, fast and cheap sample preparation using protein precipitation from blood with a mixture of acetonitrile-methanol (40:60, v/v). Chromatographic separation was performed on a ultra-high performance liquid chromatography BEH C18 1.7 μm (100 × 2.1 mm) column. Two modes of ionization, electrospray ionization and atmospheric pressure chemical ionization, were tested and compared. For validation, the electrospray ionization mode was chosen. As internal standard, isotopically labeled fingolimod-D4 was used to quantify the analytes. The method was validated according to the rules of the European Medicines Agency. The coefficients of variation for fingolimod were in the range of 1.13-11.88%, and the recovery was 98.80-106.00%. The coefficients of variation for fingolimod phosphate were in the range of 2.73-9.31%, and the recovery was 90.08-107.00%. The method is quite easy and fast and can be used for routine analysis.

Keywords:  fingolimod; fingolimod phosphate; liquid chromatography; mass spectrometry; multiple sclerosis

DOI:  https://doi.org/10.1002/bmc.5947
Int J Anal Chem. 2024 ;2024 6386570

Quantification of Rhodojaponin II and Rhodojaponin III in Rat Plasma by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Cheng Sun, Wanhang Wang, Xi Bao, Dizhong Chen, Shenshen Mei, Jianshe Ma, Xiajuan Jiang.

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to determine the concentrations of Rhodojaponin II and Rhodojaponin III in rat plasma, and their pharmacokinetic profiles were investigated. A UPLC HSS T3 (2.1 mm × 50 mm, 1.8 μm) chromatographic column was employed at a temperature of 40°C. The mobile phase consisted of acetonitrile-0.1% formic acid in water, and a gradient elution method with an elution time of 6 min and flow rate of 0.4 mL/min was utilized for analysis purposes. Methodological investigations were conducted accordingly. The plasma concentrations of Rhodojaponin II and Rhodojaponin III exhibited excellent linearity within the range of 2 ng/mL-1250 ng/mL. Moreover, both intraday and interday precision were below 15%, while accuracy ranged from 88% to 115%. Additionally, matrix effect fell within the range of 90%-110%, and recoveries ranged from 78% to 87%. These results comply with relevant regulations for drug analysis in biological samples. Therefore, this method is deemed suitable for quantifying Rhodojaponin II and Rhodojaponin III levels in rats.

DOI: https://doi.org/10.1155/2024/6386570
J Chromatogr Sci. 2024 Jun 23. pii: bmae042. [Epub ahead of print]

Problems in Commercial Kits of 25-Hydroxy Vitamin D and the Development of Simple, Robust and Faster HPLC Method.

Syed Muhammad Saad, Abdul Rafiq Khan, Khalid Mohammed Khan, Ali Al-Othaim.

Vitamin D is a lipid-soluble compound that plays a key role in bone mineral metabolism. The commercial current kits and several published assay methods (High-performance liquid chromatography (HPLC) and Immunoassay) are complicated due to the use of multiple reagents, larger sample volume, high backpressure, longer extraction time, evaporation under nitrogen after extraction, significant interference and antibody cross-reactivity. Here we report a new HPLC method for the determination of 25-hydroxyvitamin D2 (25-OHD2) and 25-hydroxyvitamin D3 (25-OHD3) that is simple (no evaporation), rapid (10-minute run time) and robust. Serum sample (300 μl) is mixed with 300 μl acetonitrile containing lauraphenone as internal standard. After vortexing and centrifugation, the supernatant was loaded into C18 extraction cartridges, washed with 70% methanol and then eluted with 200 μl of a mixture of 70% ethanol and 30% isopropyl alcohol (IPA). The eluent was mixed with 50 μl of water and injected into the HPLC-UV system for analysis. The method proved to be linear in the range of 10-750 nmol/L of 25-OHD2 and 25-OHD3. The intra- and inter-assay precision was less than 10 for both compounds at four different concentrations. The method was compared with (LC-MS/MS) and the correlation coefficients (R2) were 0.9454 and 0.9673 for 25-OHD2 and 25-OHD3 respectively. The proposed HPLC method is simple, rapid, robust and free from the most common problems encountered with commercial kits. It can be used in a high-volume laboratory that uses the HPLC method for the simultaneous determination of 25-OHD2 and 25-OHD3 in serum samples.

DOI: https://doi.org/10.1093/chromsci/bmae042
Food Chem. 2024 Jun 18. pii: S0308-8146(24)01806-5. [Epub ahead of print]457 140156

Differentiation of Cis/trans-geometrical isomers in long-chain unsaturated fatty acids based on ion mobility and theoretical calculations.

Sisi He, Lulu Shi, Zi-Wei Zheng, Fangling Wu, Chuan-Fan Ding.

  In recent years, fatty acids containing conjugated CCs have attracted extensive research attention due to their biological activities against human diseases. However, their differentiation is challenging. This study developed a comprehensive analytical solution to accurately differentiate cis/trans-fatty acid isomers using ion mobility mass spectrometry (IM-MS) and theoretical calculations. Cis/trans-fatty acids were mobility-differentiated via simple complexation with 1,5,9-triazacyclododecane (9C3N) or 1,4,8,11-tetraazacyclotetradecane (10C4N) and metal ions, obtaining baseline separation with a peak-to-peak resolution of 0.35-0.92. Moreover, the conformation of the complexes was optimized theoretically, revealing different binding modes between the cis/trans-fatty acid-9C3N/10C4N-metal ion systems, yielding in-depth structural data on the complexes and elucidating the principles of mobility separation. Furthermore, the proposed method was assessed in terms of quantification, accuracy, and precision repeatability. Finally, the method was applied to analyze oil samples. Given its simplicity, speed, and lack of chemical derivatization or chromatographic separation, this technique has potential applications in food analysis.

Keywords:  Cis/trans fatty acid; Differentiation; Ion mobility mass spectrometry; Oil sample; Theoretical calculation

DOI:  https://doi.org/10.1016/j.foodchem.2024.140156
J Chromatogr A. 2024 Jun 20. pii: S0021-9673(24)00477-1. [Epub ahead of print]1730 465103

Ultrasensitive enantiomeric barbiturate analysis in body fluids through capillary electrophoresis with large volume sample stacking and ultrasound assisted dispersive liquid liquid microextraction.

Meng-Chin Chen, Ming-Mu Hsieh, Xin-Yu Huang.

  A rapid, straightforward, and sensitive approach to quantifying enantiomeric barbiturates in serum was developed by integrating ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME) with large-volume sample stacking (LVSS) in capillary electrophoresis (CE). UA-DLLME was employed for sample preparation, and on-column preconcentration by using LVSS with polarity switching was implemented to enhance sensitivity. We thoroughly investigated and optimized various parameters influencing extraction and stacking to achieve optimal detection performance with the highest enrichment efficiencies. Under optimal extraction conditions (injection of a mixed solution containing 40 μL of CHCl3 and 200 μL of tetrahydrofuran into 1 mL of a sample solution at pH 10.0), LVSS was performed using 600 mM Tris-boric acid (pH 9.5) containing 35 mM hydroxypropyl-β-cyclodextrin and sodium taurodeoxycholate hydrate. A voltage of 20 kV was applied and a preinjection water plug was loaded at a height of 25 cm for 10 s. Subsequently, the sample solution was injected at a height of 25 cm for 480 s, after which a voltage of -20 kV was applied and the sample stacking was initiated. The stacking process was completed when 95 % of the separation current was attained. Under optimized conditions, the contraction folds of the four barbiturate analytes (R, S-Secobarbital, R, S-pentobarbital) were improved by approximately 6400-fold, achieving detection limits of 0.1 ng/mL. The limits of quantification for all analyte enantiomers were 0.5-50 ng/mL, demonstrating good linearity (r > 0.997). Migration times exhibited a relative standard deviation of less than 1.7 %, whereas peak areas for the four analytes exhibited a deviation of 8.7 %. Finally, the established method was effectively applied to the analysis of human serum samples.

Keywords:  Body fluids; Crystallographic electrophoresis; Enantiomeric barbiturate drugs; Large-volume sample stacking; Ultrasound-assisted liquid-liquid microextraction

DOI:  https://doi.org/10.1016/j.chroma.2024.465103
Adv Lab Med. 2024 Jun;5(2): 173-180

Determination of ertapenem in plasma and ascitic fluid by UHPLC-MS/MS in cirrhotic patients with spontaneous bacterial peritonitis.

Raúl Rigo-Bonnin, Alberto Amador, María Núñez-Gárate, Virgínia Mas-Bosch, Ariadna Padullés, Sara Cobo-Sacristán, José Castellote.

   Objectives: Spontaneous bacterial peritonitis is a frequent severe complication in cirrhotic patients with ascites. Carbapenem antibiotics are currently the treatment of choice for patients with hospital-acquired or healthcare-related infections. However, there is limited evidence available on the efficacy of ertapenem in cirrhotic patients with spontaneous bacterial peritonitis. As a result, the pharmacokynetics and pharmacodynamics of this antibiotic are still unknown. The objective of this study was to develop and validate measurement procedures based on liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to determine ertapenem concentrations in plasma and ascitic fluid.
Methods: Samples were pretreated by acetronile protein-precipitation. Chromatographic separation is performed on a C18 reversed-phase Acquity®-UPLC®-BEHTM column (2.1 × 100 mm id, 1.7 µm) using a non-linear gradient of water/acetonitrile containing 0.1 % of formic acid at a flow rate of 0.4 mL/min. Ertapenem and its internal standard (ertapenem-D4) are detected by tandem mass spectrometry using positive electrospray ionization and multiple reaction monitoring, and using 476.2 → 346.0/432.2 as mass transition for ertapenem and 480.2 → 350.0 for its internal standard.
Results: No significant interferences or carry-over contamination were observed. Imprecisions, absolute relative bias, matrix effects and normalized recoveries were ≤14.5 %, ≤9.3 % (92.8-104.5) % and (98.8-105.8) %, respectively. Chromatographic measurement procedures were linear from (0.50-100) mg/L.
Conclusions: The measurement procedures based on UHPLC-MS/MS developed and validated in this study could be useful in pharmacokynetic and pharmacodynamic studies in subjects with liver cirrhosis who develop spontaneous bacterial peritonitis treated with ertapenem.

Keywords:  UHPLC-MS/MS; ascitic fluid; ertapenem; plasma

DOI:  https://doi.org/10.1515/almed-2023-0168